XXVI Reunião Anual da FeSBE - FeSBE 2011 Resumo:01-001 Lima, A. M.

Transcription

XXVI Reunião Anual da FeSBE - FeSBE 2011
Resumo:01-001
BRAZIL NUT HOTSPOTS: A STRATEGY FOR QUALITY CONTROL
Lima, A. M. 1; Souza, L. M. F. 1; Costa, M. S. 1; Melo, B. M. S. 1; Oliveira, K. A. 1; Silva, A. V. C. 1;
Borges, L. M. G. 3; Gonçalves, E. C. 2; Schneider, M. P. C. 2; Teixeira, F. M. 1
1
Departamento de Farmácia, ICS - UFPA
2
Departamento de Biologia Molecular e Genética, ICB - UFPA
3
Laboratório Nacional Agropecuário no Estado do Pará, LANAGRO-PA
Objectives:
The main objective of this work was to study the potential risks, critical control points and the main problems in productive
design of Brazil nut produced and merchandized in the metropolitan area of Belém, in Pará state, and also to investigate fungi
presence with potential to produce aflatoxins in this aliment.
Methods and Results:
A list of questions based on the RDC Resolution No. 218 and the Codex Alimentarius was used to interview eight Ver-o-peso fair
vendors and eight Brazil nut gatherers living in forests areas. On this occasion cleaning and sanitizing local aspects,
instrumentation, use of personal protective equipment (PPE), waste management, appropriated time and seasonal work
conditions, temperature and humidity, pest and vectors control, and Brazil nut beneficial conditions have been observed. Samples
were collected from three localities: Brazil nut gathering place in the forest, fair of sale of this product in Ver-o-peso market and
from in-cans and in-bags hermetically packed and sealed industrial material proceeded from supermarkets. The collected samples
had been cut into 5 mm 10 pieces and placed in Petri dishes containing Sabouraud Agar with Chloramphenicol and incubated at
25°C for at least 5 days. Posteriorly, with the purpose of identifying the fungi, we used the slide culture technique followed by
with Lactophenol Cotton Blue staining to visualize the microscopic structural characteristics of fungi and the amplification of
DNA technique by polymerase chain reaction (PCR) for phylogenetic confirmation. The level of aflatoxin contamination in all
samples was evaluated by High Pressure Liquid Chromatography (HPLC). The answers to the interviews and the evaluation
made by the technical staff demonstrated that the two sites of Brazil nuts manipulation, native forests and Ver-o-peso fair, need
cares aimed at improving the sanitary quality of the product. We observed the growth of fungi in 95% of the pieces from Ver-opeso market and native forests, while there was a markedly lesser growth among the samples proceeding from industry and
purchased in supermarkets (10%). The macroscopic and microscopic characteristics and molecular biology analysis indicated the
presence of several fungi, among which we point out the genera Aspergillus, Fusarium and Penicillium, which are capable of
producing carcinogenic substances or cause infection and pathological conditions in man. The levels of aflatoxin contamination
in native forests and Ver-o-Peso fair samples were well above the limits recommended by National and International legislation.
On the other hand, samples from industry and that had been purchased in supermarkets showed no detectable levels of aflatoxins.
Conclusions:
Our results indicate that, although part of the Brazil nut handlers receive training and instruction on the need to care for ensuring
the quality of their products, there is still no commitment by part of these same workers with respect to measures required for
Good Handling Practices for Food and maintenance of hygienic and sanitary aspects needed. The index of aflatoxin
contamination in the samples studied and the presence of strains of microorganisms capable of producing mycotoxins indicate the
immediate need for control and assurance actions of the product quality in outlining Brazil nut production marketed in Belém,
Pará.
Keywords: Brazil nut, HACCP, Quality control, aflatoxin, sustainability
Financial Support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Resumo:01-002
CHARACTERIZATION OF BUTYRYLCHOLINESTERASE-LIKE ACTIVITY FROM BRAIN OF BEIJUPIRA
(RACHYCENTRON CANADUM)
Oliveira, V. M. 1,1,1,1; Assis, C. R. D. 1,1,1,1; Linhares, A. G. 1,1,1,1; Carvalho, E. V. M. M. 1,1,1,1; Carvalho
Junior, L. B. 1,1,1,1; Porto, A. L. F. 2,2,2,2; Bezerra, R. S. 1,1,1,1
1
Departamento Bioquímica/Universidade Federal de Pernambuco, UFPE
2
Morfologia e Fisiologia Animal/Uni. Fed. Rural de Pernambuco, UFRPE
Objectives:
Butyrylcholinesterase (BChE; EC 3.1.1.8) is one of the main detoxification enzymes which hydrolyze or scavenge a broad range
of xenobiotics and its rapid action is partly responsible for homeostatic conditions by protecting acetylcholinesterase (AChE; EC
3.1.1.7) against anticholinesterasic pesticides such as organophosphorus and carbamates. In some cases, these pesticides present
an inhibitory action on BChE activity which is stronger than on AChE (Hum Ecol Risk Assess 8; 165, 2002). Monitor and control
the presence of these compounds in the environment is of vital importance. To identify changes in enzyme behavior caused by
these compounds, first is necessary to investigate its normal activity. This study aimed to characterize physicochemical and
kinetic parameters of the BChE-like activity from brain of R. canadum in order to be an alternative component in biosensor
devices.
The enzymes used were from juvenile specimens of R. canadum (51.67 ± 1.5 cm; 1.575 ± 0.329 kg). The animals were sacrificed
by an ice bath and the brains were removed, pooled and weighed. The homogenates were prepared in tissue disrupter and diluted
in Tris-HCl buffer 0.5 M pH 8.0 until reach 20 mg of tissue per mL of buffer. The preparation were centrifuged at 1000 x g
during 10 min. The optimal pH and temperature were determined assaying the activity of the extracts in a pH range from 2.5 to
9.0 and temperatures from 0 to 80ºC, respectively. From temperature data was estimated the activation energy (Ea) of the
enzymatic reaction by Arrhenius plot. Kinetic parameters km, Vmax, km ratio and Vmax ratio were calculated after determinate
activity under increasing concentrations (0.8 to 20.8 mM final concentration) of the substrates S-butyrylthiocholine,
acetylthiocholine and propionylthiocholine iodides. The extracts were assayed in presence of specific cholinesterases inhibitors
(concentrations from 0.001 to 10 mM) BW284c51 for AChE, Iso-OMPA for BChE, neostigmine and eserine for total ChEs. The
enzymatic activity was determined by a colorimetric method (Biochem. Pharmacol. 7:88-95, 1961). Optimum pH was found to
be next to 8.0 and optimum temperature was 35ºC. The km and Vmax of R. canadum BChE-like activity were 4.23 mM and 31.9
mU/mg protein, respectively. The activity was almost completely inhibited at 0.01 mM eserine and was inhibited by Iso-OMPA
1mM. The Ea estimated for the enzymatic reaction was 7.1701 Kcal/mol.
Conclusions:
Part of cholinesterase activity in the brain of R. canadum can be ascribed to BChE-like activity. Several physicochemical and
kinetic features of this activity were observed and these characteristics can be useful as assay conditions for these biomolecules in
biotechnological applications such as biosensors.
Keywords: ACTIVITY, BEIJUPIRA, BUTYRYLCHOLINESTERASE, BRAIN, Rachycentron canadum
Financial Support: CNPq
Resumo:01-003
EFFECT IN VIVO AND IN VITRO OF ALUMINUM ON THE ACTIVITY OF TRYPSIN AND CHYMOTRYPSIN OF
FINGERLINGS TILAPIA (OREOCHROMIS NILOTICUS)
Silva, R. P. F. ; Oliveira, V. M. ; Assis, C. R. D. ; França, R. C. P. ; Vila Nova, M. X. ; Bezerra, R. S.
Universidade Federal de Pernambuco, UFPE
Objectives:
Digestive protease enzymes have been little explored as monitoring tools. The main digestive proteases are trypsin (EC, 3.4.21.4)
and chymotrypsin (EC, 3.4.21.1). The activities of pepsin trypsin and chymotrypsin can be influenced by the interaction with
heavy metals from industrial wastewater and domestic. In recent years the levels of contaminants in the aquatic environment have
increased as a result of anthropogenic activities. Biomonitoring is important to identify potential behavioral changes caused by
various metallic elements, it becomes necessary, therefore, to investigate its activity in normal conditions and exposure. This
study in vivo and in vitro aimed to evaluate the activity of trypsin and chymotrypsin of Oreochromis niloticus, exposed to
aluminum (Al+3) in different concentrations.
The trial lasted 14 days, with a period of 5 days of adaptation, using 90 juvenile specimens, grown in 90 L aquaria comprising
daily cleaning with dynamic exchange of water, photoperiod of 12:12 h and feeding ad libitum. The animals were divided into
three groups, control (not subjected to metal: 27.29±0.34º C; pH 6.50±0.13; 6.31±0.32 mg.L-1 DO), exposed to 1 ppm
(27.30±0.05ºC, pH 6.26±0.33, 6.09±0.29 mg.L-1 DO) and exposed to 3 ppm (27.38±0.07ºC, pH 6.13±0.10, 6.01±0.09 mg.L-1
DO), performed in triplicate. After the cultivation period, the animals were killed by heat shock, their intestinal organs were
collected, macerated and homogenized in Tris-HCl 0.01 M pH 8.0 with 0.9% NaCl (w/v), obtaining the crude extract. The
enzyme activity was determined using 170μL of Tris-HCl 0.01 M pH 8.0, 30μL and 30μL of extract BApNA and Suc-Phe-p-Nan
for trypsin and chymotrypsin, respectively, as specific substrates. After incubation for 15 min., samples were read on ELISA with
a wavelength of 405 nm absorbance. The in vitro assay was performed incubating the Al3+ in extracts from muscle of 30 juvenile
specimens of the control treatment for a period of 1h, exposed to concentrations from 1 and 3 ppm. The residual activity observed
with trypsin in vivo was 100.0 ± 0.09%, 95.0 ± 0.54% and 91.0 ± 0.59%, while that for chymotrypsin was 100.0 ± 0.60%, 92.0 ±
0.33% and 73.0 ± 1.8% for the control groups, with control, 1 ppm and 3 ppm, respectively. The in vitro results were 100.0 ±
2.10%, 89.0 ± 2.89% and 83.0 ± 0.52% for trypsin, while for the chymotrypsin was 100.0 ± 3.21%, 97.0 ± 2.07% and 91.0 ±
3.68%.
Conclusions:
The results indicated a decreased activity of proteases as studied by increasing the concentration of metal used. Thus, it is
suggested to use these enzymes as useful tools in monitoring areas impacted by this metal.
Keywords: Tilapia, Trypsin, Chymotrypsin, Aluminium, Biomarker
Financial Support: CNPq, SEAPPR, FINEP/RECARCINE, FACEPE, PETROBRAS AMBIENTAL and
EMBRAPA.
Resumo:01-004
ANTIMICROBIAL ACTIVITY OF NATIVE STINGLESS BEE PROPOLIS FROM MATO GROSSO DO SUL STATE.
Trindade, C. S. P. C. 1; Campos, J. F. 2; Eberhardt, G. N. 3; Dalbosco, S. 4; Negrão, F. J. 4; Balestieri, J. B.
P. 6; de Picoli Souza K. 7; Santos, E. L. 8
1
School of Environmental and Biological Science, UFGD
2
School of Health Sciences, UFGD
Objectives:
Stingless bees are highly eusocial found in many tropical and subtropical regions of the world. They are the major visitors and
native pollinators of blossom plants in the tropics. Propolis is formed by resin and balsamic material, it is considered a protective
barrier against bees‟ enemies. Recent studies have reported the efficiency of propolis against microorganisms pathogenic to man,
but few data have been described to stingless bees. In this way, the present study aimed to characterize the antifungal activity
against Candida albicans of propolis produced by four stingless bees‟ species (Melipona quadrifasciata, Plebeia catamarcesis,
Plebeia droryana and Scaptotrigona depilis) found Mato Grosso do Sul State, Brazil.
The extract was made with an aliquot of propolis (1 g) which was dissolved in 4.5 ml of ethanol 80 %. The antimicrobial activity
was performed against Candida albicans by two methods (A) disk diffusion – that measures the diameter of the zone of inhibition
using culture medium Agar Sabouraud and (B) determination of the minimum inhibitory concentration (MIC) - that represents
the concentration of antimicrobial which there is complete inhibition of organism growth. The MIC of extract was evaluated in
eight concentrations ranging from 0.000085 to 1.4 mg/ml in plates of 96 well, using culture medium Brain Heart Infusion (BHI).
Experiments were made in triplicate. Data were expressed as mean ± SEM and submitted to statistical analysis by Student t test.
The differences were considered significant when p≤0.05. Results: The fungus Candida albicans growth was inhibited by
propolis‟s extract of Melipona quadrifasciata in both techniques. The extract presented 8 ± 0.3 mm of inhibition halo in disk
diffusion test and was effective at concentrations from 0.087 to 1.4 mg/ml in determination of the MIC. Antifungal activity was
undetected in any propolis sample of bees Plebeia catamarcesis, Plebeia droryana and Scaptotrigona depilis.
Conclusions:
Our data suggest that the propolis from stingless bee Melipona quadrifasciata found at Mato Grosso do Sul State has antifungal
activity with potential utilization for human medicine. Complementary studies will be developed to broaden the data presented.
Keywords: Melipona quadrifasciata, MIC, disk diffusion , Candida albicans , Antifungal
Financial Support: UFGD, Fundect, CAPES and CNPq
Resumo:01-005
PHYSICOCHEMICAL AND KINETIC CHARACTERIZATION OF CHOLINESTERASES FROM BRAIN OF
TUCUNARÉ (CICHLA OCELLARIS)
Silva, K. C. C. ; Assis, C. R. D. ; Oliveira, V. M. ; Linhares, A. G. ; Silva, R. P. F. ; Nova, M. X. V. ;
Maciel Carvalho, E. V. M. ; Carvalho Junior, L. B. ; Bezerra, R. S.
Universidade Federal de Pernambuco/Dep. de Bioquímica, UFPE
Objectives:
Acetylcholinesterase (AChE; EC 3.1.1.7) is the enzyme which hydrolyzes the neurotransmitter acetylcholine and its rapid action
is responsible for modulating neuronal communication. Butyrylcholinesterase (BChE; EC 3.1.1.8) is one of the main
detoxification enzymes which hydrolyze or scavenge a broad range of xenobiotics and its rapid action is partly responsible for
homeostatic conditions by protecting against anticholinesterasic pesticides such as organophosphorus and carbamates. This study
aimed to characterize physicochemical and kinetic parameters of the AChE and BChE-like activity from brain of C. ocellaris in
order to be used in biosensor devices.
The enzymes used were from juvenile specimens of C. ocellaris (31.17 ± 2.47 cm; 322 ± 14.28 g). The animals were sacrificed
by an ice bath and the brains were removed, pooled and weighed. The homogenates were prepared in tissue disrupter and diluted
in Tris-HCl buffer 0.5 M pH 8.0 until reach 20 mg of tissue per mL of buffer. The preparation were centrifuged at 1,000 x g
during 10 min. The optimal pH and temperature were determined, respectively, assaying the activity of the extracts in a pH range
from 2.5 to 9.0 and temperatures from 0 to 80ºC. The thermal stability was determined submitting the extracts to the same
temperatures during 30 min and, after equilibration, assaying the remaining activity. Kinetic parameters km and Vmax were
calculated after determinate the activity under increasing concentrations (0.8 to 20.8 mM final concentration) of the substrate
acetylthiocholine and S-butyrylthiocholine iodide. The extracts were assayed in presence of specific cholinesterases inhibitors
(concentrations from 0.001 to 10 mM) BW284c51 for AChE, Iso-OMPA for BChE, neostigmine and eserine for total ChEs. The
enzymatic activity was determined by a colorimetric method (Biochem. Pharmacol. 7:88-95, 1961). Optimum pH was found to
be 8.0-8.5 and optimum temperature was around 35ºC. C. ocellaris retained 35% of its AChE activity after incubation at 50ºC for
30 min. The kinetic parameter km for AChE activity was 0.76 mM. BChE-like activity was negligible and only presented a peak
at pH 8.5. The extract from C. ocellaris was weakly inhibited (13%) by Iso-OMPA at 10 mM and was highly inhibited (92%) by
BW284c51 at 0.1 mM. Neostigmine and eserine strongly inhibited ChE activity at 0.001 and 0.01 mM, respectively.
Conclusions:
Some physicochemical and kinetic features of cholinesterases from brain of C. ocellaris were observed and these characteristics
can be useful as assay conditions for these biomolecules in biotechnological applications such as biosensors. Moreover, the brain
of this species has negligible BChE content or the very little BChE-like activity can be ascribed to the ability of AChE to
hydrolyze the substrate butyrylthiocholine.
Keywords: acetylcholinesterase, butyrylcholinesterase, Cichla ocellaris
EMBRAPA.
Resumo:01-006
OXIDATIVE STRESS IN RHAMDIA QUELEN EXPOSED TO AGRICHEMICALS.
Ferreira, D. 3,2; Motta, A. C. D. 2; Kreutz, L. C. 2; Toni, C. 3; Loro, V. L. 3; Barcellos, L. J. G. 2,3; Maurer, L.
4
1
Farmacologia/Universidade Federal de Santa Maria, CCS/UFSM
Universidade de Passo Fundo, Curso de Medicina Veterinária, UPF
3
Programa de Pós-Graduação em Bioquimica Toxicológica, UFSM
4
Núcleo Integrado de Desenvolvimento e Análises Laboratoriais, Nidal/UFSM
2
Objectives:
The aim of this study was determine whether methyl parathion (MP), glyphosate (Gly) and tebuconazole (Teb) to be able to
induce oxidative stress in Rhamdia quelen and whether their effects can cause liver pathologies on this specie.
Twelve Rhamdia quelen (heptaperidae, teleostei) juveniles in each box, with six months of age, average weight 115,7± 23,3g
(SEM,n= 120), were exposed by 96 hours to commercial formulation of methyl parathion (0.8 mg L_1 of Folidol 600™),
tebuconazole (0.88 mg L_1 of Folicur 200 CE™) and glyphosate based herbicide (1.21 mg L_1 of Roundup™). After the
experimental period were collected samples from liver. Moreover, the following parameters were evaluated: TBARS
(thiobarbituric acid reactive substances - nmolMDA/mg protein), carbonilated protein (nmol carbonyl/mg protein), ascorbic acid
(μmol ASA/g tissue), activity of glutathione-S-tranferase (μmol GS-DNB/min/mg) and catalase (μmol/min/mg protein), and
histopathology. Data were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett's test (p ≤ 0.05). The liver
of Rhamdia quelen exposed to MP and Teb showed highest TBARS levels than control group (56% and 59%, respectively). In
contrast, Gly did not alter the TBARS generation. The carbonylated protein content was increased only in the fish exposed to
Teb. Fish exposed to the three agrichemicals have significantly decreased on tissue liver catalase activity (52%, 48%, and 67%,
respectively, when compared with control) and increased on glutathione-S-transferase activity (57%, 46%, and 160%,
respectively). Moreover, these same groups showed high content of reduced glutathione (151%, 472%, and 130%, respectively,
when compared with control) and ascorbic acid concentrations (121%, 102%, and 184%, respectively, when compared with
control), while the non-protein thiol content increased only in the group exposed to tebuconazole. Fish exposed to MP and Teb
showed several pathological changes in the liver, including hepatocyte degeneration and bile stagnation.
Conclusions:
MP and Teb induce oxidative stress in Rhamdia quelen and the liver is a sensitive target organ. Comparing both toxicants, the
results show that Teb seems to be the more toxic of the tested substances and it is able to induce severe hepatic lesions. The data
presented here demonstrate that the sublethal concentrations of methyl parathion and tebuconazole used in agricultural fields
cause changes in oxidative stress parameters as well as hepatic cell injuries in Rhamdia quelen, and that these parameters have
the potential to be used as biomarker of exposure to these agrichemicals.
Keywords: Tebuconazol, Hepatic damage, Methyl-parathion, Oxidative stress, Silver catfish
Financial Support: Fapergs, CNPq
Resumo:01-007
CHANGES IN BEHAVIOR AND CORTICOSTERONE LEVELS AFTER ENVIRONMENTAL ENRICHMENT IN A
CAPTIVE MOUNTAIN COATI (NASUELLA OLIVACEA)
1
Corredor, C. L. 1; Soto, A. C. 1; Lozano, I. 1; Cardenas, F. P. 2
INSTITUTO DE CONSERVACIÓN, USO SOSTENIBLE Y BIENESTAR ANIMAL, INCUBA
2
UNIVERSIDAD DE LOS ANDES, UNIANDES
Objectives:
Nasuella olivacea is endemic to the north Andean highlands in South America, mainly in western Venezuela, Colombia, Ecuador
and the far north of Peru. They are very poorly known, and have been classified as “data deficient” species by the International
Union for Conservation of Nature and Natural Resources (IUCN). Among the International Species Information System (ISIS)
registered institutions, only three zoos (all in USA) reported mountain coatis in early 2011, but one of these appears to be a case
of misidentification. A mountain coati that was confiscated from poachers is kept at La Reserva Biopark in Cota, Colombia.
When this coati arrived to the Biopark, it showed stress related behaviors including some stereotypies. At La Reserva Biopark,
the mountain coati is maintained in a 36 square meters enclosure with natural stimuli including trunks, nest branches and several
types of substrates devised to promote the normal behavior. At the Biopark, where wild animals with same behavioral features
are cared, many stress reduction aimed protocols are used, including environmental enrichment. In order to assess the effect of
environmental enrichment on stress in this mountain coati, the saliva corticosterone level as well as the frequency of stress related
behaviors were assessed before and after the placement of enrichment stimuli.
In order to compare the effect of enrichment, a basic behavioral description of exploration, locomotion, foraging, inactivity,
hiding and defensive postures was obtained before and after the placing of enrichment (sensory, feeding and physical stimuli).
Feeding enrichment was achieved by modifying the way in which the food was offered, physical enrichment consisted of a new
substrate left in the enclosure and sensory enrichment consisted in placing odor tracks in some areas of the enclosure. Saliva
samples were obtained twice a day for two days before the placing of the stimuli (baseline). Great variations in the amount of
corticosterone were found along the day. Early in the morning high levels were found (222.19 mg/dl) while low levels were
found in the evening (33.52 mg/dl). Our preliminary results showed some changes in general activity characterized by an increase
in exploration, locomotion and foraging and a decrease in hiding and defensive postures. The corticosterone level found in the
evening of the second day after the placing of stimuli was even lower than the one found before (23.87 mg/dl).
Conclusions:
Environmental enrichment with natural stimuli is used at the La Reserva Biopark as part of a strategy for recovering the
wellbeing of captive wild animals. Two days after placing new stimuli both the level of corticosterone in saliva and the frequency
of stress related behaviors were found decreased. More tests have to be done in order to determine more clear correlations.
However it seems that environmental enrichment is useful in order to reduce captive wild animal stress.
Keywords: CONSERVATION, STRESS, ANIMAL BEHAVIOR, WILD LIFE
Financial Support: FAPA grant UNIVERSIDAD DE LOS ANDES - INCUBA
Resumo:01-008
BUTYRYLCHOLINESTERASE ACTIVITY IN MUSCLE OF OREOCHROMIS NILOTICUS EXPOSED IN VIVO AND
IN VITRO TO ALUMINUM
Assis, C. R. D. ; Oliveira, V. M. ; Silva, R. P. F. ; Linhares, A. G. ; Silva, K. C. C. ; Nova, M. X. V. ;
Maciel Carvalho, E. V. M. ; Bezerra, R. S. ; Carvalho Junior, L. B.
Universidade Federal de Pernambuco/Dep. de Bioquímica, UFPE
Objectives:
Aluminum has become a major pollutant in water bodies around the world. Its presence is associated with diseases such
aluminosis, osteoarthritis, Alzheimer's, Parkinson's disease and microcytic hypochromic anemia. Butyrylcholinesterase (BChE;
EC 3.1.1.8) is one of the main detoxification enzymes which hydrolyze or scavenge a broad range of xenobiotics and its rapid
action is partly responsible for homeostatic conditions by protecting acetylcholinesterase (AChE; EC 3.1.1.7) against aggressive
agents, such as heavy metals. BChE has been used as a biomarker in aquatic organisms. Nile Tilapia, Oreochromis niloticus, is
the freshwater fish most cultivated in Brazil, making it an abundant source of this enzyme. This study evaluated the in vivo and in
vitro effect of aluminum (Al3+) on BChE activity in the muscle from O. niloticus. Exposure to Al3+ was performed at the
Department of Biochemistry, Universidade Federal de Pernambuco.
The trial lasted 14 days after a period of 5 days of adaptation, using 90 juvenile specimens, grown in 90 L aquaria comprising
daily cleaning with dynamic exchange of water, photoperiod of 12:12 h and feeding ad libitum. The animals were divided into
three groups, control (not subjected to metal: 27.29±0.34º C; pH 6.50±0.13; 6.31±0.32 mg.L-1 DO), exposed to 1 ppm
(27.30±0.05ºC, pH 6.26±0.33, 6.09±0.29 mg.L-1 DO) and exposed to 3 ppm (27.38±0.07ºC, pH 6.13±0.10, 6.01±0.09 mg.L-1
DO), performed in triplicate. The animals were sacrificed by an ice bath and the muscle were excised and weighed. The crude
extracts were prepared in tissue disrupter and diluted in Tris-HCl buffer 0.5 M pH 8.0 until reach 20 mg of tissue per mL of
buffer. The extracts were centrifuged at 1,000 x g during 10 min. The in vitro assay was performed incubating the Al3+ in
extracts from muscle of 30 juvenile specimens of the control treatment for a period of 1h, exposed to concentrations from 1 and 3
ppm. The enzyme activity was determined using 20 µL of extract, 200 µL of the chromogenic reagent DTNB 0.25 mM. The
reaction was monitored on a microplate spectrophotometer at 405 nm for 3 minutes after adding 20 µL of 62 mM
butyrylthiocholine. The activity of butyrylcholinesterase in vivo, in concentrations of 1 and 3 ppm, in relation to the controls,
were 105.15 ± 2.64% and 163.60 ± 4.15%, while in vitro were 168.28 ± 5.49% and 196.17 ± 4.08%, respectively.
Conclusions:
There was an increase in enzymatic activity of BChE, both in vivo and in vitro concomitantly with increasing concentration of
exposure to aluminum sulfate. The results provide conditions for the use of muscle BChE from Oreochromis niloticus as a
biomarker for the presence of aluminum in environments impacted by such xenobiotics.
Keywords: butyrylcholinesterase, aluminum, biomarker, tilapia
EMBRAPA
Resumo:01-009
EFFECTS OF ATRAZINE ON THE GILLS OF A NEOTROPICAL FRESHWATER FISH, THE CURIMBATÁ,
PROCHILODUS LINEATUS.
Fernandes, M. N. 1; Paulino, M. G. 1; Millan, D. C. 1; Pimentel, N. Q. 1; Simonato, J. D. 2; Martinez, C. B.
R. 2
1
Depto.Ciências Fisiológicas/Universidade Federal São Carlos, UFSCar
2
Depto.Ciências Fisiológicas/Universidade Estadual Londrina, UEL
Objectives:
Atrazine is an herbicide widely used to control undesirable plants in crop fields and acts by inhibiting photosynthesis. The
environmental atrazine contamination has increased in the recent years, and the herbicide has become a common contaminant of
superficial and underground water. Fish exposed to atrazine may affect the gills which are the main organ for respiration and play
a role in ion regulation, acid-base equilibrium and nitrogen excretion. The present study evaluated the effects of atrazine exposure
in the gill tissue and cells of the Neotropical freshwater fish, Prochilodus lineatus.
Juveniles P. lineatus (n = 6; Body mass = 13.15 ± 3.8) were exposed to 2 and 100 μg⁄L of atrazine and only water (control group)
during 96 hours. The gills were removed and processed for light and scanning electron microscopy for calculation of the Mean
Alterations Value (MAV) and chloride cell analyses. Fish exposed to 2 μg⁄L atrazine did not present significant difference from
the controls. Fish exposed to 100 μg⁄L atrazine increased the gill damage frequency compared to controls but still show punctual
distributed lesions in gill tissues. Most lesions were filament cell proliferation and filament and lamellar cell hypertrophy,
lamellar telangiectasis and aneurisms. The MAV calculated for controls and fish exposed to 2 and 100 μg⁄L atrazine were 1.3 ±
0.15; 1.5 ± 0.17; 2.0 ± 0.10 and showed significant difference between the controls and fish exposed to 100 μg⁄L atrazine. The
chloride cell density in contact with environmental water did not increased at the gill surface but the fractional surface area
increased 74% in relation to controls.
Conclusions:
These results showed that the exposure to atrazine affect the gas exchange and reduce the oxygen uptake from water as the
filament cell proliferation reduces the respiratory surface area and the cell hypertrophy causes an increase of water-blood
diffusion distance. Furthermore, the increase of the fractional area of chloride cells is directly related to increase ion uptake
suggesting a possible ion unbalance caused by atrazine exposure.
Keywords: chloride cells, herbicide, histopatology, ion regulation, respiration
Financial Support: CNPq/INCT-TA, FAPESP
Resumo:02-001
MICRORNAS 133A AND 133B ARE MODULATED BY AEROBIC TRAINING IN SPONTANEOUSLY
HYPERTENSIVE RATS.
Amadeu, M. A. ; Soci, Upr. ; Mattos, K. C. ; Oliveira, E. M.
School of Physical Education and Sport, EEFE-USP
Objectives:
MicroRNAs (miRNA) are small non coding RNAs that regulate post-transcriptional expression of their target genes. MiR-133,
which is enriched in cardiac and skeletal muscle, is involved in cell specification, differentiation, and development. It is also
modulated during cardiac hypertrophy (CH), which suggested that it may play a role in the underlying pathogenesis. We
investigated the role of aerobic physical training on miRNA-133 expression leading a possible cardiac improvement in
Spontaneously Hypertensive Rats (SHR).
Male Wistar Kyoto and SHR weighing about 200 grams (n=21) were randomized into three groups: Wistar Kyoto Sedentary
(WKY-S), SHR Sedentary (SHR-S) and SHR Trained (SHR-T). Training protocol: swimming sessions of 60min, 5 days/week,
during 10 weeks, with caudal dumbbells weighing 5% of body weight. It was assessed: 349 cardiac microRNAs by microarray,
heart rate (HR) e blood pressure (BP) by caudal pletismography, VO2max by ergoespirometry, CH by left ventricle ratio body
weight (LV/BW-mg/g) or tibia length (LV/TL-mg/mm). P
Conclusions:
Aerobic physical training increased of physical capacity in SHR. The up regulation of miR-133a and miR-133b expression in
SHR was reversed by aerobic physical training. These results open perspectives to research therapeutic approaches involving
modulation of miRNAs.
Keywords: MicroRNAs, Aerobic training, Spontaneously Hypertensive Rat, Heart
Financial Support: FAPESP
Resumo:02-002
IMPACT OF DIFFERENT CONCENTRATIONS OF ROUNDUP ORIGINAL® ON THE IN VITRO HEART
FUNCTION OF BULLFROG TADPOLES
Arena, M. V. N. ; Rissoli, R. Z. ; Salla, R. F. ; Ribeiro, L. R. ; Gamero, F. U. ; Costa, M. J.
LAFISC / Universidade Federal de São Carlos, UFSCar
Objectives:
Evaluate the in vivo effect of Roundup Original® on the heart function of bullfrog tadpoles, Lithobates catesbeianus.
Ventricular strips were prepared from the hearts of bullfrog tadpoles at the 25 Gosner stage (n=15). The strips were immersed in a
bath containing physiological solution and tied to a force transducer (LETICA, Letica Corporation, USA) connected to an
acquisition system of data analyzers (ANCAD, AVS, Solução Integrada, Ltda, Brasil). Contraction tension (Fc - % of control),
time for peak tension (TPT - ms) and the time to half relaxation were registered at 0.2Hz, and a dose-response curve was outlined
for the following Roundup concentrations: 0.01mg/L, 0.1mg/L, 1mg/L, 10mg/L and 100mg/L. The maximum frequency until
which the strips were able to contract regularly was determined and compared to the in vivo heart frequency (ƒH – Hz). The Fc
and TPT decreased (P < 0.05) in the concentration of 100 mg/L of Roundup in relation to the control, in contrast to the results
observed to THR, that remained unchanged after the increase in Roundup concentration. The maximal in vitro frequency at which
strips could contract regularly showed a stepwise decrease as Roundup concentrations were elevated: 1.5Hz at control, 1.4Hz at
0.01mg/L, 1.3Hz at 0.1mg/L, 1.2Hz at 1mg/L, 1.2Hz at 10mg/L and 1.1Hz at 100mg/L, which indicates that when strips are
exposed to Roundup, specially at concentration higher than 0.01 mg/L, the in vitro heart frequency becomes very close to the in
vivo frequency (1.1Hz).
Conclusions:
The results suggest that the negative effects of Roundup on the cardiac contractility of bullfrog tadpoles may be directly
correlated to the mechanism responsible by heart contraction (specially the L-channels), since the THR remained unchanged, but
the TPT and Fc were altered. Additionally, since the in vivo heart frequency is close to the maximal stimulation frequencies
reached at higher Roundup concentrations, the species heart seems to be working near to the upper limit of their excitationcontraction coupling after the exposure to the xenobiotic.
Keywords: roundup, bullfrog, heart function, herbicide, amphibian
Financial Support: INCT in Comparative Physiology (CNPq and FAPESP)
Resumo:02-003
HEMODYNAMIC MECHANISMS OF THE ATTENUATED BLOOD PRESSURE RESPONSE AFTER RESISTANCE
EXERCISE IN SUBJECTS WITH UNTREATED STAGE 1 HYPERTENSION
Barbosa, T. P. C. ; Chrispino, T. C. ; Neves, F. J. ; Mederos, R. F. ; Nóbrega, A. C. L.
Fisiologia e Farmacologia/ Universidade Federal Fluminense, UFF
Objectives:
Aerobic exercise training reduces blood pressure in subjects with hypertension an effect that results from the temporal summation
of the hypotensive effects occurring after each exercise session with potential therapeutic impact in hypertension. However,
whether these responses occur also after resistance exercise and the potential mechanisms involved are controversial issues to
date. The aim of this study was to determine the hemodynamic mechanism of the hypotensive effect after resistance exercise in
subjects with untreated stage 1 hypertension.
Six men with stage 1 hypertension (age 42±8 yrs; SBP 140-159 mmHg and/or DBP 90-99 mmHg) were recruited. All the
subjects fulfilled the following criteria: age between 20 and 60 years, body mass index ≤ 34.9 kg/m², no medications, sedentary,
and no disease except hypertension. Forearm blood flow was assessment by venous occlusion plethysmography and blood
pressure by ambulatory blood pressure monitoring and digital infrared photoplethysmography (Finometer®). The crossover
protocol included two days in random order: time control situation when no exercise was performed and one 45-min session of
resistance exercise of 6 body movements composed of 3 series of 12 repetitions at 40% 1RM. SBP decreased after exercise
(baseline: 147±6 mmHg vs. after: 134±1 mmHg, p=0.03), but not in control day (baseline: 140±5 mmHg vs. after: 148±3 mmHg,
p=0.29). This effect lasted for 24 hours in the intervention day (baseline: 141±6 mmHg vs. 24 hours after: 133±3 mmHg,
p=0.04), but not in the control situation (baseline: 132±4 mmHg vs. 24 hours after: 135±5 mmHg, p=0.74). No differences were
observed for DBP, cardiac output, stroke volume or heart rate (P>0.05). During reactive hyperemia, vascular conductance
increased (baseline: 6.12±0.41 a.u. vs. after: 14.66±1.62 a.u., p
Conclusions:
A single session of resistance exercise in subjects with untreated stage 1 hypertension causes sustained hypotension for 24h
probably due to increased vascular vasodilatory reactivity.
Keywords: HYPERTENSION, VASCULAR REACTIVITY, EXERCISE
Financial Support: Capes, CNPq, FAPERJ, FINEP
Resumo:02-004
EFFECTS OF EXPOSURE-TIME TO 17 α – ETINYLESTRADIOL ON THE CARDIAC FREQUENCY OF
BULLFROG
Victório, J. A. ; Gamero, F. U. ; Salla, R. F. ; Arena, M. ; Costa, M. J.
Laboratório de Fisiologia da Conservação , UFSCar - Sorocaba
Objectives:
Considering that heart frequency alterations are one of the first signals of cardiac failure, the aim of this work was to analyze the
effect the exposure of bullfrog tadpoles to 10ng/L of 17 α – etinylestradiol (EE2) during 48 h (acute exposure) and 16 days
(chronic exposure) over the heart frequency of these animals.
Tadpoles at 25 Gosner stage were exposed divided into four replicated experimental groups: controls (CA – control acute; CC control chronic) and animals exposed to 10 ng/L of 17 α – etinylestradiol (concentration environmentally relevant in Brazil)
during 48h (EA) or 16 days (EC). After exposure, animals were sacrificed by cephalic concussion and the heart was exposed by a
ventral incision for the visual determination of in loco heart frequency (fH – bpm). Thereafter, ventricle strips were mounted to
isometric contraction recordings to verify which was the maximal contraction frequency at which the heart was able to contract
regularly in vitro. For both exposure periods, exposure to EE2 resulted in a tachycardia (CA = 58 ± 2,4 bpm; EA = 66 ± 1,8 bpm;
CC = 84 ± 2,3 bpm; EC = 92 ± 1,9 bpm). The in vitro experiments indicated that the animals are not working in vivo close to the
upper limit of their excitation-contraction coupling, even after the exposure to EE2 (maximal in vitro heart frequency: CA = 96
bpm; EA = 120 bpm; CC = 108 bpm; EC = 96 bpm).
Conclusions:
EE2 seems to act directly over the auto-depolarization frequency of the cardiac pacemaker and results in an increase on energetic
expenditure. This highest energetic cost of the heart can shift energy that would be employed to promote morphogenic processes,
as animal development, which reflect the negative impact of the exposure of amphibians to realistic concentration of EE2.
Keywords: CARDIAC FREQUENCY, 17 α – ETINYLESTRADIOL, BULLFROG , EE2, HEART
Financial Support: INCT-Fisc, FAPESP
Resumo:02-005
QUANTITATIVE MORPHOLOGICAL EVALUATION OF FIBROELASTIC COMPONENTS IN THE ABDOMINAL
AORTA OF RATS SUBMITTED TO CHRONIC PASSIVE SMOKING
Alvarez, D. A. P. 1; Genovez, P. S. D. S. A. 1; Pissolato, M. 1; Fabrega-carvalho, C. A. 1,2
1
Departamento de Morfologia e Patologia Básica, FMJ
2
Departamento de Anatomia/ Instituto de Ciências Biomédicas , USP
Objectives:
Considering the power of environmental pollution, it is expected that individuals exposed to the secondhand smoke coming from
the lighted end of a cigarette suffer the same consequences as active smokers. Therefore, the objective of this study was to
evaluate possible alterations in the fibrillary components of the abdominal aorta of rats submitted to chronic passive smoking.
Male rats were divided into a control group (CG, n=10) and a smoking group (SG, n=10). SG animals were exposed daily to the
smoke of three cigarettes (high content of tar [10 mg], nicotine [0.8 mg], and carbon monoxide [10 mg]) for a period of 30 weeks.
CG animals were handled under the same conditions as SG animals but were not exposed to cigarette smoke. At the end of the
experimental period, the animals of the two groups were sacrificed with an overdose of the anesthetic. The abdominal aorta was
dissected, processed by routine histological procedures, stained with HE, picrosirius and Verhoeff stain, and examined by normal
and polarized light microscopy. A 100-point system was used for the determination of the relative area density (Vv/µm²) of
collagen and elastic fibers. Significant differences between groups (P
Conclusions:
Passive smoking promotes quantitative morphological and structural alterations that lead to the loss of elasticity and an increased
fragility of the wall of the abdominal aorta, which becomes susceptible to dilatations in the long term.
Keywords: abdominal aorta, tabagism, collagen fibers, elastic fibers
Financial Support: Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP
Resumo:02-006
EFFECTS OF THE ACUTE EXPOSURE TO 17-ALPHA-ETHINYLESTRADIOL ON THE CARDIAC FUNCTION OF
BULLFROG TADPOLES
Salla, R. F. ; Victório, J. A. ; Gamero, F. U. ; Costa, M. J.
LabFisc / Universidade Federal de São Carlos, UFSCar - Sorocaba
Objectives:
The aim of this study was to evaluate if the exposure of Lithobates catesbeianus tadpoles to a concentration of the xenoestrogen
17-alfa-ethinylestradiol (EE2) normally found in Brazilian inland waters (10 nM) can exert any affects on the cardiac function of
these animals.
For this, the animals were tested in duplicate, and divided into two experimental groups: exposed animals (EE2; n=12), and the
control group (CT; n = 12). During the exposure, the activity level (AL) displayed by tadpoles of each group was similar (p >
0,05). After sacrifice by cephalic concussion, in loco heart frequency was determined (ƒH – beats per minute, bpm). EE2 animals
presented a tachycardia (ƒH ~ 66 bpm) when compared to the controls (ƒH ~ 52 bpm, P < 0,05).
Conclusions:
These results indicate that EE2 seem to act directly on the cardiac muscle, rather than acting indirectly as a result of an increase
of the cardiac demand due to an increased activity level (escape response or aversive effect to the xenoestrogen). Moreover, the
comparison between the contraction force (Fc) developed between the two experimental groups showed the positive inotropic
effect of the xenoestrogen of bullfrog tadpoles.
Keywords: xenoestrogen, 17-ALPHA-ETHINYLESTRADIOL , bullfrog, cardiac function, amphibians
Financial Support: This study was supported by INCT-FisC and CNPq.
Resumo:02-007
OUABAIN INDUCES NITRIC OXIDE RELEASE IN RAT AORTA WITH AND WITHOUT SIGNS OF HEART
FAILURE: INVOLVEMENT OF THE PI3K/AKT-DEPENDENT PATHWAY.
Siman, F. D. M. 1; Silveira, E. A. 1,2; Fernandes, A. A. 1; Stefanon, I. 1; Vassallo, D. V. 1,2; Padilha, A. S. 1
1
Departamento de Ciências Fisiológicas- UFES, UFES
2
Escola Superior de de Ciências da Santa Casa de Misericórdia, EMESCAM
Objectives:
To investigate the acute effects of 3nM of ouabain (OUA) on the vascular reactivity of rats that developed or not heart failure
(HF) after myocardial infarction (MI), and propose the possible underlying mechanisms of action of this digitalis compound.
MI was induced by coronary ligation and the rats were divided in three groups: fictitious surgery (SHAM), MI (rats without signs
of HF) and HF (rats with signs of HF). Four weeks after MI, the weight and hemodynamic parameters of the three groups were
evaluated. Vascular reactivity to phenylephrine (PHE) of aortic rings were also evaluated in the presence and absence of the
OUA, in rings with or without endothelium and after incubation with N(G)-nitro-L-arginine methyl ester (L-NAME),
wortmannin (WORT) and tetraethylammonium (TEA). The HF group showed decreased body weight (BW), increased lung/BW
ratio and right ventricle/BW ratio, compared to other groups. The MI group showed an increased lung/BW ratio and right
ventricle/BW ratio, compared to SHAM. Infarct size was similar in both groups. The left ventricular end diastolic pressure
increased in the HF group compared to the others. The positive and negative rates of pressure development reduced in the MI and
HF groups. Vascular reactivity to PHE increased in HF group and remained unchanged in the MI group. OUA reduced the PHE
contractile response in all groups. This effect was mediated by endothelium. L-NAME increased the reactivity to PHE in all
groups, but, this response was smaller in MI and HF groups. The incubation with L-NAME+OUA potentiated the PHE response
in the three groups. WORT did not alter the response to PHE in the three groups. However, the incubation with WORT+OUA
increased the response to PHE in HF and MI groups. TEA increased the reactivity to PHE in all groups, but this effect was
smaller in MI group. The coincubation with TEA+OUA increased this response in HF and MI groups. OUA increased Akt
phosphorylation in HF and MI groups.
Conclusions:
Our results suggest that OUA decreases vascular reactivity to PHE in SHAM, MI and HF. This reduction in PHE response is
associated with an increased bioavailability of nitric oxide (NO). Therefore, OUA is able to increase NO production, but acts
through different mechanisms. In the SHAM rats, OUA increases NO production by a mechanism independent on PI3K/Akt
patways. In the MI and HF rats, OUA increases NO production by a mechanism dependent on PI3K/Akt patways. Moreover, in
these latter groups, OUA also increases potassium channels activation. These results suggest a beneficial effect of the OUA after
MI and HF, since this digitalis is capable of reversing, at least partially, the reduction of vasodilators factors, such as NO, and
releases a factor that seems to open potassium channels.
Keywords: Heart failure , Nitric oxide , Ouabain, Vascular reactivity
Financial Support: CNPq, CAPES, FAPES/FUNCITEC
Resumo:02-008
INCREASED SENSITIVITY TO RHO-KINASE INHIBITION IN SEPSIS INDUCED BY KLEBSIELLA
PNEUMONIAE
Corrêa, T. ; Sordi, R. ; Santos, J. E. S.
Dep. de Farmacologia - CCB, Universidade Federal de SC , FMC - UFSC
Objectives:
Activation of the RhoA/Rho-kinase pathway plays an important role in the vascular effects of drugs able to increase arterial
pressure, such as angiotensin II and alfa-1-adrenergic agonists. This pathway acts inhibiting the activity of myosin phosphatase,
improving the interaction between actin and myosine. Sepsis is frequently associated with hypotension and reduced vascular
responses to vasoconstrictors, two hallmarks of septic shock. Nevertheless, the relationship between the vascular dysfunction
found in septic states and the RhoA/Rho-kinase pathway has been scarcely studied. We have investigated the hypothesis that a
reduced functionality of this pathway may explain, at least in part, the cardiovascular failure seen in sepsis.
All procedures were approved by CEUA/UFSC (PP00344) Male Swiss mice (3-4 months) were anesthetized and inoculated with
Klebsiella pneumoniae (KP, 1.107 CFU; septic groups) into their trachea (a sub-lethal dose). Six, 24 or 48 h after these animals
were anesthetized (ketamine/xylazine, 90 and 75 mg/kg, i.m.), and had their femoral vein and carotid artery cannulated for drug
injection and mean arterial pressure (MAP) measurement, using a pressure transducer coupled to a Powerlab® system (ADI
Instruments, Australia). In these experiments, naïve mice were used as control. After the surgical procedures and an interval of 10
min for MAP stabilization, different groups received an intravenous injection of Y-27632 (0.1 mg/kg) or isotonic saline (10
µL/10 g), followed by phenylephrine (PE, 3, 10 and 30 nmol/kg, i.v.). Each subsequent drug was injected only when the arterial
pressure had returned to basal values (usually 10-15 min). Blood samples collected at the end of the experiments were subjected
to Griess reaction. Animals exposed to KP presented MAP values of 49.8 ± 8.3 and 57.5 ± 4.0 mm Hg, at 6 and 24 h after
bacterial inoculation, respectively. These values were significantly lesser than those found in the control group (91.6 ± 3.0 mm
Hg). Nitrate + nitrite levels were increased in plasma samples of KP inoculated mice (70.6 ± 0.7, 92,3 ± 6.8 and 370,0 ± 31.3 μM,
6, 24 and 48 h, respectively) when compared to control values (17,9 ± 2.5 μM). Taken together, these data confirm that
inoculation of KP induced a septic shock like state in our experiments. Interestingly, despite the absence of effects of Y-27632
(0,1 mg/kg, i.v.) on PE-increased MAP in control mice , the hypertensive effects of PE in KP groups at 6 and 24 h after bacterial
inoculation were deeply reduced by this Rho-kinase inhibitor (injection of PE at 10 nmol/kg increased MAP by 26.8 ± 1.9 and 22
± 2.6 mm Hg in control mice and 20.7 ± 2.1 and 8.3 ± 1.1 mm Hg in the group KP 24 h, before and after Y-27632 administration,
respectively; p < 0.01).
Conclusions:
Our results show an increased sensitivity to Y-27632 in K. pneumonia-induced a septic shock like state in mice, when its effects
on PE-increase MAP were evaluated in vivo. This finding suggests that changes in the expression or function of the Rho-A/Rhokinase pathway may take place in the pathophysiology of septic shock. Financial support: CNPq. Key Words: Sepsis, Klebsiella
pneumoniae, Rho-A/Rho-Kinase, Blood Pressure.
Keywords: Blood Pressure, Klebsiella pneumoniae, Rho-A/Rho-Kinase, Sepsis
Resumo:02-009
HYPOTENSIVE EFFECTS OF 6-GINGEROL FROM ZINGIBER OFFICINALE ROSCOE
Neto, A. G. D. S. 1; Ximenes, R. M. 1; Jorge, R. J. B. 1; Jorge, A. R. C. 1; Alves, N. T. Q. 1; Alves, R. D. S.
1
; Sousa, P. C. P. D. 1; Silva, J. A. D. 2; Vieria, P. C. 2; Monteiro, H. S. A. 1
1
Departamento de Fisiologia e Farmacologia, UFC
2
Departamento de Química, UFSCAR
Objectives:
In traditional medicine, Zingiber officinale Roscoe (ginger) has been used to treat a wide array of ailments including arthritis,
respiratory disorders, infectious diseases, diabetes and hypertension. These properties of ginger are attributed to the presence of
certain pungent vallinoids, [6]-gingerol, its most active constituent, and [6]-paradol, as well as some other constituents like
shogaols and zingerone. Thus, this work aims study the effects of 6-gingerol in blood pressure of rats Wistar normotensive.
Wistar male rats were used between after birth (270–300 g), n=6, anesthetized with sodium pentobarbital (50 mg/kg). The trachea
was isolated and a cannula of polyethylene (PE 120) was inserted into the animal through a tracheostomy, allowing for
spontaneous breathing easy and possible aspiration of bronchial secretions. After tracheostomy, two polyethylene catheters (PE
10 connected to PE 50) pre-filled with heparin (500 IU / ml in saline) were implanted through the femoral vessels in the
abdominal aorta to record mean arterial pressure and inferior vena cava to drug administration. At the time of the experiment, the
aortic catheter was connected to a pressure transducer piezo-electric (Braile BXSN) coupled to the signal amplification system
with USB interface (DI-148U DATAQ) and using the data acquisition software WinDaq / Lite. The mean arterial pressure was
calculated directly by software. Were used four doses to perform the dose-response curve 30, 100, 300 and 1000 mcg / kg and the
results were expressed as percentage of decrease compared to baseline. The percentage of decrease in each of the doses were:
11,85 ± 3,05 % (30 mcg/Kg), 16,43 ± 3,64 % (30 mcg/Kg), 27,56 ± 6,09 % (300 mcg/Kg) and 46,27 ± 3,86 % (1000 mcg/Kg).
Conclusions:
There was a drop in blood pressure at all doses studied and at the highest dose the reduction was on average 46.27%, thus
showing the hypotensive effects of 6-gingerol in anesthetized animals. However, more studies are needed to elucidate the
mechanisms involved in blood pressure.
Keywords: Gingerol, ginger, hypertension
Financial Support: FUNCAP, CNPq, CAPES
Resumo:02-010
PARTICIPATION OF THE ENDOTHELIUM AND K+ CHANNELS ON VASODILATION INDUCED BY NEW
PYRAZOLE-BASED COMPOUND.
Martins, D. R. 1; Pazini, F. 3; Lião, L. M. 3; Menegatti, R. 2; Rocha, M. L. 1
LAB. DE FARMACOLOGIA CARDIOVASCULAR. FACULDADE DE FÁRMACIA, LFC - UFG
2
LAB. QUÍMICA FARMACÊUTICA E MEDICINAL. FACULDADE DE FÁRMACIA, LQFM -UFG
3
LAB. DE RESSONÂNCIA MAGNÉTICA NUCLEAR. INSTITUTO DE QUÍMICA, LRMN - UFG
1
Objectives:
The pyrazole compounds are known to rarely occur in nature. Many synthetic derivatives of pyrazole compounds are of
importance as medicinal drugs and are used for their analgesic, anti-inflamatory, antiarrhythmic, antidiabetic, tranquilizing, etc.,
activities. In this study, we investigated some mechanisms involved in the relaxation induced by a new pyrazole-based compound
(LQFM021) in isolated rat aortic.
Male Wistar rats (180-200g) were killed by cervical dislocation and thoracic aortic was isolated, cut into 4mm long rings and
mounted for isometric tension recording in an organ bath. The experiments were approved by the Animal Care Committee of the
Federal University of Goiás. Thus, the isolated aortic rings with (E+) or without (E-) functional endothelium pre-contracted with
phenylephrine were exposed to LQFM021 (1μM to 300μM ) or vehicle (DMSO). In preparations E+, the participation of
endothelial prostanoids and NO/sGC pathway were analyzed through the use of such drugs as indomethacin (10μM ), L-NAME
(100μM ) and ODQ (1μM ) on relaxation induced by LQFM021. Additionally, the influence of K+ channels were verified using
some K+ channel blockers, such as TEA (non selective K+ channel blocker, 5 mM), 4-AP (Kv blocker, 1 mM), glibenclamide
(KATP blocker, 10μM ), BaCl2 (KIR blocker, 30μM ) and clotrimazole (KCa blocker, 10μM ). Relaxation induced by LQFM021
was higher in preparations E+ (Emax: 88.1¡±2.1%, n=5) than in E- (55.0¡±6.0%, n=5). Indomethacin, L-NAME and ODQ
reduced significantly (p
Conclusions:
From these results, we conclude that LQFM021-induced relaxation of the rat aortas is potentiated by endothelium and involves
participation of NO/sCG pathway and stimulation of prostanoids synthesis. Furthermore, the relaxation induced by LQFM021
appears to be extremely dependent of K+ fluxes across the membrane.
Keywords: AORTIC, K+ CHANNELS , ENDOTHELIUM, LQFM 021, VASODILATION
Financial Support: CAPES
Resumo:02-011
PARTICIPATION OF ADENOSINE RECEPTORS IN CARDIOPROTECTION INDUCED BY ENDOGENOUS
FACTORS RELEASED FROM HEARTS DURING THE ISCHEMIC PRECONDITIONING.
Caciano, A. R. ; Oliveira, D. F. . ; Cunha, N. R. D. ; Maciel, L. ; Nascimento, J. H. M.
Instituto de Biofisica Carlos Chagas Filho-UFRJ, IBCCF
Objectives:
Evaluate the involvement of adenosine A1 receptors in cardioprotection against ischemia/reperfusion injuries by endogenously
released factors released form hearts during the ischemic preconditioning.
METHOD: Hearts from male Wistar rats were cannulated in a Langendorff system and perfused at constant flow (10ml/min) with
Krebs-Henseleit solution. A latex balloon was inserted into the left ventricle and connected to a pressure transducer to record
intraventricular pressure. Experimental groups were: Control (n=5): Isolated hearts subjected to 30 min global ischemia followed
by a 60-min of reperfusion (I/R); IPC (n=5): Hearts subjected to ischemic preconditioning, (3 cycles of 5-min ischemia and 5-min
reperfusion), before I/R; RECEP (n=5): Hearts perfused with IPC coronary effluent collected for 15 min before I/R; DPCPX
(n=5): Hearts perfused with IPC coronary effluent plus 20 çM DPCPX, before I/R. At the end of reperfusion, the hearts were
sectioned and incubated with triphenyltetrazolium chloride (1%), to determine the planimetric area of infarct. RESULTS: The
infarct area of RECEP group (7,6 ± 1,6 %) was lower than the Control (39,7 ± 6,1 %) and similar to the IPC (9,7 ± 1,5 %).
DPCPX (27,2 ± 2,3%) increased the infarct area, compared to IPC and RECEP groups. The postischemic recovery of developed
pressure (PD) was increased in IPC (75%) and RECEP groups (71%), compared to the Control group (18%). DPCPX impaired
the increase of postischemic recovery of DP induced by perfusion of IPC coronary effluent (26%).
Conclusions:
The cardioprotection induced by endogenous factors released in coronary effluent during the ischemic preconditioning is
sensitive to DPCPX, an a A1 adenosine receptor antagonist.
Keywords: CARDIOPROTECTION , ISCHEMIC PRECONDITIONING, ADENOSINE RECEPTORS , DPCPX, HEART
Financial Support: FAPERJ, CNPq e CAPES.
Resumo:02-012
TRAINING-INDUCED PLASTICITY OF BRAIN DERIVED NEUROTROPHIC FACTOR (BDNF) IN THE RVLM OF
NORMOTENSIVE AND SPONTANEOUSLY HYPERTENSIVE RATS
Zampieri, T. T. 1; Silva Júnior, S. D. 1; Ruggeri, A. 1; Alves, T. P. 1; Ceroni, A. 1; Stern, J. E. 2; Michelini,
L. C. 1
1
Physiology and Biophysic/Biomedical Sciences Institute , ICB/USP
2
Physiology/Medical College of Georgia, MCG
Objectives:
It was shown that BDNF deficiency in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are
located, plays a role in the etiology hypertension. In this study we investigate the effects of exercise training (T) on time course
plasticity of BDNF in the RVLM of spontaneously hypertensive (SHR) and normotensive rats (WKY).
Male WKY and SHR were submitted to low-intensity training protocol (55% of maximal exercise capacity on treadmill, 1
hour/day, 5 days/week) or kept sedentary (S) for 8 weeks. Resting pressure (AP) and heart rate (HR) were measured in 6-8
rats/group at weeks zero (S0), one (T1), two (T2), four (T4) and eight (S8 and T8). For gene expression, rats (8/group) were
deeply anesthetized, perfused with PBS and kept on dry ice for ventral brain stem (VBS) removal. Total RNA was extracted
(Trizol®) and treated with DNAse for cDNA synthesis. BDNF and hypoxanthine phosphoribosyl transferase (housekeeping
gene) were estimated by semi-quantitative real time PCR. For immunofluorescence, brains (5 rats/group) were perfused (PBS and
4% PFA) and cryoprotected. Sequential RVLM coronal slices (30 ìm) were incubated with anti-BDNF (1:500) for 48 hours at
4°C and secondary antibody CY3 (1:100) for 4 hours at room temperature. Images were acquired with a Confocal Microscope (6
sections with 2 ìm interval between them, 182x). T was effective to reduce resting HR in both groups (-12% in WKYT8, -10% in
SHRT8 vs. respective S controls), but MAP fall was observed only in SHR (-6%, T8 vs. S8). T did not change WKY BDNF
mRNA expression, but determined a maintained elevation in SHR (+74% T8-S0). In S groups, BDNF mRNA expression was
reduced in WKY (-37%), with no change in SHR (S8 vs. S0). T caused a significant increase of BDNF-ir density in both groups
(+76% +48% from T2-T8 in WKY and SHR), with no difference in S controls. Similar to PCR data only SHR showed higher
levels of BDNF-ir intensity after T (+86%, T8 vs. S8).
Conclusions:
The increased BDNFmRNA expression only within RVLM of SHR to maintain protein content suggest that molecular pathways
involving in BDNF turnover are deficient in SHR. Data also suggest that aerobic training is an important tool to drive beneficial
plastic changes within this important area for cardiovascular control in both normotensive and hypertensive individuals.
Keywords: RVLM, BDNF, exercise training, hypertension
Financial Support: FAPESP, CNPq
Resumo:02-013
CARDIAC FUNCTIONS ALTERATIONS ON WISTAR RATS HEARTS INDUCED BY DOCETAXEL AND
CYCLOPHOSPHAMIDE
Salata, C. 1,2,1; Ferreira-machado, S. C. 1,3; Campos, V. M. A. D. 1; Andrade, C. B. V. D. 1,4; Silva, C. M. 1;
Barreto, I. R. L. 5,1; Lima, A. M. 5,1; Peregrino, A. A. F. 1; Mandarim-de-lacerda, C. A. 2; Dealmeida, C. E. 1
1
LCR/Universidade do Estado do Rio de Janeiro, UERJ
2
LMMC/Universidae do Estado do Rio de Janeiro, UERJ
3
Dep de Biologia Geral/Universidade Federal Fluminense, UFF
4
LUBT/Universidade do Estado do Rio de Janeiro, UERJ
5
Dep de Fisiol e farmacologia/Universidade Federal Fluminense, UFF
Objectives:
Studies show that the incidence of breast cancer has increased in women aged approximately 35 years. Among the treatment
strategies used for BC treatment, the most used are radiotherapy and chemotherapy. As a result of ionizing radiation fibrosis has
been observed in different tissues such as skin, lung, heart and liver. Endothelial cells seem to play an important role in fibrosis
development. When damaged, they release chemical mediators, provoking a series of events, including increased vascular
permeability, recruitment and proliferation of inflammatory cells in the lesion site, activation and differentiation of fibroblasts.
These cells, together start to release mediators (Growth factors, cytokines and chemokines) responsible for fibrosis development.
There are now several regimens of chemotherapy used for the treatment of BC. Docetaxel, which belongs to the family of
taxanes, is one of the most widely used anticancer drugs. Recent studies suggest that schemes involving docetaxel may be less
toxic compared to other schemes, especially those involving doxorubicin, another widely used drug for anticancer
treatment.Many studies related to cardiotoxicity tend to report the effects of isolated radiotherapy or chemotherapy, and yet, there
are little information including both treatment modalities. In this study it was compared the cardiac function of rats by
echocardiography, after being treated with chemotherapy and radiotherapy, and only radiotherapy.
Wistar rats, three months old, were divided into three groups: control (G0), chemotherapy (cyclophosphamide + docetaxel) +
radiotherapy (G2) and radiotherapy only (G3). Chemotherapeutic agents were applied intraperitoneally, 4 cycles, with an interval
of seven days between them. Each animal received 50 mg / kg cyclophosphamide and 12.5 mg / kg of docetaxel. The dose
administered to each drug in each cycle was equivalent to the dose per cycle chemotherapy in humans. The rats of groups G2 and
G3 were irradiated with single dose (20 Gy), 6Mev X-ray energy beam, 240 cGy dose rate. The animals were euthanized 5
months after the treatment. The day before euthanasia, the rats underwent echocardiography for analysis of cardiac function. The
echocardiogram revealed that the animals in group G2 showed a significant decrease (p
Conclusions:
The initial results obtained allow us to say that concomitant chemotherapy and radiation alters the cardiac function of Wistar rats.
These results will be confirmed in future studies in this through other techniques such as RT-PCR and stereology of the heart
tissue.
Keywords: breast cancer, cardiotoxicity, radiotherapy, chemotherapy, docetaxel
Financial Support: Faperj
Resumo:02-014
POSSIBLE ROLE OF NTS GLIAL CELLS IN BASAL AUTONOMIC AND RESPIRATORY CONTROL
Costa, K. M. ; Moraes, D. J. A. ; Machado, B. H.
Dep. of Physiology/School of Medicine of Ribeirão Preto, FMRP-USP
Objectives:
Glial cells are involved in a variety of neurophysiological processes, including synaptic regulation, neural network
synchronization, respiratory rhythm generation and central chemoreception. In this context, we hypothesized that glial cells in the
nucleus tractus solitarii (NTS), an important nucleus for the processing of visceral input, autonomic regulation and respiratory
pattern control, may play an important role in modulating baseline respiratory and autonomic activity. In order to test this
hypothesis, we evaluated whether metabolic glial inhibition in this region affects baseline respiratory parameters and sympathetic
nerve activity.
We performed bilateral microinjections of fluorocitrate (FC; 1 nmol/20nL), a metabolic glial inhibitor, in the caudal and
intermediary aspects of the NTS in the working heart-brainstem preparation (WHBP) of juvenile Wistar rats (n=6) and changes in
phrenic and thoracic sympathetic nerve activities were evaluated. In the control group (n=4), FC was replaced by saline solution
(SAL; NaCl 0.9%). Results are expressed as mean ± SEM. Statistical analysis was performed using one-way and two-way
ANOVA and the significance level was set at p
Conclusions:
Our data show that FC microinjections in the caudal and intermediate NTS, and the consequent inhibition of glial activity in this
nucleus, does not change baseline respiratory frequency and sympathetic activity, suggesting that NTS glial cells do not play a
major role in autonomic and respiratory regulatory mechanisms in basal conditions. However, the possibility that these cells
participate in pathophysiological processes or in responses to acute or chronic physiological stressors, such as acute hypoxia,
remains open for further investigation.
Keywords: autonomic neuroscience, neuron-glia interactions, nucleus tractus solitarii, respiratory control, working heart-brain
preparation
Financial Support: CAPES, CNPQ e FAPESP.
Resumo:02-015
CHRONIC EXPOSURE TO LOW-LEVEL OF LEAD ACETATE INCREASE ARTERIAL PRESSURE AND
ENDOTHELIUM-DERIVED VASOCONSTRITOR FACTORS IN RAT AORTA.
Silveira, E. A. 1,2; Siman, F. D. M. 1; Faria, T. D. O. 1; Lizardo, J. H. F 1; Vescovi, M. V. A. 1; Jesus, H. C. 1;
Stefanon, I. 1; Padilha, A. S. 1; Vassallo, D. V. 1,2
1
Departamento de Ciências Fisiológicas - UFES, UFES
2
ESCOLA SUPERIOR DE CIÊNICAS DA SANTA CASA DE MISERICÓRDIA, EMESCAM
Objectives:
We investigated the effects of 30 days exposure to a low dose of lead on blood pressure and on vascular reactivity to
phenylephrine (PHE) in rings from thoracic aorta of rats.
Rats were daily treated with lead acetate (Pb2+) (1st dose 4 µg/100g, subsequent dose 0,55 µg/100g/day i.m) or vehicle (Control)
for 30 days. The systolic blood pressure (SBP) was assessed by tail plethysmography before and during the exposure period.
After 30 days of treatment, rats were anesthetized to perform hemodynamic measurements and after that blood samples were
collected for analysis of blood concentration of Pb2+ by atomic absorption spectrometry. In both groups, vascular reactivity was
evaluated from concentration-response curves to PHE (1010 – 10-4 M) in the presence and absence of endothelium and after 30
min incubations with with L-NAME; indomethacin; apocynin (APOC); co-incubation with L-NAME and APOC; superoxide
dismutase (SOD); catalase; enalapril and losartan. We evaluated the local release of NO and superoxide anion (O2•-). Western
Blot was performed to measure the protein expression of eNOS, iNOS, COX-2 and AT1 receptor. At the end of treatment the
concentration of Pb2+ in the blood was 11.96 µg/dL. The SBP of the Pb2+ group increased from the 7th day of exposure while
maintaining elevated to the end of treatment. These values were confirmed by the direct measurement of SBP. Pb2+ increased
PHE contractile responses in aortic rings. This effect was greater in the absence of endothelium. The contractile response to PHE
increased in the presence of L-NAME in both groups, but was higher in animals treated with lead. However, the co-incubation of
the L-NAME and APOC, abolished the vasoconstrictor effect observed in the aortic rings of the Pb2+ group incubated only with
L-NAME. The lead effects on the Rmax were diminished in the presence of indomethacin. In the presence of APOC and SOD
lead effects on Rmax were reduced, when compared to controls. Enalapril and losartan diminished pD2 and Rmax, respectively,
in the Pb2+ group in relation to the control group. The basal and stimulated release of NO was lower in the Pb2+ group. The local
liberation of O2•- was increased in the Pb2+ group compared to the control group. The protein expressions of eNOS, iNOS and
of the AT1 receptor were increased in the lead exposed animals.
Conclusions:
This study is the first to demonstrate that blood concentrations of Pb2+ (11.96 µg / dL), well below the allowable values for an
exposed population (40µg/ dL), increased the SBP and vascular reactivity to PHE. These responses were associated with reduced
bioavailability of NO induced by the increase in oxidative stress with greater involvement of ROS, COX-derived contractile
prostanoids and the renin-angiotensin system. Our study supports the idea that lead should be considered a risk factor for
developing cardiovascular diseases and their acceptable values, considered to be free of harmful effects to humans, should be
reduced
Keywords: hypertension, inorganic lead, vascular reactivity
Financial Support: CNPq, CAPES, FAPES/FUNCITEC
Resumo:02-016
HEMODYNAMIC EFFECTS OF NEW FORMULATION OF DANTROLENE AND AZUMOLENE.
Carmo, P. L. 1; Mendes, T. C. F. 1; Debom, R. 2; Rizzi, M. D. 2; Silva, A. M. S. 1; Silva, M. C. S. 1; Zapatasudo, G. 1; Sudo, R. T. 1
1
Programa de Desenvolvimento de Fármacos, UFRJ
2
Cristália Produtos Químicos e Farmacêuticos Ltda, Cristália
Objectives:
Azumolene (Az) is more water-soluble than the prototype dantrolene (DS) used for malignant hyperthermia (MH) treatment. Our
previous results showed that the potency of Az to cause muscle relaxation in comparable to DS (Basic Clin. Pharmacol. Toxicol.
102:308, 2008), effectively reversed porcine MH crisis (J. Vet. Intern. Med. 24:1224, 2010) and did not present significant
toxicity (Fundam. Clin. Pharmacol. 24:491, 2010). New formulations of DS and Az are necessary to facilitate the treatment of
MH crisis. We investigated formulations of DS and Az in cyclodextrin, in wich increase the solubility in 3 times and 15 times,
respectively, on muscle relaxation and on hemodynamic parameters.
Methods: 1) Male guinea pigs (300-400 g) were anaesthetized with pentobarbital sodium (50 mg/kg, i.p.) and the lungs were
mechanically ventilated. A catheter was introduced into right carotid artery for arterial pressure recording, and another catheter
into jugular vein for intravenous injection of DS or Az. The sciatic nerve was dissected for electrical stimulation (5 V, 1 msec.
duration, 0.2 Hz frequency) and the twitches of gastrocnemius muscle recorded. DS or Az in cyclodextrin were i.v. injected at
doses of 0.5, 1, 2.5, 5, 7.5 and 10 mg/kg. The dose-response curve was used for determination of dose to cause 50% of maximal
twitches inhibition (IC50). 2) In another set of animals, the left ventricular pressure was recorded before and after bolus injection
of regular DS or Az (2.5 mg/kg) or both prepared in cyclodextrin. Data were expressed as mean±S.E.M (n= 6/group) with
significance level to P
Conclusions:
The potency to cause muscle relaxation was not changed after incorporation of DS and Az in cyclodextrin. DS, Az and both in
cyclodextrin significantly increased MAP, VSP and the rate of contraction and relaxation of cardiac muscle after i.v. injection
with no significant changes of heart rate and VDP. More works are required to clarify these hemodynamics effects.
Keywords: azumolene, dantrolene, malignat hyperthermia, cyclodextrin
Financial Support: Cristália Produtos Farmacêuticos Ltda, CNPQ, CAPES, INCT-INOFAR, FINEP
Resumo:02-017
L-ARGININE SUPPLEMENTATION IMPROVES POST EXERCISE HYPOTENSION IN RESPONSE TO EXERCISE
PERFORMANCE IN POSTMENOPAUSAL WOMEN
Puga, G. M. ; Novais, I. D. P. ; Zanesco, A.
EducaÃ§Ã£o FÃsica, Unesp-Rio Claro
Objectives:
The post exercise hypotension (PEH) is an important mechanism for cardiovascular risks protection, especially in
postmenopausal women. Our hypothesis was to verify whether PEH is improved by combination of exercise performance with Larginine (a Nitric Oxide precursor) supplementation.
Six women with normal resting blood pressure (BP) (114 Â± 13 and 70 Â± 8 mmHg for systolic and diastolic, respectively), 56
Â± 6 yrs, BMI of 27 Â± 4 kg/m2 took part of this study. In four different days in a random order, all the participants came to the
laboratory in the morning after having their regular breakfast. After 20 min of resting, BP was measured and 9g of L-arginine was
given orally for the volunteers (Ajinomoto, Japan). Forty-five minutes after, the participants performed 30 min of exercise on
treadmill at the maximal lactate steady state intensity. The BP was measured every 15 min during 90 min after the end of the
exercise. Blood venous were collected before the L-arginine supplementation, previously and after the exercise performance.
Blood samples were also taken at 45 and 90 min after the end of the exercise (ARG-EXE day). During the others three days, the
volunteers performed the same procedure, but it was given placebo pills instead (EXE day), or only L-arginine without exercise
(ARG day), or did not take L-arginine nor performed exercise (CON day). One way ANOVA analysis obtained by incremental
area under the curve (AUC) showed that systolic BP (SBP) during the ARG-EXE (-392.5 Â± 108.7 mmHg/90 min) and EXE (251.3 Â± 203.0 mmHg/90 min) days were lower comparing with CON day (540.0 Â± 253.0 mmHg/90 min). Moreover, the AUC
of the diastolic BP (DBP) during the ARG-EXE day (-5.0 Â± 99.1 mmHg/90 min) was lower comparing with CON (301.3 Â±
194.8 mmHg/90 min), ARG (342.5 Â± 166.0 mmHg/90 min) and EXE (248.8 Â± 164.6 mmHg/90 min) days.
Conclusions:
We can conclude that L-arginine supplementation in combination with exercise performance promotes a greater reduction in DBP
as compared to EXE or L-arginine alone in postmenopausal women. Interestingly, SBP was affected by EXE only and no
additional effects were found in L-arginine supplementation group.
Keywords: Exercise, L-arginine, Post exercise hypotension, Postmenopausal Women
Financial Support: Fapesp, Capes
Resumo:02-018
INTRAVENOUS INJECTION OF DOPAMINE IN AWAKE RATS PRODUCES CHEMORREFLEX-LIKE
RESPONSES
Bonagamba, L. G. ; Moraes, D. J. ; Costa, K. M. ; Machado, B. H.
Dept. Physiology, School of Medicine of Ribeirão Preto, USP
Objectives:
To investigate the cardiovascular and respiratory alterations induced by intravenous injections (i.v.) of dopamine (DA) in awake
rats and explored the underlying mechanisms of these responses.
DA injections (0.01, 0.1 and 1µMol in 5µl; i.v.) produced a dose-dependent transient increase in mean arterial pressure (MAP)
(6±5, 25±3 and 46±8 mmHg, n=14), decrease in heart rate (HR) (-24±19, -62±26 and -292±23 bpm, n=14) and a consistent
tachypneic response (62±22, 49±14 and 82±26 cpm, n=9). These cardiovascular and respiratory responses to 1 µMol dose were
similar to those observed during peripheral chemoreflex activation (58±4 mmHg , -283±12 bpm, n=14) and (135±18 cpm, n=9).
The increase in MAP induced by DA (i.v.) was abolished by previous administration of Prazosin (i.v.), a á1 receptor antagonist,
indicating that this response is due to the activation these receptors. Previous bilateral carotid body removal (CBR), produced no
effect on the cardiovascular (51±3 mmHg and -228±40 bpm n=8) and respiratory responses (68±22, n=8) to i.v. 1 µMol DA
injection.
Conclusions:
Our data show that the cardiovascular responses to DA (i.v.) are dependent of adrenergic á1 receptor activation but not are not
generated in carotid body glomus cells.
Keywords: adrenergic receptors, arterial pressure, dopamine, glomus cells, peripheral chemoreflex
Financial Support: FAPESP, CAPES E CNPQ
Resumo:02-019
AMBULATORY BLOOD PRESSURE MEASUREMENT IN MIDDLE-AGED WOMEN AFTER EXERCISE
PERFORMANCE AND L-ARGININE SUPPLEMENTATION
Novais, I. D. P. ; Puga, G. M. ; Zanesco, A.
DEPARTAMENTO DE EDUCAÇÃO FÍSICA, UNESP
Objectives:
L-arginine plays a key role in the nitric oxide synthesis and its supplementation has been reported to improve endothelial
dysfunction as well as to ameliorate high blood pressure. Therefore, our hypothesis was that middle-aged women who take Larginine supplementation in combination with exercise were more responsive to post exercise hypotension than those underwent
exercise or L-arginine supplementation alone.
Seven women (55 ± 5 yrs, 27 ± 4 of BMI and 115 ± 12 and 71 ± 8 mmHg of systolic and diastolic blood pressure, respectively)
were submitted to four different experimental designs, separated by 72 hours and in a random order. On two of these days,
volunteers performed 30 min of exercise at the maximal lactate steady state, previously determined, on treadmill, after intake 9g
of L-arginine (ARG-EXE day) or placebo (PLA-EXE day). During the other two days, volunteers did not perform the exercise,
but received L-arginine supplementation (ARG day) or nothing (CON day). During all the four days, blood pressure was
measured during 24 hours by the ambulatory blood pressure measurement (Dyna-mapa+). ANOVA analysis showed no
difference in both mean systolic (113 ± 4, 112 ± 4, 110 ± 5 and 108 ± 4 mmHg for CON, ARG, PLA-EXE and ARG-EXE days,
respectively) and diastolic blood pressure (73 ± 4, 72 ± 4, 71 ± 4 and 72 ± 4 mmHg for CON, ARG, PLA-EXE and ARG-EXE
days, respectively) during 24 hours of measurement. During awake period, the mean systolic blood pressure had a tendency to be
lower in ARG-EXE group (110 ± 4 mmHg), comparing with the CON group (116 ± 3 mmHg).
Conclusions:
We conclude that L-arginine supplementation given orally did not improve the mean 24 hour blood pressure after the exercise
performance in middle-aged women. On the other hand, this combination reduced systolic blood pressure during the awake time.
Keywords: BLOOD PRESSURE, EXERCISE, L-ARGININE
Financial Support: Fapesp, Capes
Resumo:02-020
ORAL ADMINISTRATION OF N-ACYLHYDRAZONE DERIVATIVE REVERSES THE DEVELOPMENT OF
PULMONARY HYPERTENSION IN MONOCROTALINE-TREATED RATS.
Pereira, S. L. 1; Peixoto, M. V. C. 1; Carneiro, T. R. 1; Kummerle, A. E. 2; Fraga, C. A. M. 2; Barreiro, E. J.
2
; Sudo, R. T. 1; Zapata-sudo, G. 1
1
Farmacologia Básica e Clínica , UFRJ
2
Faculdade de Farmácia , UFRJ
Objectives:
Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance associated with endothelial
injury of pulmonary arteries, vascular remodeling, right ventricular hypertrophy and increased right ventricular pressure. The aim
of this study was to investigate the effects of LASSBio-1289, a novel N-acylhydrazone with potent vasodilatory activity in rats
with PAH induced by monocrotaline (MCT).
The protocols used in the present study were approved by Animal Care and Use Committee at Universidade Federal do Rio de
Janeiro. Male Wistar rats (200-250 g) received a single intraperitoneal injection of monocrotaline (60 mg/kg) for PAH induction.
Experimental groups were: saline (control), MCT and saline (MCT), MCT and vehicle (DMSO), MCT and LASSBio-1289
(LASSBio-1289). The animals were treated with saline, DMSO or LASSBio-1289 (50 mg/kg, p.o.) for 15 days after the onset of
disease (n=6). The following parameters were analyzed: right ventricular systolic pressure (RVSP), right ventricle weight (RV),
relation between right ventricle weight and body weight (RV/BW), RV contractility (dp/dtmax) and RV relaxation (dp/dtmin). In
addition, endothelial dysfunction of aorta and pulmonary artery was evaluated by the observation of acetylcholine-induced
relaxation. RVSP (mmHg) increased from 25.3 ± 1.9 (control) to 49.0 ± 6.1 (MCT, p
Conclusions:
LASSBio-1289 effectively reversed cardiac hypertrophy, ventricular dysfunction and reduced vascular reactivity in
monocrotaline-induced PAH in rats suggesting a new perspective for the treatment of PAH.
Keywords: LASSBio-1289, MONOCROTALINE , N-ACYLHYDRAZONE , PULMONARY HYPERTENSION
Financial Support: CNPq, FAPERJ, INCT, PRONEX
Resumo:02-021
EFFECTS OF CHRONIC COFFEE CONSUMPTION ON CARDIAC FUNCTION AND STRUCTURE IN RATS
Silva, G. 1; Melo, D. S. 1; Morais, P. L. 4; Almeida, P. W. M. 5; Almeida, C. A. S. 1; Silva, A. A. 2; Fonseca,
S. C. G. 5; Ferreira, A. J. 4; Riul, T. R. 3; Peixoto, M. F. D. 2
1
Depto de Ciências Biológicas/Facul. Ciências Bio. e da Saúde, UFVJM
2
Dep. Ed. Física / Facul. Ciências Bio. e da Saúde, UFVJM
3
Depto de Nutrição/Facul. Ciências Bio. e da Saúde, UFVJM, UFVJM
4
Depto de Morfologia/ Instituto de Ciências Biológicas, UFMG , UFMG
5
Depto de Fisiologia e Biofísica /Inst. Ciências Biológicas, UFMG
Objectives:
Coffee is a very popular drink, it contains a series of substances, notably caffeine. Caffeine has a stimulatory function on the
central nervous system, being associated with appetite inhibition, besides effects on other organs like the heart. The present study
had as objective to evaluate the effects of chronic treatment with coffee on cardiac function and structure in rats.
Mother rats in the experimental group received coffee in dilution of 3% during lactation period, whereas the control group
received conventional treatment. After weaning, the young rats still received the same treatment as the mothers up to the age of
90 days at which time they were killed for analysis of body weight, tibia length, heart weight. To evaluate cardiac function we
utilize the Langendorff analysis. In control group (n = 11) and in coffee group (n = 10). Data are presented as average values ±
standard error, and for comparison between groups test t student was performed. Significant level of *p < 0,05 was established.
Coffee treatment inhibited weight gain (292,6 ± 10,93 vs. 326,9 ± 9,45g), reduced reason heart weight /tibia length (0,0027 ±
0,0020 vs. 0,0029 ± 0,002g/mm) and heart weight (1,09 ± 0,03 vs. 1,21 ± 0,03g), demonstrating that coffee interferes with
organism development, possibly by appetite reduction and increase of basal metabolic rate due to caffeine action. As for cardiac
function and structure, coffee modified only the systolic tension (8.87 ± 0.55 vs. 7,34 ± 0,45g), increasing its value.
Conclusions:
With this study we conclude that coffee consumption can interfere with weight gain and growth, and increase the strength of
myocardium contraction.
Keywords: caffeine, cardiac function, cardiomyocytes diameter
Financial Support: UFVJM, UFMG, CNPq
Resumo:02-022
EFFECT OF CHRONIC MILD AND UNPREDICTABLE STRESS ON THE ACTIVITY OF RENIN- ANGIOTENSIN
SYSTEM IN RATS
Sanches, A. 1; Costa, R. 1,2; Almeida, B. S. 2; Ronchi, F. A. 3; Jara, Z. P. 3; Casarini, D. E. 3; Cunha, T. S.
1,3,4
; Marcondes, F. K. 1,3
1
Physiological Sciences Department, FOP/UNICAMP
2
Institut of Biology /University of Campinas, IB/UNICAMP
3
Nephrology Division/Federal University of São Paulo, UNIFESP
4
Science and Technology Institute/Federal University of SP, UNIFESP
Objectives:
Our group has already shown that CMUS induced endothelial dysfunction evidenced by lower relaxation response to
acetylcholine, endothelial NO production and increased superoxide anion production in thoracic aorta. Since increased activity of
renin-angiotensin (RAS) and catecholaminergic systems have been associated with endothelial dysfunction, the aim of this study
was to evaluate the influence of chronic mild and unpredictable stress (CMUS) on RAS, in male Sprague-Dawley rats.
Animals (60 days old) were randomly assigned into two groups: control and CMUS (n=12⁄group). Briefly, the CUMS protocol
consisted in applying different stressor stimuli (immobilization, overnight illumination, water and food deprivation for 18h
followed by restricted access to food for 2h, water deprivation for 18h followed by exposure to empty water bottle for 2h, wed
bedding for 17h, and reversed light⁄dark cycle throughout the weekend) 7 days⁄wk during three consecutive weeks (from the 3rd
to 5th wk) of the experimental protocol, 7 weeks long. Fifteen days after CMUS, animals were killed by decapitation. Trunk
blood was collected for evaluation of corticosterone (Assay Designs™) and catecholamines (HPLC) levels, and ACE and renin
activity. ACE activity was also determined in thoracic aorta, heart and kidney, using ZPhe-HL as substrate. Renin activity was
measured through the quantification of angiotensin I, the reaction product of renin on tetradecapeptide, by HPLC. Data were
analyzed by Student´s t test (Mean ± SEM; p< 0.05). All procedures were approved by UNICAMP Committee on Animal
Research Ethics (Protocol no. 1829-1). CMUS increased corticosterone (46.4 ±4.5 vs. 1.4 ± 0.4 ng⁄mL), noradrenaline (786.4 ±
49.1 vs. 131.6 ± 7.7 pg⁄mL) and adrenaline (670.6 ± 50.2 vs. 94.8 ± 16.9 pg⁄mL; p<0.05) levels. Stressed rats presented increased
serum ACE activity (253.9 ± 24.3 vs. 176.5 ± 3.5 mU⁄mL) and in thoracic aorta (231.9 ± 75.9 vs. 80.2 ± 13.6 mU⁄mg) in
comparison with control group, without differences in the heart (0.44 ± 0.02 vs. 0.57 ± 0.06 mU⁄mg) and kidney (3.6 ± 0.78 vs.
3.44 ± 0.77 mU⁄mg). CMUS also increased plasma renin activity (1.1 ± 0.3 vs. 0.4 ± 0.06 nmol⁄mL) in comparison with control
group.
Conclusions:
Our data show that CMUS increased RAS activity in blood and aorta, and this adaptation could be related to the endothelial
dysfunction induced by the stressor stimuli.
Keywords: chronic stress, renin-angiotensin system, angiotensin converting enzyme, catecholamines
Financial Support: FAPESP, CNPq, CAPES
Resumo:02-023
ON A CARDIAC EXTERNAL WORK INDEX BASED ON VENTRICULAR PRESSURE MEASUREMENT
Camilo, L. M. 1; Vassallo, D. V. 2; Aquino, R. M. 3
2
Department of Physiological Sciences, UFES
1
Carlos Chagas Filho Institute of Biophysics, UFRJ
3
Department of Mathematics, UFES
Objectives:
We previously stablished an algebraic model for the Phase Plane Area (PPA) considering pressure and its peak derivative
measurements as an index of cardiac external work index (CEWI). We evaluated parameters that are known to alter cardiac work
based on pressure and its positive and negative peak derivatives to correlate CEWI and PPA.
Male Wistar rats (C, n=7) and spontaneous hypertensive rats (SHR, n=7) were studied ex vivo using isolated hearts perfused by
the Langendorff technique. Isovolumic intraventricular pressure (IP) was accessed and phase plane, a plot of dP/dt X P, was
performed using the Biopac Student Lab software, keeping a scale of 50 mmHg and 500 mmHg/s for P in the X axis and dP/dt in
the Y axis, respectively, for all measurements. The values of PPA were obtained with AutoCad 2004 software. We applyed the
software Mathlab to simulate an algebraic description of IP curve and its PPA, which is defined by a plane curve with coordinates
P and dP, where P=P(t) represents the pressure function on the variable of time t and dP its differential, meaning its derivative
function of order one. Positive inotropic interventions induced by changes in diastolic pressure (0 to 30 mmHg), by isoproterenol
(10 μM) and by changes of extracellular calcium concentration (0.62 to 2.5 mM) increased IP and dP/dt. Hearts from SHR
compared to controls also developed higher systolic pressures and dP/dt, therefore higher PPA (C measured PPA: 148380 ±
19936 mmHg/s.s; C estimated PPA: 150465 ± 25853 mmHg/s.s; SHR measured PPA: 382230 ± 65084 mmHg/s.s; SHR
estimated PPA: 395413 ± 69070 mmHg/s.s). These inotropic interventions that are known to increase cardiac work, both in C and
in SHRs made possible a strong correlation of the proposed model and the obtained data (r=0,99).
Conclusions:
This mathematical model based on the PPA showed to be a good index of cardiac external work in isolated heart rats.
Keywords: cardiac external work, isolated heart, phase plane
Financial Support: FAPES, CAPES, CNPq
Resumo:02-024
INTERMITTENT HYPOXIC TRAINING AFFECTS THE HEMATOLOGICAL PARAMETERS IN WISTAR RATS
Simões, R. R. ; Dutra, A. L. ; França, R. T. ; Lopes, S. T. D. A. ; Portela, L. O. C. ; Zanchet, E. M.
Depto Fisiologia e Farmacologia-CCS, UFSM
Objectives:
Goals: The intermittent hypoxic training (IHT) appeared in the decade of 30 in the Soviet Union. IHT is defined as repeated
episodes of hypoxia interspersed with episodes of normoxia (21% O2). It is used for acclimatization to the altitudes and in the
treatment or prophylaxis of several diseases (High Altitude Medicine & Biology;3:205-221, 2002). Some studies show that IHT
promotes hematological adaptations, like stimulate erythropoietin production and increase red cells but there is disagreement
between researchers as for other parameters as hemoglobin mass, plasma volume and hematocrit. Additionality, there are few
studies about the effects of IHT under blood white cells. The aim of this research was to evaluate the effects of 30 sessions of
IHT on hematological parameters in Wistar rats.
Methods: Two groups of adults Wistar rats (230-250 g, eight animals) were submitted to intermittent hypoxia (IH) or normoxia
(N) sessions during four weeks. The hypoxia was conditioned in custom-made acrylic chamber. The atmosphere within the
chamber to be ajusted quickly and precisely using compressed gas (GO2 Altitude Hypocator equipment). IH program was
administered for 30 days and consisted of 15 minutes of hypoxic exposure with 5 minutes reoxygenation and a total duration of 2
hours per day. The normoxia group was subjected to the same conditions, but was exposed to normal O2 concentrations. After
this period, on 31th day the rats were anesthetized with halothane and blood was collected by cardiac punction for hematological
parameters analysis.Results: The statistical analysis did not show significant differences in red cell volume (RCV), haemoglobin
(Hbmass), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), total
protein plasma (TPP), eosinophils and monocytes. Although the value of total leukocytes (TL)did not presented difference
significant, in hypoxia situation the TL increased 16% compared with normoxia group. In hypoxia conditions had significative
differences in segmented neutrophyls (decrease 41%)and lymphocytes (increased 54%)compared with normoxia group. The
values found were:RVC(x106):IH-7,6±0,43; N-7,7±0,37; Hbmass (g/dL):IH-14,5±0,67;N-15,0±0,67;Ht(%):IH-47,4±2,79;N49,3±2,69;MCV(fL):IH-62,6±1,76;N-63,4±1,24;MCHC(%):IH-30,5±0,80;N-30,5±0,61;TPP(g/dL):IH-7,9±0,2;N7,8±0,41;TL(uL):IH-14050±2001;N-12100±2124;Segmented neutrophyls(uL):IH-2463±634*;N3486±811;Lymphocytes(uL):IH-11048±1939*;N-7167±913;Eosinophils(uL):IH-143±158;N-109±139;Monocytes(uL):IH195±118;N-228±157; * Difference between IH and N p
Conclusions:
Conclusion: Our findings indicate that 30 sessions of IHT in Wistar rats significantly changed the white cells,specially total
leukocytes,segmented neutrophyls, and lymphocytes. These results stimulate us to research more the effects of IHT about
hematological parameters and a possible therapeutic alternative use in imunossupression situations.
Keywords: Intermittent Hypoxia, wistar rats, Hematological parameters
Financial Support: No financial support
Resumo:02-025
MORPHOMETRIC ANALYSIS OF CORONARY ARTERY DIAMETER AND EXTERNAL PART OF THE
ASCENDING AORTA ARTERY
Silva, J. M. L. 1; Reis, R. B. D. 1; Nardeli, C. R. 1; Silva, M. A. D. S. 1; Nagato, A. C. 1; Bezerra, F. S. 2
1
Laboratório de Biomorfologia e Patologia Experimental, Uss
2
Universidade Federal de Ouro Preto, UFOP
Objectives:
This study aimed to analyze the right coronary artery, left coronary artery and part of ascending aorta artery by measuring its
external diameter and investigate a possible correlation between the diameter of coronary arteries and part of ascending aorta
artery.
39 adult human hearts and dissected in block, from the Institute of Human Anatomy, University Severino Sombra / VassourasRJ. The external diameters of the ascending part of the aorta (APA), anterior superior view, and left coronary artery (LCA) and
right coronary artery (RCA), upper-lateral view were measured in millimeters with the help of a digital caliper (Mitutoyo). The
sample homogeneity was confirmed by the Archimedes method, which estimated the cardiac masses. Comparison of outer
diameters was performed using the Mann Whitney test and the correlation between LCA and the RCA with the diameter of the
APA was investigated by Pearson correlation. Was considered significant at P
Conclusions:
It was demonstrated that the left coronary artery has greater external diameter of the right coronary artery and did not find
statistically significant differences to establish a relationship between the outside diameters of APA with outer diameters of both
RCA and LCA.
Keywords: CORONARY ARTERY DIAMETER, MORPHOMETRIC ANALYSIS , PART OF THE ASCENDING AORTA
ARTERY
Financial Support: Nenhum
Resumo:02-026
IS THERE A ROLE FOR SEROTONIN IN THE VASCULAR HYPOREACTIVITY ASSOCIATED TO SEPSIS?
Silva, A. K. L. ; Guarido, K. L. ; Silva-santos, J. E. ; Linder, A. E.
Farmacologia / Universidade Federal de Santa Catarina, UFSC
Objectives:
Increased platelet aggregation in sepsis is associated with microvascular failure (Am J Respir Crit Care Med. 178: 1148, 2008). In
the periphery, platelets are the largest store for serotonin (5-hydroxytryptamine; 5-HT) and this monoamine when infused to rats
lowers blood pressure (J Pharmacol Exp Ther. 325:1031, 2008). Hypothesizing that 5-HT released by platelet aggregation during
sepsis contributes to vascular hypo-responsiveness, a putative protective effect by the 5-HT receptor antagonist ketanserin was
investigated in an experimental septic model induced by the administration of lipopolysaccharide (LPS).
Male Wistar rats (~300g) received intraperitoneally (i.p.) vehicle (1 mL/kg) or ketanserin (5 mg/kg) 10 min before i.p.
administration of vehicle (1 mL/Kg) or LPS (10 mg/kg). The animals were divided into four experimental groups according to the
treatment received: 1) vehicle followed by vehicle (V+V); 2) vehicle followed by LPS (V+L): 3) ketanserin followed by vehicle
(K+V) and 4) ketanserin followed by LPS (K+L) (PP00463 experimental protocol approved by the ethics committee).
Endothelium-intact (E+) and –denuded (E-) aortic rings were isolated and mounted in an isolated organ chamber for isometric
tension recordings. Cumulative concentration-effect curves (CCEC) to PE (1 nM to 0.1 uM) and to 5-HT (1 nM to 0.1 uM) were
performed on aorta from the four experimental groups. From the concentration-effect curves the effective concentration that
induced 50% of the maximal contraction (EC50) and the maximal contraction (MC) were obtained. Data were expressed as pD2
(-log EC50) values or as g of contraction +/- standard error of the media. N represents the number of the experiments. PE and 5HT induced concentration-dependent contractions in aorta from all experimental groups. In E+ aorta from the group V+V, the
CCEC to PE were shifted to the right (pD2= 6.8 ± 0.03; N=4) when compared to those in which the endothelium was removed
(pD2= 7.6 ± 0.11; N=6; P 0.05). CCEC induced by PE in E+ aorta from the V+L group were shifted to the right (pD2= 6.4 ±
0.05; N=6) and had the MC reduced (0.9170 ± 0.2742 g; N=6) when compared to those obtained in E+ aorta from the V+V group
(pD2= 6.8 ± 0.03; N=4; P < 0,05; MC= 1.966 ± 0.1455 g; N=4). Similar results were observed for 5-HT (V+L pD2= 5.3 ± 0.05;
MC= 0.3673 ± 0.08334 g, N=5 vs. V+V pD2= 5.7 ± 0,17; MC= 1.521 ± 0.1950 g, N=4; P < 0.05). No significant effect was
observed in the CCEC induced by PE and 5-HT in E+ aorta from rats that received ketanserin previously to the administration of
either vehicle or LPS (groups K+V and K+L, respectively).
Conclusions:
Treatment with LPS was effective in impairing the response to vasoconstrictor agents in rat aorta and ketanserin does not seem to
retrieve this LPS-induced vascular hypo-reactivity. However, further studies are needed to address the influence as well as to
evaluate the effects of 5-HT in septic animals.
Keywords: Serotonin, Ketanserin, Lipopolysaccharide, Sepsis
Financial Support: FAPESC/CNPQ, CAPES, FUNPESQUISA
Resumo:02-027
LEFT VENTRICULAR HYPERTROPHY HEART INDUCED BY VOLUNTARY EXERCISE IN MICE IS
MODULATED BY MICRORNAS EXPRESSION.
Martinelli, N. C. 1,2; Cohen, C. R. 1,2; Schneider, S. I. R. 1,2; Santos, K. G. 1,2; Andrades, M. E. 1,2; Clausell,
N. 1,2; Biolo, A. 1,2; Rohde, L. E. 1,2
1
Hospital de Clínicas de Porto Alegre, HCPA
2
Universidade Federal do Rio Grande do Sul, UFRGS
Objectives:
Physiological and pathological left ventricular hypertrophy (LVH) are distinct processes that have specific gene expression.
microRNAs are small non-coding RNAs that regulate gene expression. Their profile has not been explored in models of
physiological LVH. The aim of this study was to evaluate the expression of microRNAs in mice heart of subjected to
physiological LVH.
LVH was induced by voluntary exercise. Balb/c mice were kept in cages with exercising wheels (Exercise group – EXE; n=15
sacrificed on day 7 and n=17 sacrificed 35 days after training) or in cages without wheels (Sedentary group – SED; n=12 in each
time point). Mice were subjected to functional capacity tests on a motor treadmill at baseline, 7 days and 35 days of training to
evaluate their improvement on exercise capacity. All animals underwent 5 minutes of adaptation on treadmill at 7.7 m/min speed.
The intensity of exercise was increased by 3 m/min (15–45 m/min) every 2 min at 0% inclination until exhaustion. This test
provided the total distance run by each animal. LVH was evaluated by the left ventricular weight/body weight ratio (LVW/BW).
LV mass was also estimated by two-dimensional echocardiography using an EnVisor HD System (Philips Medical, Andover,
MA, USA) and a 12–13 MHz linear transducer. A microRNA microarray based on the MIRBASE version 16 (LC Sciences,
Houston, Texas, EUA) was carried out using a pool of microRNAs (n = 4 per group) extracted from the LV. Comparisons
between groups were performed by Student t test. Results: After 7 days of training there was an increase of 17% in LVW/BW
ratio on EXE compared to SED group (3.8±0.1 vs. 3.3±0.1, respectively; p=0.0004) and this result was sustained at 35 days (18%
increase; 3.9±0.2 vs. 3.3±0.04, respectively; p=0.002). Echocardiography based LV mass was increased in the EXE [35 days]
group when compared to the SED [35 days] group (58±5.0 vs. 34±16 mg, respectively; p = 0.01). Both EXE [7days] and EXE
[35 days] group improved their functional capacity tests when compared with their respective SED groups (1550±1108m vs
522±124m, p=0.01 and 1858±789m vs 557±141m, p=0.003; respectively). In the microarray analysis we observed that the EXE
[7 days] group had 35 microRNAs deregulated and the EXE [35 days] also had 25 microRNAs deregulated when compared to the
SED groups (p value < 0.001 for both analyses). The most upregulated miRNAs were miR-149*, miR-341*and miR-1224 and
the main downregulated miRNAs were miR-21, miR-26b, miR-150 e miR-499.
Conclusions:
The experimental protocol was able to induce LVH in adult mice and improved physical performance in the trained groups.
Physiological LVH was associated to significant modulation of heart microRNA expression. The altered miRNAs by this model
have target genes evolved on cellular and molecular pathways of cardiac hypertrophy, such as VEGF and MAPKs pathways.
These data might help to understand the specific role of microRNAs on cardiac adaptation.
Keywords: left ventricular hypertrophy, microRNA expression, microRNA microarray, voluntary exercise
Financial Support: CNPq and FIPE-HCPA.
Resumo:02-028
HEART REMODELING INDUCED BY THE NANDROLONE AND RESISTANCE TRAINING: ROLE OF FETAL
GENES AND IMPLICATIONS FOR CARDIAC PATHOPHYSIOLOGY.
Neves, V. J. 1; Tanno, A. P. 1; Rosa, K. T. 2; Cunha, T. S. 3; Giordano, F. C. L. 1; Calil, C. M. 1; Fernandes,
T. 4; Oliveira, E. M. 4; Novaes, P. D. 1; Irigoyen, M. C. 2; Moura, M. J. C. S. 5; Marcondes, F. K. 1
1
Departamento de Ciências Fisiológicas, FOP/UNICAMP
2
Unidade de hipertensão, InCor-USP
3
Departamento de Nefrologia, UNIFESP
4
Laboratório de Bioquímica, EEFE-USP
5
Centro de Ciências da Vida, PUC-Campinas
Objectives:
To study the isolated and combined effects of nandrolone and resistance training on cardiac morphology, function, and mRNA
expression of pathological cardiac hypertrophy markers.
Wistar rats (2m.o.) were randomly assigned into 4 groups (n=12⁄group): Non-trained plus vehicle (NTV) or nandrolone (NTN),
Trained plus vehicle (TV) or nandrolone (TN), and submitted to treatment with nandrolone (5 mg⁄Kg) and⁄or training. All
procedures were approved by the Institutional Committee for Ethics in Animal Research (protocol no. 944-1). Adaptation (five
days) consisted of sessions of weight lifting in water with an incremental number of sets and repetitions, carrying a load of 50%
body weight strapped to the chest. After adaptation, high-intensity exercise training for 5 weeks was applied. During the first 2
weeks: 4 sets of 10 jumps (lifts) per set carrying a load of 50% body weight. During the 3rd and 4th weeks: 4 sets of 10 jumps
(load: 60% of body weight), and in the 5th week: 4 sets of 10 jumps (load: 70% of body weight). Left ventricle mass and body
weight were registered. Cardiac parameters were determined by echocardiography. Heart was analyzed for collagen infiltration
(histological analysis). Real-time RT-PCR was used to assess pathological cardiac hypertrophy markers. Data were analyzed by
Two-way ANOVA and post-hoc Tukey (p< TV: 1.95±0.13 = NTN: 1.92±0.06 < TN: 2.24±0.09 g⁄Kg). Nandrolone increased
cardiac collagen content in NTN (10,32±0,75 µm2) compared to the NTV (2,93±0,22 µm2), and the association between
nandrolone and training increased collagen content in TN (34,34±2,78 µm2) compared to all groups. Relative left ventricle wall
thickness was increased in TV (0.46±0.01 mm) compared to NTV (0.38±0.01 mm), in TN (0.56±0.02 mm) compared to NTN
(0.46±0.01 mm), and in NTN compared to NTV. Nandrolone reduced cardiac index in NTN (184.1±18 mL⁄min⁄g) compared to
NTV (213.5±11 18 mL⁄min⁄g), and in TN (150.6±14 mL⁄min⁄g) compared to the TV (204.3±19 mL⁄min⁄g). Nandrolone also
reduced the ratio of maximum early to late transmitral flow velocity both in the NTN (1.46±0.08) compared with NTV
(1.91±0.10), and in the TN (1.26±0.10) compared with TV (1.50±0.05). Nandrolone reduced alpha-myosin heavy chain mRNA
levels in both NTN (26% of NTV) and TN (43% of NTV). Training reduced beta-myosin heavy chain mRNA levels in TV (60%
of NTV) and nandrolone (23% of NTV). Only the association between training and nandrolone increased skeletal alpha-actin and
atrial natriuretic peptide (125% and 88% increase respectively, as compared with NTV) mRNA levels in left ventricle.
Conclusions:
Nandrolone treatment, whether associated with resistance training or not, induces cardiac hypertrophy, in association with
enhanced collagen content, re-expression of fetal genes in left ventricle, and impaired diastolic and systolic function.
Keywords: Cardiac hypertrophy, Gene expression, Echocardiography, Resistance training, Nandrolone decanoate
Financial Support: FAPESP, CNPq.
Resumo:02-029
ADHERENCE OF PATIENTS IN AN EDUCATION AND CONTROL PROGRAM OF HYPERTENSION IN THE
CITY OF JATAÍ – GO
Ferreira, N. S. 1; Andrade, D. O. 1; de Lira, C. A. B. 1; Ferri, L. P. 2; Morais, L. C. 2; Cintra, C. E. 2; Beniteribeiro, S. A. 1
1
Universidade Federal de Goiás, UFG
2
Secretaria Municipal de Saúde de Jataí, SMS
Objectives:
Hypertension is a major public health problem affecting more than one billion individuals worldwide, besides it is an important
risk factor for cardiovascular diseases. The patient with hypertension may benefit from intervention programs that focus on drug
treatment and non-pharmacologic procedures, such as regular physical exercise (PE), diet, and body mass (BM) control. So,
adherence (AD) to therapy is essential to reach such control. AD can be defined as the extent to which a person performs
recommendations from a health care provider. In Jatai GO, there is a multiprofessional health care program of education and
control of hypertension (PECH). In this program, patients are given drug therapy and are advised to practice PE, hypocaloric
(HCD) and low sodium diet (LSD). Thus, this study aimed to assess patients AD to the PECH and the effects of counseling done
by the multiprofessional health care team (MHC).
The study included patients with hypertension treated by the PECH, registered between the years 1998 to 2010. We evaluated the
number of patients who after being registered in the program (1st visit, n = 561) attended all the queries until the 15th and 20th
visits. We analyzed the prevalence of patients with: blood pressure (BP) decompensate (BP ≥ 140/90 mmHg- SP= systolic
pressure, DP= diastolic pressure), overweight (BMI ≥ 25kg/m2) and the patients reports about the practice of PE, HCD, and LSD.
The prevalence was tested by Chi square test and the effect of AD on BM and BP was tested by ANOVA with Bonferroni test.
The significance level was p
Conclusions:
In the present study was observed low AD of the patients to the PECH, both by reducing the number of patients who attended all
visits, as by no patients AD to advice from the health team. Problems with AD to therapeutic recommendations are common in
almost all diseases, which impact negatively the effectiveness of the treatment. Probably the factors related to the AD are due to
the complexity of the therapeutic schedule and the adaptability of the recommendations to the usual habits of the person. Indeed,
the knowledge of the patients about disease, the relationship with the health care team, and the patients perception of health and
the benefits of treatment are factors associated with AD. So, we suggest that the interventions need to be easier to apply and that
the health care time combined cognitive and behavioral strategies, planning activities of community meetings to increase patients
knowledge about the disease and the benefits of AD to the PECH. Nevertheless, patients who attended the consultations had
better control of BP, demonstrating the importance of the MHC in the control of BP.
Keywords: Body mass , Hypertension, Hypocaloric diet, Low sodium diet , Physical exercise
Financial Support: National Council for Scientific and Technological Development - CNPq
Resumo:02-030
OXIDATIVE, INFLAMMATORY AND ISQUEMIC BIOMARKERS TO THE METABOLIC SYNDROME.
Gottlieb, M. G. V. 1; Bodanese, L. C. 1; Duarte, M. F. 2; Moresco, R. N. 2; Wiehe, M. 3; Cruz, I. B. M. D. 2
1
Faculdade de Medicina, PUCRS
2
Centro de Ciências da Saúde, UFSM
3
Ambulatório de Risco Cardiometabólico, ARC
Objectives:
Aim: to investigate the association among lipids, oxidatives and inflammatory biomarkers potentially associated with ischemic in
patients with Metabolic Syndrome (MS).
Methods: prospective, case-control study that included 32 (30.2%) healthy subjects (control - C) and 74 (69.8%) subjects with
MS (case group - MS). Individuals with MS were recruited at cardiometabolic risk center of Cardiology Service, Hospital São
Lucas da Pontificia Universidade Catolica do Rio Grande do Sul and the healthy were recruited from the Universidade Federal de
Santa Maria (UFSM - students, staff and teachers). The diagnostic criteria of MS was used National Cholesterol Education
Program Expert Panel on Detection, Evaluation,and Treatment of High Blood Cholesterol in Adults (NCEP-ATPIII). Glucose,
total cholesterol (TC), high-density lipoprotein (HDL) and triglycerides (TG) were determined by enzymatic colorimetric method
(Ortho-Clinical Diagnostics), low-density lipoprotein (LDL) was determined by the Friedewald equation; ultra C-sensitive
protein (CRP-us) was measured by nephelometry, autoantibody anti-oxidized LDL (anti -oxLDL) and oxidized LDL (oxLDL)
were determined by ELISA; ischemia modified albumin (IMA) was measured by colorimetric assay with cobalt. The Research
Ethics Committee of the Pontifícia Universidade Católica do Rio Grande do Sul approved the study protocol (Nº 07/04069) and
informed consent was obtained from all individuals whose information was collected prospectively. Results: The sample included
34 men and 72 women. The average age of group C was 55.2 ± 8.8 years and the MS group 57.6 ± 8.3 years (p = 0.142). The MS
group had a higher Body Mass Index (BMI) (32.6 ± 6.1 kg/m2, p = 0.0001), systolic blood pressure-SBP (147 ± 26.8 mmHg, p =
0.001), diastolic blood pressure-DBP (85.9 ± 15.9 mmHg, p = 0.01), glucose (133.7 ± 68.7 mg/dL, p=0.001), TC (204 ± 58.1
mg/dL, p=0.001), LDL (114.4 ± 49.5 mg/dL, p=0.001), TG (214.9 ± / 34.8 mg/dL, p=0001 ) compared with C group (BMI=
22.1± 4.6; SBP=117.5± 8.0; DBP=74.6± 18.5; glucose=81.7± 7; TC=148.5± 17.8; LDL=78.2± 11.5 and TG=99.4± 43.5),
respectively. IMA levels, CRP, IL-6, oxLDL and anti-oxLDL were significantly higher (0.618 ± 0.1355, p=0.0001; 1.72 ± 0964,
p=0.0001; 53.20 ± 14.52, p=0.0001; 0662 ± 0461, p= 0.001; 27,822 ± 17,010, p=0.001) respectively in MS group, compared to C
group (IMA=0,338±0,0486; CRP=0,172±0,0716, IL-6=17,06±3,17, oxLDL=0,193±0,261, anti-oxLDL=3,359±2,277),
respectively. Multivariate analysis showed an association between high levels of IMA and MS and this was independent of sex,
age, type 2 diabetes (DM2) and hypercholesterolemia (p=0, 0405).
Conclusions:
Conclusion: This study reports an association between IMA, inflammatory biomarkers of oxidative and lipid metabolism with
MS, indicating that those individuals with MS are more likely to trigger the atherothrombotic events. Additional studies involving
genetic and environmental interactions in patients with MS can help to elucidate the potential clinical role of the IMA, to become
a predictor of atherothrombotic events, aiming to reduce the morbidity and mortality by cardiovascular disease (CVD).
Keywords: Inflammatory biomarkers, oxidative biomarkers, Metabolic Syndrome, Isquemic Biomarker, Cardiometabolic Risk
Factor
Resumo:02-031
INFLUENCE OF ENOS GENE POLYMORPHISMS ON THE CARDIOMETABOLIC PARAMETERS IN RESPONSE
TO EXERCISE TRAINING IN CLIMATERIC WOMEN
Esposti, R. D. ; Sponton, C. H. G. ; Malagrino, P. A. ; Fernandes, R. A. ; Puga, G. M. ; Novais, I. D. P. ;
Rodovalho, C. ; Bacci, M. ; Zanesco, A.
Depto de Educação Física - Universidade Estadual Paulista, UNESP/RC
Objectives:
Endothelial nitric oxide gene (eNOS) polymorphisms are associated with risk factors for cardiometabolic diseases (CMD)
development such as dyslipidemia, hypertension and atherosclerosis. Conversely, physical exercise training (ET) is recommended
as an important non-pharmacologic therapy to prevent the deleterious effects of CMD. Therefore, the aim of this study was to
analyze the influence of association of eNOS polymorphisms (-786T>C, Glu298Asp and Intron4 a/b) on the blood pressure, total
cholesterol (TC), low density lipoprotein (LDL-C), nitrite/nitrate (NOx-), superoxide dismutase (SOD) and malondialdehyde
(MDA) levels in trained women.
Forty-nine climacteric women (50±0.9 yrs) were divided into four groups: without polymorphisms – G1 (n=5), with only one
polymorphism – G2 (n=19), with two polymorphisms – G3 (n=19) and with three polymorphisms – G4 (n=6). Aerobic ET
consisted of 3 days/week, 30-40 min/session at 50-70% of heart rate reserve, on cycle ergometer during 8 weeks. Systolic blood
pressure (SBP), diastolic blood pressure (DBP), TC, LDL-C, NOx-, SOD and MDA were measured before and after ET. The
eNOS gene polymorphisms were evaluated by PCR-RFLP and genotyping in MegaBACE sequencer. The VO2max values
increased significantly after ET (baseline: 27±0.99 and after ET: 33±1.13 ml/kg/min). Both SBP and DBP were reduced in all
studied groups after ET, approximately 4 to 10 %. On the other hand, only group G1 and G2 showed a significant reduction in TC
(G1: 200.6±13.5 to 155.4±12.7 and G2: 216.3±10.4 to 177.2±8.5 mg/dL) and LDL-C (G1: 132.4±17.2 to 77.7±13.4 and G2:
148.0±11.3 to 99.3±8.2 mg/dL) after ET. Plasma SOD levels increased only in G3 (from 4.8±0.4 to 5.8±0.4 U/mL) whereas
NOx- and MDA levels were not affected by ET in all studied groups.
Conclusions:
We can conclude that the presence of two or more eNOS polymorphisms affects negatively the beneficial effects of ET on the
metabolic responses in climacteric women.
Keywords: PARAMETERS CARDIOMETABOLIC, eNOS GENE, EXERCISE TRAINING
Financial Support: CNPq/FAPESP
Resumo:02-032
CARDIORESPIRATORY EFFECTS PRODUCED BY INJECTION OF MOXONIDINE INTO THE COMMISSURAL
NUCLEUS OF THE SOLITARY TRACT IN RATS.
Totola, L. T. ; Neto, H. C. F. ; Antunes, V. R. ; Takakura, A. C. ; Moreira, T. S.
Departamento de fisiologia e biofísica/ farmacologia-USP, ICB-1/USP
Objectives:
Moxonidine (á2-adrenergic/imidazoline agonist) is a central acting anti-hypertensive drug that reduces arterial pressure due to
decreasing sympathetic nerve activity. The rostral ventrolateral medulla (RVLM/C1) contains the sympathetic pre-motor neurons
involved in cardiovascular regulation and has been implicated as one of the most important central sites for the anti-hypertensive
action of moxonidine. In the present study, we sought to evaluate the involvement of the commissural nucleus of the solitary tract
(cNTS), an important brainstem nucleus strictly related to autonomic outflow, on the cardiovascular and respiratory effect
produced by moxonidine in comparison to those effects elicited by this substance within the RVLM/C1 region.
Mean arterial pressure (MAP), splanchnic sympathetic nerve activity (sSNA) and phrenic nerve activity (PNA) were recorded in
urethane anaesthetized and artificially ventilated male Wistar rats (280-320 g, n=6-7). Injections of moxonidine (2.5 and 5
nmol/50 nl) into the cNTS reduced MAP (Ä = -12 ± 3 and -22 ± 4 mmHg, vs. saline: Ä = +3 ± 2 mmHg), HR (Ä = -19 ± 6 and 28 ± 5 bpm, vs. saline: Ä = +5 ± 2 bpm) and sSNA (Ä = -8 ± 2 and -18± 3%, vs. saline: Ä = 2 ± 2%). Injection of moxonidine (5
nmol/50 nl) reduced PNA (Ä = -15 ± 4%, vs. saline: Ä = 2 ± 3%). Bilateral injection of moxonidine (1, 2.5 and 5 nmol/50 nl) into
the RVLM/C1 region produced a dose-dependent hypotension (Ä = -15 ± 5, -27 ± 4 and -37 ± 5 mmHg, vs. saline: Ä = 6 ± 4
mmHg), bradycardia (Ä = -19 ± 4, -32 ± 5 and -45 ± 7 bpm, vs. saline: Ä = 4 ±4 bpm) and sympathoinhibition (Ä = -15 ± 6, -22
± 4 and -33 ± 7%, vs. saline: Ä = 5 ± 5%) without any significant changes in the PNA.
Conclusions:
These data suggest that the effect of moxonidine as an anti-hypertensive drug is due to its action in cNTS and RVLM neurons
eliciting the cardiorespiratory changes.
Keywords: CARDIORESPIRATORY, MOXONIDINE, NUCLEUS OF THE SOLITARY TRACT
Financial Support: FAPESP.
Resumo:02-033
USE OF SELECTIVE SEROTONIN RE-UPTAKE INHIBITOR DURING LACTATION AND RISK
CARDIOVASCULAR DISEASE.
Barros, M. D. L. D. 1; Silva, A. I. D. 2; Novaes, L. . C. M. G. 1; Martimiano, P. 1; Lagranha, C. J. 3
1
departamento de anatomia, ufpe
2
departamento de nutrição, ufpe
3
nucleo de educação física e ciências do esporte, CAV-UFPE
Objectives:
The association between antidepressant use and risk of cardiovascular disease (CVD) remains controversial. Given that
antidepressants such as selective serotonin reuptake inhibitors (SSRIs) are now prescribed not only for depression, but also for a
wide range of conditions, this issue still has important relevance. It is known that cardiovascular disease is induced in part by
oxidative imbalance, thereby increasing the production of reactive oxygen species (ROS), and leading to increased oxidative
stress in heart, vessels or brainstem. The brainstem nuclei, nucleus tractus solitarius (NTS) and the rostral ventrolateral medulla
(RVLM) play an important role in the regulation of autonomic cardiovascular system. Many studies relating ROS and
cardiovascular disease have been done in vasculature, however there are data suggesting that an increase in ROS in central
nervous system is involved in neural abnormalities of cardiovascular disease (i.e. hypertension). With this our aim was evaluate
the effect of Fluoxetine (also known by the tradename Prozac) treatment in the levels of oxidative stress in heart and brainstem,
both tissues important for the cardiovascular control.
The treatment was carry-out during the lactation time (21days) with concentration at 10mg/Kg; 1µl/g body weight in female
Wistar rats (via subcutaneo). Control group receive saline 10ml/Kg body weight during the same period. Samples from heart and
brainstem were obtained from control (6) and treated-rats (6). At 60 days of age the animals from both group was sacrificed, and
heart and brainstem were quickly removed and saved in -80ºC until biochemical analysis. We evaluate protein concentration by
Bradford (Anal Biochem. 72:248-254, 1976), levels of oxidative stress (Malondialdeyde-MDA previously described by Draper &
Hadley in Methods Enzymol 186:421-31) and antioxidant enzyme (catalase activity-CAT previously described by Aebi et al. in
Methods Enzymol 105:121, 1984). We observe that in brainstem an increase in MDA in the group that receive fluoxetine (0,6117
+/- 0,04 vs Control group: 0,363 +/- 0,04 nmol/mg protein; p<0,05).
Conclusions:
Our data suggest that chronic treatment with fluoxetine during the critic period of development induce significant increase in
oxidative stress in brainstem and heart which could be associated with cardiovascular disease in adulhood.
Keywords: antioxidant enzymes, neurogenic hypertension, oxidative stress, pharmacological manipulation
Financial Support: CNPq, FACEPE
Resumo:02-034
ANGIOTENSIN II VASODILATOR RESPONSE IN EXPERIMENTAL PREECLAMPSIA
Amaral, T. A. S. 1; Carvalho, L. C. R. M. 1; Ognibene, D. T. 2; Soares Moura, R. 1; Resende, A. C. 1
1
Departamento de Farmacologia e Psicobiologia - IBRAG, UERJ
2
Colegiado de Ciências Biológicas e da Saúde, UEZO
Objectives:
Preeclampsia, a systemic syndrome of pregnancy clinically characterized by proteinuria and hypertension, is associated with
significant morbidity and mortality to both mothers and fetuses. Despite an expressive increase in renin-angiotensin system
(RAS) activity in the normal pregnancy, blood pressure does not increase. On the other hand, litle is known about the
contribution of RAS to the maternal cardiovascular regulation at the pregnancy that is accompanied by hypertension. In the
present study, we investigated the vascular reactivity to angiotensin II and angiotensin 1-7 in a condition similar to preeclampsia
which was induced by chronic inhibition of nitric oxide synthesis by L-NAME.
The experiments were approved by the Ethics Committee of Animal Experiments of the UERJ (protocol: CEA/023/2010).
Pregnant Wistar rats were treated with L-NAME (60 mg/kg/day, orally, LNP) or vehicle (CP) from day 13 to day 20 of
pregnancy and non-pregnant rats were treated with L-NAME (CNP) or vehicle (LNNP) during 7 days. Blood pressure (systolic,
mean and diastolic) was measured by plethysmography. Urinary protein content was measured by Bradford method. The number
of fetus alive and placenta weight were also estimated at day 20 of pregnancy. The serum estradiol level was measured by
radioimmunoassay kit. The vasodilator effect of acetylcholine (ACh 10 pmol), nitroglycerine (NG 10nmol), angiotensin II (Ang
II 30-300 nmol) and angiotensin 1-7 (Ang 1-7 30-300nmol) was studied in mesenteric arterial bed (MAB) pre-contracted with
norepinephrine (3-10 µM).Sistolic (S), diastolic (D) and mean (M) blood pressure was increased in LNP and LNNP (S:194,7±5,1
and 191,9±3,8; D: 144,7±6,3 and 144,7±5,7; M: 162,8±5,0 and 159,3±5,1) compared to CP and CNP (S:122,6±1,1 and
122,1±2,2; D: 89,5±4,3 and 88,9±4,1; M: 100,1±3,1 and 104,6±3,2). Urinary protein content was increased in LNP (1,1±0,2 vs
0,38±0,1). The number of fetus alive was reduced in LNP (6,5±0,9 vs 9,3±0,8). Placenta weight was reduced in LNP (350,5±13,5
vs 436,3±7,3). The serum estradiol level increased in CP and LNP (61,9±5,3 and 65,9±5,0) compared to CNP and LNP (45,6±4,5
and 46,1±3,1). The vasodilator effect (% of relaxation) of ACh was reduced in LNNP rats compared to CNP and CP (20,7±2,6 vs
36,9±3,4 and 41,2±6,5). Otherwise, the vasodilator effect of NG is increased in both LNP and LNNP compared to CP and CNP
(58,2±6,1 and 60,5±4,3 vs 32,7±2,3 and 30,7±3,5). The vasodilator response of Ang II was reduced in LNP and LNNP compared
to CP and CNP (16,4±1,9 and 17,0±1,4 vs 28,6±4,2 and 25,8±1,5). The vasodilator response of Ang 1-7 was not significantly
different between the four groups.
Conclusions:
The results demonstrate that hypertension is associated with an endothelial dysfunction in non-pregnant rats characterized by the
reduction of ACh and Ang II vasodilator responses. The pregnancy in normal condition does not modify the vasodilator response
to Ang II, but this response is reduced in hypertensive pregnant rats which may contribute to the increased blood pressure.
Keywords: renin-angiotensin system, L-NAME , preeclampsia
Financial Support: CNPq and FAPERJ
Resumo:02-035
EFFECTS OF ORCHIDECTOMY AND TESTOSTERONE REPLACEMENT ON THE VENOCONSTRICTION
INDUCED BY ADRENOCEPTORS STIMULATION
1
Rossignoli, P. D. S. 1,2,3; Pereira, O. C. M. 2; Chies, A. B. 1,2
Laboratory of Pharmacology, Faculty of Medicine of Marília, FAMEMA
2
Department of Pharmacology, Institute of Biosciences , IBB UNESP
3
Pharmacy, University of Marília, Marília/SP - Brazil, UNIMAR
Objectives:
The cardiovascular system has been considered an important target of androgen actions. However, androgenic actions upon this
system remain controversial especially in venous bed. It has been suggested that orchidectomy increases Rmax to phenylephrine
in portal vein. This phenomenon appears to be territory specific since it was observed in portal vein but not in vena cava.
Moreover, it appears to be agonist-specific since it was not observed in portal vein preparations challenged with noradrenaline.
Thus, the aim of the present study was to assess the influence of orchidectomy and testosterone replacement on the
venoconstriction of other vascular beds challenged with adrenergic agonists.
Male Wistar rats (350-400g) were sham-operated (CONT) or orchidectomized (ORX). In the 23th day, the treatment (for 3
weeks, with 5-day intervals between the doses) was started with vehicle (CONT and ORX) or testosterone propionate (ORX+T)
(10mg/kg, i.m.). The effectiveness of the orchidectomy and testosterone replacement was evaluated through the weight of sexual
acessory hormone-dependent organs and by serum testosterone measurement. For vascular reactivity experiments, rings of renal,
pulmonary, femoral, and mesenteric veins were obtained of killed animals and were set up in organ bath containing KrebsHenseleit solution, 37°C, pH 7.4, gassed with 95% O2/5% CO2. Contractions under 0.5g of basal tension were registered in
preparations by isometric transducers and expressed as concentration-response curves to phenylephrine, noradrenaline, clonidine
and methoxamine. Log of EC50, maximal response (Rmax) and area under curve (AUC) (n=8-10) were compared by one way
ANOVA/Bonferroni (values presented as mean ± SE and significance when P
Conclusions:
Orchidectomy was effective in reducing the weight of sexual acessory hormone-dependent organs and serum testosterone level,
and testosterone replacement was efficient to revert this alterations. However, orchidectomy, following or not by testosterone
replacement, does not modify the contractile effects of phenylephrine, noradrenaline, clonidine and methoxamine either in renal,
pulmonary, femoral or mesenteric veins.
Keywords: ADRENOCEPTOR, ORCHIDECTOMY, PORTAL VEIN, TESTOSTERONE, VENA CAVA
Financial Support: FAPESP (Proc. 09/08012-2).
Resumo:02-036
ANAEROBIC THRESHOLD DETECTION GOING FROM A MATHEMATICAL TOOL BASED ON THE ARMA
MODEL
Neder Issa, F. L. 1; Giannetti, M. R. S. 1; Gomes, M. E. D. 1; Criollo, C. J. T. 1; Britto, R. R. 2; Neder Issa,
A. M. 1; Pereira, D. A. G. 2; Azeredo, G. O. C. 1; Zampa, C. C. 2
1
Dep. de Engenharia Eletrônica / Escola de Engenharia, UFMG
2
Departamento de Fisioterapia / EEFFTO, UFMG
Objectives:
Anaerobic threshold (AT) has been more and more used to classify functional limitations and stratify risk in cardiac patients. It is
also extensively used to prescribe physical activity on an individual basis in order to improve aerobic capacity and muscle
strength and to prevent cardiovascular diseases. Two AT identification methods are currently recommended. The invasive one
analyses arterial blood lactate and bicarbonate measurement. The noninvasive method examines the change in the behavior of
ventilatory and metabolic variables. However, both methods are not available to the public. It is restricted to institutions directed
to scientific research as it requires sophisticated equipment and highly skilled staff to implement them. Therefore the object of
this research is the application of a mathematical tool derived from linear modeling techniques, which is easy to use and
inexpensive, to determine AT from heart rate variability (HRV) signals.
Forty-six healthy young adult male participated in this study, age of 24.87 ± 4.6 years old, weight of 72.15 ± 10.90kg, height of
1.77 ± 0.07m, body mass index of 22.89 ± 3.12 Kg/m². The sample size calculation was estimated at 38 men. This study was
approved by the Ethics Committee of the Federal University of Minas Gerais as an addendum of a previous project, opinion
092/05 November, 2007. All volunteers signed a consent form. Initially, the AT was determined by analyzing the change of
behavior between the graphs of VCO2 and VO2. These data were collected and computed by an ergospirometry system (Medical
Graphics ® CPX Ultima, Miami, FL, USA) during a standard ramp protocol on treadmill. Also, a series of R-R intervals from the
electrocardiogram (HRV signal) were collected (Welch Allyn, Skaneateles Falls, NY, USA). This graphical method of AT
determination was considered the gold standard in this study and it was compared to the AT detection method based on linear
modeling. Recursive autoregressive moving average (ARMA) models were used to compute the AT from the series of R-R
intervals. The order of the models were determined by Akaike information criterion and the parameters were estimated by
standard least square regression. The AT was evaluated by analysing the change of behavior of the models parameters. The
Shapiro-Wilk test was used to analyse the data distribution. Spearman correlation test was used to compare the AT estimates
obtained by the gold standard method and the developed mathematical method and it was considered significant for p
Conclusions:
The estimate of the AT obtained by the ARMA modeling method was appropriate. This result suggests that the analysis of HRV
signals by linear modeling methods may contribute to compute the AT for clinical practice, expanding its use, since it is cost
effective and easy handling.
Keywords: Anaerobic threshold, Arma Model, Aerobic Capacity
Financial Support: FAPEMIG, CAPES
Resumo:02-037
THE HYPOTENSIVE EFFECT OF [RU(TERPY)(BDQ)NO+]3 (TERPY) IS INCREASED IN HYPERTENSIVE RATS
AND IS SLOWER IN MALES THAN IN FEMALES SHR.
Hildebrand, M. C. 1; Munhoz, F. C. 1; Potje, S. R. 1; Ramos, L. C. B. 2; Bendhack, L. M. 2; Antoniali, C. 1
1
DEPARTAMENTO DE CIÊNCIAS BÁSICAS, FOA - UNESP
2
DEPARTAMENTO DE FÍSICA E QUÍMICA, FCFRP - USP
Objectives:
The spontaneously hypertensive rat (SHR) is an experimental model of hypertension and oxidative stress. Sexual dimorphism
associated to oxidative stress could affect the bioviability of nitric oxide (NO) and the action of NO donors drugs. The NO donor
sodium nitroprusside (SNP) has potent hypotensive action, but releases toxic metabolites. Nytrosil ruthenium compounds have
been studied as alternative drugs, once they are not toxic and release NO in a controlled way. Recent data have shown that the
relaxation induced by the compound [Ru(terpy)(bdq)NO+]3 (TERPY) is impaired in aortic rings of renal hypertensive rats (2R1C) and is normalized by the association with antioxidant drugs. So, the aim of the present study was to evaluate the sexual
dimorphism in the hypotensive responses of TERPY in SHR.
The arterial blood pressure of SHR and normotensive Wistar rats was evaluated before, during and after the infusion of TERPY
(5 e 7 mg/Kg) and SNP (35 ìg/Kg). The basal MAP was analyzed and compared between females and males normotensive
(Female: 103 ± 5 mmHg, n= 4; Male: 111,7 ± 1,97 mmHg, n=7) and hypertensive rats (Female: 147 ± 3,7 mmHg, n= 4; Male;
161,8 ± 2 mmHg, n=8). Our results show that the sexual dimorphism altered the hypotensive effect of 7mg/Kg TERPY in SHR.
In this dose, the hypotensive effect of TERPY was increased in males compared to females SHR (Males: -33,4±2,2 mmHg, n=8;
Females: -15,4 ± 2,5 mmHg, n=4). There was no sexual dimorphism in the hypotension induced by SNP. Differences were
observed in time associated to the hypotensive effect of both drugs studied. In males SHR the hypotensive effects of SNP or
TERPY were slower than in females.
Conclusions:
These results demonstrated that sexual dimorphism altered the time associated and the hypotensive effects of the NO donor,
TERPY. More experiments will be necessary to understand the mechanisms involved with these differences.
Keywords: NITRIC OXIDE DONORS, SHR, SEXUAL DIMORPHISM, TERPY, SODIUM NITROPRUSSIDE
Financial Support: FAPESP (proc.no. 2010/12004-2)
Resumo:02-038
ENDOTHELIUM-INDEPENDENT RELAXATION TO EQUILIN IN RAT MESENTERIC ARTERIES AND THE
ROLE OF CALCIUM INFLUX
Filgueira, F. P. 1,2; Lobato, N. S. 1,2,3; Ceravolo, G. S. 1; Dantas, A. P. V. 1; Fortes, Z. B. 1; Webb, R. C. 2;
Tostes, R. C. 1,2; Carvalho, M. H. C. 1
1
Department of Pharmacology, University of Sao Paulo, USP
2
Department of Physiology, Georgia Health Sciences University, GHSU
3
Dept. of Biological Sciences, Federal University of Goias, UFG
Objectives:
Several studies have demonstrated the vasorelaxant effects of different estrogenic compounds. Equilin (3-hydroxyestra-1,3,5,7tetraen-17-one) is an example of an equine estrogen and it is a common component of Premarin (about 25%), the leading
prescribed pharmaceutical for hormonal replacement therapy for postmenopausal women in the United States. However, its direct
vascular effects have not been studied to date. Therefore, the present study aimed to investigate the direct relaxing effects of
equilin in resistance mesenteric arteries from female spontaneously hypertensive rats (SHR).
The experiments were performed using rat mesenteric arteries (RMA) (200-300µm) obtained from female SHR (16-18 week-old,
n=21) and mounted in isometric myographs. Different concentrations of equilin (10nM-100µM) were added cumulatively to
isolated RMA precontracted with either thromboxane mimetic (U46619 – 1µM) or potassium chloride (KCl – 120mM). Equilin
induced concentration-dependent relaxation in RMA. This effect was similar between vessels precontracted with U46619 and
KCl (99.94% and 99.15%, respectively), indicating that the vasorelaxation induced by equilin is mediated by effects on both
receptor-operated and potential-operated calcium (Ca2+) channels. Furthermore, incubation with equilin (10µM and 100µM) for
30 minutes decreased the maximal response of endothelium-denuded RMA to increasing concentrations of Ca2+ in high K+
(60mM) depolarizing medium. Additionally, equilin (10µM and 100µM) inhibited the effect of (S)-(–)-Bay K8644 (L-type Ca2+
channel activator) in a concentration-dependent manner. On the other hand, the transient contraction induced by caffeine
(20mM), which activates Ca2+ release from intracellular stores, was not inhibited by equilin in endothelium-denuded RMA.
Equilin effects were not different in tissues with endothelium compared with those without endothelium. Incubation (30 minutes)
with the specific estrogen receptor antagonist (ICI 182,780 – 10μM) failed to inhibit equilin-induced relaxation in U46619precontracted RMA. To determine the contribution of vasodilator prostanoids and/or nitric oxide (NO) to the equilin response,
RMA were incubated with indomethacin (10µM), an inhibitor of prostanoid synthesis and L-NAME (100µM), a non-specific NO
synthase inhibitor. The incubation (30 minutes) with indomethacin, L-NAME, or indomethacin plus L-NAME did not modify the
response promoted by equilin. Blockade of voltage-dependent (KV), Ca2+-activated (KCa) and ATP-sensitive (KATP) K+
channels did not change equilin-induced responses, showing that vasorelaxation to equilin seems not to be mediated by increasing
K+ efflux through KCa, KATP, and KV channels. Experiments elucidating the signaling pathways that could lead to equilininduced vasorelaxation indicated that adenylyl cyclase, guanylyl cyclase, protein kinase A, and protein kinase G are not involved,
since the relaxation to equilin in RMA was not affected by SQ22536 (100µM), ODQ (10µM), KT5720 (1µM), and KT5823
(1µM), respectively.
Conclusions:
Our results demonstrate that acute equilin-induced vasodilation can be explained by Ca2+ antagonistic property. Further studies
using electrophysiological recordings of voltage-gated Ca2+ currents in RMA smooth muscle cells are needed to substantiate the
Ca2+ antagonist effect of equilin.
Keywords: equilin, vasorelaxation, calcium channels, rat mesenteric arteries, hypertension
Financial Support: FAPESP, CNPq, NIH (HL074167,HL071138)
Resumo:03-001
EFFECT OF PHYSICAL TRAINING OF TWO AND THREE-WEEK SESSIONS ON PARAMETERS OF OXIDATIVE
STRESS
Tromm, C. B. 1,2; Silva, D. M. D. 1,2; Rosa, G. L. D. 1,2; Bom, K. 1,2; Mariano, I. F. 1,2; Pozzi, B. 1,2; Tuon, T.
1,2
; Silva, L. A. 1,2; Pinho, R. A. 1,2
1
Universidade do Extremo Sul Catarinense, UNESC
2
Laboratório de Fisiologia e Bioquímica do Exercício, LAFIBE
Objectives:
During the exercise-induced muscle contraction is an increase in the production of reactive oxygen species, causing oxidative
stress (OS) in several organs, including liver and heart. The well-planned exercise can increase antioxidant defenses and
decreasing OS in these organs. However, there are doubts about how many weekly sessions are necessary to improve parameters
of OS. To investigate the effect of the frequencies of two and three times per week of exercise on changes in markers of OS in the
liver and heart.
We used 18 male mice (CF1), young, weighing 30 to 35g were divided into three groups (n = 6): not trained (NT) training twice a
week (T2) and trained three times per week (T3 ). The animals underwent training for eight weeks. Forty-eight hours after the last
exercise session, the animals were killed by decapitation. The liver and heart were surgically removed and stored in a freezer 70ºC for further analysis. Thiobarbituric acid reactive substances (TBARS), protein carbonyls (CP) content of total thiols (TT),
superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) were analyzed. Our findings showed that only
the group T3 reduced oxidative damage (TBARS and CP) in the liver (0.13 ± 0.02, 0.19 ± 0.049 nmol / mg / protein) and heart
(0.20 ± 0, 01, 0.15 ± 0.011 nmol / mg / protein) compared to NT (0.25 ± 0.02, 0.36 ± 0.06 and 0.35 ± 0.041, 0.26 ± 0.017 nmol /
mg / protein , Respectively). There was an increase in these organs in TT (71.08 ± 4.79, 97.7 ± 14.2 DTNB / mg / protein), SOD
(0.37 ± 0.05, 0.23 ± 0.02 UdeSOD / mg / protein) and CAT (0.17 ± 0.03, 0.45 ± 0.04 UdeCAT / mg / protein) in the T3 group
compared to NT (41.7 ± 4.07, 41.6 ± 8.33 DTNB / mg / protein, 0.15 ± 0.01, 0.12 ± 0.01 UdeSOD / mg / protein, 0.07 ± 0.02,
0.02 ± 0.001 UdeCAT / mg / protein) respectively. The GPX activity in these organs showed no significant difference between
groups (0.7 ± 0.1, 0.8 ± 0.06, 1.0 ± 0.1 and 0.6 ± 0.04, 0.7 ± 0.1, 0.9 ± 0.1 mM / mg / protein).
Conclusions:
This study demonstrated that only the frequency of training three times a week reduces oxidative damage and increases the
efficiency of antioxidant system in liver and heart of mice.
Keywords: exercise, oxidative stress, training frequency
Financial Support: CNPq e UNESC
Resumo:03-002
EFFECTS OF A SINGLE BOUT OF EXERCISE ON CIRCULATING ENDOTHELIAL PROGENITOR CELLS IN
SUBJECTS AT INCREASED CARDIOMETABOLIC RISK
Rocha, N. G. 1,2; Sales, A. R. K. 1,2; Medeiros, R. F. 1,2; Silva, J. F. R. 1,2; Silva, B. M. 1,2; Neves, F. J. 1,2;
Silva, M. S. 2; Santos, A. A. 2; Nóbrega, A. C. L. 1,2
1
Graduate Program in Cardiovascular Sciences, UFF
2
Laboratory of Exercise Sciences, UFF
Objectives:
Endothelial progenitor cells (EPC) have a role in ongoing endothelial repair and depletion of these cells seems to contribute to
endothelial dysfunction and progression of cardiovascular disease. On the other hand, regular physical activity is associated with
reduced cardiometabolic risk and one potential mechanism is the improvement of endothelial function through the mobilization
of EPC from bone marrow. However, whether this mechanism is present in subjects with cardiometabolic risk is still unknown.
Thus, this study aimed to evaluate the number of circulating EPC after a single bout exercise in subjects with cardiometabolic
risk.
Four healthy subjects (CT: 25±4 years, 4 men) and 2 subjects with metabolic syndrome (MS: 43±3 anos, 1 man) were enrolled.
The subjects with MS had at least three of the following five criteria: waist circumference ≥90cm (man) or ≥80cm (woman);
triglycerides ≥150mg/dL; HDL cholesterol < 40mg/dL (man) or < 50cm (woman); systolic blood pressure ≥130mmHg or
diastolic blood pressure ≥85mmHg; fasting glucose ≥100mg/dL. The subjects of the CT group did not have any of these criteria.
All evaluations were conducted in the morning and the woman was evaluated during in the follicular phase of the menstrual
cycle. The exercise was performed on a cycle ergometer for 40minutes at 60rpm and at the workload where the anaerobic
threshold occurred. A peripheral blood sample (25mL) was collected before and immediately after exercise. The mononuclear
cells were isolated by Ficoll-Hypaque centrifugation and the EPC were quantified by CyAn™ flow cytometry
(CD34pos/VEGFR2pos). The EPC phenotype was confirmed by immunocytochemistry as double positive cells for Dil-labeled
acLDL and FITC-labeled Ulex europaeus agglutinin 1. All data of EPC were expressed as percentage of the number of cells
within lymphocyte gate and multiplied by 10,000 for convenience.The Cohen's d test was used to determine the effect size of the
presence of MS and of performing the exercise bout. This study was approved by the Institutional Ethics Committee (013/2010).
The preliminary results showed that subjects with MS had a reduced number of EPC at baseline compared healthy subjects (MS:
3.0±5.0% vs. CT: 39.0±44.0%, Cohen's d: 1.64, large effect). The increase in the number of EPC after exercise was lower in
subjects with MS (MS: 2.0±4.0% vs. CT: 18.0±97.0%, Cohen's d: 0.36, small effect). Although exercise increased the number
EPC in subjects with MS (before exercise: 3.0±5.0%, after exercise: 5.0±1.0%, Cohen's d: 0.97, moderate effect), the difference
between the groups persisted after exercise (CT: 45.0±98.0%, MS: 5.0±1.0%, Cohen's d: 1.21, large effect).
Conclusions:
Albeit preliminary, these results suggest that subjects with MS have a reduced number of circulating EPC at baseline and a
blunted increased in the number of these cells after a single bout of dynamic exercise.
Keywords: exercise, endothelial progenitor cells, cardiometabolic risk
Financial Support: CAPES, CNPq, FAPERJ, FINEP, Labs DÓr.
Resumo:03-003
THE ANTIDYSKINETIC EFFECT OF EXERCISE ON L-DOPA-TREATED HEMIPARKINSONIAN MICE
Aguiar, A. S. Jr1; Hoeller, A. 2; Moreira, E. L. 2; Oliveira, P. A. 1; Matheus, F. C. 1; Cordova, F. C. 2; Castro,
A. 2; Leal, R. B. 2; Latini, A. 2; Prediger, R. D. 1
1
Departamento de Farmacologia, UFSC
2
Departamento de Bioquímica, UFSC
Objectives:
The motor symptoms of Parkinson‟s disease respond satisfactory to dopamine replacement with L-3,4-dihydroxyphenylalanine
(L-DOPA), but its long-term administration is associated with aggressive motor adverse effects, such as dyskinesia and wearing
off. There is evidence that a „dopamine and cAMP-regulated phosphoprotein 32-kDa‟ (DARPP-32) misbalancing in the striatal
medium spiny neurons modulates the dyskinetic effects of L-DOPA. In the current study we described original findings that
exercise influences DARPP-32 signaling and attenuates L-DOPA-induced dyskinesia in hemiparkinsonian mice.
Four weeks after unilateral striatal lesion with 6-hydroxydopamine (6-OHDA), C57BL6 mice (male, 25-30 g) presented a
dramatic assimetry in forelimb use and apomorphine-induced rotations, and reduced striatal tyrosine hydroxylase
immunoreactivity on dopaminergic nigrostriatal pathway in the 6-OHDA-lesioned side. At this time, all animais received oncedaily intraperitoneal treatment with L-DOPA (25 mg/kg) and the peripheral DOPA decarboxylase inhibitor benserazide (12.5
mg/kg) for two additional weeks, while half of the animals had simultaneously free access to voluntary running wheels. The LDOPA administration produced a partial recovery in the forelimb use, with similar effects in sedentary and exercised animals.
Sedentary mice presented severe dyskinesias observed until 2-h after the L-DOPA administration. However, these dyskinesias
were almost completely abolished in the exercised animals. This significantly decrease was attributable to a marked exerciseinduced reduction of axial, limb, orolingual and locomotor abnormal voluntary movements. The dyskinetic sedentary mice
presented signaling and neurotransmisiom misbalancing to consecutive the L-DOPA treatment, such as increased cAMP levels,
glutamate release, and phosphorylation of DARPP-32-Thr34 and -Thr75 sites in the 6-OHDA-lesioned striatum. Furthermore, we
found that exercise prevented these signaling and neurotransmission alterations.
Conclusions:
Antidyskinetic agents also interfere in these signaling pathways, but only lessen the dyskinesia severity, and have hard-limiting
adverse effects. However, our results suggest that instead of reducing L-DOPA-induced dyskinesia levels, exercise can prevent
the development of these abnormal movements, through normalization of DARPP-32 signaling and glutamatergic
neurotransmission.
Keywords: Exercise, Parkinson's disease, L-DOPA, Dyskinesias, DARPP-32
Financial Support: CNPq, CAPES and PRONEX-UFSC.
Resumo:03-004
POSSIBLE NEUROPROTECTIVE EFFECT OF PHYSICAL TRAINING ON OXIDATIVE STRESS PARAMETERS
IN CORTEX OF RATS WITH PARKINSON’S DISEASE
Tuon, T. ; Valvassori, S. S. ; Ferreira, G. K. ; Rosa, G. B. ; Tromm, C. B. ; Silva, L. A. ; Streck, E. L. ;
Quevedo, J. ; Souza, C. T. D. ; Pinho, R. A. D.
Programa de Pós graduação em Ciências da Saúde- PPGCS/ UNESC, UNESC
Objectives:
The beneficial effects of exercise on Parkinson‟s disease (DP) has been suggested (Adv Clin Chem. 46:1-50, 2008., J
Neurol.10:1648, 2010., Circ J.75:437, 2011), but the mechanisms responsible for these effects are poorly understood. Aim of this
study was to evaluate the effect of physical training on oxidative stress markers in the cortex of rats with DP.
Twenty-four male Wistar rats, 2-month-old, were divided into untrained+sham-operated (USO), untrained+DP (UDP),
trained+Sham-operated (TSO), trained+DP (TDP), n=6. The animals were submitted to training on the treadmill (8-week, 4
days/week on alternate days, 50 min/day, 13-17 m/min). Twenty-four hours after training, DP was induced by lesion unilateral on
the left hemisphere with an injection of 6-OHDA (8 ìg in 1 ìl in 0.2% ascorbic acid). Seven days after the lesion the animals
underwent rotational test (rotameter) and euthanasia by decapitation ensued. The cortex was homogenized in specific buffer for
Western Blot and immunoblotting with anti-tyrosine hydroxylase (TH), anti-superoxide dismutase (SOD), anti-glutathione
peroxidase (GPX), and specific buffer for complex I and oxidative damage in lipid (TBARS) and protein (carbonyl and
sulphydril content). The UDP and TDP groups showed a clear rotational asymmetry when compared to sham groups and TDP
group showed less asymmetry in relation to UDP group, apart from a significant reduction in the complex I, expression SOD and
GPX as well as an increase in TBARS and carbonyl content and sulphydril groups. The expression of TH was not significantly
altered by exercise, but the TDP group increased the expression of SOD and GPX as well as prevented the oxidative damage in
lipids and protein.
Conclusions:
The effects of exercise on DP observed in the present study indicate the possibility that exercise, to a certain extent, modulates
redox status in the cortex of rats, possibly by improving the antioxidant defense system and electron transport chain.
Keywords: Cortex, Oxidative stress, Exercise, Parkinson disease, Neuroprotective
Financial Support: UNESC, FAPESC, CAPES e CNPq.
Resumo:03-005
EIGHT-WEEK OF CONTINUOUS OR INTERMITTENT TRAINING WITH EQUIVALENT LOAD CAUSES
SIMILAR ADAPTATION IMPROVING AEROBIC CAPACITY IN OBESE RATS
Brandão, B. B. 1; Souza, L. M. D. 2; Trombetta, B. N. O. 2; Papoti, M. 2; Seraphim, P. M. 1
1
Depto. Fisioterapia / Universidade Estaual Paulista, FCT - UNESP
2
Depto. Educação Física / Universidade Estaual Paulista, FCT - UNESP
Objectives:
To investigate the effect of 8-week continuous and intermittent training with load equivalent on the metabolic and lactacidemic
responses from obese-induced rats.
Ninety day-old male Wistar rats were divided into 6 groups: sedentary control (SC), sedentary obese (SO), continuous exercise
control (CEC), continuous exercise obese (CEO), intermittent exercise control (IEC), intermittent exercise obese (IEO). The
hyperlipidic diet was composed by sausage, cheese, bacon, and soft drink ad libitum. The intensities of continuous (CT) and
intermittent training (IT) were based on the values of anaerobic threshold (AT) obtained by the test of Chassain with velocities of
8, 13, 18 and 22 m.min-1. The IT was composed by 11 efforts with 2 min of duration at 120% of AT with 1 min of passive
interval. The CT lasted 30 min at 90% of AT. In the 2nd and 6th week of run, blood samples were collected at the beginning
(T1), middle (T2), and at the end (T3) of IT and CI to follow the behavior of lactate concetration. To evaluate the adaptations of
training, the lactate concentrations that corresponds to the 18 m.min-1 speed were obtained pré and pós IT and CT. For the lactate
analysis, 25μl of blood was collected from the distal proximity tail of the animal and measured in the electrochemical lactimeter
(YSI-1500,Yellow Springs Co.,EUA). The results were showed as mean ± SEM. The analysis of variance (ANOVA. One-Way)
was used for comparing the values of SC, SO, CEC, CEO, IEC and IEO, and if necessary Bonferroni post-test was performed.
The difference among the groups was considered significant when P value < 0.05. The diet caused increase of body weight
(SC=478.33±16.67; SO=561.25±17.17**; CEC=444.83±15.16; CEO=464.20±33.62; IEC=481.00±15.68; IEO=528.80±35.48* g,
**P< 0,01 vs IEO-0). At the 6th week of training, the lactate concentration showed similar behavior to the concentrations of 2nd
week. There was no statistical difference among the CEC-0=2.47±0.15; CEC-15=4.45±0.73*; CEC-30=4.76±1.71; CEO0=2.08±0.16; CEO-15=3.42±0.56; OE-30=3.46±0.58; IEC-0=2.38±0.45; IEC-15=2.71±0.31; IEC-30=2.86±1.23; IEO0=2.18±0.16; IEO-15=3.06±0.53; IEO-30=3.12±1.24. *P
Conclusions:
The continuous training caused higher effect on the body weight suggesting that this protocol of training is better in promoting
loss of weight. Both training protocols, even though in different intensities, showed similar adaptation, suggesting that IT and CT
were efficient for improving the aerobic capacity.
Keywords: obesity, aerobic capacity, intermittent training , continuous training, lactate concentration
Financial Support: CAPES e Fapesp: 2011/00472-2
Resumo:03-006
ROLE OF STRENGTH TRAINING IN RATS: OXIDATIVE STRESS IN HEART AND LIVER
Santos, J. A. 1; Costa, G. L. G. 2; Fernandes, M. P. 3; Lima, C. O. R. 4; Silva, R. B. P. 4; Falcão-tebas, F. 5;
Bento-santos, A. 1; Silva, L. L. 2; Lagranha, C. J. 3; Leandro, C. V. G. 3
1
Programa de Pós-Graduação em Neuropsiquiatria, POSNEURO/UFPE
2
Núcleo de Educação Física e Desporto, NEFD/UFPE
3
Núcleo de Educação Física e Ciências do Esporte, NEFCE/UFPE
4
Núcleo de Nutrição, CAV/UFPE
5
Programa de Pós-Graduação em Nutrição, POSNUTRI/UFPE
Objectives:
The association between strength training and oxidative stress are not clear. It is known that cardiovascular and hepatic disease
are induced in part by oxidative imbalance, thereby increasing in the production of reactive oxygen species (ROS) and decrease
in antioxidant enzymes may lead increase in oxidative stress which could induce pathological effects in heart and liver. With this
our objective was to evaluate the effects of strength physical training in ladder in oxidative stress biomarker in heart and liver in
rats.
Male Wistar rats were trained to climb a 129cm vertical (70° incline) ladder with weights secured to their tail. In start of training
week was realized a performance test in both groups, increasing progressively the weight carried during until the rats no more to
climb. The rats were trained once every 5 days for 8 weeks. Each training session consisted of 7-10 climbs requiring 10-12
dynamic movements per climb with 30%, 50% and subsequent climbs with 80% of maximal overload maximal accessed in
performance test. Samples from heart and liver were obtained from control (6) and trained rats (6). At 120 days of age the
animals from both group was sacrificed, and heart and liver were quickly removed and saved in -80oC until biochemical analysis.
We evaluate protein concentration by Bradford method (Anal Biochem. 72:248-254, 1976), levels of oxidative stress
(Malondialdeyde-MDA previously described by Draper & Hadley in Methods Enzymol 186:421-31) and antioxidant enzyme
(catalase activity-CAT previously described by Aebi et al. in Methods Enzymol 105:121, 1984). No difference in maximal
overload in start of the training was observed (trained: 286.3 ± 9.058; control: 290.0 ± 33.4 grams) but the last overload in trained
was increase (1009 ± 17.28 vs control: 656.0 ± 21.69 grams). In the same way no difference was observed in body weight
(trained: 361.7 ± 10.85; control: 391.0 ± 22.28 grams; p<0,002).
Conclusions:
The daily strength physical training increases the force in rats, but no change the body weight. Our data suggest that chronic
strength physical training can induce significant modulation in catalase activity which could block the oxidative stress in heart
and liver.
Keywords: STRENGTH TRAINING, OXIDATIVE STRESS, RATS
Financial Support: The present study receives financial support from REUNI/UFPE, Capes and CNPq
Resumo:03-007
RESPONSE TO FRACTIONED AND CONTINUED RACE TRAINING ON OXIDATIVE STRESS MARKERS IN
AGED ANIMALS
Bom, K. ; Silva, D. M. D. ; Tromm, C. B. ; Rosa, G. L. D. ; Mariano, I. F. ; Pozzi, B. ; Tuon, T. ; Silva, L.
A. ; Pinho, R. A.
Objectives:
To compare the responses of physical training and continuous fractionated in aged animals on oxidative stress parameters.
Eighteen older animals (24 months) were randomized into three groups: non-trained (NT); training fractional (TF), consisting of
three sessions 15minutos/dia; continuous training (CT) in a 45-minute session / days, with n = 6. Both groups (TC and TF) were
subjected to moderate running exercise on a treadmill with constant speed (1.0 mph), five times a week for eight weeks. 48 hours
after the last exercise session, the animals were sacrificed and quadriceps muscles removed and stored in a freezer -80 ◦ C. We
analyzed the level of protein carbonyls (CP), lipid peroxidation (LP), superoxide dismutase (SOD), catalase (CAT) and
glutathione peroxidase (GPX). Results: Our findings demonstrate that both models increased carbonylation and lipid peroxidation
decreased significantly (p <0.05).
Conclusions:
We affirm that both training (PT and CT) show similar response, causing oxidative stress in aged animals.
Keywords: oxidative stress, elderly animals, physical training
Financial Support: UNESC, FAPESC, CAPES e CNPq
Resumo:03-008
HIPPOCAMPAL PLASTICITY INDUCED BY PHYSICAL EXERCISE DURING THE POSTNATAL BRAIN
DEVELOPMENT OF RATS
Gomes da Silva, S. 1; Unsain, N. 2; Mascó, D. H. 2; Toscano-silva, M. 1; Simões, P. S. R. 3; Naffahmazzacoratti, M. D. G. 3; Mortara, R. A. 3; Scorza, F. A. 3; Cavalheiro, E. A. 3; Arida, R. M. 1
1
Department of Physiology. Universidade Federal de São Paulo, UNIFESP
2
Universidad Nacional de Córdoba, UNC
3
Universidade Federal de São Paulo, UNIFESP
Objectives:
In the last decades many studies have dedicated to the understanding of neurobiological bases of physical exercise to the
maintenance and improvement of neural function in adults and elderly subjects. Although the effects of exercise are well
documented in the mature brain, the influence of exercise in the developing brain has been poorly explored. The purpose of
present study was to investigate the effects of physical exercise on postnatal brain development. For this purpose, we evaluated
the hippocampal plasticity of rats submitted to an aerobic exercise program during the adolescent period (between 21th and 60th
postnatal day-old).
Male Wistar rats aged 21 postnatal day-old (P21) were divided into two groups: exercise group (n=27) and control group (n=27).
Animals of the exercise group were submitted to daily exercise in the treadmill between P21 and P60. Running time and speed
gradually increased during the subsequent days, until reach 18 m/min during 60 min. After the aerobic exercise program,
histological and behavioral analyses were performed. The results showed that the physical exercise program during the postnatal
development increased the mossy fibers density and hippocampal expression of parvalbumin, brain-derived neurotrophic factor
(BDNF) and receptor tropomyosin-related kinase B (TrkB), reduced cannabinoid receptor type 1 (CB1) expression, improved
spatial learning and memory, and enhanced the capacity to evoke spatial memories in later stages. It is important to note that the
adequate intensity and duration of exercise performed during brain development are not well established. While physical exercise
induces hippocampal plasticity, degenerative effects could appear in undue conditions of physical or psychological stress. In this
regard, we showed that the exercise protocol used in our study did not induce inflammatory response and degenerating neurons in
the hippocampal formation of adolescent rats.
Conclusions:
Our findings demonstrate that physical exercise during postnatal development results in positive changes for the hippocampal
formation, both in structure and function. Researches in this topic are relevant to stimulate physical exercise programs for people,
particularly for children and teenagers.
Keywords: Plasticity, Development, Brain, Exercise, Memory
Financial Support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).
Resumo:03-009
PREDICTION OF WORKLOAD OF ANAEROBIC TRAINING PROTOCOL BASED ON THE BODY WEIGHT
MAINTAINS THE BLOOD LACTATE CONCENTRATIONS SIMILAR BETWEEN CONTROL AND OBESE RATS
Pinto Júnior, D. A. C. ; Florido Neto, A. R. ; Moreira, R. J. ; Papoti, M. ; Seraphim, P. M.
Departamento de Fisioterapia FCT UNESP Campus Pres. Prudente, FCT UNESP
Objectives:
The target of this study was to verify if the concentrations of blood lactate during an anaerobic training program with workload
prediction based on the body weight change between obese and control rats.
Male adult Wistar rats were caged in two groups, exercise control (EC) and exercise obese (EO). A hyperlipidic diet (cafeteria
diet) composed by chocolate cookie, mortadella, sausage, bacon and soft drink was offered for EO group. Both groups received
standard chow and water ad libitum. All groups were subjected to anaerobic exercise training and active recover in a treadmill
(aerobic exercise). The anaerobic exercise was based on TAMAKI, T. et al. 1992 exercise protocol. The rats were immobilized
by an adapted vest over a metallic platform. The animals received electrostimulations to perform squat jump series. The workload
was characterized based on the body weight (60 – 70% of weight).The recovering (aerobic) was performed in a treadmill at a
speed of 9.45 m/minute. Exercise training was composed by 3 series of 12 reps each, with 1 minute for rest among the series
(anaerobic). After anaerobic training, rats rested for 2 minutes and ran for 10 minutes on the treadmill for 45 days, 3 times a
week. Twenty five µl of blood sample by caudal puncture was collected in heparinized capillary and transferred to microtubes
containing 50 µl of NaF 1% for lactacidemic analysis. Blood samples were collected 8 times: before the exercise, after first
series, after second series, after third series, 3 minutes of running, 5 minutes of running, 7 minutes of running and 10 minutes of
running (final). The body weight was significantly different between the EC and the EO groups since the 4th week (EC =
288.6±10.8g; EO = 344.8±10.5g* N = 10, P < 0.001) until the end of study (EC = 408.7±13.4g; OE = 478.2±16.6g** N=10, P <
0.004). Blood lactate concentrations were proportionally increasing in both groups without significant difference. Before training
the concentrations remained subthreshold (EC = 1.53±0.33mmol/dL e EO = 1.74±0.51mmol/dL). Results of lactacidemia test
showed that both groups behaved the same way when compared (after 1st series =1.68±0.29 and 1.91±0.26mmol/dL; after 2nd
series = 3±0.75 and 3.02±0.64mmol/dL; after 3rd = 4.39±1.6 and 4.26±0.42mmol/dL; 5th minute of running = 5.08±2.1 and
6.18±0.49mmol/dL; 7th minute of running = 5.65±1.37 and 4.89±1.43mmol/dL; 10th minute of running = 4.32±1.54 e
5.52±1.52mmol/dL; EC and EO respectively, N = 5), however in the 3rd minute of running, lactate concentration of EO was
significantly higher than control (EC = 4.22±0.22mmol/dL; EO = 5.8±0.26mmol/dL*, N=5 P < 0.002). Peak blood lactate
concentration was detected in control group at 7th minute of running (5.6±0.61mmol/dL) and in obese group at 5th minute of
running (6.18±0.24mmol/dL).
Conclusions:
Predicting the workload of training through the relative workload to the body weight did not influence on the blood lactate
concentrations, even that the absolute load had been higher for obese, showing that work performance was similar among them.
Keywords: Exercício Anaeróbio, Lactato, Obesidade
Financial Support: FAPESP 2009/13475-1
Resumo:03-010
EFFECTS OF THERAPEUTIC PULSED ULTRASOUND (TPU) AND DIMETHYLSULFOXIDE (DMSO)
PHONOPHORESIS ON PARAMETERS OF OXIDATIVE STRESS AFTER LESION INDUCED BY ECCENTRIC
EXERCISE.
Rosa, G. L. D. ; Silveira, P. C. L. ; Silva, L. A. D. ; Tromm, C. B. ; Silva, D. M. D. ; Bom, K. F. ; Pinho, R.
A. D.
Objectives:
Measure the effects of the therapy with therapeutic pulsed ultrasound (TPU) + dimethylsulfoxide (DMSO) about the oxidative
stress parameters after the eccentric exercise (EE).
Methods and Results: It was used 36 male rats, weighing from 250 to 300g, and divided on the following groups (n=6): control
group (CG); (EE); (EE+Gel+saline 0,9%); (EE+TPU 0.8W/cm2); (EE+DMSO); (EE+TPU+DMSO). The animals was submitted
to the EE protocol (16° negative inclination) followed by treatment. Forty-eight hours later of EE the animals was sacrificed and
the gastrocnemius was removed for posterior analysis. It was measured the Creatine Kinase (CK), Superoxide Dismutase (SOD),
Catalase (CAT), Superoxide Anion radical production, Carbonylation of Proteins (CP) and the reactive species to Thiobarbituric
Acid (TBARS). The significance level was set in 95% (p
Conclusions:
Conclusion: According to results, the TPU combined with the Gel DMSO can decrease the oxidative stress in injuries induced by
eccentric exercises.
Keywords: dimethylsulfoxide, eccentric exercise , gastrocnemius, oxidative stress , pulsed ultrasound
Financial Support: University of the South End of Santa Catarina-UNESC. Federal University SC- UFSC
Resumo:03-011
BLOOD LACTATE RESPONSES TO HIGH-INTENSITY INTERMITTENT TRAINING IN RATS
Moreira, R. J. ; Costa, A. C. P. ; Seraphim, P. M.
UNIVERSIDADE ESTADUAL PAULISTA "Julio de Mesquita Filho", UNESP
Objectives:
To investigate the blood lactate responses during intermittent training of high intensity squat jump type in Wistar rats
Ten adults male Wistar rats of 4 month-old were kept in the biotery of FCT/UNESP, Campus of Presidente Prudente, fed with
standard chow (Supra Lab – Alisul Ind. Alimentos LTDA., São Leopoldo, RS) and water ad libitum. The training protocol was
performed following the adapted model of force from Tamaki et al. (1992). It consisted on the sessions of 3 times a week,
interposed by 24 hours among sessions composed by 3 series of 12 repetitions of 60 seconds of intervals among series with a
strength load equivalent to 50% of the body weight (BW) during 6 weeks. Every 15 days samples of blood (T) were collected to
determinate the blood lactate concentrations ([La-]), immediately after 1st ([La-]1ª), 2nd ([La-]2ª) and 3rd ([La-]3ª) series and in
the 3rd, 5th and 7th minutes after the last serie of jumps, considering the highest obtained blood lactate value in the end of the
stimulations as concentration lactate peak ([La-]pico). The normality of the data was confirmed with the Shapiro Wilk test. For
comparison of the values of body weight as well as the blood lactate responses, the test of analysis of ANOVA - One Way was
used with Tukey Post-hoc test when necessary. The level of significance was predetermined for P
Conclusions:
We can conclude that the intermittent exercise of high intensity was effective in activating the lactic anaerobic way instead of
others energy sources, however there was a reduction in the levels of blood lactate during the training protocol suggesting an
adaptation to used model.
Keywords: Exercise, Lactate, Wistar rats
Financial Support: Fapesp Process # 2010/17078-4
Resumo:03-012
MORPHOLOGICAL FEATURES OF NEUROMUSCULAR JUNCTION IN RATS (ETHANOL 10% VOLUNTARY
DRINKERS) AFTER RESISTANCE TRAINING.
Matheus, S. M. M. ; Santos, T. D. M. ; Teixeira, G. R. ; Pinheiro, P. F. F. ; Martinez, F. E.
Anatomy/Institute of Bioscience, Unesp
Objectives:
Physical exercises are characterized by a situation that alters the body homeostasis, causing an instantaneous increase in energy
demand of the exercised muscles, and therefore of the organism as a whole. The neuromuscular junction (NJ), that is a chemical
synapse between motor neurons and muscle fibers, is remodeled and amplified by neuromuscular activity. The ingestion of toxic
substances such as ethanol causes severe muscle diseases. Only a small percentage of ethanol is metabolized in the skeletal
muscle, but its chronic use, affects muscle health. Thus, the aim of this study was to examine the influence of resistance training
in soleus NJ of UChB rats (ethanol 10% voluntary drinkers).
Male adult rats (100 days) from Wistar and UChB varieties were randomly divided into six groups with five animals each (83/07
CEEA): Sedentary Wistar (WAS), Wistar Trained (WT), UChB trained with ethanol consumption (UChBt), UChB sedentary
with ethanol consumption ( UChBs), UChB trained without ethanol consumption (UChBta) and UChB sedentary without ethanol
consumption (UChBsa). The training consisted of four sets of 10 jumps in liquid medium in a frequency of three days a week
during 14 weeks with overload equivalent of 50% to 70% of body weight. After training, the animals were weighed and
euthanized with pentobarbital sodium (40mg/kg IP). The soleus muscles of both antimeres were removed and weighed. The right
soleus was processed for morphological and morphometric analyses of NJ through an unspecified esterase technique. The left
soleus muscle was submitted to a procedure in order to get an ultrastructural study of muscle fibers and associated NJ. Data were
analyzed with Tukey test, and it was concluded that body weight was higher in the W group both in the trained (T) (537 ± 53.57),
and sedentary group(S) (531 ± 67.95).This result was accompanied by the increase of soleus muscle weight (WS = 0.266 ± 0.041,
and WT = 0.260 ± 0.029) compared to the other groups. There was an increase in the UChBT group (0.054 ± 0.006) relative
weight compared to the weight of the UChBS group(0.047 ± 0.002). Morphometric analysis of NJ showed a significant
interaction among the groups and types of activities, what highlights a decrease in the length of NJ UChBt group (50.44± 2.99)
compared to WT (59.36 ± 4, 0) and UChBta groups (58.35 ± 0.96).The ultrastructural study showed the presence of central
nucleus and myelin figures in the UChB group muscle fibers, and these figures were also observed in the terminal axons of these
animals. These morphologic aspects indicate signs of demyelination.
Conclusions:
The results show that the training was effective in Wistar rats to promote an increase in NJ length, suggesting a possible
hypertrophy of muscle fibers. In the variety UchB, the interaction between ethanol and resistance training promotes a deficit of
neuromuscular activity, and ethanol caused changes that suggest damage to the fibers and associated NJ. Therefore, the chronic
consumption of ethanol is harmful to the muscle, even if physical exercises are practiced.
Keywords: neuromuscular junction, ethanol, resisitance training
Financial Support: Fapesp 09/54256
Resumo:03-013
INFLUENCE OF EXERCISE ON VENOCONSTRICTION INDUCED BY NORADRENALINE
Chies, A. B. 1,2; Rossignoli, P. S. 2,3,1
Faculdade de Medicina de Marília, FAMEMA
2
Dpto de Farmacologia - Instituto de Bioc. de Botucatu-UNESP, IB-UNESP
3
Fac. de Ccs. Farmacêuticas - Universidade de Marília , UNIMAR
1
Objectives:
Training in rats adapts the portal vein to respond vigorously to noradrenaline when the animal is re-exposed to exercise. This
phenomenon appears to be territory-specific since it was not observed in vena cava. Thus, the aim of the present study was to
assess the influence of exercise on noradrenaline effects in other venous bed.
Male Wistar rats (350-400g) were trained in treadmill (60% of maximal capacity of each animal), 1 hour per day, 5 day/week,
during 10 weeks. Later, sedentary and trained animals were killed at rest or after a single bout of exercise (15 min and 1h,
respectively) and rings (3-4mm) of pulmonary, renal, mesenteric and femoral veins were obtained to be set up in organ bath
containing Krebs-Henseleit solution, 37•C, pH 7.4, gassed with 95% O2/5% CO2. Contractions (under 0.5g) were registered in
preparations (n=8-10) incubed with vehicle or L-NAME (10-4M) by isometric transducers and expressed as concentrationresponse curves. Log of EC50 and maximal response (Rmax) were compared by two way ANOVA/Bonferroni (significance
when P
Conclusions:
Exercise does not modify the effects of noradrenaline on pulmonary, renal mesenteric and femoral veins.
Keywords: Exercise, Noradrenaline, Nitric Oxide, Responsiveness, Vein
Financial Support: FAPESP - proc. 09/09788-4
Resumo:03-014
EFFECT OF RUNNING AT 0.8 AND 1.2 KM/H ON SIRT1 AND PGC-1 ALPHA PROTEIN LEVELS AND AMPK
PHOSPHORYLATION IN SKELETAL MUSCLE OF AGED RATS
Luciano, T. F. ; Nimitti, F. ; Marques, S. O. ; Souza, D. R. ; Zeferino, D. S. ; Vitto, M. F. ; Cesconetto, P. A.
; Souza, C. T.
Objectives:
Aging is associated with an overall loss of function at the level of the whole organism that has origins in cellular deterioration.
Most cellular components including mitochondria require continuous recycling and regeneration throughout their lifespan. Recent
studies have demonstrated a strong relationship between aging-associated reductions in mitochondrial function. Multiple
endogenous and exogenous factors regulate mitochondrial biogenesis through the peroxisome proliferator-activated receptor
gamma coativator-1 alpha (PGC-1 alpha), sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK), including physical
activity. Exercise is known to induce metabolic adaptations in skeletal muscle via activation of these molecules. However, the
molecular age-related alterations in cells exposed to exercise at different intensities remain unknown. The present study
investigates the potential effect of running at 0.8 and 1.2 km/h in skeletal muscle of aged rats.
Male Wistar rats aged three (young) and twenty-four (aged) months of age, weighing 325 ± 18 and 575 ± 35 g, respectively, were
used in this experiment. The young and aged animals were divided in the following groups (n=6): Non-Exercised (NE); exercised
at 0.8 km/h (0.8 km/h) and exercised at 1.2 km/h (1.2 km/h). The animals in exercise groups were submitted to one running
session at constant velocity of 0.8 km/h or 1.2 km/h without inclination for 50 min. Immediately after exercise, blood was
collected (to measure blood lactate) and animals were killed by decapitation. The gastrocnemius (red portion) was removed,
homogenized, and Western blot analysis was performed. Our results showed that aging reduces SIRT1 (45%) and PGC-1 alpha
(39%) expression, and AMPK phosphorylation (40%), when compared with young rats. On the other hand, the running protocol
at 0.8 km/h increases the levels of these molecules when compared with non-exercised group, but this increase was more
significant in young rats. The 1.2 km/h group showed increased SIRT1 and PGC-1 expression and AMPK phosphorylation levels,
when compared with 0.8 the km/h intensity group, but no statistical difference was observed between young and aged rat groups.
Conclusions:
The data show that PGC-1 alpha and AMPK phosphorylation are higher after intense exercise (1.2 km/h), and that the increase of
the expression of these enzymes differs between young and aged rats submitted to 0.8 km/h, but not 1.2 km/h intensity.
Keywords: AGE, AMPK , PGC-1 ALPHA , RUNNING, SIRT1
Financial Support: UNESC and CNPq
Resumo:03-015
REVERSAL HEPATIC STEATOSIS BY EXERCISE TRAINING IN OBESE MICE: THE ROLE OF SREBP-1C
Marques, S. O. ; Luciano, T. F. ; Souza, D. R. ; Zeferino, D. S. ; Vitto, M. F. ; Cesconetto, P. A. ; Souza, C.
T. D.
Objectives:
The worldwide prevalence of non-alcoholic fatty liver disease (NAFLD) is presently estimated to affect 30% of the general
population (Hepatology 40:1387, 2004). Sterol regulatory element binding proteins (SREBPs) have been described as master
transcription factors that regulate enzymes responsible for the synthesis of fatty acids, and triglycerides (Cell 75:187, 1993). In
addition, SREBP-1c plays a pivotal role in the dietary regulation of most hepatic lipogenic genes such as FAS, ACC, and
stearoyl-CoA desaturase-1 (SCD-1). However, effects of exercise on SREBP-1c protein level in liver not have been investigated.
Thus, in this study we investigated the SREBP-1c expression and proteins related in obese mice submitted at endurance training
exercise.
For this, Swiss mice ( n= 8 per group) were submitted a control diet (CT) or high fat diet (HF) plus exercise training (HF+EXE)
for 8 wk, running (0.8 Km/h, five days/week) in treadmill without inclination. Fourth eight hours after the last bout of training
protocol, mice were sacrificed, liver sample homogenized and analyzed for immunoblotting. Liver sample were evaluated for
SREBP-1c, fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (SCD-1) expression and acetyl-CoA carboxylase (ACC)
phosphorylation. Livers of HF mice show increases SREBP-1c (3.3 -fold), FAS (4.6 -fold), SCD-1 (2.4 -fold), CPT1 (2.9 -fold)
expression and activity of ACC (5.1-fold), as measured by [Ser79]-phosphorylation when compared to CT. In the liver of
HF+EXE group, SREBP-1c was reduced 2.0 -fold, FAS (1.8 -fold), SCD-1 (1.6 -fold), CPT1 (2.0 -fold) expression and ACC
activity (3.0 -fold) and reduces lipids hepatic content by 1.9 –fold when compared with sedentary DIO group. Our study reported
that hepatic SREBP-1c and lipogenic enzymes protein levels are significantly lower in obese mice after chronic exercise.
Conclusions:
There are few and ineffective pharmacologic strategies for nonalcoholic fatty liver disease (NAFLD), and thus, the reduction of
SREBP-1c demonstrated that exercise could be excellent non pharmacologic treatment to the diseases of fatty liver.
Keywords: EXERCISE TRAINING, HEPATIC STEATOSIS, LIPOGENIC ENZYMES, OBESE MICE, SREBP-1C
Resumo:03-016
EFFECTS OF THE L-ARGININE ADMINISTRATION, RESISTANCE EXERCISE AND L-ARGININE
ADMINISTRATION COMBINED WITH RESISTANCE EXERCISE ON ENDOTHELIUM-DEPENDENT
VASORELAXATION OF DIABETIC RAT MESENTERIC ARTERY.
Araujo, J. E. S. ; Fontes, M. T. ; Santos, A. C. V. ; Soares, A. P. S. ; Silva, T. L. T. B. ; Mota, M. M. ;
Barreto, A. S. ; Santos, M. R. V.
DEPTO. FISIOLOGIA/ UNIVERISADE FEDERAL DE SERGIPE, UFS
Objectives:
This study evaluated the effects of L-arginine (L-Arg), resistance exercise (RE), and association between them on endotheliumdependent vasorelaxation of mesenteric artery from alloxan-induced diabetic rats.
Male Wistar rats (250 - 300g) were divided into 5 groups: Sedentary Healthy (Control; CON, n = 4), Sedentary Diabetic (SD, n =
5), Sedentary Diabetic + L-Arg (SD + L-Arg, n = 5),Trained Diabetic (TD, n = 5) and Trained Diabetic + L-Arg (TD + L-Arg, n
= 5). The diabetes was induced by alloxan (40 mg/kg, i.v.) 2 weeks before the treatment. The RE protocol consisted of 3 sets of
10 squats. The intensity was set at 50% of the maximal load established by the one repetition maximum test. Animals treated with
L-Arg received 1.25 mg/ml daily in the drinking water. After 8 weeks of treatment, endothelium-dependent relaxation was
assessed through of concentration-response curves to acetylcholine (ACh, 10-9 - 10-4 M; cumulatively) in rings pre-contracted
with L-phenylephrine (Phe; 10 ìM), in the absence or presence of L-NAME (100 ìM). Endothelium-dependent relaxation in SD
(101.5 ± 3.2%) was significantly reduced (p < 0.05) when compared with CON (114.2 ± 4.0%). Furthermore, the SD + L-Arg
potency was significantly (p < 0.001) reduced from 6.8 ± 0.12 to 6.2 ± 0.11, when compared with the SD. In the presence of LNAME, endothelium-dependent relaxations from all groups were inhibited (SD: 85.7 ± 4.9%; SD + L-Arg: -2.9 ± 4.1%; TD: 75.3
± 6.2%, and TD + L-Arg: 69.2 ± 4.3%), moreover, in the SD + L-Arg and TD + L-Arg groups, this inhibition was more
pronounced.
Conclusions:
L-arginine appears to increase the participation of nitric oxide on endothelium-dependent relaxation in mesenteric artery of
diabetic rats, while resistance exercise did not induce alterations.
Keywords: DIABETIC, ENDOTHELIUM, MESENTERIC , NITRIC OXIDE
Financial Support: CNPq, CAPES, FAPITEC-SE, Brazil
Resumo:03-017
LOW-INTENSITY AEROBIC EXERCISE COUNTERACTED THE INCREASED OF PRO-INFLAMMATORY
CYTOKINES LEVELS AFTER SCIATIC NERVE CRUSH INJURY IN MICE
Bobinski, F. ; Bratti, T. ; Martins, D. F. ; Mazzardo-martins, L. ; Santos, A. R. S.
Departamento de Ciências Fisiológicas, UFSC
Objectives:
To evaluate both the profile of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6R) in the sciatic nerve and spinal cord of mice
after sciatic nerve crush injury and the effect of three different protocols of low-intensity aerobic exercise on levels of these
cytokines after nerve injury.
The experiments were performed after the approval of the protocol by the Institutional Ethics Committee for Animal Research of
the Federal University of Santa Catarina (PP404). Male Swiss mice (25 to 35 g) were distributed in five groups (n=6): 1) shamoperated (Sham); 2) sciatic nerve crush control non-exercised (Non-exer); 3) exercise-preoperative for 2 weeks (Exer 1); 4) a
combination of preoperative-postoperative training programs for 4 weeks (Exer 2); 5) exercise-postoperative for 2 weeks (Exer
3). Surgical procedures were performed under deep anesthesia that was induced with a premixed solution containing ketamine
(80 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). The sciatic nerve was exposed in the right thigh and crushed once for 30 s. The
Sham group was subjected to the same surgical procedures, but the sciatic nerve was not crushed. The exercise was performed on
a treadmill designed for human use (Athletic Advanced 2) and modified for mice. The animals ran on the treadmill for 30 minutes
at a speed of 10 m/min with no inclination five days per week, began on the third postoperative day. In the 14th day postoperative the animals were anesthetized with isoflurane (1-2%) and sacrificed by decapitation. Both the sciatic nerve and the
lumbar portion of the spinal cord (L1 to L6) were removed for measure the TNF-α, IL-1β and IL-6R levels using mouse cytokine
Enzyme Linked Immunosorbent Assay (ELISA) kits from R&D Systems (Minneapolis, MN) according to the manufacturer‟s
instructions. These tissues were homogenized, centrifuged at 3000 g for 10 min at 4 °C, and the supernatant obtained was stored
at -70 °C until further analyses. Total protein content was measured in the supernatant using the method of Bradford and sample
aliquots of 100 µl were used to measure the pro-inflammatory cytokines levels. The absorbance for all of the cytokines studied
was measured using a microplate reader at 450 and 550 nm. Statistical analysis were performed using a one-way ANOVA
followed by Student-Newman-Keuls test, P values less than 0,05 were considered to be indicative of significance. The crush
procedure increased the levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6R) in both the sciatic nerve and the spinal
cord compared to the Sham group. The concentrations of IL-1β in both sites and the IL-6R in the spinal cord were reduced in all
of the exercised groups (P < 0,05) compared to the Non-exer group. Nevertheless, a significant reduction in the concentration of
TNF-α in the sciatic nerve (P < 0,05) and spinal cord (P < 0,01) was observed in the Exer 2 and Exer 3 groups compared to the
Non-exer group.
Conclusions:
These data suggest that the protocol of low-intensity aerobic exercise can reduce the levels of some pro-inflammatory cytokines
involved in the pathogenesis of neuropathic pain, both at the site of nerve injury such as spinal cord level. Therefore, physical
exercise may be a promising strategy for the treatment of pain caused by peripheral nerve injury.
Keywords: Hypersensitivity, neuropathic pain, neuroprotection, physical exercise, pro-inflammatory cytokines.
Financial Support: CAPES and UFSC
Resumo:03-018
THE EXERCISE INTENSITY AFFECTS THE RIGHT FRONTAL CORTEX TEMPERATURE OF RATS AT THE
MOMENT OF VOLUNTARY INTERRUPTION OF THE EFFORT.
Kunstetter, A. C. ; Madeira, L. G. ; Lima, M. M. R. ; Wanner, S. P. ; Rodrigues, L. O. C. ; Lima, N. R. V.
Departamento de Educação Física/EEFFTO, UFMG
Objectives:
Body temperature influences exercise performance. Since neurons are the most heat-sensitive cells of the organism, we
hypothesized that brain temperature is important for determining the interruption of exercise. Therefore, the aim of this study was
evaluate the cortical brain temperature during exercise at three different intensities performed until voluntary interruption of the
effort.
Methods: Male Wistar rats (250-300 g) were housed in a room with an ambient temperature of 25°C, on a 14h:10h light/dark
cycle, with free access to food and water. The animals (n=4) were implanted with a cerebral guide cannula (13 mm/21 gauges) in
the right frontal cortex (AP, + 2.5 mm; ML, - 3.2 mm; DV -2.0 mm; relative to bregma). After a recovery period, rats were
familiarized to exercise for 5 days. Immediately before each trial, the thermistor for brain temperature measurement was inserted
trough the cannula. During the experimental protocol, the animals were submitted to a control trial, where they were allowed to
rest in their home cages, or to exercise until voluntary interruption of the effort at three different intensities: 18 m/min (V18), 21
m/min (V21), and 24 m/min (V24) (5% inclination). Cortical temperature was measured during exercise, and thirty minutes after
exercise, and throughout the control experiments. Total exercise time (TET) was measured from the start of exercise until the
voluntary interruption of the effort. The rate of rise in cortical temperature was calculated by dividing the change in temperature
by the TET. Ambient temperature was maintained at 25.1 ± 0.9°C. Data are expressed as mean ± SE and were analyzed by
analysis of variance with repeated measurements. Significance level was set at p≤0.05.
Results: The TET was shorter during the exercise at 24 m/min compared with the two other exercise intensities (V24: 35.0 ± 2.2;
vs. V18: 193.8 ± 25.6; V21 148.0 ± 32.0 min, p<0.05). During the three exercise conditions, it was observed a rise in cortical
temperature, but, when exercise was performed at 21 and 18 m/min the animals presented a higher temperature at the moment of
voluntary interruption of the effort (V18: 40.90 ± 0.24; V21: 40.41 ± 0.35; vs V24: 39.77 ± 0.40°C, p<0.05). During the thirty
minutes post-exercise, brain temperature remained elevated compared with the initial values, and higher at 18 m/min compared
with 24 m/min (minute 0: V18: 37.22 ± 0.19; V21 37.35 ± 0.14; V24: 37.04 ± 0.10 °C vs minute 30 post-exercise: V18: 39.21 ±
0.35; V21: 38.53 ± 0.22; V24: 38.55 ± 0.20 °C, p<0.05). The rate of rise in cortical temperature was the greatest in exercise
performed at 24 m/min (V24: 0.08 ± 0.01 vs V18: 0.02 ± 0.004; V21: 0.02 ± 0.004 °C/min , p<0.05).
Conclusions:
The higher exercise intensity produced a higher rate of rise in cortical temperature, but a lower cortical temperature at the
voluntary interruption of the effort was observed in comparison to the lower exercise intensities. Thus, it is likely that the rate of
rise in cortical temperature has determined the TET.
Keywords: brain temperature, exercise, fatigue
Financial Support: FAPEMIG, CNPq, CAPES
Resumo:03-019
L-ARGININE PROMOTES DECREASE IN METABOLIC STRESS MARKERS AND UP-REGULATE VEGF AND
TGF-β EXPRESSION IN SKELETAL MUSCLE TISSUE AFTER RESISTANCE TRAINING
Morais, S. R. L. 1,2; Mello, W. G. D. 1,2; Oliveira, S. H. P. 1,2
1
Multicentric Graduate Studies Prog in Physiological Science, SBFIS
2
Dept Basic Sciences , UNESP-Araçatuba-SP
Objectives:
A single bout of high intensity resistance training (RT) promote a number of changes in homeostasis, in specially, in skeletal
muscle tissue (SMT), promoting the release of inflammatory mediators (myokines), affecting directly the immune system, as
demonstrated in resume 17.012 of XXV annual meeting FESBE 2010. The aim of this study was to analyze the effectS of the Larginine supplementation upon the metabolic stress markers and regenerative factors released by SMT after a high intensity RT
session.
Male Wistar rats, 180 - 200 g were divided into five groups (n = 10) (hours post-exercise (hpe)) (control group, post-exercise (0),
8 hpe (8), 24 hpe (24) and 48 hpe (48)), with L-arginine (L) (1g/kg) one hour before the start of training or Aminoguanidine (A)
(50mg/kg) (only 24 and 48 hpe), 30 minutes before start the training, both by gavage. The animals were subjected to RT-oriented
practice for four days, through a staircase with 80° tilt, with the overload apparatus corresponding to 80% relative to the body
weight in the fourth day. We collected peripheral blood, skeletal muscle (extensor digitorum longus), for analysis of Reactive CProtein (CRP), Creatine Kinase (CK) and TGF-β by ELISA and VEGF expression by RT-PCR. Statistical analyzes was
performed by ANOVA with Bonferrone correction (p
Conclusions:
Take together, the L-arginine supplementation promote a protective role in skeletal muscle tissue, by reduction of metabolic
stress markers at 24 and 48 hpe, and, up-regulate the regenerative factors (TGF-β and VEGF) in the skeletal muscle tissue. The
NO pathway is involved in the protective process on SMT.
Keywords: L-arginine, resistance training, skeletal muscle, metabolic stress markers
Financial Support: FAPESP/CAPES
Resumo:03-020
ENDURANCE EXERCISE TRAINING AMELIORATES INSULIN RESISTANCE AND RETICULUM STRESS IN
ADIPOSE AND HEPATIC TISSUE IN OBESE RATS.
Cesconetto; P. A. ; Vitto, M. F. ; Marques, S. O. ; Luciano, T. F. ; Santos, D. Z. ; Souza, D. R. ; Luz, G. ;
Souza, C. T.
Universidade do Extremo Sul Catarinese, UNESC
Objectives:
Obesityâ€“induced endoplasmatic reticulum (ER) stress has been demonstrated to underlie the induction of obesity -induced JNK
and NF-kB activation inflammatory responses, and generation of peripheral insulin resistance. On the other hand, exercise has
been used as a crucial tool in obese and diabetic patients and may reduce inflammatory pathway stimulation. However, the ability
of exercise training to reverse endoplasmatic reticulum stress in adipose and hepatic tissue in obesity has not been investigated in
the literature. In the present study we investigated potential effect of exercise training to reverse ER stress in adipose and hepatic
tissues in the obese rats.
The 4-week-old Wistar rats were divided (5 rats per group) into four groups: control rats (C) fed on standard rodent chow, control
rats submitted to 8-week endurance training with workload (C+ET), obese rats fed on an obesity-inducing diet for 2 months
(DIO), and DIO rats submitted to 8-week endurance training with workload (DIO+ET). The control rats were maintained
sedentary throughout the experimental period and the trained rats were submitted to 1-hour daily swimming sessions, with an
attached weight corresponding to 5% of body weight. Twenty four hours after training protocol, the rats were killed and samples
from epididymal fat and liver were obtained and Western blot analysis was performed. Our results showed that swimming
protocol reduces pro-inflammatory molecules JNK, IkB and NF-kB in adipose (48Â±7%, 65Â±11% and 40Â±8%, respectively)
and hepatic (40Â±5%, 48Â±4% and 41Â±9%, respectively) tissues. In addition, exercise leads to a reduction in ER stress, by
reducing PERK (58Â±10% and 47Â±6%, adipose and liver, respectively) eIF2α (64Â±11% and 58Â±9%, adipose and liver,
respectively) phosphorylation. In parallel, an increase in insulin pathway signaling was observed, as confirmed by increases in IR
(72Â±12% and 70Â±14%, adipose and liver, respectively), IRS-1 in adipose tissue (57Â±6%) and IRS-2 in hepatic tissue
(60Â±9%) and Akt (44Â±5% and 59Â±7%, adipose and liver, respectively) phosphorylation following exercise training in DIO
rats.
Conclusions:
The results suggest that exercise can reduce ER stress, improving insulin resistance in adipose and hepatic tissue.
Keywords: Exercise training, Hepatic tissue, Inflammatory pathway, Insulin resistence, Reticulum stress
Resumo:03-021
EFFECTS OF DIFFERENT PROTOCOLS OF PHYSICAL TRAINING ON OXIDATIVE STRESS IN RATS
INDUCED TO VENTRICULAR INFARCTION
Silva, D. M. D. 2; Pinho, C. A. 4; Tromm, C. B. 2; Rosa, G. L. D. 2; Bom, K. 2; Tuon, T. 2; Silva, L. A. D. 2;
Benetti, M. 4; Pinho, R. A. D. 2
1
Laboratório de Fisiologia e Bioquímica do Exercício, LaFiBE
2
3
Centro de Cardiologia e Medicina do Exercicio, CEFID
4
Universidade do Estado de Santa Catarina, UDESC
Objectives:
Cardiovascular diseases are a major cause of death in Brazil and physical exercise is considered an important agent in the
prevention and treatment of these conditions. Studies related to the type, intensity and volume of physical training required to
trigger biochemical protective material are still controversial. This study investigated the effects of two exercise protocols on
parameters of ventricular oxidative stress in rats after myocardial infarction.
Thirty-six male Wistar rats of two months were divided into two groups (n = 18): Sham and Acute Myocardial Infarction (AMI).
The rats were anesthetized and the AMI was induced by coronary artery occlusion. Thirty days after AMI induction, the rats were
subdivided into six groups (n = 6): Untrained sham, US, Sham + continuous training, SCT; Sham + interval training, SIT, (10x5
min with 1 minute interval); Untrained+AMI, UI; AMI + continuous training, ICT and AMI + interval training, IIT. The exercise
sessions were conducted in water (30-32 º C), 5 times/wk for six-wk, 60 min. Forty-eight hours after the last exercise session, the
rats were killed and the left ventricle was surgically removed for analysis of oxidative stress. Mean±SEM was calculated and the
comparisons were performed using one-way ANOVA with Tukey post hoc tests, p value
Conclusions:
These results suggest a significant improvement in the redox state of the myocardium of AMI-induced rats exposed to different
training models, but the continuous training seems to be more efficient for the evaluated parameters.
Keywords: Free radicals, Heart, Myocardial infarction, Oxidative stress, Physical exercise
Financial Support: UNESC, FAPESC, CAP‟s, CNPq
Resumo:03-022
EXERCISE TRAINING REDUCES INSULIN RESISTANCE AND UP REGULATES THE MTOR/P70S6K PATHWAY
IN CARDIAC MUSCLE OF DIET-INDUCED OBESITY RATS.
Vitto, M. F. ; Souza, D. R. ; Marques, S. O. ; Luciano, T. F. ; Santos, D. Z. ; Cesconetto, P. A. ; Avila, P. R.
M. ; Souza, C. T.
Universidade do Extremo sul Catarinense, UNESC
Objectives:
Obesity and insulin resistance are rapidly expanding public health problems. These disturbances are related to many diseases,
including heart pathology. Acting through the Akt/mTOR pathway, insulin has numerous and important physiological functions,
such as the induction of growth and survival of many cell types and cardiac hypertrophy. However, obesity and insulin resistance
can alter mTOR/p70S6k. Exercise training is known to induce this pathway, but never in the heart of diet-induced obesity human
and rodents.
The 4-week-old Wistar rats were divided (6 rats per group) into four groups: control rats (C) fed on standard rodent chow, control
rats submitted to 12-week endurance training with workload (C+ET), obese rats fed on a high-fat diet for 2 months (DIO), and
DIO rats submitted to 12-week endurance training with workload (DIO+ET). The control rats were maintained sedentary
throughout the experimental period and the trained rats were submitted to 1 hour of swimming daily, in water at 32°C, with an
attached weight corresponding to 5% of body weight. Training occurred on 5 days/week for 12 weeks. Exercise training reduced
epididymal fat (32±5%, p
Conclusions:
Results demonstrate a pivotal regulatory role of exercise training on the Akt/mTOR pathway, in turn, promoting protein synthesis
and antagonizing protein degradation.
Keywords: Akt/mTOR pathway, CARDIAC MUSCLE, EXERCISE, INSULIN RESISTANCE, OBESITY
Financial Support: UNESC, FAPESC and CNPq
Resumo:03-023
COMPARISON OF TWO INTENSITIES OF EXERCISE ON INFLAMMATORY MARKERS AND LIPID
PEROXIDATION IN SUBJECTS WITH HEART FAILURE
Ferreira, A. C. 1; Ribeiro-samora, G. A. 2,1; Pereira, D. A. G. 1; Rodrigues, R. S. 1; Favero, M. 1; Guedes, G.
D. S. 3; Rabelo, L. A. 3; Pereira, L. S. M. 1; Parreira, V. F. 1; Britto, R. R. 1
3
Universidade Federal de Alagoas, UFAL
1
Departamento de Fisioterapia, Universidade Federal de Minas, UFMG
2
DCBAS / Centro Universitário de Belo Horizonte, UniBH
Objectives:
Compare the effects of two intensities of exercise (low and moderate) on the inflammatory markers (IL-6 and sTNFR1) and
levels of lipid peroxidation in patients with heart failure (HF).
Twenty-two patients with HF (aged 45.91 ± 1.91 years, classes I-III of New York Heart Association-NYHA, ejection fraction
29.91 ± 2.03% and peak of oxygen consumption 22.36 ± 1.39 mL/kg/min), in clinical stability for at least six months, did a
cardiopulmonary exercise test. Then, they were randomized to two sessions of physical exercise on a treadmill ergometer: low
intensity (LI) at 40% VO2peak and moderate intensity (MI) at 60% VO2peak, both lasting 30 minutes. Blood samples were
obtained pre-exercise, immediately after the exercise and one hour after the interruption of it. The serum levels of IL-6 and
sTNFR1 were measured using the method of ELISA (enzyme-linked immunosorbent assay) and lipid peroxidation was assessed
by the formation of malondialdehyde (MDA), from the breakdown of polyunsaturated fatty acids, by detecting of thiobarbituric
acid reactive substances (TBARS; thiobarbituric acid reactive substances). Statistical analysis: ANOVA mixed factorial, post hoc
Bonferroni test and significance level p
Conclusions:
The exercise of moderate intensity (MI), performed for 30 minutes, leaded to a reduction of lipid peroxidation and an increase of
sTNFR1 immediately after exercise, followed by a reduction in levels of sTNFR1 and increased IL-6, 1h after end of the session.
Therefore, the exercise of moderate intensity, lasting 30 minutes, was superior to promote the immunological adjustments and
lower lipid peroxidation.
Keywords: exercise, heart failure, inflammation, lipid peroxidation
Financial Support: Pesquisador Mineiro da Fapemig e Edital Universal CNPq.
Resumo:03-024
ENVIRONMENTAL ENRICHMENT VERSUS FORCED PHYSICAL EXERCISE EFFETCS ON PARVALBUMIN
HIPPOCAMPAL EXPRESSION OF DEVELOPING RATS
Maia, A. F. 1,3; Gomes da Silva, S. 1; Blazechi, L. F. 1; Fernandes, J. 1; Rachetti, A. L. F. 1; Dona, F. 2;
Fernandes, M. J. S. 2; Scorza, F. A. 2; Cavalheiro, E. A. 2; Arida, R. M. 1
1
Departmento de Fisiologia. Universidade Federal de São Paulo, UNIFESP
2
3
Depto de Desportos. Universidade Federal do Espírito Santo, UFES
Objectives:
Purpose: Several models of experience have been suggested to influence molecular systems important for maintaining neural
function and plasticity. The stimuli required to elicit plasticity are thought to be activity-dependent. In this context, physical
activity models have been used to explore experience effects on brain function. The purpose of the present study was to verify
whether environmental enrichment or a physical exercise program would promote similar changes in the hippocampal formation
of developing rats. For this aim, we performed an immunoblotting study using the calcium-binding protein parvalbumin (PV) as a
neuroplastic marker.
Methods: Male Wistar rats at 21 days of postnatal life (P21) were divided into four groups: enriched environment (EE, n=4), nonenriched environmet (NEE, n=4), exercise (EX, n=4) and non-exercise (NEX, n=4) groups. Animals of the EE and NEE groups
were placed in large cages between P21 and P60, however only the cage of EE group consisted of objects for environmental
stimulation. Animals from EX group were submitted to daily exercise on a treadmill between P21 and P60. Running time and
speed gradually increased over this period, reaching a maximum of 18 m/min for 60 min. At P61, animals of all groups were
killed and immunoblot analysis was performed. Results: No difference in the PV hippocampal expression among EE, NEE and
NEX groups was detected. However, PV expression was enhanced significantly in the hippocampal formation of EX group when
compared to the NEX group.
Conclusions:
Conclusion: These findings suggest that the brain in the maturational process may be differentially sensitive to some physical
activity models.
Keywords: exercise, enriched environment, brain, plasticity, development
Financial Support: FAPESP, CAPES, CNPq.
Resumo:03-025
INCIDENCE OF METABOLIC SYNDROME FEATURES AND RELATED-RISK FACTORS IN ADULTS.
Kano, H. T. ; Moreto, F. ; Manda, R. M. ; Burini, R. C.
CeMENutri/Faculdade de Medicina de Botucatu-UNESP, UNESP
Objectives:
To verify the incidence of MS features and related-risk factors in beginners of a Lifestyle-Modification Program (LSMP).
117 adults (99 women and 18 men, 51 ± 13 years old) beginners in the LSMP “Mexa-se Pró-Saúde” (in Botucatu city, Sao Paulo
State), were submitted to clinical, anthropometric and laboratorial assessments. The assessments included systolic and diastolic
blood pressures (BP) measurements as well as waist circumference (WC), body mass and height for the body mass index (BMI)
calculation. The laboratorial analysis included glucose (GLU), total (CT), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C,
Friedewald, 1972), triglycerides (TG), C-reactive protein (CRP), uric acid, urea, creatinine and gamma-GT, by the Dry Chemistry
method (Vitros System, Johnson & Johnson). MS diagnoses followed the modified NCEP-ATPIII and the used statistical
analyses were descriptive and the Student‟s t test, with 5% as significance criteria (p
Conclusions:
In addition to central hyperadiposity, blood dyslipidemia seems to be the next factor for the MS development with consequent
pro-inflammatory and pro-atherogenic states.
Keywords: DYSLIPIDEMIA, HIPERADIPOSITY, METABOLIC SYNDROME
Financial Support: PROEX, CAPES, CNPq
Resumo:03-026
CAN MATERNAL MODERATE PHYSICAL TRAINING RE-PROGRAM THE EFFECT OF PERINATAL
UNDERNUTRITION ON THE GLUCOSE HOMEOSTASIS IN ADULT OFFSPRING?
Moura, J. P. F. D. 1; Ferreira, D. J. S. 6; Silva, R. A. D. D. 1; Morais, D. S. C. 1; Barreto, M. D. P. 1; Lima,
C. D. O. R. 3; Silva, R. B. P. D. 3; Tebas, F. F. 2; Castro, R. M. D. 1; Amorim, M. A. F. 3
1
DEPTO. EDUCAÇÃO FÍSICA/UNIVERSIDADE FEDERAL DE PERNAMBUCO, UFPE
2
DEPTO. DE FISIOTERAPIA/ UNIVERSIDADE FEDERAL DE PERNAMBUCO, UFPE
3
DEPARTAMENTO DE NUTRIÇÃO/CENTRO ACADÊMICO DE VITÓRIA, CAV/UFPE
4
DEPARTAMENTO DE NUTRIÇÃO/UNIVERSIDADE FEDERAL DE PERNAMBUCO, UFPE
5
NÚCLEO DE ED. FÍSICA E CIÊNCIA DO ESPORTE/ CAV-UFPE, CAV/UFPE
6
Escola Superior Educação Física/Universidade de Pernambuco , UPE
Objectives:
The maternal lifestyle can induce physiological changes during pregnancy and affect the fetal growth and development.
Unbalanced nutrient intake during pregnancy and lactation has been associated to subsequent risk of develop disease in the
offspring. However, studies on physical exercise and the metabolic and physiological changes in offspring are still few and
controversial. The aim of the present study was to verify the effects of a maternal moderate-low physical training on glucose
homeostasis in adult offspring whose mothers were submitted to a low protein diet during the perinatal period.
Male Wistar rats were divided into four groups according to their mother´s manipulation: untrained (NTp, n = 8); trained (Tp, n =
8); untrained with low-protein diet (NT+LPp, n = 8); and trained with low-protein diet (T+LPp, n = 8). Trained mothers were
submitted before pregnancy to a protocol of moderate physical training over a period of 4 weeks (65% VO2max, 5 days/week,
and 60 min/day) on a treadmill. At pregnancy, the intensity and duration of the exercise was reduced (30% VO2max, 5
days/week, and 20 min/day). Low-protein groups received an 8% casein diet, and their peers received a 17% casein diet. The
glucose and insulin tolerance test (GTT and ITT, respectively), fasting blood glucose and serum cholesterol were determined at
150th day of life. Pups were submitted to GTT and ITT, respectively, and the areas under glycemic curve were evaluated and
groups were compared. Pups from trained mothers did not change the response to GTT and ITT when compared to the NT group.
On the other hand, NT+LPp showed an increase (p0,05) between T+LPp animals and NTp. At 150th day of life, pups from low
protein offspring showed a higher glycaemia (NT+LPp=108.4± 2.0; NTp=91.1± 1.6; p
Conclusions:
Our data confirm the negative consequences of perinatal undernutrition in the metabolic parameters in adult rats, however,
moderate physical training at the pregnancy can re-programms the effects of perinatal undernutrition.
Keywords: GLUCOSE HOMEOSTASIS , GESTATIONAL EXERCISE , PERINATAL UNDERNUTRITION
Financial Support: CNPq, CAPES and FACEPE
Resumo:03-027
SPATIAL MEMORY IS IMPROVED BY AEROBIC AND RESISTANCE EXERCISE THROUGH DIFFERENT
CELLULAR MECHANISMS.
Cassilhas, R. C. 1; Lee, K. S. 2; Fernandez, J. 3; Oliveira, M. G. M. 1; Tufik, S. 1; Meeusen, R. 4; Mello, M.
T. D. 1
1
Psicobiologia, UNIFESP
2
Bioquímica, UNIFESP
3
Neurofisiologia, UNIFESP
4
Human Physiology & Sports Medicine, Brussel - Belgium, Vrije Universiteit
Objectives:
The purpose of this study was to examine spatial learning and memory, the BDNF/TrkB and IGF-1/IGF-1R signalling pathways
in the hippocampus of rats subjected to either aerobic or resistance exercise.
Forty-four adult male Wistar rats underwent eight weeks of aerobic training on a treadmill (AERO group, n=11) or resistance
training on a vertical ladder (RES group, n=11); control (n=11) and sham (n=11) groups were also included. The spatial learning
and memory was evaluated by Morris water maze task. In the hippocampus were measured the BDNF (by ELISA method), IGF1 (by RIA method), TrKB/ IGF-1R/ CaMKII/ AKT/ synapsin 1 (by western blot analysis). After eight weeks of exercise training,
the animals in the AERO and RES groups spent less time (Group effect, F(3, 40)=48.57; P<0.001).
Conclusions:
In light of these results, one might conclude that aerobic or resistance training for 8 weeks increases learning and spatial memory
in rats by a similar manner. However, it seems that the mechanisms by which this occurs are to some extent divergent.
Specifically, aerobic exercise modulates neuroplasticity in the hippocampus selectively via the BDNF/TrkB pathway by
activating CaMKII and stimulating the synthesis of synapsin 1. In contrast, resistance training appears to increase synapsin 1
expression via the IGF-1/IGF-1R pathway, activating the AKT pathway.
Keywords: memory, hippocampus, BDNF, IGF-1, neuroplasticity
Financial Support: FAPESP (grant number 2008/03083-6), AFIP, CEPE, CEMSA and CNPq
Resumo:03-028
EFFECTS OF INTENSE AND EXHAUSTIVE EXERCISE ON THE GASTRIC CONTRACTILITY AND OXIDATION
PRODUCTION.
Pepineli, R. 1; Ribeiro, R. F. 2; Nouailhetas, V. L. A. 2; Rosa, E. F. 1,2
1
Centro Universitário São Camilo, CUSC
2
Objectives:
The effects of strenuous and intense exercise on skeletal muscle have been extensively studied, while much less is known about
the effects of exercise on the gastrointestinal smooth muscle. The observation that intense exercise increases intestinal oxidative
stress and impairs intestinal contractility raised the possibility that it could also cause important alterations of functionality in
other parts of the gastrointestinal tract. The aim of this study was to assess the effect of strenuous and intense exercise on both the
gastric contractility and oxidative stress level.
Male Balb/c mice, aged 3 mo, with 30 ± 2 g, were randomly ascribed into four groups: control (CT, n = 5), exercised for 4 (E4, n
= 5), 10 (E10, n = 5) or 15 (E15, n = 5) days of treadmill running. All animals were previously submitted to an adaptation period,
consisting of 5 days a 15-min daily treadmill running session at 10 m/min, for 5 successive days. To evaluate individual maximal
running capacity, animal performed a maximal incremental test (MIT), which consisted of 3 min warming-up running at 5 m/min,
followed by setting treadmill speed at 10 m/min followed by 1 m/min increments every minute until animal exhaustion. MIT was
repeated after 4, 10 and 15 days of exercise. Gastric contractility was assessed by the construction of concentration-isometric
contractile response curves of isolated segments of the fundus, in response to either carbachol or KCl for the evaluation of EC50
and Emax. Gastric oxidative stress was assessed by the level of protein oxidation, through the measurement of tissue carbonyl
radical level by Reznick technique. A significant impairment of animal physical performance in E10 group was observed in
comparison with the control group (CT: 45 ± 1 m/min; E4: 39 ± 1 m/min; E10: 33 ± 3 m/min; E15: 36 ± 2 m/min). Both E4 and
E15 animal groups displayed a lower Emax in response to KCl than the control group (CT: 4 ± 1 g; E4: 2.8 ± 0.3 g; E15: 2.7 ±
0.2 g), but a higher Emax in response to carbachol (CT: 1.8 ± 0.2 g; E4: 2.9 ± 0.3 g; E10: 0.80 ± 0.02 g; E15: 3.8 ± 0.9 g). It was
observed a significant increase in logEC50 stimulated by KCl in E4 group in comparison with CT group (CT: -0.5 ± 0.3 x E4: 1.3 ± 0.2); but not with E15 group (-0.9 ± 0.2). Differences were detected between CT and E15 groups, E4 and E15 groups and
between E10 and E15 groups in the logEC50 stimulated by CCh (CT: -6.2 ± 0.1; E4: -6.6 ± 0.2; E10: -6.45 ± 0.08; E15: -5.2 ±
0.4).The intense exercise significantly increased oxidative stress on the gastric fundus (CT: 3.6 ± 0.1 nmol/ml; E4: 8.13 ± 0.09
nmol/ml; E10: 12.6 ± 0.8 nmol/ml; E15: 11.9 ± 0.9 nmol/ml).
Conclusions:
It can be concluded that both reactivity (pharmacomechanical and eletromechanical coupling) and tissue redox status are sensitive
to intense and exhaustive exercise in murine gastric fundus.
Keywords: Intense exercise, oxidative stress, isometric contraction , stomach
Financial Support: FAPESP, Centro Universitário São Camilo
Resumo:03-029
CREATINE SUPPLEMENTATION DOES NOT DECREASE OXIDATIVE STRESS IN SKELETAL MUSCLE AFTER
ECCENTRIC EXERCISE
Silva, L. A. D. ; Tromm, C. ; Moreira, D. ; Bom, K. ; Tuon, T. ; Souza, C. T. ; Pinho, R. A.
Laboratorio de Fisiologia e Bioquimica do Exercicio , UNESC -
Objectives:
Eccentric exercise-induced damage leads to increased oxidative stress in skeletal muscle. Creatine monohydrate's (Cr) ergogenic
potential is well established; however few studies have directly examined the effects of Cr supplementation on recovery after
damage. We examined the effects of Cr supplementation on biomarkers of oxidative stress after eccentric exercise.
Third six male rats were divided into the following groups (n=6): saline; creatine; eccentric exercise plus saline 24 hours (Saline
+ 24h); eccentric exercise plus creatine 24 hours (creatine + 24h); eccentric exercise plus saline 48 hours ( Saline + 48h); exercise
eccentric plus creatine 48 hours. Creatine supplementation was administered with solution of 1mL water 300mg/kg/ body weight
(BW)/day by gavage, for two weeks before and 2, 12 and 24 hours after the exercise session. The animals were submitted to one
downhill run session with duration of 90min and velocity constant of 1.0km.h-1. Forty-eight hours after the exercise session, the
animals were killed and the quadriceps muscles were surgically removed. Creatine kinase levels, lipoperoxidation, carbonylation,
total thiol content, superoxide dismutase enzyme and glutathione peroxidase were analyze. Data were analyzed using repeated
measures ANOVA with an alpha of 0.05. Results: Creatine supplementation does not decrease lipoperoxidation (0.06±0.004
nmol TBA/mg protein), carbonylation (0.29± 0.03nmol/mg protein), total thiol (19.8±2.1nmol TNB/mg protein) or alter enzyme
activity superoxide dismutase (0.90±0.1 U SOD/mg protein) and glutathione peroxides (0.02±0.003 U GPX/mg protein-1) in
relation saline group ( 3311±22 U/L; 0.08±0.001 nmol TBA/mg protein; 0.21±0.02 nmol/mg protein; 15.6±2.3 nmil TNB/mg
protein; 0.83±0.09 U SOD/mg protein; 0.018±0.003 U GPX/mg protein-1)respectively, in skeletal muscle after eccentric exercise
(p<0.05)
Conclusions:
The present study suggests that creatine supplementation does not decrease oxidative stress after eccentric contraction.
Keywords: creatine, exercise eccentric, stress oxidative
Financial Support: UNESC, CAPES, FAPESC and CNPq
Resumo:03-030
EFFECTS OF DIFFERENT PROTOCOLS OF PHYSICAL TRAINING ON MITOCHONDRIAL MODULATION IN
RATS INDUCED IN RATS INDUCED BY MYOCARDIAL VENTRICLE
Pozzi, B. G. 3; Pinho, C. A. 4; Tuon, T. 3; Rosa, G. L. D. 3; Tromm, C. B. 3; Bom, K. 3; Silva, L. A. D. 3;
Benetti, M. 3; Souza, C. T. D. 3; Pinho, R. A. 3
3
4
Universidade do estado de santa catarina, UDESC
Objectives:
Cardiovascular diseases stand out as the leading cause of death in Brazil and the exercise has been considered as an important
agent in the prevention and treatment of these diseases. However, studies are still controversial, the type and intensity of effort
required can cause significant biochemical protective. Besides being able to check out the effects of two exercise protocols on
parameters of ventricular oxidative metabolism in rats after myocardial infarction.
We used 36 male Wistar rats, two months old, weighing 200-250g and were divided randomly into two groups (n = 18): Sham
and Acute Myocardial Infarction (AMI). The animals were anesthetized and the IM induced by coronary occlusion second
protocol Pfeffer (1979). Thirty days after induction of MI animals were divided into six subgroups (n = 6): Sham, Sham +
ongoing training (60 minutes), Sham + Training fractionated (10 x 5 minutes with 1 minute interval), myocardial infarction, AMI
+ MI + ongoing training and training fractionated. The exercise sessions were conducted in water (30-32 º C), 5 times a week for
six weeks. Forty-eight hours after the last exercise session, the animals were killed by decapitation, the left ventricle was
surgically removed and stored at -80 ° C freezer for later análises.Os data were expressed as mean and average standard deviation
and statistically analyzed by analysis of variance (ANOVA) one way, followed by post hoc Tukey test. The level of significance
for the statistical test was p
Conclusions:
These results suggest a significant improvement in the redox state of the myocardium of rats induced by MI and exposed to
different training models, but the ongoing training seems to be more efficient on the parameters.
Keywords: Exercise, Free Radicals, Heart, Infarction, Oxidative metabolism
Financial Support: UNESC, FAPESC, CAPES e CNPq
Resumo:03-031
THE ANTIOXIDANT EFFECTS OF EXERCISE IN THE HIPPOCAMPUS OF MICE ARE DOSE-DEPENDENT
Mariano, I. F. 1; Junior, A. A. 2; Tuon, T. 1; Silva, L. A. D. 1; Rosa, G. L. D. 1; Tromm, C. B. 1; Bom, K. 1;
Souza, C. T. D. 1; Pinho, R. A. 1
1
2
Universidade Federal de Santa Catarina, UFSC
Objectives:
The antioxidant benefits of exercise on the central nervous system are well known. In general, moderate amounts of exercise
improves the brain redox status and cause beneficial neuroplasticity changes, as excessive amounts of exercise can oxidize the
brain and bring functional impairment. Here, we evaluated whether these effects behave in a dose-dependent manner.
Were Used mice Swiss male the 8-18-week-old, weighing 35-40 g. Were randomly assigned four groups: untrained controls and
animals to exercise: two, three or four times a week. All mice were habituated to the exercise room for 1 h before each exercise
session. After the last day of physical training was mensured lactate concentration. The hippocampus was removed. The lipid
peroxidation was evaluated, the protein carbonyl content, Sulfhydryl content and Protein content. The results shown are Simple
linear correlation of Pearson and one-way analysis of variance (ANOVA). Following significant ANOVA, multiple post-hoc
comparisons were performed using the Newman–Keuls test. The accepted level of significance for the tests was P
Conclusions:
According to literatures, while low doses of moderate exercise induce benefits to brain redox state of animals, high doses cause
damage. Our current evidence strongly suggests that the antioxidant effects of exercise have properties of dose-dependency in the
hippocampus of rodents.
Keywords: exercise, hippocampus, oxidative stress
Financial Support: UNESC, FAPESC, CAPES e CNPq.
Resumo:03-032
EFFECT OF DIFFERENT EXERCISE PROTOCOLS ON HISTONE ACETYLTRANSFERASE AND HISTONE
DEACETYLASE ACTIVITIES IN RAT HIPPOCAMPUS
Elsner, V. R. 1; Lovatel, G. 2; Bertoldi, K. 1; Vanzella, C. 4; Moysés, F. 1; Spindler, C. 1; de Almeida, E. F. 3;
Nardin, P. 4; Siqueira, I. R. 3,2,1
1
PPG Fisiologia, Universidade Federal do Rio Grande do Sul, Fisiolgia-UFRGS
2
PPG Neurociências, Universidade Federal do Rio Grande do Sul, Neurociências-UFRGS
3
Dep. Farmacologia, Universidade Federal do Rio Grande do Sul, Farmacologia-UFRGS
4
PPG Bioquímica, Universidade Federal do Rio Grande do Sul, Bioquímica-UFRGS
Objectives:
Regular and moderate physical exercise has been considered an attractive neuroprotective strategy, whereas the mechanisms by
which exercise alters brain function are not completely clear. Our work hypothesis was that neuroprotective properties can be
related to epigenetic modifications, specifically altering the histone acetylation levels through modulation of Histone
Acetyltransferase (HAT) and Histone Desacetylase (HDAC) enzyme activities. The aim of the present study was to investigate
the effect of exercise on global HDAC activity, HAT activity on H3 and H4 histones and the balance of HAT/HDAC activities in
rat hippocampus at different times after treadmill.
Adult male Wistar rats were assigned to a non-exercised group (sedentary, SED, n=30) and an exercised group (EXE, n=30) on
different exercise neuroprotective protocols: a single session of treadmill (running for 20 min) and a chronic treadmill (running
once daily for 20 min, for 2 weeks).The effects of exercise on HDAC and HAT activities were measured immediately, 1 h and 18
h after the single session or the last training session of chronic treadmill exercise using specific assay kits (Upstate
Biotecnology®). Data were analyzed by two-way analysis of variance (ANOVA) followed by Duncan‟s multiple range test and a
value of p
Conclusions:
Conclusion: The exercise neuroprotective effects may be related, at least in part, to epigenetic mechanisms, specifically by HAT
and HDAC activities modulation in rat hippocampus. Our data also indicated that circadian rhythm can modify epigenetic
parameters. Sources of research support: Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq; Graduate
Research Group (GPPG/FIPE) at Hospital de Clínicas de Porto Alegre (Grant # 09-123).
Keywords: exercise, neuroprotection, HAT, HDAC, rat hippocampus
Resumo:04-001
AMINOPEPTIDASE ACTIVITY IN THE SMALL INTESTINE OF GOAT KIDS FED LYOPHILIZED BOVINE
COLOSTRUM
Moretti, D. B. ; Nordi, W. M. ; Lima, A. L. ; Pauletti, P. ; Machado-neto, R.
Dep. de Zootecnia/ESALQ/Universidade de São Paulo, ESALQ/USP
Objectives:
In the first hours of life, the intestinal tract especially those of ruminants undergoes major changes that will have important
implications for the animals development (Liv. Prod. Sci. NL 66; 161, 2000). Besides the exchange of fetal-type enterocytes,
which have the capacity to absorb macromolecules, the activities of intracellular and extracellular enzymes are changed (Liv.
Prod. Sci. NL 66; 161, 2000; R. Bras. Zootec. BR 31; 2314, 2002). The aim of this investigation was to evaluate the capability of
protein degradation in the small intestine of goat kids fed a heterologous colostrum, bovine, prepared from powder form, as an
alternative source of passive protection.
At 0, 7 and 14 hr of life 15 newborns male goat kids received 5% of body weight of lyophilized bovine colostrum (LBC) and 14
goat colostrum (GC), both with 55 mg/mL of IgG. Samples of duodenum, jejunum and ileum were collected at 18, 36 and 96 hr
of life. Three animals were sampled at birth, without colostrum intake (0 hr). The homogenized samples were incubated with the
substrates L-leucyl-p-nitroanilide, glycyl-L-prolyl-p-nitroanilide hydrochloride and L-glutamic acid 1-(4-nitroanilide) for
enzymatic analyses of aminopeptidase N (APN), dipeptidyl peptidase IV (DPPIV) and aminopeptidase A (APA), respectively
(Biochim. Biophys. Acta NL 321; 282, 1973; Anal. Biochem. USA 74; 466, 1976). Enzyme activity was calculated considering
one international unit (one mol of substrate hydrolyzed per minute at 37°C) per gram of tissue. A completely randomized design
was used and data were arranged in a 2X3+1 scheme, treatment and sampling time as main effects, plus the 0 hr group. Values
are presented as least-square means and standard errors. In the duodenum, activity of APA was not observed, and to APN and
DPPIV activity was present and related to sampling time. The highest activity was observed for both enzymes at 36 hr, 1.89±0.20
and 0.71±0.07, respectively. In the jejunum, effect of sampling time was also observed to APN and DPPIV, the lowest activity
was observed at 96 hr, 2.60±0.24 and 1.40±0.24, respectively. In the ileum, activity of DPPIV showed effect of sampling time, at
36 hr (3.08±0.20) enzyme activity was higher than 96 hr (1.60±0.38). In this segment activity of APA was affected by treatment
and sampling time, LBC showed higher enzyme activity than GC, 0.58±0.04 and 0.25±0.04, respectively, and the highest activity
was observed at 36 hr (0.53±0.05), following by 18 hr (0.31±0.04) and 96 hr (0.10±0.06). The 0 hr group, 2.83±0.44, differed
only to DPPIV in the jejunum from LBC 96 hr, 1.12±0.26.
Conclusions:
The ingestion of colostrum, with high concentrations of proteins stimulated the activity of aminopeptidases in the first 36 hr of
life. The change of cells in the intestinal epithelium and ingestion of milk, following the colostrum intake, with less total solids,
determined decrease of aminopeptidase activity in the gut at 96 hr. Thus, we observed that enzyme activity in the first four days
of goat kids‟ life is still not fully stimulated and that the lyophilized bovine colostrum showed a viable source of passive
protection for these small ruminants.
Keywords: enzyme, colostrum, intestinal epithelium, caprine, newborn
Financial Support: São Paulo Research Foundation/FAPESP
Resumo:04-002
EARLY WEANING REGULATES PLASMA CORTICOSTERONE LEVELS AND GLICOCORTICOID RECEPTOR
IN THE GASTRIC MUCOSA OF RATS UNDER POSTNATAL DEVELOPMENT
Ghizoni, H. ; Figueiredo, P. M. ; Gama, P.
Depto. Biologia Celular e do Desenvolvimento, USP
Objectives:
Corticosterone is part of the complex mechanism that controls gastric development, and because its functions are dependent on
dietary pattern, nutritional status and glicocorticoid receptor (GR) availability, we aimed to study corticosterone levels and GR
concentration in the gastric mucosa during early weaning in rats.
On the 15th postnatal day, Wistar rats were separated into 2 groups, according to the diet: suckling (S) and early weaning (EW)
(at least 4 pups were used for each group/age; protocol approved by CEEA – ICB nº 86/08). In the EW group, the pups were
separated from their dams and suckling was substituted by semi-solid and solid diet. In order to evaluate daily variations of
plasma corticosterone, pups were euthanized on postnatal day 15, 16, 17 and 18 after anesthesia with isoflurane. Blood samples
were collected from abdominal aorta into heparinized tubes. This procedure was conducted within 3 minutes to avoid the increase
of corticosterone levels due to handling. To prevent circadian variations, blood collection was conducted at 17h00. Corticosterone
levels were assessed through radioimmunoassay using a commercial kit (intra-assay coefficient variation was 6.2% and interassay coefficient variation was 11.6%). To determine GR concentration in the corpus region of stomach throughout EW, the
gastric mucosa was scrapped and maintained in TBS-PMSF for western blot analyses. Protein was extracted using RIPA lysis
buffer containing protease and phosphatase inhibitors and concentration was determined by Bradford method. Thirty micrograms
of total protein were separated by SDS-PAGE, transferred and immunoblotted for GR detection. All data were expressed as
means+-SD and were compared by ANOVA followed by Tukey‟s test (significance set as P <0,05).
Conclusions:
We demonstrated that the stress induced by early weaning elevates circulating corticosterone levels, concomitant with lower GR
concentration in the gastric mucosa. Because corticosterone function depends on its action through GR, we can suggest that
during early weaning, the hormone effects could be reduced, which might attenuate glucocorticoid inhibitory actions over
growth.
Keywords: Corticosterone, Corticosterone Receptor, Early weaning, Stomach, Rat
Financial Support: FAPESP and CNPq
Resumo:04-003
FOOD INTAKE AND BODY COMPOSITION IN DYSTROPHIC (MDX) MICE
Alves, G. A. 1; Silva, L. R. 1; Silva, S. M. A. 2; Ribeiro, R. F. 1; Aboulafia, J. 1; Souccar, C. 3; Luz, J. 2;
Nouailhetas, V. L. A. 1
1
Departamento de Biofísica / Escola Paulista de Medicina, UNIFESP
2
Departamento de Fisiologia / Escola Paulista de Medicina, UNIFESP
3
Departamento de Farmacologia/ Escola Paulista de Medicina, UNIFESP
Objectives:
Mdx mice is the animal model for Duchenne muscular dystrophy, the most prevalent of dystrophies, which is an X-linked
hereditary disease chacaterized by the absence of the protein dystrophin whose lack leads recurrent muscular injury. We have
previously observed (unpublished results) that dystrophic mice are heavier (15.4%) than control (non-dystrophic C57BL/10)
mice. We thus investigated the origin of such increase in body weight by evaluating food ingestion and body composition
(protein, fat and water content) in dystrophic and control mice.
Male mdx and control mice (3 month old N=10 for each group) were individually housed to allow food ingestion evaluation by
weighing ration consumption and the leftovers. Body weight control was done twice a day for 7 successive days. Food ingestion
in mdx was reduced of 16% in comparison to that from control mice. Animals were killed by cervical dislocation; the gut was
removed, emptied from its content and homogenized together with the carcasses with equal volume of water. Fat content and
protein contents were measured in fresh 4 g-samples of the homogenized carcasses by the chloroform-methanol method and
Lowry method, respectively. The remaining homogenized material was dried to constant weight at 60°C. Water content was
calculated by the difference between the wet and dry carcasses weights. Total body protein was 10% higher (162.3 ± 4.7 mg/g
and 147.9 ± 4.5 mg/g per body weight, respectively) and total fat content was 28% lower (10.4 ± 0.3 and 14.5 ± 0.7 mg/g per
body weight, respectively) in mdx mice than in control. Water content in mdx mice was 72.7 ± 0.4% and 69.8 ± 0.5% in control
mice, which means a remarkable 4% water content enhancement in the mdx mice. Student‟s t test (P
Conclusions:
Increased body weight in mdx mice might be related to enhanced water and protein contents, probably due to accelerated
degeneration/regeneration cycles, involving several cycles of muscle inflammation/regeneration (protein synthesis). The lower fat
content is intriguing as it is known that mdx mice moves physically less than control, which theoretically would lead to increased
fat content. The lower food ingestion in mdx mice is in contrast to the energy cost of its higher protein synthesis, thus suggesting
that mdx has greater capacity of nutrient digestion and absorption, associated with a pronounced anabolism. There is yet no
consensus concerning mdx body composition, which could be ascribed to differences in age and gender, and/or experimental
conditions so that investigations are still required for the full understanding of mdx metabolism.
Keywords: dystrophy, dmd, metabolism, food intake, body composition
Financial Support: Fapesp
Resumo:04-004
ROLE OF ALPHA-2 ADRENORECEPTORS AND SULFHYDRYL GROUPMENTS ON GASTROPROTECTIVE AND
ANTIULCEROGENIC ACTION OF EPICATECHIN
Tanimoto, A. 1; Rozza, A. L. 1; Gregório, B. B. 1; Moraes, T. M. 2; Kushima, H. 2; Hirmua-lima, C. A. 2;
Pellizzon, C. H. 1
1
Morfologia/IBB UNESP - BOTUCATU, M-UNESP
2
Fisiologia/IBB UNESP BOTUCATU, F-UNESP
Objectives:
Gastric ulcers etiology is still not completely known and the majority of available therapies provides significant adverse effects.
Epicatechin (EC) is an antioxidant substance present in many medicinal plant species used in the treatment of gastrointestinal
disorders. Therefore, our aim was to investigate the role of EC in the cytoprotection of gastric mucosa and its main
gastroproctetive mechanisms.
The involvement of alpha-2 adrenoreceptors, nitric oxide (NO) and non-proteic sulfhydryl compounds (SH) were determined by
the administration (i.p.) in male Wistar rats (200 – 250 g) of yohimbine (YO, an alpha-2 adrenoreceptor antagonist), N-Nitro-LArginine-Methyl Ester (L-NAME, a NO-sintase inhibitor) and N-Methyl-Maleinide (NEM, a SH depleting agent) 30 min before
the treatment‟s administration (vehicle – 10 mL/kg (V), carbenoxolone – 100 mg/kg (C) or EC – 50 mg/kg (EC50)), the lesions
area were expressed in mm². Gastric juice parameters and mucus secretion were quantified 4h after pylorus ligature by titration
and ELISA, respectively, and expressed in mEqH+/mL/4h and in μg/g of tissue. Statistical values attributed for significance were
P
Conclusions:
Through the results, we can conclude that the gastroprotective and antiulcerogenic actions promoted by EC are directly related to
an increase in the gastric production of mucus and in the maintenance of mucus structure, both promoted by SH and also by acid
secretion inhibition rooted in the modulating action that EC exert on alpha-2 adrenoreceptors.
Keywords: Gastroprotection, Epicatechin, Alpha-2 adrenoreceptors, Sulfhydryl groupements
Financial Support: FAPESP (2008/53798-1 and 2006/55542-9)
Resumo:04-005
SYMPATHETIC OUTFLOW AND PROTEIN EXPRESSION IN THE MOUSE SUBMANDIBULAR GLAND
Heluany, C. S. ; Luna, M. S. ; Yamanouye, N.
Laboratório de Farmacologia - Instituto Butantan, IBu
Objectives:
Venom gland of Bothrops jararaca snake is an oral exocrine gland related to salivary glands. We have shown that stimulation of
noradrenergic innervation by venom extraction is the key activator of venom gland. However, data in the literature show that
sympathetic outflow has only a role in stimulating the synthesis and secretion of the saliva proteins in mammals. The new
function of the sympathetic nervous system was discovery just because the venom gland can assume distinct quiescent and
activated stages, in contrast to the salivary gland that is constantly in the activated stage. The aim of this study is to verify
whether the sympathetic innervation is also important to keep the submandibular gland in activated stage.
Adult Swiss male mice (25-30g) were divided into 3 experimental groups: 1) control – treated with vehicle (n=3); 2) treated with
reserpine (n=3) for 6 days (0,5mg/kg – i.p.); 3) treated with reserpine for 6 days (0,5mg/kg – i.p.) and phenylephrine (PHEN) plus
isoprenaline (ISO) (20mg/kg – i.p.) in the last day of treatment (n=3). Extracts of submandibular glands were prepared and the
proteins were analyzed by bidimensional gel electrophoresis (2-DE). Gels were run in triplicate, stained with Coomassie Blue G
and and the quantification of the density of the spots and comparison among groups were made using ImageMaster 2D Platinum
7. The difference of density of the spots was analyzed by one-way analysis of variance (ANOVA) and the probability of less than
0.05 was considered statistically significant (p
Conclusions:
Our results obtained from 2-DE analysis showed that the impairment of sympathetic innervations caused by reserpine promotes
drastic changes in protein profile of submandibular gland and the stimulation of both α- and β-adrenoceptors restores partially the
reserpine effect in accordance with our previous results using unidimensional electrophoresis. These results are very similar to
obtained with Viperidae snake venom gland, suggesting that sympathetic innervation could be important to keep the mouse
submandibular gland in activated stage.
Keywords: protein expression, submandibular gland, mouse
Resumo:04-006
GASTROPROTECTIVE EFFECTS OF RIPARIN III ON ETHANOL-INDUCED GASTRIC LESIONS IN MICE:
ROLE OF PROSTAGLANDINS, NITRIC OXIDE AND KATP+ CHANNELS
Vasconcelos, L. F. 1; Carvalho, A. M. R. 1; Rocha, N. F. M. 1; Rios, E. R. V. 1; Moura, B. A. 1; Fernandes,
M. L. 1; Silva, M. I. G. 1; Filho, J. M. B. 2; Gutierrez, S. J. C. 3; Sousa, F. C. F. 1
1
Dpto. Fisiologia e Farmacologia / Faculdade de Medicina, UFC
2
Laboratório de Tecnologia Farmacêutica , UFPB
3
Departamento de Bioquimica e Farmacologia, UFPI
Objectives:
The ethanol-induced gastric lesions model is considered to be a reliable tool to study the pathogenesis of acute gastric mucosal
ulceration. Riparin III (ripIII) is an alkamide compound isolated from Aniba riparia, collected from the state of Amazonas, Brazil.
In previous studies, including those conducted by our group, this substance has presented a broad spectrum antimicrobial activity,
antianxiety and antidepressant-like effects, as well as potent smooth muscle relaxant activity. In this study, we decide to evaluate
the gastroprotective effect of riparin III against ethanol-induced lesions and verify the role of nitric oxide, ATP-dependent
K+channel and prostaglandins in this action.
METHODS: Male Swiss mice (20-30g) were divided in four groups: vehicle (3% Tween 80 in distilled water; p.o.), ripIII-25
(riparin III 25 mg/kg; p.o.), ripIII-50 (riparin III 50 mg/kg; p.o) and NAC (N-acetylcysteine 300 mg/kg; p.o.), used as a
gastroprotective reference drug. One hour later, absolute ethanol (0.2 mL/animal) was administrated orally to all groups. Thirty
minutes after the administration of ethanol, the mice were killed and their stomachs were removed and opened along the greater
curvature for examination. The total and injured stomach areas were measured by computer program and expressed in terms of
percent (%) of ulcerated gastric area. To study the possible mechanism of action, separate experiments were conducted using the
following drugs: L-NAME, an inhibitor of the NO synthase activity (10 mg/kg; i.p.), glibenclamide, an antagonist of KATP+
channels (10 mg/kg; i.p.) and indomethacin, a nonselective cyclooxygenase inhibitor (10 mg/kg; p.o.). L-NAME and
glibenclamide were administered 30 min before animals receiving riparin III (50 mg/kg; p.o.), while indomethacin (10 mg/kg;
p.o.) was administrated 2 h before the drug test. One hour later, absolute ethanol 0.2 mL was applied in each animal. Thirty
minutes after the administration of ethanol, the mice were killed and their stomachs were removed for examination as previously
described. Data are presented by mean ± S.E.M and analyzed by ANOVA followed by Student Newman Keuls as the post hoc
test. RESULTS: The administration of absolute ethanol produced lesions in the gastric mucosa (16.94±1.580), which were
reduced in the animals pretreated with ripIII-25 mg/kg (4.018±0.8936; p
Conclusions:
The results provide evidence that Riparin III reduces the gastric damage induced by ethanol and this gastroprotective effect
appear to be mediated, at least in part, by endogenous NO and KATP+ channels opening
Keywords: riparin III, gastric lesions, nitric oxide, prostaglandins
Financial Support: CNPq/ CAPES
Resumo:04-007
ANTI-INFLAMMATORY AND ANTIOXIDANT EFFECT OF BOSWELLIA SERRATA IN EXPERIMENTAL
MODEL OF COLITIS
Hartmann, R. M. 1; Licks, F. 2; Schemitt, E. G. 3; Martins, M. I. M. 3; Willand, E. 3; Fillmann, H. 4; Marroni,
N. P. 1,2,3
4
Pontífica Universidade Católica do Rio Grande do Sul, PUCRS
3
Universidade Luterana do Brasil, ULBRA
2
Pós-graduação em Ciências Biológicas: Fisiologia, UFRGS
1
Pós-graduação em medicina: Ciências Médicas, FAMED/UFRGS
Objectives:
Ulcerative colitis (UC) is an inflammatory disease involving the colon and rectum, characterized by leukocyte infiltration and
mucosal ulcers. The oxidative metabolism as the defective increase of reactive oxygen species (ROS may have great importance
in the activity of UC. Therefore, this study purpose to evaluate the anti-inflammatory and antioxidant effects of Boswellia serrata
(B. serrata) in animals with UC induced by acetic acid.
22 male Wistar rats ±350g were divided into groups: 1: Control (CO), 2: Control+B. serrata (CO+B) 3: Colitis (CL), 4:
Colitis+B. serrata (CL+B). The B. serrata extract (34.2 mg/kg) was administered by oral route after induction of colitis for 48
hours. The anal sphincter pressure was measured, the lipoperoxidation(LPO) evaluated by TBARS, the activity of antioxidant
enzymes: superoxide dismutase (SOD), glutathione peroxidase (GPx), total glutathione (GSH) and histological analysis of the
intestine by hematoxylin and eosin. The statistical analysis used was ANOVA followed by Student-Newman-Keuls(mean±SEM)
with significance at pCO: 59.8±0.4, CO+ B: 61.0±1.1, CL: 29.5±0.6, CL+B: 47.7±1.0). In assessing the LPO by TBARS
(nmol/mgprot) there was a significant decrease in CL+B than in group CL (CO: 2,7±0,7; CO+B: 2,8±0,8; CL: 9,0±1,1; CL+B:
3,6± 0,6). In antioxidant enzymes, SOD (U SOD/mgprot) showed a significant decrease in group CL+B than in group CL, where
the average was very close to the CO group (CO: 2.8±0.8; CO+B: 2.8±0.4; CL: 11.8±0.6; CL+B: 3.2±1.0). The GPx activity
(nmol/mgprot) showed a significant increase in group CL + B than in group CL (CO: 1.2±0.1; CO+B: 1.1±0.1; CL: 0.5±0.2;
CL+B: 1.2±0.1) in the same way that the enzyme GSH (µmol/mgprot) (CO: 0.013±0.003; CO+B: 0.013±0.003; CL: 0.005 ±
0.001; CL+B: 0.013±0.002). The averages of the enzymes in group CL+B equals the average of the CO group. Histological
evaluation in the groups CO and CO+B showed no changes in the normal architecture. In CL we observed destruction of the
crypts, submucosal edema (E) and inflammatory infiltrate (IF). However, in group CL+B we observed a preservation of crypts
and a minor edema.
Conclusions:
This study suggests that the extract of B. serrata has anti-inflammatory action, judging by the increase in anal sphincter pressure
and the results shown in histology. Also suggests the antioxidant activity of the extract, because LPO decreased in treated animals
and antioxidant enzymes when given the plant extract maintained their average close to the CO group.
Keywords: oxidative stress , ulcerative colitis , Boswellia serrata, lipoperoxidation , antioxidant enzymes
Financial Support: FIPE/ULBRA
Resumo:04-008
GASTROINTESTINAL PARASITES AND ANTIPARASITIC TREATMENT IN CAPTIVE WILD ANIMALS.
Marinho, A. V. ; Valdes, S. A. C.
Universidade Federal de Uberlândia, UFU
Objectives:
This study intended to identify endoparasites in fecal samples obtained from captive giant ant-eaters (Myrmecophaga tridactyla),
amazon parrots (Amazona aestiva) and red tail boas (Boa constrictor constrictor), and the effectiveness of the treatment with
antiparasitic drugs commonly used in domestic animals. The investigation was accomplished at the Laboratório de Pesquisas em
Animais Silvestres / Universidade Federal de Uberlândia (LAPAS/UFU), where seven giant ant-eaters, eight red tail boas, and
thirty amazon parrots were kept during all the research time.
The fecal samples from the animals in study were collected from the enclosures and kept in plastic recipients properly identified.
The collection was realized monthly. The parasitological exam was done always before 24 hours from the collection.
Parasitological exams of sedimentation (Hoffman) and fluctuation (Willis) were performed in the fecal samples. Among the giant
ant-eaters, three were infected by Strongyloides spp and Entamoeba spp, two presented nothing, one was parasitized by
Ancylostoma spp., Strongyloides spp., Ascarididae eggs, and Giganthorhyncus echinodiscus, and one was parasitized by
Entamoeba spp. The individuals that presented parasitic infection were medicated orally with metronidazole 40 mg•kg-1 during
three days, and 50 mg•kg-1 of praziquantel and 144 mg•kg-1 of pirantel on a single dose. Despite the treatment, at the exams
made one month after medication, all of the giant ant-eaters that were previously infected by Strongyloides and Giganthorhyncus
echinodiscus were still infected, probably due to reinfections caused by the lack of prophylactic procedures. The other parasites
were eliminated with those drugs. At the parrot‟s pool examined, no endoparasites were found. Among the red tail boas, two were
infected by eggs of Strongyloides spp. or Rhabdias spp. (eggs with high similarity), and were treated with febendazole 25 mg•kg1, orally, during three days. One month after treatment, no red tail boas were releasing parasitic eggs on feces.
Conclusions:
In the samples obtained from giant ant eaters the parasitological exam of sedimentation (Hoffman) and the parasitological exam
of fluctuation (Willis) were successful in diagnosing eggs of Ancylostoma spp; Strongyloides spp, eggs of the Ascarididae
family, Acantocephala eggs, probably Giganthorhyncus echinodiscus and Entamoeba spp. The medication with metronidazole
(40 mg•kg-1, 3 days) was successful in treating Entamoeba spp. The medication with praziquantel (50 mg•kg-1) and pirantel (
144 mg•kg-1) was not effective to fully eliminate Strongyloides spp. and Giganthorhyncus echinodiscus, probably because the
contamination of environment with parasitic eggs and larvae. In the red tail boas, the parasitological exams of sedimentation
(Hoffman) and the parasitological exam of fluctuation (Willis) were successful in diagnosing eggs of Strongyloides spp. or
Rhabdias spp. The use of fedendazole (25 mg•kg-1, 3 days) successfully eliminated the parasites, with no eggs in feces thirty
days after medication.
Keywords: Antiparasitic Drugs, Endoparasites, Wild Animals
Financial Support: Instituto de Ciências Biomédicas / UFU and CNPq.
Resumo:04-009
STUDY OF THE P2X7 RECEPTOR IN THE ILEUM ENTERIC NEURONS OF RAT SUBMITTED TO
ISCHEMIA/REPERFUSION
Palombit, K. 1; Mendes, C. E. 1; Tavares de Lima, W. 2; Castelucci, P. 1
1
Anatomia/Instituto de Ciências Biomédicas III/USP, ICB-III
2
Farmacologia/Instituto de Ciências Biomédicas I/USP, ICB-I
Objectives:
Intestinal ischemia-reperfusion (I/R-i) is a frequently occurring phenomenon during abdominal and thoracic vascular surgery,
small bowel transplantation and hemorrhagic shock, carrying high morbidity and mortality. In the present work, we analyzed the
effects of ischemia/reperfusion on neurons immunoreactive for the P2X7 receptor in the myenteric plexus of the rats ileum.
Tissues were obtained from male Wistar (Rattus Novergicus) rats (250-300 g). The ileal artery was occluded for 35 minutes with
an atraumatic vascular clamp and animals were sacrificed 6, 24, 72 h and 1 week (w) later (I/R-i 6h, I/R-i 24h, I/R-i 72h and I/R-i
1w groups). The false operated group (Sham group - SG) was not occluded the ileal artery. Myenteric neurons were evaluated for
immunoreactivity against the P2X7 receptor, nitric oxide synthase (NOS), calretinin (Calr), calbindin (Calb) and choline acetyl
transferase (ChAT). Data were compared by analysis of variance (ANOVA) and Tukey‟s test for multiple comparisons, as
appropriate. P
Conclusions:
These data demonstrate that I/R-i affects the expression of the P2X7 receptor in neurons of the myenteric plexus, as well as
density and size of neurons in this population. Our findings indicate that I/R-i induces changes in P2X7-IR enteric neurons that
could result in alterations in intestinal motility.
Keywords: Ischemia and reperfusion, Myenteric neurons, P2X7 receptor
Financial Support: FAPESP/Capes
Resumo:04-010
MORPHOMETRY OF THE MYENTERIC PLEXUS NEURONS OF THE STOMACH OF RATS SUBMITTED TO
CHRONIC ALCOHOLISM
Quevedo, S. 1; Pereira, V. R. 1; Bianchi, F. J. 2; Oliveira, L. R. 1
1
Instituto de Ciências Biológicas e da Saúde, UNIPAR
2
CCBS/ODONTOLOGIA, UNIOESTE
Objectives:
The stomach has the structure according to the livelihood and nutrition of living beings. To perform functions such as storage,
mixing, mechanical digestion and gastric emptying there are peristalsis movements. These movements are controlled mainly by
the intrinsic innervations‟ neurons in the myenteric plexus. Since, chronic alcoholism can cause changes in the neurons of the
stomach, this work has been the purpose of verifying the effects of alcohol on aspects of the morphology of neurons.
14 rats (Rattus norvegicus) males with 90 days were used in experiment. The control rats (n = 7) water and feed and the
experimental group and the alcoholic group (n = 7) diet and sugar cane liquor diluted with water at increasing concentrations.
After 120 days, the animals, under laparotomy, had the stomach removed. After the separation of regions of the stomach,
dissection of membranes and assembly of blades according to the Giemsa method. Based on the sampling method of counting,
we measured the sum of neurons in 40 microscopic fields (8.82 mm2) of each region of the stomach of all animals in both
groups, with optical microscope with lens 40X. The average sum area of neurons found in each animal as well as standard
deviation were analyzed by Student t test, with significance 5%. In the lower curvature gastric of the glandular stomach found
that the soma area of control animals have an average of 40.6 ± 1.1 ìm2, the experimental ìm2 58.3 ± 2.1, a difference significant,
p = 0.0001. Neurons experimental gastric fold showed an area of 51.3 ± 1.6 µm2 and control 45.33± 1.7 µm2 showed a statiscally
significant (p=0.0017). In the central region of the glandular stomach was also statistical difference (p = 0.0001), where in control
animals was 37.8 ± 0.7 µm2, and in the experimental animals was 45.4 ± 1.3 µm2. In this study there was a statistically
significant difference where the mean area of neurons in the experimental group was higher than the control group independent of
the region studied.
Conclusions:
In animals subjected to chronic alcoholism was an increase in neuronal area possibly because of being related to the energy
supply that neuronal cells need to perform their role. As chronic alcoholism leads to a lower food intake and consequently a
malnutrition, neurons compensated for the lack of nutrients becoming more sparse and with greater neuronal area.
Keywords: alcoholism, myenteric, neurons
Financial Support: UNIPAR
Resumo:04-011
SEX AS A FACTOR IN INFLUENCING THE BACTERIAL TRANSLOCATION IN MICE CHRONICALLY
INFECTED WITH
Galvão, B. H. A. ; Lima, K. D. M. ; Almeida, T. M. ; Aires, A. D. L. ; Sobrinho, C. R. W. ; de Castro, C. M.
M. B.
Medicina Tropical, UFPE
Objectives:
The aim of this work was to relate secondary infection and chronic-phase esquistosomosis by evaluating the intestinal bacterial
translocation
Two groups of Swiss webster mice were set up: schistosomotic (10 male/10 female) and control (10 male/ 10 female). All
individuals were weighted at 35th, 80th, 125th and 132nd day after birth. Following the weighing, mice were anesthetized and
euthanized in order to remove samples of the middle region of the small intestine tissue and feces to perform the histopatological
and morphometric studies and the microbiological analysis, respectively.Major numbers of adult worms recovered from the small
intestine as well as its eggs in the feces were detected in female mice compared to male mice in the schistosomotic group
(p=0.0001). Moreover, it was observed several amounts of eggs (grams of feces) at 45th day after infection (p=0.005) in both
sexes. When schistosomotic group were compared to its respective control, a decrease in weight at 80th, 125th and 132nd day
after birth (p=0.0001) and an increase of splenic weight at 132nd day after birth (p=0.0001) were found in both infected male and
female mice. Schistosomotic group also showed major positive coproculture prevalence related to the bacterial density (both
number of species and colony-forming units). Duodenal morphometry revealed a significant reduction in the area and height of
villi, as well as the mucosa surface perimeter in both infected group (p=0.0001). Major bacterial translocation occurred in all
schistosomotic groups and major incidence was observed in female mice.
Conclusions:
Results suggest that chronic schistosomosis in mice may change the ponderal and splenic weight, as well as the intestinal
morphometry and intestinal microbiota, favouring translocation and sepsis, specially in female rats
Keywords: Schistosoma mansoni, bacterial translocation, sex, intestinal microbiota, mesenteric lympho nodes
Resumo:04-012
EFFECTS OF QUALEA PARVIFLORA METHANOLIC EXTRACT IN THE TNBS-INDUCED COLITIS IN RAT
Mazzolin, L. P. 1,2; Maia, E. O. 2; Fernandes, L. T. L. 2; Hiruma-lima, C. A. 1
1
Departamento de Fisiologia, IB - UNESP
2
Faculdades Integradas Regionais de Avaré, FIRA
Objectives:
Inflammatory Bowel Disease (IBD) include two distinct diseases of great clinical importance: ulcerative colitis and Crohn's
disease. The causal factors these diseases have yet were clarified. However, studies suggest that reactive oxygen and nitrogen
species play important role in initiation and progression of the IBD, and that, antioxidant compounds have been an alternative to
new therapies. Thus, the aim this study was investigate the preventive effect of Qualea parviflora methanolic extract (QP) in the
TNBS-induced colitis in rat.
Male Wistar rats received orally 50, 25 and 12.5 mg/kg of QP, saline (SAL) and dexamethasone (DEXA) (0.5 mg/kg) at 96, 72,
48, 24 and 2 h before colitis induction with 10 mg of TNBS dissolved in 0.25 mL 50% ethanol (v/v) by means of a cannula
inserted 8 cm into the anus. Rats from the non-colitic group (SHAM) received 0.25 mL of saline. All groups (n = 6 - 7) were died
and the colonic segments obtained after laparotomy were cleaned of fat and mesentery, weighed and length measured, and lesions
evaluated with image analyzer AVSoft BioView®. After that, were divided longitudinally into different pieces for the
biochemical determinations of total glutathione (GSH) content and myeloperoxidase (MPO) activity. Results were expressed as
mean ± standard error (ANOVA-Dunnett, * p < 0.05 and ** p < 0.01). After intracolonic administration the majority of animals
showed diarrhea. The inflammation induced by TNBS is associated with decreased food intake and corporal weight. After killing,
colon macroscopic lesions were quantified with the image analyzer reaching a value of SAL = 610.84 ± 47.35 mm2 and
significantly reducing in the groups DEXA (425.47 ± 76.03 mm2**), QP 25 mg/kg (293.81 ± 57.64 mm2**) and QP 50 mg/kg
(337.27 ± 47.39 mm2*). The inflammatory changes the intestinal tract were also associated with a significant increase of
weight/length of the colon, an indicator of inflammation. In this parameter, significant differences were observed between the
groups SAL (164.06 ± 3.69 mg/cm) and SHAM (123.61 ± 4.98 mg/cm**). Morphological damage was accompanied by
alterations in the biochemical parameters as total GSH content and MPO activity in the colon. Treatment with QP (25 and 50
mg/kg) and DEXA (0.5 mg/kg) were able to prevent the colonic GSH depletion that occurre as a result of the colonic oxidative
damage induced by TNBS (SAL = 1085.95 ± 109.64 nmol/g; DEXA = 1416.40 ± 193.84 nmol/g*; QP 25 = 1404.32 ± 170.30
nmol/g* and QP 50 = 1394.91 ± 263.12 nmol/g*). The increase in colonic MPO activity, a marker for neutrophil infiltration,
characterizes the inflammation caused by TNBS. The QP doses (25 and 50 mg/kg) showed significant reduction in this activity,
indicating a decrease in the degree of neutrophil infiltration (SAL = 2740.13 ± 94.42 mU/g; QP 25 = 1478.50 ± 173.58 mU/g**
and QP 50 = 1899.29 ± 215.54 mU/g*).
Conclusions:
The present study shows that QP treatment prevent colonic damage induced by TNBS supposedly for their antioxidant activity,
evidenced by the maintenance of glutathione levels and reducing of activation of neutrophils.
Keywords: Colitis, TNBS, Rat, Qualea parviflora
Financial Support: Biota/Fapesp
Resumo:05-001
CHANGES IN IMMUNOREACTIVITY OF ANGIOTENSIN-(1-7) AND ITS RECEPTOR MAS IN THE FUNCTIONAL
AND REGRESSING CORPORA LUTEA IN THE RAT OVARY THROUGHOUT THE ESTROUS CYCLE
Pereira, V. M. 1; Reis, F. M. 2; Santos, R. A. 2; Reis, A. M. 2
1
Universidade Federal de Sergipe, Departamento de Fisiologia, UFS
2
Universidade Federal de Minas Gerais, Depto de Fisiologia , UFMG
Objectives:
In mammalian ovaries, the life span of corpus luteum (CL) depends on several endocrine and paracrine factors, which may act as
angiogenic/ mitogenic agents in early development and maturation, or mediating apoptosis during luteolysis. Ovarian ReninAngiotensin System (OVRAS) has been described in many species, however its precise role in ovarian physiology is unclear yet.
We have shown the presence of Angiotensin-(1-7) in rat and human ovaries, as well as its participation on steroidogenesis and
ovulation in rats and rabbits (Endocrinology 144:1942, 2003; Reprod. Sci. 16:1165, 2009; Fertil. Steril. 95:176, 2011). The aim
of this study was to investigate the immunolocalization of Angiotensin-(1-7) and receptor Mas in CL from different stages of
development and regression throughout the estrous cycle.
Twenty adult Wistar rats weighing 220-250 grams were used after monitoring of the estrous cycle for at least three consecutive
regular cycles by daily vaginal smears. Five rats of each phase were selected. Animals were submitted to laparotomy and
thoracotomy under anesthesia (sodium pentobarbital, 30 mg/kg). Followed, they were perfused through the left ventricle with
PBS solution containing protease inhibitors. After, they were perfused with 4% paraformaldheyde. Ovaries were removed,
cleaned of fat, and fixed in Bouin´s solution. Immunohistochemistry was carried out in paraffin-embedded slices by the avidinbiotin-peroxidase method with specific primary antibodies: rabbit polyclonal anti-Ang-(1-7)[1:30000]and mouse polyclonal antiMas receptor [1:1000]. The immunostaining was visualized with DAB and counterstained with hematoxylin. The intensity of the
immunoreactivity was analyzed with use of a semiquantitative method according to a gradient range of absence, faint, moderate,
and strong staining. Ang-(1-7) and Mas immunoreactivity was detected in CL in all phases of cycle. However, the intensity of
labeling was different in relation to CL status. Functional CL presented weak to moderate immunolabelling for Ang-(1-7) and
Mas, whereas regressing CL shown moderate to strong staining for both.
Conclusions:
Our results demonstrated the local production of Ang-(1-7)/Mas receptor by CL in all phases of development. The higher
expression in regressing stages could indicate a possible participation in luteolysis process. This finding also suggest that some
discrepant reports about the role of Angiotensin II in the progesterone secretion by luteal cells could be explained for these new
OVRAS active members playing essential roles in the regulation of luteal function.
Keywords: Corpus luteum, Renin-angiotensin system, immunohistochemistry, luteolysis
Financial Support: FAPITEC/SE, CAPES, CNPq
Resumo:05-002
EFFECTS OF SHORT-TERM TREATMENT WITH ROYAL JELLY ON REPRODUCTIVE PARAMETERS IN
OVARIECTOMIZED RATS
Braz, L. B. S. ; Viana, S. M. O. ; Estevam, C. S. ; Pereira, V. M.
Departamento de Fisiologia/Universidade Federal de Sergipe, UFS
Objectives:
Royal Jelly (RJ), produced in the hypopharyngeal and mandibular glands of worker honeybees, is the exclusive food of the queen
honeybees, and contains proteins, free amino acids, lipids, vitamins and sugars. RJ is known to have some diverse nutritional and
pharmacological functions in humans and animals, such as antioxidant, antiinflammatory and antiproliferative activities. In the
reproductive system, the precise effect of RJ is unclear yet. An estrogenic activity was demonstrated in vitro using human breast
cancer cell line MCF-7 (eCAM 5:295, 2008). In vivo studies had shown discrepant results about reproductive performance and it
seems to be related to animal species, and dose and time of treatment. In ewes, RJ treatment increased plasma progesterone and
pregnancy rate (Anim. Reprod. Sci. 92:75, 2006), but in female mice RJ had none effect on embryo quality (Rev. Bras. Saúde
Prod. An. 10:146, 2009). The aim of this study was to investigate a possible estrogenic activity of RJ, by determination of uterine
and pituitary weight of ovariectomized (OVX) rats treated with RJ.
Thirty female Wistar adult rats (200-250 g) were submitted to bilateral ovariectomy surgery (OVX) under anesthesia (xylazine+
ketamine). Six rats were not ovariectomized and used as control. After 21 days, rats were separated in six groups and received
different treatments by daily subcutaneous injections, as followed: 1) Control (n=6); 2) OVX + corn oil (0.1 ml/100g, n=6); 3)
OVX + estradiol cypionate (EC: 5µg/0.1ml/100g, n=6); 4) OVX + RJ 100 mg/kg (n=6); 5) OVX + RJ 250 mg/kg (n=6); 6) OVX
+ RJ 500 mg/kg (n=6). Rats were injected for 4 consecutive days and then were sacrificed. After laparotomy, uterus and pituitary
glands were immediately removed, cleaned and weighed. The weight of the glands was standardized to 100 mg. The efficiency of
the model was confirmed by the reduction of uterine weight after ovariectomy (group 2: 126 mg ±15.5) and by the increase after
EC treatment (group 3: 277mg ±24.2). Statistical analysis was made by ANOVA followed by Student Newman-Keuls. Results
demonstrated that RJ reduced significantly (p
Conclusions:
Our data showed an inhibitory effect of RJ upon the uterus and pituitary weight. These preliminary findings pointed to a possible
antiproliferative or apoptotic effect of RJ on different tissues related to reproductive functions in the female rat. However, further
investigation, including morphological analysis, may be performed to clarify the role of RJ compounds in the reproductive
system.
Keywords: Royal Jelly, Ovariectomy, Uterus, Pituitary
Financial Support: FAPITEC/SE
Resumo:05-003
STRUCTURAL ORGANIZATION OF THE PLACENTA IN PREGNANT RATS FED WITH PROTEIN
RESTRICTION DIET
Rebelato, H. J. ; Catisti, R. ; Esquisatto, M. A. M.
Programa de Pós Graduação em Ciências Biomédicas, UNIARARAS
Objectives:
Placental weight is highly correlated with birth weight and therefore, survival and growth rate of the newborn mammal (Reprod.
Fertil. Dev. 7:333, 1995). Placental growth, in terms of cellular proliferation, is maximal in the first half of gestation suggesting
that placental size and even function later in gestation may be substantially influenced by protein restriction in this time. Thus, the
aim of this study was to evaluate the effect of low protein diet intake, during pregnancy, on the structural organization of placenta
in female Wistar rats.
Twenty pregnant female Wistar rats, with 65 days old, were equally divided in control group, started to be fed with standard diet
(NP, n = 10, 17% protein basal diet), and protein-restricted group (low protein diet, LP, n = 10, 6% protein). The animals were
kept under controlled temperature (22oC), humidity, light (12 h light/dark cycles) and pressure. At day 19 of gestation, the
females were anesthetized, by xylazine hydrochloride (0.2 ml/kg) and ketamine hydrochloride (1 ml/kg), and the placentas were
removed. After removal, the organs were weighed and measured and immediately immersed in fixative solution (10%
formaldehyde in Millonig buffer, pH 7.4, 24 hours at room temperature) and the standard procedures for immersion in Paraplast
® were made. Longitudinal sections, with 6 µm thick, of the samples were treated with Picrossirius-hematoxylin, for the collagen
fibers analysis, under polarized light microscopy; Toluidine blue in McIlvaine buffer pH 4 0, for acid glycosaminoglycans
detection, and Verhoeff method for analysis of elastic fibers. The morphometric analyses were made using three slices from each
middle region of the five placentas from the three control and three protein-restricted animals. All images were captured and
digitized using Leica DM2000 photomicroscope. The measures were implemented in scanned images using Sigma Scan Pro™
6.0, and the data analyzed by ANOVA and Tukey post-test (Excel spreadsheets in Windows XP™). Macroscopical evaluation
showed significant decrease in the LP group in relation to wet weight (NP, 378.5 ± 24.9 mg; LP, 283.7 ± 15.7 mg) and organ
volume (NP, 147.2 ± 17.4 cm3; LP, 44.5±17.7 cm3). Morphometrical analysis of the chorionic thickness did not detect
alterations (NP, 547.2 ± 87.4 µm; LP, 534.5±75.7 µm). Toluidine blue analysis showed no cellular and morphological alteration.
When stained by Verhoeff, showed no difference on the elastic fibers, the LP and NP placentas have the same proportion and
organization of elastic fibers. In placentas stained with picrosirius-hematoxylin is possible to affirm that there is presence of
collagen fibers is more important in decidual plate. In this region, the rats LP showed, apparently, high content of collagen fibers.
Conclusions:
The current data led us to hypothesize that maternal protein restriction altered the size but not placental structural organization.
Keywords: protein restriction, placenta, structural organization, gestational, rat
Financial Support: FAPESP, FHO/UNIARARAS and PROSUP/CAPES
Resumo:05-004
EFFECT OF SOYBEAN OIL ON THE MORPHOMETRY SEMINIFEROUS TUBULES OF WISTAR RATS.
Dias, F. C. R. 1; Campos Jr, O. 1; Cabral, S. P. 2; Harand, W. 2; Neves, E. M . 1
1
departamento anatomia/universidade federal de pernambuco, UFPE
2
centro de tecnologias estrategicas do nordeste, CETENE
Objectives:
To investigate the effect of soybean oil in the testis of adult mice by morphological and morphometric analysis of this body,
considering that this oil is usually used as a vehicle for fat-soluble substances.
12 male Wistar rats with 90 days of age were divided into two groups: control (C), consisting of six rats, and treated group (T),
consisting of 6 rats. Throughout the treatment, the animals were kept in cages with the maintenance diet (Labina ® - Purina) and
water ad libitum and were subjected to a reversed cycle of 12h light/ dark at a temperature of 22  2 ° C. The treated group was
submitted to the ingestion of 5mg of soybean oil by gavage for thirty consecutive days. Control animals received no treatment. 24
hours after the last gavage treated group, animals in both groups were sacrificed by perfusion fixed with paraformaldehyde (4%)
buffered orchiectomized and their testes were weighed. Studies of testicular biometric parameters are frequent in animals, since
they are important in assessing andrological (Brazil. J. of Vet. Research and Ani. Sci. 40; 154-160, 2003). After obtaining the
body and testicular weights on the day of sacrifice, we calculated the gonadosomatic index (GSI), which corresponds to the ratio
between the testicular mass and body weight, morphometric analysis were also made, among them the seminiferous tubule
diameter, height of the seminiferous epithelium, tubular lumen diameter, percentage of testicular compartments and length of the
seminiferous tubule. Statistical analysis used the nonparametric Mann-Whitney. The level of statistical significance was
considered p ≤ 0.05. The use of soybean oil caused a significant reduction of 15% of body weight (C = 414.000 [410.000 468.000] gr and T = 376.000 [336000-402000] gr) treated animals compared to control, without however interfere with testicular
weight (C = 1.672 [1.632 to 1.773] gr and T = 1.679 [1.672 to 1.698] g). The IGS, which determines the percentage of body mass
allocated in the testes had a significant increase (p
Conclusions:
The results of this study suggest that soybean oil is not soluble vehicle more suitable for use in experimental protocols that target
analysis of testis, and consequently the spermatogenesis and sperm production.
Keywords: testis, soybean oil, morphometry, seminiferous tubules
Resumo:05-005
STEREOLOGICAL ANALYSIS OF THE ANTERIOR PROSTATE STROMA IN RATS SUBMITTED TO CHRONIC
PASSIVE SMOKING
Pissolato, M. 1; Alvarez, D. A. P. 1; Rightti, M. M. D. S. 2; Fabrega-carvalho, C. A. 1,2
1
MORFOLOGIA E PATOLOGIA BÁSICA, FMJ
2
ANATOMIA, USP
Objectives:
Chronic nicotine use has been shown to provoke epithelial alterations, stromal alterations such as an increase in collagen and
reticular fibers, and changes in the secretory phenotype of smooth muscle cells in the ventral prostate lobe. However, individuals
passively exposed to cigarette smoke do not only inhale nicotine, but practically all components present in cigarettes. Therefore,
in view of the importance to identify the different factors that can cause alterations in the prostate gland, the objective of this
study was to evaluate collagen fiber density and smooth muscle cell volume in the prostate stroma of rats submitted to chronic
passive smoking.
Male rats (Rattus norvegicus albinus) were divided into a control group (CG, n=5) and a smoking group (SG, n=5). SG animals
were kept in a 180cm³ container (developed by the first author) simulating the environment of smokers. The animals were
exposed daily to the smoke of three cigarettes (high content of tar: 10mg; nicotine: 0,8mg, and carbon monoxide: 10mg) for 30
weeks. Control animals were manipulated in the same manner as SG animals but were not exposed to cigarette smoke. At the end
of the experimental period, the animals of the two groups were sacrificed with an overdose of the anesthetic (Francotar/Virbaxyl;
1:1; 0,25mL/100 g body weight). Samples of the anterior prostate lobe were collected and processed using routine histological
procedures. Semi-serial sections (5 to 10 µm) were stained with Hematoxylin & Eosin and Picrosirius Hematoxylin. The study
was approved by the Ethics Committee of Faculdade de Medicina de Jundiaí (protocol 336/2010). An integration grid of 100
points was superimposed on the scanned images to determine the relative stroma/parenchyma area (Vv/µm²) and the density of
collagen fibers (Vv/µm²) (final magnifications of 40X and 850X, respectively). A 100-point integration grid and a metric ruler
placed on a 10X eyepiece and 100X objective were used for the determination of the volume of smooth muscle cells. No
significant differences (P>0,05) were observed in the relative stroma/parenchyma area between the two groups (CG:
36,88±10,74; SG: 42,19±4,63). With respect to collagen fiber density, significant differences (P0.05) were observed in mean
cytoplasmic volume (CG: 123,47±20,40; SG: 97,64±18,99) or total cell volume (CG: 126,81±20,49; SG: 102,73±19,18). The
results permit to infer that passive smoking results in 1) an inflammatory process associated with glandular fibrosis, demonstrated
by a significant increase in the density of type I collagen fibers, and 2) increased nuclear synthesis demonstrated by a significant
increase of nuclear volume.
Conclusions:
Passive smoking provokes structural morphoquantitative changes that lead to fibrosis of the anterior prostate lobe.
Keywords: COLLAGEN FIBERS, PASSIVE SMOKING, PROSTATE GLAND, STROMA
Financial Support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Resumo:05-006
EFFECTS OF MATERNAL PROTEIN RESTRICTION ON OFFSPRING OVARIAN FUNCTION IN ADULT RAT
Canale, V. 1; Juliani, C. 2; Gontijo, J. A. R. 1; Boer, P. A. 2; Mesquita, F. F. 1
1
Faculdade de Ciências Médicas, UNICAMP
2
Instituto de Biociências de Botucatu, UNESP
Objectives:
Maternal nutrient restriction impairs overall fetal development with a long-term outcomes, including reproductive system
physiology alterations in the adult offspring. The gestational food restriction may acts as stress factor that induces abnormal
development/differentiation of ovarian cells in late life leading to infertility. The activation of several intracellular pathways in
this disorder could affect in adulthood the follicular cells survival and proliferation. The follicular development and atresia seem
to depend upon a sophisticated balance between survival and atretogenic molecules. The heat shock proteins (HSPs), acts as
molecular chaperon, allowing cells to adapt to graded environment changes and to survive in adverse conditions. Leptin is satiety
and reproductive hormone secreted by adipose tissue and could be a regulator of hypothalamic-pituitary-ovarian function. Aim:
To investigate the effects of protein restriction in utero on ovarian expression and localization of key proteins that regulate
follicular development.
Wistar Hannover female rats were fed either normal protein diet (NP) or a low protein diet (LP) during pregnancy (n=6). Pup
weight was recorded at birth. Females at 8th (n NP=2 LP=4) and 16th (n NP=3 LP=4) week old were anesthetized and their
ovaries collected, weighed, fixed and processed in paraffin, cut to 5μm and processed for immunohistochemistry for visualization
of HSP90 and 70 and leptin receptor (ObR), or processed for Western blot to analyze the expression of ObR, HSP90 and 70. At
8th weeks the HSP90 was decreased on LP group. The leptin receptor ObR expression was lower in LP than NP rats. The study
in older rats (16th week)showed a higher expression of HSP90 and also a 78% higher expression of ObR. The HSP70 expression
didn‟t show difference by Western Blot. Immunohistochemistry labeling to HSP90, HSP70 and ObR confirmed the Western blot
results.
Conclusions:
This study corroborates our thesis that the maternal environment could be reflected in other generations, leading the offspring to
show physiological and pathological adaptations. The HSP90 is a proteomic chaperon, however at a stress situation it can act as
an apoptotic factor. In younger rats, this chaperon down regulation reflects the first attempts to keep the cell survive, but when we
studied the older rats (16th week) we observed that this protein is highly expressed in LP animals. These results added to our
previous results lead us to suggest that HSP90 is acting as apoptotic factor at 16th week age. The study of leptin receptor showed
an opposite regulation between younger and older rats. The leptin deficiency is associated with suppression of ovarian
folliculogenesis and increase in ovarian granulose cell apoptosis. We can hypothesize that the down regulation at 8-weeks-age
could have contributed to diminished folliculogenesis, while the followed up regulation is an adaptation in order to keep the
ovarian function.
Keywords: Ovarian development, Maternal protein restriction, Folliculogenesis, Hypothalamic-Pituitary-Ovarian axis
Financial Support: FAPESP and Capes
Resumo:05-007
ESTROGEN RECEPTOR ERALFA AND ERBETA-5 ARE ASSOCIATED WITH PROSTATE DISEASES.
Seibel, F. E. R. 1; Martiny, P. B. 1; Araujo, A. S. R. 1; Branchini, G. 1; Neto, B. S. 2; Berger, M. 2; Brum, I.
S. 1
1
Departamento de Fisiologia, UFRGS - RS
2
Serviço de Urologia, HCPA - RS
Objectives:
Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are major diseases involved with ageing in masculine sex. The
pathogenesis of these diseases is yet unknown. On the other hand, it has been reported the key role of sex steroid hormones in the
development and progression of BPH and PCa. Androgen hormones appear to be important in the pathogenesis of prostate
diseases. Nevertheless, it is paradoxical that manifestation of these diseases occurs when androgen levels, specially the
testosterone, are declining. Serum testosterone levels decrease in ageing men, conversely, estradiol levels can increase or remain
constant with age. However, the role of estrogen and its receptors, in the etiology and progression these diseases, is poorly
understood. This study was designed to test whether isoforms of estrogen receptors (ERα, ERβ2 and ERβ5) could play an
important role in the understanding of health and diseases prostate status.
The study included prostate specimens obtained from patients undergoing transurethral resection for the treatment of BPH or
radical prostatectomy for PCa, during a 12-month period (Urology Service, Hospital de Clínicas de Porto Alegre-HCPA). 25
patients for each group (BPH and PCa), whose ages ranged from 45 to 90 years, were selected for the study. We evaluated men
with untreated BPH and PCa. The diagnosis of BPH and PCa was based on anatomopathologic examination. Samples were
immediately frozen in liquid nitrogen and then transferred to −80ºC freezer for posterior RNA and protein extraction. On the
same day, in which excisional biopsy was performed, venous blood samples were drawn for total estradiol, total testosterone and
total PSA determination by chemiluminescence. Tissues specimens from 25 cases of PCa and 25 cases of BPH were examined
for ERα, ERβ2 and ERβ5 gene expression by reverse transcriptase-PCR. Protein analysis for ERα and ERβ was performed by
Western blot. All analyses were performed using the Statistical Packages for the Social Sciences. Differences were considered
significant statistically to P < 0.05. The majority of patient had mean age of 64 years old nearly in both groups. PCa showed 2
fold PSA levels higher than BPH group. Serum total estradiol and total testosterone levels did not change. ERalpha; mRNA
(P=0.001) and protein (P=0.038) expression were higher in PCa than in BPH group. ERβ5 mRNA expression was increased
(P=0.001) in hyperplastic tissues as compared to tumor group. Expression ERβ2 mRNA and ERβ protein did not change.
Conclusions:
Our study showed that expression of ERα and ERβ5 are differents between the groups. Our data suggested that both receptors are
involved in prostate diseases, mediating cellular growth and differentiation.
Keywords: estrogen receptor, prostate cancer, Benign prostatic hyperplasia
Financial Support: CNPq, CAPES, FIPE and FAPERGS.
Resumo:05-008
SLEEP LOSS DURING PREGNANCY IN RATS: EFFECTS ON FERTILITY
Mazaro-costa, R. 1,2; Alvarenga, T. A. 1; Maia, L. O. 1; Tufik, S. 1; Andersen, M. L. 1
1
Depto. de Psicobiologia / Universidade Federal de São Paulo, UNIFESP
2
Depto. Ciências Fisiológicas / Universidade Federal de Goiás, UFG
Objectives:
This study evaluated the consequences of sleep restriction during pregnancy on fertility parameters in rats.
Thirty three pregnant Hannover rats at 3 months of age were distributed into 2 groups: control (CTRL, n=19) and sleep restriction
(SR, n=14). Animals were submitted to SR (6 hours/day of sleep) through the modified multiple platform method during to the
entire pregnancy (21 days). At the end, the females were weighed and euthanized. Laparotomy was performed to evaluate the
fertility. Number of corpora lutea, implantations sites, embryonic reabsorptions, fetal deaths and live fetuses were recorded. Live
fetuses were weighed, sexed and euthanized by freezing. The parameters evaluated were: mother final weight, number of pups,
number of male and female pups, sex ratio, litter weight/sex, pre- and post-implantation embryonic loss. Blood samples of the
pregnant rats were collected to determine the plasmatic concentration of prolactin (PRL), follicle-stimulating hormone (FSH),
luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) by Multiplex technology. Parametric data was analyzed by
ANOVA and non-parametric data was analyzed by Mann-Whithney test. The level of significance was set to p ≤ 0.05. Data
expressed in mean ± SE. The results showed no significant differences among the groups in the parameters evaluated: mother
final weight (CTRL: 289.5 ± 7.7; SR: 285 ± 8.9), number of offspring (CTRL: 9.3 ± 0.5; SR: 10.3 ± 0.4), number of males
(CTRL: 4.0 ± 0.43; SR: 5.4 ± 0.6), number of females (CTRL: 5.2 ± 0.4; SR: 4.8 ± 0.4), sex ratio (CTRL: 1.0 ± 0.1; SR: 1.4 ±
0.2), weigh of male litter (CTRL: 4.5 ± 0.1; SR: 4.4 ± 0.0), weigh of female litter (CTRL: 4.3 ± 0.1; SR: 4.1 ± 0.7), preimplantation loss (CTRL: 3.1 ± 0.6; SR: 3.0 ± 0.8), post-implantation loss (CTRL: 0.9 ± 0.2; SR: 1.1 ± 0.2). The hormonal
analysis showed that there was a significant increase in the PRL and LH (pg.ml-1) plasmatic concentrations in the SR group
compared to CTRL rats (PRL, CTRL: 524143 ± 205331.34; SR: 5899199.43 ± 1765405.8; p = 0.003; LH, CTRL: 753229.5 ±
136767.2; SR: 1678104.5 ± 303410.6; p = 0.03). TSH and FSH plasmatic concentrations were unchanged.
Conclusions:
Although prolactin and LH were increased after SR in pregnant rats, the SR condition did not affect the fertility parameters. Thus,
sleep loss might not promote short term fertility outcomes in adult rats.
Keywords: fertility, LH, pregnancy, prolactin, sleep deprivation
Financial Support: AFIP, CNPq, FAPESP (09/01030-5 to TAA and CEPID 98/14303-3 to ST).
Resumo:05-009
MORPHOLOGY OF THE OVIDUCT OF COMMERCIAL LAYING HENS SUPPLEMENTED WITH ORGANIC
MINERALS
Medeiros, J. P. D. 1; Maia, L. M. S. D. S. 1; Estevão, L. R. M. 2; Borba, L. B. D. C. 2; Bastos, F. J. F. 2;
Mendonça, F. D. S. 2; Evêncio, L. B. 1; Evêncio-neto, J. 2
1
Departamento de Histologia e Embriologia/UFPE, UFPE
2
Departamento de Morfologia e Fisiologia Animal/UFRPE, UFRPE
Objectives:
Selenium, initially classified as a toxic mineral, plays an important role in the body. It is an essential element in human nutrition
and animal, found in different concentrations in the tissues constituting the body part of at least 25 selenoproteins (Br. Poult. Sci.
47:65, 2006). Studies have shown an impressive improvement in productive output from layers, broilers and broiler breeder
flocks with providing selenomethionine in the diet. The aim of this study was to analyze the morphology of the oviduct of
commercial laying hens supplemented with organic minerals.
2400 Dekalb hens with 42 weeks of age were used. Throughout the period of the experiment (40 weeks, divided into four periods
of 10 weeks each) the supplement was given. The hens received natural light (12 hours) during the experiment. Diets offered to
animals were based on corn and soybean meal were isocaloric and isoproteic, one without the supplement as a differential test
diet and the other containing the product, at a rate of 0.1%. Being administered to group II, 800g of product for every one ton of
feed. The animals Were divided at random into two groups, each consisting of 1200 hens, called Group I - Control, hens without
administration of the supplement; Group II - hens submitted to the administration of the supplement with a combination of
selenium (0.3 ppm ), zinc (200g) and manganese (200g). The morphology of the oviduct was evaluated; all these tests were
performed during 52 to 82 weeks. After orthotanasia birds by cervical dislocation, was made to collect the fragments of the
oviduct with 10 birds per group were then fixed in Bouin liquid for 6 hours, processed for light microscopy and analyzed. In the
morphological analysis of the oviduct of birds, it was found that in group I, the lining mucosa showed longitudinal folds and a
pseudo-ciliated, with a lack of uniformity and lower lashes, supported by a disorganized tissue conjunctive. However in group II
also observed a pseudo-ciliated, with higher and uniform cilia preserved, with well vascularized tissue conjunctive and uniform,
probably due to the supplementation of organic minerals, related to the reduction of oxidation and greater cellular integrity.
Conclusions:
Based on our results we can conclude that the supplementation of organic minerals causes an improvement in morphology of
hens oviduct.
Keywords: poultry, reproductive system, Gallus gallus, histology, supplementation
Financial Support: CNPQ
Resumo:05-010
PHYSICAL STRESS CHANGES THE BEHAVIOR OF UTERINE NATURAL KILLER CELLS
Rodrigues, K. L. ; Paffaro, A. M. A. ; Paffaro-junior, V. A.
Instituto de Ciências Biomédicas, Unifal
Objectives:
Aerobic exercise demands increasing of blood flow in the skeletal muscle and may cause decreasing of blood flow in other
organs, including uterus-placental blood circulation leading to reduction of uteruplacental oxygen and nutrients transportation to
the embryo, besides increasing immunologic suppression during pregnancy.Furthermore, the continuous exercise can be stressful,
changing the hypothalamic-pituitary-adrenal axis leading to secretion of glucocorticoids that may cause complications during
pregnancy.Uterine natural killer cells (uNK) are the largest population of decidual leukocytes from humans and rodents. The
mouse uNK have been extensively investigated by the use of DBA lectin cytochemistry, which has affinity for N-acetyl-DGalactosamia selectively expressed in the granules and plasma membrane of these cells. This selectivity allowed the
characterization of four subtypes of uNK related to their differentiation stages. The most accepted function of uNK is the IFN-y
secretion leading to uterine arteries dilation, maintaining the decidua and embryo nutrition. The goal of this study was to
investigate whether stress induced by strenuous exercise has effect in the embryo implantation and viability, as well as, in the
number, differentiation, distribution and DBA lectin reactivity pattern of uNK cells.
Ten female Swiss mice were sedentary (group 1). Other 10 (group 2) were submitted to swimming, from 1st to 10th gestation day
(gd) for 60 minutes daily, been the last 20 minutes conducted with a steel caudal dumbbell with 4% of their body masses,
attached in alternately days from the 1st to 5th gd, and 10% of this mass from the 6th to 10th gd. The animals from group 3 were
submitted a training program of 3 weeks before pregnance, 5 days a week, being 15 minutes/day in the first week, increasing
gradually by 10 minutes a day up to 60 minutes in the second week and 60 minutes a day in the third week. After pregnancy were
submitted to swimming in the same way as group 2. All mice were anesthetized and perfused with 4% PFA at 10th dg. The rates
of embryo implantation, viability resorption were evaluated and the implantation sites of these animals were collected and
embedded in paraffin to obtain histological sections that were submitted to DBA lectin. We quantified four DBA reactive uNK
cells subtypes in three areas of the mesometrial region of the implantation sites. Our results showed that strenuous exercise causes
a decrease in embryo implantation and viability and increased fetal loss, as well as increase in number and differentiation of uNK
cells, without apparent activation of its cytotoxicity. The exercise performed prior to pregnancy decreased these effects, since
restored the normal rates of implantation and the total number of UNK, as well as increased viability and decreased embryonic
loss, without completely prevent the deleterious effects of exercise during pregnancy.
Conclusions:
Our results indicate that this stress model established here may be useful for future studies about the neuroimmunoendocrinology
of preterm labor and abortion caused by often unknown stress factors.
Keywords: exercise, physical stress, pregnant mice, uterine natural killer
Financial Support: Unifal-MG, CAPES
Resumo:06-001
HISTOLOGICAL ANALYSIS OF HEPATOPANCREAS AND MANDIBULAR MUSCLE OF NEOHELICE
GRANULATA CRABS FED WITH A HIGH PROTEIN (HP) OR HIGH CARBOHYDRATE (HC) DIET.
Inohara, E. T. S. 1; Pinto, C. B. 1; Fraga, L. S. D. 1; Silva, R. S. M. D. 1; Wiilland, E. D. F. 2; Vinagre, A. S. 1
1
DEPARTAMENTO DE FISIOLOGIA , UFRGS
2
CENTRO DE CIÊNCIAS DA SAÚDE, ULBRA
Objectives:
The objective of this work was to investigate the effects of the nutritional treatment with a high protein (HP) or high carbohydrate
(HC) diet on histological characteristics of the hepatopancreas and mandibular muscle of the crab Neohelice granulata.
Methods: Male crabs were maintained in standardized conditions and fed with raw bovine meet (HP diet) or boiled rice (HC diet)
during 15 days. After this treatment period, the HC and HP crabs were crioanesthesized to collect samples of hepatopancreas and
mandibular muscle to the histological analysis. Samples were fixed in Bouin´s solution for 24h at room temperature. After
fixation, the tissues were slowly washed and dehydrated with alcohol, cleared with xylene, and included in paraffin. Serial
sections (7 µm) were obtained by means of a microtome. The slides were stained with hematoxylin-eosin (HE) and Periodic Acid
Schiff (PAS) reaction for glycogen investigation. Some slides were treated with amylase before PAS staining. Subsequently, they
were analyzed under optical microscope (Leica CME). Photographs were taken using a digital camera (SONY DSC-W1) attached
to the microscope. Results: Hepatopancreas presented typical crustacean morphology, consisted of numerous blind-ended tubules,
composed of an epithelial tissue and separated by a connective tissue, rich in inclusions. The epithelium is composed of a single
layer of cells, with several cell types, distributed in a simple or a pseudostratificated manner, varying according to the region of
the tubule (distal, medial or proximal). In the distal end of the tubules, embryonic cells were found. These cells are cylindrical
and aligned at their base forming a simple epithelium. They have rounded nuclei, granular chromatin, prominent nucleolus and
acidophilic cytoplasm. In the medium and proximal zones, tree types of cells were found: secretory, absorptive and fibrillar.
Secretory or goblet-like cells have a narrow basal region and an enlarged apical region with a cromophobous vacuole that
compresses the nuclei. Absorptive cells are characterized by small nuclei with visible nucleolus in the medium or basal region of
the cell, apical cytoplasm with numerous vacuoles, and a brush border in the apical region. Fibrillar cells are thin and long, are
intensely pigmentated, and have enlarged and basal nuclei with prominent nucleolus. These differences in the position of the
nuclei in the cells are responsible for the pseudostratificated aspect of the epithelium. No positive PAS reaction were detected in
the hepatopancreas epithelium, not even in the crabs fed with HC diet. Mandibular muscle share many features of vertebrate
skeletal muscle, being composed of bundles of long, cylindrical muscle fibers inserted in a chitinous peace by a delicate
connective tissue, distributed between the fibers. The fibers are oriented in longitudinal and transversal directions and have
typical vertebrate sarcomere organization presenting a striated acidophilus cytoplasm and peripheral nuclei. A weak PAS
reaction, similar in HC and HP groups, was detected in muscle fibers.
Conclusions:
The histological analysis of the hepatopancreas and mandibular muscle suggests that the morphology of the tissues is not affected
by the diet of the crabs.
Keywords: HEPATOPANCREAS AND MANDIBULAR MUSCLE , HISTOLOGICAL ANALYSIS , NEOHELICE
GRANULATA CRABS , DIET
Financial Support: CAPES and CNPq.
Resumo:06-002
EFFECTS OF ANOXIA AND POST-ANOXIA RECOVERY ON THE ARGININE PHOSPHATE RESERVES IN
TISSUES OF THE CRAB NEOHELICE GRANULATA SUBMITTED TO TWO DIFFERENT DIETS
Rossetti, C. L. ; Gonçalves, A. S. ; Kucharski, L. C. ; Silva, R. S. M.
Departamento de Fisiologia/ICBS, UFRGS
Objectives:
The burrowing crab Neohelice granulata lives in estuaries where it undergoes daily episodes of lack of oxygen during its raids on
land. Moreover, in winter N. granulata stays in its holes for long periods with low oxygen supply. Studies suggest that the
maintenance of high concentrations of phosphagen in tissues would be an invertebrate strategy to bear high variations of the
oxygen levels in the environment. In addition, N. granulata is highly adaptable to different types of diet and its composition
influences the carbohydrates metabolism patterns. Therefore, the aim of this project was to evaluate the effect of anoxia and postanoxia recovery on the arginine phosphate concentration in tissues of N. granulata crabs previously fed on a high-protein or
carbohydrate rich diet.
Male crabs were divided into two groups with two different diets for 15 days: a high-protein diet (HP) and a carbohydrate-rich
diet (HC). Within each group the animals were divided into three subgroups: a control group that remained under normoxic
conditions (HP n=5, HC n=3), an experimental group with animals that remained under anoxia for 1h (HP n=5, HC n=3); and a
post-anoxia recovery group that was kept for 3h in normoxic water after 1h in anoxic conditions (HP n=5, HC n=3). After these
treatments the crabs were anesthetized by chilling on ice, and jaw muscle and hepatopancreas tissue samples were immediately
collected. The concentrations of arginine phosphate were assessed by the Bergmeyer‟s method (Enzymat 3:425, 1985). Results
are expressed as mean. Crabs fed with the HP diet, when compared with the control group, had a significant increase in arginine
phosphate concentration during anoxia in jaw muscle (8,2 and 171,2µmoles/g-1, respectively. ANOVA F=42,186 p-1. ANOVA
F=46,926 p-1, respectively. ANOVA F=35.392 p-1, respectively. ANOVA F=111.731 p-1, respectively. ANOVA F=36.532 p-1,
respectively. ANOVA F=289.821 p<0.001).
Conclusions:
Those results show that an hour of environmental anoxia increases the arginine phosphate concentration in jaw muscle and
hepatopancreas of crabs that received the HP diet. During this same experimental period there is a reduction in the glycogen
content and an increase in piruvate kinase activity (J Exp Mar Bio Ecol 322:198, 2006). Therefore, the present data suggest that
the transition from normoxia to anoxia could be stimulating an increase of phosphagen reserves in HP crabs. The aim of this
mechanism could be to increase ATP concentration in order to use it in case of longer anoxic periods. The absence of differences
in the arginine phosphate concentration in crabs fed with the HC diet suggests that the arginine phosphate reserves are not
necessary during anoxia for crabs that received carbohydrates.
Keywords: anoxia, arginine phosphate, carbohydrate rich diet, crustacean, high-protein diet
Financial Support: CNPq; PIBIC-UFRGS
Resumo:06-003
THE INFLUENCE OF PINEAL AND THE DIGESTION ON TERMOFILIC RESPONSE OF BOA CONSTRICTOR
AMARALI (SNAKES: BOIDAE)
Sunti, D. M. 1,4; Santos, L. G. 1,4; Armelin, V. A. 1; Abe, A. S. 2,4; Rantin, F. T. 3,4; Florindo, L. H. 1,4
1
Department of Zoology and Botanic-UNESP, IBILCE/UNESP
2
Departament of Zoology-UNESP, UNESP-RC
3
Departament of Physiological Sciences-UFSCar, UFSCar
4
INCT-Comparative Physiology., INCT
Objectives:
Unlike endotherms, ectotherms don´t have just one optimal body temperature (T B). Different activities such as digestion and
locomotion ability are related to distinct optimal T B. These animals depend primarily on behavior to regulate their T B, what can
be clearly observed in reptiles when they select thermally suitable microhabitats or change body postures generating an adequate
TB. However, the physiological mechanisms that controls these behaviors are not fully understood. It is known that the
thermoregulatory behavior in reptiles can be controlled by endocrine pathways that are regulated by the circadian system, being
that pineal and its main hormone, melatonin, has been associated with thermoregulation in this group. Thus, the most probable
mechanism of action of melatonin in reptiles T B is the adjustment of set point established in the hypothalamus. The boa snake,
Boa constrictor amarali, has hunting habits mostly nocturnal. However, they can present activity during the day, what makes it a
good model for studies of thermoregulation. The aim of this study was assess the influence of melatonin on thermoregulation of
B. constrictor amarali fed and fasting.
Methods: The snakes were placed in a box with a heated rock providing a thermal gradient (25°C to 37ºC) for observing the
thermoregulatory behavior (thermal preference and body postures) and checking of T B using probes inserted beneath the skin.
The experiments were conducted between 12 h and 14 h (a period of reduced plasma concentration of MEL) with males, adult
individuals of B. constrictor amarali (n = 9, 1.11 ± 0.41 Kg). All subjects were used in four experimental groups: Control Fasting
(1); Control Fed (2); MEL Fasting (3) and MEL Fed (4). Groups 4 and 2 were fed with Rattus norvegicus (27% of body weight) 1
and 2 days before, respectively, of observations and groups 3 and 4 received intraperitoneal melatonin (5 mg kg -1) 1 h before the
experiments. We used a saline group (fasted and fed) to check the effects of the injection. Results: Among the fasted groups (1
and 3) there was no significant difference in the evaluated parameters: T B (1)= 29,5 ± 0,28ºC and (3)= 30 ± 0,19ºC; stay in 37ºC:
(1)= 86 ± 7,5% and (3)= 75 ± 14%; and stay leaning in the rock: (1)= 65 ± 14% and (3)= 58 ± 14%. Fed snakes (2 and 4)
presented higher TB (2= 31 ± 0,31ºC; 4= 32 ± 0,43ºC) than in fasted groups and the T B of group 4 was significantly higher than
TB of group 2. It was also found that the group 2 remained longer at 37ºC (100%) and leaning in the rock (93 ± 4%) than the
other groups, while group 4 remained 84 ± 5% of the time at 37°C and 48 ± 13% leaning in the rock. There were no significant
differences between control and saline.
Conclusions:
We conclude that fed boas exhibit higher thermophilic responses with consequent higher T B than fasted snakes, and melatonin
influences the selection of higher body temperatures only in snakes in digestion process, and this temperature gain was probably
mediated by physiological adaptations.
Keywords: Digestion, Melatonin, Pineal, Snakes, Thermoregulation
Financial Support: CNPq/FAPESP ; INCT - Comparative Physiology
Resumo:06-004
PHYLOGEOGRAPHY OF POPULATIONS OF ZYGOTHRICA VITTIMACULOSA IN SOUTHERN BRAZIL
Fonseca, P. M. 1; Gottschalk, M. S. 2; Loreto, E. L. S. 3; Robe, L. J. 2,3
1
Graduação em Ciências Biológicas , UFSM
2
Instituto de Ciências Biológicas - ICB, FURG
3
Departamento de Biologia/CCNE, UFSM
Objectives:
The genus Zygothrica currently encompasses a total of 124 described species, most of which appear to be essentially
mycophagous and/or anthophilous. So far, 54 species have been identified in Brazil. However, despite all this diversity, various
ecological and evolutionary aspects of these species are as yet unknown. Zygothrica vittimaculosa, for example, was found in Rio
Grande do Sul, Santa Catarina and São Paulo, demonstrating a considerable gap in its known distribution. This organism can be
considered an ideal model for investigating the evolutionary and ecological factors which have affected diversification of
drosophilids associated with fungi and flowers, as it presents useful adaptations to both niches. This study aims to contribute to
the understanding of the phylogeographic structure of these populations in southern Brazil, trying to evaluate the evolutionary
patterns associated with radiation of these species into the Neotropical region.
Until now, inflorescences of Cestrum spp. were collected in the municipalities of Agudo, Cachoeira do Sul, Cruz Alta,
Horizontina, Itaara, Porto Alegre, Saldanha Marinho, Santa Maria, Santiago and Santo Cristo. After collected, the flowers are
stocked in artificial conditions simulating the natural ones for completing the development of flies. After hatching, the adult flies
are identified through examination of the male aedeagus, the DNA is extracted and PCR (DNA - Polymerase Chain Reaction) are
performed in order to obtain copies of the mitochondrial genes COI and COII. The preliminary molecular analysis consists on
making a DNA barcoding of the obtained mitochondrial sequences. After it, networks and diversity analysis are conducted. At
the moment, 21 specimens had their mitochondrial genes sequenced and it was observed the presence of eight distinct haplotypes
in COI which differ by a total of 29 variable sites. These haplotypes encompass two different haplogroups in the network
analysis, which differ by 21 sites. These findings, coupled with the barcoding analysis, raise the possibility that we are actually
dealing with two sympatric sister species. This hypothesis is further supported by the aedeagus morphological analyses, which
demonstrate the presence of two different structures, one of which is identical to that already registered for Z. vittimaculosa
individuals.
Conclusions:
Continuation of the study associated with the collection of flies associated either with Cestrum spp. flowers or with macroscopic
fungi in places not yet sampled should help to clarify the hypothesis of sympatric speciation and other issues related to the
evolution of Z. vittimaculosa. In this sense, collections from fungal fruiting bodies will also help to assess the levels of structure
between different populations, clarifying if geographic distance or substrate segregation are affecting population structure.
Keywords: Phylogeography, Zygothrica vittimaculosa, Southern Brazil, Barcode, Cestrum
Financial Support: CAPES, CNPq.
Resumo:06-005
NILE TILAPIA (OREOCHROMIS NILOTICUS) SUBMITTED TO ABRUPT SALINITY INCREASES: PLASMA
OSMOLALITY AND MUSCLE WATER, AND NA,K-ATPASE AND CARBONIC ANHYDRASE ACTIVITIES IN
GILLS AND KIDNEY.
Ceron, F. J. M. ; Freire, C. A.
Departamento de Fisiologia/SCB, UFPR
Objectives:
The Nile tilapia is considered one of the most important species in aquaculture, in Brazil and several other countries. Although it
is recognized as one real threat to the conservation of native freshwater fishes, its culture is largely supported and stimulated,
even by State agencies. A fine characterization of its capacity for plasma osmotic homeostasis upon sudden increases in salinity
was lacking in the literature. Knowledge about the osmoregulatory plasticity of an introduced species aids in the prediction of its
potential for invasion. Further, it means a contribution to the knowledge of comparative physiology of osmoregulation. Cichlids
as the tilapia are recent freshwater dwellers, thus very euryhaline.
The tilapias (~12 cm) were purchased from a culture facility located in Araucária, State of Paraná, and were transported (~1-hour
drive) to the Laboratory in Curitiba, where they were kept in a stock tank (250 liters) containing filtered fresh water, at 20-22ºC,
under constant aeration. The fishes (n= 3-6) were submitted for 6, 12 or 24 hours to water of 0 (control, fresh water), 20, 35, and
40‰ salinities in plastic aquaria of 12 liters capacity, at 20ºC, containing 3 fishes. After the experiments, fishes were
anesthesized with benzocaine (80 mg/L, in 3-5 min they show loss of equilibrium and swimming activity). A blood sample was
obtained through caudal vein or heart puncture. Plasma samples were kept in the freezer (-80ºC) until assayed for osmolality. One
sample of axial muscle was dissected to allow determination of its water content (drying for 24 hours at 60º C). Gills and kidney
slices were homogenized and assayed for the activities of the Na,K-ATPase (NAK, coupling ouabain-sensitive ATP hydrolysis
with NADH oxidation), and carbonic anhydrase (CA, rate of pH drop in cold CO2-saturated water). Data obtained were analyzed
through Student´s t-tests or the non-parametric Mann-Whitney U test (P
Conclusions:
Mortality in the higher salinities was associated with total failure of osmo- and tissue volume- regulatory capacities. On other
hand, the survival and regulation of muscle hydration when in brackish water of salinity 20‰ shows that the tilapia can easily
invade adjacent river basins separated by common estuaries. The enzymes NAK and CA are apparently not directly involved in
the mechanisms of adaptation to increased salt, under the protocol employed here. These results are preliminary, and an increase
in sample sizes is necessary in order to strengthen the conclusions.
Keywords: carbonic anhydrase, muscle water, Na,K-ATPase, osmoregulation, tilapia
Financial Support: CNPq and CAPES-REUNI
Resumo:06-006
EFFECT OF CAPTOPRIL AND LOSARTAN ON THE VASCULAR RESPONSE INDUCED BY ANGIOTENSIN II
(ANG II) IN THE SNAKE OXYRHOPUS GUIBEI (SERPENTES, COLUBRIDAE)
Costa, M. B. D. ; Breno, M. C.
Lab. Farmacologia - Instituto Butantan, IBU
Objectives:
The renin-angiotensin system (RAS) is an important endogenous system to the process of the cardiovascular homeostasis. We
have previously shown, in two species of snakes from Viperidae family, the presence of a RAS relatively conserved compared
with other vertebrates species. The angiotensin converting enzyme (ACE) is a rate-limiting step on the generation of the active
peptide Ang II, by removing two amino acids from the inactive angiotensin I (Ang I). The Ang II receptor characterized in the
Viperidae snakes is pharmacologically distinct from the AT1 and AT2 receptors present in the mammalian vertebrates. The aim
of this study is to investigate the presence of a functional RAS in vascular tissue, aorta, of the snake Oxyrhopus guibei, which
belongs to a family of non poisonous snakes, named Colubridae. We analysed the presence of an active ACE on this tissue, and
also the Ang II receptor subtype.
By using in vitro functional assay with vascular smooth muscle, we obtained cumulative concentration-effect curves for Ang I
and Ang II (10-10 - 10-6M) in the absence and in the presence of the inhibitor of ACE, Captopril (10-6M). Cumulative
concentration-effect curves for Ang II (10-10 - 10-6M) were also constructed in the absence and in the presence of the non
selective antagonist of the Ang II receptor, [Sar1, Ile8] Ang II (10-7-10-5M), or a selective AT1 receptor subtype, Losartan (106-10-4M). Dithiotreitol (DTT - 3x10-3M), an agent that reduce disulfide bridges in the receptor structure, was also used in some
assays. Pre-treatment with Captopril shifted the Ang I curves to the right (pD2 6.9 to 5.9, n= 6), but was not able to displace the
Ang II curves (pD2 7.1 to 7.1, n= 5). [Sar1, Ile8] Ang II (pKB 5.04; 5.03 n= 4) and Losartan (pKB 5.57; 4.74; 4.01 n= 2) shifted
to the right the Ang II curves and reduced the maximum effect, while DTT did not modify the pD2 value (7.0 to 7.2, n= 4) but
strongly reduced Ang II response (55%).
Conclusions:
These results indicate the existence of angiotensin converting enzyme in the vascular tissue of the snake Oxyrhopus guibei, which
is functionally active and responsible to convert Ang I into Ang II. The presence of an Ang II receptor in the aorta of this snake
was identified by using the non selective Ang II antagonist, but it could not be classified as an AT1 subtype. DTT results suggest
the presence of at least one disulfide bridge functionally important in the Ang II receptor structure of the snake Oxyrhopus guibei.
Similar results with DTT were also reported for the Ang II receptor in mammalian species and in the snakes from Viperidae
family.The presence of two important key proteins of RAS on the vascular muscle from Oxyrhopus guibei point to a functional
tissue renin-angiotensin system. This Colubridae specie of snake has also an atypical Ang II receptor as characterized previously
in two Viperidae species. Our results contribute to the knowledge of RAS in vertebrate.
Keywords: angiotensin II receptor, angiotensin converting enzyme, vascular smooth muscle, snake
Financial Support: Fapesp (08/ 54351-0).
Resumo:06-007
THE ROLE OF MELATONIN IN THE THERMOREGULATION OF BOA CONSTRICTOR AMARALI (SNAKES:
BOIDAE).
Santos, L. G. 1,4; Sunti, D. M. 1,4; Teixeira, M. T. 1,4; Abe, A. S. 2,4; Rantin, F. T. 3,4; Florindo, L. H. 1,4
1
Zoology and Botanic-UNESP São José do Rio Preto, UNESP/IBILCE
2
Departament of Zoology, UNESP-Rio Claro, UNESP-RC
3
Departament of Physiological Sciences, UFSCar, UFSCar
4
INCT-Comparative Physiology., INCT-CP
Objectives:
The body temperature (TB) regulation of reptiles is mainly carried out by behavioral adjustments, such as the preference for
thermally suitable habitats and changes in body postures that favor or difficult heat loss. In many ectotherms, behavioral
thermoregulation is regulated by circadian rhythm, with melatonin acting as a neuroendocrine transducer by converting the
environmental information in appropriate endogenous signals. However, in some reptiles, this relationship (melatoninthermoregulation) has not been established, leaving many questions about the influence of this hormone in ectotherms
thermoregulation. Snakes are a good model for studies with thermoregulation, because, being cylindrical, they have a large
surface/volume ratio, therefore, gain and lose heat rapidly if not occurring behavioral and physiological adjustments. And the
Jibóia, Boa constrictor amarali, is a particularly interesting in this study by presenting diurnal and nocturnal activity.
Methods: During the experiments, individuals of B. constrictor amarali (n= 9, 1.11 ± 0.41 Kg) were kept in a box with two
thermal gradients: 1= 25,5ºC and 2= 37ºC, formed by a hot rock at one end. Thermocouples were inserted under the skin of
snakes to monitoring TB, and monitored, through a video camera, the time spent on each gradient and on differents body postures
of each snake. The experiments were carried out during the day (between 12h and 14h) on groups Diurnal Control (C D), Diurnal
Saline (SD) and Luzindol (L), and at night (between 00h and 02h) in the groups Nightly Control (C N), Nightly Saline (SN) and
melatonin (MEL), being that, the nine animals were used in all groups. To MEL and L groups were administered,
intraperitoneally, solutions of these substances (5 mg.kg-1 and 1.5 mg.kg-1, respectively) 1 h before the experiments. Results: The
results showed that all groups had a T B between 29ºC and 30ºC (CD = 29,5 ± 0,28ºC; SD = 29,5 ± 0,22ºC; L= 29,5 ± 0,21ºC; CN =
20 ± 0,16ºC; SN = 29,5 ± 0,12ºC and MEL= 30 ± 0,19ºC), all groups preferred the warmer gradient (37ºC) (C D = 86 ± 7,5%; SD =
80 ± 11%; L= 85 ± 11%; CN = 81 ± 12%; SN = 86 ± 10% and MEL 75 ± 14%) and all groups remain longer leaning against the
rock than another body posture (CD = 65 ± 14%; SD = 69 ± 6%; L= 77 ± 6%; CN = 70 ± 11%; SN = 68 ± 10% and MEL= 58 ±
14%). There were no significative differences in the parameters evaluated between any groups of snakes.
Conclusions:
The hormone melatonin has no effect on thermoregulation of fasting B. constrictor amarali.
Keywords: Boas, Melatonin, Pineal, Snakes, Thermoregulation
Financial Support: CNPq/FAPESP; INCT- Comparative Physiology
Resumo:06-008
PHYSIOLOGY OF NATIVE SPECIES WITH POTENTIAL TO BE USED IN AQUACULTURE:
OSMOREGULATION OF THE DIADROMOUS FRESHWATER PALAEMONID SHRIMP MACROBRACHIUM
ACANTHURUS
Amado, E. M. ; Freire, C. A.
Departamento de Fisiologia - Universidade Federal do Paraná, UFPR
Objectives:
Brazil has several native species with great potential for aquaculture, but the lack of scientific information and support to research
renders Brazilian aquaculture to be dominated by exotic species. This work is part of a project that aims to generate knowledge
on the physiology of native species, to foster their use in aquaculture, as an alternative to the use of exotic species. The objective
of this study was to provide detailed information on the tolerance and response of a freshwater diadromous shrimp of reasonable
size, M. acanthurus, to salinity increases.
Adult specimens of either sex (n=6 for each experimental group) were exposed to fresh water (controls), or to diluted seawater of
salinities 5, 10, 15, 25, and 35‰ for 3, 6, 24, and 120 hours. After exposure, shrimps were crioanesthesized, and hemolymph,
gills and abdominal muscle were removed and stored for later analysis of osmolality, branchial Na,K-ATPase (NaK) and
carbonic anhydrase (CA) specific activities, and muscle tissue hydration. Mortality occurred at 24 and 120 hours in 35‰ salinity;
only one animal from each group survived. From fresh water up to 15‰ salinity, animals were able to maintain stable
hemolymph osmolality (~400-440 mOsm/kg H2O) and tissue hydration (~78%) until 120 hours of exposure. At higher salinities
hemolymph osmolality increased and tissue water content decreased. After 6 and 24 hours at 25‰, osmolality increased to 536 ±
23 and 614 ± 27 mOsm/kg H2O, respectively, and tissue hydration decreased to 76.0 ± 0.6% and 75.0 ± 0.8%, respectively.
Interestingly, after 120 hours in 25‰ animals returned to control levels of hemolymph concentration and tissue water
content.However, at 35 ‰ salinity a physiological limit was reached, and hemolymph osmolality and muscle water content have
shown progressive changes until the 120 hours of experiment. Values were: 704± 26 mOsm/kg H2O and 73.0 ± 0.7% after 6
hours, 963 mOsm/kg H2O and 73.2% after 24 hours, 793 mOsm/kg H2O and 71.9% after 120 hours, n = 1 due to mortality.
Exposure to increased salinity up to 120 hours lead to no changes in NaK and CA activity.
Conclusions:
Up to salinity 15‰, the maintenance of a constant haemolymph osmolality offered no osmotic challenge to muscle tissue, which
thus kept its hydration. This may have been achieved by decreased apparent permeability, in order to prevent the entry of salt or
efflux of water. In 25‰, hemolymph osmolality increase was directly related to the decrease in muscle water content along the
time course of experiment. The recovery of tissue hydration after 120 hours reflected the recovery of hemolymph osmotic
concentration, although mechanisms of tissue water regulation are also possibly involved. The enzymes NaK and CA appear not
to be up- or down-regulated to allow M. acanthurus to cope with this high salinity change.
Keywords: aquaculture, carbonic anhydrase, crustaceans, Na,KATPase, tissue hydration
Financial Support: CAPES/PRODOC
Resumo:06-009
EXPRESSION OF HEAT-SHOCK PROTEINS (HSP70) IN THE MUSCLE TISSUE OF THE FRESHWATER
DIADROMOUS SHRIMP MACROBRACHIUM ACANTHURUS SUBMITTED TO INCREASED SALINITY AND
TEMPERATURE
Maraschi, A. C. ; Prodocimo, V. ; Freire, C. A.
Departamento de Fisiologia/SCB, UFPR
Objectives:
Variations in abiotic parameters, such as salinity and temperature, directly influence physiological processes of aquatic animals,
and possibly induce the expression of heat shock proteins (HSPs), as a cellular defense mechanism. Palaemonid shrimps such as
Macrobrachium acanthurus are freshwater species as adults, but migrate to the estuaries for development, thus facing salinity
challenges during their life cycle. Previous studies in our laboratory using this species have shown increases in hemolymph
osmo-ionic levels only after ~24 hours of exposure of the shrimps to diluted seawater of salinity 20‰. However, muscle
hydration was maintained until 120 hours of exposure. A clear whitening of the abdominal muscle during saline stress was also
noted in the shrimps. This study was then conducted in order to verify whether the increase in hemolymph concentrations and
change in texture of the muscle would be related to protein denaturation or the expression of HSP70. A temperature shock was
also offered to the shrimps, to serve as positive control for the expression of HSP70.
Shrimps were exposed to high salinity (20‰) or high temperature (30°C) for 1, 24, and 120 hours. Controls were kept in fresh
water (temperature of 20°C) for 1 hour. After the exposure periods, shrimps were anesthesized in ice and a sample of abdominal
muscle was dissected for the assay of HSP70 expression, with n=7-9 for each time of exposure to 20‰, and n=4 for each time of
exposure to 30°C. Total protein content of the muscle homogenates was determined using the Bradford assay. Samples were then
submitted to SDS-PAGE electrophoresis under denaturing conditions. After the run the proteins were transferred to a
nitrocellulose membrane in a Western Blot chamber. Membranes were then incubated with primary antibody anti-HSP70
(Sigma), and secondary antibody conjugated with alkaline phosphatase. Band intensity (arbitrary units) was quantified through
the ImageJ software, and data were compared with controls in fresh water using Student´s t-test (P
Conclusions:
Results indicate that the expression of HSP70 was not induced in the muscle tissue of M. acanthurus submitted to high salinity
for up to 120h, under the conditions reported here. However, expression of HSP70 was clearly induced upon high temperature
shock, after 24 and 120h. It seems that this euryhaline species can deal with the salinity challenge employing its constitutive
levels of HSP70.
Keywords: HSP70, osmoregulation, Palaemonid, salinity, temperature
Resumo:06-010
A COMPARISON OF RESISTANCE TO OXIDATIVE STRESS IN AN INVERTEBRATE AND A VERTEBRATE
Welker, A. F. 1; Campos, E. G. 2; Hermes-lima, M. 2
1
Faculdade de Ceilandia, UnB
2
Departamento de Biologia Celular, UnB
Objectives:
Reperfusion after ischemia causes severe injury and death in mammals due to overproduction of reactive oxygen species during
reoxygenation. Recent studies showed that the induction of specific antioxidant enzymes in rats, such as catalase, may protect
tissues from reperfusion damages. Several hypoxic-tolerant invertebrates and vertebrates increase antioxidant enzymes before
episodes of reoxygenation. However, the contribution of each antioxidant enzyme for their tolerance to reoxygenation is
unraveled. We compared the mechanisms responsible for the high resistance to oxidative stress in the land snails (Helix aspersa)
and the tilapia fish (Oreochromis niloticus).
The animals were submitted to two combined treatments: firstly, catalase activity was suppressed by the injection of 3-amino1,2,4-triazole (ATZ, 1g/kg); and secondly, the animals with depleted catalase activity were subjected to a cycle of oxygen
restriction followed by reoxygenation. We analyzed data from the hepatopancreas and foot muscle of the snails (n = 6) and the
liver of male fish (n = 6). ATZ injection inhibited catalase activity in both snails (by 69% in hepatopancreas and 60% in foot
muscle) and tilapia (by 90% in liver) and,did not cause changes in glutathione levels, oxidative stress markers and the activities of
most other antioxidant enzymes under normoxia. Oxygen restriction alone (saline-injected animals) caused activation of snail
foot muscle Se-GPX (200%), which also tended to increase in snail hepatopancreas (213%) and in fish liver (170%). Increased
levels of SOD (by 170%), GSSG/GSH-eq ratio (140%) and carbonyl protein (140%) were found in ATZ-injected fish during
hypoxia exposure, indicating the occurrence of oxidative stress, while no changes were observed in the snails. Most importantly,
Se-GPX induction was abolished in snail hepatopancreas and fish liver by ATZ injection upon oxygen restriction. Reoxygenation
alone caused increases in SOD (202%), GSH (127%) and carbonyl proteins (155%) in fish and no alterations in snails. ATZinjection augmented oxidative stress during reoxygenation: higher levels of GSSG/GSH-eq ratio (134%), TBARS (146%) and
carbonyl protein (142%), and diminished levels of GST activity (by 18%) were present in fish; and there was a small increase of
carbonyl protein (116%) in snail hepatopancreas.
Conclusions:
Tilapia fish showed more oxidative stress under disturbances of oxygen availability than Helix aspersa snails, which present
higher tolerance to anoxia. Inhibition of catalase activity caused a redox imbalance in fish and almost no change in snails during
reoxygenation, indicating that this enzyme helps to minimize oxidative damages in fish. This comparative study allowed us to
conclude that in both, H. aspersa and tilapia fish, resistance to oxidative stress is linked to Se-GPX stimulation during oxygen
restriction.
Keywords: free radicals, antioxidant, oxygen, metabolism, reperfusion
Financial Support: Redoxoma-CNPq and FINATEC
Resumo:06-011
COMPARISON OF PROTEINS SETS PRESENT IN THE CRUDE VENOM AND IN THE MICROVESICLES OF THE
VENOM OF THE CROTALUS DURISSUS TERRIFICUS SNAKE.
Souza, A. ; Sakai, F. ; Carneiro, S. M. ; Sant'ana, S. S. ; Fernandes, W. ; Yamanouye, N.
Instituto Butantan, IBU
Objectives:
The Crotalus durissus terrificus venom is constituted by toxins that are responsible for the clinical complications caused by
envenoming. These toxins are not the only components presents in the venom; numerous electron-dense microvesicles (40 – 80
nm in diameter) are observed in venom gland and in venom. These structures have intramembranous particles on the cytoplasmic
leaftet, suggesting the presence of transmembrane proteins. Microvesicles are known structures that shed from the surface of
many cells and have important physiological and pathological function. The aim of this study is to compare the proteins present
in the crude venom and in the microvesicles in order to confirm previous data.
The venom used was manually extracted from Crotalus durissus terrificus maintained in the Laboratory of Herpetology at
Instituto Butantan. 7.5 ml of venom was diluted with cold PBS (1:4) and centrifuged at 150 g for 15′ at 4oC to eliminate cell
debris. The supernatant (S1) was ultracentrifuged at 200,000 g for 60′ at 4oC, and the pellet was ressuspended in 10ml of cold
PBS and ultracentrifuged again at the same condition. The resulting pellet was processed for morphological analysis by electron
microscope or for bidimensional electrophoresis (2-DE). Proteins in the crude venom, in the supernatant of the first
ultracentrifugation (S2) and microvesicles extract were compared by 2-DE. Morphological analysis showed that after
ultracentrifugations the microvesicles kept their morphology. The protein spots in the 2-DE images were analyzed by
ImageMaster Platinum 7.0 software. The 2-DE images showed that 18 spots were present only in the microvesicle extract. The
spots were 1) approximately 180 kDa with PI around 4; 2) ranging from 60 to 70 kDa with PI ranging from 7 to 9; 3) 11 kDa with
PI 4; 4) approximately 10 kDa with PI ranging from 5 to 7 or 9 to 10. Ten spots were in common among crude venom, S2 and
microvesicles extract. Between crude venom and microvesicles extract or S2 and microvesicles extract 22 spots and 10 spots
were in common, respectively.
Conclusions:
Our results showed that after ultracentrifugations the microvesicles kept their morphology, but some of them seem to be lysed
since 10 spots were found only in S2 and microvesicles extract, but not in the crude venom. In addition, we showed that
microvesicles haves specific proteins that could have important role such as regulating the activity of toxins from the venom, or
even having a biological activity that contribute to the pathology of the envenoming.
Keywords: Crotalus durissus terrificus, Venom, Microvesicles, Proteins
Financial Support: FAPESP, CAPES, CNPq
Resumo:08-001
COMPARATIVE STUDY OF THE MECHANICAL PROPERTIES OF THE LUNG SURFACTANT IN AN ANIMAL
MODEL OF VENTILATOR-INDUCED LUNG INJURY
Munoz, D. M. M. 1; Rivas, J. M. V. 2; Gomes, S. 1; Moriya, H. T. 2; Amato, M. B. P. 3; Rebello, C. M. 1;
Alencar, A. M. 5
1
Departamento de Fisiopatologia Experimental , FMUSP
2
Laboratório de Engenharia Biomédica , EPUSP
3
Departamento de Pediatria, FMUSP
4
Divisao Pneumologia-INCOR, FMUSP
5
Departamento de Física Geral, IFUSP
Objectives:
Aim: The aim of this project was to study the mechanical properties of the lung surfactant extracted during several moments in a
protocol of an animal model of ventilator-induced lung injury (VILI).
Methods and Results:Pulmonary surfactant is a lipid-protein complex, synthesized and secreted by the epithelium of airways in
the lungs and the alveolar spaces, whose primary function is to reduce the surface tension at air-liquid interface to minimize
respiratory work. The forces of attraction between adjacent liquid molecules are much stronger than those between the liquid and
gas, resulting in a trend to decrease the net surface. The lung surfactant acts through an imaginary line on the interface liquid-gas,
reducing this trend, preventing the alveoli collapse during expiration, and also assisting the inspiration (J Appl Physiol. 16:444,
1961). This study analyzed samples of surfactant from three pigs (35.0  3.2 kg, mean  standard deviation) submitted to
mechanical ventilation. The surfactant‟s samples were extracted by the procedure of bronchoalveolar lavage (BAL) with saline
(0.15 M NaCl) during the protocol of ventilator-induced lung injury (VILI). The BAL procedures were performed at different
times of the ventilation protocol: the control sample was obtained after 30 minutes of ventilation (Basal), and after 180 minutes of
ventilation the injury sample was taken (After VILI). First, the samples were centrifuged at 3,500 rpm to remove cells. After that,
centrifugation was performed at 10,000 rpm and two types of extraction were separated: supernatant and pellet. Surface tension
of the samples (supernatant and pellet) were assessed using a Wilhelmy Balance (Minitrough, KSV Instruments, Finland) that
compress and expands the surface area (6400 cm2-1650 cm2) available for the surfactant added in a through using Milli-Q water
as a substract (175 mL). After five continues cycles of expansion and compression, the minimum surface tension in the last
compression was recorded. The minimum surface tension of the supernatant samples was 54.5  2.3 mN/m (mean  standard
deviation) (Basal) and 50.4  2.9 mN/m (mean  standard deviation) (After VILI). The minimum surface tension of the pellet
samples was 44.1  5.6 mN/m (mean  standard deviation) (Basal) and 51.0  12.5 mN/m (mean  standard deviation) (After
VILI). There was significant statistical difference only between supernatant samples Basal and After VILI using t-student test (p
Conclusions:
Conclusion: After induction of the ventilator-induced lung injury, the respiratory mechanics of the pigs deteriorated very fast.
Therefore, we believe that the mechanical properties of the surfactant also deteriorated. So far, our results showed significant
statistical difference in the surface tension of supernatant surfactant samples. Two future actions are going to be taken. First, the
centrifugation of 10,000 rpm will be replaced by a centrifugation of 17,000 rpm in order to have a better concentration in the
pellets. Another action is to carry out quantification of proteins and lipids in the samples in order to have correlation between
mechanical and chemical properties.
Keywords: Lung surfactant, Surface tension, Mechanical ventilation
Financial Support: CNPq and FAPESP
Resumo:08-002
RHO KINASE INHIBITION ATTENUATES CHANGES ON AIRWAY AND LUNG TISSUE MECHANICS INDUCED
BY CHRONIC ALLERGIC INFLAMMATION IN GUINEA PIGS.
Righetti, R. F. 1; Pigati, P. A. S. 1; Possa, S. S. 1; Habrum, F. C. 1; Charafeddine, H. T. 1; Reis, R. A. 1;
Saraiva, B. M. 1; Xisto, D. G. 3; Antunes, M. A. 3; Rocco, P. R. M. 3; Prado, C. M. 2; Leick-maldonado, E.
A. 1; Martins, M. D. A. 1; Tibério, I. F. L. C. 1
1
Faculdade de Medicina da Universidade de São Paulo, FMUSP
2
Universidade Federal de São Paulo, Unifesp
3
Universidade Federal do Rio de Janeiro, UFRJ
Objectives:
Evaluated if Rho kinase inhibition (Y-27632) modulates airway and distal lung responsiveness in guinea pigs (GP) with chronic
allergic inflammation.
Methods: GP were submitted to seven ovalbumin (OVA) or saline (SAL) inhalations (2x/wk/4wks). From 5th inhalation, GP in
Rho kinase groups were submitted to Y-27632 inhalation (1mM; 2min) 10 minutes before each inhalation with OVA or SAL.
72hs after the 7th inhalation, GP were anesthetized and the respiratory system resistance (Rrs) and elastance (Ers) as well as the
oscillatory distal lung mechanics (Rt and Et) were evaluated (baseline and maximal responses after ovalbumin challenge).
Results: Ovalbumin-exposed GP had an increase in maximal Rt, Et, Rrs and Ers compared to control groups (p<0.05).
Conclusions:
The Rho kinase inhibition contributes to the control of airway and distal lung parenchyma mechanical responsiveness in an
animal model of chronic allergic inflammationattenuated values of Rt an Et, suggesting the constrictor papel of Rho kinase in
airways and distal lung parenchyma.
Keywords: Rho kinase, chronic allergic inflammation , Y-27632 , Asthma
Financial Support: FAPESP, LIM-20-FMUSP
Resumo:08-003
LASSBIO 596 PER OS AVOIDS PULMONARY AND HEPATIC INFLAMMATION INDUCED BY MICROCISTINLR.
Casquilho, N. V. ; Carvalho, G. M. C. ; Alves, J. L. C. R. ; Machado, M. N. ; Soares, R. M. ; Azevedo, S.
M. F. D. O. ; Lima, L. M. ; Barreiro, E. J. D. L. ; Valença, S. S. ; Carvalho, A. R. S. ; Faffe, D. S. ; Zin, W.
A.
Objectives:
Cyanobacterial blooms that generate microcystins are increasingly recognized as an important health problem in aquatic
ecosystems. In the present study we aimed to evaluate the usefulness of LASSBio 596 (a new symbiotic agent that modulates
TNF-α level and is a potent inhibitor of phosphodiesterases 4 and 5) in the treatment per os of pulmonary and hepatic injuries
induced by MCYST-LR.
Swiss mice (25-35 g) received an intraperitoneal injection of 40 μL of saline CTRL, n=8) or a sublethal dose of MCYST-LR (40
μg/kg). After 6 h, the animals received either saline (TOX, n=8 and CTRL, n=10) or LASSBio 596 (50 mg/kg, LASS) by gavage.
8 h after the first instillation, pulmonary elastance and its viscoelastic component (cmH2O/mL), viscoelastic and total pressures
(cmH2O) were determined by the end-inflation occlusion method. Left lung and liver were prepared for histology. In lung and
hepatic homogenates MCYST-LR, TNF-α, IL-1β and IL-6 were determined by ELISA. TOX group showed significantly higher
lung mechanics parameters (40.3 ± 2.6, 8.5 ± 1.0, 0.5 ± 0.05, 1.7 ± 0.1, 2.2 ± 0.1, respectively) compared to CTRL (22.6 ± 0.8,
5.7 ± 0.5, 0.4 ± 0.04, 1.1 ± 0.1, 1.5 ± 0.1, respectively) and LASS (24.0 ± 1.0, 5.4 ± 0.2, 0.5 ± 0.03, 1.0 ± 0.05, 1.5 ± 0.06,
respectively). Furthermore, TOX group presented a significantly higher amount of polymorphonuclear cells and alveolar collapse
in lung parenchyma than CTRL and LASS. Moreover, pro-inflammatory mediators (TNF-α, IL-1β and IL-6) in TOX group, were
significantly higher in lung and liver homogenates compared to CTRL and LASS. Likewise, liver structural deterioration
(necrosis and steatosis) were less evident in the LASS than TOX group. Microcystin was found in the livers of all animals
receiving MCYST-LR. LASS and CTRL did not differ in any studied parameter (p < 0.05).
Conclusions:
Orally administered LASSBio 596 prevented lung and hepatic inflammation and completely blocked pulmonary functional and
morphological changes induced by MCYST-LR.
Keywords: Inflammation, anti-inflammatory, cyanobacteria, lung mechanics, microcystin-LR
Financial Support: PRONEX-MCT, FAPERJ, CNPq, MCT
Resumo:08-004
PARACRINE FACTORS OF BONE MARROW-DERIVED MONONUCLEAR CELL THERAPY AMELLIORATE
LUNG INJURY IN A MURINE MODEL OF EMPHYSEMA.
Cruz, F. F. 1; Antunes, M. A. 1; Abreu, S. C. 1; Xisto, D. 1; Ornellas, D. S. 1,2; Maron-gutierrez, T. 1,2;
Fujisaki, L. C. 1; Capelozzi, V. L. 3; Morales, M. M. 2; Rocco, P. R. M. 1
1
Laboratory of Pulmonary Investigation - IBCCF, UFRJ
2
Laboratory of Cellular and Molecular Physiology - IBCCF, UFRJ
3
Department of Pathology, USP, USP
Objectives:
We hypothesized that bone marrow-derived mononuclear cell (BMDMC) therapy improves lung mechanics and histology in a
murine model of emphysema through the release of growth factors.
28 female C57BL/6 mice were randomly assigned into two groups. In control (C) animals, saline was intratracheally (it) injected
(50 &mul), whereas emphysema mice received porcine pancreatic elastase (EL, 0.1 UI). Saline and elastase were intratracheally
injected once a week during 4 weeks. C and EL groups were further randomized into subgroups receiving saline (50 &mul, SAL)
or BMDMC (2x106, 50 &mul, CELL) intravenously 3 hours after the first saline or elastase instillation. Seven days after the last
instillation, in vivo (airway resistance, viscoelastic pressure, static elastance) and in vitro (elastance, resistance, and hysteresivity)
lung mechanics, morphometry, quantitative analysis of collagen and elastin in alveolar septa, lung epithelial cell apoptosis, light,
confocal and electron microscopy, mRNA expressions of transforming growth factor (TGF)-&beta], platelet-derived growth
factor (PDGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF) and gene detection of Sexdetermining region Y (SRY) in lung tissue were analyzed. At day 7, BMDMC led to: 1) significant reduction in static lung
elastance (40%), fraction area of alveolar collapse (52%), alveolar hyperinflation (298%), lung tissue cellularity (31%), collagen
fiber content (45%), number of apoptotic cells in lung, and elastic fiber disruption, 2) less damage in the alveolar septa
membrane, epithelium, and endothelium, 3) an increase in mRNA expressions of VEGF and a decrease in IGF, PDGF, and TGF&beta], and 4) no SRY gene was detected in the groups treated with BMDMC.
Conclusions:
BMDMC therapy was effective at modulating inflammatory, fibrogenic, and elastogenic processes, thus improving lung
mechanics. These beneficial results may be attributed to paracrine effects.
Keywords: cell therapy, emphysema, paracrine effects, remodeling
Financial Support: PRONEX-FAPERJ, CNPq, FAPERJ, CAPES
Resumo:08-005
PULMONARY AND UPPER RESPIRATORY TRACT IMPAIRMENT AFTER CHRONIC EXPOSURE TO
MICROCYSTIN-LR
Oliveira, V. R. 1; Mancin, V. G. L. 1; Pinto, E. F. 1; Soares, R. M. 1; Azevedo, S. M. F. O. 1; Pires-neto, R.
C. 2; Mauad, T. 2; Carvalho, A. R. S. 1; Zin, W. A. 1
1
Instituto de Biofísica Carlos Chagas Filho, UFRJ
2
Departamento de Patologia, USP
Objectives:
Microcystin-LR (MCYST-LR) is a toxin commonly released by cyanobacteria in water reservoirs. It can damage the lung if
absorbed by oral, venous or respiratory routes. To verify the putative lung mechanical and histological impairment and the upper
airways of mice after chronic intra-nasal instillation of MCYST-LR.
Male Swiss mice (25-30 g) were daily intranasally instilled with 10 µL of distilled water (CTRL group, n=10) or 6.7 ng/kg of
MCYST-LR diluted in 10 µL of distilled water (TOX, n=8) 7 days/week for 1 month. Lung mechanics was determined 24 h after
the last instillation. Lungs and nasal cavity were prepared for histological analysis (hematoxylin-eosin and alcian blue,
respectively). TOX showed higher static elastance and viscoelastic component of elastance (33.9 ± 1.2 and 5.2 ± 0.4 cmH2O/mL,
respectively), viscoelastic/inhomogenous pressure and total resistive pressure (1.0 ± 0.1 and 2.0 ± 0.2 cmH2O, respectively) than
CTRL (26.4 ± 2.6 and 3.7 ± 0.3 cmH2O/mL, 0.7 ± 0.1 and 1.5 ± 0.1 cmH2O, respectively). Alveolar collapse and
polymorphonuclear cell content were significantly larger in TOX group (41.2 %, and 8.1 x 10-3 cells/µm²) than in CTRL (3.1 %
and 2.3 x 10-3 cells/µm²). TOX displayed a significantly higher volume proportion of mucous substances in the nasal epithelium
(61.5 %) than CTRL (38.5 %). T-test was used (α = 5 %).
Conclusions:
Prolonged exposure to low levels of microcystin-LR triggered pulmonary tissue mechanical impairment, damage to lung structure
and secretory changes in the nasal cavity of mice. Thus, frequent exposure to low levels of MCYST-LR can damage the
respiratory system and should be avoided.
Keywords: lung mechanics, lung histology, upper airways, mucous substances, microcystin-LR
Financial Support: CNPq, FAPERJ, MCT
Resumo:08-006
EFFECT OF PROTEINASE INHIBITOR FROM CRATAEVA TAPIA (CRATABL) IN DISTAL LUNG MECHANICAL,
INFLAMMATORY AND REMODELING ALTERATIONS INDUCED BY ELASTASE IN MICE.
Oliva, L. V. 2; R. A. Reis2; Theodoro, O. A. 2; Oliveira, B. M. 2; Ferreira, R. 3; Pailva, P. 3; Lopes, F. D. T.
Q. S2; Brito, M. V. 3; Tibério, I. D. F. L. C. 2; Oliva, M. L. V. 3
3
Universidade Federal de São Paulo - UNIFESP , Unifesp/EPM
2
Faculdade de Medicia da a Universidade de São Paulo, FMUSP
Objectives:
to evaluate if a plant Kunitz proteinase inhibitor EcTI contributes to inactivation of elastase-induced mechanical, inflammatory
and extracellular matrix remodelling alterations.
Methods: C57Bl6 mice received elastase (50ml/animal- IN-E group). Control group received saline (Ve group). Mice were
treated with EcTI (2mg/kg) at days 1, 7, 14, 21, 28 and 35 after elastase instillation (I-E group). At day 40, mice were
anesthetized, mechanically ventilated and we analyzed respiratory system resistance and elastance, tissue elastance, tissue
damping, and airway resistance. Afterwards, BAL was performed and lungs were removed. By morphometry, we quantified the
mean linear intercept (Lm), collagen and elastic fibers in distal lung. Results: There were no differences in pulmonary mechanics
comparing all groups. In E-group, there was an increase in BAL-total cells, BAL-lymphocytes, BAL-neutrophils, collagen,
elastic fibers and LM compared to control (p<0.05).
Conclusions:
This proteinase inhibitor (EcTI) reduced elastase-induced pulmonary inflammatory and extracellular matrix remodeling
alterations induced by elastase. Although more studies need to be performed, this inhibitor may contribute as potential therapeutic
tool for COPD management.
Keywords: inflammatory, lung, elastase, remodeling, emphysema
Financial Support: FAPESP, CNPq, LIM-20 HCFMUSP.
Resumo:08-007
EARLY EFFECTS OF TIDAL VOLUME AND POSITIVE END-EXPIRATORY PRESSURE IN ANESTHESIAINDUCED ATELECTASIS
Cruz, L. F. 1; Spieth, P. M. 2; Abreu, M. G. D. 2; Carvalho, A. R. 1
1
2
Department of Anesthesiology and Intensive Care Therapy, UHCGC
Objectives:
To evaluate the acute effects of positive end-expiratory pressure (PEEP), tidal volume (VT) and ventilatory mode on pulmonary
function and distribution of aeration in pigs under general anesthesia.
Eight pigs weighing 33 to 43 kg were anesthetized and mechanically ventilated with a VT of 6 or 12 ml/kg in volume or pressure
controlled ventilation, randomly assigned, and with PEEP sequentially set at 0, 6 and 12 cmH2O, 15-min per step. At each
ventilatory strategy, hemodynamic, respiratory variables and static whole lung as well as juxta-diaphragmatic dynamic computed
tomography (CT-scan) images were acquired. Tidal recruitment and overdistension was also assessed by means of a volumedependent elastance model fitted to the airway pressure and flow data. No differences among ventilatory modes were observed.
At low VT, increase of PEEP decreased tidal recruitment and the amount of non-aerated areas while increased normally and
hyperinflated areas, plateau pressure, respiratory system elastance, tidal overdistension and the arterial pressure of carbon dioxide
(PaCO2). At high VT, tidal recruitment and overdistension was always higher at comparable PEEP levels and PaCO2 as well as
respiratory system elastance decreased with PEEP with comparable oxygenation levels.
Conclusions:
At low VT, increased PEEP levels promote alveolar recruitment with some overdistension and a progressive increase in PaCO2
levels. At high VT, increased PEEP promotes even more tidal recruitment and overdistension but with progressive decrease of
PaCO2 levels, suggesting an improvement in alveolar ventilation.
Keywords: anesthesia induced-atelectasis, computed tomography imaging, positive end-expiratory pressure, protective
ventilation
Financial Support: CNPQq, FAPERJ and DFG
Resumo:08-008
ASSOCIATION BETWEEN URIC ACID LEVELS, HYPOXIA AND SLEEP APNEA: A POPULATION-BASED
SURVEY
Hirotsu, C. ; Tufik, S. ; Bittencourt, L. R. A. ; Santos-silva, R. ; Andersen, M. L.
Departamento de Psicobiologia, UNIFESP
Objectives:
Recurrent hypoxia associated with obstructive sleep apnea syndrome leads to an increase in the degradation of adenosine
triphosphatase into xanthine, which in turn increases uric acid concentrations. The current study aimed to determine whether a
correlation exists between uric acid levels in the peripheral blood and the arterial oxyhemoglobin saturation (SpO2) from a
representative population of Sao Paulo.
A population-based survey adopting a probabilistic three-stage cluster sample of Sao Paulo was used to represent the population
according to gender, age and socioeconomic class (CEP 0593/06). A total of 1,021 volunteers underwent polysomnography
recordings, blood pressure assessment and biochemical blood analysis. The results indicated that 30.3% of women and 48.3% of
men with an apnea-hypopnea index (AHI)>5 had higher uric acid levels compared with normal gender-matched individuals. Men
presented higher levels of uric acid than women, independent of AHI. Uric acid levels were significantly correlated with AHI,
systolic blood pressure, SpO2, arousal index, percentage of slow-wave sleep, creatinine, sodium, cholesterol, triglycerides and
glucose levels. A linear regression model revealed that the predictors for uric acid concentration were gender, creatinine,
triglycerides, systolic blood pressure, SpO2 and HDL. However, when the model was controlled for body mass index, the
influence of SpO2 decreased in the model.
Conclusions:
Uric acid levels were positively correlated with the number of obstructive respiratory episodes during sleep and present a possible
marker for sleep apnea. Nevertheless, this association seems to be influenced by a confounding factor: obesity.
Keywords: apnea-hypopnea index, sleep apnea, uric acid, oxyhemoglobin saturation
Financial Support: This work was supported by grants from AFIP, FAPESP and CNPq.
Resumo:08-009
EXPERIMENTAL MODEL OF PULMONARY EMPHYSEMA IN MICE: MEDIUM-TERM EFFECTS OF
EXPOSURE TO CIGARETTE SMOKE
Toledo, A. C. ; Suehiro, C. L. ; Caleman-neto, A. ; Magalhães, R. M. ; Mello, P. ; Camilo, B. F. ; Olivo, C.
R. ; Lopes, F. D. Q. D. S. ; Martins, M. A.
Lab. Terapeutica Experimental/ Faculdade de Medicina da USP, LIM20/FMUSP
Objectives:
The major environmental factor that predisposes patients to COPD is long-term cigarette smoking. In mice cigarette smoke (CS)
exposure induce inflammation, changes in respiratory mechanics and airspace enlargement that is similar to those found in
humans just in response to prolonged exposure (> 24 weeks) to cigarette smoke which expend time and costs. The objective of
this study was to evaluate the medium-term effects of twice daily exposure to cigarette smoke in mice.
Methods: Male C57Bl6 mice (6-8 weeks old) were divided in six groups. Smoke groups: S4, S8 and S12 groups were exposed to
cigarette smoke for 30min/day, twice a day, 5days/week for 4, 8 or 12 weeks respectively (n=8 in each group). Control groups
were exposed to room air at the same time points (C4, C8 and C12, n=8 in each group). Respiratory mechanics, total and
differential cells count in bronchoalveolar lavage (BAL) and mean linear intercept (Lm) were evaluated. Results: Exposure to CS
for 4 weeks resulted in an increase in total cells and macrophages in BAL (p
Conclusions:
Conclusion: We showed that exposure to CS for 12 weeks was enough to induce inflammation, respiratory mechanics changes
and pulmonary emphysema in mice.
Keywords: emphysema, smoke, inflammation, respiratory mechanics
Financial Support: FAPESP - Fundação de Amparo à Pesquisa no Estado de São Paulo
Resumo:08-010
5,4’-DIHYDROXY-7-METHOXYFLAVANONE FROM BACCHARIS RETUSA (ASTERACEAE) ATTENUATES
ELASTASE-INDUCES EMPHYSEMA IN MICE.
Medeiros, L. T. P. 1,3; Pinheiro, N. M. 2; Olivo, C. R. 2; Grecco, S. S. 1; Toledo, A. C. 2; Lopes, F. 2; Tibério,
I. F. L. C. 2; Martins, M. A. 2; Lago, J. H. G. 1; Prado, C. M. 1,2
1
2
Universidade São Paulo , USP
3
Universidade Santa Cecília, UNISANTA
Objectives:
COPD with emphysema is characterized by lung tissue inflammation and parenchyma destruction, and one of mechanisms
involved is the physiopathology of this disease is the oxidative stress pathway and persistence of inflammation. It has been shown
that flavonoids present anti-inflammatory and anti-oxidant properties. Our aims was to detect if 5,6,7-trihydroxy-4´-methoxyflavanone (5,6,7-FLA), derived from Baccharis retusa (Asteraceae) attenuates elastase-induced emphysema in C57BL6/J mice.
C57BL-6 male mice (6-8weeks) received intranasal elastase (o.667 UI) or saline on day 0. Both groups received vehicle
(DMSO+Saline) or flavonoid treatment (2mg/Kg/animal-intranasal)one hour after the instillation. This treatment was repeated on
days 14,21 and 28. After 1h of the last treatment, mice were anesthetized and bronchoalveolar lavage (BAL)was collected.
Animals were exsanguinated, lungs were removed and stained with HE, Picrossirius and RFO. We analyzed the total and
dofferential cell count in BAL, mean linear intercept (Lm), and elastic and collagen fibers content in alveolar septa by
morphometry. Emphysema was confirmed in elastase animals by the increase in Lm compared to saline groups (P
Conclusions:
5,4'dihydroxy-7-methoxyflavanone exhibits an anti-inflammatory activity, reducing lung injury and remodeling in a murine
emphysema model, probably related to antioxidant properties of flavonoids.
Keywords: emphysema, flavonoid, lung inflamation, lung remodeling, experimental model
Financial Support: FAPESP, CNPq, FMUSP-LIM20
Resumo:08-011
THE IMPACT OF AEROBIC EXERCISE ON LUNG INFLAMMATION AND REMODELING IN EXPERIMENTAL
EMPHYSEMA
Guimarães, I. H. 1; Padilha, G. A. 1; Lopes-pacheco, M. 1; Marques, P. S. 1; Antunes, M. A. 1; Magalhães,
R. F. 1; Rocha, N. N. 2; Assis, E. F. 3; Xisto, D. G. 1; Rocco, P. R. M. 1
1
Laboratório de Investigação Pulmonar/ IBCCF, UFRJ
2
Universidade Federal Fluminense, UFF
3
Laboratório de Imunofarmacologia/ Instituto Oswaldo Cruz, FIOCRUZ
Objectives:
This study investigated the impact of aerobic exercise on lung inflammation and remodeling in experimental emphysema.
Thirty-two BALB/c (20-25 g) mice were randomly assigned into two groups. In control (C) animals, saline was intratracheally
(it) injected (50 µl), whereas emphysema mice received porcine pancreatic elastase (emphysema, 0.1 UI, 50 µl, it). Saline and
elastase were intratracheally injected once a week during 4 weeks. After the last week, C and emphysema groups were further
randomized into sedentary and exercise groups. Exercise protocol was conducted 3 times a week for 4 weeks. Animals ran on a
motorized treadmill, at moderate intensity (8-12 m.min-1), 5% grade, 30 min/day. Twenty-four hours after the last session, lung
mechanics and morphometry, as well as total and differential cell count, cytokines and chemokines in bronchoalveolar lavage
fluid (BALF) and blood were measured. Echocardiographic analysis was done before and after the induction of emphysema (5
weeks), and at the end of the experiment (9 weeks). Sedentary emphysema group presented, compared to C: 1) reduced lung
static elastance (28%) 2) increased lung hyperinflation (80%), alveolar collapse (40%), as well as elastic fiber content; 3) elevated
levels of KC [murine interleukin (IL)-8 homolog], tumour necrosis factor (TNF)-alpha, interferon (INF)-gamma, IL-4 and IL-10;
and 4) pulmonary arterial hypertension, evidenced by increased pulmonary flow acceleration. Aerobic exercise: 1) improved lung
mechanics, 2) reduced the fraction area of hyperinflation and alveolar collapse, as well as the levels of these mediators, 3)
increased the total number of cells in the bronchoalveolar lavage fluid and blood due to an augment in mononuclear cells, 4)
diminished elastic fiber content, and 5) restored the pulmonary flow acceleration to control values.
Conclusions:
In the present elastase-induced emphysema model, four weeks of aerobic exercise modulated the inflammatory process and acted
on lung remodelling, improving pulmonary morphofunction.
Keywords: emphysema, physical exercise, lung remodeling , inflammation
Financial Support: CNPq, CAPES, INCT-INOFAR, FAPERJ, PRONEX
Resumo:08-012
THERAPEUTIC EFFECTS LASSBIO596 THROUGH ORAL ROUTE OF ADMINISTRATION ON AIRWAY AND
LUNG PARENCHYMA REMODELING IN A MURINE MODEL OF CHRONIC ALLERGIC ASTHMA.
Siqueira, R. E. B. 1; Silva, J. D. 1; Abreu, S. C. 1; Samary, C. S. 1; Silva, A. L. 1; Padilha, G. A. 1; Lima, L.
M. 1; Jesus-barreiro, E. 1; Capelozzi, V. L. 2; Rocco, P. R. M. 1
1
Laboratório de Investigação Pulmonar - IBCCF, UFRJ
2
Departamento de Patologia , USP
Objectives:
Both proximal and distal airways are implicated in the pathophysiology of asthma. Although inflammatory cell infiltrate and
activation appear similar in proximal and distal airways in most cases of asthma, both the magnitude of inflammation and
activation of inflammatory cells appear to be more important in distal airways in severe and uncontrolled asthma. However, distal
airways were poorly attained by conventional aerosol of asthma medications, and thus, studies have stimulated the discovery of
novel molecules through the oral route. The present study investigated the efficacy of LASSBio596, which exhibits potent
inhibitory effects on TNF- and TGF-, through the oral route, on airway and lung parenchyma remodeling in a murine model of
chronic allergic asthma.
Forty-eight BALB/c mice were randomly assigned to four groups. In the asthma group, mice were immunized by intraperitoneal
injection of ovalbumin (OVA, 10 µg) on each of 7 alternate days. After day 40, they were challenged with 3 intratracheal
instillations of ovalbumin (20 µg) at 3-day intervals. Controls (C) received saline using the same protocol. Twenty-four hours
after the last challenge, animals were further randomized into 2 subgroups to receive orally either LASSBio596 (10 mg/kg) or
saline (SAL, 0.05 ml) for 8 consecutive days. After seven days, lung mechanics and histology (light and electron microscopy)
were evaluated. OVA-SAL group presented higher airway resistance (57%), viscoelastic pressure (50%), static elastance (52%),
alveolar collapse (46%), neutrophil and eosinophil infiltration, bronchoconstriction index, collagen and elastic fiber content in
airways and lung parenchyma, and smooth-muscle actin expression in alveolar duct and respiratory bronchiole higher than CSAL. The oral therapy with LASSBio596 resulted in a reduction in the inflammatory (total and differential number of total cells
in lung tissue) and remodeling (subepithelial fibrosis, mucus metaplasia, myocyte hyperplasia and hypertrophy, and epithelial
hypertrophy) processes resulting in a decrease in airway resistance (40%), viscoelastic pressure (30%), static elastance (40%) and
airway hyperresponsiveness.
Conclusions:
LASSBio596 through oral route reduced the inflammatory and remodeling as well as acted on both proximal and distal airways
improving lung mechanics.
Keywords: remodeling, inflammation, chronic allergic asthma, treatment, respiratory function
Financial Support: INCT-INOFAR, CAPES, PRONEX, CNPq, FAPERJ
Resumo:08-013
BUFFERING OF HYPERCAPNIC ACIDOSIS MODULATES INFLAMMATORY PROCESS ON LUNG AND DISTAL
ORGANS IN EXPERIMENTAL ACUTE LUNG INJURY
Garcia, C. S. N. B. 1; Nardelli, L. 1; Rzezinski, A. 1; Silva, J. D. 1; Maron-gutierrez, T. 1,2; Ornellas, D. D. S.
1,2
; Capelozzi, V. L. 3; Morales, M. M. 2; Pelosi, P. 4; Rocco, P. R. M. 1
4
Department of Surgical Sciences and Integrated Diagnostics, University of Genoa
1
Laboratory of Pulmonary Investigation, IBCCF, UFRJ
2
Laboratory of Cellular and Molecular Physiology, IBCCF, UFRJ
3
Department of Pathology, School of Medicine, USP
Objectives:
Protective effects of hypercapnic acidosis (HCA) as well as buffering HCA on lung and distal organs are controversial. We
investigated the effects of inspired carbon dioxide and buffering HCA on lung, kidney, liver, and heart in experimental acute lung
injury (ALI).
Paraquat-induced ALI rats (n=21) were studied. At 24h, animals were anesthetized and mechanically ventilated (VT=8ml/kg and
PEEP=5cmH2O). ALI groups were further randomized into 3 subgroups: (1) normocapnia(NC, PaCO2=35-45mmHg): ventilated
with a gas mixture of 0.03%CO2+ 21%O2+balanced N2; (2) Hypercapnia (HC,PaCO2=60-70 mmHg): gas mixture of
5%CO2+21% O2+balanced N2; and (3) Buffered Hypercapnia (BH): ventilated similar to HC, but treated with sodium
bicarbonate. After 1h mechanical ventilation, lung mechanics, lung and distal organs histology, gas-exchange, and interleukin
(IL)-1β, IL-6, caspase-3 and type III procollagen (PCIII) mRNA expressions (RT-PCR) in lung tissue were analyzed. Lung
mechanics were similar in all ALI groups. ALI-HC group showed higher liver and kidney cell apoptosis, increased PCIII (37%)
and caspase-3 (13%) mRNA expressions, and lower IL-6 (31%) and IL-1β (37%) compared to NC. Conversely, in ALI-BH, cell
apoptosis in lung, diaphragm, liver and kidney and PCIII (53%) and caspase-3 (48%) mRNA expressions were significantly
reduced and IL-6 (44%) and IL-1β (41%) mRNA expressions presented a further decrease in relation to ALI-HC.
Conclusions:
In the present ALI model, hypercapnic acidosis increased lung and distal organs cell apoptosis and type III procollagen and
caspase-3 mRNA expressions while attenuating IL-6 and IL1-β mRNA expression in lung tissue. However, buffering
hypercapnia reduced lung and distal organ cell apoptosis and mRNA expression in lung tissue of some inflammatory and
fibrogenic mediators.
Keywords: mechanical ventilation, cytokines, type III procollagen, acute lung injury, hypercapnic acidosis
Financial Support: CNPq, PRONEX-FAPERJ, FAPERJ, CAPES
Resumo:08-014
TREATMENT WITH LASSBIO 596 CAN ATTENUATE PULMONARY FUNCTIONAL IMPAIRMENT IN MICE
EXPOSED TO CYLINDROSPERMOPSIN
Avila, M. B. 1; Oliveira, V. R. 1; Carvalho, G. M. C. 1; Alves, J. L. C. R. 1; Soares, R. M. 1; Azevedo, S. M.
O. 1; Lima, L. M. 2; Barreiro, E. J. 2; Carvalho, A. R. 1; Zin, W. A. 1
1
Carlos Chagas Filho Institute of Biophysics/UFRJ, IBCCF/UFRJ
2
Faculty of Pharmacy/Federal University of Rio de Janeiro, FF/UFRJ
Objectives:
Cyanobacteria blooms can release toxins, such as cylindrospermopsin, into water supplies. These toxins are potentially harmful to
human health. LASSBio 596 is a new drug candidate with an anti-inflammatory profile. We aimed at studying the therapeutic
potential of LASSBio 596 to treat lung injury in mice intratracheally instilled with cylindrospermopsin.
: Female BALB/c mice (20-26 g) were submitted to a single intratracheal instillation of 50 μL of saline solution (NaCl 0.9%) or
semi-purified extract of cylindrospermopsin (70 μg/kg). 18 hours after exposure the animals that received saline were treated by
gavage with saline (SAL group, n=5) or 200 mg/kg of LASSBio 596 diluted in saline (SLB group, n=4) while the animals that
received cylindrospermopsin were treated with saline (TOX group, n=6) or 200 mg/kg of LASSBio 596 (TLB group, n=7).
Pulmonary mechanics (resistive pressure, ΔP1; viscoelastic/inhomogenous pressure, ΔP2; pulmonary total pressure, ΔPtot; static
elastance, Est; and the elastic component of viscoelasticity, ΔE) was measured 6 hours after the treatment. Two-way Analysis of
Variance was used (p < 0.05). We observed a significant increase in Est, ΔE, and ΔP2 in TOX group (35.7 ± 2.5, 6.0 ± 0.3
cmH2O/mL, and 1.2 ± 0.1 cmH2O, respectively) compared to SAL group (25.9 ± 1.2, 4.6 ± 0.2 cmH2O/mL and 0.9 ± 0.1
cmH2O, respectively). TLB group showed smaller mechanical parameters (25.6 ± 1.1, 5.1 ± 0.4 cmH2O/mL and 1.0 ± 0.1
cmH2O, Est, ΔE e ΔP2, respectively) than TOX group, but did not differ from SAL mice. The SLB group also was similar to
SAL group in all parameters (24.45 ±1.01, 4.83±0.66 cmH2O/mL, 0.57±0.04, 0.98±0.14 and 1.55±0.14 cmH2O; Est, ΔE, ΔP1,
ΔP2 and ΔPtot, respectively).
Conclusions:
Cylindrospermopsin altered pulmonary elastic and viscoelastic/inhomogenous components, suggesting a lesion located at the
lung distal airways/alveoli. Moreover, LASSBio 596 reversed the mechanical impairment produced by the acute exposure to
cylindrospermopsin.
Keywords: Cyanobacteria , lung injury, inflammation, lung mechanics
Financial Support: CNPq, FAPERJ, MCT
Resumo:08-016
WHICH IS THE BEST DOSE OF MESENCHYMAL STEM CELLS TO TREAT CHRONIC ALLERGIC ASTHMA?
Barcelos, P. R. S. 1; Abreu, S. C. 1; Pacheco, M. L. 2; Morales, M. M. 2; Rocco, P. R. M. 1; Xisto, D. G. 1,2
1
1Laboratory of Pulmonary Investigation - IBCCF, UFRJ
2
Laboratory of Cellular and Molecular Physiology - IBCCF, UFRJ
Objectives:
Even though there are many studies analyzing the effects of mesenchymal stem cells (MSCs) in respiratory diseases, so far, no
report investigated the best dose of MSCs to provide lung mechanical improvement in experimental chronic allergic asthma. The
present study aimed to analyze the impact of three different doses of MSCs on lung mechanics and morphometry in experimental
chronic allergic asthma.
25 female C57BL/6 mice were randomly assigned into five groups. In the OVA group, mice were sensitized and challenged with
ovalbumin while control group (C) received saline using the same protocol. OVA group were further randomized into subgroups
receiving three different doses of MSCs (10 3, 104, 105 intravenously) 24 hours after the last challenge. Airway resistance,
viscoelastic pressure, and static elastance were analyzed by end-inflation occlusion method. Lungs were then removed en bloc,
fixed and stained with hematoxylin-eosin to quantify the fraction area of alveolar collapse. When the dose of MSCs was 10 5, lung
static elastance (20%), viscoelastic pressure (30%), airway resistance (28%), and fraction area of alveolar collapse minimized
compared to OVA group. Doses of 10 3 and 104 MSCs did not improve lung mechanics and histology.
Conclusions:
In the present model of chronic allergic asthma, the best dose of MSCs to improve lung mechanics and histology was 1x10 5
Keywords: Mesenchymal Stem Cells, Chronic Allergic Asthma , Lung Mechanics
Financial Support: PRONEX, FAPERJ, CAPES, CNPq
Resumo:08-017
THE SPASMOLYTIC EFFECT OF GLUCAGON ON AIRWAY SMOOTH MUSCLE DEPENDS OF NITRIC OXIDE
AND CYCLOOXYGENASE DERIVATES
Insuela, D. B. R. ; Coelho, L. P. ; Cordeiro, R. S. B. ; Martins, M. A. ; Silva, P. M. R. ; Carvalho, V. F.
Laboratório de Inflamação/Instituto Oswaldo Cruz, IOC/FIOCRUZ
Objectives:
Glucagon is a polypeptide hormone that is secreted by the pancreatic α-cells. The main physiological action of glucagon is to
stimulate the synthesis and mobilization of hepatic glucose, carrying in an increase of blood glucose levels, which
counterbalances the actions of insulin. Further, glucagon also plays several effects in extrahepatic tissues, through its receptor
activation. The expression of glucagon receptor was detected in lungs of mice and rats, and the effect of glucagon on
bronchospasm in asthmatic patients is controversy. However, some studies suggested that glucagon may have bronchodilator
effects. In this study, we investigated the effect of glucagon on airway smooth muscle contractility and possible mechanism
involved.
The animals were obtained from Oswaldo Cruz Foundation breeding colony and used in accordance with the guidelines of the
Ethic Committee on Use of Laboratory Animals of the Oswaldo Cruz Foundation (CEUA-FIOCRUZ, License LW – 23/10).
Tracheas of A/J mice were removed and inserted in an isolated organ bath system, and the contractile response to carbachol was
measured in the presence or absence of glucagon (0.1 or 1.0 ìM) in vitro. In some experiments, we previous incubated the
tracheas with glucagon receptor antagonist (des-His1-[Glu9] glucagon amide), NOS inhibitor (L-NAME), COX inhibitor
(indometacin) or HO inhibitor (ZnPP IX), 30 minutes before glucagon treatment in vitro. Moreover, the epithelium of some
tracheas was removed mechanically by rubbing the internal trachea surface with a fine silver wire, before glucagon treatment in
vitro. We also tested the effect of glucagon (0.1 - 10 µg/kg, i.n) on methacholine-evoked airways obstruction in A/J mice using
invasive barometric plethysmography. The treatment was realized 3 hours before the stimulation with methacholine. The
treatment with glucagon inhibited tracheal contraction induced by carbachol stimulation in vitro in a concentration-dependent
manner (from 137.0 ± 3.03 in saline to 107.7 ± 4.38 and 86.4 ± 3.70 emax (% of carbachol 2.5 µM) in glucagon 0.1 and 1 µM,
respectively; mean ± SEM, n = 7). In addition, the intranasal treatment with glucagon at all doses was able to reduce the
bronchoconstriction induced by methacoline in vivo, as attested by measurement of airways resistance and lung elastance. The
glucagon receptor antagonist (des-His1-[Glu9] glucagon amide) was able to inhibit the spasmolytic effect of glucagon in vitro.
Moreover, this effect of glucagon was abrogated by epithelium removal. The pre-incubation with L-NAME or indometacin
inhibited the spasmolytic effect of glucagon in vitro, however this inhibition was not observed in the group of tracheas that were
pre-incubated with ZnPP IX.
Conclusions:
Our results show that glucagon presents a spasmolytic effect on airway smooth muscle in vitro and in vivo, and this action
appears to occur in an indirectly way, through activation of its receptor present in epithelium with consequent release of nitric
oxide and products of COX activation.
Keywords: Airway, Cyclooxygenase, Glucagon, Nitric Oxide, Smooth muscle
Financial Support: CNPq, FAPERJ and FIOCRUZ
Resumo:08-018
EFFECTS OF URBAN POLLUTION IN MICE CHRONICALLY EXPOSED TO CIGARETTE SMOKE
Freire, C. ; Magalhães, C. B. ; Machado, M. N. ; Zin, W. A.
Instituto de Biofísica Carlos Chagas Filho, IBCCF
Objectives:
Cigarette smoking is responsible for 95% of cases of chronic obstructive pulmonary disease. Epidemiological studies show an
association between pollution and morbidity and mortality from lung disease. We evaluated the effects of exposure to total
suspended particulate (TSP) from the city of São Paulo in animals chronically exposed to cigarette smoke.
C57BL/6 (n = 21) mice were exposed to air or light cigarette smoke (4 cigarettes/day, 5 days/week for 2 months) in a 10x15x20
cm chamber. The animals received "puffs" of smoke alternating 60-s exposures to smoke with 60 s of ambient air. In the last
week of exposure the mice received a 15-mL intranasal instillation of saline (CTRL and FUM groups) or 15 mg of TSP dissolved
in 15 mL of saline (FUM-TSP and TSP groups). 72 h after the last exposure we measured pulmonary mechanics, i.e., pressures
(cmH2O): resistive (ΔP1), viscoelastic (ΔP2) and total (ΔPtot), elastance (Est) and the viscoelastic component of elastance (ΔE,
cmH2O/mL). At the end of the experiment, the functional residual capacity (FRC) was determined by volume displacement and
the lungs were prepared for histology. TSP and FUM-TSP showed higher Est (34.1 ± 3.5 and 40.6 ± 5.2), ΔE (6.2 ± 1.4 and 5.4 ±
0.8) and ΔP2 (1.2 ± 0.2 and 1.1 ± 0.1) than CTRL (25.8 ± 1.5, 3.5 ± 0.4 and 0.7 ± 0.1, respectively), Est being significantly
higher in FUM-TSP than in TSP and FUM. FUM and CTRL presented similar values of Est, ΔE and ΔP2. There was no
significant change in FRC between animals exposed to cigarette smoke versus CTRL and TSP. Histologically, the percentage of
collapse was significantly greater in TSP, FUM and FUM-TSP (25.7 ± 4.1, 33.3 ± 3.1 and 37.0 ± 1.9 %, respectively) than in
CTRL (5.2 ± 2.6 %). Collapse in FUM-TSP (37.0 ± 1.9 %) was greater than in TSP (25.7 ± 4.1 %); however FUM-TSP and
FUM did not differ (37.0 ± 1.9 and 33.3 ± 3.1 %). Animals exposed to cigarette smoke lost weight during the 2-mo experimental
period.
Conclusions:
The association between exposure to particulate matter and cigarette smoke exacerbated the impairment of lung function,
determined by increased Est, thus indicating deterioration of the elastic component. Pulmonary morphometry showed an increase
in alveolar collapse in animals exposed to cigarette smoke.
Keywords: cigarrete, pollution, pulmonary mechanics
Financial Support: PRONEX, FAPERJ, CNPq, MCT
Resumo:08-019
THE INVOLVEMENT OF PURINERGIC P2X7 RECEPTORS IN MURINE SILICOSIS.
Monção-ribeiro, L. C. 1,2; Santana, P. T. 1,2; Borojevic, R. 2; Takiya, C. M. 1; Coutinho-silva, R. 1
1
2
Instituto de Ciências Biomédicas, UFRJ
Objectives:
P2X7 receptor (P2X7R) activation is involved in a number of proinflammatory responses in macrophages and other immune
cells. Silicosis is an occupational lung disease resulting from the inhalation of silica particles over prolonged periods of time,
which causes chronic inflammation and progressive nodular pulmonary fibrosis. The aim of this study was to characterize the
importance of P2X7 activation in lung inflammatory response of silicotic animals.
C57BL/6 wild-type and P2X7R knockout mice were intratracheally instillated with silica (20mg-50μL/animal) (WT-SIL/KOSIL) or saline (50 μL-CTRL-WT/CTRL-KO). After 14 days of silica instillation, the animals were sacrificed and lungs collected
for histological analysis, immunohistochemistry and histomorphometry. The silica instillation increased significantly the P2X7R
immunoexpression in WT animals. The immunoreactivity was presented in inflammatory cells mostly, bronchiolar cells, and
alveolar epithelial cells. Animals WT-SIL and KO-SIL showed alveolitis and formation of nodular structures. However, there
was a significant decrease in the number of inflammatory cells (mononuclear cells, and polymorphonuclear cells) both in the in
nodules in animals KO-SIL (222±3 and 197±2 respectively) when compared to WT-SIL animals (389±3and 279±3 respectively).
Silica particles quantification in the lungs was lower in KO-SIL (874±2) animals when compared with WT-SIL animals
(1005±4). Collagen fibers amount was similar between all genotypes. The nodular area was significantly diminished in KO-SIL
animals (9894±27) when compared to WT-SIL animals (20254±155). The KO-SIL animals exhibited a significant decrease in
iNOS and NFkB/p65 immunoreactivity. The immunoreactivity for p-smad2/3 (19±6) (TGF-β-signaling activator) was diminished
when compared with WT-SIL animals (32±9) . The immunoreactivity for FAS (CD95) was incresead in KO-SIL animals when
compared with all others groups.
Conclusions:
Therefore, the P2X7 receptor appears to modulate the pulmonary inflammatory response in experimental silicosis.
Keywords: Silicosis, Purinergic Receptors, P2X7 Receptor, Lung Fibrosis
Financial Support: FAPERJ-CNPQ-PRONEX
Resumo:08-020
IMPACT OF AUTOLOGOUS BONE MARROW DERIVED MONONUCLEAR CELL TRANSPLANTATION IN
EXPERIMENTAL PULMONARY AND EXTRAPULMONARY ACUTE LUNG INJURY.
Miranda, G. F. 1; Silva, J. D. 1; Araújo, I. M. 1; Lopes-pacheco, M. 2; Oliveira, M. V. 1; Morales, M. M. 2;
Rocco, P. R. M. 1
1
2
Laboratório de Investigação Pulmonar - IBCCF, UFRJ
Laboratório de Fisiologia Celular e Molecular - IBCCF, UFRJ
Objectives:
The idea of treating acute lung injury (ALI) using autologous adult stem cells is very attractive and also helps prevent
immunological rejection by the host. The goal of this transplantation is to transfuse healthy bone marrow cells. However, a severe
obstacle to the clinical application of bone marrow derived mononuclear cell transplantation is the possibility of altering adult
stem cells ex vivo due to ALI before their autologous delivery. The aim of this study was to investigate the effects of bone
marrow derived mononuclear cells (BMDMCs) originated from healthy and acute lung injury animals on lung mechanics and
histology in experimental pulmonary and extrapulmonary acute lung injury presenting similar lung morpho-function.
Fourty-two female C57BL/6 mice were randomly assigned into three groups. In control group (C), sterile saline solution was
intratracheally (0.05 mL) or intraperitoneally (0.05 mL) injected. Acute lung injury mice received Escherichia coli
lipopolysaccharide (LPS) intratracheally (40 µg, ALIp) or intraperitoneally (400 µg, ALIexp). Twenty-four hours after saline or
LPS administration, 5x106 whole bone marrow cells obtained from C, ALIp and ALIexp donors were intravenously injected. At
day 7, lung mechanics and histology were measured. ALIp and ALIexp animals presented increased static lung elastance (25%)
as well as resistive (20%) and viscoelastic (30%) pressures. BMDMCs originated from C, ALIp and ALIexp animals led to a
reduction in lung static elastance (20%), resistive (20%) and viscoelastic pressures (23%) and reduced the fraction area of
alveolar collapse but this decrease was greater with BMDMCs originated from ALI animals (p < 0,05).
Conclusions:
In the present LPS-induced ALI model, BMDMC therapy originated from ALI animals was more effective in attenuating lung
mechanical and histological changes than those from C mice.
Keywords: cell therapy, acute lung injury, bone marrow, inflammation, lung function
Financial Support: CAPES, IM-INOFAR, PRONEX, CNPq, FAPERJ
Resumo:08-021
TEMPORAL ANALYSIS OF OXIDATIVE EFFECTS ON PULMONARY INFLAMMATORY RESPONSE IN MICE
EXPOSED TO CIGARETTE SMOKE
Gonçalves, E. G. 1; Dourado, V. A. 1; Ferreira, B. S. B. 1; Lopes, A. D. A. 3; Lanzetti, M. 3; Silva, M. E. 2;
Valença, S. S. 3; Lima, W. G. 1; Costa, D. C. 1; Bezerra, F. S. 1
1
Departamento de Ciências Biológicas (DECBI), UFOP
2
Escola de Nutrição (ENUT), UFOP
3
Instituto de Ciências Biomédicas (ICB), UFRJ
Objectives:
This study aimed to analyze and compare the influx of inflammatory cells into the lung parenchyma in animals exposed to
cigarette smoke, as well as investigate the oxidative damage in the form of lipid peroxidation in lung extracts and the activity of
antioxidant enzymes: Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT).
We used 36 C57BL / 6 mice, 8 weeks, divided into groups of six animals exposed to cigarette smoke for 1 (GF1D), 2 (GF2D), 3
(GF3D), 4 (GF4D) and 5 (GF5D) days respectively, 3 times a day and the control group (CG) was exposed to air. Each cigarette
was attached to a plastic syringe of 60 ml, which is inflated after the smoke was expelled in the inhalation chamber 40 cm long,
30 cm wide and 25 cm high. Each cigarette produces about 1 liter of smoke diluted with 30 liters of ambient air present in the
chamber, the final concentration of 3%. After euthanasia by cervical dislocation, were collected bronchoalveolar lavage fluid
(BALF) through the tracheostomy, the peripheral blood by cardiac puncture and lung tissue removed through an incision in the
anterior chest wall for analysis of thiobarbituric acid reactive substances (TBARS ) and activity of SOD, GPx and CAT. There
was an increase in total leukocytes in GF2D groups (1.35 ± 0.53x10 5 cels/ml) GF3D (2.33 ± 0.33x105 cels/ml) GF4D (3.05 ±
0.29x105 cels/ml) and GF5D (3.4 ± 0.39x105 cels/ml) compared to control (0.31 ± 0.09x105 cels/ml) (p 4 cels/ml) GF4D (7.2 ±
0.23x104 cels/ml) and GF5D (9.3 ± 0.883x104 cels/ml) compared to the control group (2.6 ± 0.43x10 4 cels/ml) (p -1) x 10-5],
GF2D [182.3 ± 14.25 (mM / min / mg ptn-1) x 10-5] compared to the control group [115.0 ± 4.509 (mM / min / mg ptn -1) x 10-5]
(p=0,0001) was increased. well as the increased activity of catalase in GF3D groups (24.71 ± 5.923 U / mg ptn) GF4D (23.96 ±
3.524 U / mg ptn) compared to GC (12.31 ± 3.125 U / mg ptn) (p = 0.0001).
Conclusions:
There was a growing influx of inflammatory cells in bronchoalveolar lavage, blood periferies as well as oxidative damage and an
increased activity of antioxidant enzymes in animals exposed to cigarette smoke. Further studies are necessary to assess the
cellular profile of this experimental model.
Keywords: Temporal analysis , oxidative stress, lung, cigarette smoke, inflammatory
Financial Support: CNPQ,UFOP
Resumo:09-001
EUGENOL BLOCKS HOMOMERIC KV3.1 IN L929 CELLS
Carvalho-de-souza, J. L. ; Cassola, A. C.
Fisiologia e Biofísica/ICB, USP
Objectives:
Eugenol (EUG) is a low molecular weight terpenoid used in dentistry as analgesic and antimicrobian agent. Previous studies
stated that EUG is a voltage-gated Ca2+ channels blocker. Recently, we demonstrated that EUG interacts with native voltagegated Na+ channels expressed in sensory neurons from rats, blocking the Na+ currents passing through them in a dose-dependent
manner. As those neurons express many types of Na+ channels the analysis of the blockade mechanism was limited. Beside this,
the attempt to simulate the drug-protein interactions by physicochemical models was limited by the fast inactivation process of
the Na+ currents. Therefore, we have initiated a study with a cell lineage (L929, a murine fibrosarcoma cell line) with a stable
expression of KV3.1. These voltage activated K+ channels are slowly inactivated and the study of EUG effects on this protein
can disclose aspects of the channel-drug interaction.
We have used the patch clamp technique to record a macroscopic K+ current in control conditions and after applying EUG. Until
the time of the experiments, the L929 cells were kept in a MEM/EBSS supplemented with 10% fetal horse serum in a water
jacket incubator with 5% CO2 atmosphere and 80% humidity. The K+ currents expressed by these cells were characterized by a
I-V relationship, which was typical for voltage activated K+ channels. EUG was applied at 2, 4, 5 and 6 mM which reduced the
peak of K+ currents to 69.3 ± 4.89 (n=17), 78.0 ± 2.60 (n=21), 64.9 ± 5.11 (n=18) and 83.9 ± 1.61 % (n=7) of control,
respectively. Tetraethylamonium Chloride 20 mM was used as a standard K+ channels blocker and blocked the currents to 10.4 ±
2.48 % (n=19) of control values.
Conclusions:
Heretofore we can state that EUG reversibly interacts with K+ channels formed by KV3.1, blocking the channel. Presently our
results are not sufficient for a full definition of the dose-effect relationship. Therefore, we are carry out new experiments to
elucidate this question and to advance toward the simulation trials.
Keywords: eugenol, K+ channels, L929 cells, patch clamp, K+ currents
Resumo:09-002
NANOBIOTECHNOLOGY APPROACH AGAINST LEISHMANIA AMAZONENSIS INFECTION: GPI-ANCHORED
PROTEINS ASSOCIATED WITH LIPOSOMES PARTIALLY PROTECTS BALB/C MICE
Colhone, M. C. 1; Jardim, I. S. 3; Stabeli, R. G. 2,3; Ciancaglini, P. 1
1
Depto. Química/ FFCLRP, USP
3
Fundação Oswaldo Cruz do Noroeste do Brasil, FIOCRUZ / IPEPATRO
2
Núcleo de Saúde (NUSAU) / UNIR, UNIR
Objectives:
The reconstitution of membrane proteins into liposomes is a powerful tool to prepare antigenic components to induce immunity
effects. Considering the properties of liposomes and the proteins antigenicity of the Leishmania membrane, the main goal of this
work was to evaluate if liposomes carrying GPI-anchored proteins of Leishmania amazonensis promastigotes could induce
protective immunity against this parasite infection in BALB/c mice model.
To assay protective immunity, five BALB/c mice per group (male, 8-12 weeks old and 19-22g) were injected intraperitoneally
with a buffer, liposomes, Triton X-114 solubilized GPI-protein extract (EPS-GPI) as controls and the proteoliposomes previously
GPI-proteins prepared and three weeks later, were challenged in the footpad with 10 4 L. amazonensis promastigotes. The
protective immunity was evaluated weekly for up to 14 weeks by measuring the size of lesions caused by the parasite. BALB/c
mice inoculated with 0.120 mg (total protein) in constitutive proteoliposomes showed a protection of about 90%, as compared to
mice that received only the buffering solution as control. Also, mice immunized with 0.010 or 0.020 mg of GPI-solubilized total
protein extract (EPS-GPI) displayed a moderate protection of about 70% post-infection. In another essay, murine peritoneal
macrophages (1 × 10 6 cells/ mL) (n=3) stimulated with liposomes (0.05mL), proteoliposomes (0.001 mg) and EPS-GPI (0.005
mg) for 20 h and subsequently infected with amastigotes of L. amazonensis (3:1 parasite: cell), showed a reduction in the
percentage of infected cells (42, 39 and 99%, respectively) and in the number of intracellular parasites per cell (29, 32 and 99%,
respectively). Moreover, studies of cellular viability of murine peritoneal macrophages show that our system does not present
cytotoxic effect for the macrophages and still stimulates their nitric oxide (NO) production, in which EPS-GPI had the highest
NO production (0.042 µ M) when compared to control cultures (0.015 µ M), that had received no stimuli.
Conclusions:
Thus, our results indicated that either EPS-GPI or proteoliposomes may be used to induce protective immunity against L.
amazonensis in mice. These systems seem to be a promising vaccine system for immunization against leishmaniasis.
Keywords: vaccine, nanobiotechnology, Liposomes, GPI-proteins, Leishmania amazonensis
Financial Support: CAPES, CNPq and FAPESP.
Resumo:09-003
INFLUENCE OF CHOLESTEROL ON THE THERMAL STABILITY AND ENZYMATIC ACTIVITY OF NA,KATPASE RECONSTITUTED IN DPPC:DPPE-LIPOSOMES
Yoneda, J. S. ; Rigos, C. F. ; Ciancaglini, P.
Depto de Química / FFCLRP, USP
Objectives:
The objective of the study was to verify the influence of different lipidic microenvironment in the denaturation and enzymatic
activity of Na,K-ATPase.
The (αβ)2 form of Na,K-ATPase was purified as previously standardized in our laboratory (Braz. J. Med. Biol. Res. 35: 277-288,
2002). Liposomes of DPPC, DPPE and cholesterol were prepared by the extrusion method. The lipids were dissolved in
chloroform:methanol (2:1) and dried with nitrogen flow, forming a lipid film, and then dried overnight under vacuum. Then it
was re-suspended in 5 mM Tris-HCl buffer, pH 7.0, containing 1 mM EDTA, 150 mM KCl, by incubation for 1 h, at 70 °C and
stirred using a vortex at 10 min intervals. The mixture was extruded (11 times) through a 100 nm polycarbonate membrane under
hot air flow. Na,K-ATPase was reconstituted in liposome system by the co-solubilization method as described in Colloids Surf B:
Biointerfaces 41: 239-248 (2005) using DPPC, DPPE with different proportions of cholesterol (0, 10, 20, 30 and 40 mol %).
ATPase activity was assayed discontinuously at 37 °C. DSC experiments were performed using a Nano-DSC II - Calorimetry
Sciences Corporation, CSC (Lindon, Utah, USA); all samples were degassed under vacuum for 30 min prior to use, and scanned
from 10 to 100 °C at an average heating and cooling rate of 0.5 °C/min, under 3 atm pressure. The addition of cholesterol to an
equimolar mixture of DPPC and DPPE promoted the gradual increase of the main phase transition temperature and the
broadening of the peaks of liposome. The thermal profile of the solubilized protein exhibited three peaks between 50 and 70 °C.
For proteoliposome systems three peaks were also observed; however, the transition temperatures were altered as well as the
enthalpy variation values. Moreover, the presence of cholesterol in the proteoliposome preparation resulted in reconstitution with
slightly higher protein recovery and with a maximum hydrolytic activity at 20 mol % cholesterol.
Conclusions:
We can propose that cholesterol has an influence on packing and fluidity of lipid bilayers and, changes in lipid microenvironment
alters the thermostability as well as enzymatic activity of Na,K-ATPase.
Keywords: Cholesterol, Liposome, Na,K-ATPase, Proteoliposome, Thermal Stability
Financial Support: CNPq, CAPES AND FAPESP
Resumo:09-004
POSSIBLE CORRELATION BETWEEN THE P2X7-ASSOCIATED PORE AND THE PORE OPENED BY
CAPSAICIN
Braga, L. ; Faria, R. X. ; Alves, L. A.
FUNDAÇÃO OSWALDO CRUZ / LABORATÓRIO DE COMUNICAÇÃO CELULAR, FIOCRUZ
Objectives:
The P2X7 receptor (P2X7R) belongs to P2X family which are ionotropic ligand-gated channels, having seven subtypes cloned up
to now (P2X1-7). The P2X7R is an intriguing receptor which posses two conductance states: a low conductance ion channel (±10
pS) and high conductance non-selective pore formation that is activated by high ATP concentrations (100 uM) allowing the
passage of molecules up to 900Da. To our knowledge, there is a lack on the literature regarding the mechanism of the pore
formation. There are two main hypotheses that might explain the pore formation event: 1) the P2X7R dilates itself or 2) a distinct
protein is responsible for the formation of the high conductance channel (non-selective pore). Based on the latter, the Transient
Receptor Potential Vanilloid subtype 1 (TRPV1) might be a putative pore candidate. The TRPV1 is the most studied member of
the TRP channels and despite of being predominantly distributed on the central nerve system it is also found in immune system.
This receptor is a non-selective cationic channel that is activated by polimodal stimuli such as high temperature, tact and
osmolarity. The pungent compound found in Capsicum sp, Capsaicin, its agonist which one promotes the activation of the
TRPV1 and non-selective pore. Both pores described above have similar biophysical and functional characteristics. In this regard,
in this present study we evaluated a possible correlation between the P2X7-associated pore and the capsaicin activated pore.
Primary mouse macrophage cells were extracted from peritoneal cavity of 4-week-old Swiss webster. Electrophysiology
experiments were developed using cell-attached and whole-cell patch clamping configurations, flow cytometry, cellular
permeabilization as well as microfluorimetry assays. Capsaicin induced ionic current and dye uptake similar to the current
observed in the pore formation associated to P2X7R. This event was blocked by a general TRP antagonist, Ruthenium Red (RU2). In addition, we observed that the P2X7R antagonist, Brilliant Blue B (BBG) inhibited the pore activated by Capsaicin. The
reverse effect also was observed, when the RU-2 blocked the pore formation activated by ATP via P2X7R activation.
Conclusions:
Our data suggests that there is a correlation between the P2X7-associated pore formation and the one activated by capsaicin. This
is an interesting results, because both of the described receptors are related to participate in the pro-inflammatory cytokines
release and are involved in several pathologic pathways as ischemic tissue injury progressing and death.
Keywords: P2X7 RECEPTOR, NON-SELECTIVE PORE, ATP, TRPV1, CAPSAICIN
Financial Support: Instituto Oswaldo Cruz, Faperj, CNPq
Resumo:09-005
SPECIFIC ANION EFFECT ON DNA TRANSLOCATION THROUGH PROTEIN NANOPORE
Brito, A. M. S. ; Machado, D. C. ; Rodrigues, C. G. ; Krasilnikov, O. V.
Departamento de Biofísica e Radiobiologia, UFPE
Objectives:
In all currently used methodologies nucleic acids need to be marked and/or amplified before sequencing. The use of nanopore
technology (that is believed to be a base of a new generation sequencers) does not demand amplification and/or labeling. Unitary
α-hemolysin nanopore in planar lipid bilayers is supposed to be a key element of such sequencers where the “reading” of the
nucleotide sequence will occur during electrophoretically driving translocation of single-stranded DNA (ssDNA) through the
pore (Nanoscale, 2:468-483, 2010). However, the extremely high speed of ssDNA-translocation prevents a secure identification
of nucleotides by this type of sequencer. The main objective of this study was to evaluate the influence of different anions on
translocation speed of ssDNA molecules.
Planar lipid bilayer membranes of 40 pF capacitance were formed by the lipid monolayer apposition technique (Proc Nat Acad
Sci USA, 69:3561-3566, 1972), using 1,2-diphytanoyl-sn-glycero-3-phosphocholine. S. aureus α-hemolysin was added from the
cis side of the membrane in a concentration sufficient to form unitary protein nanopore in planar lipid membranes. The
experiments were carried out at voltage clamp at room temperature of 25±1•C. The membrane-bathing aqueous solution
contained 4 M KCl or KF (both 99.99% purity) in 5 mM Tris-citric acid buffer (pH 7.5). Poly-dA 50b ssDNA was used as a
probe added in the cis compartment of the experimental chamber. As expected, the residence time (τoff) of the DNA inside of the
pore was found to decrease with voltage. We have established the values of τoff depended strongly on the salt species
demonstrating the specific (Hofmeister) effect. τoff was found to be significantly larger (22±2 times) in KF solution in compare
with KCl. So a simple change in ion composition leads to considerable, more than one order of magnitude, improving the
detection and analysis of nucleic acids.
Conclusions:
Modification of ion composition is the simple maneuvers considerably improves the resolution of kinetic parameters of DNA/αhemolysin nanopore interaction and could be helpful for developing of nanopore based new generation DNA sequencers.
Keywords: alfa-hemolysin, DNA sequencing, Hofmeister, protein nanopore
Financial Support: CNPq, FACEPE, INAMI
Resumo:09-006
EVALUATION OF THE BONE REPAIR IN CRITICAL SIZED DEFECTS GRAFTED WITH HYDROXYAPATITE
AND COLLAGEN MEMBRANE COMBINED WITH LASER THERAPY IN RATS.
Figueira, L. A. ; Ribeiro, D. C. P. ; Cunha, M. R.
Faculdade de Medicina de Jundiaí, FMJ
Objectives:
The implantation of autogenous grafts may be necessary in cases of bone mass loss due to high impact trauma. The disadvantages
of the latter approach include morbidity of the donor area. Biomaterials represent an alternative for bone restoration. The most
widely used compounds are collagen or hydroxyapatite membranes because of their biocompatibility and osteoconductivity.
Laser therapy has been applied in combination with these implants to accelerate bone regeneration. The objective of this study
was to evaluate the effects of low-power laser therapy on the healing of rat left tibial bone defects filled with hydroxyapatite or
collagen membrane.
Twenty male rats (Rattus norvegicus), with approximately 12 weeks and weight between 350-380g, were used. Surgical bone
defects were created in the proximal third of the left tibia, with 2 mm thickness, and the animals were divided into four groups
according to treatment: animals receiving hydroxyapatite implants (group H), animals receiving collagen implants (group C),
animals treated with hydroxyapatite plus laser therapy (group HL), and animals treated with collagen membrane plus laser
therapy (group CL). Was used Gallium arsenide laser an intensity of 5,0 J/cm2, with wavelength of 940nm, by 02 months, 03
times in the week. The time of each application was 1 min and 55 sec. The animals were sacrificed 8 weeks after surgery and
samples were obtained for analysis. Stereology was used to quantify the volume of newly formed bone in the recipient area
according to the principle of Delesse. Data were analyzed by ANOVA (p0.05) in the volume of newly formed bone in the
recipient area between the groups studied.
Conclusions:
The hydroxyapatite and collagen membrane used in this research stimulated the new bone formation in the recipient area adjacent
to the implants, confirming the osteoconductivity of these biomaterials even without total closure of bone defects. Moreover, no
significant osteogenic stimulus was observed using these laser parameters in the advanced process of bone regeneration. Thus,
the osteogenic function of laser therapy is totally dependent of the dose and type of laser, and this application had no significant
effect in the advanced stages of bone repair associated with the same implants of hydroxyapatite or collagen.
Keywords: Bone, Collagen, Hydroxyapatite, Laser, Rats
Financial Support: NAPED/FMJ and PIBIC/FMJ
Resumo:09-007
THE ACTIVATION OF ERK1/2 BY 1,25(OH)2 VITAMIN D3 IS DEPENDENT OF PKA AND PKC AND RESULTS IN
RAPID CALCIUM INFLUX IN IMMATURE RAT TESTIS
Gonçalves, R. ; Zanatta, L. ; Zamoner, A. ; Silva, F. R. M. B.
Objectives:
The active form of Vitamin D3, 1,25(OH)2 vitamin D3 (1,25D), plays important role in calcium homeostasis, bone metabolism
and cell differentiation. The aim of the present work was to study the effect of 1,25D on rapid plasma membrane calcium influx
and the pathways involved in this process in immature rat testis.
For this study, 30-day-old Wistar rats were used. The testes were pre-incubated in KRb buffer for 15 min at 32°C, pH 7.4 with
O2:CO2 (95:5 v/v). After that, the testes were transferred to another series of wells with fresh KRb containing 0.1 μCi/ml
45Ca2+, during 60 min. Finally, the tissues were incubated for 5 min in the presence or absence of 1,25D (10-9 M). 1 μM digoxin
(Na+-K+ ATPase inhibitor), 50 μM BAPTA-AM (intracellular calcium chelator), 10 μM H-89 (PKA inhibitor), 50 μM
theophyline (phosphodiesterase inhibitor), 500 μM dibutyryl cAMP (cAMP analog), 1 μM RO 31-8220 (PKC inhibitor), 1 μM
U73122 (PLC inhibitor) and 10 μM PD-98059 (MEK inhibitor), were added 20 min before the hormone. After incubation, the
testes were washed with LaCl3 (washing solution) in order to prevent release of the intracellular 45Ca2+. From testicular
homogenate, aliquots were used for radioactivity measurement and for total protein quantification. For the Western Blot analysis,
rat testes were pre-incubated in the presence or absence of PKC and PKA inhibitors for 20 min followed by incubation
with/without 10-9 M 1,25D for 5 min at 32°C in KRb. After incubation, the tissues were processed in SDS buffer to the analysis
of mitogen-activated protein kinases (phospho and total levels of ERK1/2). Statistical evaluation was done using one-way
analysis of variance (ANOVA), followed by Bonferroni post-test. Data were expressed as % of control and differences were
considered to be significant at p < 0.05 (n=4). The stimulatory effect of 1,25D (154.35±15.64) on calcium influx was
significantly inhibited by BAPTA-AM (105.80±10.37), H-89 (104.24±6.38), RO 31-8220 (110.04±6.92), U73122 (95.00±1.58)
and PD-98059 (115.10±9.26) when compared to control group (100.00±6.24). In addition, digoxin (155.10±13.95), theophyline
(141.14±13.86), and dibutyryl (163.62±24.46) produced a stimulatory effect similar to 1,25D. The basal levels of phosphorylated
ERK1/2 were not influenced by neither H-89 (91.01±1.30/ 94.51±0.93) nor RO 31-8220 (87.71±3.71/ 99.73±3.45). However, the
stimulatory effect on phospho ERK1/2 immuno-content was totally blocked by these inhibitors respectively
(91.47±2.51/102.01±3.21;92.05±4.53/82.32±3.17). The total immuno-content was not altered.
Conclusions:
The stimulatory effect of 1,25D on calcium influx in immature rat testis depends on intracellular calcium stocks, PKA, PKC and
MAPK. The inhibition on Na+/K+ ATPase can induce a rise in sodium concentration inside the cell, culminating in calcium
influx via Na+-Ca2+ exchanger activation in a reverse mode.
Keywords: CALCIUM INFLUX, ERK1/2, TESTIS, VITAMIN D3
Financial Support: CAPES/Cofecub; CNPq; PGFAR-UFSC
Resumo:09-008
THE ROLE OF LEUKOTRIENE ON SODIUM REABSORPTION IN RENAL PROXIMAL TUBULE
Freitas, E. L. D. 1; Landgraf, S. S. 1; Portella, V. G. 1; Pinheiro, C. S. 1; Canetti, C. A. 1; Takiya, C. M. 2;
Pinheiro, A. A. S. 1,4; Caruso-neves, C. 1,3
1
Instituto de Biofísica Carlos Chagas Filho, IBCCF
2
Instituto de Ciências Biomédicas, ICB
3
Instituto Nacional de Biologia Estrutural e Bioimagem, INCT-INBEB/CNPq/MCT
4
Pesquisa Translacional em Saúde e Ambiente na Reg. Amazônica, INCT-INPeTAm/CNPq/MC
Objectives:
Leukotrienes (LTs) are lipidic inflammatory mediators. The rate-limiting step in their synthesis is the 5-lipoxygenase (5-LO)
activity, together with its activating protein (FLAP). All LTs molecules are derived from a common precursor, LTA4. It has been
shown that LTs are augmented in traumas and infections. The pharmacological blocking of LTs receptors, as well as the
inhibition of 5-LO in different models of nefrotoxicity, ischemia/reperfusion and during renal injury have a protective effect.
Thus, in this work, we further assess the mechanisms underlying the role of LTs during acute renal failure (ARF).
Male SV129 (wild-type=WT) and 5-LO knockout(5-LO-/-) mice were submitted to i.p. injections of bovine serum albumin
(10g/kg/day) for 7 days to develop the acute renal failure (ARF) or saline (control). During this period of time, mice were
allocated in metabolic cages and their urines were collected to assess the renal sodium fraction excretion (FENa+). After
euthanasia, their kidneys were collected and homogenates were used to analyze (Na++K+)ATPase activity (nmol-Pi.mg-1.min-1)
and expression (arbitrary units) in the cortex and medulla. Protein expression was assessed by immunoblotting, and the enzymatic
activity was measured in the presence and in the absence of ouabain (a specif inhibitor) according to the method described by
Grubmeyer & Penefsky (J Biol Chem 256; 3718, 1981). The cortical (Na++K+)ATPase α1-subunit expression decreased in ARF
model in both WT (0,66 ± 0,13) and 5-LO-/- (0,54 ± 0,12) when compared to WT/saline mice (2,05 ± 0,25). Interestingly, the
same profile was found in cortical (Na++K+)ATPase activity. In renal medulla, (Na++K+)ATPase expression decreased in ARF
animals (WT= 0,77 ± 0,24; 5-LO-/-= 0,32 ± 0,04) when compared to WT mice (1,20 ± 0,15). Furthermore, it was observed that
5-LO-/- saline also presented lower (Na++K+)ATPase expression (0,75 ± 0,18) than WT control. A similar profile was observed
for medullary (Na++K+)ATPase activity. The FENa+ was augmented in both 5-LO-/- groups (saline=1,88 ± 0,38%; ARF=1,63 ±
0,11%), as well as in the WT/ARF (1,33 ± 0,07%) when compared to WT/saline (0,73 ± 0,12%).
Conclusions:
5-LO depletion augments the renal sodium fraction excretion (FENa+), which is a result of the diminished activity and expression
of (Na++K+)ATPase in the renal cortex and medulla.
Keywords: leukotriene, kidney, sodium pump
Financial Support: FAPERJ, CAPES, INBEB, INPeTAm and CNPq
Resumo:09-009
REGULATION OF WILSON DISEASE ATPASE (ATP7B) ACTIVITY BY CAMP-DEPENDENT PROTEIN KINASE
Hilário-souza, E. ; Valverde, R. H. F. ; Britto-borges, T. ; Vieyra, A. ; Lowe, J.
Instituto de Biofísica Carlos Chagas Filho, UFRJ, IBCCF
Objectives:
Copper homeostasis and molecular studies of the active transport of this metal are essential to understand the copper-related
diseases (Menkes and Wilson diseases). The main objective of this study was to analyze the modulation of the Cu(I)-ATPase
(Atp7b) activity in porcine liver by cyclic AMP-dependent protein kinase (PKA) and investigate the direct effects of this
regulatory phosphorylation on the catalytic cycle of Atp7b.
Golgi enrichment in the membrane fractions (Int. J. Biochem. Cell. Biol. 43:358, 2011) was confirmed by immunoblotting of
GM130 and TGN46 (the markers for the cis- and trans-Golgi regions, respectively). Anti-Atp7b, a specific antibody, was also
used to identify mammalian Cu(I)-ATPase. The acyl-phosphate intermediate formation during the catalytic cycle was confirmed,
indicating that it is a P-type ATPase. Catalytic phosphorylation was inhibited by 40% in the presence of BCS (copper chelator)
when a steady level was reached (1 min). The catalytic phosphorylation was copper dependent. Golgi-enriched preparations
contain a membrane associated PKA (as indicated by immunobloting using a specific antibody for this protein). The activity was
assayed by measuring the release of Pi from ATP. The Atp7b activity was inhibited by 50% when PKA pathway was stimulated
using forskolin (1 nM), cAMP (100 nM), cholera toxin (1 nM) or exogenous PKA α -catalytic subunit (2000 U/mL). The Atp7b
activity was increased by 50% when incubated with a specific PKA inhibitor (10 nM PKAi5-24 peptide). This mirror image of
the inhibition obtained with activators indicates that endogenous cAMP produced by endogenous adenylyl cyclase is sufficient to
partially inhibit the control activity by activating the membrane-associated PKA, which was detected in membrane fractions. The
addition of the exogenous PKA α -catalytic subunit increased the K0,5 for free copper (2.5 x 10-17 M and 6.2 x 10-17 M in the
absence and presence of PKA, respectively), although the Hilll coefficient for free copper was not changed (2.7 ± 0.4 and 2.3 ±
0.7 in the absence and presence of PKA, respectively). The Vmax value found for the ATP curve with no exogenous PKA was
69.1 ± 4.3 nmol Pi x mg-1 x min-1 and on the presence of PKA α -catalytic subunit, the Vmax decreased to 42.1 ± 3.6 nmol Pi x
mg-1 x min-1. The Km for ATP was the same when exogenous PKA was present (0.97 ± 0.22 mM and 1.00 ± 0.31 mM in the
absence and presence of PKA, respectively).
Conclusions:
In conclusion, PKA-mediated regulatory phosphorylation inhibited Cu(I)-ATPase activity by 50%. This phosphorylation did not
change the affinity of this enzyme for ATP and the Cu(I) ions cooperativity, but it affected the enzyme turnover because of the
reduction of Cu(I)-ATPase affinity for copper ions. The possibility of hormones acting on protein kinases in liver to modulate
active copper transport opens new horizons in our understanding of how cells and organisms handle intracellular copper.
Keywords: Copper ATPase, Copper affinity, Cell signaling, Regulatory phosphorylation, cAMP-dependent protein kinase
Financial Support: FAPERJ, CNPq and CAPES
Resumo:09-010
ANTINOCICEPTIVE EFFECT OF THE ARMED SPIDER TOXIN TX3-5 ON MICE PAIN MODELS
Silva, C. R. 1; Oliveira, S. M. 1; Vilarinho, J. G. 1; Trevisan, G. 1; Gomes, M. V. 2; Ferreira, J. 1
1
Centro de Ciências Naturais e Exatas, UFSM
2
Instituto de Pesquisa da Santa Casa de BH, IEP
Objectives:
Ziconotide (Prialt®), a synthetic form of the calcium channel blocker, ω-conotoxin MVIIA, from the marine snail Conus magus
has been an exceptional lead for drug development in the management of severe and chronic pain. Similarly, the venom of the
Brazilian “armed” spider “Phoneutria nigriventer” is a rich source of biologically active peptides, including the toxin Tx3.6 and
Tx3.3, which are active on neuronal calcium channels demonstrating analgesic effects in different pain models. This study
evaluates the antinociceptive effect of Tx3.5 in acute and chronic pain models in mice, by intrathecal (i.t) administration.
The experiments were conducted on adult male Swiss mice (25-35 g, n= 6-8) or C57BL/6 mice (20-30g, n= 7-8 to plantar
inoculation of melanoma, Federal University of Santa Maria, Brazil). The animals were acclimated approximately 1.5 h before
measuring sensitivity after mechanical stimulation with von Frey filaments, using the "Up-and-Down” method. The toxin Tx3-5
(3-300 fmol/site) or vehicle (phosphate-buffered solution - PBS) were administered by intrathecal (i.t.) route (5 µl/site). The
antinociceptive effect of Tx3-5 was evaluated in models of postoperative pain after plantar surgery, neuropathic pain induced by
partial sciatic nerve ligation (PNSL) and in a model of cancer pain induced by inoculation of B16–BL6 cells. Tx3-5 was
administered before or after the surgical procedure and after the installation of neuropathic pain. Seventeen days after expose the
animals at inoculation of B16-BL6 cells, they were divided into two groups. One group of animals received morphine during
three consecutive days for tolerance development (10-10-15 mg/kg in day 18; 15-15-20 mg/kg in day 19 and 20-20-25 mg/kg in
day 20) and other group received saline (10 ml/kg) also for three consecutive days. Morphine or saline were administered by
subcutaneous (s.c.) route. In 21st the two groups of animals were treated with PBS or Tx3.5 (30 fmol/site). Was observed that
plantar surgery caused nociception in the animals (reduction of mechanical threshold of 83 ± 22%). The toxin Tx3-5 (30
fmol/site) was able to reduce this nociception induced by plantar surgery when administered before (increase of mechanical
threshold of 57 ± 12%) or after surgical procedure (increase of mechanical threshold of 67 ± 12%). Likewise, the PNSL promoted
nociception in the animals 7 days after surgical procedure (reduction of mechanical threshold of 90 ± 10%) and Tx3-5 (30
fmol/site) was able to increase the threshold in 50 ± 15%. Furthermore, the toxin (30 fmol/site) reduced the nociception of
animals in the cancer pain model (maximal inhibition of 94 ± 21%), and completely abolished the nociception in the group of
animals that presented morphine tolerant (inhibition of 100%).
Conclusions:
Our results suggest that Tx3-5 is a promising drug for the treatment of acute and chronic painful conditions such as postoperative,
neuropathic and cancer-related-pain.
Keywords: Calcium channels, Cancer pain, Neuropathic pain, Phoneutria nigriventer, Postoperative pain
Financial Support: CAPES, CNPq, PRONEX, FAPERGS
Resumo:09-011
CARACTERIZATION OF THE HEPATIC CU(I)-ATPASE FROM RATS.
Britto-borges, T. ; Gomes, F. ; Hilário-souza, E. ; Valverde, R. R. F. H. ; Vieyra, A. ; Lowe, J.
Instituto de Biofísica Carlos Chagas Filho/UFRJ, IBCCF/UFRJ
Objectives:
Wilson disease is a monogenic inherited disorder characterized by liver failure and neurological symptoms. The molecular basis
of these events are related with mutations and consequent loss of function of ATP7B, one of the mammal the hepatic Cu(I)ATPases. Liver is responsible for copper plasma distribution and the excretion of its excess, being the most import organ in
mammals copper homeostasis. Likewise, this organ is central in energetic metabolism by controlling glucose levels when
signalized about of the nutrition state by insulin and glucagon, both hormones that trigger different kinase-mediated signaling
pathway including PKA, PKC and tyrosine kinases. Recent studies of our group demonstrated that PKA phosphorylates specific
residues that modifies catalytic behavior of Cu(I)-ATPases when heterologously expressed in a Sf9 cells (J. Biol. Chem.
286;6879, 2011) and in pig liver enriched Golgi fractions (Int. J. Biochem. Cell Biol. 43;358, 2011). This work concentrates not
only in rat Cu(I)-ATPase characterization, but an understanding in vivo animal model of interaction between energetic
metabolism and copper homeostasis.
Golgi-enriched membrane fractions were obtained from Wistar rat liver. ATP7B protein was detected by Western Blotting. The
Cu(I)-ATPase specific activity was evaluated by release of Pi from ATP. The results were given in nmol × mg-1 × min-1 (means
± SE) and was measured by the difference in the absence and presence of 0.3 M BCS, a specific Cu(I) chelator. Optimal
temperature, time reaction and pH curve were determined, showing that the maximum Pi release was obtained in acidic pH (5.0)
at 37 °C. The Cu(I)-ATPase activity was 42.83 ± 6.39 nmol Pi × mg-1 × min-1 (n=5). Catalytic behavior from this protein
resembles Ccc2, the yeast Cu(I)-ATPase, with maximum catalytical phosphorylation in 45 s followed by dephosphorylation after
3 min.
Conclusions:
Conclusion: This protein, a P-type ATPase, is responsible for active copper transport to the lumen of trans-Golgi network. In
vitro studies shows that insulin is responsible for stimulation of ATP7B activity and therefore the kinases-mediated regulatory
phosphorylations are being studied in this in vivo model.
Keywords: Cell Signaling, P-type ATPase, Liver
Financial Support: Sources of research support: FAPERJ and CNPq.
Resumo:09-012
STUDY OF THE OSTEOCONDUCTIVE CAPACITY OF HYDROXIAPATITE IMPLANTED INTO THE FEMUR OF
OVARIECTOMIZED RATS
Ribeiro, D. C. P. ; Figueira, L. A. ; Cunha, M. R.
Faculdade de Medicina de Jundiaí, FMJ
Objectives:
Osteoporosis is a global public health that affects postmenopausal women due to the deficiency of estrogen, a hormone that plays
an important role in the microarchitecture of bone tissue. Osteoporosis predisposes to pathological bone fracture that can be
repaired by conventional methods. However, depending on the severity and quantity of bone loss, the use of autogenous grafts or
biomaterials such as hydroxyapatite might be necessary. The latter has received increasing attention in the medical field because
of its good biological properties such as osteoconductivity and biocompatibility with bone tissue. The objective of this study was
to evaluate, using histologic and radiographic analyses, the osteogenic capacity of hydroxyapatite implanted into the femur of rats
with ovariectomy-induced osteoporosis.
Fifteen female rats (Rattus norvegicus),with 10 weeks and 300g of weight, were divided into three groups with five animals in
each: group non-ovariectomized (NO), bilaterally ovariectomized not receiving estrogen replacement therapy (BOWHR), and
bilaterally ovariectomized submitted to estrogen replacement therapy (BOHR). Defects were created experimentally in the distal
epiphysis of the femur with a surgical drill and filled with porous hydroxyapatite granules. The animals were sacrificed 8 weeks
after surgery. The volume of newly formed bone in the implant area was quantified by morphometrical methods of Delesse. The
results were analyzed by ANOVA followed by the Tukey test (p
Conclusions:
Bone neoformation can be expected even in bones compromised by estrogen deficiency, but the quantity and velocity of bone
formation are lower.
Keywords: bone loss, bone defect, estrogen, hydroxiapatite
Financial Support: NAPED/FMJ and PIBIC/FMJ
Resumo:09-013
ANESTHETIC EFFICACY OF UNI AND MULTILAMELLAR LIPOSOMAL PRILOCAINE FORMULATIONS IN
INFRAORBITAL NERVE BLOCK, IN RATS
Nolasco, F. P. 1; Caldas, C. S. 1; Serpe, L. 1; Paula, E. D. 2; Ranali, J. 1; Volpato, M. C. 1; Groppo, F. C. 1
1
Departamento de Ciências Fisiológicas, FOP/UNICAMP
2
Departamento de Bioquímica, IB/UNICAMP
Objectives:
Compare the anesthetic efficacy of unilamellar and multilamellar prilocaine solutions in infraorbital nerve block with a
commercial solution of 3% prilocaine with 0,03UI of Felipressin, in rats.
A total of 30 adult male wistar rats, weighing between 300-400g, were divided into 3 groups: group 1 received prilocaine
commercial solution (PRL), group 2 received unilamellar prilocaine solution (LUV) and group 3 received multilamellar solution
(MLV) all infiltrations were in the right side near the infraorbital foramen. The duration of anesthesia was measured in the region
through vigorous cross-clamping of the upper lip, every 5 minutes, until a first sign of aversive response of the animal, indicating
the end of anesthesia. All formulations were effective (100% success) in promoting the upper lip anesthesia (immediately
injection), indicating the block of infraorbital nerve. Data were submitted to Tukey test (p < 0.05) and the results showed that
PRL and LUV had a statistical difference of p < 0.01. The other groups didn‟t show any statistical difference between them when
the test was applied.
Conclusions:
It can be concluded that the unilamellar solution was the only solution that showed a slightly difference when comparing to
commercial prilocaine. But when comparing the other formulations the efficacy was similar when blocking the rat infraorbital
nerve.
Keywords: Prilocaine, Anesthetics, Liposomal Solution
Resumo:09-014
THE EFFECTS OF TWO CARRIERS ON THE PRODUCTION OF CYTOKINES INDUCED BY LIDOCAINE IN
HUMAN ORAL EPITHELIAL CELLS.
Muniz, B. V. 1; Ferreira, L. E. N. 1; de Paula, E. 2; Groppo, F. C. 1
1
Depto. de Ciências Fisiológicas , FOP - Unicamp
2
Depto. de Bioquímica - Instituto de Biologia, IB - Unicamp
Objectives:
To evaluate the effects of free lidocaine and lidocaine coupled with liposomes or cyclodextrin on the production of IL-1α and IL6 in oral epithelial cells.
HaCaT cells were exposed to 1 µM, 10 µM and 100 µM of free lidocaine or lidocaine associated with the carriers during 6 hours
and 24 hours. Cytokines were measured by ELISA and cells were quantified by counting in a Neubauer chamber. The data were
submitted to Kruskal-Wallis or Wilcoxon tests. The higher concentrations (10 and 100 µM) in both free and liposomal solutions
caused significant decrease (p
Conclusions:
Lidocaine was able to induce toxicity to oral epithelial cells causing a decrease in the number of cells or inducing cytokines IL-6
and IL-1α. Both liposome and cyclodextrin carriers significantly reduced the lidocaine effects on these cells.
Keywords: cytokines, epithelial cells, lidocaine, liposomes, cyclodextrin
Financial Support: Fapesp # 2009/16225-6
Resumo:09-015
ANESTHETIC EFFICACY OF MEPIVACAINE UNI AND MULTILAMELLAR LIPOSOMAL FORMULATIONS IN
INFRAORBITAL NERVE BLOCK IN RATS
Caldas, C. S. 1; Nolasco, F. P. 1; Serpe, L. 1; Groppo, F. C. 1; Ranali, J. 1; de Paula, E. 2; Volpato, M. C. 1
1
Departamento Ciências Fisiológicas, FOP/ UNICAMP
2
Departamento de Bioquímica, IB/ UNICAMP
Objectives:
The purpose of this study was to compare the anesthetic efficacy of two formulations of liposome encapsulated mepivacaine with
a commercial solution of 2% mepivacaine and 1:100.000 epinephrine in the rat infraorbital nerve block model.
Multilamellar and unilamellar vesicle suspensions were obtained from egg phosphatidylcholine, cholesterol and α-tocopherol, to
which the mepivacaine hydrochloride salt was incorporated (2% concentration). A total of 30 adult male Wistar rats (Rattus
norvegicus albinus) weighing 300-400g were used. The animals were divided into 3 groups (10 rats/group) and received 0.1 ml
of the following formulations near the infraorbital foramen: group 1: 2% mepivacaine with 1:100.000 epinephrine; group 2: 2%
mepivacaine encapsulated in multilamellar liposomes and group 3: 2% mepivacaine encapsulated in unilamellar liposomes. The
duration of anesthesia was assessed by upper lip pinching every 5 minutes until observation of aversive response of the animal,
indicating the end of anesthesia. Data were submitted to ANOVA (t LSD test), α=5%. The solutions tested resulted in immediate
anesthesia of the upper lip in all rats. The control side did not show upper lip anesthesia. The duration of anesthesia (mean ±
standard deviation, in minutes) for groups 1, 2 and 3 were, respectively: 106 ± 43.8; 59.5 ± 18.6 and 69.5 ± 40.1. Group 1
presented longer anesthesia duration (p
Conclusions:
Liposomal formulations presented lower anesthesia duration than the solution containing epinephrine.
Keywords: anesthetics, liposome, mepivacaine
Financial Support: FAPESP 2006/00121-9; 2009/11848-5
Resumo:10-001
NUTRITION AND REPRODUCTIVE EFFECTS OF ENVIRONMENTAL STRESS BY LIGHT IN SWISS MICE
Dias, H. L. M. ; Sales, I. S.
FACULDADE SANTA TEREZINHA, CEST
Objectives:
To verify the impact of environmental stress by lighting during pregnancy in female Swiss mice;identify morphological changes
in offspring of Swiss mice on light stress;Check Changes of cholesterol,triglycerides and glucose in offspring of Swiss mice on
light stress; Verify variation of doses of cholesterol,triglycerides and glucose in offspring of Swiss mice to environmental stress
by light with the sample of young Swiss mouses the Control Group.
48 animals were used in this study,with 3 females and 3 males that mated and reproduced 42 pups males(21) and females
(21).The pairs were mated and the Control Group was the default group, Group 9 / 15 was induced to stress by light,9 hours light
and 15 hours dark,Group 15 / 9 was induced to stress by light,15 hours light and 9 hours dark.The induced stress involved the
gestation period of females and birth,weaning and the beginning of adulthood for the offspring,thus having a duration of 70 days.
After birth the pups were weighed, and examined the weights of pups at birth and weaning,and thus selected the 7 heaviest pups,
7 males and 7 females of each couple.After 60 days of the birth of pups was performed blood collection that was made at the
height of 3 cm above the anus with the aid of a lancet, then used the meter Glucose - Cholesterol - Triglycerides (Accutrend
trademark ROCHE).The values of total cholesterol (TC) and triglycerides (TG) were obtained using the method of photometry of
reflection. The collection method followed the instructions recommended by the manufacturer, the time interval required for
measurement was 180s for total cholesterol and 68-174S for triglycerides. Blood glucose was measured with the portable monitor
Accutrend (Roche) using test strips following the instructions provided by the manufacturer and after about 26 seconds,the blood
glucose result is displayed in mg / dl.Results:The average weight of pigs at birth was: control = 1.16 g; Group 9 / 15 = 0.95 g;
Group 159 = 0.94 g,and the average weight of animals at weaning was: Control Group = 11 ,44 g;Group 9 / 15 = 10.4 g; Group
15 / 9 = 10.07 g. The average results of the glycemic index of females in the Control Group = 94.85 mg / dl, Group 9 / 15 = 81.57
mg / dl,Group 15 / 9 = 116.14 mg / dl and the male control group = 111.14 mg / dl,Group 9 / 15 = 89.71 mg / dl, Group 15 / 9 =
133mg/dl,and mothers in the Control Group = 95mg/dl, Group 9 / 15 = 85mg/dl, Group 15 / 9 = 115 mg / dl. The average results
of the cholesterol of the female Control Group = 103mg/dl; Group 9 / 15 = 84.14 mg / dl, Group 15 / 9 = 122.71 mg / dl, and
average males in the Control Group = 113.42 mg / dl,Group 9 / 15 = 100.42 mg / dl,Group 15 / 9 = 130.57 mg / dl and that the
mothers in the Control Group = 99mg/dl, Group 9 / 15 = 83mg/dl, Group 15 / 9 = 125mg / dl. The average results of the
triglycerides of the females in the Control Group = 81.28 mg / dl,Group 9 / 15 = 67.85 mg / dl, Group 15 / 9 = 96mg/dl, and
average males in the Control Group = 92mg/dl;Group 915 = 82.85 mg / dl; Group 159 =103.42 mg / dl and that the mothers in the
Control Group = 85mg/dl; Group 915 = 66mg/dl; Group 159 =94mg/dl
Conclusions:
In this study,the pups and the mother who had peak glycemic index, cholesterol and triglycerides were exposed to a longer period
of activity and a reduction in resting time,so that we might have thought that tiny changes in the circadian cycle of these animals
in the pre-trial or trial could interfere with any results of an experimental protocol.
Keywords: glucose, cholesterol, triglycerides, mice, stress by light
Resumo:10-002
DISCRIMINATION REVERSALS REDUCES COGNITIVE INFLEXIBILITY OF EARLY MALNOURISHED RATS.
Silva, E. M. ; Almeida, S. S.
Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, FFCLRP-USP
Objectives:
Investigate the effects of operant techniques on learning tasks performance of rats submitted to early postnatal protein
malnutrition.
METHODS: Thirty-two male Wistar rats were divided in 4 groups: a) DNF ï¿½ early protein malnourished (5.1% protein) not
submitted to fading in of S-; b) CNF ï¿½ well-nourished (13.6% protein) not submitted to fading in of S-; c) DF ï¿½ early
malnourished submitted to fading in; d) CF well-nourished submitted to fading in. When the animals were 80 days old, they were
water deprived, until their body weight were equal to 85% (ï¿½ 5%) of ad lib weight, and started a discrimination training
between a vertical line (S+) and a triangle (S-), the training was divided into phases. One phase of acquisition, and 3 phases of
consecutive reversions of the discrimination. Fading in procedure consisted in the gradual introduction of the S- of each phase, to
facilitate the discrimination learning. RESULTS: A significant effect of fading factor [F(1,28) = 54.9, p
Conclusions:
The comparison between well-nourished and early malnourished animals in the number of sessions necessary to meet the
learning criteria discrimination and reversals has showed no evidence of lower capacity to learn from malnourished ones. In the
first reversal malnourished animals required more sessions training than in the discrimination, these data would be consistent with
the hypothesis of cognitive inflexibility, where the control animals reached learning criteria faster, and only two rats from the DF
group met the learning criteria. The rise on the number of training sessions on first reversion, when compared to discrimination
training, for all groups is consistent with the hypothesis that the task of discriminating between line and triangle is difficult for
rats. Thus, the successive reversals of simple discrimination facilitated the learning of new discriminations and served to indicate
that the differences in performance between groups could not be attributed to sensory deficits resulting from poor nutrition.
Groups subject to fading in needed more sessions on reversals 2 and 3 of the animals groups that were not subject to fading, this
is due to the fact that the groups subject to fading experienced at least 18 sessions, 15 for stimuli changes and three to reach the
learning criterion with the stimuli in their maximum intensities.
Keywords: PROTEIN MALNUTRITION, LEARNING, COGNITIVE INFLEXIBILITY
Financial Support: CAPES.
Resumo:10-003
FREE RENAL LEVELS OF FLUCONAZOLE IN HEALTHY AND CANDIDA ALBICANS INFECTED WISTAR
RATS DETERMINED BY MICRODIALYSIS.
Azeredo, F. J. ; Araújo, B. V. ; Dalla Costa, T.
Faculdade de Farmácia, UFRGS
Objectives:
Fluconazole (FCZ) is a triazolic antifungal agent used for treating systemic candidiasis in immunocompromised patients.
Infection eradication is dependent on free drug concentration at the infection site rather than total plasma concentration (Liu, P. et
al, J Clin Pharmacol 45: 564, 2005). Furthermore, infection can alter drug penetration into specific tissues (Joukhadar, C. et al,
Antimicrob Agents Chemother 47: 103, 2003). The aims of this study were to evaluate free levels of FCZ in the kidney of healthy
and C. albicans infected Wistar rats using microdialysis and to establish the relationship between free renal and total plasma
levels in both conditions.
Animal experiments were approved by UFRGS Ethics in Research Committee (2008187). An HPLC/UV and an LC-MS/MS
method were developed and validated to quantify FCZ in the microdialysate and in plasma samples, respectively. CMA/20
microdialysis probes (4 mm, cutoff 20 kDa) were used to determine recoveries in vitro by dialysis and retrodialysis an in vivo by
retrodialysis (n = 3/experiment). Probes recoveries were around 50% independent of method, drug concentration or matrix used.
FCZ kidney penetration in healthy and C. albicans infected male Wistar rats was investigated after administration of 10 mg/kg
i.v. and 50 mg/kg p.o. (n = 6/group). After dosing, blood and microdialysate samples were collected at predetermined time points
up to 24 and 18 h, respectively. FCZ plasma pharmacokinetic (PK) parameters after i.v. dosing to healthy and infected animals
showed t1/2 of 6.8 ± 1.0 and 6.9 ± 0.4 h, AUC0-∞ of 6621 ± 1592 and 7900 ± 2007 μg•h/mL, and MRT of 5.3 ± 0.7 and 4.9 ±
0.9 h, respectively. Plasma parameters after oral dosing to healthy and infected animals showed t1/2 of 6.8 ± 0.7 and 6.2 ± 0.6 h,
AUC0-∞ of 27887 ± 4530 and 30214 ± 1583 μg•h/mL, and MRT of 10.9 ± 1.5 and 8.6 ± 0.5 h, respectively. No statistical
difference between PK parameters determined in plasma and kidney for the same dose regimen for both conditions was observed
(α= 0.05). FCZ renal penetration was similar for both doses and conditions investigated (ranging from 0.77 to 0.84). Unbound
FCZ plasma fraction, determined by microdialysis, was concentration-independent (86.0 ± 2.0%). The PK parameters were
determined by individually fitting plasma concentration-time profiles to two-compartment model after i.v. and one compartment
model after oral dosing using the software Scientist v. 2.0 (MicroMath). Using appropriate equations, PK hybrid constants
determined by fitting plasma concentration-time profiles were able to predict free renal levels determined by microdialysis.
Conclusions:
FCZ easily penetrates the kidneys. PK parameters determined in plasma can be used to predict free FCZ kidney levels assuming
the plasma protein binding is known. Furthermore, C. albicans infection does not interfere with the antifungal renal penetration,
indicating that plasma concentrations can be used to establish and optimize FCZ dosing regimens to treat disseminated
candidiasis.
Keywords: fluconazole, microdialysis, renal penetration, Candida albicans
Financial Support: CNPq/Brazil for scholarship and financial support (Process 554229/2009-9)
Resumo:10-004
BLADDER AND URETHRA UNRESPONSIVENESS TO PROLONGED USE OF HIGH-DOSE OF TIBOLONE IN
OVARIECTOMIZED RATS
Henriques, H. N. 1; Carvalho, A. C. B. 1; Pantaleão, J. A. S. 2; Guzmán-silva, M. A. 1
1
Departamento de Patologia / Faculdade de Medicina, UFF
2
Departamento Materno-Infantil / Faculdade de Medicina, UFF
Objectives:
Tibolone is a synthetic steroid with selective tissue action and has beneficial effects on the urogenital tract in women. The aim of
this study was to evaluate the effect of prolonged use of high-dose of tibolone on the bladder and urethra of ovariectomized rats.
Bilateral ovariectomy was performed on 14 rats weighing 250g, which were randomly divided in two groups. Experimental rats
(n=9) received orally 1mg tibolone/day; control rats (n=6) received placebo (carboxymethylcellulose). After 150 days, all rats
were sedated and euthanized by cervical displacement. Bladder and urethra were removed, fixed in 10% buffered formalin,
fragmented, processed and embedded in paraffin. Histological sections were stained with hematoxylin and eosin, picrosirius red,
Weigert‟s resorcin fuchsin, PAS and PAS-diastase. The bladder cell proliferation was analyzed by immunohistochemistry for Ki67. Histomorphometric analyses were performed for epithelial thickness, number of mast cells, percentage area of collagen fibers
and blood vessels. Mean and standard error of the mean were calculated and data were compared using the Mann-Whitney test
and significance level at P
Conclusions:
Long-term administration of high-dose of tibolone has no effect on the epithelial trophism, collagen fibers, number of blood
vessels and cell proliferation, in urethra and bladder of ovariectomized rats.
Keywords: tibolone, bladder, urethra , rats, menopause
Financial Support: OFFICILAB, PROPPI/UFF.
Resumo:10-005
ACTIVITY OF ENZYMES THAT HYDROLYZE ADENINE NUCLEOTIDES IN LYMPHOCYTES FROM RATS
EXPERIMENTALLY INFECTED WITH SPOROTHRIX SCHENCKII
Rocha, B. C. 1; Castilhos, L. G. 1; Noal, C. B. 1; Souza, V. D. C. G. 1; Jaques, J. A. D. S. 2; Bertoldo, T. M.
D. 1; Oliveira, D. C. D. 3; Alves, S. H. 1; Leal, D. B. R. 1
1
Microbiologia e Parasitologia / Ciências Farmacêuticas, UFSM
2
Microbiologia e Parasitologia / Bioquímica toxicológica, UFSM
3
Farmacologia, UFSM
Objectives:
Sporotrichosis is a mycosis with the fungus Sporothrix schenckii as the etiological agent, affecting humans and several animal
species. The infection caused by this disease, activates the immune and inflammatory response which is modulated by the
purinergic signaling. This study aimed to determine the activity of the enzymes ectonucleoside triphosphate diphosphohydrolase
(NTPDase) and adenosine deaminase (ADA) in lymphocytes from rats with systemic and lymphatic sporotrichosis induced.
Wistar rats were obtained from the Central Bioterium of Universidade Federal de Santa Maria (UFSM), 8 weeks old and
weighing about 160g. They were divided into three groups (C-control; CS-infected with systemic strain of S. schenckii; and CLinfected with lymphatic strain of S. schenckii), and each group consisted of 10 animals. The strains of S. schenckii used in the
experiment were from the mycology collection of the Research Laboratory of Mycology (LAPEMI-UFSM) and they were stored
in an appropriate culture medium. Inocula of the strains were composed of a conidial suspension in water for injection and the
administration was performed intraperitoneally (1mL). The enzymatic activity of NTPDase was determined by the amount of
inorganic phosphate released in the reaction of nucleotide hydrolysis, expressed as nanomoles of inorganic phosphate per minute
per milligram of protein (nm Pi/min/mg protein). To determine the ADA activity, the method was based on the production of
ammonia during the degradation of adenosine and the activity was expressed in units per milligram of protein (U/mg of protein).
The protein content of samples was determined using serum albumin as standard. No significant difference was observed in the
activity of the enzyme NTPDase (ATP as substrate) between the groups (p ¡Ý 0.05), but when ADP was used as the substrate, we
observed a significant difference between groups C and CS (40,6% increased) and between CL and CS groups (19, 43%
decreased in CL group) (p
Conclusions:
This study demonstrated that NTPDase (ADP as substrate) activity was increased in the state of infection, possibly as a
compensatory response against the body¡¯s inflammatory process that occurs in this pathology. Furthermore, the increase in ADP
hydrolysis could contribute to raise the levels of adenosine, a major immunosuppressive molecule, which can downregulate the
inflammatory response, signalling the production of inflammatory cytokines.
Keywords: adenosine deaminase , lymphocytes, NTPDase , Sporothrix schenckii
Financial Support: PROAP CAPES
Resumo:10-006
TREATMENT OF PERITONEAL ENDOMETRIOSIS WITH LOCAL INJECTION OF ACID ACETYLSALICYLIC EXPERIMENTAL STUDY IN RABBITS
Barretto, A. B. ; Saad-hossne, R.
Departamento de Cirurgia e Ortopedia, FMB - UNESP
Objectives:
The aim of this study is evaluate the effects of the bicarbonate solution of acetyl salicylic acid (aspirin) in autologous peritoneal
endometrial implants of mature rabbits to base any therapeutic technique for the treatment of endometriosis.
We used 20 female rabbits, aged virgins, weighing approximately 3.5 kg, being 10 treated with local injection of acetylsalicylic
acid (treatment) and 10 with local injection of physiological saline (control), partial results indicate that spots of endometriosis
were destroyed, leading to a new analysis for certification of treatment was 100% effective.
Conclusions:
According to partial results, acetylsalicylic acid is effective in destroying the endometrial spots.
Keywords: endometriosis, acid acetylsalicylic, rabbits
Financial Support: Capes and FAPESP
Resumo:10-007
PROTECTIVE EFFECTS OF ANTIOXIDANTS AND IRON CHELATOR ON DYSTROPHIC MUSCLE CELLS
Bollineli, R. C. 1; Moraes, L. H. R. 1; Silveira, L. R. 2; Curi, R. 3; Minatel, E. 1
1
Anatomia, Biologia Celular, Fisiologia e Biofísica/IB, UNICAMP
2
Bioquímica e Imunologia/Escola de Educação Física e Esporte, USP
3
Fisiologia e Biofísica/ICB, USP
Objectives:
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by the absence of the protein dystrophin.
Oxidative stress plays an important role in the pathogenesis of DMD. In the present study the effects of ascorbic acid (AA), Nacetylcysteine (NAC) and deferoxamine (DFX) or NAC-DFX in combination on primary cultures of dystrophic muscle cells
were investigated.
Primary cultures of skeletal muscle cell from mdx mice, an experimental model of DMD, were treated with AA (100 μM), NAC
(10 mM), and DFX (5 mM) or NAC plus DFX for 24 h. Muscle cells of C57BL⁄10 mice were used as controls. The H2O2
production was measured by using the Amplex red assay kit. The levels of 4-HNE (a marker of lipid peroxidation) and of TNF-α
(a pro-inflammatory cytokine) were analyzed by western blotting. H2O2 production was significantly higher in mdx muscle cells
compared to C57BL⁄10 muscle cells (p≤0.05, Student′s t-test). Treatments with NAC, DFX or NAC plus DFX significantly
decreased H2O2 production (by 24%, 58% and 72% respectively; p≤0.05, Student′s t-test) in dystrophic muscle cells. In contrast,
AA treatment did not change H2O2 production by mdx muscle cells. A significant increase of the 4-HNE (9%) and TNF-α (40%)
levels in mdx muscle cells compared to C57BL⁄10 muscle cells was observed (p≤0.05, Student′s t-test). Treatments with NAC,
NAC plus DFX and AA significantly decreased 4-HNE levels (p≤0.05, Student′s t-test) in dystrophic muscle cells. The TNF-α
levels were decreased in mdx muscle cells after all treatments mentioned (p≤0.05, Student′s t-test).
Conclusions:
Our results suggest that the antioxidants agents used and the iron chelator exerted protective effects on mdx muscle cells and may
be therapeutically used for the treatment of patients with muscle dystrophinopathies.
Keywords: skeletal muscle cells, mdx mice, antioxidant, iron chelator
Financial Support: FAPESP (#2010/07775-0; #2010/ 01087-4; #2008/50731-3); FAEPEX
Resumo:10-008
MODELING THE OLFACTORY FEAR CONDITIONING IN MICE
Back, F. P. ; Carobrez, A. P.
Departamento de Farmacologia, UFSC, UFSC
Objectives:
The olfactory fear conditioning (OFC), derived from Pavlovian classical conditioning, is a reliable protocol for evaluation of
neurobiological processes involved in fear and anxiety in rats [Neurobiol Learn Mem, 91(1):32-40, 2009]. The increasing
sophistication in molecular/genetic approach for the study of the biological bases of fear and anxiety prompt us to develop a
similar model of OFC in mice, considering that the majority of studies employing genetic approaches use this rodent species.
Based on these facts, the present study was designed to develop and characterize a mice OFC model, analyzing possible
procedural and organismic variables that could interfere with its functioning.
Male Swiss mice were submitted to the OFC protocol comprising two consecutive phases: acquisition (2 days) and expression (3
days). The acquisition session was performed in a conditioning chamber, where subjects received footshocks in the presence of
amylacetate odor as the conditioned stimulus (CS). Conditioned emotional response (CER) expression occurred in a two
compartment (open and enclosed) odor box where, 24h after a 5 min familiarization session, a CS1 test was performed by
exposing the mice for 5 min in the odor box saturated with the CS. On day 5, a context CS2 5min-session was performed in the
odor box without the amylacetate odor. The experimental groups were divided according to the age (3 or 5 month-old) and
footshock+odor (paired/non paired). Hence, four groups were formed: 3-mo/non-paired (n=12), 3-mo/paired (n=4), 5-mo/non
paired (n=15) and 5-mo/paired (n=9). CER includes the percentage of time spent in the enclosed compartment (%CLOSED), the
frequency of crossing (CROSS; number of entries in the enclosed compartment plus the number of approximations to the odor
source) and latency to explore the open compartment (LATENCY). Statistical analyses performed using the ANOVA-repeated
measures, followed by Newman-Keuls, compared the CER data obtained during the CS1 and CS2 sessions. For %CLOSED, both
paired groups (92.2±6.4%, MEAN±SEM) spent more (p
Conclusions:
Preliminary results indicate that OFC protocol for mice is a reliable animal model of fear conditioning, susceptible to
standardization and therefore, to be developed as a tool to study gene targeting manipulations so far available only in this species.
Different from the OFC rat protocol, variables such as the number of crossings and the latency to emerge from the enclosed
compartment together with the time spent in the hidden compartment represent the CER during the CS presentation (CS1 test)
and during the CS2 test characteristic of second-order conditioning.
Keywords: Animal Model, Anxiety, Fear , Conditioning, Odor
Financial Support: CNPq, PROEX-CAPES, FAPESP
Resumo:10-009
AN ALTERNATIVE METHOD TO INDUCE EXPERIMENTAL RESPIRATORY PARACOCCIDIOIDES
BRASILIENSIS INFECTION
Ribeiro, R. S. A. ; Cardoso, R. A. ; Teixeira, G. A. P. B. ; Dias, E. P. ; Junior, I. F. ; Vericimo, M. A.
Departamento de Imunobiologia/Instituto de Biologia, UFF
Objectives:
In experimental protocols of pulmonary infection, for example, infection by Paracoccidioides brasiliensis (Pb), a common
procedure to access the airways of the mouse is the tracheal intubation through surgical puncture. Besides being a timeconsuming procedure it is very traumatic for the animals which constantly lead to loss of animals in our experience. Thus our aim
was to develop a modified model of intubation of mice for the induction of pulmonary infection by Pb.
A total of ten C57BL/6 (8-10 week old) mice were divided into two groups, infected and control which received one million Pb
yeast, or saline (0,9% NaCl) respectively. After 14 days, the mice were sacrificed for histological analysis of the tongue, trachea
and lungs. Hematoxylin-eosin stain was used for standard histology and Grocott stain for identification of the fungi. We
developed a vertical platform that allowed adequate placing of the animals to perform orotraqueal intubation. The platform was
composed of a cork board fixed on a base on which a nylon cord was loosely fixed with cork board pins. After anesthetizing the
animals the nylon cord was passed behind the animals front upper teeth and then pinned to the cork board. While the thumb and
index Finger of one hand lowered the tongue and the lower jaw the middle finger was positioned on the back of the animal to
straighten the cervical spinal cord. After positioning the animal a second person transilluminated the neck with a optic fiber in
order to visualize the vocal cords. The next step consisted in the introduction of the tip of a catheter (intravenous BD angiocath
20Gx1,16”-1,1x30 mm – 49 ml/min: cat 388333, MG, Brasil) in the orotraqueal cavity until overcoming the vocal cords and
introducing another 5-7 mm. This is a measure that avoids selective pulmonary infection and lesions of the trachea. Once the
catheter was positioned in the trachea 50 μl saline or Pb solution was slowly inoculated afterwhich the catheter was immediately
removed. The mean time used in the technique proposed is significantly shorter than the surgical technique approximately 2 min.
for each mouse compared to the 15 min. necessary for the surgical puncture. After recovery from anesthesia animals were housed
in their cages and were observed every two days. The study was registered with Animal Research Ethics Committee (NAL / UFF
0138/2009).On animal died following tracheal intubation nor presented any systemic or behavioral changes. On the other hand
histopathology of the lung revealed a bilateral infection with the presence of numerous yeast forms typical of Pb infection
accompanied by an intense inflammatory infiltrate. No significant differences were observed between the right and left lungs. No
histological aggression in the oral cavity or trachea was observed.
Conclusions:
We suggest that the surgical puncture should be replaced by oral intubation in performing experimental pulmonary infection in
mice once it is far less traumatic for the animals besides being faster.
Keywords: Paracoccidioides brasiliensis, OROTRACHEAL INTUBATION, MICE INFECTION,
PARACOCCIDIOIDOMYCOSIS
Financial Support: APQ1/FAPERJ – 2010/2011
Resumo:10-010
DIAGNOSTIC OF PNEUMOCYSTIS CARINII IN MICE AND RAT COLONIES FROM BRAZILIAN ANIMAL
HOUSES
Andrade, L. A. G. D. 1,1; Demolin, D. M. R. 1; Minagawa, C. Y. 1; Moreira, J. C. D. O. 1; Santos, S. R. C. D.
1
; Nicklas, W. 2; Gilioli, R. 1
1
CEMIB , UNICAMP
2
Microbiological Diagnostic and Laboratory Animal Medicine, DKFZ
Objectives:
The main goal of this work consists in establish a PCR assay in our routine of laboratory animal health in order to perform an
initial trial to detect P.carinii in different mice and rat strains. Pneumocystis carinii (Pc) is classified as a fungal pathogen and it‟s
an opportunistic organism that can cause a lethal pneumonia in mammals with compromised immune system. Animal models
have been infected and all published data confirm that Pneumocystis species are strictly specific for their hosts and there‟s no
cross-species transmission. P.carinii and P.wakefieldiae sp.nov., are species found in laboratory rats while laboratory mice can
be infected with Pneumocystis murina sp., formerly known as P.carinii f. sp.muris. Immunodeficient mice usually suffer of a
subacute or chronic illness, characterized by wasting, debilitation and weight loss. Microscopically,it‟s characterized by a
progressive intersticial pneumonia. In immunocompetent rats, P.carinii has been found to cause an idiopathic intersticial
pneumonia, but organisms are difficult to find, except by PCR. Immunocompetent mice become infected with P. murina, but
clear the infection without developing lesions. The diagnostic is based on histopathology with specific silver stains and PCR of
lung tissues.
In this study, specified pathogen free and non barrier mouse and rat colonies from nine Latin America animal facilities were
screened. We tested 55 animals representing 27 rats (8 strains) and 28 mice (19 strains), both sexes. The animal ages ranged from
one month to one year and many of them were immunodeficient strains. The mean weight of mice was 20 grams (g) to 35 g and
rats was 110g to 400g. Animals exhibiting or not typical signs of chronic pneumonia were examined. Lung tissue was collected
for DNA extraction prior to PCR and histological preparations. Specific stains were used and consists of Hematoxylin-Eosin
along with Grocott‟s methenamine silver (GMS) and PAS to verify the presence of Pc cysts. DNA was extracted using a QIAmp
DNA mini kit (QIAgen®) and a specific primer sequence directed to 355bp of the rRNA gene as described by Wakefield et al.
(Mol.Biochem.Parasitol. 43(1);69-76,1990). P. carinii was detected by PCR in the lungs of immunocompetent, immunodeficient
and genetic modified animals analyzed. Thus, we have confirmed by PCR 07 rats and 10 mice, representing 30,9% of positive
animals. Immunodeficient animal tissues examined through histopathology was able to demonstrate the presence of Pc organisms
characterized by an alveolar aggregate of cysts and honeycombed foamy material along with an intersticial pneumonia,
confirming the clinical disease.
Conclusions:
The present study confirmed that PCR is able to detect the Pc infection, even the animal have no clinical signs. Screening of
immunodeficient and immunocompetent animals withouth apparent disease may be accomplished by PCR, a sensitive and
specific technique to establish a definitive diagnosis.
Keywords: animal houses, mice, PCR, Pneumocystis carinii, rat
Resumo:10-011
EFFECT OF ADMINISTRATION OF NORETHISTERONE ENANTHATE AND ESTRADIOL VALERATE ON THE
BODY WEIGHT AND FOOD INTAKE IN RATS
Couto, B. B. 1; Henriques, H. N. 1; Boaventura, G. T. 2; Pantaleão, J. A. S. 3; Guzmán-silva, M. A. 1
1
Departamento de Patologia / Faculdade de Medicina, UFF
2
Departamento de Nutrição e Dietética / Faculdade de Nutrição, UFF
3
Departamento Materno-Infantil / Faculdade de Medicina, UFF
Objectives:
Combined progestogen-estrogen preparations have shown marked differences between species and in their responses to diverse
ratios of these two steroids. The aim of this study is to evaluate the effect of a combined injectable hormonal preparation on the
variation in body weight and food intake in rats.
Fifteen Wistar female rats, aged eight weeks and weighing in average 250g were randomly divided in three groups. The group 1
(n=5) received one i.m. injection (0.02ml) of Mesigyna® (norethisterone enanthate and estradiol valerate) and subsequent shots
of sterile water. The group 2 (n=5) received one i.m. injection/week (0.02ml) of Mesigyna®. The group 3 received one i.m.
injection/week (0.02ml) of sterile water. The treatments were performed on days 0, 7, 14, 21, and 28 of the experiment. The body
weight was monitored every seven days and food intake was evaluated every 2-3 days. Mean and standard error of mean were
calculated and data were compared using the ANOVA one way test (significance level at P
Conclusions:
The administration of norethisterone enanthate and estradiol valerate only once or one time/week does not interfere on the body
weight and food intake in rats during 28 days.
Keywords: injectable hormone, food intake, body weight, rat
Financial Support: PROPPI/UFF
Resumo:10-012
BEHAVIOR AND WELFARE OF GROWING RABBITS IN ENRICHED CAGES
Barros, T. F. M. D. ; Moura, A. S. A. M. T. ; Fernendes, S. ; Oliveira, L. V. D. D. ; Siqueira, E. R. D.
FACULDADE DE MEDICINA VETERINARIA E ZOOTECNIA, UNESP
Objectives:
The objective was to evaluate the effect of the environmental enrichment on the beharvior and welfare of growing rabbits in
different social groups: males only, females only, or mixed.
The experiment, which was conducted in two phases, was initiated at weaning with five weeks and was finished at 11 weeks of
age. Seventy two rabbits from the Botucatu genetic group, 36 males and 36 females, were involved in each phase. The animals
were assigned to a completely randomized design, in a 2 x 3 factorial arrangement (with or without cage enrichment and three
types of social groups – males only, females only or both sexes) totaling six treatments with two replicates in each phase. They
were housed in 12 wire cages (80 x 60 x 45 cm), six animals per cage. Two eucalyptus sticks (15 x 3 x 3 cm) were hanged with
wire to the cage ceiling, one on the right side and another on the left side, from 20 to 30 cm from the cage floor. Six cameras were
used for image recording during 24 h, once a week, on weeks 7, 10 and 11. Four black light bulbs were turned on at night. The
following behavioral activities were registered during five out of 20 minutes of recording, every time the occurred: ludic,
agonistic, stereotypes, interaction with enrichment and social interaction. For welfare evaluation, the frequencies of ludic and
stereotyped activities as well as the interaction with the enrichment were used. At 11 weeks the number of skin lesions caused by
fighting in the ears and other body parts was recorded before slaughter, when the brains were collected and weighed. In enriched
cages, females at seven weeks of age and males at 10 and 11 weeks showed higher frequencies of ludic behavior than the
corresponding groups in non-enriched cages. Mixed social groups, both enriched or not, showed higher frequency of exploratory
behavioral activity. However, at seven weeks, mixed groups in non-enriched cages showed higher frequency of social interaction.
Males in enriched cages had heavier brains than those in non-enriched cages (8.52 ± 0.21 vs. 7.86 + 0.22 g, P
Conclusions:
Cage enrichment with eucalyptus sticks improved the welfare of animals by increasing the frequency of ludic behavioral
activities, apart from contributing to the reduction of occurrence of skin lesions caused by older animals‟ fighting.
Keywords: BEHAVIOR, WELFARE, ENRICHED CAGES, ORYCTOLAGUS CUNICULUS, AGRESSION
Financial Support: FAPESP e CNPq
Resumo:10-013
EFFECT OF THE KALLIKREIN AND FACTOR XIIA INHIBITOR ON MICE ARTERIAL THROMBOSIS
Salu, B. R. 1; Brito, M. V. 1; Oliveira, C. 1; Ottaiano, T. F. 1; Vicente, C. P. 2; Werneck, C. C. 2; Sampaio,
M. U. 1; Maffei, F. H. A. 3; Oliva, M. L. V. 1
1
Biologia Molecular/ Universidade Federal de São Paulo, UNIFESP/ EPM
2
Depto de Proteômica/ Universidade Estadual de Campinas, UNICAMP
3
Dpto Cirurgia e Ortopedia / Facul de Med de Botucatu, UNESP
Objectives:
The effect of Enterolobium contortisiliquum trypsin inhibitor (EcTI) on a mouse arterial thrombosis model.
The effect of EcTI was investigated on a mouse arterial thrombosis model. Mice were randomly allocated into three groups
(n=6), being EcTI, 2 mg/kg and 4 mg/kg and control 0.15 M NaCl, administered intravenously, 10 minutes prior to the thrombus
induction. The right carotid artery was dissected free by a lateral incision in the neck and a flow meter probe was positioned
around it, Rose Bengal solution in the concentration of 50 mg/kg was injected in the lateral caudal vein of the animal. After this
procedure, a green laser beam at 540 nm with 1.5MW, positioned at a distance of 6 cm of the incision was applied on the artery.
Light sensitive rose Bengal is degraded, releasing oxygen singlet able to injure endothelium, inducing thrombus formation,
detected by the arrest of the artery flow.
Conclusions:
EcTI in the used concentrations prolonged the time of total occlusion of the carotid artery in 58% and 96%, respectively, in
comparison to that of NaCl group. These data show that EcTI was able to prevent arterial thrombosis in mice. Further studies are
to be performed in order to clarify the mechanisms involved in EcTI action. This study was authorized by the ethics committee,
CEP 0193/06 (UNIFESP)
Keywords: plasma kallikrein , factor XIIa, thrombosis, Enterolobium contortisiliquum , mice
Financial Support: Fapesp,Capes, Cnpq,
Resumo:10-014
ACUTE EXPERIMENTAL TRYPANOSOMA CRUZI INFECTION: ESTABLISHING A MURINE MODEL USING
NON INVASIVE PARAMETERS.
da Silva, D. R. 1; de Castro, S. L. 1; Alves, M. C. D. S. 1; Batista, W. S. 1; de Oliveira, F. S. 2; Oliveira, G.
M. 1
1
Laboratório de Biologia Celular, LBC/IOC - FIOCRUZ
2
Laboratório de Inovações em Terapias, Ensino e Bioprodutos, LITEB/IOC FIOCRUZ
Objectives:
Infection by Trypanosoma cruzi has a higher impact on public health in Latin America countries. Although vectorial and
transfusional transmission has sharply declined in the past 20 years due to policies of the Southern Cone countries, there is still
and a large people number at risk of infection. Since 1909, has description of the basic features of T. cruzi infection in animal
models. However, at the moment, there is not a consensual experimental model and descriptors of clinical symptoms during the
course of murine acute infection. The aim of this study is through noninvasive methodologies described clinical signs presented
by Balb/c lineage infected with Y strain of T. cruzi.
Eight-week-old specific pathogen-free (s.p.f.) male isogenic BALB/c were infected by Y strain of T. cruzi (n10/experiment). Was
evaluated the parasitemia, physical aspects and food consumption, behavioral activity (grooming, immobility and rearing), motor
and exploratory activity by NOLDUS Ethovision XT6 system and body temperature. The results showed an evident change in
infected mice (when compared to non infected) mainly in the third week of infection in physical appearance, as hunched posture,
mainly thoracic region, with closed eyes (in some individuals, conjutivitis was detected), skin peeling and higher piloerection.
Also observed significants decreased motor and exploratory activities, rearing and grooming behaviour, food (chow and water)
consumption and body temperature.
Conclusions:
The applications of non-invasive methodologies are effective in the evaluation and description of clinical signs displayed by mice
during the course of experimental acute infection T. cruzi. From the results of physical activity (motor and exploratory),
behaviour (rearing and grooming), food consumption and body temperature is possible to describe a murine model for Chagas
disease. Correlation of our results with those of the literature suggests that acutely infected mice have similar signals clinical with
toxemia (sepsis).
Keywords: Mice, Trypanosoma cruzi, Non Invasive parameters
Financial Support: FAPERJ and FIOCRUZ/RJ
Resumo:10-015
EFFECTS OF SCAFFOLD BIOSILICATE CONSOLIDATION OF BONE DEFECTS INDUCED
Tim,1; Zambone Pinto, 1; Rossi, B. R. 1; Fernandes, K. 2; Oliani, V. 1; Crovace, M. 1; Peitl, O. P. 1; Rennó,
A. C. M. 2
1
Fisioterapia, Universidade Federal de São Carlos, UFSCar
2
Ciencias Sociais Universidade Federal de São Paulo, UNIFESP/Santos
Objectives:
Biomaterials are defined as materials capable of producing a biological and specific response and an interface between materials
and tissues, forming a link between them, without being toxic or promote immune responses. They have applications in different
areas of medicine such as cardiovascular implants, dental, soft tissue, ophthalmic, orthopedic. Currently, the most studied amomg
bioactive materials is the bioactive glass. Which in contact with living tissues reacts and becomes Hidroxicarbonatoapatita
equivalent to the bone mineral phase. Recently, biomaterials such as scaffolds, are being used in bone regenerative strategies,
since these serve as a guide to the growth of bone tissue and allow the penetration of blood supply and nutrients to the cells that
are within the scaffolds. Within this context, this project aimed to investigate the effects of scaffold Biosilicate ® in the
consolidation of induced tibial bone defect in the rats.
In the study 20 male Wistar rats (3 months ± 250 grams) were used divided into 2 groups of 10 animals each: a control group of
bone defect without any treatment (GC) and a group submitted to the bone defect and implantation of the scaffold Biosilicate ®
(GB). The euthanasia of animals occurred on day 15 after surgery.This experimental study was conducted in accordance with the
ethical principles of animal experimentation and was approved by the Ethics Committee on Animal Experimentation of Federal
University of São Carlos (protocol 002/2009). Morphological analysis revealed that the control group had complete demarcation
of the margin of the lesion, a mild inflammatory infiltration, a mild amount of granulation tissue, a moderate amount of new bone
formation, with little interconnectivity between trabecula, representing an early stage of repair. In the same way, animals treated
with scaffold Biosilicate ® showed mild inflammatory infiltrate, a moderate amount of granulation tissue, bone trabecular mainly
in small amount on the edges of the lesions with concentric organization and a moderate presence of biomaterial. Still, it was
possible to observe new bone formation in both macropores and micropores in the scaffold implantation, we also observed the
presence of bone cells on the surface of biomaterial. However, the results of immunohistochemical analysis demonstrated that the
COX-2 expression was more intense in the group implantation of a scaffold Biosilicate ® when compared to controls groups,
demonstrating the high osteogenic potential of biomaterials. In the same way, the immunoreactivity Runx-2 was moderately
detected in the group scaffold Biosilicate ®, in comparison to the control group.
Conclusions:
From the results obtained in this study, it is possible to suggest that the scaffold Biosilicate ® has a high osteogenic potential and
is capable of stimulating bone repair in the tibia of rats.
Keywords: Bone repair, bioactive material, bone defect
Resumo:10-016
MORPHOFUNTIONAL ACTIONS OF ENVIRONMENTAL ENRICHMENT IN YOUNG AND ADULT RATS.
Lucas, F. F. ; Bandeira, R. ; Batista - Jr, A. M. B. ; Silva, J. C. S. ; Lacchini, S.
Depto. de Anatomia - ICB III, USP
Objectives:
To monitor the physical changes in laboratory rats maintained with environmental enrichment.
We used 20 Wistar rats, between 30 and 75 days old, wrapped and kept in accordance with traditional, therefore, in plastic boxes
under free access to water and diet, with controlled lighting and ventilation. The animals were divided into control group - C (which received no stimulus) and treated group - EA - (received periodical conical tubes of paper, recyclable and nontoxic).
Measures of body weight, consumption of water and food was purchased, it was the stress test on a treadmill and after sacrifice
by guillotine, the tissues and blood were collected for biochemical and meso and microstructure datas. We have seen at the end of
experimental cycle the animals C and EA showed similar values and their water consumption (105 x 101 mL daily), ration (61 x
65 g daily) and body weight gain (39 vs.40 g day) daily. The stress test showed an animal with EA increased physical capacity, as
ran longer (11.91 ï¿½ 3.17 x 7.31 ï¿½2.16 min.), at a higher velocity (1.32 ï¿½ 0.34 x 0.96 ï¿½ 0.25 m / min.) and also the heart
morphometry showed a higher heart weight in treated animals (0.624 ï¿½ 0.558 ï¿½ 0.0484 x 0.0585 g). It is noteworthy that in
the dissection of animals was not apparent accumulation of fat in animals EA, which gives a view to increasing the physical
potential of these animals, and still not even considered, it was noted behavior change their view of facilitating the manipulation
by the attendant.
Conclusions:
Access to environmental enrichment, as in this work, using a simple paper tube proved to be useful in facilitating the
management of decreasing anxiety and increasing the docility animal for the treatment of laboratory tests. However, was variable
in the microenvironment of animal facilities, increasing physical activity and overall energy expenditure of the animal, by contact
with the article by EA, as seen in combining physical activity associated with absent macroscopic deposition of fat and also with
increasing heart weight. Thus, the AE should be viewed as part of processing, but also qualitative and quantitative variable to be
considered in research protocols, such as those who study physical activity or cardiac morphology.
Keywords: LAB ANIMAL, ENVIRONMENTAL ENRICHMENT , MORPHOLOGY, HEART
Financial Support: Funding for assistance to the Department of Anatomy/ USP.
Resumo:10-017
LONG-TERM TREATMENT WITH SURAMIN IMPROVES CARDIAC FIBROSIS IN OLDER MDX MICE
Moreira, D. O; Pereira, J. A. ; Taniguti, A. P. T. ; Ramos, L. A. F. ; Areas, M. A. ; Santo Neto, H. ;
Marques, M. J.
Anatomia, Biologia Celular e Fisiologia e Biofísica, Unicamp
Objectives:
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease that causes respiratory or cardiac failure and
results in death at about 20 years of age. Diaphragm and heart are severely affected and exhibit fibrosis similar to that observed in
DMD. The objective of this study was to investigate the effects of long-term treatment with suramin, an anti-fibrotic drug, on
diaphragm and cardiac muscles of aged mdx mice.
Mdx mice (n=12; 8 months old) received intraperitoneal injections of suramin (60 mg/kg body weight), twice a week for 3
months. Control mdx mice (n=12; 8 months old) were injected with saline. Blood was obtained to determine creatine-kinase (CK)
levels. Forelimb muscle force was measured by a grip strength meter (New Primer). Urine samples were analyzed to determine
total protein and ketones. All mice were subjected to electrocardiogram (ECG) at the beginning and at the end of the
experimental protocol. Our results show that suramin decreased CK (1049,65±134,02 U/L in control vs. 268,03±123,13 U/L in
suramin-mdx, p≤0,05) and reduced the loss of muscle force (0,77±0,12g/g in control vs. 0,97±0,2g/g in suramin-mdx, p≤0,05). In
diaphragm, suramin significantly reduced the area of fibrosis (31,92±5,70% in controls vs. 23,60±6,93% in suramin-mdx,
p≤0,05), increased the percentage of fibers with peripheral nuclei (63,44±7,04% in control vs. 73,67±9,49% in suramin-mdx,
p≤0,05) an reduced central nucleated fibers (33,51±3,38% in control vs. 21,10±3,87% in suramin-mdx, p≤0,05). In the heart,
electrocardiogram analysis showed that suramin led to a 3-fold increase in the S/R amplitude ratio, with a reduction in total
fibrosis (6,0±1% in control vs 2,8±1,5% in suramin-mdx, p≤0,05) and inflammation (12,0±9,5% in control vs 3,0±2,0% in
suramin-mdx, p≤0,05, test T de Student). Urine total protein content was similar in control and suramin-mdx mice (0,3 g/L). Most
urine samples were negative for ketones.
Conclusions:
These results indicate that suramin exerts a positive effect during later stages of the disease by alleviating fibrosis in cardiac and
diaphragm muscles of aged mdx mice.
Keywords: electrocardiogram, fibrosis, heart, mdx, suramin
Financial Support: CAPES-Proex; CNPq; FAPESP
Resumo:10-018
OMEGA-3 AS A POTENTIAL ALTERNATIVE THERAPY FOR DYSTROPHINOPATHIES
Mauricio, A. F. ; Machado, R. V. ; Neto, H. S. ; Marques, M. J.
Depto.de Anatomia,Biol.Cel. e Fisiol. e Biof. - IB UNICAMP, UNICAMP
Objectives:
In Duchenne muscular dystrophy (DMD) and in the mdx mice model of DMD, the lack of dystrophin leads to sarcolemma
changes in permeability and progressive myonecrosis. Muscle fiber damage activates an inflammatory response that plays a
central role in the progression of myonecrosis. Omega-3 has anti-inflammatory activity by inhibiting the production of
proinflammatory cytokines. Previously, we demonstrated that purified eicosapentaenoic acid ameliorated the dystrophic
phenotype of the mdx mice. In the present study, we were interested to see whether over-the-counter omega-3 capsules for human
use would also protect skeletal muscles of the young mdx mice against dystrophy and inflammation.
Mdx mice (14 days of age; n=5), received omega-3 (FDC, EUA; 300mg/kg) by gavage. Control mdx (n=5) received an equivalent
dose of nujol. Animals were treated for 16 days. Forelimb muscle force (grip strength) was evaluated at the beginning and at the
end of the treatment. After treatment, blood was collected to determine creatine-kinase (CK) levels for biochemical analysis of
myonecrosis. Diaphragm (DIA) and biceps brachii (BB) muscles were removed for histological analysis of inflammation with
hematoxylin-eosin and of changes in sarcolemma permeability with Evans blue dye (EBD).
Conclusions:
At the young age studied, control mdx mice did not show loss of forelimb muscle force overtime (1.8 ¡À 0.2 g/g - beginning and
1.7 ¡À 0.2 g/g - end) and omega-3 did not change this profile (2.0 ¡À 0.3 g/g - beginning and 2.0 ¡À 0.2 g/g - end). CK levels
were decreased in omega-3-treated mice, but this was not statistically different from control mice (control: 1284 ¡À 248 U/L vs
treated: 953 ¡À 265 U/L; p > 0.05, Student's t test). Omega-3 significantly decreased the inflammatory area in DIA (control: 13.2
¡À 1.5 vs treated: 3.0 ¡À 2.4; p ¡Ü 0.05) and BB (control: 21.8 ¡À 18.9 vs treated: 4.2 ¡À 2.2; p ¡Ü 0.05). The number of muscle
fibers with peripheral cell nuclei, indicative of fibers that did not undergone muscle degeneration, was increased by omega-3 in
both DIA (control: 67.4 ¡À 3.5 vs treated: 79.5 ¡À 6.0; p ¡Ü 0.05) and BB (control: 69.0 ¡À 7.7 vs treated: 77.0 ¡À 9.1; p ¡Ü 0.05)
muscles. Central nucleated fibers, indicative of muscle degeneration-regeneration, did not change in omega-3-treated compared
to control mice (DIA control: 12.2 ¡À 3.5 vs DIA treated: 12 ¡À 3.5; BB control: 20.4 ¡À 7.7 vs BB treated: 19.6 ¡À 9.1; p >
0.05). Omega-3 lead to a decrease in the number of EBD-positive fibers in DIA (control: 20.3 ¡À 8.2 vs treated: 10.2 ¡À 8.2; p ¡Ü
0.05) and BB (control: 10.4 ¡À 4.6 vs treated: 3.3 ¡À 2.8; p ¡Ü 0.05). The use of over-the-counter omega-3 capsules decreased
inflammation and protected dystrophic fibers against changes in permeability and myonecrosis. This suggests that omega-3 may
be potentially useful for preclinical trials of dystrophinopathies.
Keywords: Duchenne muscular dystrophy , inflammation, mdx, muscle, omega-3
Financial Support: CAPES; CNPq; FAPESP
Resumo:10-019
VARIATION OF BONE STRENGTH IN FEMURS OF RATTUS NORVEGICUS ALBINUS FEMALES OF 2, 6, 12
AND 14 MONTHS OF AGE
Apolinário, J. C. 1,1,1; Yamamoto, A. P. M. 2; Coelho, W. M. D. 3; Vendrame, K. E. 4; Florindo, P. L. 5;
Louzada, M. J. Q. 6
1
Ciências Fisiológicas , FOA-UNESP Araçatuba
2
Ciências Fisiológicas, FOA-UNESP Araçatuba
3
Faculdades de Ciências Agrárias e Veterinárias, FCAV-UNESP
4
Clínica, Cirurgia e Reperodução Animal, FMVA-UNESP Araçatuba
5
Apoio, Produção e Saúde Animal , FMVA-UNESP Araçatuba
6
Apoio, Produção e Saúde Animal , FMVA-UNESP Araçatuba
Objectives:
Knowing the variation of biophysical parameters of laboratory animals used in testing during development is important because
of its routine use in research projects, so this study was conducted on the determination of bone strength in femurs of Rattus
norvegicus Albinus, Wistar lineage, females, 2, 6, 12 and 14 months of age.
The bone strength, established by the Stiffness and Maximum Force allowed by the head of the femur was analyzed by
destructive mechanical testing. The results demonstrated for the Maximum Force (N) admitted to the average femoral head and
standard deviation of 123.75±5.71, 138.33±6.61, 141.45±10.61, 128.16±6.96 for groups of 2, 6, 12 and 14 months respectively.
And for Stiffness (N / mm) of the femoral head 87.09 ± 8:51, 185.53 ± 18.60, 240.56 ± 29.27, 207.63 ± 2:45 for the groups of 2,
6, 12 and 14 months respectively. It was used ANOVA statistical analysis and Tukey test. There was statistical difference in
maximum Force between groups of 2 and 6 months (p <0.01).
Conclusions:
These results provide an idea that bone strength in Wistar female rats increases during the development of the animal, peaking at
12 months of age, being reduced to 14 months of age compared to 12 months.
Keywords: bone strength, maximum force , stiffness
Financial Support: CNPq, FAPESP, CAPES
Resumo:10-020
THE EFFECT OF AMMONIA AND METHYLMALONATE ON MITOCHONDRIAL VIABILITY IN BRAIN
CORTEX OF MICE
Ribeiro, L. R. ; Della-pace, I. D. ; Stamm, D. N. ; Gerbatin, R. R. ; Mota, B. C. ; Rodrigues, F. S. ; Royes,
L. F. F.
Universidade Federal de Santa Maria, UFSM
Objectives:
Mitochondrial enzyme methylmalonil-CoA mutase (MCM, EC 5.4.99.2), is responsible for converting methylmalonyl-CoA, that
is generated during the metabolism of amino acids isoleucine, methionine, threonine and valine, and odd-chain fatty acids and
cholesterol, to succinyl-CoA. Hereditary methylmalonic acidemias comprise a group of inborn autosomal recessive genetic
disorders characterized by deficient activity of the MCM, or defects in the synthesis of 5‟-deoxyadenosylcobalamin, the cofactor
of MCM. The inhibitory effects of the enlarged mitochondrial pool of acyl CoA esters on N-acetylglutamate synthetase,
consequently decrease the conversion of ammonia to urea, causing hyperammonemia in approximately 70% of patients. Studies
show that ammonia and methylmalonate reduces mitochondrial efficiency which results in excessive free radicals generation and
a deficient antioxidant system, creating a vicious circle that has been associated with an increased risk of seizure recurrence and
central nervous system degeneration as observed in patients with methylmalonic acidemia and/or hyperammonemia. Based on
these evidences, it would be interesting to verify the effect of ammonia and MMA on mitochondrial viability in the cerebral
cortex of mice.
Adult male Swiss mice (25-35 g) kept under controlled environmental conditions with free access to food and water were used in
all experiments. Mice were underwent to surgery for implantation of a cannula in the right brain ventricle and after a recovery
period of five days, the animals were subcutaneously injected with sodium chloride (NaCl, 0.9%) or ammonium chloride
(NH4Cl, 6 mmol/kg) and received intracerebroventricular (i.c.v.) injection of vehicle (NaCl, 0.9%) or MMA (0,66 μmol/2 μL) 5
minutes later. Fifteen minutes later, the animals were killed by decapitation and the brain cortex were removed and homogenized
to perform the biochemical analysis. Two-way ANOVA revealed a significant reduction in succinate dehydrogenase (SDH)
activity in animals injected with NH4Cl + MMA [F(3,28)=3,80; p
Conclusions:
Since it has been suggested that MMA induces convulsions through impairment of mitochondrial function and that both ammonia
and MMA increase free radical generation, we concluded that in the presence of ammonia may occur potentiation of
mitochondrial inefficiency and the consequent increase in the levels of free radicals, leading to central nervous system damage, as
observed in patients with methylmalonic acidemia.
Keywords: Ammonia, Brain Cortex, Methylmalonate, Mice, Mitochondria
Financial Support: CAPES, REUNI, CNPq, PIBIC
Resumo:12-001
THREE-DIMENSIONAL CULTURE OF MESENCHYMAL STEM CELLS EXTRACTED FROM RABBIT ADIPOSE
TISSUE WITH THE USE OF BIONEXT®, A CELLULOSE MATRIX.
Sousa, I. O. P. 2,1; Piñero-eça, L. 1; Vaccari-mazzetti, M. 1; Bruni-piana, A. M. 1; Ferreira, A. T. 2,1; Pontes,
P. 2
1
Instituto de Pesquisas de Células Tronco, IPCTRON
2
Universidade Federal de São paulo, UNIFESP
Objectives:
The pluripotentiality of mesenchymal cells, together with their ease of acquisition and their abundance in adipose tissue,
encouraged us to study the fixation and maintenance capacity of these cells, evaluating their morphology, proliferation and
viability in two- and three-dimensional culture systems.
Adipose tissue was collected from five rabbits (n = 5) for extraction of mesenchymal cells, the counting and viability test with
tipan blue, these cells was performed later using methods two and three dimensional culture(with cellulose matrix) in DMEM
containing 10% Serum Fetal bovine (FBS) 1% penicillin / streptomycin (PE). To show that they were of mesenchymal cells
induced to differentiate into adipocytes, chondrocytes and middle prefabricated Advanced Stem Cells - HyClone to prove their
multipotency. The growth of mesenchymal cells was observed within 20 days and the quantification and viability rate compared
statistically by histogram. For statistical analysis we used the Student t test.
Conclusions:
The simulation of a three-dimensional cell culture showed that this type of cultivation using the biomaterial of cellulose for
packaging cells, provided an environment very similar to that found in tissues in vivo, because there was a proliferation of
mesenchymal cells three times higher than in medium-dimensional.
Keywords: mesenchymal stem cells , rabbit, cellulose matrix, Three-dimensional culture
Financial Support: IPCTRON
Resumo:12-002
MICRORNA-208 ARE INVOLVED IN REGULATION OF CARDIAC HYPERTROPHY INDUCED BY AEROBIC
TRAINING IN RATS
Soci, U. P. R. 1,2; Fernandes, T. 1; Amadeu, M. A. 1; Hashimoto, N. Y. 1; Rosa, K. T. 2; Mota, G. F. 1;
Irigoyen, M. C. 2; Oliveira, E. M. 1
1
Escola de Educação Física e Esporte - USP, EEFE-USP
2
Faculdade de Medicina- USP, FM-USP
Objectives:
MicroRNAs (miRNA) are small non coding RNAs that regulate post-transcriptional expression of their target genes. The
miRNA-208 is exclusively expressed in heart and regulates gene expression of slow myosin heavy chain (MHC). We investigated
the role of miRNA-208 in cardiac hypertrophy (CH) induced by aerobic exercise training (TF).
Female Wistar Rats (n=21) were randomized into three groups: sedentary (S), Trained 1 (T1): swimming sessions of 60min, 5
days/week, during 10 weeks, with caudal dumbbells weighing 5% of body weight. Trained 2 (T2): similar to T1 until 8th week.
On the 9th week rats swan twice/day and on the 10th week 3 times/day. Were assessed: 349 cardiac microRNAs by microRNA
array, heart rate (HR) e blood pressure (BP) by caudal pletismography, VO2max by ergoespirometry and ventricular function and
CH by echocardiography, CH by LV/BW ratio (mg/g) and cardiomyocyte diameter, gene expression of pathological markers of
CH (ANF, skeletal α-actin; α/β-MHC) and miRNA-208 by Real-time PCR. P
Conclusions:
Aerobic Training promoted physiological CH, decreased MiRNA-208 and β-MHC gene expression. The inhibition of miR-208
exercise-induced can be a viable therapeutic strategy to repress the β-MHC gene expression in pathological CH. These results
open perspectives to research therapeutic approaches involving modulation of miRNAs.
Keywords: MicroRNAs, Hipertrofia Cardíaca, Treinamento Aeróbio, Beta-Miosina de Cadeia Pesada, Marcadores Patológicos
Financial Support: FAPESP/CAPES
Resumo:12-003
VINBLASTINE EFFECTS ON THE PROLIFERATIVE INDEX IN THE ODONTOGENIC REGION OF THE RAT
INCISOR SUBMITTED TO A HYPOFUNCTION CONDITION.
Carmo, E. R. D. 1; Omar, N. F. 2; Novaes, P. D. 2; Gomes, J. R. 1,1
1
Universidade Estadual de Ponta Grossa, UEPG
2
DEPARTAMENTO DE MORFOLOGIA, FOP-UNICAMP
Objectives:
To evaluate the vinblastine effects on the cell proliferation in rat incisor tooth under a hypofunctional condition.
Rats were divided in two groups: hypofunctional control (group I) and hypofunctional vinblastine (group II). Group I received an
intraperitoneal injection of saline as drug vehicle and Group II received a single intraperitoneal vinblatine injection (2mg/Kg) on
8o day of the experiment. Groups I and II had their right lower incisors intact which were considered in a normal eruption while
that in both groups the left inferior incisor were shortened at intergingival level using a diamond high-speed rotating instrument
each two days until 12 day producing the hypofunctional condition eruption. On the 12o day all groups received a single
intraperitonial injection of 5-bromo-2-deoxyuridine 60 minutes before death. All rats were killed by cervical dislocation and the
hemimandibles were dissected, immersed in 4% paraformaldehyde, decalcified in 4.13% EDTA, reduced to small fragments
containing the apical end of the incisor which were placed into Paraplast. Cross-sections (5µm) of the odontogenic region were
incubated with anti-BrdU monoclonal antibody (1:20) and after the incubation with DAB the sections were counterstained with
hematoxylin. Cell labeled and no labeled were counting in three sections of the right and left incisor per group to obtain the
proliferative index which was analyzed using the ANOVA and Tukey post-test. A significant reduction in proliferative index was
observed in the shortened incisor (left) submitted to the vinblastine (group II) when compared to all the intact incisors (right) in
both groups.
Conclusions:
In a hypofunction condition the rat incisor double its eruption rate. We conclude that since vinblastine which is a stathmokinetic
drug decreased the cell proliferation in odontogenic region of the rat incisor in a hypofunctional eruption condition it suggested
that cell proliferation may be an important factor involved in the eruption process.
Keywords: Erupção, Incisivo , Proliferação, Rato, Vimblastina
Financial Support: Fundação Araucaria-PR
Resumo:12-004
ARUCANOLIDE INDUCES APOPTOSIS IN U-118 MG GLIOBLASTOMA CELLS AND ACTIVATES CASPASES
Marchetti, G. M. 1,2,3; Melo-lima, S 3; Silva, K. A. 4; Foglio, M. A. 4; Mollinedo, F. 3; Carvalho, J. E. 1,2
1
Cellular and Structural Biology/ Biology Institute, IB-UNICAMP
2
Pharmacology and Toxicology Department/CPQBA, DFT-CPQBA
3
Cancer Research Centre/Universidad de Salamanca, CSIC-USAL
4
Phytochemistry Department/CPQBA, DFito-CPQBA
Objectives:
Arucanolide is a germacranolide obtained from Calea pinnatifida aerial parts. This compound has been shown to have
antiproliferative activity and to induce apoptosis in human acute myeloid leukemia HL60 cells (J. Pharmacol. Sci., 97: 242,
2005), but its effects on other cancer cell types have not been tested yet. A glioma is a malignant brain tumor originating in glial
cells; these cells constitute the sustentacular tissue that surrounds and supports neurons in the central nervous system. The U-118
MG cell line derives from a malignant glioma, classified as a grade IV astrocytoma (glioblastoma multiforme). Programmed cell
death is essential for the development and maintenance of multicellular organisms, and apoptosis is one of the mechanisms by
which cell death might be executed.
U118 cells were seeded in 6-well plates and treated with arucanolide 12 &muM, diluted in DMSO, during 6, 12 or 24 hours.
Drug-untreated control cells incubated with the same volumes of DMSO were run in parallel. After treatment, cells were fixed
overnight in 70% ethanol at 4¨¬C, and then incubated with 1 mg/ml RNAse A and 5 &mug/ml propidium iodide (PI) for 1 hour
at room temperature in the dark, and analyzed for cell cycle with a Becton Dickinson (San Jose, CA) FACSCalibur flow
cytometer. Quantitation of apoptotic cells was calculated as the percentage of cells in the sub-G1 region (hypodiploidy) in cell
cycle analysis. To evaluate mitochondrial transmembrane potential and the generation of reactive oxygen species (ROS), cells
were resuspended in PBS with 20 nM DiOC6 (3,3¡Ç-dihexyloxacarbocyanine iodide) and 2 &muM DHE (dihydroethidine)
during 15 minutes at 37¡ÆC, and then analyzed by flow cytometry. Cell cycle analysis revealed a progressive increase in the subG1 population following arucanolide treatment: from 2% in the untreated control cells to 17.0 ± 1.0 % and 38.2 ± 1.3 % after 12
and 24 hours of treatment, respectively, indicating the induction of apoptosis following arucanolide treatment. No apoptosis was
detected before 6 hours of arucanolide incubation. U118 cells treated for 24 hours showed an increase in the generation of ROS
(15.3 ± 3.0 %), but no loss of mitochondrial transmembrane potential was detected. For protein extraction, U118 cells were plated
in 10-cm culture plates and treated with arucanolide 12 &muM during 1, 6, 12 or 24 hours. Western blotting of the extracted
proteins revealed activation of caspase-3 and caspase-9, as well as cleavage of the caspase-3 substrate PARP, after 6 hours of
arucanolide treatment.
Conclusions:
The results indicate that arucanolide induces apoptosis in U118 glioblastoma cell line, as assessed by DNA degradation in cell
cycle analysis, and caspase activation. The generation of ROS and caspase-9 activation suggest that both ROS and the intrinsic
mitochondrial pathway could be involved in the proapoptotic action of arucanolide on U118 cells. Experiments are in progress in
order to unveil its mechanism of action.
Keywords: Arucanolide, Apoptosis, Glioblastoma
Financial Support: FAPESP, CNPq, MCI España, Junta de Castilla y León.
Resumo:12-005
HUMAN MENSTRUAL BLOOD-DERIVED MESENCHYMAL CELLS: CHARACTERIZATION AND EVIDENCE
FOR ENHANCED RESISTANCE AGAINST OXIDATIVE STRESS
Asensi, K. D. 1; Fortunato, R. S. 1; Souza, E. C. 1; Rodrigues, D. C. 1; Kasai-brunswick, T. H. 1; Silva dos
Santos, D. 1; Carvalho, A. C. C. 1,2; Carvalho, D. P. 1; Carvalho, A. B. 1; Goldenberg, R. C. S. 1
1
2
Instituto Nacional de Cardiologia, INC
Objectives:
Mesenchymal stem cells transplantation (MSCs) prove to be useful to treat diseases in which tissue damage is linked to oxidative
stress (OS). Recently, it has been demonstrated that stem cells can be isolated from menstrual blood. This is an important new
source of stem cells, since they can be easily obtained, do not require invasive procedures, and availability is virtually endless.
Thus, the aim of this work was to isolate, characterize, differentiate menstrual blood-derived stem cells (MBMC) and evaluate
whether MBMC can manage oxidative stress (OS).
Human menstrual blood was collected from eleven healthy female subjects when menstrual flow initiated. All experiments below
were approved by our local institutional review board (HUCFF, UFRJ, RJ, Brazil) protocol no: 056/09. All donors signed an
informed consent form. Human menstrual blood-derived cells presented a subpopulation of adherent cells with spindle-shape
morphology. Population doubling time was 38.0 ± 1.9 hours of women not using contraception while MBMC from women who
use contraceptives double their population every 35.9 ± 1.7 hours, with no statistical difference between samples. After five
passages, the presence of surface markers was evaluated by flow cytometry. These cells were positive for the mesenchymal cell
markers CD90 (93.3%), CD73 (85.5%), CD105 (100%); and negative for the hematopoietic markers CD34, CD45, CD14, CD19
and HLA-DR, endothelial markers CD31, CD133. At the mRNA level, MBMC expressed the core embryonic stem (ES) cell
regulators Oct4, Sox2, nanog and Klf4. At the protein level, these cells stained positive for the pluripotency markers SSEA-4,
TRA1-60 and TRA1-81. Moreover, when adipogenic and osteogenic differentiation was induced, these cells formed fat vacuoles
demonstrated by Oil Red O staining and showed calcium deposits when stained with Alizarin Red, respectively. MBMC were
cultured with increasing H2O2 concentrations and its viability was evaluated by MTT assay. These cells showed high resistance
to OS-induced death (IC50: 1.8mM). Moreover, MBMC can produce three times more extracellular H2O2 than iPS-MBMC
(induced pluripotent stem cell from MBMC using retroviral vectors containing human OCT4, SOX2 and KLF4 genes) and H9
human embryonic stem cells (2.37; 0.74; 0.74 nmol H2O2/h/105 cells), respectively. This extracellular production of H2O2 by
MBMC was inhibited by 1µM of diphenyliodonium (DPI), indicating that some NADPH oxidases are responsible for this
production. To measure intracellular ROS production, cells were incubated with 10 µM of CM-H2DCFDA, an intracellular
fluorescent probe, and signal was acquired by flow cytometry. The mean fluorescence intensity was two fold higher in MBMC in
comparison with iPS-MBMC and H9.
Conclusions:
In conclusion, MBMC are highly proliferative, express mesenchymal stem cell surface markers and differentiate into osteocytes
and adipocytes in vitro, indicating that they are multipotent mesenchymal cells. These cells express pluripotency markers,
suggesting that they are a powerful source of progenitors and could potentially be differentiated in other mesodermal tissue types.
Moreover, these cells presented high resistance to OS and might be considered a tool for cell therapy strategy aimed to treat
patients with diseases in which onset and progression is associated with OS.
Keywords: Menstrual blood stem cells, Mesenchymal stem cells, Resistance against oxidative stress.
Financial Support: CNPq, Capes, FAPERJ, Ministério da Saúde
Resumo:12-006
ACTION OF HYDROALCOHOLIC EXTRACT OF SOLIDAGO CHILENSIS MEYEN (ASTERACEAE) ASSOCIATED
WITH MICROCURRENT ON THE REPAIR OF EXPERIMENTAL SKIN INJURY IN ADULT WISTAR RATS.
Gameiro, E. B. ; Felizatti, R. ; Neves, L. M. G. ; Esquisatto, M. A. M. ; Gaspi, F. O. D. G. D. ; Mendonça,
F. A. S. ; Santos, G. M. T. D.
Programa de Pós-graduaçao em Ciências Biomédicas, PPGCB-UNIARARAS
Objectives:
The use of herbal medicines in the repair of cutaneous wounds has been known in the literature (Clin Dermatol. 27:502, 2009), as
well as the action of microcurrent in the same lesions (Low Extrem Wounds Int J 4:23, 2005). Yet little is known about the
combination action of these agents in skin excisional lesion. Thus, the aim of this study was to evaluate the effects of topical
application of hydroalcoholic extract of Solidago chilensis leaves and associated with the application of microcurrent (MC) on
the repair of surgically induced skin wounds in rats.
The hydroalcoholic extract of S. chilensis (EHS) was obtained by maceration in 70% hydroalcoholic solution, followed by
filtration and evaporation. We used 48 male Wistar rats 120 days old. The animals were anesthetized (xylazine hydrochloride (0.2
ml/kg) and ketamine hydrochloride (1 ml/kg) and underwent surgical punch (8 mm) lesion in the skin of the back and divided
into groups: Group A – Control, Group B - treated MC (10 μA/2 min/day), group C - treated with EHS, and Group D - treated
with EHS and microcurrent. Three animals from each group were sacrificed at 2, 6, 10 and 14 days for removal and preparation
of tissue for structural and morphometrical analysis. In each experimental day, were measured in the tissue repair region: the total
area of reparative tissue (x 10000 μm2), number of inflammatory and fibroblastic cells (N in 10000 μm2), total number of vessels
(N in 10000 μm2) and the thickness of the epithelium (μm). Data were compared by ANOVA and Tukey post-test (p
Conclusions:
Microcurrent application alone or combined with the extract hydroalcoholic of S. chilensis exerted significant effects on wound
healing in the experimental model used. This was probably due to the efficacy of microcurrent application since the extract alone
did not significantly accelerate the healing process. S. chilensis extract probably participates in the wound healing process as a
result of its anti-inflammatory properties.
Keywords: Injury, Microcurrent, Repair, Skin , Solidago
Financial Support: Fundaçao Herminio Ometto
Resumo:12-007
EXPRESSION OF CONNEXINS AND CYCLINS IN NEURODEGENERATION CAUSED BY MECHANICAL
TRAUMA IN THE RAT RETINA
Higa, G. S. V. 1,2; Paschon, V. 1,2; Casado, O. A. N. 1; Kihara, A. H. 1
1
Núcleo de Cognição e Sistemas Complexos, UFABC
2
Depto. de Fisiologia e Biofísica - ICB, USP
Objectives:
The signaling in the nervous system can occur by direct coupling between cells via gap junctions channels (GJ). These channels
allow the passage of molecules up to 1 kDa and are composed of protein subunits called connexins (Cxs). The cellular
communication via GJ plays an important role during development and in visual signaling. Moreover, the cell coupling provided
by JCs has been linked to processes of survival / cell death. Similarly, cyclins and cyclin dependent kinase (CDK), besides its
classical role in regulating cell cycle and differentiation, are involved in neurodegenerative processes. Recent studies have
observed the re-entry into cell cycle in post-mitotic neuronal cells undergoing apoptosis. In this context, the objective of this
work is to evaluate changes in gene expression of cyclins D1, E1 and B1 and Cxs 36 and 43, as well as protein modulation of Cxs
at different times after the induction of neurodegenerative processes.
The induction of cell death was performed using the technique of mechanical focal lesion through a needle in the visual system,
specifically in the retina of rats (Rattus novergicus) Long-Evans strain.Our experiments were conducted in accordance with NIH
and Institute of Biomedical Sciences guidelines.Using the technique of polymerase chain reaction in real time (Real-Time PCR)
we analyzed the changes of gene expression at different times of exposure to injury for Cxs (Cx36 and 43) and cyclins (D1, E1
and B1). To evaluate the significance of the results we use the statistical paired t test (P ¡Ü 0.05, n = 8 ~ 10). Preliminary results
of Real-time PCR revealed a modulation of gene expression of Cx36 but not for the different post-injury examined. The Cx43
showed an increase of transcripts after 3 days of injury (delta ct= 2,83, Erropad= 0,39). Cyclin D1 showed a significant
modulation after 1 (delta ct = 2.91, Erropad = 0.44), 3 (delta = 4.14 ct, Erropad = 0.47) and 7 (delta ct = 3.29, Erropad = 0.31)
days post injury. Cyclin E1, in turn, showed a significant modulation (delta ct = 1.12, Erropad = 0.38), 3 (delta ct = 2.25, Erropad
= 0.41), and 7 (delta ct = 1.75, Erropad = 0.35), days post injury. The cyclin B1 showed a significant modulation after (delta ct =
2.35, Erropad = 0.52), 3 (delta ct = 5.54, Erropad = 0.54) and 7 (delta ct = 4.09, = Erropad 0.34) days post injury. Since the
analysis of immunohistochemistry demonstrated a modulation of the spatiotemporal Cx36 in different areas of injury in different
times of exposure to injury. The Cx43 in turn, showed an apparent increase of its expression in focus and the penumbra of the
lesion at 1, 3 and 7 days post-injury.
Conclusions:
Connexins and cyclins examined showed increased gene expression after induction of the neurodegenerative process induced in
the rat retina. Connexins showed apparent change in its distribution in response to injury, suggesting that changes in the pattern of
coupling.
Keywords: Ciclina, Conexina, Junções Comunicantes, Neurodegeneração, Retina
Financial Support: FAPESP; CNPQ; UFABC.
Resumo:12-008
EFFECT OF SULFASALAZINE AND VALPROIC ACID ON INHIBITION OF HUMAN MULTIFORM
GLIOBLASTOMA VIABILITY
1
Garcia, C. G. 1; Alves, Tr. 2; Kahn, Sa. 2; Moura Neto, V. 2; Cossenza, M1
DEPT FISIOLOGIA FARMACOLOGIA,UNIVERSIDADE FEDERAL FLUMINENSE, UFF
2
ICB, UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, UFRJ
Objectives:
Human multiform Glioblastomas (GBMs), aggressive cerebral tumors (WHO IV degree), lead patients to death in 12-36 months
with bad prognosis due to tumor‟s resistance to chemo and radiotherapies with no therapeutic alternatives. Overexpression of the
aminoacid transport system xc-, an exchanger of cystine (uptake) and glutamate (release), consisted on catalytic (xCT) and
regulatory (4f2HC) subunits, has been described. Such exchange is a rate-limiting process on gluthatione (GSH) synthesis, an
essential molecule involved in protection against oxidative stress, proliferation and DNA synthesis. Sulfasalazine (SAS) is an
antinflamatory drug recently described as an inhibitor of xc-, leading to inhibitory effects on GSH synthesis. Valproic acid (VPA)
is an anticonvulsivant drug largely used clinically. Recently, it was observed an inhibitory activity on histone deacetylase(HDAC)
induced by VPA. This activity has correlations to some tumor‟s viability decrease, such as GBM. This work had goals on
verifying the antineoplasic potential of SAS and VPA isolated or in combination on in vitro models of GBM cell lines.
Human GBM cells (CONEP nº 2340) were harvested and treated with different concentrations of SAS, VPA or both drugs for
24h or 48h, followed by cell counting with Trypan Blue or Western blotting assay to detect xCT protein levels. Results: GBM
cell lineages (C6, U87, GBM-95 and GBM-02) revealed highest levels of xCT compared to mouse‟s cortical astrocytes. Neurons
and microglias exhibited undetectable levels of xCT. GBMs treated with SAS 1mM presented a decrease of approximately 60%
(24h) and 75% (48h) in viability and VPA 1mM did not present significant effect in 24h, although its inhibitory effect in 48h was
of 75%. When GBMs were treated with both drugs, the inhibitory effect was of approximately 90%. Both drugs isolated or in
conjunct, were tested in a dose-dependent manner.
Conclusions:
Results suggest that xc- system is important to maintain GBM‟s viability, and that SAS and VPA have summative effect in a
dose- and time-dependent manner on the inhibition of this viability.
Keywords: Glioblastoma, Sulfasalazine, Valproic Acid
Financial Support: PROPPI-UFF, AGIR-UFF, INNT, FAPERJ
Resumo:12-009
CONNEXIN MODULATION DURING THE DEVELOPMENT OF THE CHICK OPTIC TECTUM (GALLUS
GALLUS)
Paschon, V. 1,2; Higa, G. S. V. 1,2; Kihara, A. H. 1,2
1
Universidade Federal do ABC, UFABC
2
Universidade de SÃ£o Paulo, ICB - USP
Objectives:
GJ channels couple adjacent cells, allowing transfer of second messengers, ions and molecules up to 1 kDa. These channels are
composed by a multigene family of integral membrane proteins called connexins (Cx). In the nervous system, besides being
essential circuit element in the visual processing, GJ channels also play important roles during the development. Although the Cx
expression was described in the retina (Dev. Neurobio. 1287:02, 2008), their expression in the visual areas of the chick brain is
largely unknown. The aim of this study was elucidate how GJ channels act during the diverse periods of development of the chick
optic tectum (OTe).
Experiments were carried out with Gallus gallus, ranging from embryonic day 5 (E5) to adult (E5, E10, E15, and P15), n=5.
Animals were euthanized with an overdose of ketamine and xylazine. The chick brains were homogenized for different
methodologies, such as Western Blot and real-time PCR. These experiments were conducted in accordance with NIH and
Biomedical Sciences Institute / USP guidelines. By using purified antibodies raised against chick Cxs, we analyzed the protein
levels of Cx36, Cx43 and Cx45 during the chick OTe development. The Cx43 was mostly expressed in the adult chick OTe (P15
ï¿½ 100%) while in E5 it was only 15-30% compared with control P15 (P
Conclusions:
Our results suggested an important modulation of Cxs during the development of the chick OTe. Cx45 may have important roles
during the neurogenesis and in the control of proliferative processes. On the other hand, Cx36 and Cx43 may have important
roles after cell differentiation.
Keywords: Connexins, Gap Junctions, Development, Optic Tectum, Visual System
Financial Support: FAPESP, CNPq e UFABC.
Resumo:12-010
MYC TRANSCRIPTION FACTORS REGULATE RETINA AND LENS DEVELOPMENT
Almeida, ; Martins,
Instituto de Ciências Biomédicas, UFRJ, UFRJ
Objectives:
N-myc, c-myc and L-myc are members of a proto-oncogene family encoding basic helix-loop-helix (bHLH) proteins that regulate
gene expression through a variety of mechanisms, including transcriptional activation and repression. By regulating the
expression of multiple target genes, Myc family proteins play crucial roles in cell proliferation, differentiation and survival during
development, adulthood and tumorigenesis. Using Nestin–Cre transgenic mouse line it was observed that N-myc regulates cell
proliferation in an autonomous manner in developing retina. Interestingly, N-myc inactivation also affected eye growth. (Genes
Dev. 22(2): 179–193, 2008). In this work, we studied how the developmental growth of various eye tissues is regulated by both cmyc and N-myc genes using different Cre-lines.
Initially, we tested whether the inactivation of Myc genes in mice Cre lines in which the Cre recombinase is expressed in the
developing retina would affect eye growth as previously observed. We generated Pax-6-Cre; N-myc lx/lx and Chx10-Cre; N-myc
lx/lx mice and measured eye, retina and lens volume at different developmental stages. No difference in the volume of these
ocular structures was observed. We also generated mice with double-deficient retinas by inactivating c-myc and N-myc (Pax-6Cre; N-myc lx/lx; c-myc lx/lx and Chx10-Cre; N-myc lx/lx; c-myc lx/lx). Again, inactivation of both Myc family members did
not affect eye or lens growth. To test whether the eye hypotrophy phenotype observed in Nestin-Cre mice may result from
inactivation of Myc genes in developing lens tissue, we used another mouse Cre-line, in which genetic inactivation occurs
exclusively in the lens. In the Le-Cre; N-myc lx/lx mice, inactivation of N-myc in the lens severely affected eye and lens growth.
N-myc-deficient lens are 50% smaller then as compared to their respective controls.
Conclusions:
Inactivation of both c-myc or N-myc by Nestin-Cre severely affected eye and lens growth. For our surprise, retina-specific
inactivation of Myc genes by the use of Pax6-Cre and Chx10-Cre lines did not affect eye growth. Interestingly, we have found
that lens-specific genetic inactivation of N-myc replicates the observation of eye hypotrophy previously observed in Nestin-Cre
mouse line. These data reveal for the first time a role of Myc genes in lens development in vivo and confirm previous findings
that both N-myc and c-myc are important regulators of eye development.
Keywords: CNS, Cell cycle, Eye , Morphogenesis, Oncogenes
Financial Support: IBRO, IRRF, FAPERJ, CNPQ
Resumo:12-011
GROWTH OF GLIAL CELLS IN MECHANICAL-INDUCED LESIONS OF RETINAL CELL CULTURES: RELEASE
OF ATP AND INVOLVEMENT OF GLUTAMATE RECEPTORS
Guimarães, G. H. C. ; Loiola, E. C. ; Ventura, A. L. M.
Neurobiology Department, UFF
Objectives:
Previous data from our laboratory demonstrated that mechanical injury of chick embryo retinal monolayer cultures induces glial
growth over the wounded area that is dependent on ATP signaling. Cultured Müller cells can release ATP by activation of
glutamate receptors (Neurochem. Int. 58; 414, 2011). Here, we investigated if mechanical lesion of retinal cell cultures modify
extracellular ATP levels and if the growth of glial cells over the healed area is related to glutamatergic signaling.
Retina cell cultures of 8-day-old chick embryos incubated for 7 days (E8C7) were rinsed twice with Hanks‟ solution and
incubated for different periods of time when aliquots of the extracellular medium were collected (basal levels). Thereafter,
cultures were scratched once with a pipette tip and incubated again for different periods of time (stimulated levels). Some cultures
were rinsed after wounding and incubated during five min. ATP content of the samples were determined by bioluminescence
using the ATP determination kit and following the manufacture‟s instructions (Invitrogen). For estimation of cell growth,
wounded cultures in E8C7 were photographed along the wounded areas during 3 days. The area devoid of cells was measured
(mm² x 100) through the SCION IMAGE program. In cultures that were washed after injury with the pipette tip, no significant
increase in extracellular ATP levels was observed at any time studied. However, in cultures where no change of medium was
performed, a significant increase in extracellular ATP levels was observed 5 min after they were wounded with the tip (%
control: 291.7 ± 82.0%; p
Conclusions:
Our results suggest that ATP is released when retinal cultures are mechanically injured. Although the glutamate antagonist
DNQX did not block ATP release after lesion, our data suggest that glutamate receptors are also involved in the growth of glial
cells over the area of lesion.
Keywords: ATP, Cell Growth, Glutamate Receptors, Mechanical Lesion
Financial Support: CNPq, FAPERJ, MCT-Pronex, PROPPi-UFF, CAPES
Resumo:12-012
EVIDENCES FOR ACTIVATION OF UNFOLDED PROTEIN RESPONSE AFTER AXOTOMY OF RETINAL
GANGLION CELLS: ANALYSIS OF CHOP/GADD153 AND JNK.
Vieira-barroso, L. ; Gonçalves, B. S. ; Ribas, V. T; Torres, D. N. M. ; Chiarini, L. B.
Objectives:
Studying the mechanisms that control retinal ganglion cell death is relevant for neurodegenerative conditions of the inner retina,
such as glaucoma. It has been described that neonatal retinal ganglion cell death induced by axotomy is blocked by anisomycin,
an inhibitor of protein synthesis. This result indicates that transcription factors are involved with ganglion cell death induced by
axotomy. It has been recently reported that the Unfolded Protein Response (UPR) mediates neuron death after deprivation of
target-derived neurotrophic factors. Our hypothesis is that inhibition of protein synthesis can block axotomy-induced ganglion
cell death by alleviating endoplasmic reticulum stress which activates UPR in ganglion cells after axotomy. It has been described
that UPR activated by endoplasmic reticulum stress induces cell death by both an increase of expression of transcription factor
CHOP/GADD153 and the activation of c-Jun N-terminal Kinase (JNK). In order to analyse whether UPR occur after axotomy of
retinal ganglion cells we analysed both the expression of CHOP/GADD153 and the role of JNK in neonatal axotomized retinal
ganglion cells.
The retinal explants from newborn rats were maintained in vitro for 3 hours or 18 hours in distinct conditions (1-control medium,
2-inhibitor of JNK, 3-inhibitor of protein synthesis anisomycin). Retinal ganglion cells were axotomized during preparation of the
explants. Protein were then extracted from retinal explants for Western blot. The level of CHOP/GADD153 protein was then
analyzed by western blot. Transcription factor c-Jun and pro-apoptotic protein Hrk were analysed by immunofluorescence.
Programmed cell death was detected by staining with sytox green (condensation of chromatin), TUNEL assay (DNA
fragmentation) and identification of cleaved caspase-3. We found that 3 hours after axotomy there is an increase of
CHOP/GADD153 content in the retina. Anisomycin, an inhibitor of protein synthesis blocks the increase of CHOP/GADD153
induced by axotomy. The inhibitor of JNK completely blocks axotomy-induced ganglion cell death. We also found an increase of
c-Jun (transcription factor) and Hrk (pro-apoptotic protein) in axotomized ganglion cells. Finally, an inhibitor of JNK blocked the
upregulation of both c-Jun and Hrk induced by axotomy of retinal ganglion cells.
Conclusions:
There is an increase of CHOP/GADD153 expression after axon damage on retinal ganglion cells. We found that retrograde
degeneration of axotomized-ganglion cells in neonatal retina is dependent of JNK. These results are consistent with our
hypothesis that axon damage of retinal ganglion cells activates Unfolded Protein Response which in turn contributes to death of
retinal ganglion cells.
Keywords: Unfolded Protein Response, Retinal ganglion cells, endoplasmic reticulum stress, JNK, programmed cell death
Financial Support: FAPERJ, CNPq, PIBIC-UFRJ-CNPq, CAPES.
Resumo:12-013
EFFECTS OF MICROCURRENT ON THE HYALINE CARTILAGE REPAIR IN INMATURE MALE RATS
(RATTUS NORVEGICUS).
Ciccone, C. C. ; Zuzzi, D. C. ; Morais, L. M. G. ; Passarini, J. R. ; Barbieri, R. ; Mendonça, J. S. ; Zorel, V.
J. ; Esquisatto, M. A. M.
Programa de Pós-graduaçao em Ciências Biomédicas, PPGCB- UNIARARAS
Objectives:
This study aims to analyze the effects of continuous electrical current on the structural organization of the extracellular matrix in
hyaline cartilage, during the repair process, in an injury model from not loaded cartilage in young rats (Rattus norvegicus).
Thirty male Wistar rats, aged 45 days, were obtained from the CEA-UNIARARAS and divided in two groups: Control group
(CG) and Treated group (TG). The animals are kept in individual cages and treated with commercial chow and water ad libitum.
The animals were anesthetized (xylazine (0.2 ml/kg) and ketamine (1.0 ml/kg) and underwent surgical procedure to access
xyphoid cartilage. After isolation of structure, a punch (3 mm) lesion was made. After that, the surgery procedures were finished
and the animals were treated with dipyrone (0.5% at a ratio of 1:1000) during 24 hours. Animals from TG group were treated
daily with continuous electrical current of low frequency (1 Hz) and low intensity (20 μA) for 5 minutes. To this end, each animal
is lightly anesthetized using 50% of the dose used for the surgical procedure. The CG was submitted to the same procedure
without electrical current treatment. Five animals from each group were sacrificed at 7, 21 and 35 days for removal and
preparation of xyphoid cartilage to macroscopic, histological and morphometrical analysis. Data were compared by ANOVA and
Tukey post-test (p
Conclusions:
The use of microcurrent seems to represent a useful therapy for cartilage healing, with stimulation of tecidual components
deposition and positive effects on cartilage tissues.
Keywords: Cartilage, Microcurrent, Repair, Inmature, Rats
Financial Support: Fundaçao Hermínio Ometto
Resumo:12-014
EVALUATION OF THE EFFECTS OF ELECTRICAL STIMULATION ON CARTILAGE HEALING IN ADULT
MALE RATS.
Zuzzi, D. C. ; Ciccone, C. C. ; Morais, L. M. G. ; Passarini, J. R. ; Barbieri, R. ; Mendonça, J. S. ; Zorel, V.
J. ; Esquisatto, M. A. M.
Programa de Pós-graduação em Ciências Biomédicas, PPGCB-UNIARARAS
Objectives:
This study objective to characterize the organization of the hyaline xyphoid cartilage during repair, induced by electrical current,
providing understanding of the biomolecules and cellular dynamics associated with changes in non-loaded anatomical sites from
adult animals
Thirty male Wistar rats (Rattus norvegicus), aged ninety days, were obtained from the CEA-UNIARARAS and divided in two
groups: Control group (CG) and Treated group (TG). The animals are kept in individual cages and treated with commercial chow
and water ad libitum. The animals were anesthetized (xylazine - 0.2 ml/kg) and ketamine - 1.0 ml/kg) and underwent surgical
procedure to access xyphoid cartilage. After isolation of structure, a punch (3 mm) lesion was made. After that, the surgery
procedures were finished and the animals were treated with dipyrone (0.5% at a ratio of 1:1000) during 24 hours. Animals from
TG group were treated daily with continuous electrical current of low frequency (1 Hz) and low intensity (20 ìA) for 5 minutes.
To this end, each animal is lightly anesthetized using 50% of the dose used for the surgical procedure. The CG was submitted to
the same procedure without electrical current treatment. Five animals from each group were sacrificed at 7, 21 and 35 days for
removal and preparation of xyphoid cartilage to macroscopic, histological and morphometrical analysis. Data were compared by
ANOVA and Tukey post-test (p
Conclusions:
The repair process of the cartilage tissue was stimulated by microcurrent treatment, but the standard of tecidual components
deposition was different among them.
Keywords: Cartilage, Electic field, Healing, Stimulation
Financial Support: Fundação Herminio Ometto
Resumo:12-015
HUMAN ENDOTHELIAL CELLS MODULATION BY TUMOR MICROENVIRONMENT: A ROLE FOR LIPOXIN
SYNTHETIC ANALOGS
Vieira, A. M. 1,2; Neto, E. H. 1,2; Figueiredo, C. C. 1; Barja-fidalgo, T. C. 2; Fierro, I. M. 2; Morandi, V. 1
1
DEPARTAMENTO DE BIOLOGIA CELULAR, DBCEL
2
DEPARTAMENTO DE FARMACOLOGIA E PSICOFARMACOLOGIA, DFP
Objectives:
Tumors represent a pathological disorder of cell growth characterized by an incessant and excessive cell proliferation. Melanoma
is a tumor that originates from melanocytes. The process of angiogenesis is necessary for tumor growth and dissemination.
Lipoxins (LX) and 15-epi-LX are lipids with a potent inhibitory effect on angiogenic processes in different models in vivo and in
vitro. Our group recently demonstrated that 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 (ATL-1), a synthetic analog of 15-epiLX, inhibits multiple angiogenic actions stimulated by vascular endothelial growth factor (VEGF). However, the actions of LX
on endothelial cells in a tumor microenvironment are presently unknown. Therefore, we investigated a possible modulation of
ATL-1 on endothelial cells using a model mimicking melanoma cell intravasation.
All the experiments in this study were submitted to the Ethics Committee under the registration number SISNEP/CONEP
0009.0.325.000-09. VEGF secretion by human umbilical vein endothelial cells (HUVEC), melanocytes (NGM) and melanoma
cells (MV3) was analyzed at different times (4h, 8h, 16h, 24 h) using ELISA. Melanoma cells highly secreted VEGF in a timedependent manner, what could be related to the metastatic potential of these cells. As expected, VEGF secretion was not observed
for both HUVEC and NGM. In transmigration assays, HUVEC monolayers were pretreated with ATL-1 (100 nM) for 30 minutes
and stimulated with VEGF (10 ng/mL) for 1 hour before the addition of MV3 for further 4 hours. ATL-1 completely inhibited
MV3 transmigration through VEGF-stimulated endothelial layer.
Conclusions:
ATL-1 has a potent inhibitory action on VEGF-induced MV3 migration, by modulating HUVEC-tumor cells interaction.
Keywords: Endothelial cells , Lipoxins, Tumor microenvironment , Vascular endothelial growth factor
Financial Support: FAPERJ, CNPq and CAPES.
Resumo:12-016
COLLAGEN TYPE V FACILITATES DIFFERENTIATION OF ADIPOSE TISSUE-DERIVED STEM CELLS OF
RABBITS INTO A CHONDROCYTE-LIKE PHENOTYPE “IN VITRO”
Brindo da Cruz, I. C. 1; Velosa, A. P. P. 1; Carrasco, S. 1; Pompeu, E. 3; Capelozzi, V. L. 2; Yoshinari, N. H.
1
; Teodoro, W. R. 1
1
2
Disciplina de Reumatologia da Faculdade de Medicina da USP, FMUSP
Departamento de Patologia da Faculdade de Medicina da USP, FMUSP
3
Centro de Bioterismo da Faculdade de Medicina da USP, FMUSP
Objectives:
Osteoarthrite (OA) is a syndrome of joint pain and dysfunction caused by joint degeneration that affects more people than any
other joint disease and with difficulties in its treatment. Currently treatments for OA have no regenerative aspects and provide
little positive impact on the disease. The use of anti-inflammatory drugs with effect on pain reduction and control of
inflammatory processes are insufficient in more advanced stages of the cartilage degeneration. Collagen V (COLV) regulates the
diameter of collagen fibers showing an important role in the development of functional tissue. The purpose of this study is to test
if COLV can induce differentiation of adipose tissue-derived stem cells in the chondrocyte-like phenotype.
We report here the isolation and preliminary characterization of mesenchymal stem cells (MSCs) obtained from the rabbit New
Zealand adipose-tissue. Collagen I, II, III and CD34 immunostaining analysis confirmed mesenchymal lineage differentiation
into chondrocyte-like phenotype. After 2 and 3 weeks, cell aggregates were fixed for 2 hours in 4% formaldehyde, then
dehydrated with etanol, washed with xylene and embedded in paraffin. The sections were stained with Toluidine blue, Alcian
blue and Picrosirius that revealed positive for proteoglicans and collagen by Picrosirius staining resulted positive for collagen
fibers. Moreover, after COLV culture stimulation the MSCs was capable to increase collagen I and II expression without
chondrogenic defined medium when compared with control.
Conclusions:
According to the comparison of results obtained from the culture with and without collagen V we suggest that this protein
stimulate the expression of collagen I and II and facilitates the differentiation of adipose tissue-derived stem cells of rabbits.
Keywords: Chondrocyte-like phenotype, Collagen V, Mesenchymal stem cells, Osteoarthrite
Financial Support: FAPESP – 2010/17824-8, Federico Foundation Grants, FFMUSP, LIM 05, LIM 17.
Resumo:12-017
ROLE OF GLUTAMATE AND D-SERINE ON NEURAL PROGENITORS OF CEREBRAL CORTEX
Sousa, V. O. 1; Panizzutti, R. 2; Gomes, F. C. A. 3
1
Campus Macaé, UFRJ
2
Programa de Neurociência Básica e Clínica, UFRJ
3
Programa de Biologia Celular e do Desenvolvimento, UFRJ
Objectives:
Neuron-glia interactions play an important role in nervous system development. Radial glia (RG) cells are neural progenitors that
constitute the major part of the glial population in embryonic cortex and guide neuronal migration. Durant migratory period, RG-
neuron interactions are important to the development of these cells, once intimate contact provides molecules that influence both
phenotypes. We investigated the effect of synaptic transmitters (glutamate and D-serine, which together activate NMDA
receptors) as mediators of such interactions during radial glia cells differentiation.
Cerebral cortex progenitor cultures were prepared from 13 days embryonic swiss mice. One culture was called radial glia (RG)like enriched because of morphology and the high amount of nestin-GLAST positive cells (RG markers) as described in Glia 55:
1026, 2007. Approximately 70–80% of the total progenitors double labeled for nestin and GLAST, both markers of RG cells.
Dissociated cells (200,000 cells/well) were plated onto 13 mm diameter glass coverslips, previously coated with polyornithine.
Cells were incubated in DMEM/F12 medium supplemented with 10% fetal bovine serum, at 37°C in a humidified 5% CO2, 95%
air chamber for 24 h and then for an additional day in different conditions: (1) in serum-free medium; (2) glutamate; (3) D-serine;
(4) glutamate and D-serine. Neural progenitors were also cultured as neurospheres. 100,000 cells were plated in a 25 cm bottle.
After one week, the neurospheres were incubated in the same conditions of the other culture for 48 h. Using
Immunocytochemistry and Western Blotting assays, we have shown that RG express Serine Racemase (SR), an enzyme that
converts L-serine in D-serine. As RG is involved in neuronal migration, the identification of SR in this population is important
because D-serine has been related to migration. In vitro data have shown that glutamate and D-serine induce progenitors to take a
radial phenotype and increase BLBP expression, a radial glia cell marker, in the cerebral cortex from embryonic mice. We
showed that glutamate and D-serine increased 50% the BLBP positive cells (compared to control).
Conclusions:
Our data suggest a role for glutamatergic receptors NMDA in RG specialization during cerebral cortex neuronal migration.
Keywords: D-serine, Glutamate, Neural progenitors, NMDAR, Radial Glia
Financial Support: CAPES, CNPq, FAPERJ
Resumo:12-018
THE PHYTOSTEROL PROMOTES INCREASE OF NESTIN-POSITIVE CELLS IN THE SUBGRANULAR ZONE OF
HIPOCAMPAL DENTATE GYRUS OF ADULT MICE
Nascimento, M. V. L. 1; Magalhães, R. C. 1; Santos, I. V. F. 1; Carvalho, L. 3; Reis, R. A. M. 3; Silva, E. O.
1
; Tavares, D. V. 2; da Silva, M. N. 2; do Nascimento, J. L. M. 1; Bastos, G. N. T. 1
1
Lab. de Neuroquimica Instituto de Ciências Biológicas, UFPA
2
Lab de Cromatografia Instituto de Ciências Exatas e Naturais, UFPA
3
Lab de Neuroquímica Instituto de Biofísica, UFRJ
Objectives:
Newborn neurons emerge from neural stem cells (NSCs) from niches in the mammalian adult brain. These cells are incorporated
into functional circuits and may be important to acquisition and retention of memory. Therefore, the search for new compounds
that enhance proliferation and differentiation of neural stem cells in the hippocampus represent a significant scientific challenge
with great promise. This study was designated to examine the neurogenic activity of phytosterol (PH) on subgranular zone of
hipocampal adult mice.
Here we have used PH on the neurogenesis in the subgranular zone of hipocampal dentate gyrus of adult mice using 5`-bromo-2`deoxyuridine (BrdU)-pulse chase method. Increased doses (0.1; 1; 5mg/Kg) of PH were given to adult male BALB/c mice with 6
to 8-weeks-old; or 0.9% NaCl (control). Mice were sacrificed at 24 hours or 7 days after the BrdU administration, and
hippocampal slices were processed for immunohistochemistry. We found that PH did not modify the mice behavior at any dose
used, but increased the number of BrdU-positive cells in the subgranule zone of hipocampal dentate gyrus 24 hours or 7 days
after injection. Using PH treatment in vitro, the compound promoted the increase of nestin expression in neural progenitors cells
when compared with control. PH not showed BrdU-positive cells out subgranule cell layer (ectopic neurogenesis). All procedures
involving animal care and experimentation were performed in accordance with the guidelines of the Ethical Committee for
Research with Experimental Animals of the UFPA (BIO001-09).
Conclusions:
These results suggest that PH increase the proliferation of nestin-positive cells and also BrdU-positive cells in differentiation
process in the sub granular zone of hippocampus of adult mice. In conclusion, we have evidence that phytosterol induce
proliferation of newborn cells in hipocampal neurogenic niche.
Keywords: hippocampus, neurogenesis, proliferation, stem cells
Financial Support: FAPESPA, CNPQ, UFPA, CAPES
Resumo:13-001
SMOKING DURING PREGNANCY/LACTATION: ROLE FOR HIF1Á ON GLUT4 GENE MODULATION AND
CAUSE OF INSULIN RESISTANCE STATE IN RATS
Gomes, P. R. L. ; Seraphim, P. M.
Fisioterapia - Univerisdade Estadual Paulista, UNESP
Objectives:
To investigate the effect of cigarette smoke exposure during pregnancy and lactation on the insulin sensitivity, fasting glycemia,
morphometric parameters and glucose transporter (GLUT4) expression in skeletal muscle from rats correlating with SOCS3
(signaling and HIF1á expression analysis.
Forty 2-month-old Wistar rats were divided into four groups: control pregnant (CP) and smokers during pregnancy (SP) – both
constituted by rats sacrificed after the birth of offspring; control lactant (CL) and smoker during lactation (SL) – both constituted
by rats sacrificed after the breastfeeding period. SP and SL groups were submitted to the exposure of combustion of 4 cigarettes
during 30 minutes, 2x a day, 5 days a week for 40 and 61 days, respectively. RT-PCR was performed for GLUT4, SOCS3 and
HIF1á mRNA expression analysis. Blood was collected for serum analysis of glycemia and insulinemia by colorimetry and
ELISA immunoenzymetric assay, respectively. HOMA IR was calculated. One-way ANOVA, parametric, with post hoc
(Bonferroni) was used for comparisons. The content of GLUT4 mRNA was correlated with SOCS3 and HIF1á mRNA results by
Pearson correlation. Differences among groups were considered significant when P < 0.05. Results: At the end of pregnancy there
was a decrease on body weight in CL, SL and SP groups compared to CP group (CP=350.7±13.2*; CL=298.2±24.2;
SP=293.2±16.7; SL=304.6±23.3g, *P
Conclusions:
We concluded that exposing rats to cigarette smoke during pregnancy and lactation causes: a) weight loss in pregnant and lactant
since it is known that nicotine promotes elevation of neurotransmitters that inhibit food intake; b) hyperglycemia and increased
HOMA IR, probably because of increases in plasma concentrations of sex steroids and activation of the sympathetic nervous
system that are known to change glucose metabolism; c) increased GLUT4 mRNA expression without changing HIF1á and
SOCS3 mRNA content, however associated with a positive correlation between the expression of HIF1á and GLUT4 mRNA.
This suggests that the modulation of glucose transporter gene was influenced by intermittent hypoxia generated by exposure to
cigarette smoke in this model of insulin resistance.
Keywords: Smoking, GLUT4, Insulin Resistance, HIF1a
Financial Support: FUNDUNESP 795/2010
Resumo:13-002
EFFECTS OF FOOD PATTERN CHANGE AND PHYSICAL EXERCISE ON CAFETERIA DIET INDUCED
OBESITY IN FEMALE RATS
Goularte, J. F. ; Sanvitto, G. L.
Fisiologia/Universidade Federal do Rio Grande do Sul, UFRGS
Objectives:
Obesity affects a large number of people around the world and appears to be the result of poor eating habits and low physical
activity levels. To treat obesity and its complications, changes in dietary patterns and exercise are recommended. Thus, this study
evaluated the effect of food pattern change and exercise on cafeteria diet induced obesity in female rats.
Fifty-six 3-week-old female Wistar rats were divided in five groups: control chow diet sedentary (CON-CON-SED) for 34 weeks,
cafeteria diet sedentary (CAF-CAF-SED) for 34 weeks, control chow diet for 26 weeks and after the cafeteria diet sedentary
(CAF-CON-SED)for 8 weeks, cafeteria diet for 34 weeks and exercise since 26 week for 8 weeks (CAF-CAF+EX) and cafeteria
diet for 26 weeks and after control chow diet and exercise (CAF-CON+EX) for 8 weeks. Cafeteria diet groups were submitted to
palatable foods and exercise groups were submitted to 8-week of swimming training. At week 34 were analyzed body weight (g),
visceral adipose tissue (g), fasting glucose (mmol/L) and insulin (µIU/ml). We used the HOMA-IR index to assess insulin
resistance. The results were analyzed using a one-way ANOVA with Student Newman-Keuls post hoc test. Values are expressed
as mean ± SEM. Significance level was established as p
Conclusions:
Exposure to palatable food caused obesity and insulin resistance, and diet change was sufficient to prevent cafeteria diet induced
obesity and insulin resistance. In addition, exercise prevented the development of insulin resistance in obese rats. These results
may help in developing new approaches to the treatment of obesity and T2DM.
Keywords: Obesity, Cafeteria diet, Physical exercise, Food pattern change
Financial Support: Conselho Nacional de Pesquisa (CNPQ)
Resumo:13-003
INTRACELLULAR MECHANISMS UNDERLYING INCREASES IN GLUT4 EXPRESSION IN RESPONSE TO
INSULIN IN SKELETAL MUSCLE
Moraes, P. A. ; Machado, U. F.
Instituto de Ciências Biomédicas, USP
Objectives:
Skeletal muscle is a target tissue for approaches that can improve insulin sensitivity in insulin-resistant states such as obesity and
type 2 diabetes mellitus. The increase of GLUT4 content in the insulin-sensitive tissues is related with improvement in insulin
sensitivity and has been proposed as possible treatment for obesity and type II diabetes mellitus. It‟s known that insulin stimulates
the translocation of GLUT4, but it´s role in GLUT4 expression is uncertain. Thus, the aim of this research was to evaluate a
possible mechanism involved in regulation of GLUT4 expression by insulin.
Rats were submitted to 48 hours of food deprivation, then the soleus muscles were extracted and submitted to 180 minutes of
incubation in 25ml of Krebs–Henseleit bicarbonate buffer (pH 7.4) containing 0,1% bovine serum albumin and continuously
oxygenated with 95% O2–5% CO2 at 37°C. The muscle were treated or not with insulin, wortmannin and ML-9 during the
incubation. In these muscles were evaluated the GLUT4 protein content and Akt activity by western blotting. The involvement of
PI3k and Akt activity on GLUT4 expression was analyzed using the inhibitors wortmannin and ML-9, respectively. The GLUT4,
MEF2A, MEF2B, MEF2D and HIF1-a mRNAs were analyzed by reverse transcription-PCR. The binding activity of the nuclear
proteins into the DNA sequences containing the binding site of At-rich and E-box into the GLUT4 gene promoter was analyzed
by eletrophoretic mobility shift assay (EMSA). To identify the transcription factor present in the E-box complex, a supershift
assay was performed. The incubation with insulin induced an increase in GLUT4 protein content (72%, p
Conclusions:
These findings indicate that insulin plays an important role in regulation of GLUT4 expression. Insulin increases the expression
of GLUT4 mRNA and protein. It is possible that this regulation occurs through activation of the PI3K/Akt pathway and the
transcription factors MEF2 and MyoD.
Keywords: GLUT4, Insulin, Akt, Muscle
Financial Support: FAPESP 2007/57873-5.
Resumo:13-004
ENDOCRINE ASPECTS OF OPIOIDERGIC STIMULATION IN FEMALE RATS
Cruz, A. M. 1; Sukikara, M. H. 2; Felippe, E. C. G. 1; Anselmo-franci, J. A. 3; Canteras, N. S. 2; Oliveira, C.
A. 1; Felicio, L. F. 1
1
FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA, USP
2
INSTITUTO DE CIÊNCIAS BIOMÉDICAS, USP
3
FACULDADE DE ODONTOLOGIA - FISIOLOGIA, USP RIBEIRÃO PRETO
Objectives:
Opioid peptides play an important role in maternal behavior as well as in physiological and pathological phenomena involving
motivation. This study was designed to investigate the endocrine aspects of opioidergic stimulation induced by morphine
treatment during late pregnancy. Because this treatment induced a clear increase in serum progesterone concentrations, another
set of animals was treated with progesterone during late pregnancy and tested for morphine-induced inhibition of maternal
behavior during lactation.
Adult Wistas nulliparous female rats were timed-mated with sexually experienced males. First experiment: daily 3.5 mg/Kg doses
of morphine or saline from day 17 to 21 of pregnancy are able to change the expression of maternal behavior patterns. On day 5
of lactation, animals pretreated were challenged with saline or morphine 1.5 mg/Kg, and maternal behavior was evaluated. 100%
of animals from groups SS (n=10), SM (n=10) and MS (n=10) displayed full maternal behavior, while 0% of MM animals group
(n=10) displayed this behavior. Second experiment: 30 minutes after saline or morphine injections from day 17 to 21 of
pregnancy, a blood sample was collected. Corticosterone, progesterone, estradiol and prolactin serum concentrations were
measured after each morphine or saline injection. No significant differences were found in corticosterone, estradiol, or prolactin
serum concentrations. In morphine-treated animals, however, progesterone concentrations were consistently and significantly
increased from days 18 to 20 of treatment. A third experiment investigated the effects of daily progesterone injections from day
17 to 21 of pregnancy on sensitivity to morphine inhibitory effects on maternal behavior during lactation. Pregnant rats were
treated with progesterone 400&mug or peanut oil once per day, beggining on day 17 of pregnancy, during 5 days. On day 5 of
lactation, dams were challenged with saline or 1.5 mg/Kg morphine, and maternal behavior was evaluated. Progesterone
pretreated dams were more sensitive to morphine-induced inhibition of maternal behavior. Fisher test revealed a significant lower
number of rats showing full maternal behavior in group PM as compared to groups PS and OS, p
Conclusions:
The results suggest that the treatment was unable to promote more stressor effects than those caused by saline injections.
Progesterone emerges as a potential mediator for morphine-induced changes in opioid sensitivity during late pregnancy and early
lactation. Increased serum progesterone concentrations may account for behavioral effects observed in lactating rats treated with
morphine during late pregnancy.
Keywords: MATERNAL BEHAVIOR, OPIOID, PROGESTERONE, RAT
Financial Support: FAPESP 2009/53194-1; 2010/50415-4 and CNPQ 351107/92-4
Resumo:13-005
1ALPHA,25(OH)2-VITAMIN D3 REGULATES THE GAMMA GLUTAMYL TRANSPEPTIDASE ACTIVITY IN RAT
TESTIS VIA PKA ACTIVATION
Zanatta; L. 1,2; Gonçalves; R. 1; Zamoner; A. 1; Bouraima-lelong; H. 2; Bois; C. 2; Carreau; S. 2; Silva; F. R.
M. B. 1
1
UNIVERSIDADE FEDERAL DE SANTA CATARINA, UFSC
2
UNIVERSITÉ DE CAEN BASSE-NORMANDIE, UNICAEN
Objectives:
1α,25-dihydroxyvitamin D3 (1,25D3) is critical for the maintenance of normal reproduction since reduced fertility is observed in
male rats on a vitamin D deficient diet. The aim of the present work was to localize, by immunohistochemistry, the vitamin D
receptor (VDR) in immature rat testis as well as to study the effect of 1,25D3 on gamma glutamyl transpeptidase (GGTP) activity
and on lactate secretion levels.
For this study, 30-day-old Wistar rats were used. The immunolocalization of VDR in testis slices was performed by confocal
microscopy using the rabbit polyclonal anti-VDR (C-20) and anti-rabbit IgG conjugated to Alexa Fluor 488. For GGTP and LDH
activity assays and lactate determinations, the testes were pre-incubated in KRb buffer for 15 min at 34°C, pH 7.4 with O2:CO2
(95:5 v/v). After that, they were incubated for 5, 15, 30, 60, 120 or 180 min in the presence or absence of 1,25D3 (10-9 M). In
GGTP experiments 10 ìM H-89 (PKA inhibitor) or 10 ìM PD-98059 (MAPK inhibitor) were added 20 min before the hormone.
After incubation, testicular tissue was homogenized in cold 0.1 M Tris buffer, pH 8.5 (10% homogenate w/v). Adequate aliquots
were saved for total protein determinations by Lowry method and 50 ìL were incubated with the enzyme substrate. The
enzymatic reaction was allowed to proceed for 60 min at 34 °C and stopped by addition of acetic acid. The GGTP activity was
expressed as % of control. The lactate levels in the incubation medium were estimated by the lactate oxidase method and the
results were expressed as mg of lactate/ìg of protein. The tissue viability was determined by the LDH activity present in the
incubation medium and the results were expressed as IU/L/mg of tissue. Statistical evaluation was done using one-way analysis
of variance (ANOVA), followed by Bonferroni post-test. The results showed the VDR immunoreactivity in the cytoplasm of
numerous testicular rat cells. The 1,25D3 increased the GGTP activity after 30 and 60 min of incubation and this increment was
38% (19.66 ± 1.08; n=4) and 69% (91.68 ± 6.84; n=4) compared to control groups (14.25 ± 1.80; 54.06 ± 10.58; n=4),
respectively. In addition, the stimulatory effect of 1,25D3 on GGTP activity (91.68 ± 6.84; n=4) was dependent of PKA since H89 (56.71 ± 4.18; n=4) was able to block the 1,25D3 effect (61.52 ± 2.01; n=4). On the other hand, PD-98059 (55.92 ± 2.04; n=4)
did not change significantly the hormone effect (71.23 ± 5.11) on the enzyme activity. Moreover, the lactate levels and the tissue
viability was not altered by 1,25D3 in all studied times.
Conclusions:
These findings demonstrate that the VDR is present in 30-day-old rat testis and that the 1,25D3 increase testicular GGTP activity
via PKA activation suggesting that the hormone may be involved in male reproductive functions.
Keywords: 1alpha,25(OH)2-Vitamin D3, Gamma Glutamyl Transpeptidase, PKA, Testis
Financial Support: CAPES/Cofecub (Projeto 554/07); CNPq; FAPESC, PGFAR-UFSC.
Resumo:13-006
αVβ3 INTEGRIN RECEPTOR SEEMS TO MEDIATE THE ACTION OF THYROXINE IN IMMATURE RAT
TESTIS: AMINO ACID ACCUMULATION AND THYMIDINE INCORPORATION
Zanatta, A. P. ; Zanatta, L. ; Zamoner, A. ; Silva, F. R. M. B.
Objectives:
The aim of this work was to study the mechanism of action of thyroxine (T4), 3,5,3‟-triiodo-L-thyronine (T3) and reverse T3
(rT3) on amino acid accumulation (a plasma membrane specific amino acid transport system) and to investigate the role of αVβ3
integrin receptor in this event. Also, to clarify if the stimulatory effect of T4 on amino acid accumulation and on rapid calcium
influx (nongenomic effects) culminates in an intracellular cross-talk pathway able to alter DNA activation (genomic effect), in
immature rat testis.
11-day-old Wistar rat testes were used. For amino acid accumulation and thymidine DNA incorporation, whole testes were preincubated (30 min) and incubated (60 min) with 0.2 µCi/mL of [14C]-MeAIB or 1 µCi/ml of [methyl-14C] thymidine,
respectively. Also, 45Ca2+ influx was studied in the presence of T4 with/without verapamil. For amino acid accumulation T4,
T3, and rT3 (10-11 to 10-5 M), tetraiodothyroacetic acid (tetrac; 10-9 M) and/or RGD peptide (500 nM) were added to the preincubation and incubation media. For thymidine experiments T4 (10-9 M), EGTA (2 mM) and BAPTA-AM (50 µM) were added
to the pre-incubation and incubation media. After incubation, tissues were washed in Krebs Ringer-bicarbonate (KRb). For
45Ca2+ influx, testes were pre-incubated in KRb for 15 min, pH 7.4 at 34°C. Then, the testes were transferred to fresh KRb with
0.1 µCi/ml 45Ca2+ and preincubated for 60 min. Verapamil (10-4 M) was added 15 min before incubation with T4. Finally, the
gonads were incubated for 1 min with/without T4 (10-9 M). Amino acid accumulation, thymidine DNA incorporation and
calcium influx were expressed as tissue/medium, cpm/µg of protein and pmol of 45Ca2+/µg of protein, respectively. T4 and rT3
stimulated amino acid accumulation, reaching 50% (3.3 ± 0.11; n=4) and 138% (3.95 ± 0.95; n=3) of increase at 10-9 M,
respectively. Similar stimulatory effect on amino acid accumulation was observed with T3 (10-6 M) (49%; 3.28 ± 0.18; n=4)
compared with control (2.2 ± 1.0; n=8). In addition, stimulatory effect of T4 (10-9 M) on amino acid accumulation was blocked
by tetrac and RGD peptide. Also, T4 stimulated 45Ca2+ uptake around 126% (210.72 ± 24.98; n=4) compared with control
(93.11 ± 8.75; n=5) and this effect was blocked by verapamil. Also, T4 stimulated the thymidine DNA incorporation around 72%
(3.76 ± 0.22; n=3) compared with control (2.18 ± 0.22; n=6) and this action was blocked by EGTA and BAPTA-AM.
Conclusions:
The stimulatory effect of thyroxine on amino acid accumulation seems to be mediated by αVβ3 integrin receptor since tetrac (an
inhibitor of T4 action at the integrin) as well as RGD peptide (blocker of T4 binding on αVβ3 receptor) were able to nullify the
hormone effect. Surprisely, the thyroid hormone metabolite, rT3 (10-9 M), showed more potent effect on amino acid
accumulation than T4. In addition, the stimulatory effect of thyroxine in calcium influx culminates in alteration in DNA activity
since as much EGTA (calcium chelator) as BAPTA-AM inhibited the stimulatory effect of T4 on thymidine incorporation into
DNA. Taking it in account, it is clear that T4 acts on plasma membrane by αVβ3 integrin receptor and also modify nuclear
activity in immature rat testes. Studies are underway to understand if the shuttling of amino acid accumulation induced by T4 can
culminate in an interface between nongenomic and genomic hormonal action.
Keywords: amino acid accumulation, immature rat testis, integrin receptor, thimidine incorporation, thyroxine
Financial Support: CNPq; CAPES, FAPESC, UFSC
Resumo:13-007
PROCASPASE-3 AND CLEAVED CASPASE-3 EXPRESSION IN MAGNOCELLULAR NEURONS DURING
POLYMICROBIAL SEPSIS
Oliveira-pelegrin, G. R. 1; Riester, K. D. 2; Rocha, M. J. A. 1
1
Faculdade de Odontologia de Ribeirão Preto, USP
2
Eberhard Karls Universität Tübingen , EKUT
Objectives:
During sepsis, the complex interaction between host and infection results in a massive production of inflammatory mediators as
cytokines such as interleukin-1beta (IL-1β), which may directly or indirectly affect the central nervous system and cause
neuroendocrine alterations. An important alteration observed is the biphasic secretion pattern of vasopressin (AVP), which is
synthesized in the magnocellular neurons of the hypothalamus. In the initial phase of sepsis, when hypotension starts, the
organism responds with a high AVP secretion in an attempt to maintain normal blood pressure and tissue perfusion. However, in
the late phase, AVP plasma levels are inappropriately low, despite progressive hypotension. In this study, we investigated
whether the relative deficiency of AVP in the late phase of sepsis may be related to apoptosis in the magnocellular neurons of the
supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus.
Male Wistar rats (250±30g) were divided into two groups: a sham operated and one subjected to cecal ligation and puncture
(CLP) to induce sepsis. After surgery, one set of animals were decapitated at 4, 6, 24 or 48 h. Blood was collected for IL-1β
plasma level measurement by ELISA. The brains were quickly removed and frozen in dry ice for hypothalamus dissection. The
PVN and SON were carefully punched out by using stainless steel needles of 1400μm and 1200μm diameter, respectively. The
hypothalamic nuclei were processed for procaspase-3 mRNA quantification by RT-PCR, and cleaved caspase-3 and cytochromec analysis by Western Blots. Another set of animals was perfused at 24 h after sham or CLP surgery and the brains were
removed, post-fixed and processed for TUNEL assay. IL-1β plasma levels increased progressively until 24 h after CLP (P
Conclusions:
Our results suggest that apoptosis of hypothalamic magnocellular neurons may be caused by elevated levels of IL-1β, and this in
turn would compromise vasopressin secretion in the late phase of sepsis.
Keywords: apoptosis, vasopressin, interleukin-1beta, hypothalamus
Financial Support: FAPESP, PROCAD/CNPq
Resumo:13-008
EIGHT WEEK OF INTERMITTENT TRAINING PROGRAM IMPROVES THE PERIPHERAL INSULIN
RESISTANCE WITHOUT MUSCLE MORPHOLOGIC ALTERATIONS IN OBESE-INDUCED RATS
Souza, L. M. 1; Brandão, B. B. 2; Trombeta, B. N. O. 1; Castoldi, R. C. 2; Araujo, R. G. 1; Malheiro, O. C.
M. 1; Seraphim, P. M. 2
1
DEPTO. EDUCAÇÃO FÍSICA, FCT/UNESP/P.PRUDENTE
2
DEPTO DE FISIOTERAPIA, FCT/UNESP/P.PRUDENTE
Objectives:
To evaluate the alterations caused by a intermittent training program on the insulin sensitivity and morphological adaptations in
gastrocnemius skeletal muscle of obese rats induced by hyperlipidic diet.
Male Wistar rats were divided into 4 groups: sedentary control (SC); sedentary obese (SO); intermittent exercise control (IEC);
intermittent exercise obese (IEO). The hyperlipidic diet was composed by sausage, biscuit, bacon and soft drink. Intermittent
training (IT) protocol was performed in a treadmill from the third month of age once a day, 3 times a week, during 8 week, 11
bouts of 2 minutes of duration on 120% of the anaerobic threshold (AT), 1 minute of passive interval, totalizing 32 minutes of
training. Exercise intensity was determined by Chassain test by the detection of the AT. Lactate analysis was performed using
25μl of blood samples collected by the distal proximity of the animal tail in heparinized capilar, immediately transferred to 1.5
mL tubes containing 50 μL of NaF solution (1%) and stored on ice for posterior electroenzymatic reading (YSL 2700 STAT,
Yellow Springs Co., EUA). For the assessment of insulin sensitivity “in vivo” a Insulin Tolerance Test was performed (ITT).
Glucometer and glucose strips Biocheck TD4225 (Bioeasy) were used for measuring the glucose concentration. After 1 hour the
last training session, animals were sacrificed for removal of the gastrocnemius skeletal muscle and periepidydimal adipose tissue.
Ten slides were produced with cuts of 5 mm thick and stained with hematoxylin and eosin (HE). We evaluated the cross section
of 200 muscle fibers using the software AxioVision 4.8. For analysis of biochemical and morphometric data ANOVA,
parametric, and post-test when necessary (Bonferroni) was used. The results are presented as mean ± SEM. The differences
among the groups were considered significant when P < 0.05.The hyperlipidic diet induced body weight gain (SC=478.33+/16.67; SO=561.25+/-17.16**; IEC=481.00+/-15.67; IEO=528.80+/-35.48 g, **P< 0.001 vs IEC e IEO; #P< 0.001 vs IEO, µm²
n=2). Although the hyperlipidic diet has reduced the insulin sensitivity, the exercise was efficient for improve this condition (SC
= 1.530+/-0.121; SO=1.00+/-0.190*; IEC=1.509+/-0.120 e IEO=1.44+/-0.102. *P
Conclusions:
The imposed hyperlipidic diet was efficient to induce an obesity state associated with lower insulin sensitivity. On the other hand,
the intermittent training has improved this condition for obese group, even without changing the periepididymal adipose tissue
weight compared to non-exercised obese group. But this protocol was not sufficient for improving the size of muscle fiber area in
this model of obesity, suggesting that it is necessary to increase the exercise intensity and/or schedule time for best results.
Furthermore the reduced muscle fiber area in exercised obese group suggests that the improvement on insulin sensitivity did not
involve higher capacity on glucose disposal per muscle area.
Keywords: intermittent training, obesity, insulin resistance , hyperlipidic diet, morphologic alterations
Financial Support: FAPESP nº 2011/00472-2 and CAPES.
Resumo:13-009
EFFECTS OF THE HIGH-FAT DIET AND ESTROGEN REPLACEMENT THERAPY IN LEPTIN LEVELS IN
OVARIECTOMIZED SPONTANEOUSLY HYPERTENSIVE RATS
Santos, C. S. ; Gusmão, D. O. ; Souza, C. L. S. ; Neves, J. V. ; Marinho, C. S. ; Belo, N. O.
Universidade Federal da Bahia , UFBA-IMS
Objectives:
There is a complex interaction between sex steroids and leptin on body weight regulation.The hormone replacement therapy
attenuates the fat mass increase in postmenopausal women and seems to regulate the leptin secretion. In this study, we examined
changes in leptin levels in high fat diet-fed spontaneously hypertensive rats (SHR) and the role of estrogen in these changes.
Twenty-four 12-wk-old female SHR were ovariectomized or sham-operated. The animals were divided (6 per group) according
to the diet received: high-fat diet (54.4% of fat) and standard diet (groups: OHFD: ovariectomized high-fat diet-fed rats, OSD:
ovariectomized standard diet-fed rats, SHFD: sham-operated high-fat diet-fed rats and SSD: sham-operated standard dietfed rats).
The rats were placed in metabolic cages to quantify the food intake. Body weight and blood pressure were measured weekly. In
the last two weeks, the ovariectomized rats were submitted to hormone replacement therapy with 17β-estradiol (E2:
5μg/0.1ml/100g weight) and control rats received vehicle (corn oil-VEH: 0.1ml/100g weight). After, the rats were sacrificed and
blood was collected to determination of estradiol and leptin plasma levels by enzyme linked immunosorbent assay. High-fat dietfed rats decreased food intake compared to control diet-fed animals (OHFD:12.6±0.6; SHFD: 10.8±0.7; OSD: 16.3±0.5; SSD:
17.2±1.2 g/day, p
Conclusions:
Both ovariectomy and high-fat diet increased weight gain and systolic blood pressure of female spontaneously hypertensive rats.
The 17β-estradiol replacement therapy had a beneficial effect on body weight and blood pressure only in ovariectomized rats fed
with control diet. Also, we observed that ovariectomy decreased plasma leptin levels, while the high-fat diet increased. These
results show the deleterious effect of high-fat diet, which may impair the estradiol effect in ovariectomized high-fat diet-fed rats.
Furthermore, our findings indicate that estradiol has a positive effect on leptin production independent of diet type.
Keywords: ESTROGEN REPLACEMENT THERAPY, HIGH-FAT DIET, LEPTIN
Financial Support: CNPq and CAPES
Resumo:13-010
PROGRAMMING OF LEPTIN PRODUCTION BY ADIPOCYTE AND MUSCLE IN NICOTINE EXPOSURE
DURING LACTATION: GENDER DIFFERENCES IN RATS
Pinheiro, C. R. ; Santos-silva Ap ; Oliveira, E; Trevenzoli, I. H. ; Claudio-neto, S. ; Manhaes, Ac. ; Moura,
E. G. ; Lisboa, P. C.
Depto. ciências fisiológicas/IBRAG, UERJ
Objectives:
Aim: The risk of developing chronic diseases during adult life may be influenced by the exposure to chemical pollutants and/or
diets in early life that may interfere with hormonal action, as an endocrine disruptors (Mol Endocrinol. 475:82, 2006). Previously
we have demonstrated that nicotine exposure during lactation affects the future endocrine-metabolic development of the
offspring. This phenomenon characterized by alterations in early life that result in diseases at adulthood is known as
programming (Biosci Rep. 251:69, 2005). Neonate male rats whose mothers were nicotine-treated during lactation have higher
adiposity and hyperleptinemia. At adulthood, they present central obesity, hyperleptinemia and leptin resistance (J
Endocrinol.397:405, 2009). To understand the tissue contribution for higher leptin levels, here we evaluated the leptin content in
white adipose tissue and skeletal muscle of male and female adult offspring whose mothers were nicotine-treated during lactation.
Methods and Results: At the 2nd postnatal day (PN2), Wistar dams were subcutaneously implanted with osmotic minipumps
releasing nicotine (NIC, 6mg/Kg/day) or saline for 14 days. At least, 8 male and female offspring (one from each litter) were
killed on PN180. Serum leptin was determined by radioimmunoassay. Leptin protein expressions in visceral adipose tissue
(VAT), subcutaneous adipose tissue (SAT) and soleous muscle were measured by Western blotting. Significant data were p
Conclusions:
Conclusion: For the first time, a sex dimorphism on leptin production was evidenced in the model of programming by maternal
nicotine exposure during lactation. Sources of research support: CAPES, CNPq and FAPERJ.
Keywords: GENDER DIFFERENCES, LEPTIN, NICOTINE
Financial Support: CAPES, CNPq and FAPERJ.
Resumo:13-011
17 β-ESTRADIOL REDUCES THE CONNEXIN40 GENE EXPRESSION IN CELL CULTURES OF RAT ATRIUM
Almeida, L. S. 1; Zeca, S. G. 1; Souza, L. M. 1; Louzada, R. A. N. 2; Cordeiro, A. 3; Pazos-moura, C. C. 3;
Almeida, N. A. S. 1
1
DEPARTAMENTO DE CIENCIAS FISIOLÓGICAS, IB - UFRRJ
2
DEPARTAMENTO DE BIOCIENCIAS E ATIVIDADE FISICA, EEDF - UFRJ
3
DEPARTAMENTO DE FISIOLOGIA DE ÓRGÃOS E SISTEMA, IBCCF - UFRJ
Objectives:
Several cardiac genes are regulated by 17 β-estradiol acting via the estrogen receptors. The propagation of electrical activity in
the myocardium depends on the intercellular transfer of current at gap junctional channels and Connexin40 (Cx40) is the
predominant junctional protein in the atrium and conduction system. The present study investigated the effect of 17β-estradiol on
mRNA Cx40 expression in vitro and in vivo in correlation to ECG studies.
Primary cultures of neonatal rat atrium were treated with 17 β-estradiol (10-6M) for 8 hours. Total mRNA was extracted and the
Cx40 mRNA were analyzed by semi-quantitative RT-PCR, using specific primers for Cx40 and GAPDH as an internal control. A
fragment comprising 1200 bases pairs from mouse Cx40 promoter, subcloned upstream of the coding region of the luciferase
gene (-1190/+121Cx40Luc), were used in transient transfection assays on rat smooth muscle cells (A7r5) treated with 17 βestradiol (10-6M) for 24 hours . ECG was performed in unanesthetized mice. Mice were subjected to ovariectomy (OVX), and
one group subjected to ovariectomy was treated with subcutaneous injections of 0.1 mg/kg BW estradiol benzoate for 15 days
(OVX+R). These groups were compared to false-operated mice (sham). Statistical analysis was performed using Student T test
and one-way ANOVA. Results were expressed in mean±SEM and p
Conclusions:
Ours findings showed that estrogen reduces the expression of mRNA Connexin40 in vitro and suggest that E2 modulates the
Cx40 gene expression acting at Cx40 promoter.
Keywords: Connexin40, ECG, Heart, P wave duration, 17 beta-estradiol
Financial Support: FAPERJ, CNPq
Resumo:13-012
SHORT-TERM INHIBITION OF SREBP-1C EXPRESSION REVERSES DIET-INDUCED STEATOSIS, BUT NOT
INSULIN RESISTANCE HEPATIC IN MICE
Souza, D. R. ; Vitto, M. F. ; Cesconetto, P. A. ; Marques, S. O. ; Luciano, T. F. ; Zeferino, D. S. ; de Souza,
C. T.
UNIVERSIDADE DO EXTREMO SUL CATARINENSE, UNESC
Objectives:
The non-alcoholic fatty liver disease (NAFLD) is caused by triglyceride accumulation in the liver, and recent reports indicate that
this disease is responsible for hepatic insulin resistance (Hepatology 40:1387, 2004). For example, increased hepatic lipid stores
in mice by overexpression of lipoprotein lipase in the liver (Proc Natl Acad Sci USA 98:7522, 2001) and in rats by short term
high-fat feeding (J. Biol. Chem. 279:32345, 2004) have been shown to result in liver-specific fat accumulation and hepatic insulin
resistance. On the other hand, the hypothesis that fatty liver causes insulin resistance or insulin resistance drive in fatty liver is
shrouded in controversy. Thus, in this study, we investigated whether the reversion diet –induced steatosis ameliorates insulin
resistance hepatic in mice.
Swiss mice became obese after 8 weeks of feeding with chow containing 61kJ% saturated fat. After the dose-response curve (3.0
nmol/day) was determined, the animals were separated (n = 6) in Lean, obese mice (DIO) and obese mice were treated for 14
days with antisense (DIO/ASO). Insulin tolerance test, immunoblotting assays and histology were used in this investigation. For
immunobloting, Aliquots of 0.2 mg of protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes
and blotted with anti-phospho IR, anti-phospho IRS-2, anti-phospho Akt and anti-phospho Foxo1 antibodies. Antisense
oligonucletide did not change glucose, the constant of glucose decay (kITT), body weight nor insulin blood levels. In addition, no
differences (ANOVA) between the DIO and DIO/ASO group after treatment was observed in the molecules of the insulin
pathway (IR, IRS-2, Akt and Foxo1). The treatment inhibits SREBP-1c and decreases expression of lipogenic enzymes, reducing
the accumulation of triglycerides, but not reverse insulin resistance hepatic.
Conclusions:
Thus, SREBP-1c inhibition reverses steatosis, but does not ameliorate insulin resistance hepatic, at least, in this used model
animal.
Keywords: ANTISENSE, INSULIN RESISTANCE, SREBP-1C, STEATOSIS, TRIGLYCERIDE ACCUMULATION
Resumo:13-013
EFFECTS OF NOVEL ARYLPYRAZOLE GLUCOCORTICOID RECEPTOR MODULATORS ON SKELETAL
MUSCLE STRUCTURE AND FUNCTION
Guilherme, J. P. L. F. 1; Artioli, G. G. 2; Baptista, I. L. 1; Ramos, G. V. 1; Miyabara, E. H. 3; Scanlan, T. S. 4;
Moriscot, A. S. 1
1
Departamento de Biologia Celular e do Desenvolvimento, ICB/USP
2
Escola de Educação Física e Esporte, EEFE/USP
3
Departamento de Anatomia, ICB/USP
4
Departamento de Fisiologia e Farmacologia, OHSU
Objectives:
Glucocorticoids are the most commonly used anti-inflammatory drugs. Although they are widely used to treat a variety of
diseases, a great number of important side-effects are often observed as a consequence of glucocorticoid treatment. Notably,
skeletal muscle atrophy is one of the most evident side-effects. In order to isolate the anti-inflammatory effect, selective
glucocorticoid receptor modulators (SGRMs) have been designed and tested in vivo and in vitro. Recently, a group of novel
arylpyrazole compounds have been identified as potential SGRMs. Each of these compounds carries a different substituent at a
single position in a common arylpyrazole backbone forming 15 variations (named L1–L15). Among them, L5 and L7 were found
the most promising in terms of anti-inflamatory effects in vitro. Nonetheless, the biological impact of those compounds in various
tissues remains elusive. In this ongoing study, we analyzed the effects of the compounds L5 and L7 on skeletal muscle function.
Future analysis include histological features and gene expression profile.
The rats were treated for 7 days with three different doses of L5 and L7, which equimolarly correspond to 200µg, 600µg and
1200µg/kg body weight of dexamethasone. After treatment, the animals were anesthetized, the distal tendon of the tibialis
anterior muscle was exposed and attached to the lever arm of a servomotor. Then, muscle twitch contractions were induced by
electrical stimulation via a platinum hook electrode placed under the sciatic nerve. Maximum tetanic force were measured and
normalized by naso-tail length. No significant difference was found between L5, L7 and control group in any of the doses
studied, suggesting that L5 and L7 do not negatively affect muscle function.
Conclusions:
The present data showed that the intraperitoneal injection of L5 or L7 did not affect the maximum tetanic force, as is expected by
dexamethasone treatment. This data indicates those glucorticoid modulators are unable to decrease skeletal muscle function and
as previously shown in literature able to exert anti-inflamatory effects.
Keywords: Glucocorticoid, Dexamethasone, Skeletal muscle, Arylpyrazole compounds
Resumo:13-014
SWIMMING INTRODUCED DURING GROWTH PROMOTES CHANGES IN THE ACETYLCHOLINE RESPONSE
IN PANCREATIC ISLETS FROM OBESE RATS.
Rickli, S. 1; Barros, V. B. 2; Leite, N. D. C. 3; Borck, P. C. 4; Alípio, J. C. D. L. 5; Machado, M. J. R. 1;
Pereira, D. K. V. 1; Gonçalves, D. C. D. O. N. 4; Ferreira, T. R. 2; Grassiolli, S. 10
1
Dep Enf. Saúd Pública, Universidade Estadual de Ponta Grossa, DENFSP-UEPG
2
Dep Ed Física, Universidade Estadual de Ponta Grossa, DEDUFIS-UEPG
3
4
Dep C. Farmacêuticas, Universidade Estadual de Ponta Grossa, DEFAR-UEPG
5
6
7
8
Dep C. Farmacêuticas, Universidade Estadual de Ponta Grossa, DEDUFIS-UEPG
9
10
Dep Biologia Geral. Universidade Estadual de Ponta Grossa, DEBIO-UEPG
Objectives:
Obesity is associated with altered autonomic nervous system (ANS) activity which modulates energetic metabolism. High
parasympathetic (PNS) and simultaneous low sympathetic (SNS) nervous firing contributed to adipose tissue accumulation.
Insulin hypersecretion, frequently founded in the obesity is related to changes in insulinotropic Acetylcholine (Ach) and
muscarinic receptor responses in pancreatic islets. Neonatal monosodium glutamate (MSG) administration induces obesity onset
accomplished by hyperinsulinemia. Pancreatic islets obtained from MSG-obese rats show reduced cholinergic response. Exercise
activates SNS and increase energetic expenditure promoting adipose tissue reduction. Our study evaluated the effect of exercise
realized during growth in the cholinergic response in isolated pancreatic islets from MSG-obese rats.
Male Wistar rats received during 5 first days postnatal intradermic injection of MSG (4g/Kg). Control (CON) rats received saline.
After weaning both groups were divided in Sedentary (S) and Exercised (E), originating 4 groups: CON-S, CON-E, MSG-S and
MSG-E. The exercised rats swan 3 times/weeks/30min during 70 days. All groups were sacrificed at 90 old-days and body
weight and adipose tissue evaluated. Pancreatic islets were isolated by collagenase technique and incubated in 1mL of Krebs
solution contained glucose (8.3mM) in the presence of Ach (0.001 - 1000µM) plus neostigmine (10µM), an inhibitor of
acetylcholinesterase enzyme. Insulin was measured by radioimmunoassay. Neonatal MSG-treatment induced obesity onset.
MSG-S rats presented reduction of 14% in body weight and increase of 3 fold in the retroperitoneal fat pad deposit in relation to
CON-S (p
Conclusions:
Neonatal MSG-treatment induces obesity onset accomplished of insulin hypersecretion and weak cholinergic response.
Swimming changed cholinergic action in pancreatic islets from both groups. Nevertheless, isolated pancreatic islets from MSG-E
presented more accented alteration in the Ach response. Muscarinic composition or function may be involved in the dual
cholinergic response founded in isolated pancreatic islets from MSG-obese rats.
Keywords: acetylcholine, islets, obesity, MSG
Resumo:13-015
EFFECTS OF NEONATAL TOBACCO SMOKE EXPOSURE ON BODY COMPOSITION AND GLUCOSE
HOMEOSTASIS METABOLISM AT WEANING
Santos-silva, A. P. ; Pinheiro, C. R. ; Silva, L. F. ; Souza, G. T. ; Santana, A. C. ; Nascimento-saba, C. C. A.
; Abreu-villaça, Y. ; Moura, E. G. ; Oliveira, E. ; Lisboa, P. C.
Depto de Ciências Fisiológicas, UERJ
Objectives:
Overall, approximately 40% of children around the world are exposed to tobacco smoke at home (WHO, 2009). Children born
from smoking mothers present several development impairment (WHO, 2009; Acta Obstet Gynecol Scand 85(6): 675, 2006;
Tohoku J Exp Med 218(3): 221, 2009). Tobacco smoke exposure in infants has high correlation with the presence of
autoantibodies against pancreatic islet cells which could be the first step towards the development of type 1 diabetes (Medicina
(Kaunas) 44(1): 56, 2008). As few data relate tobacco smoke exposure in children and its effects upon weight maintenance and
glycemic control, here we studied the effects of tobacco smoke during lactation on body composition and glucose homeostasis.
After 72 h from birth, nursing rats and offspring were divided into 2 groups (n=8 dams/group, 2 pups/litter): SE: smoke-exposed
of cigarettes (1.7mg nicotine/cigarettes for 1h, 4 times/day) and C: air filtered-exposed. Pups were killed at weaning (21 days of
lactation). Body weight (BW) and nose-rump length were monitored. Visceral fat mass (VFM) were collected. Total body fat and
body protein were measured by carcass method. Adipocyte area was analyzed by morphometry. Fasting glucose was assessed by
glucometer and serum insulin was measured by radioimunoassay. Total adrenal catecholamine content was determined by
triindoxindol method. SE offspring showed lower BW (-7%,43.50±0.40 vs 40.55±0.54, p<0.05).
Conclusions:
Tobacco smoke exposure only during lactation led to growth failure of neonate pups. The lower insulinemia associated with
higher catecholamine in SE pups can explain the lower VFM and weight loss. At weaning, SE offspring had higher insulin
sensitivity. However these animals may have a lower insulin secretion, indicating a failure of pancreatic beta cells, which could
be caused by the known inhibitory effect of adrenaline. We suggest that tobacco smoke exposure in early periods of life affects
the fat tissue development and glucose homeostasis. Thus, smoking should be avoided during lactation.
Keywords: smoke, lactation, glucose, body composition
Financial Support: CNPq and FAPERJ
Resumo:13-016
PARTICIPATION OF ISOFLAVIN BETA IN THE MARKERS OF OXIDATIVE STRESS AND LOSS OF MUSCLE
MASS IN GASTROCNEMIUS MUSCLE OF THYROTOXIC RATS.
Marinello, P. C. ; Bernardes, S. S. ; Guarnier, F. A. ; Cecchini, R. ; Cecchini, A. L.
Ciências Patológicas / Universidade Estadual de Londrina , UEL
Objectives:
Previous studies in our laboratory has demonstrated that oxidative stress is related to muscle mass loss in hyperthyroidism. Based
on this, the objective of this work was to investigate if Isoflavin-beta (Iso-B) is efficient against oxidative stress related to muscle
waste in thyrotoxic rats. This approach may contribute to a possible introduction of Iso-B as an association in the treatment of
hyperthyroidism.
Male Wistar rats weighing 250 g was treated with i.p injections of Iso-B (10mg/Kg). The animals were weighed daily to calculate
the rate of mass loss (RML). The gastrocnemius muscle lipid peroxidation was avaliated by chemiluminescence (CL) induced by
tert-butyl, malondialdehyde (MDA) through the technique of TBARs, carbonilated proteins by reaction with dinitrophenyl
hydrazine, and quantification of total glutathione (GSH) and oxidized (GSSG). The results were analyzed by unpaired t-student
using the program GraphPad Prism 4, adopting as significative p<0.0001).
Conclusions:
The Isoflavin-beta (10mg/Kg) exerted pro-oxidant effect in animals with hyperthyroidism, increasing the loss of muscle mass and
modification of proteins, facilitating its degradation. Though, Iso-B is not indicated as an adjuvant on the treatment of
hyperthyroidism. However, further studies are needed with other doses for this effect is confirmed.
Keywords: Isoflavin beta, Loss of muscle mass, Oxidative stress, Thyrotoxicosis
Resumo:13-017
INVOLVEMENT OF B-TYPE NATRIURETIC PEPTIDE (BNP) IN HEART HYPERTROPHY IN A FEMALE RAT
MODEL OF OBESITY INDUCED BY HIGH-FAT DIET.
Gonçalves, G. K. N. ; Belo, N. O.
INSTITUTO MULTIDISCIPLINAR EM SAÚDE, UFBA
Objectives:
The B-type natriuretic peptide (BNP) acts in the heart promoting antiproliferative and antifibrinolytic effects. Estrogen has been
reported to prevent development of cardiac hypertrophy in female rodent models and in humans. In fact, premenopausal women
are much less likely to develop cardiovascular disease compared with men of similar age, suggesting that estrogen has a
cardioprotective effect. However, the mechanisms of sex steroid action are incompletely understood. The objective of this work
was to evaluate the participation of BNP in the cardiac hypertrophy in ovariectomized high-fat diet-fed rats.
Twenty-four 10-wk-old female Wistar rats intact (sham-operated) or ovariectomized were housed in 12-h light, 12-h dark
lighting. They were divided into four groups (n=6) according to diet: diet containing 54.4% of total calories from fat (high fat
diet) or control/standard diet (Regul Pept. 167:149, 2011). During 24 weeks we measured body weight and blood pressure by
plethysmography (LE5001, Panlab, Barcelona, Spain). At the end of the experiment the rats were decapitated and the hearts were
removed for gene expression determination by RT-PCR and histological analysis. Also, trunk blood were removed for plasma
BNP level determination by ELISA. The high-fat diet associated with ovariectomy was able to produce a greater weight gain than
control group (510±20 vs. 270±19g, p
Conclusions:
This study showed that BNP is associated with cardiac hypertrophy in a female animal model of obesity induced by high-fat diet
even in the hypertension absence.
Keywords: BNP, HYPERTROPHY, OBESITY
Financial Support: CAPES and CNPq
Resumo:13-018
T3 UP REGULATES SEVERAL E3 LIGASES IN RAT SKELETAL MUSCLE.
Ramos, G. V. 1,3; Rosansky, A. 1,3; Carneiro-ramos, M. S. 2; Yamate, G. 1,3; Baptista, I. L. 1,3; Guilherme, J.
P. L. F. 1,3; Artioli, G. G. 4; Moriscot, A. S. 1,3
1
INSTITUTO DE CIENCIAS BIOMEDICAS I, ICBI
2
UNIVERSIDADE FEDERAL DO ABC, UFABC
3
DEPARTAMENTO DE BIOLOGIA CELULAR E DO DESENVOLVIMENTO, BIOL CEL. DESENV.
4
Escola de Educação Física e Esporte, EEFE
Objectives:
T3 is associated with muscle wasting and weakness due to atrophy. Recent studies have shown that muscle specific E3-ligases
such as Atrogin-1 and Murf-1 could be responsible for muscle mass loss in hyperthyroid rats. However, we found other E3-ligase
that were also responsive to treatment of T3. In this sense, the ultimate goal of this study is to understand the relationship between
these new E3 ligases with muscle wasting induced by T3.
The responsiveness of E3-ligases by T3 was first analysed by Microarray and later by RT-PCR. Rats were induced to
experimental hyperthyroidism (6µg T3/100g BW/day), corresponding to 20X physiological doses for 12, 24 hours and 7 days. In
Microarray was considered responsive by T3 E3-ligases that presented ≥1.2 fold change (FC) in comparison with the control
group. According to the overall analyses by Microarray we noted three new E3-ligases responsive by T3 (Smurf-1, Nedd4 and
MDM2) in addition to Atrogin and Murf-1. Since MDM2 was the most resposive to T3, we decided to focus our efforts on this
E3 ligase. We observed that the gene expression of MDM2 in hyperthyroid rats is dose-dependent. Rats treated with 20X and
50X physiological doses of T3 showed an increased mRNA of 70% and 80% vs. control respectively.
Conclusions:
T3 upregulates several E3 ligases, especially MDM2. We intent to submit rats to other models leading to atrophy, such as
immobilization, starvation and glucorticoid treatment in order to better understand the role of MDM2 in skeletal muscle atrophy.
Keywords: E3 LIGASES, MUSCLE, T3
Financial Support: FAPESP and CNPq
Resumo:13-019
NEONATAL TOBACCO SMOKE EXPOSURE AFFECTS ADIPOSITY AND LEPTIN LEVELS IN THE RAT ADULT
OFFSPRING
Viana, A. I. C. ; Santos-silva; Pinheiro, C. R. ; Silva, L. F. ; Villaça, Y. A. ; Moura Eg; Oliveira E; Lisboa,
P. C.
DCF/UERJ, IBRAG
Objectives:
Environmental changes in a critical period of development can lead to permanent alterations in the metabolism and cause disease
at adulthood; a phenomenon called metabolic programming(1). Maternal smoking in pregnancy is often associated with the
development of obesity in childhood(2). In our laboratory, we observed a relationship between increased leptin concentration and
adiposity in adult offspring whose mothers were exposed to pure nicotine during lactation(3) and the development of leptin and
insulin resistance(4), however little is known about the effects of the exposure to cigarette smoke in neonatal life upon adiposity.
Then, we evaluated the body composition and serum leptin levels in adult animals programmed by neonatal exposure to cigarette
smoke.
After 72 h from birth, nursing rats and offspring were divided into 2 groups (n=8 dams/group, 2 offspring/litter): SE: smokeexposed of cigarettes (1.7mg nicotine/cigarettes for 1h, 4 times/day) and C: air filtered-exposed. Offspring groups were killed at
postnatal day 180 (PN180). Body weight (BW) and food intake (FI) were monitored. Visceral fat mass (VFM) were collected.
Total body fat and body protein were measured by carcass method. Serum leptin levels were measured by radioimunoassay. At
PN180, SE group showed no significant difference in BW or FI compared to C group. However, these animals had higher VFM
(+65.5%, p
Conclusions:
Taken together our data suggest that neonatal exposure to cigarette smoke programs for the development of central obesity and
hyperleptinemia at adulthood, possible due to leptin resistance and mainly due to the effect of nicotine. Therefore, smoking
should be avoided in households with infants since it can be a risk factor for the programming of metabolic disturbances in adult
life.
Keywords: exposure to ciagarette, hyperleptinemia, leptin, lactation
Financial Support: FAPERJ and CNPq
Resumo:13-020
ANTIOBESITY EFFECT OF YERBA MATÉ (ILEX PARAGUARIENSIS) IN RATS PROGRAMMED BY EARLY
WEANING
Lima, N. S. ; Franco, J. G. ; Moura, E. G. ; Maia, L. A. ; Nogueira-neto, J. F. ; Rodrigues, V. S. T. ;
Felzenswalb, I. ; Kaeser, A. ; Oliveira, E. ; Lisboa, P. C.
Departamento de Ciências Fisiológicas/IBRAG/UERJ, UERJ
Objectives:
Recently, we have shown that early weaning (EW) with no use of pharmacological substances or maternal separation programs
for obesity, hyperleptinemia and leptin and insulin resistance at adulthood (Br J Nutr, 1, 2011). The yerba maté (Ilex
paraguariensis) has been suggested to be important in the management of obesity, contributing in the weight loss, even with the
use of high fat diets (Obes, 17; 2127, 2010). The aim of the present study was to evaluate the effects of yerba maté extract on
weight management, visceral fat mass, lipid profile and fast glycemia.
After birth, lactating rats were separated in: EW (early weaning) - dams were wrapped with a bandage to interrupt lactation
during the last 3 days of lactation, and C (control) - dams whose pups had free access to milk throughout lactation (21 days). At
least 8 litters were studied. At 150 days-old, two programmed EW offspring from the same litter were subdivided into: EW (8)
and EW + maté (8), according to the treatment (water or yerba maté extract 1g/kg body weight, respectively for gavage, daily)
during 30 days. C offspring (8) also received water for gavage (daily/30 days). At 180 days-old, pups were sacrificed by
decapitation and blood was collected. The visceral fat mass was weighed. Lipid profile and glycemia were determined by
colorimetric assay. At 150 days-old (first day of treatment) all groups showed no difference in body weight. However, at 180
days-old (end of treatment), EW + maté group presented lower weight (-10%, p
Conclusions:
As the therapy with yerba maté extract was capable to reverse abdominal obesity and hypertriglyceridemia in offspring
programmed by early weaning, we suggested that yerba mate has an important antiobesity action in this experimental model.
Keywords: Early Weaning, Obesity, Yerba Maté, Visceral Fat Mass, Lipid Profile
Financial Support: FAPERJ, CNPq and CAPES
Resumo:13-021
SERUM THYROID HORMONE LEVELS AFTER SLEEP LOSS IN RATS.
Rodrigues, N. C. 1; Cruz, N. S. 1; Nascimento, C. D. P. D. 1; Conceição, R. R. D. 1; Olivares, E. L. 1; Silva,
A. C. M. D. 1; Cavalho, D. P. . 2; Andersen, M. L. 3; Marassi, M. P. 1
1
Departamento de Ciências Fisiológicas - UFRRJ, UFRRJ
2
Instituto de Biofísica Carlos Chagas Filho - UFRJ, UFRJ
3
Departamento de Pisicobiologia - UNIFESP, UNIFESP
Objectives:
Modern life is shortened sleep time and the consequences of the sleep decrease or deprivation has been study these last years.
Thyroid hormones, thyroxine (T4) and triiodothyronine (T3), have effects in virtually all tissues and are influenced by other
endocrine signals like cortisol and gonadal hormones, but only a few studies relating thyroid hormone and sleep loss. The aim of
this study was to analyze thyroid economy during paradoxical sleep deprivation and sleep restriction and after 24 hours of
rebound period.
Male Wistar rats (200-250g) from the UFRRJ animal facility were housed at controlled temperature (22ºC), with 12 hours of light
(7am to 19pm), with food and water ad libitum. The sleep restriction and sleep deprivation protocols used were based on the
modified multiple platform methodology. The rats were randomly assigned in 7 groups: control (n=11) rats kept in the
experimental room, with normal sleep pattern; paradoxical sleep deprivation (PSD) for 24h (PSD24, n=15); PSD24 with rebound
of 24h (PSD24R24, n=12), same protocol of group PSD24 but the animals can sleep freely in last day; PSD for 96 hours (PSD96,
n=13), rats underwent paradoxical sleep deprivation for 96 hours; PSD96 with rebound of 24h (PSD96R24, n=14), the sleep
restriction for 21 days (SR21, n=14), animals were allowed to sleep 6 hours a day (10am to 4pm); and SR21 with a rebound of
24h (SR21R24, n=15). All animals were euthanized on the same day, thyroid gland was weighed and blood samples were
collected for analysis of T3 and T4 by electrochemiluminescence. The results were expressed as mean ± SEM and were
considered to be significantly different when P
Conclusions:
Serum T3 increased after acute and chronic sleep loss not being the recovery able to normalize these changes. Conversely, T4
decreased only in acute sleep deprivation; in addition, the sleep rebound normalized T4 values after paradoxical sleep deprivation
for 24h, suggesting differential thyroid function modulation in chronic and selective sleep loss.
Keywords: Rats, sleep, thyroid hormone
Financial Support: FAPERJ; CNPq; AFIP
Resumo:13-022
SWIMMING INTRODUCED AFTER WEANING AND MAINTAINED DURING THE GROWTH ATTENUATES
OBESITY INDUCED BY HYPOTHALAMIC LESIONS.
Barros, V. B. 1; Leite, N. D. C. 1; Borck, P. C. 3; Rickli, S. 4; Alípio, J. C. D. L. 5; Machado, M. J. R. 6;
Venturelli, A. C. 2; Vellosa, J. C. R. 9; Mathias, P. C. D. F. 10; Grassiolli, S. 11
1
Dep. Ed. Física, Universidade Estadual de Ponta Grossa, DEDUFIS-UEPG
2
3
DP. C FARMACEUTICAS, Universidade Estadual de Ponta Grossa, DEFAR-UEPG
4
DP ENF E SAÚDE PÚBLICA, Univer Estadual de Ponta Grossa, DENFSP-UEPG
5
6
7
8
DP. C FARMACEUTICAS, Universidade Estadual de Ponta Grossa, DEFAR-UEPG
9
DEP ANALISES CLÍNICAS, Universidade Estadual de Ponta Grossa, DECLIN-UEPG
10
Dep. Biologia Cel e Gen, Universidade Estadual de Maringá, DBC, UEM
11
Dep Biologia Geral, Universidade Estadual de Ponta Grossa, DeBio - UEPG
Objectives:
Obesity is a complex pathology intimately associated at development of type 2 diabetes. High caloric food intake and sedentary
lifestyle have a central role in the epidemic obesity in the worldwide. Physical exercise increases energetic expenditure, reduces
adipose tissue accumulation and improves insulin response contributing to glycemic control. Neonatal glutamate monosodium
(MSG) administration induces hypothalamic lesions that determine obesity onset, insulin resistance, glucose intolerance,
hypertension and dyslipidemia. Thus, MSG-obese rats show metabolic syndrome profile. This work evaluated the effect of
chronic exercise realized after weaning in the MSG-obesity progression.
Male Wistar rats received during 5 first days postnatal subcutaneous injections of MSG (4g/Kg). Control (CON) rats received
saline. At 21 days of life the rats were divided in sedentary (S) or exercised (E) resulting in 4 groups: CON-S; CON-E; MSG-S
and MSG-E. Swimming was realized 3 times/weeks/30min during 70 days. At 90 days of life the rats were sacrificed, body
weight (g), nasoanal length (NAL) were evaluated to calculate Lee index (LI). Visceral (epididymal and retroperitoneal) and
subcutaneous inguinal fat pads, liver, heart, adrenal gland and gastrocnemius muscle were weighed to comparative analyze
(g/100g). The total blood was collected, the plasma used to insulin dosage (radioimmunoassay) and glucose, triglycerides, total
cholesterol evaluated by enzymatic methods (kits). MSG administered during neonatal phase induces obesity development. MSGS rats presented reduction of 9.5% in the body weight and NAL, promoting increase of 8% in the LI in relation to CON-S group
(p<0.05).
Conclusions:
Neonatal MSG treatment provoked adipose tissue accumulation and body growth reduction. High adipose tissue content is
associated with hyperinsulinemia and dyslipidemia observed in MSG-obese rats. Swimming maintained during growth could
attenuate and/or blockage the installation of obesity, just as improve glucose homeostase control. This action can avoid diabetes
type 2 development.
Keywords: obesity, adipose tissue, Msg, exercise
Resumo:13-023
IMPLICATIONS OF THYROID AND GONADAL HORMONES IN THE GENESIS OF OBESITY AFTER
OVARIECTOMY.
Teixeira, M. J. 1; Marassi, M. P. 1; Olivares, E. L. 1; Ferreira, A. C. F. 2; Carvalho, D. P. 2; Silva, A. C. M. 1
1
Departamento de Ciências Fisiológicas/Instituto de Biologia, UFRRJ
2
Laboratório de Fisiologia Endócrina Doris Rosenthal / IBCCF, UFRJ
Objectives:
Obesity is a public health concern and the influence of gonadal hormones as regulators of corporal weight is well described but
little is known about the possible correlation between gonadal hormones and thyroid function in the genesis of obesity, since
thyroid hormones are important regulators of energetic metabolism. The aim of this study was to analyze serum thyroid hormones
and type 2 deiodinase (D2) activity after 15 days of ovariectomy.
Female Wistar rats (200-250g) from the UFRRJ animal facility were housed in individual cages at controlled temperature (22ºC),
with 12 hours of light (7am to7pm), with water ad libitum and controlled food. Treatment was initiated 24 hours after
ovariectomy Eb (0,7&mug/100g pc,sc), PG (250&mug/100g pc,sc) ou PG+Eb and finished 15 days after surgery. The
experimental protocol was approved by the institutional committee of ethics. Rats were assigned in 5 groups: sham (n=9); Ovx
(n=9), Ovx+Eb (n=8), Ovx+PG (n=9) and Ovx+Eb+PG (n=9). All animals were euthanized in the same day, blood samples were
collected for analysis of T3, T4, estradiol and progesterone by RIA and thyroid gland was weighed. The D2 activity was
measured in the brown adipose tissue (BAT), pituitary and hipothalamus. The results were expressed as mean ± SEM and were
considered to be significantly different when P
Conclusions:
Treatment with Eb and PG may not interfere with thyroid function acutely, suggesting that thyroid hormones are not involved in
the genesis of obesity after ovariectomy, at least in the schedule protocol used herein.
Keywords: Fisiologia, Glândulas, Metabolismo, Tiroxina
Financial Support: FAPERJ and CNPq
Resumo:13-024
UP-REGULATION OF TYPE 2 DEIODINASE RESTORES SERUM T3 LEVELS AFTER MYOCARDIAL
INFARCTION IN RATS
Império, G. E. 1,2; Araujo, I. G. 4,2; Marassi, M. P. 2; Mcelligott, A. 3; Paul, J. T. 3; Henderson, K. K. 3;
Olivares, E. L. 2
1
Laboratory of Molecular Endocrinology, IBCCF, UFRJ
2
Department of Physiological Sciences, Institute of Biology, UFRRJ
3
Department of Medicine and The Cardiovascular Institute, LUMC
4
Dep. of Physiology, Medicine College of Ribeirão Preto, USP
Objectives:
Recently, we demonstrated acute decrease of serum triiodothyronine (T3) and thyroxine (T4) levels in an animal model of
myocardial infarction (MI) that was mainly attributed to an acute induction of cardiac type 3 deiodinase (D3) activity. In spite of
persistently low T4 concentrations, serum T3 returned to control levels by 8 weeks post-MI. Considering the lack in literature
regarding the underlying mechanism of T3 normalization post-MI, we aimed to verify if type 2 deiodinase (D2) participates of T3
restoration after MI in rats. Also, we investigated the D2 and D3 expression profile on rat infarcted hearts.
Adults male Wistar rats and Sprague Dawley rats underwent experimental MI through permanent occlusion of coronary artery or
sham-operation. The animals were submitted to echocardiogram (ECO) and serial blood collection, via jugular vein, to measure
serum T3 and T4 (MI, n=20 and Sham, n=18) at 1, 8 and 12 weeks after surgery. At the same time points animals were randomly
euthanized (n=4-10/time/group) and brown adipose tissue (BAT) and pituitary were collected for D2 activity measurement.
Animals from a separate group have their heart left ventricle (LV) isolated from Sham (n=12), or separated into the infarcted
region (INF), border-zone (BZ), and non-infarcted posterior wall (PW) from MI animals (n=26) to measure D2 and D3
expression by quantitative real-time PCR. A subset of animals was daily supplemented with T4 (1µg/100g b.w.) or vehicle
(0.02M NaOH) s.c. from 1-day to 6 wks after surgery and they were submitted to the same procedures described before. ECO
demonstrated a dramatic and sustained reduction (40-50%) in LV ejection fraction at all time points in MI vs. Sham animals.
Serum T4 concentrations had significant and sustained reduction in MI vs. Sham animals at 1, 8 and 12 wks. Serum T3
concentrations (ng/dL) fell transiently at 1 (23.47±3.07vs.56.50±5.34, p0.05) and 12 wks (44.89±6.98vs.61.40±4.35, p>0.05) in
MI vs. Shams. The MI or BZ D3 expression increased 1-day and remained elevated at 14 days post-MI. D2 expression was
significantly attenuated at 1-day, but was significantly elevated at 7 and 14 days post-MI vs. sham (p
Conclusions:
Our findings suggest that changes in myocardial D2 and D3 expression may play immediate and important roles in the early
stages of myocardial remodeling and up-regulation of D2 activity in BAT in rats constitutes an important mechanism for serum
T3 normalization in the later phases of a myocardial infarction.
Keywords: Infarction, Myocardium, Thyroid, Deiodinase, Rats
Financial Support: CNPq, FAPERJ, Dr. Ralph and Marian Falk Trust for Medical Research, AHA.
Resumo:13-025
ACTIVATION OF PPAR-GAMMA BY ROSIGLITAZONE RESTORES MAST CELL NUMBERS AND REACTIVITY
IN ALLOXAN-DIABETIC RATS
Torres, R. C. ; Telles, T. S. ; Cordeiro, R. S. B. ; Martins, M. A. ; Silva, P. M. R. ; Carvalho, V. F.
Laboratório de inflamação/ Instituto Oswaldo Cruz, IOC/ Fiocruz
Objectives:
Diabetes mellitus is a metabolic disorder associated with chronic hyperglycemia. Alloxan-induced diabetic rats present a deficient
inflammatory response in association with a decrease in the number and responsiveness of mast cells. This reduction in the mast
cell numbers and reactivity noted in diabetic rats is related to a hyperreactivity of the hypothalamic-pituitary-adrenocortical
(HPA) axis and increased levels of glucocorticoids. Some authors have showed that the nuclear receptor PPAR-gamma is
expressed on mast cells and its activation increases the viability and proliferation of these cells. In addition, agonists of PPARgamma have been effective in reducing the hypercortisolism in diseases associated with hyperreactivity of HPA axis, including
Cushing disease. Therewith, our aim was to investigate the effect of the PPAR-gamma agonist rosiglitazone on mast cell numbers
and reactivity in diabetic animals.
The animals were obtained from the Oswaldo Cruz Foundation breeding colony and used in accordance with the guidelines of the
Committee on Use of Laboratory Animals of the Oswaldo Cruz Foundation (CEUA-FIOCRUZ, license LW 23/10). Diabetes was
induced by one single intravenous injection of alloxan (40 mg/Kg) into fasted rats and rosiglitazone (0.5 mg/Kg) was
administered after 3 days of diabetes induction, once daily for 18 consecutive days. Mast cell numbers were evaluated after
staining of the pleural washed and mesenteric tissue with toluidine blue dye. Plasma corticosterone levels were quantified by
radioimmunoassay and adrenocorticotropin hormone receptor (MC2-R) expression in adrenal was assessed by
immunohistochemistry. Treatment with rosiglitazone restored mast cell numbers in the pleural cavity and in mesenteric tissue of
diabetic rats. Rosiglitazone also significantly reversed the diabetes-induced reduction in histamine release, as measured by
fluorescence, following activation with antigen in vitro. Diabetic rats presented an increase in the plasma corticosterone levels
with non-diabetic rats (from 247.32 ± 27.87 to 751.00 ± 159.55 plasma corticosterone ng/ml, respectively; mean ± SEM, n = 5).
Treatment with rosiglitazone restored plasma corticosterone levels in diabetic rats (355.57 ± 78.26 plasma corticosterone ng/ml,
mean ± SEM, n = 5). Furthermore, we noted that the increase in MC2-R expression in adrenal glands of diabetic rats was
inhibited by the drug.
Conclusions:
In conclusion, our findings showed that rosiglitazone restored the number and reactivity of mast cells in diabetic rats,
accompanied by reduction in plasma corticosterone levels and adrenocorticotropin hormone receptor expression in adrenal
glands, evidencing that PPAR- gamma activation have a critical role in mast cell behavior of diabetic rats.
Keywords: Diabetes, Glucocorticoid, HPA axis, Mast cell, PPAR-gamma
Financial Support: CNPq, FAPERJ and FIOCRUZ.
Resumo:13-026
EFFECT OF CONGENITAL HYPOTHYROIDISM IN HIPPOCAMPAL CELLS
Goulart, P. B. 1; Cattani, D. 1; Rieg, C. E. H. 1; Wohel, V. M. 2; Duarte, E. C. W. 2; Silva, F. R. M. B. 1;
Zamoner, A. 1
1
2
Depto de Bioquímica/ CCB, UFSC
Depto de Ciências Morfológicas/CCB, UFSC
Objectives:
Thyroid hormones modulate central nervous system development and function. Congenital hypothyroidism is associated with
delay in cell migration and proliferation in brain tissue, impairment of synapse formation, dysregulation of neurotransmitters,
hypomyelination and mental retardation. However, the effect of hypothyroidism in hippocampus is still unknown. The aim of this
study was to investigate alterations in glial and neuronal cell number in hippocampus of hypothryroid of immature rats and the
involvement of cholinergic system as well as oxidative stress in the pathophysiology of this hormonal dysfunction.
Congenital hypothyroidism was induced in rats by adding 0.05 % 6-propyl-2-thiouracil in the drinking water during gestation and
suckling period. Morphological analysis was carried out by using cresil violet. The levels of GFAP were determined by
immunoblotting. Interestingly, hypothyroidism leads to inhibition of cholinesterase activity in hippocampus that might account
for increased cholinergic transmission and neural cell damage. We also analyzed the neuronal and glial damage in hypothyroid
hippocampus. Neurons and astrocytes were counted in the dentate gyrus, CA1, CA2, CA3, and CA4 hippocampal regions. It was
found an important neuronal damage in the hypothyroid group. Also, the glial cell number was altered in some of the
hippocampal regions. GFAP immunoreaction confirmed the altered number of glial cells. In addition, glutathione (GSH) levels
were reduced and accompanied by increased gamma-glutamyl transferase (GGT) activity in hypothyroid hippocampus.
Conclusions:
The modulation of GGT activity could be involved, at least in part, as a tentative to maintain GSH turnover. The results observed
herein demonstrate a misregulation of cholinergic system, cell death and/or diminished cell proliferations, which are probably
related to oxidative stress and neurotoxic damage in hypothyroid brain.
Keywords: hypothyroidism, hippocampus, morphological alterations, cholinesterase, gamma-glutamyl transferase
Financial Support: CAPES, CNPq, FAPESC, PGFAR-UFSC.
Resumo:13-027
AN INHIBITOR OF LEUKOTRIENE SYNTHESIS AFFECTS THE VASOPRESSINERGIC HYPOTHALAMICNEUROHYPOPHYSEAL SYSTEM
Santos, J. F. D. 1; Pelegrin, G. R. 2; Rocha, M. J. A. 2
1
Escola de Enfermagem de Ribeirão Preto, EERP
2
Faculdade de Odontologia de Ribeirão Preto, FORP
Objectives:
Previous work of our laboratory suggests the involvement of centrally produced leukotrienes (LTs) in vasopressin secretion
during sepsis. Our aim was to analyze the effect of central injection of MK 886 (LTs synthesis inhibitor) on arginine vasopressin
(AVP) release and c−fos expression in vasopressinergic neurons following osmotic stimulation in rats.
Male Wistar rats received an intracerebroventricular injection of MK 886 (1, 2 or 4μg/kg) or vehicle (DMS0 5%), 1 hour prior to
an intraperitoneal injection of hypertonic or normal saline (2M and 0,01M NaCl, respectively). Thirty minutes after the stimulus,
one group of animals (n= 28) was decapitated and blood collected for plasma osmolality and AVP determination. A second group
(n= 28) was anaesthetized and perfused with PBS 0,01M followed by paraformaldeyde 4%. Brains were then removed and
processed for Fos protein detection by immunocytochemistry. The osmotic stimulus caused a significant increase in osmolality
(P≤0,05), plasma AVP concentration (P≤0,05) and c-fos expression in the SON and PVN (P≤0,05). The central administration of
MK 886 did not affect plasma osmolality but induced a dose-dependent increase in circulating AVP levels (P≤0,05) and a
reduction of c−fos expression in both nuclei already at a dose of 1μg/kg (P≤0,05).
Conclusions:
Blocking LT synthesis in the context of an osmotic stimulus caused a decrease in hypothalamic neural activation (c−fos
expression), but an increase in vasopressin secretion. These finding are strong evidence for a modulatory role of leukotrienes in
neuroendocrine system functions
Keywords: cis-leukotrienes, c-fos, vasopressin, hypothalamus, hyperosmotic
Resumo:13-028
THE SMOKING DURING PREGNANCY AND LACTATION CAUSES ELEVATION ON FASTING GLUCOSE
LEVELS WITHOUT CHANGING GLUT4 PROTEIN CONTENT IN MUSCLE
Santos, L. M. 1; Gomes, P. R. L. 2; Oliveira, M. A. N. 2; Machado, U. F. 2; Seraphim, P. M. 1
1
Fisioterapia, UNESP
2
Fisiologia e Biofísica., USP
Objectives:
To investigate the effect of smoking during pregnancy and/or lactation on GLUT4 protein content in the gastrocnemius muscle
from Wistar rats offspring.
Wistar rats were divided by gender and treatment type in 8 groups: FNP – female offspring of nonsmoking mothers sacrificed
after pregnancy; MNP - male offspring of nonsmoking mothers sacrificed after pregnancy; FSP - female offspring of smoking
mothers sacrificed after pregnancy; MSP - male offspring of smoking mothers sacrificed after pregnancy; FNB - female offspring
of nonsmoking mothers sacrificed after breast-feeding; MNB - male offspring of nonsmoking mothers sacrificed after breastfeeding; FSB - female offspring of smoking mothers sacrificed after breast-feeding; MSB - male offspring of smoking mothers
sacrificed after breast-feeding. After anesthesia gastrocnemius muscle tissue and blood were collected for analysis. Fraction of
total cellular membranes was obtained after different centrifugation and the quantification of GLUT4 protein content was
performed by Western Blotting. The fasting glucose was analyzed by colorimetry. Data are expressed as Mean+/-SEM. The body
weight did not change among the groups at birth (FNP/MNP= 6.48+/-0.67; FNB/MNB= 6.02+/-0.16; FSP/MSP= 5.4+/-0.22;
FSB/MSB= 5.33+/-0.66g, n=7-10). At 2 months of age, a significant increase was observed in the body weight of males probably
due to natural sexual dimorphism (FNP=132.77+/-5.65; MNP=165.25+/-4.99*; FNB=117.0+/-3.47; MNB= 170.5+/- 8,28***;
FSP=129.77+/-5.14; MSP=165.57+/-12.21*; FSB=121.0+/-8.97; MSB=155.1+/-12.02* g, *P
Conclusions:
The smoking during pregnancy and lactation increases fasting glucose of offspring without changes the GLUT4 protein content.
The sexual difference of GLUT4 protein expression suggests minor capacity of glucose uptake in male rats compared to female
rats. However considering the glucose levels among the genres, we can deduce that or 1) there must be some compensation in the
intrinsic activity of transporter protein in males; or 2) the lack of adipose tissue in female can provoke an increase of GLUT4 per
gram of tissue in the muscle, highlighting its important participation to maintenance of glucose homeostasis.
Keywords: GLUT4, LACTATION, PREGNANCY, SMOKING
Financial Support: FAPESP #2010/09020-6; FUNDUNESP #0795/2010.
Resumo:13-029
&DELTA-AMINOLEVULINATE DEHYDRATASE ACTIVITY AND REACTIVATION INDEX IN TYPE 2
DIABETIC PATIENTS
Bonfanti, G. ; Ceolin, R. B. ; Lucca, L. D. ; Bona, K. D. S. D. ; Cargnelluti, L. D. O. ; Gonçalves, T. D. L. ;
Moretto, M. B.
Departamento de Análises CLínicas e Toxicológicas, UFSM
Objectives:
Aim:&delta-Aminolevulinate dehydratase (&delta-ALA-D) is a sulfhydryl-containing enzyme that is extremely sensitive to
oxidizing agents (Toxicol Lett 66; 139:55, 2003). The decreased activity this enzyme has been associated with chronic diseases
and diabetes mellitus (Am J Kidney Dis 40(1); 43-50, 2002), where the condition of oxidative stress also was observed. The
reactivation index of &delta-ALA-D activity has been used in human studies that evaluate the connection of thiol groups in
&delta-ALA-D inhibition by oxidizing agents (Clin Biochem 40(9-10); 591-4, 2007) and has been considering a good tool to
evaluate oxidative stress in a chronic exposure process like diabetes mellitus (Clin Chem 38; 1071-5, 2005). The present study
was designed to investigate the possibility of the involvement of –SH groups in &delta-ALA-D activity of type 2 diabetes
mellitus (T2DM) patients and correlate the enzymatic activity with oxidative status.
Methods: Blood samples of 63 T2DM patients, (26 males and 37 females, age 58,71 ± 9,35 years) attended in University Hospital
of Santa Maria (HUSM) were collected and 63 blood samples of healthy people (31 males and 32 females, age 55,98 ± 9,57
years) were collected as control. The present study was approved by the Human Ethical Committee of the Federal University of
Santa Maria (0199.0.243.000-09). &delta-ALA-D activity was assayed in whole blood (Z Klin Chem Klin Biochem. 12(8); 38990, 1974). The reactivation index was estimated using dithiothreitol (DTT), a reducing agent, and measured using: A-B/A*100
where A=absorbance of assay with DTT and B=absorbance of assay without DTT. Lipid peroxidation was estimated in plasma
by measurement of Thiobarbituric Acid Reactive Substances (TBARS) (Free Radic Biol Med. 31(3); 331-5, 2001). Following the
distribution of the variables, a non-parametrical Mann-Whitney U-test was performance and the results were expressed as median
(lower/ upper quartiles). Results: Blood &delta-ALA-D activity was significantly lower in diabetic patients (3,01 (1,47/4,26)
nmol PBG/ml blood/h) than control group (3,27 (2,16/6,37) nmol PBG/ml blood/h) (p
Conclusions:
Conclusion: These findings may contribute to the consideration of the existence of a decreased efficiency of the cellular
mechanism against reactive oxygen species in T2DM, leading to lipid peroxidation and reduced activity of &delta-ALA-D. Thus,
we can suggest that this enzyme, through its enzymatic activity and reactivation index, could be a good biomarker for assessing
oxidative stress, a condition that can play an important role in impaired metabolic process.
Keywords: &delta-Aminolevulinate dehydratase, oxidative stress, type 2 diabetes mellits
Financial Support: FIPE, CAPES
Resumo:13-030
EFFECT OF PPAR AGONISTS ALPHA AND GAMMA ON RENAL GLUCOSE TRANSPORTERS EXPRESSION OF
INSULINOPENIC DIABETIC RATS
Okamoto, M. M. ; Miyake, G. M. ; David-silva A; Marques, M. F. D. S. ; Sabino da Silva R ; Freitas, H. S.
; Machado, U. F.
Departamento de Fisiologia /Instituto de Ciências Biomédicas, ICB-USP
Objectives:
There is growing evidence that inflammatory processes play important role in diabetic nephropathy. Both PPAR alpha and
gamma are expressed in the kidney, and their agonists exhibit pivotal role in tissue inflammation and repair. The repercussion of
these agonists on glucose transporters expression is still unclear. In the present study we investigated the effect of PPAR agonists
pioglitazone (PPAR-gamma) and fenofibrate (PPAR-alpha) on renal glucose transporters protein expression in insulinopenic
diabetic rats.
Male Wistar rats were rendered diabetic at 3 month of age. Diabetic rats were treated with saline (DS), insulin (DI- 4U/day),
insulin + pioglitazone (DIP- 20mg/Kg bw), insulin + fenofibrate (DIF -100mg/Kg bw) or insulin + pioglitazone + fenofibrate
(DIPF) during 5 weeks. Non diabetic rats (ND) were studied as a control group. We evaluated in these groups after treatment: a)
glycosuria and proteinuria; b) glycemia; c) renal cortex and medulla glucose transporters protein expression by Western blotting.
As expected, DS showed in comparison to ND, hyperglycemia, glycosuria, proteinuria, increased expression of renal glucose
transporters in cortex (GLUT2 - ~26%, SGLT2- ~55%, P< 0.01) content; DIF and DIPF increased GLUT2 (DIF~19%,
DIPF~21%) but only DIF decreased SGLT2 (~41%, P
Conclusions:
Our results suggest that PPAR agonists gamma and alpha play an important role on glucose transporters expression in kidney of
diabetic rats.
Keywords: kidney, diabetic nephropahty, glucose transporters
Resumo:13-031
TIMING OF ESTROGEN THERAPY AFTER OVARIECTOMY MODIFIES ITS CARDIOPROTECTIVE ACTIONS
IN SHR FED WITH HIGH FAT DIET.
Neto, A. J. A. R. ; Souza, C. L. S. ; Neves, J. V. ; Santos, C. S. ; Nogueira, E. F. ; Gusmão, D. O. ; Belo, N.
O.
Institutuo Muldisciplinar em Saúde, UFBA - IMS - CAT
Objectives:
Estrogen exerts a wide variety of actions involving many target tissues such as adipose tissue and heart. We studied the effects of
long-term ovariectomy, short-term ovariectomy and 17beta-estradiol treatment on the level of atrial natriuretic peptide (ANP) and
leptin in plasma of female spontaneously hypertensive rats (SHR) fed high fat diet. Methods: This study included two
experimental paradigms. In the first paradigm (short-term ovariectomy), 10-wk-old female SHR were ovariectomized or shamoperated and 8 weeks later received hormone replacement therapy with 17β-estradiol (E2: 5μg/0.1ml/100g weight, subcutaneous
injections) and control rats received vehicle (corn oil-VEH: 0.1ml/100g weight) for 14 days. In the second paradigm (long-term
ovariectomy), 10-wk-old female SHR were ovariectomized or sham-operated and 24 weeks later received daily injections
containing either oil or E2 for 14 days. Also, the rats were divided according to the diet received, high fat diet (54.4% of fat) and
standard diet. Body weight and blood pressure were measured weekly. After the hormone replacement therapy, the rats were
sacrificed and blood was collected to determination of estradiol, ANP and leptin plasma levels by ELISA.
As expected, ovariectomy induced a marked reduction in plasma estradiol levels in all groups. But, the E2 treatment promote a
higher plasma estradiol levels in short-term ovariectomized rats (increase of more than 30 times) compared to long-term
ovariectomized animals (increase of approximately 3 times). The short-term ovariectomized standard diet-fed rats showed higher
plasma ANP levels than the long-term ovariectomized standard diet-fed rats (312±4 vs. 174±34 pg/ml, n:4, p<0.05).
Conclusions:
These data show that the long-term ovariectomy associated with the high fat diet was able to reduce some cardioprotective E2
effects such as increased plasma ANP levels as well as lowering blood pressure.
Keywords: ESTROGEN, MENOPAUSE, TIME
Resumo:13-032
SWIMMING EFFECT ON K+ATP CHANNEL-DEPENDENT AND K+ATP CHANNEL-INDEPENDENT PATHWAYS
IN PANCREATIC ISLET FROM OBESE RATS
Borck, P. C. 1; Rickli, S. 2; Barros, V. B. 3; Alípio, J. C. D. L. 2; Machado, M. J. R. 2; Gonçalves, D. C. D.
O. N. 1; Leite, N. D. C. 3; Costa, I. C. G. D. 8; Mathias, P. C. D. F. 9; Grassiolli, S. 10
1
Dep. Ciencias Farmacêuticas, Univer. Estad. de Ponta Grossa, DEFAR UEPG
2
Dep. Enfermagem , Universidade Estadual de Ponta Grossa, DENFSP UEPG
3
Dep. Ed. Física, Universidade Estadual de Ponta Grossa, DEDUFIS
4
5
6
Dep. Ciencias Farmacêuticas, Univer. Estad. de Ponta Grossa, DEFAR UEPG
7
Dep. Ed. Física, Universidade Estadual de Ponta Grossa, DEDUFIS
8
Dep. Odontologia , Universidade Estadual de Ponta Grossa, DEODON
9
Dep. Bio. Celular e Gen., Universidade Estadual de Maringá , DBC, UEM
10
Dep. Bio. Geral, Universidade Estadual de Ponta Grossa, DEBIO UEPG
Objectives:
The World Health Organization considers the obesity one of the ten most important risks to health closely related to early onset
of type 2 diabetes. Insulin hypersecretion is a remarkable feature of obesity and insulin resistance syndrome which culminates
with failure of insulin release by beta cells. Physical exercise reduces adipose tissue; improve insulin secretion and action
maintaining glucose homeostasis. The neonate administration of monosodium glutamate (MSG) to rodents induces hypothalamic
lesion that lead to development of obesity reproducing clinic profile of metabolic syndrome. Pancreatic islets isolated from MSG
- obese rat shown hypersecretion of insulin in the presence of glucose. This study investigated the effect of physical activity in the
ATP sensitive potassium (K+ATP) channel-dependent and K+ATP channel-independent pathways in pancreatic islets from MSG
obese-rats.
Males Wistar rats received in the 5 first days of life, injections intradermal MSG (4g/Kg). Control received saline. After 21 days
the animals were divided into sedentary (S) and Exercised (E) resulting 4 groups: MSG-S, MSG-E, CON-S and CON-E. The
swimming was held for 70 days lasting 30 minutes/3 times/week. Body weight and the retroperitoneal fat were evaluated. Islets
were isolated by a technique of collagenase and incubated in Krebs solution with glucose (16.7mM). K+ATP channel-dependent
pathway was evaluated in the presence of agonist diazoxide (Dz-250 µM) and the K+ATP channel-independent pathway in the
presence of high K+ (30mM) plus Dz (250µM). Insulin was measured for radioimmunoassay. The data was evaluated by Test t of
Student‟s (p
Conclusions:
Physical exercise realized after lactation and maintained during growth attenuates the development of obesity and improves
glucose-induced insulin secretion. Pancreatic islets from MSG obese rats show low response the depolarization and absence of
K+ATP channel-independent pathways. Chronic exercise reduces the depolarizing effect of K+ and also the activation of K+ATP
channel-independent pathways in pancreatic islets.
Keywords: K+ATP CHANNEL , MSG-OBESE, INSULIN SECRETION
Resumo:13-033
EFFECTS OF EARLY WEANING ON BONE MINERAL DENSITY AND BONE MINERAL CONTENT OF RATS AT
WEANING
Maia, L. A. ; Santos, A. S. ; Costa, C. A. S. ; Lima, N. S. ; Lisboa, P. C. ; Moura, E. G.
Departamento de Ciências Fisiológicas / IBRAG, UERJ
Objectives:
Neonatal environmental changes play an important role in programming the susceptibility in later life to chronic degenerative
diseases, such as obesity, cardiovascular diseases, diabetes mellitus, cancer and osteoporosis. Leptin has important functions in
early life regulating growth and development. Recent findings also support a role of leptin in the process of bone remodeling. It
has been proposed that leptin may modulate the proliferation of osteoblasts via sympathetic stimulation. Exclusive and prolonged
breast-feeding has also been associated with protection against chronic diseases at adulthood. We showed recently that
precocious weaning can lead to hypoleptinemia at weaning (Brit J Nutr. 28:1, 2011). The present study was designed to evaluate
the immediate repercussion of precocious weaning without the use of pharmacological substances or maternal separation upon
body weight, length, total fat body content, bone mineral density (BMD), bone mineral content (BMC), serum ionized calcium
(Ca) in rats at weaning.
Lactating rats were separated into two groups: EW – dams were wrapped on the 18th of lactation with a bandage to interrupt
lactation in the last 3 days (n=8); Control – dams whose pups had free access to milk during all the 21 days of lactation (n=8).
Body weight and length were daily monitored. Total fat body content, BMD and BMC were evaluated by DEXA. Data were
significant at P
Conclusions:
Lower serum ionized calcium could be due to a failure in the transfer of calcium throughout milk, which is the main source of
calcium to offspring. As EW offspring showed hypoleptinemia, mainly to the lack of milk‟s leptin, those animals are deficient on
the leptin direct osteogenic effects on stromal precursor cells. Thus, hypoleptinaemia and reduction of calcium intake may have
caused lower BMD and BMC in offspring.
Keywords: Early weaning, Bone mineral density, Bone mineral content, Leptin
Financial Support: FAPERJ, CNPQ and CAPES.
Resumo:13-034
EFFECT OF CINNAMON ON LIPID PROFILE OF EUTHYROID AND HYPOTHYROID RATS
Lopes, B. P. 1; Gaique, T. G. 2; Souza, L. L. 1; Paula, G. S. M. 1; Neto, J. F. N. 3; Pazos-moura, C. C. 1;
Oliveira, K. J. 2,1
1
Instituto de Biofísica Carlos Chagas Filho , UFRJ
2
Departamento de Fisiologia e Farmacologia , UFF
3
Faculdade de Ciências Médicas , UERJ
Objectives:
Recent studies have demonstrated the hypolipidemic effect of cinnamon and its ability to modulate Peroxisome ProliferatorActivated Receptors alpha (PPAR alpha) expression, known to be involved in hepatic lipid metabolism. However, this action has
not been investigated in eu- (EU) and hypothyroid (HYPO) animals, an important model of dyslipidemia. Our objective was to
analyze the effect of chronic ingestion of cinnamon on lipid profile and expression of hepatic PPAR alpha in EU- and HYPO rats.
Male Wistar rats EU and HYPO (0.03% methimazole in drinking water, 21 days) received cinnamon powder in standard chow (P
- 7g/kg of chow) or oral administration with cinnamon water-extract (WE - 400mg/kg body weight) for 28 days. Methimazole
was maintained during cinnamon treatment. Lipid profile was evaluated by biochemical analysis and protein expression of
hepatic PPAR alpha by Western Blot, and n=4-8. EU rats showed no changes in serum lipids levels. Cinnamon-WE reduced
serum triglycerides in HYPO rats (p<0.05).
Conclusions:
Cinnamon-P intake promotes an increase in cholesterol levels in hypothyroid rats, suggesting that beneficial hypocolesterolemic
effect of cinnamon-P is, at least in part, dependent of thyroid hormone levels. Cinnamon-WE, but not cinnamon-P, reduced serum
triglycerides in HYPO rats. This suggests that different components in cinnamon-P or WE exhibited hypo or hyperlipidemic
effects on HYPO model. The changes observed in lipid profile have no correlation with protein expression of hepatic PPAR
alpha.
Keywords: Cinnamon, Lipid profile , PPARalpha, Hypothyroidism
Financial Support: CNPq, FAPERJ, CAPES
Resumo:14-001
PRODUCTION, DISTRIBUTION AND USE OF EDUCATIONAL MATERIAL ON THE EFFECT OF DRUGS IN
CENTRAL NERVOUS SYSTEM
Agostini, A. G. 1,1,1,1; Marra, C. 1,2,1,2; Fernandes, R. A3,3,3,3; Melo, L. A. S. , . 5,4,4,4; Santos, T. L. D. 2,5,4,4;
Sholl-franco, A. 6,6,6,6
1
2
3
Escola de Belas artes , UFRJ
4
5
6
Objectives:
Drugs use by children and adolescents is a worldwide public health problem, affecting not only addicts, but also the society as a
whole. Thus, it becomes increasingly important to devise effective strategies that can lead the youth of this age group to know
about the real dangers of addiction. Studies analyzing the effectiveness of antidrug campaigns emphasize that, despite all the
awareness campaign, the number of users and addicts is increasing. It is important to emphasize that the approach to the drugs is
done only from the perspective of preventive models of intimidation and of moral principle, pointing out the missing individual
and collective aspects, with information provided with scientific seriousness, credibility, social and ethical reasons. The most
effective way to minimize this problem is the development of specific preventive measures for each segment and age group,
having as objective the enhancement of health and respect for life. Consequently, the objectives of our study were: (i) to produce
information booklets about drugs (legal and illegal) and how these affect the functioning of the body and, in particular, the central
nervous system (CNS) (ii) to distribute the material produced in schools of Basic Education to Public and Private schools in the
Metropolitan Region of Rio de Janeiro, (III) to give lectures to raise awareness about the effects of drugs on the CNS.
Were produced informational booklets aimed at elementary school students and II High School, where there were basic aspects of
the structure and functioning of the CNS, physiological and behavioral changes resulting from the use of different types of legal
and illegal drugs (inhalants, stimulants, nicotine , opioids, hallucinogens, marijuana, methamphetamine, steroids, alcohol). After
detailed analysis of the content in question, the material has been reproduced for distribution. Lectures on the theme, followed by
discussions were held during visitations to schools, allowing for clarification of doubts not only for students but also teachers. At
the end of the lectures the students were given information booklets, where they had access to all the content that was taught and
discussed.
Conclusions:
Our group observed that the production and distribution of textbooks, combined with lectures, are effective methods to convey
knowledge about the consequences of addiction in a simple and practical way. Thus, this set of actions works as a preventive tool
in the school environment, where the participation of students and teachers contributes to the awareness process occurs
efficiently.
Keywords: comunicação científica, dependência química , drogas , neurociências, prevenção
Financial Support: Organização Ciências e Cognição e FAPERJ.
Resumo:14-002
NEUROSCIENCE AND EDUCATION FROM THE PERSPECTIVE OF VYGOTSKY’S CULTURAL-HISTORICAL
PSYCHOLOGY
Silva, C. L. ; Rego, T. C.
Faculdade de Educação - Universidade de São Paulo, FEUSP
Objectives:
Vygotsky‟s cultural-historical psychology (CHP) is one of the most important theories of psychology of the twentieth century,
especially for the field of education, where it has yielded great fruits. In his short lifespan, Vygotsky (1896-1934) addressed a
wide variety of themes, chief among which was the brain as the material basis for the human psyche. In this work (based on the
author‟s doctoral thesis) the aim is to analyze how the brain is conceived in CHP, discussing the potential implications of this
approach for education. It is a theoretical contribution that draws on Vygotsky‟s output from 1924 to 1934, the year of his death.
From amongst the dozens of well-known works from Russian psychology, nineteen texts that discuss the brain in particular were
selected and analyzed. Based on the findings, it is argued that Vygotsky‟s theories about the brain are intimately linked to the
founding principles of CHP. Vygotsky‟s discussions of the brain were both profound and novel, and include considerations that
have been confirmed more recently by neuroscience. His main ideas include: the constitution of consciousness by the
internalization of social experience; the working of the brain through functional systems (as opposed to localizationist views); the
articulation between elementary and higher psychic functions; the complementary sense of the development and segregation of
the higher psychic functions; signs as extra-cerebral connections of cultural origin; the development of brain functions based on
the relationship between the cortical centers (rather than on structural changes per se); and the defense of psychophysical
monism, characterizing the materialistic approach to the psyche seen in Russian psychology. It was also Vygotsky‟s work that
formed the basis for the founding by his collaborator, Luria, of what is today known as neuropsychology. Some interesting
parallels can be drawn between certain ideas derived from CHP and today‟s neuroscience, such as the interaction between genes
and environment (nature vs. nurture), Vygotsky‟s concept of interaction and neural plasticity, the importance of imitation in CHP
as a basis for the zone of proximal development and mirror neurons, the direction of the learning development process, implying
that learning produces development, and others.
Conclusions:
As for its potential contributions to education, we highlight that the provision in challenging educational contexts continues to be
of the essence for schools that are keen to invest in a broader learning experience for their students, and that through a diversity of
contents they can promote a wider repertoire of learning and therefore of human development, since this is based on socially
constructed experience.
Keywords: EDUCATION , NEUROSCIENCE AND EDUCATION, CULTURAL-HISTORICAL PSYCHOLOGY,
VYGOTSKY
Financial Support: FEUSP - Faculdade de Educação - Universidade de São Paulo
Resumo:14-003
PERFORMANCE EVALUATION OF THE MEDICAL SCHOOL GRADUATES AT THE DISCIPLINE OF
RADIOLOGY INTRODUCTION: INFLUENCE OF FREQUENCY AND NUMBER OF PROFESSORS
1
Contenças, T. S. 1,3; Vieira, G. 2,3; Leite, C. C. 2,3; Amaro Jr, E. 2,3
Instituto de Ciências Biomédicas - Universidade de São Paulo, ICB-USP
2
Departamento de Radiologia da Faculdade de Medicina da USP, Inrad
3
Laboratório de Neuroimagem Funcional , NIF
Objectives:
To evaluate if the performance of medical student varies according to the number of instructors in a basic radiology course.
This study was conducted through a survey from 2004 to 2010, with 1.203 undergraduate students of Medicine, in the University
of São Paulo, at the Discipline of Radiology Introduction. The course was taught by professors through lectures, followed by
practical sessions conducted by tutors from the same institution. The main topics included medical imaging anatomy and physics.
Each class (180 students, approximately) was subdivided into four groups (45 students, approximately) according to the weekday
(Monday, Tuesday, Wednesday and Thursday). The number of teachers per class varied according to the year and according to
the weekday group. The assessment of student performance by number of teachers was obtained through the score and frequency
average reached at the end of the course. We used a multivariate linear regression model contrasting student final score with the
following variables: number of teachers per class, years and frequency of class attendance.All variables were statistically
associated with the score (R² = 0.077, significance p
Conclusions:
Contrary to our expectations, it is noted that there was no difference in scores for the number of teachers. A more predictable
result is the relationship between the class attendance and scores.
Keywords: education, graduates, radiology
Resumo:14-004
PRODUCTION OF A VIDEO LESSON: INTRODUCTION TO PHYSIOLOGY - THE INTERNAL ENVIRONMENT
Rosat, R. 1; Vivan, D. 2; Grassi, A. 2
DEPARTAMENTO DE FISIOLOGIA - ICBS, UFRGS
2
CENTRO NACIONAL DE SUPERCOMPUTAÇÃO, UFRGS
1
Objectives:
Today the development of educational technology is crucial to facilitating the access to knowledge. This video lesson shows the
relationship between physiological systems and the extracellular fluid, also called the internal environment. It points to the role of
the digestive, respiratory and urinary systems for renewal of the internal environment, and the circulatory system for reception,
transport and delivery of substances through compartments between the exchange systems mentioned above, the internal
environment and the cells.
This compact (13 min) video lesson presents an analogy of the body with a physiological aquarium where the water would be the
internal environment and the fish would be the cells. It also points to the origin of this key feature common to multicellular
organisms. The softwares Adobe CS4 (Premiere, Illustrator, Photoshop, Flash, After Effects) and Sound Forge 9.0 were used for
edition of external takes, studio recordings with chroma key, illustrations, image processing, animations and sound effects.
Conclusions:
This integrated approach enables to visualize and understand the importance of a precisely controlled environment for survival of
the cells, the role of the physiological systems and the location and movement of substances between the external and internal
environment, its different compartments (plasma, linfa and interstitial fluid) as well as the intracellular fluid. Moreover, it also
helps to associate structure to function, i.e., morphology to physiology.
Keywords: aquarium, compartments of internal environment, internal environment, physiological systems, video
Financial Support: Programa para Produção de Objetos de Aprendizagem, Edital 11, SEAD, UFRGS
Resumo:14-005
THE USE OF EXERGAMES AS AN SUPPLEMENTARY INSTRUMENT IN THE TEACH-LEARNING PROCESS
Almeida, C. E. J. C. C. 1,2,4; Barreto, T. M. 1,2,3; Assis, T. S. 1,2,5; Marra, C. 1,2,6; Barros, G. A. 1,2,4; Franco,
A. S. 1,2,6
1
Organização Ciências e Cognição, OCC
2
Núcleo de Divulgação Científica e Ensino de Neurociências, Nudcen
3
Escola de Educação Física e Desportos/ UFRJ, EEFD
4
5
Centro de Ciências Matemáticas e da Natureza / UFRJ, CCMN
Ciep 178/Secretaria de Educação do Estado do Rio de Janeiro, SEEDUC
6
Instituto de Biofísica Carlos Chagas Filho/UFRJ, IBCCF
Objectives:
Games are important allies in education, since they support the knowledge‟s construction by students, and provide an ambient
more ludic and interactive for learning. The exergames, a new genre of games, promote a powerful cognitive work through
virtual reality mechanisms, during motor tasks execution. In this way, the aim of this study is to report the conception,
organization, and application processes of an electronic exergame workshop, occurring during the Second Brain Awareness
Week of Rio de Janeiro (“II Semana do Cérebro: Desvendando a Memória”). In addition, we will evaluate the contributions of
this kind of teach-learning tool in non-formal teaching environment.
The Second Brain Awareness Week of Rio de Janeiro (15-19/03/2011), an event of scientific divulgation and neurosciences
teaching, had this year the theme of memory. Several practical-based activities have been made approaching some types or
particularities of memories. In the activity named “Memory in Motion” (“Memória em Movimento”), we analyze characteristics
of the operational memory and movement in one way using the exergame “Dance Dance Revolution” (DDR) and the console
Wii. In this game the subject need to step at the arrows of a dance platform following correct sequential signs. The workshop
counted with two coordinators (2) and three voluntary monitors (3), responsible to explain the contents approached, as well as to
apply the game passing instructions, orienting the public and controlling the number of people, since it were allowed only ten
participants per in each session. The monitors were previously trained regarding all neuroscientific content to the workshop and
the event. During the workshop‟s realization, one participant was invited to play with the DDR, using the proper sneakers, to
avoid accidents with the dance platform. Only basic level music was choosing. After the first turn, the total number of errors and
hits (“combo”) were observed. The participants were invited to play again, but in this second turn it was added distracters, since
beyond watching the arrows in the screen to execute the movement sequences in a limited time, the player should performed at
the same time specific tasks, such as spell words and perform mathematical calculations presented by monitors. The multitasks
work was used to demonstrate the importance of attention during the realization of operational memory tasks, and as result we
could observed a significant increase in the number of errors and the decrease of combos during the second and multitask
performances. It was also observed that exergame motive the participants and stimulated all public to join the workshop, what
could be explained by the attractiveness of electronic games by different age groups.
Conclusions:
The games are important tools to be explored during teach-learning processes, acting through social and affective ways to
contribute with cognitive improvement. So, the utilization of the exergame in the workshop was essential to its success, because
at the same time it: (i) explored the ludic component of the activity; (ii) contributed to the diffusion of specific knowledge, such
as operational memory and sensorimotor integration; (iii) allow the association of these contents with everyday tasks and action.
Keywords: exergame, operational memory, teach-learning process
Financial Support: Organização Ciências e Cognição, Espaço Ciência Viva and FAPERJ
Resumo:14-006
EVALUATION OF EDUCATIONAL INNOVATIONS.
Lopes, R. S. ; Lira, M. L. F. ; Faioli, C. N. ; Santos, L. F. C. ; Amaral, V. F.
Depto de Imunobiologia - Universidade Federal Fluminense, UFF
Objectives:
Aim: In Brazil, the National Council of Education (CNE) and the Committee of Higher Education recommends for any
undergraduate course there are activities that integrate academic knowledge to professional practice, encouraging the recognition
of abilities and competences acquired outside of school environment, increasing the link between theory and practice. Given
current trends in higher education are necessary new pedagogical approaches and methodologies that can assist students and
contribute significantly to their professional and personal development. Our study aimed to establish a relationship between
theoretical and practical content of Immunology discipline in order to support and facilitate student learning, and inspire respect
and pursuit of knowledge about history of national science aspects, showing importance of lifelong learning.
Methods and Results: To conduct the project, thirty students in the 3rd period of Veterinary Medicine/UFF graduation were
invited to participate in a visitation to the Vital Brazil Institute, located in Niterói / RJ, where they knew the history, work and
cultural space of local. After the visit, a satisfaction questionnaire with nine questions was delivered to students. This
questionnaire had order to evaluate how important this kind of activity is to learn from the standpoint of college students.
According to the results, we already observed in the first stage of the project, in relation to visit, the existence of interest by
students in extracurricular activities, because most of the class attended the visitation (83%). After analysis of questionnaires
answered by students, we can conclude that all were satisfied with the proposed activity, finding their achievement of great
importance, contributing to the understanding of the specific contents of Immunology particularly on issues such as production of
hyperimmune serum and vaccines (56%), stimulating interest to learning (100%) acquisition of historical information about
national science (96%). Additionally, students visitation showed that basic concepts of immunology studied in class have
practical application (54%) and they observed in the place visited opportunity to enter in professional market.
Conclusions:
Conclusion: Thus, was observed a great importance of extracurricular activities on learning providing a better understanding of
Immunology, inspiring student reflection about the importance of the history of national science and contribution in research
applied to immunology. Therefore, this pedagogic approach can be applied to students of other courses such as Biologicals
Sciences, Biomedicine, Medicine and Pharmacy.
Keywords: education, immunology, extracurricular activity
Resumo:14-007
THE PRODUCTION OF HYPERTEXTS AS A TOOL OF TEACHING-LEARNING PROCESS IN ANIMAL
PHYSIOLOGY
Ã•vila, R. ; Fernandes, H. ; Costa, M.
Universidade Federal de SÃ£o Carlos â€“ Campus Sorocaba, UFSCar
Objectives:
The project aims to: 1) monitor and evaluate students' difficulties in the production of hypertext for the popularization of science
in the field of Comparative Animal Physiology for development of an electronic journal, 2) investigate the criteria used by
students in the choice of themes and justifications, development of resumes and literature, as well as the development of scientific
texts, 3) verify that this material works as a motivational factor for learners to study.
The hypertext was produced by pairs of graduate students of the Bachelor of Sciences, for the discipline of Comparative Animal
Physiology, Federal University of SÃ£o Carlos, Campus Sorocaba, in the following steps: 1) in the first lecture, the pairs were
asked to choose a topic related to comparative physiology that they would like and / or had the curiosity to study, justifying the
choice, 2) after the summary of the text have been prepared, it was found consistency between the title and summary. In this step,
a literature review was also requested, verifying its relationship and relevance to the topic being discussed in the hypertext,3) the
final text was developed, aimed at popularizing science in a language accessible to the lay public, trying to analyze how they had
been written (ie, checking whether fundamental physiological concepts were correct). In this phase, students were also able to
provide the secondary texts (links) and images to be used in conjunction with its commitments by the authors, 4) lastly was given
a lecture about the process of emergence of hypertext and the methodology to be used for their preparation. Subsequently, the
final phase of preparation consisted of one hypertext practice session at the multimedia lab where students were able to convert
your texts into hypertexts and hyperlinks (including connections with the texts written by other teams in the process), 5), a semistructured interview was conducted with students to evaluate the efficacy of this method as a way of improving the teachinglearning process in Comparative Animal Physiology.
Conclusions:
By using a technology that by itself is attractive to the students, the present results demonstrated that the process of elaboration of
the hypertexts acted as a motivational factor and developed in the students several skills, including performing a discerning
bibliographic review, an adequate use and definition of physiological concepts, and also to establish a rational correlation
between different subjects related to the discipline. Indeed, having successfully employed this tool, the students became able to
convert a scientific text into a hypertext accessible to the lay public, becoming not only scientists, but individuals able to help
popularize the science.
Keywords: Hypertexts, Popularization of science, Comparative Animal Physiology, Learning-teaching Process
Financial Support: National Institute of Science and Technology in Comparative Physiology INCT-FisC
Resumo:15-001
THE PRACTICE OF THE NURSE IN THE ADMINISTRATION OF ANTIMICROBIAL IN PEDIATRICS
Rodrigues, F. T. S. ; Chaves, E. M. C. ; Sampaio, L. R. L. ; Araújo, D. P. D. ; Vitoriano, B. F. ;
Vasconcelos, S. M. M. ; Lobão, E. L. ; Bomfim, I. S. ; Menezes, R. R. P. P. B. D. ; Lima, C. N. D. C.
Objectives:
The knowledge of the issues surrounding the clinical pharmacology during infancy period is an important factor in the care given
by nurses for children in Pediatric Clinical Units (PCU), who are mostly suffering from infections or susceptible to infections and
require antibiotics (ATB) during the period of hospitalization. These patients are susceptible to changes related to the dose,
dosing interval, route and conditions of maturation of renal, which is responsible for drug excretion, and liver system, that
metabolizes the administered drugs. The nurse in PCU plays an important role in planning the administration of antibiotics, as
appropriate therapy helps to restore the child, minimizing the risk of complications associated with small dosage or overdose of
these medicines. This research tried to answer how pediatric nurses care for the administration of ATB and how they should act
to optimize therapy that involves scheduling, reactions and complications related to the use of these drugs. The aim of this study
was to analyze nursing practice regarding the administration of ATB in children admitted to a hospital with pediatric care. We
identified the most used ATB to treat children and their complications, and it was also defined the actions of the nurses in the
administration of ATB.
This is a retrospective, descriptive, documentary study. We analyzed public records of 180 children hospitalized from September
2008 to February 2009 at Hospital Geral de Fortaleza, Ceará. The research was conducted in October-November 2009. The study
made reference to complications such as chemical phlebitis, thrombophlebitis, extravasation, hematoma, infiltration, renal failure,
seizures, skin rash, anaphylactic reaction. The results showed that 44.7% of records do not indicate the reason for treatment with
ATB, and 64.9% of records did not contain such a form of rationalization. The study also showed that a large percentage of
children using ATB (80.7%) that left hospital with clinical improvement, but no cure. The most prescribed antibiotics were
cephalothin (27%), cefazolin (21%), ceftriaxone (10%), oxacillin (10%), penicillin b (10%), trimethoprim (9%), cephalexin (7%),
amikacin ( 5%) and vancomycin (4%). On the simultaneous use of antimicrobials, 62% used a combination of two medications,
12% used three medications simultaneously, 9% were treated with four and 2% used five types of antibiotics. The most
commonly used routes of administration were intravenous (68%) and oral (28%). Regarding the complications of ATB therapy,
92% of nursing records did not mention such events, being found in only 22% of the records the route of administration for
treatment.
Conclusions:
The research identified gaps in the involvement of nurses in the rehabilitation of the patient, the need to standardize the service
with educational and research, and other factors which could improve treatment success, reducing, thus, microbial resistance and
consequently risks to the patient.
Keywords: Anti-bacterial agents, nursing, rationalization
Financial Support: Funcap
Resumo:15-002
CREATION OF NEW PRODUCTS IN A NATIONAL PHARMACEUTICAL INDUSTRY: CASE BIOLAB.
Cafeu, M. C. ; Alário, M. M. ; Falci, M. ; Sacurai, S.
Biolab Sanus Farmaceutica, Biolab Farmaceutica
Objectives:
To create innovative medicines based on tracking trends and new drugs under development in different companies on the market.
Methods: The Study Group of New Products (Grupo de Estudos de Novos Produtos – GENP, in Portuguese) was created in 2009
at Biolab Sanus Farmacêutica, an innovative Brazilian pharmaceutical company. The mission of the group formation was to
study, propose and participate in the development of pharmaceutical and dermocosmetics incremental innovation based on
evaluation of products already marketed or currently under different phases of development in several countries. These proposed
innovations are to be aligned with strategic development plan within Biolab. This group is composed of employees and
consultants from research institutions with background/degree such as in Chemistry, Pharmacology, Toxicology and General
Practice. GENP is allocated within the Department of Knowledge Management as a branch of the R&D Directorate. As
supportive tools used by the group, there are databases such as Integrity, SciFinder, PubMed, Micromedex. The work is
performed through exhaustive search and study of drugs‟ mechanism of action, likely structural modifications and patents
evaluations that could result in a new medicinal product (“me too”) or a new fixed combination (previously marketed in separate
or possibly driven to a different therapeutic use). GENP also monitors medicines under development in pharma/biotech
companies around the world including follow-up at preclinical, PhaseI-III stages as well as collecting practical information on
these products. Relevant information on those compounds such as chemical and pharmacological data is recorded to generate an
internal database. GENP members also participate in scientific conferences, especially pharmacology ones in order to keep
contact with researchers willing to establish fruitful partnerships between University and Biolab. Results: The studies carried out
so far led to the proposal of three new medicines currently under internal diligence. These products are patentable, or meet the
three requirements of inventiveness, novelty and industrial applicability. Studies also resulted on the synthesis of nine “me too”
compounds derived from anti- cholesterol drugs, which are promising products with greater vasodilator and pleiotropic action.
The internal product information database possess full strategic information on around 50 products already registered and of at
least 100 more within the initial phases of development. Biolab also received several proposals of new medicines projects to be
carried out in partnership between University and Biolab through direct prospection within research institutions and contact with
researchers. Information presented in this summary are only a few examples of GENP‟s recent achievements, while most part of
its job cannot be disclosed for confidentiality issues.
Conclusions:
GENP has been acquiring strategic role within Biolab on the proposal and creation of innovative medicines. As such, GENP was
created as one of multiple tools in the search, creation and development of innovative drugs (either incremental or radical), thus
contributing to our mission of generating innovative medicines to Brazilian population.
Keywords: Innovative medicines, National Pharmaceutical Industry, Incremental innovation
Financial Support: Biolab Sanus Farmacêutica.
Resumo:15-003
PHARMACOKINETIC EVALUATION OF GEMIFLOXACIN IN RAT PLASMA- A PILOT STUDY
Grünspan, L. D. 1; Pires, C. C. 1; Kaiser, M. 2; Costa, T. D. 2; Tasso, L. 1
1
Universidade de Caxias do Sul, UCS
2
Objectives:
The present study aimed to determine the plasma pharmacokinetic of gemifloxacin (GEM) in rats following single-dose
intravenous administration.
The Wistar rats (n = 4, 250-350g) were purchased from FATEC (Santa Maria, Brazil) and were housed under standard conditions
with room temperature 22 ± 2°C and relative humidity approximately 64%. Each rat received a single bolus intravenous dose of
GEM (40 mgkg-1) by the lateral tail vein. At predetermined times (before dosing, 0.03, 0.5, 1.5, 3.0, 6.0 and 8.0 h) after
intravenous administration blood was withdrawn into heparinized centrifuging tubes. High plasma levels were diluted in blank rat
plasma to give concentrations into the calibration curve and the concentrations were back-calculated. Plasma was separated by
centrifugation at 6800 x g, 21 ºC for 10 min and stored at -80 ºC until analysis by HPLC. Pharmacokinetic parameters were
calculated. The protocol used for GEM pharmacokinetic evaluation was previously approved by the Ethics in Research
Committee of University of Caxias do Sul. The pharmacokinetic parameters obtained after model independent analysis were: ke
= 0.34 h-1, t1/2 β = 2.04 h, AUC 0-t = 43615.85 ng.h.mL-1 and AUC0-inf = 46420.40 ng.h.mL-1.
Conclusions:
It was possible to determine the pharmacokinetic plasma parameters of gemifloxacin in rats after intravenous administration (40
mgkg-1). The terminal phase of GEM could be well characterized.
Keywords: gemifloxacin, pharmacokinetic, rat
Financial Support: FAPERGS/Brasil
Resumo:15-004
17-BETA-ESTRADIOL PLUS PROGESTERONE REPLACEMENT THERAPY INCREASES MNSOD ACTIVITY IN
MENOPAUSAL WOMEN
Maurer, L. H. 1; Unfer, T. C. 3; Zanchi, M. 1; Figueiredo, C. G. 1; Ferreira, D. 3; Kemerich, D. M. 1; Knopka,
C. 3; Mottecy, C. D. S. 2; Emanuelli, T. 1
1
Departamento de Ciência e Tecnologia em Alimentos/ CCR, UFSM
2
Departamento de Clínica Médica /Centro de Ciências da Saúde, UFSM
3
Departamento de Fisiologia e Farmacologia/ CCS, UFSM
Objectives:
Hormone replacement therapy (HRT) is one of the most effective treatments to reduce the health problems associated with
menopause. The antioxidant enzyme superoxide dismutase (SOD) contributes to cellular protection against reactive oxygen
species (ROS). It was recently shown that different isoforms of this enzyme may be regulated by sex hormones in various tissues
and organs of animals and in the human endometrium. Thus, this study was aimed at evaluating the influence of different HRTs
on the activity of SOD isoforms (CuZnSOD and MnSOD) in the blood of postmenopausal women.
The study was approved by the local ethics committee of Federal University of Santa Maria (UFSM, Brazil) (CAAE n°
0120.0.243.000-06). The studied subjects were divided into four groups: premenopausal women (n=21), postmenopausal women
without HRT (n=28), postmenopausal women with estrogen replacement therapy (ERT) (n=9) and with estrogen plus progestin
replacement therapy (EPRT) (n=10). Serum estradiol and progesterone levels were determined by immunoassay kits. SOD
activity was measured at 560 nm, using nitroblue tetrazolium (NBT) and xanthin-xanthin oxidase system. MnSOD activity was
quantified in the presence of KCN, which inhibits only CuZnSOD. The results were expressed as SOD U/mg hemoglobin, where
one unit was defined as the amount of SOD that decreases NBT reduction to one half of the maximum. Data were analyzed by
one-way analysis of variance (ANOVA), followed by Duncan‟s test (Statistica 7.0) and the mean age was used as co-variable in
the statistical analysis. Serum levels of E2 (pg/mL) were 83,0±13.8, 52.0±13.4, 72.7±21.0 and 107.5±48.7 while the levels of
progesterone (ng/mL) were 4.7±0.7, 0.7±0.1, 0.4±0.1 and 0.7±0.2 in the premenopausal women, postmenopausal women without
HRT, postmenopausal women with ERT and postmenopausal women with EPRT, respectively. The mean age were 44.6±3.9,
61.3±7.5, 56.5±6.5 and 55.8±2.5, respectively. The antioxidants enzymes catalase, glutathione-peroxidase, CuZnSOD and totalSOD were not significantly different among the studied subgroups. However, the postmenopausal women with EPRT had higher
MnSOD activity (4.4-fold, p
Conclusions:
Since MnSOD is located in the mitochondria, our results suggest that combined estrogen plus progestin therapy can improve
mitochondrial enzymatic antioxidant defense against superoxide anion radical.
Keywords: Estradiol, Progesterone, MnSOD, Hormone Replacement Therapy, Menopause
Financial Support: CAPES, REUNI, FAPERGS
Resumo:15-005
EFFECTS OF PROPRANOLOL ON REDOX BALANCE DURING HEALING OF ISCHEMIC WOUND
Bandeira, A. C. B. ; Monte-alto-costa, A.
Departamento de Histologia e Embriologia - LRT, UERJ
Objectives:
Oxidants play an important role in wound healing providing signaling and defense against microorganisms. When the antioxidant
defense system fails or when the generation of oxidants is exacerbated, the alteration in homeostasis leads to oxidative stress. Its
has been shown that propranolol presents antioxidant properties by reducing tissue lipid peroxidation products and presenting
membrane antiperoxidative activity, and its major metabolite is even more potent as antioxidant. It is believed that oxidative
stress has an important role in delayed healing of cutaneous ischemic wounds, so our aim was to evaluate the antioxidant effects
of propranolol in the redox balance during the healing of a ischemic wound, in rats.
Male Wistar rats were subjected to an ischemic lesion in the dorso, and were treated (i.p.) with vehicle (control group) or
propranolol in doses of 3mg/kg/day (Group 3mg) or 6mg/kg/day (Group 6mg) during the experiment. Wound evaluation
(measuring and photographic record) was performed at the day of wounding, and 3, 7 and 10 days later. On day 10 the animals
were sacrificed and samples of lesion and normal skin were collected and processed for determination of malondialdehyde
(MDA) and protein carbonyls content. The group 3mg showed a smaller wound area (73.6% ± 30, 7) compared to the 6mg group
(116.0 ± 14.5%) and the control group (133.2% ± 16.0) on d10 (P
Conclusions:
Although the 3 mg dose has improved the healing of ischemic lesions, at the moment there are no clear evidences that the effect
was due to anti-oxidative properties.
Keywords: Ischemic wound, Propranolol, Oxidative stress
Financial Support: CNPq, CAPES
Resumo:15-006
DEVELOPMENT OF SIMULATION SOFTWARE FOR RATIONAL THERAPEUTICS REGIMENS DESIGN
Souza, F. C. 1; Coutinho, K. C. 1; Ribeiro, A. A. R. 1; Vicentini, M. F. B. 2; Alvim, R. N. T. 2; Sabine, K. 2;
Scaramello, C. B. V. 1
1
MFL/Laboratório de Farmacologia Experimental (LAFE), UFF
2
Faculdade de Medicina, UFF
Objectives:
Therapeutic window (TW), a security pharmacokinetic (PK) index, is the range between drug plasma concentrations (Cp)
associated with effectiveness and toxicity. Digoxin, used in heart failure (HF), is an example of insecure drug because it has
narrow TW. Cp is modulated by PK processes that depend not only on drugs chemistry but also on patients´ health status and
environmental factors. So, rational dose regimens should be based on drugs PK in different patients and situations, especially in
cases like digoxin (Can. J. Hosp. Pharm. 42: 63, 1989; Clin. Pharmacol. Ther. 48: 503, 1990; Mayo Clin. Proc. 78: 1564, 2003).
The aim of our work is to establish rational therapeutic regimens of 2 cardiotonics used in HF (digoxin and dobutamine),
elaborating Standard Operating Procedures (SOPs). Computer programs are interesting tools to attain this purpose, thus an
important step of this work is the genesis of a simulation software whose database is obtained from clinical PK studies. Aiming to
enlarge the software´s database, clinical PK studies are also being performed at the Instituto Nacional de Cardiologia (INC) and
Hospital Universitário Antônio Pedro (HUAP) (Ethics Committees approvals: respectively 0306/07-12-10 and 240/2010).
A program prototype based on Microsoft Excel (version 7-2003) was created using a preliminary database from literature. It was
included studies involving patients with HF using digoxin in 2 distinct situations: (1) different stages of renal failure (RF) (Clin.
Pharmacol. Ther. 48: 503, 1990; J. Bras. Nefrol. 30: 6, 2008), (2) concomitant use of carvedilol (Eur. J. Clin. Pharmacol. 62: 535,
2006). Classical PK equations were entered into the system in hidden sheet to be rescued and used at the beginning of the
simulation. The software variables are Cp, PK parameters and dosage. Thus, since 2 of the 3 variables are known and entered into
the computer program, it can calculate the third. The software is also able to plot Cp versus time graphics to illustrate the
simulation result. In male HF patients (40 years old, 70 kg) presenting no comorbities, the PK parameters of digoxin are: volume
of distribution (Vd)=545L, clearance (CL)=8.10L/h and plasma half life (T1/2)=46.6h. In this case, target Cp=0.001mg/L
(TW=0.0008-0.002mg/L) can be attained using digoxin daily loading dose (LD)=0.78mg and maintenance dose (MD)=0.28mg,
both p.o. Performing a simulation with male HF patients (similar age and weight) presenting RF stage 4, an administration of the
same LD and MD leads to digitalis intoxication (Cp=0.003mg/L) because digoxin PK parameters are changed (Vd=297L,
CL=2.92L/h and T1/2=70.6h). Comparing male and female patients using digoxin with and without carvedilol, it was observed
that this beta blocker raises digitalis oral biodisponibility (F) just in male patients (Fcarvedilol(+)=1 vs Fcarvedilol(-)=0.7).
Conclusions:
The results indicate that in HF patients with RF stage 4, digitalis LD and MD must be decreased to 0.42mg and 0.10mg,
respectively, to avoid intoxication. Other data shows that digoxin p.o. doses must be diminished in male patients in concomitant
use of carvedilol. Selection of patients at INC and HUAP are in course to collect blood samples and measure digoxin/dobutamine
Cp and calculate these drugs PK parameters in different situations (age, gender, comorbidities and HF functional class), enlarging
software´s database. This work highligths the relevance of a rational individualized therapy.
Keywords: CLINICAL PHARMACOKINETICS STUDIES, RATIONAL THERAPEUTICS REGIMENS, SIMULATION
SOFTWARE
Financial Support: CAPES, FAPERJ, PROAC/UFF
Resumo:15-007
PHARMACOKINECTICS OF A ISONIAZID ANALOG JVA 001: PRELIMINARY STUDIES
Roman, M. 1,2; Faria, T. D. 2; Assis, J. V. 3; Almeida, M. V. 3; Báfica, A. L. B. 2; Poli, A. 1
1
Laboratório de Farmacocinética/ Departamento de Farmacologia, UFSC
2
Lab Imunofarmacologia Doenças Infecciosas, MIP, UFSC
3
Departamento de Química, UFJF
Objectives:
We have recently synthesized an isoniazid (INH) analog JVA001, which displayed enhanced nanoencapsulation as well as
antimycobacterial activity. Therefore, the objective of this study was to develop and validate an analytical method by High
Performance Liquid Chromatography (HPLC) to measure plasma levels of JVA 001 in mice.
METHODS: The method employed a RP-C18 column ACE with mobile phase constituted by a mixture of acetonitrile: phosphate
buffer 100mM pH 6,0, at a flow rate 0.2 ml min-1, and allowed the detection an ultraviolet detector at 305nm. To validate the
method, blank plasma of mice were spiked with known concentrations of JVA 001 standards which were stored at −80°C.
Sample preparation was carried out precipitating proteins with acetonitrile, followed centrifugation and evaporation at 37ºC under
a gentle stream of N2. The residue was resuspended in mobile phase and injected in an AllianceBio HPLC system, (Waters Co).
Intraday and interday assays were evaluate in plasma samples spiked with lower, medium and upper nominal concentrations (0.1,
1 and 5 µg ml-1). Calibration curves were prepared in the concentration range of 0.05 to 10 µg ml-1, and samples concentrations
were calculate using the linear regression equation. Linearity, precision, accuracy, recovery, robustness and stability were
evaluated. The intraday precision (repeatability) was assessed by the relative standard deviation (% RSD) of five replicates of
lower, medium and upper concentration of spiked plasma and the interday precision (reproducibility) was evaluated by the
analysis of samples in tree batches (in triplicate) (n= 9); RSD= (SD/mean)×100. RESULTS: The method demonstrated excellent
linearity, expressed by correlation coefficient R2> 0,998. The intraday precision analysis (n=5) shown a RSD of 6%, 2% and 3%
for lower, medium and upper samples concentrations (0.1, 1 and 5 µg ml-1), respectively. To the same samples, the interday
assays (n= 9) revealed a RSD values of 7.3%, 4.6% and 3.1%. Accuracy was found to be 114,.6%, 102.7% and 103.8% to 0.1, 1
and 5 µg ml-1 concentrations, respectively. These results indicate that such assays are within the acceptable limits (RSD < 15%).
The limit of quantification (LOQ) and limit of detection (LOD) were calculated directly from the calibration plot, the LOD was
0.035µg ml-1 and LOQ was 0.106 µg ml-1 (n=4). In this method the recovery was around 90%. Furthermore, stability and
robustness assays were performed in order to evaluate the JVA 001 sensitivity of both standard and plasma samples to
freeze/thaw, UV light and heat. Standards displayed stability to all conditions. In contrast, samples presented significant
sensitivity to freeze/thaw and heat conditions. UV light did not alter the measurement of used samples. The robustness was tested
by little changes in flow rate, pH mobile phase, % organic solvent and buffer ionic strength. The results were satisfactories to
allow some variations in these parameters without compromising the method.
Conclusions:
A simple, fast, sensitive and suitable HPLC analytical method was develop and validate to measure plasma levels of JVA 001.
Current studies are in progress to determine pharmacokinetic parameters of this compound either soluble or nanoencapsulated.
Keywords: HPLC method, Isoniazid anolog, JVA 001, Pharmacokinetics
Financial Support: CAPES/Nanobiotec/Rede NANOTB; CNPq
Resumo:15-008
PHYTOCHEMISTRY AND ANTIBACTERIAL ACTIVITY OF HYPTIS PECTINATA L. POIT (LAMIACEA)
AQUEOUS EXTRACT
Paixão, M. S. 1,2; Oliveira, I. D. 1; Lima, A. C. B. 3; Fernandes, X. A. 3; Damascena, N. P. 3; Dias, A. S. 3;
Araújo, B. S. D. 3; Estevam, C. S. 3; Botelho, M. A. 4; Quintans-junior, L. J. 1
1
Laboratório de Farmacologia Pré-clínica de Produtos Naturais, LAPEC/UFS
2
Departamento de Odontologia, DOD/UFS
4
Laboratório de Biotecnologia da Universidade Potiguar, UNP
3
Laboratório de Bioquímica e Química de Produtos Naturais, UFS
Objectives:
Hyptis pectinata L. Poit (Lamiaceae), known as „sambacaitá‟, is used by the population in the northeast of Brazil to treat
respiratory and digestive diseases, fever, inflammation, and microbial infections. The present study evaluated the phytochemistry
and antibacterial activity of the crude aqueous extract (CAE) of H. pectinata leaves
This study evaluated the phytochemistry of the crude aqueous extract (CAE) of H. pectinata leaves by colorimetric methods and
high-performance liquid chromatography HPLC which detected the presence of phenols, tannins condensed and catechinic,
saponins, and free pentacyclic triterpenes. Antibacterial activity of H. pectinata leaf CAE was tested against strains of
Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and
Streptococcus mutans by the agar well diffusion technique. The growth of all bacterial strains tested was inhibited by the extract,
with inhibition zone diameter varying from 15 to 36 mm and minimum inhibitory concentration values ranging from 12.5 to 1000
mg mL-1. Results showed the aqueous extract of H. pectinata leaves has a strong antibacterial activity against Gram-positive
bacteria Staphylococcus aureus (ATCC#25923) and Streptococcus mutans (ATCC#27923).
Conclusions:
Our present results demonstrated that CAE possess important secondary metabolites and wide spectrum of antimicrobial activity
against the test microrganisms.
Keywords: Hyptis pectinata, activity antibacterial, phytochemistry, aqueous extract
Resumo:15-009
CHARACTERIZATION OF CLINICALLY RELEVANT ADVERSE REACTIONS TO CHEMOTHERAPY FOR
BREAST CANCER
Indio-do-brasil, V. 1,2; Martins, C. L. 1,3; Telles, Cn1; Citrangulo, S. M. T. G. 1; Koifman, S. 2; Vianna-jorge,
R1,3
1
Instituto Nacional do Câncer, CPQ/INCA
2
Escola Nacional de Saúde Pública - Fiocruz, ENSP/Fiocruz
3
Instituto de Ciências Biomédicas , ICB/UFRJ
Objectives:
The standard protocol of citotoxic chemotherapy used for breast cancer in the Brazilian National Cancer Institute (INCA) consists
of anthracycline-based combinations followed by taxanes (plus trastuzumab for HER2+ tumors), which has proven efficacy, low
risk of recurrence and high overall survival. Despite these benefits, there is a concern about the risk of several adverse drug
reactions (ADR), which can reduce the quality of life and lead to hospitalization for treatment of life-threatening conditions. The
objectives of the present work were to evaluate the incidence of ADR to antracycline/taxane regimens, identify the most
clinically relevant ADR (CRAR), characterize the correlation among the CRAR and evaluate their intra and interindividual
variability.
The study protocol (129/08) consisted of a prospective cohort of Brazilian women with first diagnosis of unilateral non-metastatic
breast cancer, who were treated with adjuvant chemotherapy. The recruitment was initiated in march/2009 and all participants
signed a written informed consent. An active search for 34 possible ADR was conducted, which included consults to medical
files and interviews to patients at each cycle of chemotherapy. The ADR were graded according to the National Cancer Institute
Common Terminology Criteria for Adverse Events v 2.0. The cumulative incidence of ADR along the chemotherapy regimens
was obtained with Kaplan-Meyer curves. The CRAR were characterized as those that presented high severity (grades 3 or 4) in
more than 5% of the patients. The results involved data from 266 (among 496 recruited). The most prevalent chemotherapy
protocols, with the distribution of patients in each protocol, were: CAF-D (cyclophosphamide, doxorubicin and 5-fluorouracil, 3
cycles, followed by 3 cycles of docetaxel - 61.6%); CAF (6 cycles - 19.2%), AC (doxorubicin and cyclophosphamide, 4 cycles 15.4%); and D-CAF (3 cycles of D, followed by 3 cycles of CAF - 3.0%). The CRAR were evaluated for the patients under
treatment with CAF-D, and their respective incidences at grades 3 or 4, after each step of the protocol were: neutropenia (34.7%;
15.4%), leucopenia (19.4%; 10.1%), muscular weakness (17.7%; 42.1%), fatigue (14.6%; 46.5%), constipation (12.5%; 7.0%),
dyspepsia (11.1%; 11.9%), nausea (10.9%; 12.1%), oral mucositis (10.7%; 7.2%), dyspnea (7.6%; 10.0%), abdominal pain
(6.35%; 15.6%), dizziness (5.8%; 11.4%), headache (5.0%; 6.6%), arthalgia (3.4%; 25.7%) and mialgia (3.4%; 31.5%). A great
intraindividual variability in the severity of the CRAR was observed along the cycles. A positive correlation was found between
neutropenia and leucopenia (CAF: 0,863; D: 0,888) and between fatigue and muscular weakness (CAF: 0,605; D: 0,567).
Conclusions:
The identified CRAR and the characterization of their inter and intraindividual variability may be useful for the evaluation of
predicitive factors of severe toxicity to breast cancer chemotherapy and for the selection of patients at higher risk, who could
benefit from a personalized attention.
Keywords: ADVERSE DRUG REACTIONS, ANTHACYCLINA, BREAST CANCER, CHEMOTHERAPY, TAXANE
Financial Support: MS/FAF, CNPQ, FAPERJ
Resumo:15-010
IN VITRO ANALYSIS OF THE ANTITHROMBOTIC AND TOXICOLOGICAL PROFILE OF NEW
ANTIPLATELETS N-ACYLHYDRAZONE DERIVATIVES
Sathler, P. C. 1,2; Silva, L. A. 2; Prevatto, J. D. P. 2; Lione, V. 3; Rodrigues, C. R. 4; Mendes, L. C. 3; Kang,
H. C. 5; Jordão, A. K. 6; Cunha, A. C. 6; Castro, H. C. 2
1
Programa de Pós-graduação em Patologia, UFF
2
Labiomol, IB, UFF
3
LabTIF, FF, UFRJ
4
ModMolQSAR, FF, UFRJ
Departamento de Patologia, UFF
6
Departamento de Química Orgânica, UFF
5
Objectives:
Cardiovascular diseases are the most frequent cause of morbidity and mortality worldwide. Among them, atherothrombosis and
venous thromboembolism are highlighted, both related to the formation of vascular thrombosis. Although antiplatelet therapy is
the cornerstone of antithrombotic treatment of ischemic cardiovascular disorders, the current available antiplatelet agents have
less than satisfactory efficacy. Therefore, the identification of novel potential target candidates for antiplatelet therapy is still an
urgent need. In a recent study, our group detected an antiplatelet effect of novel N-substituted-phenylamino-5-methyl-1H-1,2,3triazole-4-carbohydrazides (2a, 2c, 2e, 2g and 2h) onto in vitro platelet aggregation induced by arachidonic acid (2c-IC50 = 8.8
ìg/mL), adrenaline (2h-IC50 = 7.8 ìg/ mL) and ADP (2a-IC50 = 19.04 ìg/mL). However the antiplatelet, anticoagulant and
toxicity profiles of these compounds have not been determined or fully evaluated. Thus, our study aims to analyze the in vitro
antithrombotic (antiplatelet and anticoagulant) and toxicological profiles of these new antiplatelet compounds.
To identify the extended profile of the antiplatelet activity of these compounds, we tested them in collagen-induced-platelet
aggregation assays (3 ìg/mL), which revealed compounds 2a, 2c and 2e with an extended inhibitory profile (IC50 = 66.5, 91.0
and 80.0 ìg/mL respectively). Differently, the evaluation of the anticoagulant profile in human plasma using prothrombin time
(PT), and activated partial thromboplastin time (APTT) tests (research ethics committee registration - N 140/10) showed that
these compounds have no effect in these assays, revealing the derivatives exclusively antiplatelet profile. For the toxicity
evaluation, we performed tests of MTT reduction in kidney cell line (VERO) that showed compounds 2c and 2g (100 ìg/mL) with
lower toxicity than the other compounds and similar profile to aspirin (AAS), the control drug, during 2, 6 and 8 hours.
Conclusions:
Overall, 2c showed the best integrated activity-toxicological profile from the N-acylhydrazone series at the in vitro analysis. Due
to its low cytotoxicity profile similar to AAS, this compound should be prioritized for further in vitro and in vivo evaluations
leading to a better understanding of its mechanism and its full potentiality for treating thrombotic disorders.
Keywords: Antiplatelet, Haemostasis , N-acylhydrazones, Platelet Aggregation , Thrombotic disorders
Financial Support: CNPq, Capes and Faperj
Resumo:15-011
MISUSE OF ANTIHYPERTENSIVE DRUGS BY PEOPLE IN PRODUCTIVE AGES IN METROPOLITAN AREA OF
RIO DE JANEIRO STATE
Coutinho, K. C. ; Ribeiro, A. A. R. ; Scaramello, C. B. V.
Objectives:
Our study aims to evaluate the use of drugs by people in productive ages that live in metropolitan area of Rio de Janeiro state.
Some pharmacoepidemiological studies have been seeking information from specific groups such as children, elderly, school
children and pregnant women (Rev. Saude Publica 38: 228, 2004). In the state of Rio de Janeiro 58% of the people are aged from
20 to 60 years and the human development index (HDI) is 0832. In Brazil southeast, education and longevity constituted the most
incisive factors in HDI improvement (Boletim Regional do Banco Central do Brasil. janeiro: 91, 2009). In addition the number of
colleges have increased and only 16% of students have incomes below three minimum wages (São Paulo em Perspectiva 14: 41,
2000).
Data collection included a structured questionnaire with a recall period of 6 months applied through individual interviews and
informed consent. Nonsteroidal anti-inflammatories (NSAIDs, 25%), drugs used in cardiovascular diseases (13%) and
contraceptives (6%) were the most used pharmacological classes by 235 respondents. Among NSAIDs, 95% were not selective
for COX2. The diuretics (D) consumption (29%) was equivalent to angiotensin converting enzyme inhibitors/ angiotensin
receptor blockers (A, 33%) and superior to beta blockers (B, 23%) use by 186 individuals aged from 20 to 60 years. Only 10% of
these subjects were using calcium channel blockers (C) and 6% consumed nitrovasodilators (N).
Conclusions:
Pharmacoepidemiology is an important tool to avoid drugs misuse and unnecessary spending with health (Br. J. Pharm. Sci
42:475, 2006). As 61% of all respondents were reproductive age women, a significant use of contraceptives was observed. Our
findings reflect data from previous studies that shown NSAIDs as the most frequently pharmacological class consumed in Brazil.
They are medicines that do not require prescription and are present in several irrational drug combinations available in the
pharmaceutical market (Rev. Ciênc. Farm. Básica Apl. 28: 67, 2007). This observation is not restricted to people that do not have
information access because 65% of respondents in productive ages were graduated and had incomes greater than three minimum
wages. The hypertension pharmacological management indicated for the study group covers A or B monotherapy. Alternatively
C and D are drugs recommended to black people (J. Human Hypertens . 18: 139, 2004), but the genotype-phenotype relationship
is questionable in Brazil (Estudos Avançados, 18:57, 2004). It is inappropriate to use N drugs and B must be avoided such as D,
especially in combination therapy. In this case it is recommend A and C association because B and D increase diabetes incidence
mainly in patients with risk factors (BMJ 328: 633, 2004). The use of antihypertensive drugs by people in productive ages in
metropolitan area of Rio de Janeiro state is not in agreement to current consensus pointing to the necessity of government policies
discussion and educational interventions for its rational use.
Keywords: antihypertensive drugs, pharmacoepidemiology, productive ages, Rio de Janeiro
Financial Support: FAPERJ, PROPPi/UFF.
Resumo:15-012
TRITICUM VULGARE IN THE HEALING OF OPEN WOUNDS IN EXPERIMENTAL ANIMALS
Peres, W. 1; Ramos, T. 2; Tillmann, M. T. 3; Campello Felix, A. O. 3; Mueller, E. N. 3; Felix, S. R. 3; Ramos,
S. C. 3; Nobre De, M. O. 3
1
Centro de Ciências Químicas, Farmacêuticas e de Alimentos , UFPel
2
Faculdade de Odontologia, UFPel
3
Faculdade de Veterinária, UFPel
Objectives:
The aim of this study was to assess the healing evolution of open wounds treated with creams containing different concentrations
of T. vulgare. Furthermore, the resistence of the resulting tissue was assessed at the end of the experiment.
In this study, 135 open skin wounds were surgically made in New Zeland White rabbits. These were treated with aquose extreact
of T. vulgare at 2mg/ml (group I), 10mg/ml (group II) and non ionic cream (group III, control), for 21 days. Ten wounds per
group were randomly chosen to suffer clinical and histopathological evaluations on days seven, 14, and 21. The wound´s
contraction, the type and quantity of exudates, the formation of granulation and epiteliation tissue were all observed, as well as
the phases of the healing process and the amount and pattern of collagen. The analyses of healing quality (classified as
normotriphic and hypertrophic), rupture resistance, and tensiometric assays were conducted in the 21st day. The clinical assays
were carried out with 10 wounds per group, the others were made with 15 for a total of 45 wounds per group. The tensiometric
(Pa) and the rupture resistance (N) assays were conducted in a universal assay machine (DL 500) through axial traction submitted
to a tension of 100 newtons at a speed of 5mm/minute. For the clinical and histopathological evaluation the mean sum of the
scores were used and submitted to ANOVA and Tukey test. For the tensiometric and rupture assay the tukey test was used
(statitix 9.0). The results show no difference between treatments, neither in the clinical and histopathological evaluations, nor in
the quality observation. When rupture resistance is considered, group I had the highest mean (45.6 N) while groups II and III
showed no statistical difference (35 N and 33.5 N respectively). In the tensiometric evaluation, group I (1.6 Pa) was statistically
different (p=0.0295) from the control group (1.2 Pa), indicating that the wounds treated with the phytotherapic at 2mg/mL had
better resistance than the others.
Conclusions:
This study has shown that, while there is no difference in the clinical and histopathological analises among treatment groups,
wounds treated with the T. vulgare extract at 2 mg/mL had a better result in the tensiometric evaluation.
Keywords: creams, healing, rabbits, wounds
Financial Support: CNPq according to process no 481605/2010-0 (edital universal) and for researcher
Resumo:15-013
COMPARISON OF THREE VOLATILE ANESTHETIC AGENTS IN EXPERIMENTAL CHRONIC ALLERGIC
ASTHMA.
Burburan, S. M. ; Abreu, S. ; Samary, C. ; Silva, J. D. ; Lucas, I. H. ; Xisto, D. G. ; Rocco, P. R. M.
Laboratory of Pulmonary Investigation, IBCCF- UFRJ
Objectives:
The present study aimed to compare the respiratory effects of routinely used anesthetic agents: halothane, isoflurane and
sevoflurane, in a model of chronic allergic asthma. For this purpose, pulmonary mechanics, airway responsiveness and lung
morphometry were analyzed to determine whether the physiological modifications reflected underlying morphological changes.
Fifty-six BALB/c mice (20-25 g) were randomly divided into eight groups. In asthma groups, mice were sensitized and
repeatedly challenged with ovalbumin. SAL groups received saline using the same protocol. Twenty-four hours after the last
challenge, animals were anesthetized with either halothane (Halo), isoflurane (Iso), sevoflurane (Sevo) or control drug thiopental
sodium (Control). Lung static elastance (Est), resistive (ΔP1) and viscoelastic/inhomogeneous (ΔP2) pressures were analyzed by
end-inflation occlusion method. Lungs were fixed and stained for histopathological analysis. Asthma animals anesthetized with
halothane, isoflurane and sevoflurane resulted in lower ΔP1 (34%, 36%, and 52%), ΔP2 (48%, 46%, and 47%), and alveolar
collapse (48%, 25%, and 59%) compared to the Control group. Est was more reduced in Asthma-Halo (20%) and Asthma-Sevo
(22%) groups than Asthma-Control. Furthermore, sevoflurane led to greater airway dilation (24%) compared to Control.
Conclusions:
Among these volatile anesthetic agents, sevoflurane acted on central and distal airways reducing airway resistance, viscoelastic
pressure and static elastance in the present model of chronic allergic asthma.
Keywords: anesthetics, asthma, halothane, isoflurane, sevoflurane
Financial Support: PRONEX-FAPERJ, FAPERJ, CNPq, INCT-INOFAR, CAPES
Resumo:15-014
STIMULATORY EFFECT OF 17-BETA-ESTRADIOL AND PROGESTERONE ON HUMAN CUZNSOD IN VITRO
Unfer, T. C. 1; Maurer, L. H. 2; Figueiredo, C. 2; Kemerich, D. M. 2; Emanuelli, T. 3
2
Graduação em Farmácia/CCS, UFSM
1
Pós-Graduação em Farmacologia/CCS, UFSM
3
Departamento de Tecnologia e Ciência dos Alimentos/CCR, UFSM
Objectives:
Superoxide dismutase (SOD) comprises the first line of defense against reactive oxygen species (ROS) in the body by catalyzing
the dismutation of O2•- to H2O2 and O2. Menopausal women show increased oxidative stress, possibly due to the lack of
estrogen. Steroid hormones modulate cellular activity by binding to specific receptors and there is considerable evidence that
estrogens are able to decrease the production of ROS and oxidative stress in vitro and in vivo. Recently, it was suggested that 17beta-estradiol (E2) can exert antioxidant action not only by direct scavenge of reactive species and gene modulation, but also
likely by direct modulation of enzymatic activities. However, little is known about the influence of progesterone (Prog) on
antioxidant systems. The objective of this study was to evaluate the in vitro effect of different concentrations of natural hormones
E2 and Prog on the activity of CuZnSOD in human erythrocytes.
The study was approved by the local ethics committee of Federal University of Santa Maria (UFSM, Brazil) (CAAE n°
0120.0.243.000-06). Healthy woman blood donors were recruited for the study, and informed consent was obtained from all
participants. A total of four women (age range 20 – 28 years) in the follicular phase of the menstrual cycle were included in the
study. The concentrations of E2 and Prog were assessed in the blood samples. Erythrocytes were pre-incubated for 24 h at 37 °C
in the absence of hormones (control) and presence of increasing concentrations of E2 and Prog. After, the samples were
incubated with the same concentrations of these hormones to evaluate the CuZnSOD activity in the erythrocytes. CuZnSOD
activity was measured at 560 nm using nitroblue tetrazolium (NBT) and xanthin-xanthin oxidase system. Data were analyzed by
one-way analysis of variance (ANOVA) followed by Tukey‟s test. The activity of CuZnSOD increased 8 times when the samples
were incubated with E2 (2-10 nM) and 19 times after incubation with Prog (0.3 to 10 nM). The hormones had no stimulatory
effect on the enzyme at concentrations higher than the physiological level (> 10 nM). The concentration to increase 50% of SOD
activity (EC50) was 2 nM for E2 (confidence interval 1-2 nM) and 0.95 nM for Prog (confidence interval 0.3- 3 nM),
respectively.
Conclusions:
We conclude that E2 and Prog at physiological concentrations can directly activate erythrocyte CuZnSOD with similar potency.
Keywords: 17-beta-estradiol , CuZnSOD , erythrocytes, oxidative stress, progesterone
Financial Support: CAPES, REUNI, FAPERGS
Resumo:15-015
MORPHINE TREATMENT IN EARLY LIFE ALTERS NTPDASE ACTIVITY IN RAT BLOOD SERUM
Nonose, Y. 1,3; Rozisky, J. R. 1,4,3; Laste, G. 1,4,3; Santos, V. S. 1,3; Macedo, I. C. 1,4,3; Oliveira, C. M. D. 1,3;
Oliveira, C. D. 1,3; Battastini, A. M. O. 2,4; Torres, I. L. S. 1,3,4
1
Dept. Farmacologia - Instituto de Ciências Básicas da Saúde, ICBS - UFRGS
2
Dept. de Bioquímica – Instituto de Ciências Básicas da Saúde, ICBS - UFRGS
3
Unidade de Experimentação Animal - Hospital de Clínicas, UEA - HCPA
4
Programa de Pós-Graduação em Medicina – Ciências Médicas, PPGCM - UFRGS
Objectives:
Opioids such as morphine are the most common treatment for acute pain in newborns. Previous works by our laboratory have
shown that morphine exposure in early life promotes alterations in E-NTPDase activity and gene expression pattern in central
nervous structures of rats. The E-NTPDases hydrolyze ATP and ADP, while 5'-nucleotidase hydrolyzes AMP to adenosine.
These enzymes are the major regulators of purinergic signaling in the blood. It has been shown that ATP stimulates a nociceptive
response, although the adenosine mediates a component of morphine analgesia. Considering the close relationship between the
opioid and the purinergic systems, the aim of this study was to evaluate whether morphine exposure in early life, from postnatal
day 8 until postnatal day 14, alters NTPDases and 5'-nucleotidase activities in the short, medium and long term in blood serum of
rats.
The protocol of this study was approved by the Ethics Committee of Hospital de Clínicas de Porto Alegre. Eight-day-old male
Wistar rats (150-250 g) were divided into two groups: saline control (C) and morphine treatment (M). Each animal received
saline (C) or morphine (total dose of 5 &mug in the mid-scapular area) once a day for seven days. At the end of each stage of the
experiment, rats were decapitated and trunk blood was collected to obtain blood serum. The enzyme assays were carried out on
samples at P16 (C: 5; M: 4), P30 (C: 11; M: 11) and P60 (C: 7; M: 7). The statistical analysis was performed using Student's t
test. Differences between groups were considered significant at Pt test, P-1 .mg-1 of protein, M = 1.74±0.21 nmol Pi.min-1.mg-1 of
protein; Student's t test, P
Conclusions:
The difference in activities observed after morphine withdrawal in this study are possibly due to different types of NTPDases
present in blood serum, since our results showed a change in the ATPase and ADPase activities. NTPDase1 together with
NTPDase2 represents the dominant ecto-nucleotidases expressed by vascular endothelial cells. The NTPDase1 is notable for its
high preference for nucleoside triphosphates and nucleoside diphosphates, while NTPDase 2 stands out for it's high preference
for nucleosides triphosphates. When NTPDase1 is inhibited, as is the case at P60, ATP is converted to ADP by other ATPases,
and ADP will be relatively stable. It is probable that the two different NTPDases are carrying out the same function, one
hydrolyzing preferentially ATP and the other hydrolyzing ADP slowly. The nucleotide hydrolysis profile may lead to an increase
in the ADP availability at the peripheral level. Our findings highlight the importance of NTPDases in regulating nucleotide levels
in rats exposed to morphine. More studies are necessary to elucidate ADP functions at peripheral level after the end of morphine
treatment.
Keywords: NTPDase, 5'-nucleotidase, morphine, neonate, rat blood serum
Financial Support: CNPq, CAPES, FAPERGS, GPPG of Hospital de Clínicas de Porto Alegre
Resumo:15-016
THE IN VITRO CLOMIPHENE CITRATE EFFECT ON OXIDATIVE METABOLISM AND PROLIFERATIVE
LYMPHOCYTE CELL CULTURES
Costa, F. 1,3; Garcia, L. F. M. 1; Dornelles, E. 1; Mânica-cattani, M. F. 1; Filho, O. C. D. S. 1; Sagrillo, M. R.
1,2
; Montagner, G. F. S. 1; Cadoná, F. C. 1; Algarve, T. D. 1; Cruz, I. B. M. D. 1
1
Departamento de Morfologia - Laboratório de Biogenômica, UFSM
2
Centro Universitário Franciscano, UNIFRA
3
Universidade de Santa Cruz do Sul, UNISC
Objectives:
We performed analysis to test if the clomiphene citrate (CC), a non-steroidal selective estrogen receptor modulator (SERM) used
to induce ovulation or to ovarian reserve tests have some oxidative properties as previously described to other SERMs
(ex.tamoxifen).
We performed an in vitro prospective study to test the CC potential effects on oxidative metabolism and lymphocyte cell
proliferation exposed to concentrations of this drug). Blood was collected from 3 women and the peripheric blood mononuclear
cell (PBMC) were cultured in CO2 5%, at 37oC during 72h with different CC concentrations (1 µM, 5 µM, 10 µM and 20µM).
After this period the cell proliferation was measured by MTT spetrophotometric assay. An additional test to analyze if the CC
have some influence in reactive oxygen species (ROS) was performed using 2',7'-dichlorofluoresceinacetate (DCFH-DA)
fluorometry assay after 24 h exposition of CC treatments.
Conclusions:
The CC treatments increased the cell proliferation from > 5 µM concentration when compared to control group and 1 µM
concentration (F=3,382, p=0,009). The ROS was also influenced by the CC treatments when compared to negative control group.
Keywords: Clomiphene citrate, Lymphocyte, Oxidation, Proliferation
Resumo:15-017
BACTERIAL SENSITIVITY TO THE COMPOUNDS OF THE FAMILIES OF QUINOLONES, QUINONES AND
NAFTA QUINONE.
Ochioni, A. C. 1; Novais, J. 2; Moscoso, J. M. 3; Gama, I. L. 4; Souza, M. C. B. 5; Rangel, C. R. 6; Catro, H.
C. 7; Carballido, J. M. 8; Guimarães, D. A. 9; Lione, V. 10
2
DepartamentodeBiologiaCelulareMolecular/InstitutodeBiologia, UFF
5
Departamento de Química Orgânica/InstitutodeQuimica, UFF
3
1
4
Departamento de Química Orgânica/InstitutodeQuimica, UFF
6
7
8
SetordeMicrobiologia/Hospital Universitário Antonio Pedro, HUAP
9
SetordeMicrobiologia/Hospital Universitário Antonio Pedro, HUAP
10
Objectives:
The resistance of microorganisms to antibiotics and chemotherapy drugs are a problem of world order that demands the discovery
of new molecules for the infections treatments caused by multiresistant microorganisms. This challenge has encouraged the
scientific search to find alternatives to antibiotic therapy, aiming the isolation or synthesis of new and more effective antibiotics.
Based on the importance of these bacteria in the hospital area and the difficulty of treat infections caused by resistant strains, this
study has the objective identification new and more effective antibiotics with the greatest action against the 16 strains of different
bacterial species.
We performed the tests for antibiotic activity of new derivatives through the determination of bacterial susceptibility to the
compounds in the disk diffusion test, besides the of minimal inhibitory concentration (MIC). Alls procedures follow the standard
protocol used by the Clinical and Laboratory Standards Institute (CLSI). We tested 10 compounds INT and INT NH2 each,
belonging to the family of quinolones and quinones, as well as two compounds (AMINO and ACRILATOQUI) of the family of
quinone NAFTA. The results showed that only INT 12 compound formed a zone of inhibition 15mm front Escherichia coli strain
635, while the AMINO compound showed inhibition zone of 16mm sample for Staphylococcus epidermidis201. This result
enabled the realization of MIC, for we quantified their effect on the strains in study. Then, we conducted the evaluation of
cytotoxicity of both compounds in Vero cells by MTT (3 - (4,5-DimethylTiazol-2yl)- 2,5- diphenyl bromidetetrazolina) assay,
whre the compounds INT 12 and AMINO
Conclusions:
Preliminary results showed that the amino and INT 12 have potential for antibacterial activity against Staphylococcus epidermidis
201 and Escherichia coli 635. Moreover, both compounds were less toxic than the antibiotics used in clinical practice, such as
chloramphenicol and ciprofloxacin.
Keywords: Bacterial , quinolones, resistance, Sensitivity, synthetic Compounds
Financial Support: Antônio Pedro Hospital, UFF, FAPERJ, CNPq e CAPES.
Resumo:15-018
PHARMACOKINETICS EVALUATION OF SUFENTANIL-2-HYDROXYPROPYL-&BETA;-CYCLODEXTRIN
INCLUSION COMPLEX
Macedo, M. D. 1; Pedrazzoli-júnior, J. 1; Calafatti, S. A. 1; Paula, E. D. 2; Araujo, D. R. D. 3; Tofoli, G. R. 1
1
Universidade São Francisco, USF
2
Depto. de Bioquímica- Instituto de Biologia, UNICAMP
3
Centro de Ciências Humanas e Naturais, UFABC
Objectives:
Determining drug concentration in biological fluids provides the fundamental information needed for the development of new
sustained-release pharmaceutical formulations, such as sufentanil-2-hydroxypropyl-β-cyclodextrin inclusion complex (SUF:HPβ-CD). Thus, the purpose of this blinded study was to determine the plasma levels and to evaluate the pharmacokinetics induced
by SUF:HP-β-CD in comparison with its aqueous formulation (SUF) in rabbits.
The complex was obtained by mixing equimolar amounts of HP-β-CD and sufentanil (SUF). The protocol was approved by the
Institutional Committee for Ethics in Animal Research of São Francisco University (protocol #001.12.09). Twelve New Zealand
White rabbits (2500-3000g) were divided in two groups (n=6) and treated by intramuscular route with SUF or SUF:HP-β-CD
complex (10 μg.kg-1). Blood samples (2 mL) from an ear vein were collected via a heparinised cannula pre dose (0 min) and at
15, 30, 45, 60, 90, 120, 180, 240, 300, 360, 420 and 480 minutes after the injection of formulations. Immediately after each blood
collection, plasma was separated and stored at −70οC until analysis. Sufentanil plasma levels were determined using a Waters™
HPLC system (2795) coupled to a Micromass Quattro Premier XE triple stage quadrupole mass spectrometer (LC-MS/MS),
equipped with an API electrospray source. Data were submitted to unpaired t test (α=0.05). Sufentanil induced higher plasma
concentrations than SUF:HP-β-CD at all periods of time (p0.05).
Conclusions:
The ciclodextrin-based drug-delivery system of sufentanil was effective to reduce the absorption of the drug, due to the sustained
release of sufentanil from ciclodextrins.
Keywords: cyclodextrin, local anesthetics, pharmacokinetics, sufentanil
Financial Support: FAPESP (Proc 2009/17715-7)
Resumo:15-019
GROWTH INHIBITION OF PSEUDOMONAS AERUGINOSA MULTIRESISTANT HOSPITAL STRAINS BY
TRIAZOLES DERIVATIVES
Novais, J. S. 1; Ferreira, B. L. A. 1; Lannes, A. C. 3; Jordão, A. K4; Cunha, A. C. 4; Ferreira, V. F. 4;
Carballido, J. M. 2; Guimarães, D. A. 2; Santos, D. O. 1; Castro, H. C. 1
1
Biologia Celular e Molecular / Instituto de Biologia, UFF
2
Microbiologia / Hospital Universitário Antônio Pedro, UFF - HUAP
3
Patologia / Pós-graduação Patologia, UFF - Patologia
4
Química Orgânica / Instituto de Química, UFF
Objectives:
Bacterial growth is directly involved in the top ten leading causes of death worldwide, the infections. Due to the development
antibiotic resistance in virtually all clinically important pathogens, alternatives to conventional antibiotics are urgently needed
including new molecules development (Appl Microbiol Biotechnol, 88:1261, 2010). Pseudomonas aeruginosa is the most
common Gram-negative non-fermenter, which can cause opportunistic infections in immunocompromised individuals such as
patients with AIDS, cancer, burn victims and cystic fibrosis (Antimicrob Agents Chemother, 49:3281, 2005). Based on the
importance of bacteria in hospitals and difficult to treat infections caused by resistant strains, our aim is to identify new and more
effective antibiotics that have less toxicity and greater potency.
In this paper we present the profile of ten new molecules derived from acid N-arylamine-5-methyl-1H-〈1,2,3〉-triazol-4carboxilic
hydrazide by using susceptibility tests and disk diffusion methods also performing an in silico structural evaluation using
molecular modeling tools. Interestingly two compounds with fluorine and bromine substitution (N-Br-FUR-NO2 and N-F-FURNO2) formed an inhibition zone against strain of P. aeruginosa(22mm and 24 mm) similar to currently used hospital antibiotics
(i.e. Ciprofloxacin). In this work we also determined the Minimal Inhibitory Concentration (MIC) for these compounds (N-BrFUR-NO2 = 1 &mug;⁄mL and N-F-FUR-NO2 = 2 &mug;⁄mL), which pointed them as promising compounds that act similar to
other antibiotics currently in the market (i.e. Ciprofloxacin = 1 &mug;⁄mL). Initial in silico and in vitro cytotoxicity results
pointed to a safe profile against monocytes derived human macrophages similar to ciprofloxacin. The analysis of structureactivity relationship using a molecular modeling approach revealed conformational and electronic features including polar area
surface and number of acceptor hydrogen groups directly involved on the antibiotic profile of these compounds. These results
may help on proposing new chemical modifications to maximize the degree of antimicrobial activity of these derivatives against
the multidrug resistant P. aeruginosa.
Conclusions:
Our results demonstrated that Br-NO2-Fur and Fur-NF-NO2 compounds presented a potential antibacterial activity against P.
aeruginosa for further in vivo cytotoxic and structure-activity relationship evaluations molecules to pursue a new antibiotic
prototype.
Keywords: growth inhibition, bacteria, antibiotic resistance , structure-activity relationship, compounds
Financial Support: FAPERJ, CNPq and UFF-PROPPi
Resumo:15-020
STUDY OF INTESTINAL ABSORPTION AND TOXICITY IN VITRO OF DIFFERENT FORMULATIONS OF
SILDENAFIL.
Moscoso, J. M. 1; Lione, V. 2,1; Silva, J. H. 2; Silva, A. R. 2; Nasciutti, L. E. 2; Novais, J. S. 1; Castro, H. C.
1
; Cabral, L. M. 2
1
BIOLOGIA CELULAR E MOLECULAR/UNIVERSIDADE FEDERAL FLUMINENSE, UFF
2
DEPT. DE MEDICAMENTOS/UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, UFRJ
3
DEPTO. DE HISTOLOGIA/ UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, UFRJ
Objectives:
Sildenafil, which belongs to the class of inhibitors of phosphodiesterase, is an important compound used to treat erectile
dysfunction. This acts in the final phase of the biogeochemical cycle of erection enhancing effect of nitric oxide (NO) by
inhibiting the enzyme phosphodiesterase 5 (Quim. Nova, 31, No. 5; 1112, 2008.) This means that there is an increased of cyclic
GMP levels which acts by relaxing muscles in the corpus cavernosum, which allows filling of blood, favoring the erection. Some
tissues also expressed of the enzyme phosphodiesterase, resulting in new possible targets of action of sildenafil, and also
contributing to appearance of side effects. It follows that sildenafil is sold only in tablets of 25, 50 and 100mg, and has a low
biodisponibility. Thereby it is necessary to study new formulations that increase the biodisponibility through pharmaceutical
forms for faster absorption such as solutions, as well as, making the action of the drug directed through a process of vectorization
with microemulsions (ME), minimizing yours adverse effects.
For permeation testing of new formulations was used cell line Caco-2 (cells from immortalized human colon adenocarcinoma)
cultured in complete DEMEM. In 24 well plates, cells were grown on transwell filters. After 14 days, time of formation and
maturation of the monolayer, we measured the transepithelial resistance (TEER) and the cells were exposed to different
formulations of sildenafil (3.33% solution, ME 01 and ME 02) at some intervals of time (0.5h, 1h, 2h and 3h). The control of
monolayer integrity was performed by measurement of TEER, as well as by the passage of fluorescein. The results showed that
permeation of the formulation developed as a solution had the highest levels in intestinal cell permeation of the microemulsions,
being formulated as a condition more promising. A similar profile was shown in toxicity tests in vitro using MTT (3 - (4,5DimethylTiazol-2yl) -2,5-diphenyl bromidetetrazolina) where the 3,33% solution of sildenafil was less that the toxic
microemulsions. Also are being developed, morphological tests to prove that the solution of sildenafil 3,33% shows less toxicity
compared to microemulsions.
Conclusions:
From the results of the tests described above, we conclude that the develop new formulations showed promising, mainly to
sildenafil solution, because it has higher biodisponibility and low cytotoxicity.
Keywords: DRUGS ABSORPTION, BIODISPONIBILITY, CACO-2 CELLS, PHOSPHODIESTERASE-5, SILDENAFIL
Financial Support: CAPES, CNPq and FAPERJ
Resumo:16-001
INBORN HIGH AEROBIC CAPACITY CONFERS CARDIOPROTECTION AGAINST MYOCARDIAL
INFARCTION-INDUCED MALADAPTATIONS.
Ferreira, J. C. B. 1,2; Rolim, N. P. L. 3; Bacurau, A. V. N. 1; Moreira, J. B. N. 1; Monteiro, A. W. A. 1;
Wisloff, U. 3; Brum, P. C. 1
1
Esc. de Educaçao Física e Esporte/Universidade de São Paulo, EEFE/USP
2
Stanford University School of Medicine, Stanford Medicine
3
Norwegian University of Science and Technology, NTNU
Objectives:
Improved aerobic fitness has been shown to be positively correlated with life span and cardiovascular risk factors are known to
emerge after artificial selection for low aerobic capacity, therefore, we hypothesized that inborn high aerobic capacity would
promote protection against cardiac maladaptations promoted by myocardial infarction (MI).
Rats from unique strains artificially selected through several generations for inborn low (n=20) or high aerobic capacity (n=20)
(low [LCR] and high capacity runners [HCR], respectively) were submitted to either Sham or MI surgery and studied four weeks
after surgical procedures. Maximal oxygen uptake (VO 2max), left ventricle (LV) fractional shortening, inotropy (+dP/dT) and
lusitropy (-dP/dT), cardiac collagen content, ATP levels in total cardiac lysate and maximal respiration of isolated cardiac
mitochondria were evaluated. Aerobic capacity and cardiac function as evaluated by echocardiography were depressed in both
LCR and HCR after MI, however, reduced VO2max (42.3±3.5 vs. 32.0±2.3 vs. 59.8±1.8 vs. 50.8±0.9 mLO2/kg0.75 in LCRSHAM, LCR-MI, HCR-SHAM and HCR-MI, respectively) and LV fractional shortening (33.0±0.3 vs. 18.6±1.7 vs. 35.1±1.4 vs.
28.0±2.5 %) were observed in LCR-MI when compared with HCR-MI. While +dP/dT were similar among groups, HCRSHAM‟s -dP/dT values were significantly higher than LCR-SHAM‟s and were depressed in both MI groups without significant
difference between LCR-MI and HCR-MI, however, lusitropism of HCR-MI remained similar to healthy LCR (7729±1337 vs.
4667±729 vs. 11181±1045 vs. 6417±1021 mmHg/sec). LV collagen content was increased by MI in LCR rats (2.1±0.3 vs.
3.2±0.4 vs. 2.1±0.2 vs. 2.7±0.3 % of total area), suggesting that cardiac fibrosis was present only in LCR-MI. Extending findings
to the cellular level, we verified reduced cardiac ATP levels of LCR-MI when compared with HCR-MI (6.3±0.9 vs. 2.8±0.7 vs.
7.9±0.5 vs. 6.2±1.3 pM/μg of cytosolic protein), while maximal mitochondrial respiration was elevated in HCR-SHAM and
unchanged by MI in LCR and HCR animals (10.6±0.6 vs. 11.3±1.2 vs. 15.4±1.9 vs. 12.5±1.9 μmolO 2/min/g).
Conclusions:
Taken together our data suggest that inborn high aerobic capacity confers cardioprotection against MI-induced LV fibrosis and
dysfunction, which might be due to improved energy metabolism observed in HCR rats.
Keywords: myocardial infarction, cardiac dysfunction, aerobic capacity
Financial Support: FAPESP, CAPES and CNPq
Resumo:16-002
ACUTE AORTIC REGURGITATION INCREASES WATER INTAKE FOLLOWING ACUTE FLUID DEPLETION
Leme, G. A. 1; Tardivo, A. C. B. 1; Roscani, M. G. 2; Matsubara, L. S. 2; Matsubara, B. B. 2; Gobbi, J. I. F. 1
1
Departamento de Fisiologia/Instituto de Biociências , UNESP - IBB
2
Departamento de Clínica Médica/Faculdade de Medicina, UNESP - FMB
Objectives:
Aortic regurgitation (AR) is associated with a left ventricle dilation and hypertrophy in response to chronic volume overload. AR,
often secondary to rheumatic fever, is still prevalent in Brazil. The ingestive behavior has not been investigated for this pathology
yet. Therefore, our aim was explored the influence of acute volume overload, 1 week after AR, on sodium and water intake and
sodium excretion following an acute fluid depletion.
Male Wistar rats (280 - 300g) were submitted to surgical procedure for AR (n = 9) or sham operation (n = 8). A week after the
surgeries, transthoracic echocardiograms were performed to confirm the presence of AR and to collect morphological data. Two
days after the exams animals were treated with the diuretic furosemide (10 mg/kg bw) combined with a low dose of the
angiotensin converting enzyme inhibitor captopril (5 mg/kg bw), a procedure to induce water and sodium intake. The
echocardiograms revealed an increase in left ventricule diastolic diameter (7.3 ± 0.1 vs. sham: 6.3 ± 0.2 mm, P
Conclusions:
Data suggested that acute AR, a volume overload situation, increases left ventricule diastolic diameter that leads to an increase in
water intake and natriuresis following an acute fluid depletion.
Keywords: aortic regurgitation, water intake, volume overload, natriuresis, echocardiograms
Financial Support: CAPES, FAPESP-09/52547-8
Resumo:16-003
EFFECT OF SEPSIS ON THE CARDIAC AND VASCULAR FUNCIONALITY IN MICE.
Rezende, E. ; Bóf, E. R. ; Dalbó, S. ; Assreuy, J.
Departamento de Farmacologia, UFSC
Objectives:
Sepsis causes cardiovascular complications, such as cardiac collapse and reduction in vascular tone. It has been described that the
sepsis involves an initial hyperdynamic phase characterized by increased cardiac work, followed by a hypodynamic phase in
which cardiac output and arterial pressure are reduced. Considering the importance of calcium signaling, the aim of the present
study is to evaluate the vascular and cardiac consequences of the interference in the early calcium signaling by verapamil.
Female Swiss mice (3-3.5 months old, weighing 35-45 g) were submitted to cecal ligation and puncture (CLP) model to establish
sepsis. In order to evaluate the arterial pressure and the vascular and cardiac responses, dobutamine was administrated in different
doses, namely 3, 10 and 30 nmol/kg, i.v. Animals were treated with vehicle (saline) or verapamil (0.3; 1 or 3 mg/kg, p.o.), 1 hour
after CLP surgery. Twenty four hours after surgery, the arterial pressure of the animals as well as their response to dobutamine
(n=6) were recorded. All experimental procedures were approved by the institutional Ethics Committee. Normal animals show a
systolic basal pressure of 71 ± 4 mmHg and diastolic pressure of 49 ± 1 mmHg. After dobutamine administration, a dosedependent increase of systolic pressure in 78 ± 2; 87 ± 5 and 92 ± 3 mmHg, respectively and diastolic pressure of 54 ± 2; 61 ± 3;
66 ± 1 mmHg, respectively, were observed. Septic animals show basal systolic pressure of 61 ± 2 mmHg and diastolic pressure of
34 ± 3 mmHg. In addition, they become hyporesponsive, verified by of the small changes in arterial pressure after dobutamine
administration (systolic: 60 ± 4; 72 ± 2; 69 ± 1 mmHg; respectively; diastolic: 32 ± 2; 38 ± 4; 33 ± 2 mmHg, respectively).
Interestingly, animals treated with verapamil (1 mg/kg) showed a normal basal pressure (systolic 69 ± 5 and diastolic 40 ± 5
mmHg) and an increase in the arterial pressure after dobutamine administration (systolic: 79 ± 4; 85 ± 6; 89 ± 7 mmHg; diastolic:
49 ± 4; 51 ± 6; 49 ± 6 mmHg). This effect is not observed in animals treated with verapamil 0.3 or 3 mg/kg.
Conclusions:
Our data suggest that verapamil-sensitive calcium channels have an important role in the initial phase of sepsis and may
contribute to the cardiovascular dysfunction of this condition.
Keywords: sepsis, cardiac disfunction, vascular disfunction
Financial Support: CNPq, CAPES and FAPESC.
Resumo:16-004
HIGHER SALT PREFERENCE IN RATS WITH SUBCRHONIC AORTIC REGURGITATION AFTER SODIUM
DEPLETION.
Leme, G. A. 1; Siqueira, T. F. 2; Omoto, A. C. M. 1; Tardivo, A. C. 1; Roscani, M. G. 2; Matsubara, L. S. 2;
Matsubara, B. B. 2; Gobbi, J. I. F. 1
1
Depto de Fisiologia/Instituto de Biociências de Botucatu, UNESP - IBB
2
Depto de Clínica Médica/Faculdade de Medicina de Botucatu, UNESP - FMB
Objectives:
The renin-angiotensin-aldosterone system (RAAS) is one of the nerohumoral factors involved in fluid intake. Aortic regurgitation
(AR) is associated with a left ventricle dilation and hypertrophy in response to chronic volume overload, and it might increase the
RAAS. Nevertheless, fluid intake in this pathological situation has not been explored. Thus, our aim was investigate the influence
of subcrhonic overload on sodium and water intake and excretion following an acute fluid depletion.
Male Wistar rats (280 - 300g) were submitted to AR (n = 9) or sham operation (n = 8). AR procedure was shaped by retrograde
puncture of the aortic valve leaflets under anesthesia (association of ketamine and xylazine). Transthoracic echocardiograms were
performed 4 weeks after the surgical procedure. Water and 0.3M NaCl intake were daily recorded since the day following
surgeries, till the end of the fourth week. Echocardiograms at the fourth week revealed a decrease in ejection fraction (50.7 ± 2.8
vs. sham: 61.7 ± 1.9 %, P
Conclusions:
Aortic regurgitation produced a systolic disfunction of left ventricule and increases sodium preference following an acute sodium
depletion.
Keywords: sodium intake, aortic regurgitation, volume overload, systolic disfunction , angiotensin
Financial Support: CAPES, FAPESP-09/52547-8
Resumo:16-005
ROLE OF NO AND CALCIUM IN THE VASORELAXANT EFFECT INDUCED BY IM-7 IN THE MESENTERIC
ARTERY FROM L-NAME HYPERTENSIVE RATS
Almeida, M. M. 1; Anjos, R. M. 1; Oliveira-filho, A. A. 1; Alustau, M. C. 1; Furtado, F. F. 1; Queiroz, T. M.
1
; Luis, J. A. S. 2; Athayde-filho, P. F. 2; Medeiros, I. A. 1
1
Laboratório de Tecnologia Farmacêutica/UFPB , UFPB
2
Departamento de Química/CCEN/UFPB, UFPB
Objectives:
The aim of this study was to investigate the role of NO and calcium in the mechanisms by which 5-(4-isopropylphenyl)-3phenylimidazolidin-2,4-dione (IM-7) causes vasorelaxation in the superior mesenteric artery from L-NAME hypertensive rats.
Hypertension was induced in male Wistar rats, weighing 250-300 g, by oral administration of the L-NAME (50 mg/kg bw/day) in
drinking water for 7 days. In isolated hypertensive rat mesenteric artery rings (1-2 mm) were suspended by platinum hooks for
isometric tension recordings. IM-7 (10-12 to 10-3 M) elicited concentration-dependent relaxation of PHE-induced contraction
(pD2 = 5.64 ± 0.09, MR = 99.43 ± 0.57 %, n = 6). After removal of the vascular endothelium IM-7 effects were significantly
attenuated (pD2 = 3.8 ± 0.07, n = 7), suggesting the participation of endothelium derived relaxant factors in the IM-7 induced
vasorelaxant effect. Similar results were obtained in the presence of atropine (10-7 M), L-NAME (10-4 M) or ODQ (10-5 M)
(pD2 = 4.93 ± 0.17, 3.8 ± 0.06 and 3.75 ± 0.06, respectively, n = 6). Also, IM-7 caused relaxation of vessels pre-contracted with
80 mM KCl (pD2 = 3.8 ± 0.07 and MR = 83.66 ± 4.2 %). Moreover, in absence of extracellular calcium, IM-7 (10-5, 10-4 and
10-3 M) concentration-dependently depressed the contractions induced by CaCl2 (MR = 91.6 ± 6.0 %, 69.6 ± 9.9 % and 13.5 ±
4.5 %, respectively). Similar results were obtained in contractions induced by 5-HT (MR = 43,88 ± 6,83 % to 10-3 M of IM-7) or
cumulative administration of PHE (pD2 = 6.37 ± 0.03, 6.0 ± 0.02 and 10.7 ± 4.1 to 10-5, 10-4 and 10-3 M of IM-7; MR = 51.0 ±
14.6 % and 10.7 ± 4.1 % to 10-4 and 10-3 M of IM-7, respectively) and Na3VO4 (MR = 47.0 ± 6.31 %, 45.58 ± 7.61 % and
41.94 ± 4.32 %, respectively). The data suggest inhibition of calcium influx and contractile apparatus. Furthermore, no change
was observed on the transients contractions induced by PHE in without-Ca2+ medium, suggesting the no involvement of calcium
intracellular-stokes.
Conclusions:
Taken together, these data suggests that concentration-dependent relaxation induced by IM-7 in the superior mesenteric artery
from L-NAME hypertensive rats, involves activation of muscarinic receptor-NO-sGC pathway and also inhibiting calcium influx
and contractile apparatus.
Keywords: CALCIUM, HYPERTENSIVE RATS, MESENTERIC ARTERY , NO, VASORELAXANT EFFECT
Financial Support: CAPES, CNPq
Resumo:16-006
CONTRIBUTION OF THE NITRIC OXIDE-SOLUBLE GUANYLYL CYCLASE-G KINASE SYSTEM TO THE
VASCULAR EFFECTS INDUCED BY C-TYPE NATRIURETIC PEPTIDE (CNP) ON AORTAS FROM 2K-1C RATS.
Pernomian, L. 1,1; Bendhack, L. M. 2
1
Pharmacology/ School of Medicine from Ribeirão Preto - USP, FMRP-USP
2
Physics and Chemistry, Faculty of Pharmaceutical Sciences, FCFRP-USP
Objectives:
This study aimed to investigate the contribution of the NO-sGC-GK system in the vascular effects induced by CNP on aortas
from renal hypertensive 2K-1C rats. All the procedures are in accordance to the Ethical Committee of the University of São
Paulo, 071/2009.
Methods: Male Wistar rats (200 g) underwent surgery to the implantation of a silver clip in the renal artery (2K-1C) and control
rats only was subjected to laparatomy (2K). 6 weeks after the surgery, systolic arterial pressure (SAP) was measured by tail cuff
and rats were considered hypertensive when SAP > 160 mmHg. Rats were killed by decapitation and aortas were isolated and
vascular reactivity or flow cytometry studies were developed. Endothelial cells isolated from aortas from 2K and 2K-1C were
loaded with DAF-2/DA (10 μM) or FLUO-3AM (5 μM) and the cells with FLUO-3AM were stimulated with CNP (1 μM) and
the fluorescence intensity (FI) was measured. Cumulative concentration-effect curves to CNP (1 pM to 0.5 μM) were performed
on aortic rings from 2K and 2K-1C, pre-contracted with Phenylephrine (0.1 μM) with (E+) or without (E-) endothelium, in the
absence or presence of L-NAME, (100 μM), NO scavenger Hydroxicobalamine (100 μM), ODQ (1 μM) or Rp-8-Br-PETcGMPS (10 μM). Pharmacologic parameters analyzed were efficacy (Emax) and potency (pD2). Results: Basal FI of DAF-2/DA
(NO) in 2K cells was higher than in 2K-1C cells (2K: 1965.60±175.42; 2K-1C: 1286.83±110.52; n=5-6). However, basal FI of
FLUO-3AM (Ca2+) was not different between groups, but CNP induced an increase in FLUO-3AM fluorescence (2K:
1410.6±53.74; 2K-1C: 1494.86±47.31; n=5-7). CNP induced vascular relaxation in aortas from 2K and 2K-1C with similar pD2.
In denuded arteries, the Emax was greater in 2K-1C (117.5±4.1%; n=9) than in 2K (2K: 95.9±3.3%; n=11) and 2K-1C E+
(E+101.4±2.0%; E-: 117.5±4.1%; n=9). L-NAME reduced the potency of inhibition to CNP in 2K-1C (E+: 8.09±0.23; E-:
6.83±0.13; n=7-9). NO scavenger decreased the pD2 of CNP in 2K-1C E+ (6.56±0.37; n=7). The presence of ODQ decreased
pD2 in 2K E+ (7.22±0.15; n=7) and Emax in 2K-1C E- (102.6±6.9%; n=6). GK inhibitor decreased the pD2 of CNP in all groups
(2K E+: 6.76±0.12; 2K E-: 6.50±0.22, n=11; 2K-1C E+: 6.67±0.31; 2K-1C E-: 6.40±0.22; n=6). However, only the denuded
arteries had their Emax decreased (2K E-: 64.2±6.6%; 2K-1C E-: 76.1±5.3%, n=6-11).
Conclusions:
Taken together, these results suggest that renal hypertensive rats present endothelial dysfunction, which does not interfere with
the calcium mobilization induced by CNP. CNP induces relaxation of aortas from 2K and 2K-1C rats. The NO-sGC-GK system
plays a role in the vascular relaxation induced by CNP that is modified in denuded endothelium aortas.
Keywords: C-type natriuretic peptide, cGMP dependent protein kinase, Nitric oxide, Renovascular hypertension 2K-1C, Soluble
guanylyl-cyclase
Financial Support: FAPESP, CAPES and CNPq.
Resumo:16-007
EVALUATION OF ANTIPLATELET AND ANTITHROMBOTIC PROFILE OF DISINTEGRINS FROM BOTHROPS
JARARACA VENOM.
Succar, B. B. 1; David, V. C. 1; Serrão, L. W. 2,1; Frattani, F. S. 2,1; Zingali, R. B. 1
1
Instituto de Bioquimica Médica, Universidade Federal do RJ, IBqM - UFRJ
2
Faculdade de Farmácia, DACT, UFRJ, DACT - UFRJ
Objectives:
Thrombosis can occur in the arterial or venous circulation, the first one being the most common cause of death (myocardial
infarction and stroke) in the developed world. Arterial thromboembolism is the third leading cause of cardiovascular-associated
death. Nowadays, the antithrombotic drugs are effective at reducing arterial and venous thrombosis. However, the main side
effect of these drugs is bleeding, which limits their use. Progress on safety and effectiveness of antithrombotic drugs is required.
Aiming to analyze the antiplatelet profile of molecules from natural origin, we evaluated two disintegrins from snake venom
(jarastatin and jararacin) using in vitro and in vivo assays.
The two disintegrins, jararacin (JARC) and jarastatin (JAST) were isolated from Bothrops jararaca venom using two liquid
chromatography steps, including size-exclusion (Sephacryl S-200) and reverse phase (Resource RPC) columns, and purification
was also confirmed by MALDI-TOF/MS (7356 m/z). For jarastatin purification we added a SP-Sepharose cation exchange
chromatography step to finally isolate this disintegrin and confirmed the isolation using mass spectrometry (7556 m/z).
Interestingly, the disintegrin isoforms purified in this work present peaks of 7356 and 7556 m/z, which correspond to the
jararacin and jarastatin isoforms with the N-terminal reduced by four and two amino acids, respectively. The isolated disintegrins
were tested in human platelet aggregation assays using thrombin (10 nM) and ADP (5 μM) as agonists. Despite their different
activity profiles, both jararacin and jarastatin inhibited human platelet aggregation in a dose- dependent manner. Interestingly,
jararacin was more potent than jarastatin in both thrombin- (IC50 = 23.1 and 99.5 nM, respectively) and ADP- (IC50 = 89.5 and
281.0 nM, respectively) induced assays. Despite of potent effect of these peptides, none of them were able to prevent the death of
BALB-C mouse animals, previously treated with disintegrins in a dose of 1mg/kg, in the pulmonary thromboembolism model
induced by ADP and confirmed by lung histology.
Conclusions:
In this study, we isolated two disintegrins from Bothrops jararaca, jararacin (JARC) and jarastatin (JAST). The disintegrin
isoforms purified in this work possess an N-terminus that is reduced by four and two amino acids, respectively, and the mass
spectra data confirmed their molecular masses after post-translational modifications. Both disintegrins were able to inhibited
platelet aggregation induced by ADP and thrombin. Despite, disintegrins were not able to prevent the death of animals in
pulmonary thromboembolism model. Additional studies in various animal models are necessary in order to indicate the use of
these disintegrins for the treatment of cardiovascular diseases.
Keywords: disintegrins, thrombosis, platelet aggregation
Financial Support: CNPq, CAPES and FAPERJ
Resumo:16-008
EFFECT OF A FLAVONOID AND A METAL-FLAVONOID COMPLEX IN HYPERCHOLESTEROLEMIC RATS
Oliveira, D. A. F. ; Gonçalves, I. D. ; Fortes, E. R. S. ; Pereira, R. M. S. ; Okuyama, C. E.
Universidade Bandeirante de São Paulo, UNIBAN
Objectives:
Aim: The flavonoids are natural compounds which have several therapeutic activities. The rutin flavonoid has shown benefic
activity on the lipid metabolism on the hypercholesterolemic rats. It is known that flavonoids increase their biologic activity,
specially the antioxidant effect, when coordinated with transition metal ions (Cell Mol Biol. 53:62, 2007). The objective of this
project was to evaluate the effects of copper-rutin complex, in the serum levels of cholesterol and triglyceride in rats induced to
hypercholesterolemia.
Methods and Results: Male Wistar rats (Rattus norvegicus) were used at two month age and initial medium weight of 220g. The
animals were separated in seven groups (n=5; G1 – control, G2 – vehicle, G3 – sinvastatine, G4 - rutin 10mg/Kg, G5 - rutin
100mg/Kg, G6 - copper-rutin 10mg/Kg and G7 cupper-rutin 100mg/Kg). The animals were fed during two months with a diet
supplemented with cholesterol, except the control group that received standard food. The treatment of each group was performed
three times per week, by oral administration. After the treatment, the animals were submitted to euthanasia and the samples were
collected. The statistic difference between G1 and G2 groups confirmed the correct induction of the hypercholesterolemia. The
results of serum levels of total cholesterol (mean ± SEM) of the G1, G2, G3, G4, G5, G6, and G7 groups were 86.6±4.8,
174.1±23.7, 161.7±31.2, 181.0±10.3, 149.2±9.3, 149.7±21.6, 156.3±23.5mg/dL, respectively. Although there were discrepancies
between groups, these differences were not significant. Besides, hypertriglyceridemia was not observed with this diet. However,
the treatment with copper-rutin complex in G6 and G7 groups significantly reduced the triglycerides levels in 32.3 and 23.6%,
respectively, when compared with non-treated groups.
Conclusions:
Conclusion: These results demonstrated that rutin alone and cupper-rutin complex presented a small effect on preventing the
increase of serum levels of total cholesterol. The triglycerides levels can be reduced by cupper-rutin complex. Other experiments
are being performed to better understand these activities.
Keywords: flavonoids, hypercholesterolemia, metabolism, rutin, transition metal
Financial Support: Sources of research support: UNIBAN and FAPESP.
Resumo:16-009
ANTIATHEROGENIC EFFECTS OF THIENYLACYLHYDRAZONE DERIVATIVE LASSBIO-788 IN WISTAR
RATS FED HYPERCHOLESTEROLEMIC DIET
Motta, N. A. V. 1; Miranda, A. L. P. 2; Kummerle, A. E. 3; Barreiro, E. J. L. 2; Brito, F. C. F. 1
1
MFL/Laboratório de Farmacologia Experimental, UFF
2
Laboratório de Avaliação e Síntese de Substâncias Bioativas, UFRJ
3
Departamento de Química, Instituto de Ciências Exatas, UFRRJ
Objectives:
This study aims to investigate the pharmacological properties of thienylacylhydrazone LASSBio-788 derivatives analogous to
LASSBio-294 in an atherosclerosis model induced in wistar rats and contribute to the elucidation of its effects. Cardiovascular
and antiinflammatory properties have been reported for LASSBio-294 (J. Pharmacol. Exp. Ther. 299: 558, 2001), which has
shown a positive inotropic effect in cardiac and skeletal muscles (Br. J. Pharmacol. 134: 603, 2001), vasorelaxation (Br. J.
Pharmacol. 135: 293, 2002) and also in vivo antinociceptive and anti-edematogenic activities. Brito et al (Eur. J. Pharmacol.
638:5, 2010) have shown relevant antiplatelet properties to LASSBio-788 against different agonists in human and rabbit platelet
rich plasma. Our hypothesis is that LASSBio-788 could exert beneficial effects on atherosclerosis.
The use of animals was according to Ethics Committee (CEPA/UFF00116/09). Adult male Wistar rats (150-200g) were randomly
divided into 3 groups: C (control group) has received normal rat chow for 45 days. AT (atherosclerosis group) and AT+788
(compound LASSBio-788 group) have received hypercholesterolemic diet (HCD) for 45 days. To C and AT groups were
administered vehicle (0.05 ml/ Kg) and to AT+788 was administered LASSBio-788 (100 µMol/ Kg) by intraperitoneal injection
once daily for 15 days. The animals were euthanized by cervical dislocation and decapitation under anesthesia. Blood samples
were collected, the thoracic aorta, and liver were excised. Data was analyzed using one way analysis of variance (ANOVA) with
the Bonferroni´s test (p< 0.05). At the dose employed (100µMol/Kg), LASSBio-788 was able to reduce total cholesterol total
(102.0±6.0 x 435.4±28.0 mg/dL), triglicerydes (51.6±4.6 x 199.4±16.0 mg/dL) LDLc (58.9±6.0 x 335.1±12.5 mg/dL) and
VLDLc (9.3±1.2 x 45.0±5.0 mg/dL), levels when compared to atherosclerosis group. It was also able to reduce lipid peroxidation
by TBARS assay, when compared with AT group (4.0±0.5 x 9.0±0.7 nmol/mL). In the evaluation of platelet aggregation,
LASSBio-788 showed an antiplatelet activity against ADP 20µM (46.6±4.3 x 71.2±7.2%) and collagen 20µg/mL (33.4±3.2 x
56.0±6.0%) induced aggregation, when compared with AT group. In the vascular reactivity assay, AT+788 showed a decrease in
phenylephrine dependent contraction (CE50 = 3.1x10-7M), when compared with AT group (CE50 = 7.4x10-8M). In
acetylcholine dependent relaxation, was observed a statistically difference between AT+788 group (CE50 = 3.2x10-8M) and AT
group (CE50 = 1.3x10-6M). It was also able to reduce levels serum of TNF-α (10.5±1.5 x 31.0±4.0 pg/mL) and ICAM-1
(327.6±36.2 x 822.8±30.0 pg/mL) when compared with AT group.
Conclusions:
The compound LASSBio-788, presented an antiatherogenic effect in vivo, exerting antiplatelet, antiinflammatory, vasodilatory,
antioxidant and lipid lowering properties.These results contribute to elucidate the role of LASSBio-788 as an useful drug for
antiatherogenic therapy.
Keywords: Atherosclerosis, Hypercholesterolemic diet, N-acylhydrazone derivatives
Financial Support: CAPES, FAPERJ, PROPPi/UFF
Resumo:16-010
CARDIAC P-TYPE ATPASES MODULATION BY N-ACYLHYDRAZONE DERIVATIVES IN A DIET-INDUCED
ATHEROSCLEROSIS MODEL
Neves, M. S. 1; Marques, E. B. 1; Mattos, P. S. 1; Motta, N. A. V. 1; Miranda, A. L. P. 2; Kummerle, A. E. 3;
Barreiro, E. J. L. 2; Brito, F. C. F. 1; Scaramello, C. B. V. 1
1
2
Laboratório de Avaliação e Síntese de Substâncias Bioativas , UFRJ
3
Departamento de Química, Instituto de Ciências Exatas, UFRRJ
Objectives:
This study aims to investigate the in vivo activity of 2 thienylacylhydrazone derivatives analogous to LASSBio294 (LASSBio785
& LASSBio788), in cardiovascular system. Sudo et al (British J. Pharm. 134: 603, 2001) suggested that LASSBio294 positive
cardioinotropic effect is due to an increase in sarcoplasmic reticulum (SR) Ca2+ content. Previous studies showed that
LASSBio788 and LASSBio785 inhibited phenylephrine-induced contracture in aorta, being the latest more potent than the
reference compound LASSBio294 (Bioorg. & Med. Chem.13: 3431, 2005). These derivatives also presented higher potency than
the lead compound to inhibit platelet aggregation induced by different agonists (Eur. J. Pharmacol. 638: 5, 2010). Our hypothesis
is that these derivatives may mitigate atherosclerosis harm.
The use of animals was according to Ethics Committee (CEPA/UFF00116/09). Adult male Wistar rats (200g) were randomly
divided into 6 groups: G1 - normal diet for 30 days (G1´= 45 days); G2 - high-fat diet (HFD) for 30 days (G2´= 45 days); G3 HFD for 30 days and LASSBio785 treatment during 15 days; G4 - HFD for 45 days and LASSBio788 treatment during 15 days.
The animals were anesthetized and the hearts were removed to prepare ultracentrifuged homogenates. P-type ATPases activity
was determined according to Fiske & Subbarow method. Fourty five days of HFD increased SERCA ATPase activity
(G1´=1989±437nmolPi.mg-1.h, n=2; G2´=4238±412 nmolPi.mg-1.h, n=2). LASSBio788 prevented this HFD effect
(G4=1881±318nmolPi.mg-1.h, n=2). Although 30 days of HFD had no effect on SR Ca2+ ATPase activity
(G1=255±123nmolPi.mg-1.h – 24.0±5.5 %, n=4; G2=798±289,9nmolPi.mg-1.h – 33.7±9.8%, n=3), LASSBio785 was able to
increase SERCA ATPase specific activity and its contribution to total Ca2+ ATPase activity (G3=3375±170nmolPi.mg-1.h –
52.0±8.9 %, n=3). Thirty days of HFD decreased Na+/K+ ATPase activity (G1=3375±170nmolPi.mg-1.h, n=2; G2=1364±143
nmolPi.mg-1.h, n=2) and LASSBio785 did not prevent this effect (G3=1941±289nmolPi.mg-1.h, n=2). Na+/K+ ATPase activity
remained unchanged in rats fed with HDF during 45 days with or without LASSBio788 (G1´=1286±78nmolPi.mg-1.h, n=2;
G2´=1193±187 nmolPi.mg-1.h, n=2 and G3=1632±437 nmolPi.mg-1.h, n=2).
Conclusions:
Both cardiac Na+/K+ATPase impairment and SR Ca2+ATPase activity increase activity are associated to Ca2+ overload and
tachyarrhytmias (J. Pharmacol. Exp. Ther. 330:696, 2009; Pharmacol. Ther. 119: 340, 2008). A recent concept for
cardioprotection consists of ischemic Na+ accumulation and Ca2+ overload inhibition (J. Pharmacol. Exp. Ther. 330: 696, 2009;
Eur. J. Pharm. 603: 86, 2009). LASSBio788 showed a cardioprotective effect by preventing SERCA ATPase activity raise due to
45 days HFD. LASSBio785 was not able to prevent Na+ accumulation in this model, but its effect on SR Ca2+ATPase activity
suggests that this LASSBio294 derivative may be useful for patients suffering from conditions in which muscle fatigue is a
debilitating symptom (e.g., chronic heart failure).
Keywords: DIET-INDUCED ATHEROSCLEROSIS MODEL, N-ACYLHYDRAZONE DERIVATIVES, P-TYPE ATPases
Financial Support: CAPES, CNPq, FAPERJ, PROPPi/UFF, AGIR/UFF
Resumo:16-011
MOTIVATIONAL DEFICIT AND DEPRESSION AFTER MYOCARDIAL INFARCTION IN RATS: A TIMECOURSE STUDY
Sonoda Côrtes,; Silveira, ; Silva -almeida, ; Olivares,
Dep. de Ciências Fisiológicas/ Inst. de Biologia/UFRRJ, UFRRJ
Objectives:
Stress, anxiety and depression are the most common mood disorders, with high prevalence and incidence. Although their
association with cardiovascular diseases are very common, only few reports describe behavioral changes in heart failure models
and alternative therapeutic strategies for heart failure patients are elusive. The aim of this study was to evaluate the time course
behavioral changes induced by myocardial infarction (MI) in rats.
Methods: Thirty Male Wistar rats (200-250g) underwent MI induced by left coronary artery ligation (INF group, n = 13), or a
sham- surgery (SHAM group, n = 17). Twenty four hours later, ischemia was confirmed by electrocardiogram (Q pathological
wave and/or ST-segment elevation) in INF group. Three days and 2, 5 and 9 weeks after MI, the animals were evaluated in the
Open Field test, to analyze exploratory activity. Elevated Plus Maze and Tail Suspension Tests were also performed, but only at 9
wk post-surgeries, to study anxiety and the time of immobility (for diagnosis of depression) respectively. To further characterize
depression-like symptoms, it was verified the onset of anhedonia, through the Preference for Sucrose Test, and evolution of body
weights gain (%), both weekly. Results: Open Field: Three days after infarction, there was significant difference in the parameter
rearing/surveys (Sham: 43.59 ± 1.79 vs. INF: 26.77 ± 4.39, p≤0.001). Two weeks after MI, there were differences in the rearings
(Sham: 39.29 ± .39 vs. INF: 16.25 ± 191, p≤0.001) and immobility time (Sham: 98.59 ± 7.3 vs. INF: 168.8 ± 18.1, p≤0.001). In
five weeks, there were differences in rearings (Sham: 30.71 ± 3.56 vs. INF: 14.00 ± 1.57,p≤0.01), immobility time (Sham: 161.2
± 9.41 vs. NF: 213.8 ± 18.29, p≤001) and crossed quadrants (Sham 95.35 ± 3.44 vs. INF: 75.91 ± 7.78, p≤0.05, which was
reproduced at 9 week: rearings (Sham: 26.19 ± 2.82 vs. INF 13.80 ± 2.63, p≤0.01),immobility time (Sham: 185.5 ± 6.135 vs.
INF: 38.4 ± 12.32, p≤0.001) nd crossed quadrants (Sham: 81.75 ± 3.94 vs. INF: 63.0 ± 6.90, p≤0.05). Anhdonia was found three
days and 3, 4, 5, 6, 7, 9 weeks post infarction (Sham: 89.9 ± 1.99; 98.75 ± 0.13;96.06 ± 2.57; 6.31 ± 0.53; 9.46 ± 1.04; 9758 ±
1.41; 98.2 ± 1.90 vs. INF: 80.37 ± 1.5***; 87.21 ± 1.62∗∗∗; 7.72 ± 2.80∗∗; 8668 ± 2.06∗∗∗; 86.6 ± 1.44∗∗; 89.1 ± 0.10∗∗; 90.63
1.67∗, respectiely; ∗p≤0,05; ∗∗≤0,01; ∗∗∗p≤0,001). Finally, INF group had greater b.w. gain from week 2 until 9 after infarction
(week 2,3: p≤0,01; 4,5,6,7,8,9 p≤0.001) . There was no significant difference in Plus Maze Test. This study was submitted to the
ethics committee of UFRRJ (CEPBE/IB/UFRRJ-014/2008), and followed the standards proposed by the Guide for the Care and
Use of Laboratory Animals published by the National Institute of Health the United States (NIH Publication No. 85-23, revised
1996).
Conclusions:
Conclusion: Our data suggest that while signs of motivational deficits are showed early, clear depressive-like symptoms appear
only in chronic stage of myocardial infarction in rats.
Keywords: anhedonia, behavior, depression , infarcion, myocardial
Resumo:16-012
PARAOXONASE 1 ACTIVITY IN PATIENTS WITH DIFFERENT LOW-DENSITY LIPOPROTEIN LEVELS AND
ITS RELATIONSHIP WITH OXIDATIVE AND INFLAMMATORY PROCESSES.
Somacal, S. 1; Augusti, P. R. 2; Conterato, G. M. M. 1; Quatrin, A. 1; Ruviaro, A. R. 1; Emanuelli, T. 1
1
Department of Food Technology and Science, UFSM
2
Campus Itaqui, Unipampa
Objectives:
Low-density lipoproteins (LDL) are the major cholesterol carrier in the circulation and undergo oxidative modification by
vascular cells. The cellular uptake of oxidized LDL (LDLox) leads to the generation of reactive oxygen species (ROS).
Additionally, LDLox can initiate and enhance the inflammatory process, which plays a pivotal role in the development of
atherosclerotic changes. It has been suggested that high density lipoprotein (HDL) protects LDL particles from the oxidative
process and subsequent inflammation. This protective action of HDL is due to its associated enzyme paraoxonase 1 (PON1).
Considering the growing evidences about reduced PON1 activity as a predictive factor for cardiovascular diseases in humans and
the link between hypercholesterolemia (HC) and oxidative stress in atherogenesis, this study aimed at assessing PON1 activity
and its relationship with lipids, oxidative stress and inflammatory processes in subjects with different LDL levels.
Population studied consisted of patients from LABIMED (Santa Maria, RS, Brazil). The protocol was approved by the Research
Ethics Committee of the Federal University of Santa Maria (Protocol number: 23081.019182/2007-10). Subjects were divided
into three groups according to serum LDL levels, as follows: LDL levels 160 mg/dL (4.15 mmol/L; group III, n=41). Serum
LDL, LDLox, LDLox autoantibodies (LDLoxAB), highly sensitive C-reactive protein (hs-CRP) levels and PON1 activity were
measured. LDLox levels increased in groups along with the increase in LDL levels (group I=0.28±0.04; group=II 0.42±0.05 and
group III=0.71±0.07 mg/L, p0.05) in patients from the present study and the reasons need to be clarified.
Conclusions:
Oxidative events initiate when LDL levels are clinically acceptable and the actual role of serum PON1 as a predictor of
cardiovascular events remains controversial.
Keywords: Oxidized ow-density lipoprotein , Highly sensitive C-reactive protein , Hypercholesterolemia, Atherosclerosis
Financial Support: CNPq and FAPERGS.
Resumo:16-013
A NOVEL N-ACYLHYDRAZONE DERIVATIVE, LASSBIO-897, REDUCED ENDOTHELIAL DYSFUNCTION AND
CARDIAC HYPERTROPHY ON MONOCROTALINE-INDUCED PULMONARY HYPERTENSION
Pontes, L. B. 1; Silva, J. S. 1,2; Pereira, S. L. 1,2; Kummerle, A. E. 3; Sudo, R. T. 1; Fraga, C. A. M. 3;
Barreiro, E. J. 3; Zapata-sudo, G. 1
1
Programa de Pesquisa em Desenvolvimento de Fármacos, ICB, UFRJ
2
Programa de Pós-graduação em Farmacologia e Quim. Medicinal, UFRJ
3
Faculdade de Farmácia, UFRJ
Objectives:
Pulmonary arterial hypertension (PAH) is a severe and progressive disease, characterized by an increase in pulmonary artery
pressure leading to right ventricular (RV) hypertrophy, usually culminating in right heart failure, significant morbidity and early
mortality. Despite considerable studies, PAH has limited treatment and poor prognosis. Current treatments can improve
symptoms and reduce hemodynamic disorder but gradually can progress to lung transplantation. The present work investigated
the effects of LASSBio-897 a novel N-acylhydrazone derivative that exhibits potent vasodilatory activity mediated by the
NO/cGMP pathway via activation of endothelial M3 receptors in PAH model.
Protocols were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. PAH was induced by
monocrotaline (MCT, 60 mg/kg) injection i.p. in male Wistar rats. Animals were randomly divided in six groups: no MCT +
saline (control, n = 8); MCT + saline (MCT, n = 6); MCT + vehicle (n = 8, MCT + DMSO); MCT + LASSBio-897 2 mg/kg i.p.
(n = 6) during 28 days on the preventive protocol; MCT + LASSBio-897 (10 mg/kg p.o.) during 14 days. On the therapeutic
protocol, two groups were treated with LASSBio-897, one dissolved in DMSO and another suspended in an aqueous vehicle (n =
8 for each group). After 28 days of MCT injection, right ventricular systolic pressure (RVSP), heart and ventricle weights were
determined to evaluate cardiac hypertrophy. Right ventricular hypertrophy was measured using the ratio of RV weight to left
ventricle (LV) plus interventricular septum weight [RV/(LV + S)]. Aorta and pulmonary artery were removed from the
experimental groups and prepared for isometric tension recording. Vascular reactivity was observed during exposure to
increasing concentrations (1nM-10 μM) of phenylephrine and acetylcholine. Increased contractile response to phenylephrine and
reduced acetylcholine-induced vasodilation were observed in vessels isolated from MCT and MCT + DMSO groups compared to
control group. Vascular relaxation induced by acetylcholine (10 μM) was 92.3 ± 5.7%, 48.3 ± 7.0% and 54.8 ± 5.6% (P < 0.05) in
aorta from control, MCT and MCT + DMSO groups, respectively. In pulmonary arteries, the maximal acetylcholine-induced
vasodilation was reduced from 55.3 ± 4.3% in control group to 35.4 ± 3.3% and 35.8 ± 4.7% in MCT + salina and MCT + DMSO
groups, respectively (P < 0.05). LASSBio-897 treatment increased maximal acetylcholine-induced vasodilation to 80.5 ± 6.7% (2
mg/kg i.p.; P < 0.05) and 60.4 ± 10.8% (10 mg/kg p.o. dissolved in DMSO; P < 0.05). LASSBio-897 also prevented right
ventricular hypertrophy decreasing the ratio [RV/(LV + S)] from 0,59 ± 0,06 for MCT + DMSO group to 0.38 ± 0.03 for MCT +
LASSBio-897 group (2 mg/kg i.p.; P < 0.05), 0.29 ± 0.03 for MCT + LASSBio-897 10 mg/kg DMSO group and 0.33 ± 0.02 for
suspension group (P < 0.05). Administration of LASSBio-897 (10 mg/kg) in both vehicles significantly reduced VD/body weight
ratio from 1.55 ± 0.16 (MCT + DMSO) to 0.74 ± 0.09 and 0.94 ± 0.10 for DMSO and suspension group respectively (P < 0.05).
Conclusions:
LASSBio-897 prevented right ventricular hypertrophy and recovered pulmonary artery vasodilatory response to acetylcholine in
HAP model.
Keywords: cardiac hypertrophy, endothelial dysfunction, monocrotaline, pulmonary hypertension, n-acylhydrazone
Financial Support: PRONEX, FAPERJ, CAPES, CNPq, INCT-INOFAR, PENSA RIO
Resumo:16-014
IN VITRO STUDY OF TOLERANCE INDUCED BY SODIUM NITROPRUSSIDE IN MICE AORTA.
Diniz, M. C. 1; Alves, S. M. C. 1; Santos, R. A. S. 2; Bonaventura, D. 1
1
FARMACOLOGIA/INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UFMG
2
FISIOLOGIA E BIOFÍSICA/INSTITUTO DE CIÊNCIAS BIOLÓGICAS, UFMG
Objectives:
Organic nitrates, such as nitroglycerin, are commonly used in clinical cardiovascular medicine. Although nitrovasodilators are
considered as being safe and free of serious side effects, their clinical use is limited by reduction of efficacy upon long-term
treatment, resulting in a complete loss of hemodynamic effects by continuous application. This phenomenon is called nitrate
tolerance. This study aimed to verify if an inorganic nitrate, sodium nitroprusside (SNP), induces tolerance similar to
nitroglycerin.
This project was previously approved by CETEA/UFMG (037/2010). Vascular reactivity was performed in thoracic aortas
isolated from Balb/c mice. Functional studies were performed in aortic rings pre-contracted with phenylephrine (Phe 100 nM).
We have analyzed the maximal relaxation (Emax) and potency (pD2) of SNP. The in vitro tolerance was induced, in intact and
denuded mice aortic rings, by SNP (EC50: 10μM) incubation for 15, 30, 45 and 60 minutes. After this period of incubation, aortic
rings were washed for 10 minutes and then pre-contracted with Phe for SNP relaxation studies. Vascular reactivity experiments
showed that previous exposition of denuded mice aorta to SNP for 60 minutes reduced the Emax and pD2 values to SNP,
suggesting the phenomenon of tolerance (pD2→control: 8.32±0.14, n=10; pD2→tolerant(60′): 7.47±0.23, n = 06). This effect
was not observed in denuded rat aorta exposed to SNP for 15, 30 and 45 minutes, as well as in intact mice aorta in all times of
incubation studied.
Conclusions:
Taken together, our results demonstrated that in vitro development of EC50 SNP tolerance is time dependent , since tolerance
was observed only after 60 minutes of exposure and independent of endothelium.
Keywords: AORTA, NITRATE TOLERANCE, SODIUM NITROPRUSSIDE, VASCULAR REACTIVITY
Financial Support: CAPES and FAPEMIG
Resumo:16-015
RISK FACTORS FOR ATHEROSCLEROTIC DISEASE IN ELDERLY PATIENTS ASSISTED AT OUTPATIENT
GERIATRICS CLINICS IN LAVRAS – BRAZIL
Araujo, C. V. D. 1,2; Nascimento Av2; Cunha, T. D. S. D. 1,3
Department of Medicine, Translational Medicine, UNIFESP
2
Family Healthcare Program - Unit Chacrinha, FHP Chacrinha
3
Science and Technology Institute, UNIFESP
1
Objectives:
From the Framingham Study (1948), it is known that several risk factors including advanced age, hypertension, dyslipidemia and
obesity (especially in women), beyond glucose intolerance, smoking, and actually, the hyperhomocysteinemia, precipitate the
onset of atherosclerotic disease and its terrible consequences. The aim of this study was to determine the prevalence of risk
factors for atherosclerotic disease in elderly patients assisted at outpatient geriatrics clinics in Chacrinha Unit, in Lavras (MG).
Methods: Were examined in cross section, elderly patients assisted at outpatient geriatrics clinics in Chacrinha Unit in the period
from July 2009 to June 2010. Results: Were evaluated 152 patients (93 female and 59 male), aged 60-89 years (24%: 60-64 years,
28%: 65-69 years, 22%: 70-74 years, 13%: 75-79 years , 11%: 80-85 years and 2%: 86 years or more). The most prevalent risk
factor for atherosclerotic disease was hypertension (83.6%), followed by dyslipidemia (39.5%), diabetes mellitus (23.1%) and
smoking (9.2%). LDL-c ≥ 160 mg/dL was found in 58 (38.2%) and BMI ≥ 27 kg/m2 was found in 82 (54%) patients.
Conclusions:
This study describes the frequency of traditional risk factors for atherosclerotic disease in elderly assisted outpatients. Some of
the most common factors are modifiable, such as obesity, or controllable, such as hypertension and dyslipidemia. Precise
information about the importance of atherosclerosis as a factor for morbidity and mortality and the presence of risk factors should
be ongoing concerns over the monitoring of elderly people at any level of care, or to reduce their cardiovascular risk.
Keywords: Atherosclerosis, Geriatrics clinics, Health of the elderly, Risk factors
Resumo:16-016
INVESTIGATION OF PHARMACOLOGICAL MECHANISMS INVOLVED IN CARDIAC REMODELLING IN
EXPERIMENTAL HYPERTHYROIDISM IN RATS
Freitas, F. S. S. ; Bonomo, I. T. ; Estato, V. ; Lessa, M. A. ; Tibiriçá, E.
Laboratório de Investigação Cardiovascular/IOC, FIOCRUZ
Objectives:
Thyroid hormone (TH) has profound effects on cardiac muscle, the conducting system, the peripheral circulation, and the
sympathetic nervous system (SNS) that predictably alter cardiovascular hemodynamics in patients with hyperthyroidism.
Therefore, the aim of this study was to investigate the main cardiovascular changes induced in an experimental model of
hyperthyroidism, as well as the role of the SNS and renin-angiotensin system (RAS) in the pathophysiology of cardiovascular
changes in experimental hyperthyroidism.
Male Wistar rats weighing (170-250 g) were divided in two groups: euthyroid (CONT n = 8), treated with 0.9% saline solution
and hyperthyroid (HYPER n = 32), treated with thyroxine (600 μg/kg/day) during 14 days intraperitoneally (ip). On the 15th day,
the group HYPER was divided in four groups (n = 8): HYPER; HYPER + losartan (LOS); HYPER + diltiazem (DILT); HYPER
+ Propranolol (PROP), treated for 21 days with thyroxin (600 μg / kg / day ip), and respective drugs (10 mg / kg / day) orally.
Echocardiography study was performed to evaluate the alterations in cardiac structure and geometry using the following
parameters determined in M-mode: thickness of left ventricle posterior wall in diastole (PWd), internal end-diastolic diameter of
LV (EDD) and heart rate (HR). The evaluations were performed on days (0, 7, 14 and 35). We performed univariate analysis of
variance (ANOVA) followed by multiple comparison test of Bonferroni when appropriate. In all tests, the minimum degree of
significance is 95% (P
Conclusions:
This experimental model of hyperthyroidism caused a pathological form of left ventricle (LV) hypertrophy, as evidenced by the
decrease in LV internal diameter, which may be associated with a diastolic dysfunction. Specific inhibitors of the RAS, SNS and
a calcium channel blocker, in this study were able to reverse cardiac hypertrophy and remodeling induced in experimental
hyperthyroidism in rats. Taken together these results suggest that the pharmacological blockade of these systems may contribute
significantly to the treatment of patients with thyrotoxic heart disease.
Keywords: Cardiac hypertrophy, Hyperthyroidism, Renin-Angiotensin System, Sympathetic Nervous System
Financial Support: FAPERJ; INSTITUTO OSWALDO CRUZ
Resumo:16-017
MAINTAINENCE OF RENAL VASCULAR RESPONSIVINESS TO VASOCONSTRICTORS IN ENDOTOXEMIC
RATS: IS THERE A ROLE TO THE RHOA/RHO-KINASE PATHWAY?
Guarido, K. L. ; da Silva Santos, J. E.
Department of Pharmacology/ Federal University of Santa Cata, UFSC
Objectives:
It was shown that in small mesenteric arteries from endotoxemic rats, the vascular hyporeactivity is related to impaired
RhoA/Rho-kinase pathway (Crit Care Med, 37, No. 5, 2009). Nevertheless, endotoxemic mice appear to keep the renal vascular
responses to these agents (J Am Soc Nephrol 16: 117-124, 2005). This study aimed to evaluate the responses to vasoconstrictors
and the functionality of Rho-kinase (ROCK) in the renal vascular bed of rats treated with lipopolysaccharide (LPS).
All procedures were approved by the Institutional Ethics Committee from UFSC (protocol 463). Male Wistar rats (230-280g)
received by intraperitoneal route (i.p.) either saline (1 ml/kg) or LPS (10 mg/kg), 6 or 24 hours before the experiments. Animals
were anesthetized with ketamine/xylazine (100/20 mg/kg, i.m.), and heparinized polyethylene catheters were inserted into the
femoral vein and carotid artery for drug administration and measurement of mean arterial pressure (MAP). After a stabilization
period the animals received intravenous doses of phenylephrine (PE), acetylcholine (ACH; both at 3, 10 and 30 nmol/kg), and
vasopressin (VASO; 3, 10 and 30 pmol/kg). A period of 10 min was followed between each treatment. In the second set of
experiments, after anesthesia, the animals had the abdominal aorta cannulated for perfusion of the left kidney. It was removed and
attached to a perfusion system under constant flow (4 ml/min) with physiologic salt solution (PSS) kept at 37 °C, constantly
bubbled (5% CO2/95% O2). Cumulative concentration-response curves (CCRC) to either PE (0.1 to 300 nmol) or VASO (0.1 to
300 pmol) were performed. In addition, the kidneys were perfused with PSS containing PE (3 µM), and the relaxation induced by
Y-27632 (3 to 300 nmol) was measured. Endotoxemia induced by LPS reduced the responsiveness to PE, a hallmark of septic
shock. For instance, the highest dose of PE increased MAP by 79.4 ± 2.4 mm Hg in control rats, and by 37.13 ± 4.94 and 60.4 ±
6.7 mmHg in LPS groups, 6 and 24 h, respectively (p < 0.05). On the other hand, the hypertensive effects of VASO (30 ñmol/kg)
were increased from 51.5 ± 6.5 to 69.7 ± 4.2 mm Hg in control and LPS (24 h) groups. The hypotensive effects of ACh had not
been changed. Differently than the results found in vivo, there were no hyporesponsiveness to PE in the perfused kidneys from
LPS-treated rats. In addition, the EC50 obtained in CCRC to VASO was reduced from 21.4 (11.5 to 39.7 ñmol) to 6.7 (3.7-122
ñmol), in control and LPS 24 h groups, respectively. In these same groups, the maximal responses to VASO (10 ñmol) increased
from 78.6 ± 22.8 to 138.9 ± 13.7 mm Hg (p < 0.05). Moreover, the responses to Y-27632, an inhibitor of ROCK, were not
different between control and LPS groups.
Conclusions:
Despite reduced hypertensive effects of PE were found in vivo, the renal vascular bed presents unaltered (to PE) or increased (to
VASO) responses to vasoconstrictors. At least in our experiments, the vasodilatory effects obtained with the inhibition of ROCK
were not changed in kidneys from endotoxemic rats, suggesting that the activity of this pathway is not impaired in the renal
vascular bed in sepsis.
Keywords: kidney, ROCK, septic shock, vasopressin
Financial Support: FAPESC; CNPq
Resumo:16-018
ASSOCIATION BETWEEN HYPERTENSION AND BIOMARKERS OF OXIDATIVE METABOLISM IN AMAZON
RIVERINE ELDERLY.
Federle, C. B. 1; Algarve, T. D. 2; Montagner, G. F. F. S. 2; Manica-cattani, M. F. 2; Ribeiro, E. E. 3;
Bresciani, G. 5; Oliveira, A. R. D. 1; Figueira, G. C. 1; Rocha, M. I. U. M. 1,5; Cruz, I. B. M. 1,2,5
1
Universidade Federal de Santa Maria, UFSM
2
Progr. de Pos-Grad. em Ciencias Biolog.: Bioq. Toxicologica, UFSM
3
Univ. Aberta da Terceira Idade/Univ. do Estado do Amazonas, UnATI/UEA
5
Laboratorio de Biogenomica, Departamento de Morfologia, UFSM
Objectives:
Oxidative stress has been hypothesized to play a role in aging and age-related disorders, such as hypertension. Many studies have
showed non univocal data about oxidative stress status and nitric oxide metabolites in essential hypertension. However,
complementary studies need to be performed to evaluate if the oxidative stress biomarkers present alterations in different elderly
populations. In this context, we analyzed if biomarkers related to oxidative metabolism (lipoperoxidation analyzed by
TBARS,advanced oxidized protein products (AOPPs), nitric oxide and protein carbonylation) could to present differences
between hypertensive and normotensive elderly that lives in Amazon riverine region (Maués, AM, Brazil).
We included 637 Amazon riverine elderlies, 297 were hypertensive and 340 normotense. Lifestyle indicators , health,
anthropometric were evaluated. Blood was collected after fasting for biochemical profile and oxidative metabolism analysis as
well as to measure lipid and glucose levels. The results were compared between two groups using Student t Test. This study was
approved by the Universidade do Estado do Amazonas Ethics Research Committee. The protocols were implemented according
to the CONEP normative Resolution 196/1996. The results showed hypertension prevalence was higher in women (50.3%) than
men (42.4%). The hypertensive elderlies presented higher cardiovascular disease prevalence as expected. Hypertensive showed
higher levels of lipid peroxidation and protein carbonilation then normotensive elderly. These results were independent of sex,
age, and smoking habit. The other variables were similar between groups.
Conclusions:
From the results obtained and published in literature we suggest that hypertension increase the levels of some oxidative
biomarkers as lipoperoxidation and protein carbonilation. However, additional studies need to be performed to observe if these
alterations are related to risk future cardiovascular events on elderly.
Keywords: Hypertension, Oxidative Stress, Aging, Riverine Eldery, Amazon Rainforest
Financial Support: FAPEAM and CAPES.
Resumo:16-019
OLMESARTAN IMPROVES BRAIN MICROCIRCULATION IN DIABETIC HYPERTENSIVE RATS.
Obadia, ; Estato, ; Tibiriçá,
Laboratório de Investigação Cardiovascular, LIC/FIOCRUZ
Objectives:
Arterial hypertension and diabetes mellitus (DM) when associated induce brain microvascular changes more pronounced than in
the two diseases separately. Structural alterations of cerebral vascular network, resulting from systemic arterial hypertension, lead
to increased risk of cerebral hypoperfusion, decreased oxygen supply to the brain tissue and consequent increased risk of
isquemic injury. Thus, the selection of an anti-hypertensive treatment should consider the microcirculatory effects of individual
drugs, mainly when diabetes is associated to hypertension. The present study investigated diabetes-induced functional changes in
the brain microcirculation of spontaneously hypertensive rats (SHR) as well the effects of oral long-term treatment with an
angiotensin AT1 receptor antagonist, olmesartan, on brain capillary density of diabetic and nondiabetics SHR.
Methods: We used a model of type 2 diabetes in SHR (Pharmacological Res. 52: 313. 2005), induced by the combination of highfat diet with the injection of low doses of streptozotocin (35 mg/kg, ip). Twelve weeks-old male (8 per group) diabetic or nondiabetic hypertensive rats (SHRDM or SHR) were treated orally during 28 days with olmesartan (5 mg/kg/day) (SHRDM-OLM
and SHR-OLM, respectively) and compared with normotensive non-diabetics rats (WKY). The cerebral microcirculation (pial
membrane) of anesthetized (pentobarbital 75 mg/kg, ip), catetherized and artificially ventilated rats was assessed through a
cranial window, using intravital fluorescence videomicroscopy. The functional capillary density (FCD), considered as the number
of spontaneously perfused capillaries (vessels with diameters less than 10 µm) per mm², was determined in random microscopy
fields during 4 minutes. Results: Chronic treatment with olmesartan lowered systolic arterial pressure in both SHRDM-OLM
(206 ± 3 to 142 ± 11 mmHg, P
Conclusions:
Our data demonstrated that chronic treatment with olmesartan reverts brain microvascular alterations in a model of diabetes
associated to hypertension in rats.
Keywords: Brain microcirculation, hypertension, diabetes, olmesartan
Resumo:16-020
THE ROLES OF IL-6 AND TNF-ALPHA INCREASED LEVELS IN HEART DURING DENGUE VIRUS INFECTION
IN MICE.
Kangussu, L. M. G. O. ; Costa, V. V. ; Arifa, R. D. N. ; Soriani, F. M. ; Rachid, M. A. ; Souza, D. G. ;
Bonaventura, D.
Farmacologia/ICB/UFMG, UFMG
Objectives:
Dengue is one of the most important vector-borne viral diseases in the world and constitutes a serious world health problem.
Dengue virus (DENV) infection is caused by 1 of 4 antigenically distinct virus of the Flaviviridae family. This infection is
characterized by hematological alterations, increased levels of cytokines, increased vascular permeability, hemorrhage and shock.
Although clinical symptoms of this infection are well characterized, the mechanisms involved in Dengue pathogenesis are not
clearly understood. Based on the fact that infection with dengue virus is associated with increased levels of systemic or local
cytokines and enhanced vascular permeability, the aim of this study was to investigate if there is cardiac abnormalities in this
animal model of Dengue fever, as well as, the mechanisms involved in these disorders.
This project was previously approved by CETEA/UFMG (038/2010). Balb/c mice were i.p infected with 1 PFU (LD50) of the
adapted DENV-3. On day 3 and 6 after infection, survival, hematological signals: hematocrit (%) and platelets counts, hepatic
transaminases (AST/ALT) levels in serum (Bioclin/Quibasa Kits), inflammatory parameters -cytokines and chemokines (by
ELISA - R&D systems), MPO and NAG (as a marker of neutrophil and macrophage accumulation, respectively) were evaluated.
Increased vascular permeability was determined by Evans blue dye. Tissue damage was verified by histological analyses of liver,
lung and heart (H&E staining). Systolic blood pressure (SBP), and heart rate (HR) were determined by tail-cuff plethysmography,
in unanesthetized mice. Results: animals infected with DENV-3 died approximately 7 days after infection. At day 3 and 6, there
was evidence of clinical disease and tissue damage confirmed by thrombocytopenia (NI: 814.5 x 103 ± 21.19, 3 days after
infection 614.5 x 103 ± 18.3 and 6 days after infection 381.2 x 103 ± 17.7, n=12), hemoconcentration (NI: 41.9 ± 0.78, 3 days
after infection 44.3 ± 0.78 and 6 days after infection 55.7 ± 0.77, n=12) increased levels of transaminases, neutrophil and
macrophage accumulation in tissues and elevated levels of cytokines and chemokines (TNF-á, NI: 27.6 ± 2.76, 3 days after
infection 46.3 ± 5.37 and 6 days after infection 386.7 ± 88.3, n=5, IFN-ã and IL-6, NI and 3 days after infection non detected and
6 days after infection 3472.2 ± 390.0 and 70.1 ± 13.3, respectively n=6, IL-1â, NI: 63.7 ± 15.5, 3 days after infection 467.4 ± 28,9
and 6 days after infection 80.3 ± 10.5, n=5 and CXCL2, NI: 30.1 ± 3.01, 3 days after infection 49.6 ± 4.37 and 6 days after
infection 359.0 ± 28.3, n=5) in serum and (TNF-á, IL-6, IL-17) in heart. An increased expression levels of IL-6 in heart was
observed (NI: 1.00 ± 0.04, 3 days after infection 1.10 ± 0.37 and 6 days after infection 6.89 ± 1,22, n=5). These findings were
accompanied by changes in hemodynamic parameters, such as drastic reduction in systolic blood pressure (NI: 112.4 ± 1.12, 3
days after infection 108.4 ± 1.32 and 6 days after infection 71,4 ± 1.24, n=8) associated with increased vascular permeability in
lung, liver, heart, stomach and kidney.
Conclusions:
The present findings indicate that, in animals infected with Dengue virus, there is an enhancement in systemic and cardiac IL-6
and TNF-á levels, leading to myocarditis and cardiac dysfunction which can be related with changes in hemodynamic parameters.
Keywords: Dengue, IL-6, TNF-alpha, Myocarditis., Cardiac dysfunction
Financial Support: CAPES and FAPEMIG.
Resumo:17-001
EVALUATION OF ANALGESIC AND ANTIPYRETIC EFFECTS OF COMPOUND 1.5-DIPHENYL-PYRAZOLE-3HIDRAZINOPIRAZOL (DHP)
Malvar, D. C. 1; Ferreira, R. T. 2; Silva, P. H. S. 2; Castro, R. A. 2; Souza, G. E. P. 1; Freitas, A. C. C. 3;
Mafra, J. C. M. 4; Vanderlinde, F. A. 2
1
Depto Física e Química Faculdade de Ciências Farmacêuticas , USP - Ribeirão Preto
2
Área de Farmacologia, Depto Ciências Fisiológicas, IB, UFRuralRJ
3
Depto Tecnologia Farmacêutica, Faculdade de Farmácia, UFF
4
Núcleo de Pesquisas de Produtos Naturais, UFRJ
Objectives:
Oral administration (p.o.) of DHP (1-30mg/kg) produced antinociceptive effects in abdominal constriction and formalin (2nd
phase) tests, antioedematogenic activity in croton oil-induced ear oedema and reduction of leukocyte migration when submitted
to the carrageenan-induced peritonitis test (Braz. J. Pharm. Sci. 41: 1; 388, 2005). This study investigates the analgesic potential
of DHP on mechanical hypernociception (von Frey) and thermal hyperalgesia (Hargreaves) in model of plantar incision pain and
antipyretic activity (LPS-induced fever) in male Wistar rats (200-300g) (ethical commission protocol nº
011/2007/CEPEB/UFRRJ).
Measurements of baselines of paw withdrawal thresholds to mechanical (g) and thermal (s) stimuli were performed before and on
the three subsequent days after plantar incision surgery to observe the development of hypernociception and hyperalgesia (1st
step). In the 3rd day post-surgery DHP (10mg/kg, p.o.) group (InDHP) and vehicle group (InV) were administered and the
behavioural tests repeated exactly at 30 min, 1, 3, 6, 9, 12 and 24h (2nd step), 7, 10 and 14 days (3rd step) after the treatments.
Similar procedures were used with a 3rd group not incised and treated with vehicle (NInV). In the evaluation of mechanical
hypernociception InDHP (n=9) showed a significant inhibition of hypernociception (IHn) after 1h of administration, with
maximum IHn 12h (28.2%) in the 2nd step of the experiment, keeping in the 3rd step with IHn of 26.9, 43.4 and 60.4% in the
7th, 10th and 14th days respectively of evaluations, when compared with InV group (n=9). In thermal hyperalgesia InDHP also
produced significantly inhibition of hyperalgesia (IHa) after 1h of treatment, with IHa maximum of 6h (67.1%) in the 2nd step,
obtaining in the 3rd step IHa of 43.4, 28.6 and 64.0% in 7th, 10th and 14th days of evaluations respectively, compared with InV
and obtaining similar values of NInV on 14th day. In von Frey and Hargreaves NInV (n=8) showed similar records of paw
withdrawal thresholds in the three steps of the experiment. To investigate the antipyretic activity rats received vehicle (3%
DMSO, 1.5% Tween 80 in water) or DHP (30 or 60mg/kg, p.o.) 30 min before the intravenous injection (0.2mL) of LPS (5ìg/kg)
or saline (control group). The rectal temperature (rT, degrees Celsius) was measured by telethermometry every 30 min for up 6h.
LPS elicited a marked rT elevation that started at 2h and persisted up to 6h. Pre-treatment with 30mg/kg of DHP (n=5) did not
produce antipyretic effect, but 60mg/kg of DHP significantly reduced 46% the fever from 2 to 6h after LPS injection (the area
under the time-course curves were 6.1 and 3.3 for vehicle/LPS (n=11) and DHP 60mg/kg/LPS (n=12), respectively).
Furthermore, 60mg/kg of DHP did not modify the basal rT of rats injected with saline (n=5) when compared to the vehicle/saline
group (n=8).
Conclusions:
These results demonstrated that DHP is an analgesic and antipyretic drug and can be represent an effective intervention to treat
post-operative pain and fever.
Keywords: 1.5-diphenyl-pyrazole-3-hidrazinopirazol, antipyretic activity, incisional pain, hypernociception von Frey ,
hyperalgesia Hargreaves
Financial Support: FAPERJ, CAPES, FAPESP
Resumo:17-002
PARTICIPATION OF TRPV1 RECEPTOR IN THE ANTINOCICEPTIVE EFFECT OF SPINASTEROL: A STEROID
ISOLATED FROM VERNONIA TWEEDIANA BAKER LEAVES.
Trevisan, G. ; Rossato, M. F. ; Klafke, J. Z. ; Rosa, F. D. ; Athayde, M. L. ; Zanon, R. B. ; Walker, C. I. B. ;
Ferreira, J.
Química, Universidade Federal de Santa Maria, UFSM
Objectives:
The specie Vernonia tweedieana Baker, popularly known as “assa-peixe”, is an herbaceous plant widely distributed in the plains
of south Brazil. The leaves of this plant are used in Brazilian folk medicine to the treatment of the respiratory diseases. Chemical
analysis carried out with dichloromethane-soluble fraction from the ethanol extract of the leaves of Vernonia tweediana led to the
isolation of αamyrin, βamyrin, lupeol, βsitosterol, stigmasterol and spinasterol. Among this compounds, some studies have been
described the antinociceptive effects of spinasterol. Thus, the goal of this study was to evaluate the antinociceptive effect of
Vernonia tweediana Baker and some of the mechanisms underlying it antinociceptive effect.
Three-month-old male albino Swiss mice (25-35 g, n=6-9) were used. Firstly, mice were submitted to an oral (p.o.)
administration of hydroalcoholic extract (HE), diclorometane (Dcm), ethyl acetate (Act), and butanolic (But, all in the dose of
100 mg/kg) fractions, isolated compounds (0.1 mg/kg) from Dcm fraction (mixture of αamyrin and βamyrin, lupeol, βsitosterol,
stigmasterol and spinasterol) or vehicle (5% Tween 80, 20% polyethyleneglycol and 75% saline) to verify the antinociceptive
effect in the intraplantar capsaicin test. The hidroalcoholic extract, fractions, compounds and vehicle were administrated 1 hour
before capsaicin test (1 nmol/paw). For HE, Dcm, spinasterol and vehicle dose and time-response curves have been made. The
HE, Dcm, Act and But produced antinociception in the capsaicin test with an Imax of 51±4, 67±5, 44±4, and 33±9%,
respectively. When capsaicin was administered at different time points after HE and Dcm (100 mg/kg, p.o.) treatment (Imax of
54±4% and 60±6%) they had a significant effect against nociception (1 up to 4 hours). In the dose-response curve for nociception
assessed 1 hour after the treatment with HE (Imax of 66±4%) and Dcm (Imax of 70±4%) showed antinociceptive effect with
ED50 values of 51.3 (34.4-76.6) mg/kg, and 52.0 (36.7-73.9) mg/kg, respectively. Among the compounds isolated from de Dcm
fraction, the spinasterol reduced the nociception induced by capsaicin (Imax of 58±4%). In the dose-response curve á-spinasterol
(Imax of 64±6%) developed an antinociceptive effect with an ED50 value of 0.017 (0.013-0.024) mg/kg. The antinociception
observed for spinasterol (0.1 mg/kg, p.o.) started at 0.5 and last until 4 hours after administration (Imax of 60±6%). Then to
verify the mechanism of action of spinasterol (0.01-10 µM) we have done the [3H]-resiniferatoxin binding assay. The spinasterol
was able to displace [3H]-RTX binding from spinal mice membranes with an IC50 of 1.6 (0.8 – 2.9) µM. After, we evaluate the
ability of spinasterol (10-300 µM) to change calcium influx induced by capsaicin (20 µM). To it calcium Influx has assessed
using synaptosomes prepared from mice spinal cord samples. Results are expressed as the percentage to the maximum response
obtained with triton-x 100 and then compared with the influx of capsaicin. The spinasterol inhibits the influx elicited by
capsaicin, with an IC50 of 39.5 (22.9 – 68.2) µM.
Conclusions:
Then these results led us to observe that spinasterol have an important role in antinociception observed for Vernonia tweediana
Dcm fraction in the capsaicin test and this molecule might act as a TRPV1 receptor antagonist.
Keywords: capsaicin, TRPV1 receptor, Calcium influx, Resiniferatoxin, Binding
Resumo:17-003
ROLE OF ARYL HIDROCARBON RECEPTOR (AHR) IN RHEUMATOID ARTHRITIS – POSSIBLE MECHANISM
IN SMOKING-INDUCED ARTHRITIS ENHANCEMMENT -
Talbot, J. 1; Pinto, L. G. 1; Peres, R. S. 1; Oliveira, R. D. R. 1; Almeida, S. C. L. D. 1; Silva, J. R. 1; Franca,
R. F. O. 1; Cunha, T. M. 1; Ryffel, B. 3; Lyew, F. 4; Ferreira, S. H. 1; Junior, P. L. 1; Alves-filho, J. C. 1;
Cunha, F. D. Q. 1
1
Depto. de Famacologia/FMRP-USP, FMRP-USP
2
Depto de Clinica Médica/FMRP-USP, FMRP-USP
3
CNRS-Orleans, CNRS-Orleans
4
Glasgow Biomedical Research Centre - University of Glasgow , GBRC- Glasgow
Objectives:
Rheumatoid arthritis (RA) is a chronic inflammatory arthropaty. It is true that interaction between genetic and environmental
factors (eg. cigarette smoke) can enhance immune response in RA, however the mechanism by which these factors can interact
and play this enhancement is not clear. It has been showed that Th17 cells are the “mastermind” cells in generation of
autoimmune response and it was related that activation of aryl hydrocarbon receptor (AhR) may modulate Th17 differentiation.
Genetic polymorphisms in AhR can change response to aryl hydrocarbon compounds (presents on cigarette smoke). The aim of
this study was to evaluate the influence of AhR genetic polymorphisms (SNPs) in human RA susceptibility and activity, and the
interaction between smoking and genetic variants in arthritis phenotype. Addly, using experimental arthritis models we accessed
the mechanism by which AhR plays its role in arthritis development.
This study was approved by HCFMRP/USP Human Ethics Committee (2981/2009) and FMRP/USP Animal Ethics Committee
(038/2009). We evaluated the distribution of 7 AhR Human SNPs in healthy individuals (129) and in RA patients (138).
Experimental arthritis were accessed by mBSA-induced arthritis in C57BL/6 mice, AhR or IL-17RA genetic-deficient mice
(AHRKO or IL-17RAKO); and by collagen-induced arthritis in DBA1/J mice. Mice were treated i.p. with vehicle (PBS), AhR
agonist FICZ (10, 30 or 90ug/kg) or daily with AhR antagonist CH223191 (0.8mg/Kg). A set of inflammatory parameters were
evaluated to access arthritis activity. Among them: a) Articular hypernociception; b) Arthritis-associated histopathology; c) Th17
frequencies profile. Results: We found an up-regulation of AhR mRNA expression in RA patients. Genetic analysis showed an
AhR haplotype (T-A) associated with RA development (OR 2.08, CI 95% 1.1-3.8, p=0.0084). We also identified this mutant
haplotype T-A associated with higher Disease Activity Score (DAS) (p=0.0232). It has been described that the allele A is
associated with higher AhR transcriptional activity than allele G. Then, our hypothesis was that increased activation of AhR
could be related to enhancement of RA. In experimental arthritis we identified that AhR activation enhances inflammation while
AhR blockage or deficiency reduces arthritis. The enhancement of arthritis induced by AhR activation was followed by increased
frequencies of Th17 and IL-17 in affected tissue, while AhR blockage or deficiency was followed by Th17 reduction. The
adoptive transference of wild-type CD4 cells (T lymphocytes) to AhRKO mice restored arthritis. AhR activation-induced arthritis
enhancement was impaired in IL-17RAKO mice. Corroborating with literature reports, we showed that activation of AhR
enhanced in vitro differentiation of Th17. Considering that smoking is a strong RA risk factor and there are potent AhR agonists
in smoke, we accessed the effect of interaction between AhR haplotype T-A and smoking on RA activity. We found that RA
smokers that have haplotype T-A had higher DAS when than carriers of wild-type haplotype C-G. Experiments in experimental
arthritis are being performed to access if smoking-induced arthritis and Th17 enhancement is really AhR-dependent.
Conclusions:
Together, these results suggest that AhR activation is involved in RA development by enhancement of Th17-dependent response
and mainly, AhR can be the link between smoking and arthritis enhancement described in literature.
Keywords: Experimental Arthritis, Genetic Polymorphisms, Smoking, Th17
Financial Support: CNPq, FAPESP, CAPES and FAEPA
Resumo:17-004
THE INFLAMMATORY EFFECT OF VATAIREA GUIANENSIS LECTIN INVOLVES CARBOHYDRATE DOMAIN
AND MACROPHAGE ACTIVATION.
Marques-domingos, G. F. O. 1; Pires, A. F. 1; Rodrigues, N. V. F. C. 1; Silva, H. C. 2; Cavada, B. S. 2;
Assreuy, A. M. S. 1
1
Instituto Superior de Ciências Biomédicas, ISBC-UECE
2
Departamento de Bioquímica e Biologia Molecular, UFC
Objectives:
Based in the pro-inflammatory effects demonstrated by lectins with binding specificity to galactose, it was evaluated the
edematogenic effect and macrophage activation of a lectin isolated from Vatairea guianensis seeds (VgL).
Wistar rats (150-250g) and Swiss mice (25-30g) were maintained and handled in accordance with the principles recommended by
our Institutional Ethical Committee (UECE No. 10130208-8/40). Rat paw edema was induced by the subcutaneous (s.c.) injection
of VgL (0.01, 0.1 and 1 mg/kg; 0.1 mL/100 g body mass) and compared to controls injected with saline in substitution of lectin.
In order to investigate the lectin domain participation VgL (1 mg/Kg) was incubated with its binding sugar galactose (0.1 M) for
60 min at 37 °C before protocols. Lectin and sugar were also incubated in separate solutions at same conditions as controls. Paw
volume was measured immediately before (zero time) lectin injection into animal hind paws and at selected time intervals
thereafter (0.5, 1, 2- 8, 24, 32, 48 and 72 h). For the macrophage participation, mice were pretreated intraperitoneal (i.p.) with
1mL 3% thioglycolate during 4 days and sacrified for peritoneal fluid collection and macrophage isolation. The macrophages
layer was incubated with VgL at 50 μg/ml for 1 hour and cells further incubated with RPMI medium for 3 hours. Peritonits was
induced by the i.p. injection of cultured supernatants macrophages and total and differential leukocyte counts (cells/μL)
performed after 4h. Briefly, 20 μL of the fluid was diluted in 380 μL Turk's reagent for total leukocyte count in a Neubauer
chamber and differential (neutrophils and mononuclears) counts performed on slides stained with hematoxylin/eosin (HE).
Results were expressed as Mean ± SEM (n = 6) and statistical analysis performed by Analysis of Variance (ANOVA) followed
by Bonferroni‟s test. Significance was considered for P values
Conclusions:
Vatairea guianensis lectin present dose-dependent edematogenic effect via lectin domain and chemothactic activity by indirect
mechanism via macrophage activation.
Keywords: Carbohydrate domain, Inflammatory effect, Lectin, Macrophage activation, Vatairea guianensis
Financial Support: FUNCAP, UECE.
Resumo:17-005
ROLE OF THE NITRIC OXIDE IN THE LUNG FUNCTION AND AIRWAY HYPERREACTIVITY IN SILICOTIC
MICE.
Dias, D. F. ; Ferreira, T. P. T. ; Ciambarella, B. T. ; Arantes, A. C. S. D. ; Martins, M. A. ; Martins, P. M. R.
Instituto Oswaldo Cruz, Fiocruz
Objectives:
Silicosis is a restrictive pulmonary disease caused by silica particle inhalation, characterized by an inflammatory response
followed by intense fibrosis and granuloma formation. Several inflammatory mediators are involved in this disease, including
nitric oxide (NO). In this study we aimed to investigate the involvement of NO in the lung function and airways hyperreactivity
in silicotic mice. Morphological changes in the lung tissue were also evaluated.
Male (18-20g) wild-type C57Bl6 (iNOS+/+) and iNOS knockout (iNOS-/-) mice were used. Anesthetized animals received a
single intranasal instillation of silica particles (10 mg/50 µL mouse) or 0.9% saline as controls. The analyzes were made 7 and 28
days after silica provocation and included: i) lung function (resistance and elastance) and airways hyperrreactivity to
methacholine by invasive whole body plethysmography (Finepointe, Buxco System), ii) quantification of nitric oxide levels in the
bronchoalveolar lavage (BAL) by Griess method and iii) morphological and morphometric analyses by classical histology
(haematoxilyn-eosin). In another set of experiments, the animals received an intransal instilaltion of the NO donor DETANonoate
(0.25 µmol/kg), every other day during 5 days. All experimental procedures were performed in accordance with the guidelines of
the Committee on Use of Laboratory Animals of the Oswaldo Cruz Foundation (L-034/09). We noted that, at both time points (7
and 28 days), silica exposure caused alteration of lung function as attested by increased basal levels of lung resistance and
elastance. Aerosolization of methacholine led to elevation in lung resistance and elastance, clearly indicating a condition of
airways hyperreactivity. These phenomena paralleled to increased levels of NO in the BAL fluid of silicotic mice as compared to
those of the controls. Instillation of the NO donnor DETANonoate led to an increase in lung resistance and elastance to
methacholine. In addition, we observed that at 7 and 28 days post-silica, the lung function compromise and airways
hyperreactivity were abolished in the iNOS -/- mice, in close correlation with suppression of the inflammatory/ fibrogenic
responses.
Conclusions:
Our findings show that silica instillation induces lung function compromise and airways hyperreactivity, in a direct association
with inflammation and granuloma formation. These responses were markedly suppressed in iNOS knockout mice, indicating that
NO seems to importantly contribute to several features of the silicosis, including airways hyperreactivity and inflammation.
Keywords: Silicosis, Lung, Hyperreactivity, Nitric oxide, iNOS
Financial Support: PAPES 5/FIOCRUZ, CNPq e FAPERJ.
Resumo:17-006
EVALUATION OF SYSTEMIC TREATMENT WITH FLUOXETINE ON NOCICEPTION AND ANXIETY IN MICE
Costa, V. P. N. 1; Baptista, D. 1; Nunes-de-souza, R. L. 2; Canto-de-souza, A. 1
1
Dep. de Psicologia/ Universidade Federal de São Carlos, UFSCar
2
Universidade Estadual Paulista "Júlio de Mesquita Filho", UNESP
Objectives:
One of the neurotransmitters that modulate pain responses and anxiety state is serotonin. Fear situations can induce
antinociception, i.e., blocks pain (Annual Rev. Psychol., 33; 87, 1982). One of the most widely used animal models of anxiety is
the elevated plus maze (EPM) (Psychopharmacol., 92; 180, 1987), and open-arm (OA) confinement in the EPM elicits
antinociception in mice (Psychopharmacol., 150; 300, 2000). This study investigated the effects of systemic treatment with
fluoxetine, a serotonin selective reuptake inhibitor (SSRI), on nociceptive response in mice exposed to the (EPM) and anxiety.
Swiss-albino male mice (35-45g) (n= 6-11/group) received subcutaneous injection of fluoxetine (0, 5.0, 10 or 20 mg/kg) and then
were randomly divided into two experiments. Experiment 1: twenty five minutes after injection fluoxetine, each mouse received
an intraperitoneal injection of 0.6% acetic acid (0.1 ml/10g weight; nociceptive stimulus) and housed in individual cages.
Immediately after writhing beginning the mice were confined to either open arm (OA) or enclosed arm (EA) of the EPM for 5
minutes. During this period, the number of writhes was recorded. Experiment 2: twenty five minutes after injection fluoxetine,
the mice were individually exposed to the elevated plus-maze (EPM) for five minutes. Anxiety was assessed in the EPM by
recording the conventional measures [% open arms entries (%OAE) and % open arms time (%OAT)]. Locomotor activity
[frequency of closed arms entries (EAE) was also recorded]. For Experiment 1, two-way ANOVA (place of confinement x
treatment) showed significant effects for place of confinement factor [F(1.75)=109.54, P < 0.05] and treatment factor
[F(3.75)=3.09, P < 0.05] but no effect for place of confinement x treatment interaction [F(3.75)=0.40, P > 0.05]. Duncan‟s
multiple comparisons test revealed that OA-confined animals showed lower number of writhes when compared to EA-confined
mice for all groups [saline (OA: 6.6±0.88; EA: 15.36±1.48, P < 0.05); fluoxetine 5.0 mg kg-1 (OA: 5.73±1.33; EA: 15.91±1.95,
P < 0.05), fluoxetine 10 mg kg-1 (OA: 5.91±1.19; EA: 14.18±0.8, P < 0.05) and fluoxetine 20 mg kg-1 (OA: 2.0±0.71; EA:
12.72±1.11)]. OA-confined animals treated with all doses of fluoxetine exhibited lower number of abdominal writhes when
compared to the control group. For Experiment 2, one-way ANOVA showed no significant difference to the conventional
measures: %OAE [F(3.22)=1.51, P > 0.05] and %OAT [F(3.22)=0.96, P > 0.05] and EAE [F(3.22)=0.35, P > 0.05].
Conclusions:
Our results corroborate previous studies showing that OA confinement in the EPM elicits antinociception in mice
(Psychopharmacol., 150; 300, 2000). Systemic treatment with all doses of fluoxetine increased OA-induced antinociception.
These results confirm the previously reported analgesic effect of SSRI drugs (Brain Res., 915; 218, 2001), suggesting an
important role played by serotonin in the modulation of nociception. Contrasting previous findings (Pharmacol. Biochem. Behav.,
65; 209, 2000), present results showed that fluoxetine treatment did not change anxiety-like behavior.
Keywords: Dor, Antinocicepção, Antidepressivo, Fluoxetina, Contorção
Financial Support: UFSCar, FAPESP (Processo nº 2010/06654-4).
Resumo:17-007
THERAPEUTIC AND PREVENTATIVE INHIBITION OF HYPERALGESIA AND ALLODYNIA IN PERIPHERAL
NEUROPHATY BY A NEW ANTINOCEPTIVE AGENT.
Mendes, T. C. F. 1; Júnior, N. M. N. 2; Antunes, F. 4; Barreiro, E. J. 2,2,1; Fraga, C. A. M. 2,1,3; Sudo, R. T. 1;
Zapata-sudo, G. 1
1
Programa de pós-graduação em Farmacologia e Química Medicina, UFRJ
2
Programa de pós-graduação em Química Orgânica, IQ, UFRJ
3
Faculdade de Farmácia, UFRJ
4
Centro de Ciências e Tecnologias Agropecuárias, UENF
Objectives:
New pirazol[3,4-b]pirrol[3,4-d]piridine derivatives, as LASSBio-873 were structurally designed by using zolpidem as lead
compound. LASSBio-873 induced significant antinociceptive activity in protocols of inflammatory pain through activation of
cholinergic system. Based on this, LASSBio-873 was tested in animal model of chronic pain.
Methods: Spinal nerve ligation (SNL) was used to induce peripheral neuropathy in male Wistar rats (180 – 200 g) anesthetized
with the association of ketamine (100 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.). Four hours (preventive protocol) or 7 days after
surgery (therapeutic protocol), LASSBio-873 (100 mg/kg, per orum) was administered daily for 7 consecutive days. Behavioral
tests were used to assess nociceptive threshold: 1. presence of heat thermal sensitivity using a plantar analgesia meter; 2. presence
of mechanical allodynia using a digital analgesia meter (von frey). Nociceptive threshold was determined before and after nerve
ligation. Withdrawal of the hind limb was considered a positive response either to mechanical allodynia (in grams) and heat
hyperalgesia (in seconds). Values were expressed as mean ± S.E.M of 5 measurements. This study was performed in compliance
with the Animal Care and Use Committee at Universidade Federal do Rio de Janeiro (UFRJ) under the protocol number
DFBCICB017. Results: Injury resulted in the development of paw mechano-tactile allodynia assessed by significant decrease in
withdrawal threshold of hind paw 30.7 ± 1.8 to 18.3 ± 2.3 g after SNL in rats. Paw heat hyperalgesia was reflected by significant
decrease in withdrawal threshold of hind paw from 8.8 ± 0.6 s and 5.2 ± 0.6 s before and 7 days after SNL surgery. LASSBio-873
(100 mg/kg) inhibited the development of heat hyperalgesia (from 8,9 ± 0,7s to 10,7 ± 0,6 s) and mechanical allodynia (from 32,2
± 0,9 g to 34,7 ± 2,3 g) in preventive protocol. In the therapeutic protocol, animals that developed hyperalgesia and allodynia
were treated with the derivative. The control group (no treatment) has no significant changes in the latency that has already
decreased from 8.8 ± 0.7s to 5.2 ± 0.6 s 7 days after SNL which maintained reduced until 14 days after surgery (5.2 ± 0.3 s).
LASSBio-873 administered p.o. recovered latency back to control values in the neuropathic group. Latency for hyperalgesia was
altered from 5.6 ± 0.4 to 10.4 ± 0.3 s after treatment with LASSBio-873. Similar results were observed with mechanical
allodynia, the control latency 32.2 ± 0.7 g was reduced to 21.0 ± 1.3 g after SNL and was recovery to 33.2 ± 1.0 g 7 days after
LASSBio-873 treatment.
Conclusions:
Lassbio-873 administered orally could abolish the development of allodynia and heat hyperalgesia in SNL rats in both therapeutic
and preventive protocols.
Keywords: neurophaty, antinociceptive, allodynia, hyperalgesia
Financial Support: CNPQ, FAPERJ, FUJB e CAPES
Resumo:17-008
MK-801 INVOLVEMENT IN AMPHETAMINE-INDUCED EFFECTS ON PULMONARY ALLERGIC
INFLAMMATORY RESPONSE IN MICE
Hamasato, E. K. 1; Ribeiro, A. 1; Ferraz-de-paula, V. 1; Pinheiro, M. L. 1; Lino-dos-santos-franco, A. 3; de
Oliveira, A. P. 2; Damazo, A. S. 4; Tavares-de-lima, W. 3; Palermo-neto, J. 1
1
Department of Pathology/Faculty of Veterinary Medicine, FMVZ-USP
2
Department of Immunology/Institute of Biomedical Sciences, ICB-USP
3
Department of Pharmacology/Institute of Biomedical Sciences, ICB-USP
4
Department of Basic Science in Health/UFMT, UFMT
Objectives:
Amphetamine (AMPH) is a drug of abuse that exerts profound effects on behavior, biochemistry and immunity. Glutamate has
been considered an important connecting agent between the central nervous and immune systems. Since we observed that AMPH
altered the leukocyte distribution in a murine model of asthma, we searched for the effects of MK-801 (NMDA receptor
antagonist) in AMPH-induced effects on pulmonary allergic inflammatory response in mice. Specifically, we investigated the
MK-801 involvement on AMPH effects on pulmonary cellular efflux, cytokines release, airway reactivity and mast cell
degranulation.
Animals were housed and used in accordance with the guidelines of the Committee on Care and Use of Laboratory Animal
Resources of the School of Veterinary Medicine, University of São Paulo, Brazil (protocol No. 1633/2009, FMVZ –USP). Male
Balb/c mice were divided in 5 groups: basal (B), allergic saline (AS), allergic AMPH (AA), allergic MK-801+ saline (AMKS)
and allergic MK-801+AMPH (AMKA). In order to induce lung allergic inflammation mice of all groups except the group B were
sensitized (i.p.) with ovalbumin (OVA, 10µg) on days 0 and 7 and challenged on days 13 and 14 with OVA nebulization (1% in
PBS). MK-801 pretreatment was performed 30 min. before AMPH (2mg/Kg) or saline (0.9%NaCl) treatments on days 13 and 14.
Moreover, the AMPH treatment was performed 1 hour before OVA nebulization by i.p route. On day 15, 12 h after the last OVA
challenge, BAL (bronchoalveolar lavage), bone marrow and peripheral blood were collected to proceed leukocyte counting in
Neubauer chambers. Cytokines (INF-γ, IL-5, IL-13 and IL-10) levels in BAL supernatant was determined by ELISA. Tracheal
rings were mounted for the measurement of isometric force quantification by means of two steel hooks in a 15-ml organ bath and
suspended in organ bath system at 37ºC. Tissues were maintained continuously aerated (95% O2 and 5% CO2). After the
equilibrium period (40 min), cumulative dose-response curves to methacholine were constructed. Mast cell number and activation
in lung tissue samples was performed with a high-power objective (x40) and measuring the area of analysis with the software
Axiovision. AMPH treatment per se decreased both leukocyte number, and IL-5, IL-13, IL-10 levels in BAL (p
Conclusions:
Our findings showed that AMPH alters the mechanisms of degranulation of mast cells, decreasing the release of cytokines and
thus contributing to reduction in the number of leukocytes in the lung. Pretreatment with MK-801 prevented the effects of AMPH
on cell migration to the lung. For the first time to our knowledge, we suggest the involvement of the glutamatergic system via
NMDA receptors in cell migration effects of AMPH.
Keywords: Amphetamine, Inflammation, Mice, Neuroimmunomodulation, NMDA receptor antagonist
Financial Support: FAPESP (2009/01826-4, 2009/51886-3) and CNPq.
Resumo:17-009
ANNEXIN A1 REGULATES THE HUMAN LARYNX TUMOR GROWTH IN VITRO BY INTERACTING WITH THE
FORMYL PEPTIDE RECEPTOR LIKE-1
Gastardelo; T. S. 1; Rodrigues-lisoni; F. C. 2; Tajara; E. H. 3; Oliani; S. M. 4,1
1
Depto Morfologia/Universidade Federal de São Paulo, UNIFESP-EPM
2
Depto Biologia e Zootecnia. Universidade Estadual Paulista, UNESP
3
Depto Biologia Molecular. Faculdade de Medicina de Rio Preto, FAMERP
4
Depto Biologia. Universidade Estadual Paulista., UNESP-IBILCE
Objectives:
Larynx cancer is one of the most common types of tumors with high mortality rates and poor prognosis, requiring an improved
understanding of this cancer. The anti-inflammatory protein annexin A1 (ANXA1) has been associated with the progression in
cancer, suggesting a role on the regulation of tumor cell migration. Some of the ANXA1 effects can be attenuated by Boc2, an
antagonist of formyl peptide receptor like-1 (FPRL1. In the present study we investigated the effect of the exogenous ANXA1
(peptide Ac2-26) and Boc2 during the growth of the Hep-2 cells (derived from human larynx epidermoid carcinoma) and the
potencial role of the ANXA1/FPRL1 interaction in these cells.
Hep-2 cells were cultured in complete medium (control), treated with peptide Ac2-26 (1µM) alone, Boc2 (10 µM) alone or
peptide Ac2-26 (1µM) and Boc2 (10 µM). Cell proliferation was observed, using Countess Automated Cell Counter, at 6, 24, 48,
72, 96 and 120h. The growth curve showed that peptide Ac2-26 treatment significantly reduced (p
Conclusions:
Thus, this study sheds light on the receptor mechanism mediating ANXA1. This protein might contribute to the regulation of
larynx tumor growth by a paracrine mechanism mediated by FPRL1. A better understanding of the regulatory mechanisms of
ANXA1 may lead to future biological targets for the therapeutic intervention of human larynx cancer.
Keywords: Annexin A1, Cancer, FPRL1 receptor, Inflammation
Financial Support: FAPESP (2008/01655-2).
Resumo:17-010
SEEKING FOR NEW ANALGESIC DRUGS: ORTOBENZAMOL, A PARACETAMOL ANALOGOUS.
Queiroz, L. M. D. 1,3; Crespo-lópez, M. E. 1; Borges, R. S. 2; Sousa, P. J. C. 3
1
Laboratório Farmacologia Molecular, ICB, UFPA
2
Laboratório de Química Farmacêutica, ICS, UFPA
3
Laboratório de Farmacodinâmica, ICS, UFPA
Objectives:
Paracetamol (PAR)is one of the most consumed analgesic drugs in the world. However, its known and high hepatotoxicity makes
essential the seeking for new alternatives. Changes in the molecular structure became a good alternative to minimize the toxicity
of PAR and to obtain better analgesic activity. Thus, this study aimed to evaluate experimentally antinociceptive activity of
orthobenzamol (OBZ), analogous of PAR.
Molecular modelling was performed using semi-empirical and functional density theory methods with PM3 and B3LYP/6-31G*
basis set. OBZ was obtained with acylation reaction and it was characterized by melting point and MNR 1H and 13C. Ten adults
male Swiss mice (Mus musculus), weighing between 25-30g, were orally treated with of OBZ (5 - 50 mg/kg). Handling of
animals was in accordance with the rules of the ethics committee on animal research at UFPA (MED010/2008). Writhing test
(Fed. Proc. 18:412, 1959), Paw licking test (Eur. J. Clin. Pharmacol. 27:1, 1974), and Hot plate test (Pain. 30:103, 1987) were
used for the biological evaluation of nociception. Results, expressed as mean ± sd, were analyzed with ANOVA followed by
Student-Newman-Keuls or Dunn's tests when appropriated, and significant values for P were set at
Conclusions:
Our results demonstrate that OBZ shows a stronger antinociceptive activity when compared to PAR and that the opioid receptors
are involved in this effect.
Keywords: Molecular modeling, Nociception, Ortobenzamol, Paracetamol
Resumo:17-011
UVAOL, A PENTACYCLIC TRITERPENE, ATTENUATES ALLERGIC INFLAMMATION ON MURINE MODEL
OF OVALBUMIN-INDUCED PLEURISY.
Agra, L. C. ; Ferro, J. N. S. ; Albuquerque, A. L. ; Brito, F. A. ; Barreto, E. O.
Lab. de Biologia Celular/ Universidade Federal de Alagoas, ICBS/ UFAL
Objectives:
The present study was undertaken to evaluate the anti-allergic effect of Uvaol, a pentacyclic triterpenoid on pleurisy induced by
ovalbumin in sensitized mice.
Swiss mice (20-30 g, n=6) were obtained from breeding unity of Federal University of Alagoas and the experimental protocols
were submitted to the ethics committe and approved (nº 23065.12614/2006-89). Animals were sensitized by subcutaneous
injection of ovalbumin (OVA, 50 µg) and aluminum hydroxide (5mg) in 200 µl final volume. Fourteen day later, animals were
challenged with intrapleural injection of OVA (12.5 µg/cavity) at final volume of 50 µl/cavity. At 6h after allergenic challenge,
the exudates were collected to perform total and differential leukocyte counts, as well as the determination of protein
concentration by Bradford assay. Tha animals were treated by oral route with saline, Uvaol or dexamethasone 1 h before
antigenic challenge. Treatments were performed by oral route 1 h before challenge with saline, Uvaol (10-4, 2x10-4 and 5x10-4
mol/kg) or dexamethasone ( 10-3 mol/kg). Statistical analysis of the data was performed by two-way analysis of variance
(ANOVA) followed by Students′t test. A P value < 0,05 was considered to be statistically significant. Antigenic challenge
induced a significant increase in the total leukocyte count (25.3± 2.6x10 6 cells) and neutrophils accumulations (18.1± 3.4x10 6
cells) into pleural cavity. It also caused an increase in protein concentration in the exsudate (54.5± 11.0 µg/ml). Preteatment with
Uvaol (10-4, 2x10-4 and 5x10-4 mol/kg) inhibited the total leukocyte counts (to 12.3± 2.3x10 6, 10.4± 2.7x106 and 10.0± 2.6x106
cells, respectively)and neutrophils influx (to 10.5± 2.3x10 6, 5.1± 1.9x106 and 4.4± 3.3x106 neutrophils, respectively) in allergic
pleurisy. All doses of Uvaol also attenuated protein extravasation OVA-induced in sensitized mice (to 34.9± 4.9, 35.8± 13.5 and
25.9± 8.0 µg/ml, respectively). As expected, dexamethasone reduced total leukocytes count (to 8.0± 1.8x106 cells), neutrophils
influx (to 9.8± 3.6x106 cells) and protein concentration(to 3.6± 1.0 µg/ml) after antigenic challenge.
Conclusions:
These results showed that pentacyclic triterpene Uvaol may be a promising anti-allergic therapy by attenuate the inflammatory
reaction induced by antigen. Studies are underway to explain the possible mechanisms responsible for the activity.
Keywords: Pleurisy allergic, Uvaol, Inflammation
Financial Support: CNPq, CAPES and FAPEAL
Resumo:17-012
ANTI-INFLAMMATORY ACTIVITY OF RIPARIN II (N-2-HYDROXYBENZOYL TYRAMINE) IN DIFFERENT
RAT PAW EDEMA MODELS
Carvalho, A. M. R. 1; Vasconcelos, L. F. 1; Rocha, N. F. M. 1; Rios, E. R. V. 1; Dias, M. L. 1; Feitosa, M. L.
1
; Silva, M. I. G. 1; Filho, J. M. B. 2; Gutierrez, S. J. C. 3; Sousa, F. C. F. D. 1
1
Depto de Fisiologia e Farmacologia-Faculdade de Medicina, UFC
2
Laboratório de Tecnologia Farmacêutica, UFPB
3
Departamento de Bioquimica e Farmacologia, UFPI
Objectives:
Riparin II (ripII) is an alkamide compound isolated from unripe fruit of Aniba riparia, but it can be synthesized too. In previous
studies this substance presented antimicrobial, anxiolytic and analgesic effects in different animal models. In this study, we
decided to evaluate the anti-inflammatory effect of ripII on rat paw edema induced by several inflammatory agents, including
carrageenan, dextran, histamine, serotonin and bradykinin.
Methods: Male rats (180-240 g) were divided into groups (7-9 animals): controls (vehicle-tween 80 2%), ripII-25 (riparin II 25
mg/Kg), ripII-50 (riparin II 50 mg/Kg) and INDO (indomethacin 10 mg/Kg) or CIPRO (cyproheptadine 10 mg/kg). INDO and
CIPRO were used as standards for carrageenan and dextran-induced edemas respectively. All drugs were injected orally.
Treatments were conducted one hour before injection of the inflammatory stimulus.In different experiments, the paw edema was
induced by intraplantar injection of 100 μL of following agents: carrageenan 1%, dextran 1.5%, histamine 200 μg/paw, serotonin
200 μg/paw or bradykinin 10 µg/paw.The paws were measured before and 1, 2, 3, 4 and 24 hours after injection of carrageenan.
In the dextran edema measurements were made before and 1, 2, 3 and 4 hours after injection. In the histamine edema,
measurements were made before and 15, 30, 60 and 90 min after injection whereas for serotonin edema measurements were made
before and 30 and 60 min after injection. For bradykinin edema measurements were made before and 15 and 30 min after
injection. Data are here presented by mean (mL) ± S.E.M and analyzed by ANOVA followed by Student Newman Keuls as the
post hoc test. Results: The carrageenan-induced paw edema was significantly reduced by the previous administration of ripII in
times measured, when compared to respective controls [2 h : Ctrl: 3.482±0.291; ripII-25: 2.332±0.346; ripII-50: 2.142±0.251; p
Conclusions:
The results showed that ripII has anti-inflammatory effects on two different types of edema: inflammatory (carrageenan) and
vascular (dextran). Hence, corroborating with these findings, ripII decreased the histamine and the bradykinin-induced paw
edema showing that its anti-inflammatory effect is mediated, in part, by inhibiting the actions of histamine and bradykinin.
Keywords: riparin II, edema, inflammation, carrageenan
Financial Support: CNPq/ CAPES
Resumo:17-013
EVALUATION OF THE ANALGESIC EFFECT OF TRANS-CARYOPHYLLENE, AN ACTIVE PRINCIPLE FOUND
IN MANY MEDICINAL PLANTS ESSENTIAL OIL
Paula-freire, L. I. G. ; Andersen, M. L. ; Molska, G. R. ; Köhn, D. O. ; Carlini, E. L. A.
Psicobiologia, UNIFESP
Objectives:
Trans-caryophyllene is a sesquiterpene present in the essential oil of many medicinal plants, such as those of the Ocimum gender.
The aim of the present study was to evaluate the analgesic effect of trans-caryophyllene in pain animal models.
In all experiments, groups of 6 adult male C57BL/6J mice were treated with corn oil (control group/vehicle – CTRL) or transcaryophyllene (20, 40 or 80 mg/kg, p.o.). A group of 6 animals received 5mg/kg (i.p.) of morphine and was used as positive
control in the hot-plate and in the formalin tests. In the acetic acid-induced writhing test, animals received the different doses of
trans-caryophyllene and after one hour, acetic acid 0.8% was administered (i.p.) and the number of writhing was quantified. In
the hot-plate test, animals were pre-treated with the different doses of the studied drug and submitted to a 50° C heated plate. The
reaction time was defined as the latency to lick the paw(s) or jump. In the formalin test, animals received intraplantar injection of
2% formalin (20µl/animal) one hour after treatment and the licking time of the injected paw was recorded during first phase (5-10
minutes) and second phase (15-30 minutes) of test. Statistical analysis were performed using one-way ANOVA. In the hot-plate
test, all animals treated with trans-caryophyllene were capable of staying significantly longer on the hot plate when compared to
control group (mean ± standard error: CTRL: 25 ± 1.82; morphine: 47 ± 2.54; 20mg/kg: 41± 2.26; 40mg/kg: 38 ± 1.82; 80mg/kg:
38 ± 2.91; p < 0,01). The same effect was observed for the animals submitted to the writhing test, in which all tested doses of
trans-caryophyllene were capable of reducing the number of writhing when compared to control animals (mean ± standard error:
CTRL: 16 ± 1.78; 20mg/kg: 6.83 ± 1.22; 40mg/kg: 9.50 ± 1.20; 80mg/kg: 8.87 ± 1.58; p< 0,001). The dose 80mg/kg of transcaryophyllene was the only dose that inhibited the second phase of formalin test (mean ± standard error: CTRL: 141.50 ± 9.09;
morphine: 12.00 ± 4.80; 20mg/kg: 99.00± 27.18; 40mg/kg: 85.17 ± 14.39; 80mg/kg: 72.17 ± 14.55; p < 0,05).
Conclusions:
Trans-caryophyllene was capable to reduce pain in all tests performed in mice. Possibly, this active principle might be involved
in the analgesic properties observed in many medicinal plants.
Keywords: Pain, Medicinal plants, Trans-caryophyllene, essential oil, active principle
Financial Support: FAPESP and AFIP
Resumo:17-014
URINARY BIOMARKERS FOR THE IDENTIFICATION OF CYCLOSPORINE A-INDUCED RENAL INJURY
Carlos, C. P. 1; Sonehara, N. M. 2; Oliani, S. M. 2; Burdmann, E. D. A. 1
1
Lab. Fisiopatologia Renal, Fac. Medicina S.J. Rio Preto, FAMERP
2
Lab. Imunomorfologia, IBILCE, UNESP
Objectives:
The aim of this study was to identify urinary biomarkers for cyclosporine A (CsA)-induced acute and chronic renal injury in rats
and correlated them with hemodynamic and structural alterations development.
Munich-Wistar male rats weighing 250 g were divided into 6 groups with 8 animals each. The nephrotoxicity was studied 7, 14
and 21 days after CsA (15 mg/kg/day) or vehicle treatment (VH). At the end of treatment, animals were allocated in metabolic
cages and urine collected for 24 hours before glomerular filtration rate (GFR, inulin clearance), renal blood flow (RBF, Doppler
ultrasound) and RVR (renal vascular resistance) assessment. Urinary samples were used for microalbuminury measurement. Elisa
assays were used to quantify the following renal injury urinary biomarkers: KIM-1 (kidney injury molecule-1, proximal tubule
injury); NGAL (neutrophil gelatinase-associated lipocalin, tubular injury); TGF-beta and fibronectin (fibrosis); MCP-1,
osteopontin, TNF-alpha and IL-6 (inflammation and leukocyte infiltration). Results are mean ± SD. CsA caused a GFR reduction
in all periods (0.19±0.08 CsA vs 0.94±0.15 VH, 7 d; 0.18±0.06 CsA vs 0.82±0.09 VH, 14 d; 0.15±0.05 CsA vs 1.0±0.19 VH;
ml/min/100g, 21 d, p<0.01).
Conclusions:
Fibronectin, a marker of fibrosis, TNF-alpha, a pro-inflammatory cytokine, and elevated microalbuminuria, were indicators of the
early phase of CsA-induced nephrotoxicity. TGF-beta and osteopontin, markers of fibrosis and proinflammatory cytokine, were
observed in the later phase of the lesion. KIM-1, a biomarker of proximal tubular injury, was present in all stages of
nephrotoxicity, suggesting that the tubular damage is a continuous process during CsA exposure. MCP-1, monocyte attractive
protein, was probably down-regulated or exhausted by the high number of infiltrative macrophages observed in all stages of CsAinduced nephrotoxicity.
Keywords: cyclosporine, renal injury, biomarkers
Financial Support: FAPESP (09/17100-2) and CNPq (150954/2009-3)
Resumo:17-015
ENDOTOXIN-INDUCED UVEITIS PROVOKES MODULATION OF ANTI-INFLAMMATORY PROTEIN
GALECTIN-1
Zanon, C. F. 1; Girol, A. P. 1,2; Gil, C. D. 3; Oliani, S. M. 1
1
Depto Biologia, Inst. de Biociêncs, Letras e Ciêncs. Exatas , IBILCE/UNESP
2
Depto. De Ciências Básicas, Fac.. Integradas Padre Albino, FIPA/FAMECA
3
Dpto. de Morfologia e Genética, Univ. Fed. de São Paulo, UNIFESP/EPM
Objectives:
Galectins are a conserved family of glycan-binding proteins, usually found in the extracellular matrix, that have emerged as
pleiotropic regulators of innate and adaptive immune responses. Some members of the galectin family, including galectin-1 (Gal1), can regulate immune cell trafficking, activation, cytokine secretion, and apoptosis by triggering multivalent interactions with
cell surface glycoconjugates. In this study, the objective was investigate the inflammatory response and the role of Gal-1 in
ocular tissues in endotoxin-induced uveitis (EIU).
Methods: Male rats of the species Rattus norvegicus were obtained from the Vivarium of the Faculty of Medicine of São José do
Rio Preto (FAMERP) with six to eight weeks old, weighing 150 to 200g. The EIU was induced by inoculation of
lipopolysaccharide (LPS) into the right paw (1 mg/kg in 0.1 ml of phosphate-buffer saline) and animals were sacrificed after 24
and 48 hours. Enucleated left eyes were processed to histopathological analysis and the right ones for drawing samples of
aqueous humor (AH) to quantification of inflammatory cells. Gal-1 protein expression was evaluated in the ocular tissues by
immunohistochemistry and Western blotting. Results: After 24 hours of EIU, ocular tissues presented inflammatory response
characterized by the intense recruitment of neutrophils (EIU 24h 17.75 ± 5.437 cells/mm2; Control 0 ± 0), and activation of mast
cells, especially in the anterior eye segment. The inflammatory stimulus by LPS also provokes a significant inflow of neutrophils
(67.25 ± 13.27 x 106 cells/100µl) and mononuclear phagocytic cells (4.5 ± 0.326 x 106 cells/100µl) in the aqueous humor
compared to control group (7.25 ± 1.69 x 10 6 cells/100µl; 0.550 ± 0.255 x 106 cells/100µl, respectively). In the late phase of EIU,
48 hours, inflammation was mirrored by the significantly decrease of inflammatory cells in the tissues and aqueous humor. The
expression of Gal-1 was detected in the neutrophils and epithelial cells (of the cornea, ciliary body and iris) in the anterior
segment. In the posterior eye segment, the internal and external plexiform layers of retina showed Gal-1 immunoreactivity,
significant expression in the ganglion cells. Densitometric analysis of ocular tissues, after 24 hours of EIU, showed a significant
upregulation of Gal-1 expression in the epithelial cells of cornea (164.8 ± 2.426 arbitrary units), ciliary body (151.4 ± 1.726 a.u.)
and iris (144.7 ± 2.629 a.u.) compared to control (135.5 ± 1.433; 143.4 ± 1.501; and 143.1 ± 3.147 a.u., respectively) and EIU 48
hours (119.5 ± 1.507; 121.4 ± 1.614; and 119.1 ± 2.747 a.u., respectively). Total levels of Gal-1, detected by Western blotting,
were significantly increased after 24 hours of EIU, confirming the results obtained by immunohistochemistry.
Conclusions:
This data provides not only further insight into the protective effect of Gal-1 by inhibition of inflammatory cells, but also
provides a rationale for a clinical use for drugs developed from this line of research.
Keywords: anti-inflammatory, galectin-1, uveitis
Financial Support: CNPq (106357/2009-3; 472056/2009-3) and FAPESP (2010/15200-7).
Resumo:17-016
NOCICEPTION AND INFLAMMATION IN DIET-INDUCED OBESITY RATS
Carvalho, A. L. O. ; Vilela, F. C. ; Ferreira, M. S. ; Giusti-paiva, A. ; Nascimento, C. G. O.
Instituto de Ciências Biológicas , UNIFAL - MG
Objectives:
The aim of this study was to evaluate the potential changes on both inflammatory and nociceptive responses in diet-induced
obesity and control rats. The animals who received a hypercaloric diet were fed with a mix of chow, sweetened milk, sugar and
water. Lee index was used to evaluate the development of obesity. Thermal and mechanical hypernociception were considered.
We also assessed the responses to intraplantar carrageenan injection in a model of mechanical hyperalgesia and the paw volume
was measured to assess inflammation and edema.
Male Wistar rats (n=30; weight:150~200 g) with 4 weeks of age were divided in two groups. One group (n=13) was fed with
regular rat chow (CD) and the other group (n=17) was fed with hypercaloric diet (HD) for 16 weeks. After this period, we
performed thermal tests (Hot Plate and Tail Flick) to assess basal nociception. The mechanical test (Von Frey) was performed
after I.pl.Cg (0,1 ml solution 0,5mg/ml) injection in hind right paw. We evaluated edema and inflammation after I.pl. Cg by
Plestimometria. In Hot Plate test (n=5 per group), HD group presented largest latency period (15.18±1.52) than CD group
(10.76±1.02). In Tail Flick test (n=7 per group) the thermal nociceptive threshold was similar in HD/CD groups, in latency
(11.56±1.046 and 11.62±1.157,respectively) and in temperature (55.84±1.4 and 53.63±1.80,respectively). In Von Frey test (n=6
per group) obese rats presented higher intensity of mechanical hypernociception after I.pl.Cg than lean rats on 3rd hour
(52.648±2.109 and 44.440±0.918,respectively). Edema and inflammation were higher in HD group who received I.pl.Cg than CD
group with Cg, and this difference was higher on 4th hour (0.433±0.055 and 0.190±0.051, respectively).
Conclusions:
In Hot plate test, HD group had a higher latency in thermal basal nociception(*p
Keywords: hyperalgesia, inflammation, nociception, obesity, pain
Financial Support: CAPES,FAPEMIG
Resumo:17-017
VALPROIC ACID REDUCES POLYMORPHONUCLEAR CELL MIGRATION AND MYELOPEROXIDASE
RELEASE: AN IN VIVO AND IN VITRO STUDY
Gonçalves, D. O. 1; Ximenes, J. C. M. 1; Siqueira, R. M. P. 1; Neves, K. R. T. 1; Cavalcante, A. L. C. 1;
Leal, L. K. A. M. 1; Naffah-mazzacoratti, M. G. 2; Viana, G. S. B. 1
1
2
Departamento de Bioquímica, Escola Paulista de Medicina, USP
Objectives:
Valproic acid (VA) is an antiepileptic drug widely prescribed as a mood stabilizer for bipolar disorders. It may also have a
neuroprotective effect and, although this mechanism is still unclear, might involve the inhibition of histone deacetylase. We
previously showed that VA presents not only an antinociceptive action but also and predominantly an anti-inflammatory one. In
the present study, we focus on the drug anti-inflammatory actions by in vivo and in vitro experimental models.
Male Wistar rats were used for the assessment of VA (25 and 50 mg/kg, p.o.) effects on PMN cells migration to the peritoneal
cavity, in the model of carrageenan-induced peritonitis. We also measured myeloperoxidase (MPO, an inflammatory marker)
release to the peritoneal exudate. The results showed that VA inhibited by 86 and 92% PMN cells release, and similar results
were observed for indomethacin (20 mg/kg, p.o.) as the reference drug. The percentage inhibitions of MPO release to the
peritoneal exudate were 69 and 85%, respectively, for the same VA doses, and 80% for indomethacin. In addition, VA (25-100
μg/ml) inhibited by 18, 23 and 45% MPO release from humans neutrophils, while a 52% inhibition was seen with indomethacin
(10 μg/ml). Furthermore, while the positive control (0.2% Triton X-100) increased more than 2 times TBARS content (an index
of lipid peroxidation), VA results were similar to those of Vit E, an antioxidant drug.
Conclusions:
. We conclude that VA effects are probably dependent upon its inhibition of histone deacetylase, since this enzyme inhibition is
known to produce anti-inflammatory effects. These results may have important implications for future clinical uses of VA,
considering the role played by inflammation in several neurological disorders, as epilepsy, and neurodegenerative diseases as
well.
Keywords: carrageenan, inflammation, neutrophils, valproic acid
Financial Support: FUNCAP, CNPq
Resumo:17-018
ANTI-INFLAMMATORY EVALUATION OF A SULFATED-POLYSACCHARIDE EXTRACTED FROM THE
MARINE RED ALGAE GRACILARIA CAUDATA
Nicolau, L. A. D. 1; Sila, R. O. 1; Santos, G. M. P. 1; Lucetti, L. T. 2; Santana, A. P. M. 2; Chaves, L. S. 3;
Freitas, A. L. P. 3; Souza, M. H. L. P. D. 2; Medeiros, J. V. R. 1
1
Departamento de Biologia, Universidade Federal do Piauí, UFPI
2
Departamento de Fisiologia e Farmacologia, Fac. de Medicina, UFC
3
Dpartamento de Bioquímica e Biologia Molecular, UFC, UFC
Objectives:
The aim of this study was to evaluate the anti-inflammatory activity of a sulfated-polysaccharide extracted from the Marine Red
Algae Gracilaria caudate.
All animal treatments and surgical procedures were approved by the local ethics committee (protocol No 0066/10). Male Swiss
mice were divided in six groups (G1-G6) and initially treated with Saline (i.p), Indometacine (10mg/kg i.p) and the
Polysaccharide (PLS, 2.5, 5 and 10 mg/kg, i.p). The groups division was: G1-Only Saline (p.o); G2-Saline (p.o) + Carrageenan
(i.pl.); G3-Indometacine (10mg/kg i.p) + Carrageenan (i.pl.); G4, G5 and G6- Polysaccharide (2.5, 5 and 10mg/kg p.o.
respectively) + Carragenan (i.pl.). In brief, to measure the paw edema in mice, after one hour the animals received a 300µL
intraplantar (i.pl.) injection, into the right paw, of Carragenan (3%) in the groups G2-G6. Paw edema was measured with a Ugo
Basile plethysmometer during the first 4 hours. Anti-inflammatory activity was expressed as the percentage reduction in edema in
treated mice by comparison with controls. Animals treated with carrageenan induced the paw edema in the time 4 hours.
However, the animals treated with PLS 2.5, 5 and 10mg/kg had edema inhibition with 38.18%, 54.54% and 85.45%,
correspondingly. Portions of the paw of mice were removed to determine myeloperoxidase (MPO) activity. The MPO activity is
an indication of neutrophil infiltration, and carrageenan administration increased the MPO concentration (20.31U/tissue mg). PLS
seemed inhibit the neutrophil-induced inflammation and had significant inhibition of MPO (p
Conclusions:
Findings of the present study suggest that PLS extracted from the Marine Red Algae Gracilaria caudate has anti-inflammatory
effect by inhibiting the paw edema in mice and reduces MPO.
Keywords: inflammation, myeloperoxidase, paw edema
Resumo:17-019
ANTINOCICEPTIVE EFFECT OF LQFM-002, A NEW DERIVED FROM PYRAZOLE NUCLEUS.
Lino, R. C. 1; Florentino, I. F. 1; Galdino, P. M. 1; Nascimento, M. V. M. 1; Gomes, M. N. 2; Menegatti, R. 2;
Costa, E. A. 1
1
Instituto de Ciências Biológicas II - UFG, ICB-II / UFG
2
Faculdade de Farmácia - UFG, FF-UFG
Objectives:
In the search for new anti-inflammatory agents that have fewer side effects, new nucleus pyrazole derivatives have been
highlighted as possible prototypes of drugs (Eur Med Chem 40:850, 2005). The aim of this work was to evaluate the
antinociceptive effect of the pyrazole derivative LQFM-002 synthesized by the Laboratory of Medicinal Pharmaceutical
Chemistry-FF-UFG.
All experimental protocols were done with adult male Swiss mice (n=8, per group) and were carried out in accordance with the
Research Ethics Committee of UFG (Prot. No. 182/10). Data were analyzed statistically by one-way ANOVA followed by the
unpaired Student-Newman-Keuls test. Values of p ≤ 0.05 were considered significant. To evaluate the antinociceptive effect of
LQFM-002 were used the following methodologies: acetic acid-induced writhing, formalin-induced pain and hot plate tests. The
intermediate dose of LQFM-002 (200 µmol/kg) was determined from previous study (Abstract ID:86-1, FeSBE, 2009). In the
acetic acid-induced writhing test, 10 mL/kg of acetic acid (0.6 % v/v, i.p.) was injected in the mice pretreated with vehicle
(DMSO 20%, 10 mL/kg, p.o.), LQFM-002 (200 µmol/kg, p.o.) or indomethacin (28 µmol/kg, p.o.). LQFM-002 (200 µmoL/kg)
reduced the writhes to 85.7 ± 3.9 %, when compared to control group (87.60 ± 2.87). Indomethacin (28 µmoL/kg), used as
positive control, reduced the writhes to 56.0 ± 5.17 %. In the formalin-induced pain, 20 μL of the phlogistic agent (3.0% v/v) was
applied subcutaneously in the right paw of mice pretreated with vehicle (DMSO 20%, 10 mL/kg, p.o.), LQFM-002 (200 µmol/kg,
p.o.), indomethacin (28 µmol/kg, p.o.) or morphine (35 µmol/kg, s.c.); in this test, LQFM-002 (200 µmoL/kg) reduced the licking
time in both neurogenic and inflammatory phases to 59.0 ± 4.30 s and 124.0 ± 26.2 s, respectively, when compared to control
group (DMSO 20%): 93.0 ± 7.5 s and 231.6 ± 18.0 s, first and second phase, respectively. Morphine (35 µmoL/kg) reduced both
the first and the second phases of formalin-induced pain to 13.5 ± 3.5 s and 0.0 ± 0.0 s, respectively. In contrast, indomethacin
(28 µmoL/kg) reduced only the inflammatory pain to 62.7 ± 7.9 s. In the hot plate test, the mice pretreated with vehicle (DMSO
20%, 10 mL/kg, p.o.), LQFM-002 (200, 400 and 800 µmol/kg, p.o.) or morphine (35 µmol/kg, s.c.) were placed into a heated
surface at 56 ºC (Hot Plate – Insight) and the latency to pain response was recorded. 60 min after the treatment, LQFM-002 200,
400 and 800 µmoL/kg increased the latency to 190.0 ± 34.6, 213.0 ± 33.8 and 213.0 ± 40.7 % respectively; and 90 min after the
treatment, LQFM-002 400 and 800 µmoL/kg increased the latency to 153.6 ± 20.5 and 179.2 ± 25.4 %, respectively. Morphine
(35 µmoL/kg, s.c.) exhibited significant antinociceptive activity since 30 min up to 150 min after the treatment. To examine the
involvement of opioids receptors in the LQFM-002 antinociceptive activity in the formalin-induced pain, the animals were
pretreated with naloxone (8.25 µmoL/kg. i.p.). Naloxone reversed the effect of LQFM-002 (200 µmoL/kg) in the first phase of
formalin-induced pain from 33.2 ± 5.1 s to 54.2 ± 5.8 s. The licking time of the control group was 59.6 ± 6.6 s.
Conclusions:
LQFM-002, a pyrazole derivative, showed analgesic activity. This antinociceptive activity may involve the opioid receptors, as
seen in the formalin test with the pre-treatment with naloxone.
Keywords: anti-inflammatory , antinociceptive, nucleus pyrazole , opioid receptors
Financial Support: CNPq, CAPES, FAPEG, FUNAPE/UFG.
Resumo:17-020
ROLE OF NMDA RECEPTORS IN ACUTE AND PERSISTENT HYPERNOCICEPTION INDUCED BY SYSTEMIC
MAGNESUIM DEFICIENCY IN RATS SUBJECTED TO TEMPOROMANDIBULAR JOINT ARTHRITIS.
Cavalcante, A. L. C. 1; Siqueira, R. M. P. 2; Araújo, J. C. B. D. 2; Vale, M. L. 2
1
Programa de Pós-Graduação em Ciências Médicas , UFC
2
Objectives:
Study the role of NMDA receptors in hypernociception amplification induced by systemic magnesium deficiency in rats
subjected to temporomandibular joint arthritis.
The experimental protocol was previously approved by the Animal´s Ethics Committee of Federal University of Ceará (protocol
number: 3010). Male Wistar rats (180-200g) received carrageenan (Cg) intrarticular (i.a.) injection (5%, 10 ul) in the left TMJ.
We evaluated the behavioral nociceptive parameter, through the mechanical hypernociception test using electronic von Frey. The
nociceptive threshold was measured before and after (4,6,10, 24 - 168 hours) the Cg injection. The NMDA receptor antagonist,
MK-801 (0.5 mg/kg) was administered intraperitoneally 30 min before the Cg injection. Magnesium deficiency was promoted by
feeding the animals with synthetic depleted diet along with deionized water ad libitum for 14 days in 3 groups before the Cg
injection. The animals were divided into 4 groups: group A (depleted diet, injected saline i.a.); group B (depleted diet, injected
Cg i.a.); group C (depleted diet, injected Cg i.a and MK-801 pre-treated) and group D (normal diet, injected Cg i.a). The
magnesium level in the serum was checked in all groups after 9 days of the diet, which was decreased by 70% in A, B and C
groups. All the depleted diet groups (A, B and C) showed a decrease of the initial threshold, in 18% (p<0.001).
Conclusions:
Together these data suggest that magnesium deficiency has an important effect in orofacial sensitivity, lowering nociceptive
threshold. NMDA-R have a role in this effect being an important key in the acute phase of TMJ inflammatory hypernociception,
as well as in persistent hypernociception that occurs, at least, until the 5th day after a single Cg injection.
Keywords: Atrite, Dor, Trigemio, Temporomandibular, Magnésio
Resumo:17-021
ANTI-HYPERNOCICEPTIVE PROPERTY OF α -TERPINEOL IN MICE.
Prado, D. S. 1; Oliveira, M. G. B. 1; Santana, M. F. 1; Santana, M. T. 1; Sousa, D. P. 1; Siqueira, R. S. 1;
Bonjardim, L. R. 1; Almeida, J. R. G. S. 2; Lima, J. T. 2; Quintans-júnior, L. J. 1
1
DEPARTAMENTO DE FISIOLOGIA/UNIVERSIDADE FEDERAL DE SERGIPE, UFS
2
UNIVERSIDADE FEDERAL DO VALE DO SÃO FRANCISCO, UNIVASF
Objectives:
α-Terpineol (TPN) is an alcoholic monoterpene and is a major component of the essential oil of many plants. This study
examines the anti-hypernociceptive effect of TPN in mice.
Experiments were performed on male Swiss mice with weight between 25-30 g (n=6/group). To show the anti-hypernociceptive
property followed by models of mechanic hypernociception in which thirty minutes after treatments, it was injected into the
plantar region of the rear right paw: 20 μL of carrageenn (CG; 300 μg/paw), TNF-α (100 pg/paw), prostaglandin (PGE2; 100
ng/paw) and dopamine (DA; 30 μg/paw). The mice were pre-treated intraperitoneally with vehicle (saline + cremophor 0.2%) or
TPN (25, 50 and 100 mg/kg; doses A, B and C respectively). The hypernociception was checked with the analgesymeter digital
(Von Frey), which consists of a preassure transducer adapted to a digital counter force in grams (g), at times of 0.5, 1, 2 and 3
hours after the injection of CG, TNF-α, PGE2 and DA. The experimental protocols were approved by the Ethics Committee on
Animal Research at the Federal University of Sergipe (CEPA: 17/10). These results were presented with Mean ± SEM, express in
grams (g), and considering p < 0.05 as significant. Statistical comparison of the data was performed by analysis of variance
(ANOVA) followed by tukey's of test (p
Conclusions:
Thereby, these results suggest anti-hypernociceptive property of TPN, probably by a mechanism inhibition of production of
cytokines.
Keywords: α-TERPINEOL, CYTOKINES, HYPERNOCICEPTION, MICE, MONOTERPENE
Financial Support: FAPITEC-SE, CNPq
Resumo:17-022
INVOLVEMENT OF P2X3 AND P2X7 RECEPTORS IN CARRAGEENAN-INDUCED ARTHRITIS IN THE RAT
KNEE-JOINT MODEL
Teixeira, J. M. ; Parada, C. A. ; Tambeli, C. H.
Instituto de Biologia , UNICAMP
Objectives:
Aim: Osteoarthritis is a degenerative disease that causes progressive movement limitations, pain and inflammation (Radio. Clin.
North Am 47; 539, 2009). The aim of this study was to investigate the role of the P2X3 and P2X7 receptors in carrageenaninduced articular inflammatory hyperalgesia in the rat knee-joint arthritis model. To this end, the antagonist of the P2X3 and
P2X7 receptor, A-317491 and A-740003 respectively, was co-administered with carrageenan in the rat knee joint and the
hyperalgesic response (gait disturbance - articular incapacitation) was quantified.
Methods: Male Wistar rats (2 months old, 200-250g) were used in this study and all experimental procedures were approved by
the Ethics Committee in Animal Research at the UNICAMP (protocol number: 2049-1). The hyperalgesic responses (gait
disturbance) were quantified using the Knee-joint Incapacitation Test (Pain 48; 421, 1992). Results: Carrageenan (300µg/knee)
induced an articular hypernociception (Means±SEM 55±1.2s n=6) that was significantly reduced by the co-administration of A317491 (60, 180 and 540µg/knee; 33.8±1.6s, 25.0±0.7s and 14.6±0.7s, respectively, n=6, p0.05, Tukey test) and (54.8±1.2s,
55.2±1.1s, 51.4±1.4s and 54.6±1.2s, respectively, p>0.05, Tukey test), respectively, significantly reduced carrageenan-induced
mechanical hyperalgesia.
Conclusions:
Conclusion: The results strongly suggest that activation of peripheral P2X3 and P2X7 receptors by endogenous ATP not only
contributes, but is essential for the development of joint hypernociception induced by carrageenan in the rat knee-joint and that
peripheral activation of these receptors are crucial for the sensitization of primary afferent nociceptors mediated by
prostaglandins and the sympathomimetic amines, since the selective antagonists of P2X3 and P2X7 receptor blocked
carrageenan-induced joint hypernociception.
Keywords: ATP, carrageenan, hyperalgesia, P2X3 receptor, P2X7 receptor
Resumo:17-023
NEURAL MOBILIZATION FOR NEUROPATHIC PAIN CONTROL: EVALUATION OF GLIAL CELLS AND
BRAIN-DERIVED NEUROTROPHIC FACTOR
Giardini, A. C. ; Santos, F. M. ; Silva, J. T. ; Chacur, M.
Anatomia / Universidade de São Paulo, USP
Objectives:
The neural mobilization (NM) is a technique used to control the pain induced by injury which reflect on the peripheral nervous
system. This technique it is able to restore the mobility and the elasticity of the peripheral nervous system. The present study
examined the effects of the neural mobilization in response to the pain induced by the chronic constriction injury (CCI) and the
participation of the glial cells and brain-derived neurotrophic factor (BDNF) in the central neurons system.
Adult rat (170-190g) of the Wister species were submitted to the CCI of the sciatic nerve, 14 days after injury we starded sessions
the NM, every other day,with a total of 10 sessions. Sham animals were submitted by the same procedures as the operated ones,
but left unaffected the sciatic nerve and served as a control. Before and after each mobilization session, the animals were
submitted to behavioral tests for allodynia mechanical, hyperalgesia thermal and mechanical to evaluate the nociceptive threshold
of the animals. After the last session, the animals (n=4) were decapitated and Western blotting tests for BDNF and GFAP in
thalamus tissue made. The animals with CCI showed a decrease (90%) on the nociceptive threshold, when compared to the
control. In the other hand, the CCI treated with NM animals presented a positive increase (80%) on the behavioral studies when
compared to the animals that did not receive mobilization sessions. The Western blotting assays showed that after the CCI there
was an increase of BDNF (50%) and GFAP (150%) expression on the thalamus compared to the control group: After
mobilization treatment there was a decrease of the BDNF (40%) and GFAP (50%) when compared to the CCI animals.
Conclusions:
We suggest that neural mobilization is efficient in improve behavioral response and suggest the involvement of glial cells and
BDNF in such on effect.
Keywords: BDNF, Glial cells, neural mobilization, neuropathic pain
Financial Support: FAPESP, CAPES and IASP
Resumo:17-024
ANTI-NOCICEPTIVE EFFECT OF KININ B1 AND B2 RECEPTOR ANTAGONISTS ON PERIPHERAL
NEUROPATHY INDUCED BY PACLITAXEL IN MICE
Costa, R. 1; Motta, E. M. 1; Dutra, R. C. 1; Manjavachi, M. N. 1; Bento, A. F. 1; Pesquero, J. B. 2; Calixto, J.
B. 1
1
Farmacologia/Universidade Federal de Santa Catarina, UFSC
2
Objectives:
In the current study, we investigated the role of both kinin B1 (B1R) and B2 (B2R) receptors in peripheral neuropathy induced by
the chronic treatment of mice with paclitaxel (PTX), a widely used chemotherapeutic agent.
Chemotherapy-evoked hyperalgesia was induced by intraperitoneal (i.p.) injections of PTX (2 mg kg-1) over five consecutive
days. Mechanical and thermal hyperalgesia were evaluated between 7 and 21 days after the first PTX treatment. PTX treatments
induced a significant increase in both mechanical and thermal hyperalgesia in mice (C57BL/6 and CD1 strains). Kinin B1R, B2R
and B1B2R (double knockout) deficient mice presented a significant reduction in PTX-induced hypernociceptive responses in
comparison to wild-type animals (p<0.05).
Conclusions:
The present results demonstrated that kinins acting on both B1R and B2R, expressed in spinal and supra-spinal sites, play a
crucial role in controlling the hypernociceptive state caused by chronic treatment with the PTX.
Keywords: neuropathic pain, bradykinin, B1 receptor, B2 receptor, paclitaxel
Financial Support: CNPq, CAPES, FAPESC
Resumo:17-025
ANTI-INFLAMMATORY ACTIVITY OF RACEMIC GONIOTHALAMIN IN PAW EDEMA MODEL INDUCED BY
DIFFERENTS MEDIATORS
Vendramini-costa, D. B. 1,2,3; Tinti, S. V. 1; Pilli, R. A. 3; Carvalho, J. E. 1,2
1
Divisão de Farmacologia e Toxicologia - CPQBA, UNICAMP
2
Instituto de Biologia, UNICAMP
3
Depto de Química Orgânica - Instituto de Química, UNICAMP
Objectives:
The styryl lactone goniothalamin (GTN) is a secondary metabolite naturally found as the enantiomer (R)- form in plants of the
genus Goniothalamus (Annonaceae). Previous studies showed that GTN, both in racemic and in its enantiomeric pure forms, has
in vitro anticancer activity and reduces the Ehrlich solid tumor in mice (Bioorg. Med. Chem. 18; 6742, 2010). Epidemiological
evidences suggest that more than 25% of cancer diseases are related to chronic infections or other types of inflammation. Based
on this relationship and on the antitumor activity apparently with low toxicity, the anti-inflammatory activity of GTN racemic
was evaluated in mice paw edema induced by different mediators.
Balb/C mice (±30g), n=8/group were treated ip as follows: vehicle (saline 0.9%), positive control (Piroxicam 20 mg/kg), GTNrac
30, 100 and 300 mg/kg, doxorubicin 5 mg/kg. After 30 min, inflammation was induced by carrageenan (2,5%, 40µl) into the right
hind footpad and edema was evaluated by plethysmometer (1.5, 3.0, 4.5, 6.0, 24, 48 and 72 hours after induction). The activity
profile of GTNrac was dose dependent with inhibition of all phases at 300 mg/kg and inhibition of the late phase by lower doses
in a similar way by doxorubicin, probably interfering on neutrophils migration and viability. In order to evaluate the possible
mechanism of action, GTNrac at 300 mg/kg was investigated in paw edema induced by PLA2 (Naja naja poison, 5 µg, positive
control dexamethasone 1 mg/kg), histamine (Compound 48/80, 10 µg, positive control ciproheptadin 4 mg/kg) and bradykinin
(BK) (50 µg). GTNrac reduced the inflammatory process induced by PLA2, with maximal inhibition at 30 min (65.0±11.1%),
like dexamethasone (70.8±18.1%) p
Conclusions:
The anti-inflammatory activity of GTNrac, together with its antitumor activity, support the relationship between cancer and
inflammation. This activity may be related with the inhibition of BK-dependent mechanisms, but further studies will be done to
confirm this hypothesis.
Keywords: cancer, goniothalamin, inflammation, mice, paw edema
Financial Support: Fapesp, Cnpq, Capes
Resumo:17-026
THERAPEUTIC ATTENUATION OF SILICA-INDUCED PULMONARY FIBROSIS BY IMMUNOTOXIN
CHIMERIC MOLECULE IL-13PE.
Ferreira, T. P. T. 1; Arantes, A. C. S. D. 1; Rocco, P. R. M. 2; Puri, R. 3; Hogaboam, C. M. 4; Martins, M. A.
1
; Silva, P. M. R. E. 1
1
Fundação Oswaldo Cruz, FIOCRUZ
2
Laboratório de Investigação Pulmonar , LIP
3
3Center for Biologics Evaluation and Research, FDA
4
University of Michigan Medical School, UMMS
Objectives:
IL-13 appears to contribute to the development of pulmonary fibrosis, presumably due to its ability to induce irreversibly
fibroblasts activation, thus representing an attractive target in the case of fibrotic diseases. Inhalation of crystalline silica particles
leads to silicosis, a chronic lung disease characterized by granulomatous inflammation and fibronodular response. Since there is
no effective treatment that can effectively abolish the risk of the disease, in this study we examined the curative effect of the
fusion protein IL-13PE on the experimental model of silica-induced pulmonary in mice.
Male Swiss-Webster mice (18-20 g) were intranasally instilled with silica particles (10 mg/50 µL) and IL-13PE was administered
intranasally, every other day, starting on day 21 up to day 27 post silica provocation. Twenty four hours later, pulmonary function
(resistance and elastance) and airways hyperreactivity to aerosolization with the bronchoconstrictor agent methacholine were
evaluated by invasive whole body plethysmography (Buxco system). All animals were killed and whole lung samples were
prepared for biochemical and histological analyses. All experimental procedures were performed in accordance with the
guidelines of the Committee on Use of Laboratory Animals of the Oswaldo Cruz Foundation (licence L034/09). We showed that
intranasal administration of IL-13PE had a significant therapeutic effect on the increased pulmonary resistance and elastance as
well as on the airways hyperreactivity to methacholine in silicotic mice. IL-13PE reduced the inflammatory infiltrate, fibrotic
response and granuloma formation. Coherently, the expression of extracellular matrix components was sensitive to IL-13PE. In
parallel, the levels of inflammatory and profibrotic chemokines (MIP-2, JE, MIP-1α, TARC and MDC) and cytokines (TGF-ß
and TNF-α) were clearly suppressed in the lung tissue of silica-challenged mice treated with IL-13PE. A less intense labelling for
F4/80 and α-smooth muscle actin (α-SMA) was also noted, indicating reduction of macrophages and myofibroblasts presented in
the lung tissue, respectively. A decrease of the IL-4R, IL-13Rα1 and IL-13α2 receptor-positive cells was noted in silicotic mice
under condition of IL-13PE treatment. Additionally, IL-13PE suppressed TNF-α release from LPS-stimulated macrophages.
Conclusions:
Our findings show that treatment with the immunotoxin IL-13PE effectively inhibited silica-induced lung inflammation and
fibrosis, indicating that it seems to constitute a promising therapeutic approach in the case of chronic inflammatory lung diseases
such as silicosis.
Keywords: Inflammation, silicosis, lung, IL-13PE
Financial Support: FIOCRUZ, CNPq, FAPERJ (Brazil), Global REACH Michigan University (USA),
UNESCO
Resumo:17-027
EFFECT OF ACUTE STRESS IN MECHANICAL OROFACIAL SENSIBILITY AND IN NOCICEPTIVE
SECUNDARY SENSIBILITY IN RATS WITH PERSISTENT INFLAMMATION OF THE TEMPOROMANDIBULAR
JOINTS
Novaes, A. P. R. 1; Leite-panissi, C. R. A. 1,2
1Psychobiology Graduation Program - FFCLRP-USP/SP, FFCLRP-USP/SP
2
2Department of Morphology, Stomatology and Physiology - FORP, FORP/USP
1
Objectives:
This study evaluated the effect of acute stress in orofacial mechanical sensibility and in nociceptive secondary sensibility in rats
with persistent inflammation induced by administration of Complete Freund‟s Adjuvant (CFA) in the temporomandibular joint
(TMJ) bilaterally.
Wistar male rats (250g, N = 34), received administration of CFA (50 mL) or saline (0.9 %) in the region of TMJs. In two groups
were evaluated before and after induction of inflammation or not, the withdrawn threshold of mechanical sensitivity (using von
Frey filaments) and the nociceptive sensibility (by means the hot plate test, HP). After 24 hours the hot plate test the rats were
exposed to the Elevated Plus Maze (EPM) during 5 min. In the other groups, the rats were submitted to the same experimental
procedures described above but before the HP test they were submitted to acute restraint stress for 2 hours. Statistical analysis
showed differences (F1.21 = 15.03, p < 0.001, ANOVA) in the mechanical withdrawal threshold in CFA group, before and after
induction of inflammation in TMJs, which did not happen in the control group. Regarding the nociceptive sensibility there was a
significant hyperalgesia in the CFA group (F1.103 = 63.0, p < 0.01, ANOVA) when compared with the control group. The same
happened to the CFA group that has been previously exposed to acute stress (F1.103 = 45, p < 0.001) when compared to the
control group previously exposed to acute stress. Statistical analysis did not show difference in the EPM analyzed categories.
Again, there was a significant antinociception in the CFA group that has been previously exposed to acute stress (F1.103 = 15.4,
p < 0.001) when compared to the CFA group that was not submitted to the stressful situation
Conclusions:
The results have shown that the CFA administered at TMJs produced widespread mechanical allodynia and hyperalgesia but did
not promote any behavioral changes. Again, the results have shown that acute stress situations are able to reduce the nociceptive
sensibility in the HP test
Keywords: Orofacial pain, Temporomandibular inflammation, stress
Financial Support: CAPES/PROEX, FAPESP, CNPq
Resumo:17-028
DEVELOPMENT OF MECHANICAL ALLODYNIA AND OROFACIAL HYPERALGESIA DEPEND ON
METALLOPROTEINASES AND PROMOTE ALTERED EXPRESSION OF MMP-2 AND MMP-9 IN THE RAT
TRIGEMINAL GANGLION
Nascimento, G. C. ; Gerlach, R. F. ; Leite-panissi, C. R. A.
Dep. Morphology, Stomatology and Phisiology, FORP-USP, FORP-USP
Objectives:
The temporomandibular joints (TMJs) inflammation is considered to be the main cause of pain in patients with
temporomandibular disorders. In laboratory, there are animal models used for studying the TMJ‟s inflammation, such as
intraarticular administration of Complete Freund's Adjuvant (CFA). The purpose of this investigation was to evaluate whether
mechanical allodynia and orofacial hyperalgesia induced by the injection of CFA into the TMJ are dependent on
metalloproteinases (MMPs). In addition, this study characterized the expression of MMP-2 and MMP-9 in the trigeminal
ganglion in different stages of inflammation.
Wistar male rats (250 g, n = 136), received administration of CFA or saline 0.9 % in the TMJs (0.1 mL). In two groups were
evaluated before and after induction of inflammation or not, the mechanical allodynia by the von Frey test. In three other groups,
the rats received intraarticular administration of CFA or saline in TMJs and after 10 days, was injected saline or formalin (2 %) in
the vibrissae region. Immediately after injection, was analyzed the time spent by the rat scratching orofacial area for 45 min. In
two groups were removed the trigeminal ganglion for the quantification of gelatinases by the conventional zymography and
zymography in situ. Finally, four groups of animals received administration of doxycycline or vehicle of doxycycline for 10 days
and were evaluated the mechanical allodynia and orofacial hyperalgesia. Statistical analysis showed differences (p < 0.05,
Newman-Keuls) in the mechanical withdrawal threshold, before and after induction of inflammation in TMJs, which did not
happen in the control group (F1,11 = 7.59, p < 0.05, ANOVA). Regarding the hyperalgesia, the CFA+FORM group revealed an
increase of the rubbing time to the orofacial region when compared with other groups (F28, 299 = 4.935, p < 0.001). Again, there
was a significant increase of MMP-9 at 1 and 3 days after injection of CFA (F3,54 = 3.715, p < 0.01) and at 3, 7 and 10 days after
inflammation, was observed an increase of MMP-2 in the ganglion compared with the control group (F3,62 = 6.259, p < 0.001,
F3,67 = 3.409, p < 0.05, Two-Way ANOVA). Also, there was a statistically significant increase in gelatinase activity in the four
periods analyzed (F3,63 = 4.123, p = 0.01, Two-Way ANOVA). Additionally, systemic treatment with doxycycline promoted a
significant reduction of mechanical (F1,15 = 17.308, p < 0.001) and nociceptive orofacial sensitivity (F42,359 = 2.191, p < 0.001,
ANOVA).
Conclusions:
The results have shown that the CFA administered at TMJs produced local inflammatory response that promotes mechanical
allodynia and orofacial hyperalgesia. Besides that, the results suggested that while the MMP-9 in the trigeminal ganglion is
related to the early-stage of the inflammation development, the MMP-2 is associated with in late stage. In addition, the alteration
of the mechanical and nociceptive orofacial sensibility induced by TMJs inflammation are depending on MMP expression.
Keywords: Mechanical allodynia, Metalloproteinases, Orofacial hyperalgesia, Orofacial pain, Temporomandibular inflammation
Financial Support: FAPESP, CAPES/PROEX, CNPq
Resumo:17-029
MINOCYCLINE, A SECOND GENERATION TETRACYCLINE, SHOWS ANTI-INFLAMMATORY AS WELL AS
ANTINOCICEPTIVE PROPERTIES IN RODENTS.
Leite, L. M. B. D. F. ; Viana, G. S. D. B. ; Cruz, G. M. P. D. ; Carvalho, E. T.
Faculdade de Medicina de Juazeiro do Norte - CE, FMJ - CE
Objectives:
Previously we showed that minocycline (Mino) presents a potent peripheral anti-inflammatory effect, unrelated to its
antimicrobial action. In the present work we attempted to study the possible antinociceptive activity of minocycline in three
experimental models of nociception in rodents.
In the writhing test and in the formalin tests, male Swiss mice (25 g) were used. Animals were distributed (8-14 animals per
group) into untreated controls and groups treated with Mino (5, 10, 25 and 50 mg/kg, i.p.). The observed parameters were number
of contortions/20 min for the Writhing test and licking time/5min (sec) measured 5 min after the test initiation (1st phase,
neurogenic) and 20 min later (2nd phase, inflammatory). In the Hargreaves test, male Wistar rats (200 g) were used ( 6-14
animals per group) and the response (sec) for the thermal stimulus was measured in the following groups: carrageenan in the
absence and in the presence of Mino (10 and 25 mg/kg, i.p.). The latency times were measured 1 h later. Results: Mino
significantly reduced by the number of writhing 31, 48 and 77% at doses of 5, 10 and 25 mg/kg, i.p. respectively, as compared o
controls. In the formalin test Mino (5, 10, 25 and 50 mg/kg) reduced the licking time (sec) in the 1st phase from 45 to 63% ,
respectively. However it acted predominantly at the 2nd phase of the formalin test reducing the licking time by 75, 86, 95 and 95
at the same doses. Morphine (2 mg/kg, i.p.) and indomethacin (25 mg/kg, p.o.) were used as reference drugs and reduced the
licking time by 68 and 57% and by 48 and 78% at the 1st and 2nd phases of the formalin test respectively. In the Hargreaves or
plantar test the latency time to thermal stimulus (sec) was the observed parameter. Mino at the doses of 10 and 25 mg/kg, i.p.,
increased in 1.9 and 2.2 times, respectively the latency time as compared to controls (carrageenan only).
Conclusions:
We showed that Mino besides presenting an anti-inflammatory effect shows also a potent antinociceptive effect as detected by the
experimental models in vivo.
Keywords: ANTINOCICEPTIVE, MINOCYCLINE, INFLAMMATION, EXPERIMENTAL MODELS
Resumo:17-030
LIPOPOLYSACCHARIDE IN VIVO INCREASES REACTIVE OXYGEN SPECIES GENERATION IN RAT
PLATELET MAINLY VIA NADPH-OXIDASE ACTIVATION
Lopes-pires, Me. ; Cardeli, N. J. A. ; Anhê, G. F. ; Antunes, E. ; Marcondes, S. ; Naime, C.
Farmacologia/Universidade Estadual de Campinas, Unicamp
Objectives:
The septic responses can be reproduced by LPS injection such as decrease of circulating platelets, along with increased
production of reactive oxygen species (ROS). The Src kinase and PI3K are important to platelet activation, modulate the
formation of ROS and are also responsible for mediating some effects of LPS. We propose investigate the sources of ROS and
the modulatory role of Src and PI3K on ROS formation in platelets of rats treated with LPS
Methods: The present study was approved by the Committee for Ethics in Animal Research (State University of Campinas –
UNICAMP, protocol number 2097-1). Wistar rats (250-320 g) were injected i.p. with saline or LPS (from E. coli, 1 mg/kg) and
after 6 or 48h arterial blood was collected in ACD-C (9:1 v/v). Production of ROS in washed platelets was measured by flow
cytometry using DCFH-DA (5 µM). The platelets were activated with ADP (20 µM) in absence or presence of the inhibitors of
cyclooxygenase (acetylsalicylic acid, ASA), xanthine oxidase (allopurinol), NADPH-oxidase (DPI), electron transport chain
(rotenone), PI3K (wortmannin) and Src kinase (PP2). Aggregation assay was performed using a two channel ChronoLog
aggregometer. Platelets were activated by ADP (20 µM) and in some experiments the platelets were pre-incubated for 3 min with
wortmannin or PP2. Results: The ROS production in platelets of LPS group was significantly higher than in control animals
(increase by 2- and 3-fold at 6 and 48h after LPS injection, respectively). Stimulation of platelets with ADP (20 µM) did not
affect the production of ROS in both groups saline- and LPS-treated rats. Incubation of ADP-stimulated platelets from LPStreated rats with DPI (5 µM) significantly reduced the ROS generation (27% and 60% reduction at 6 and 48h after LPS treatment,
respectively). The increased ROS production in non-stimulated or ADP-stimulated platelets of LPS-treated rats (at 6h or 48h) was
diminished nearly 20% in presence of ASA (100 µM) or Allopurinol (100 µM). Rotenone (100 µM) did not affect ROS
production in platelets of LPS group. ROS production in non-stimulated or ADP-stimulated platelets of saline-treated rats was not
significantly modified by DPI, rotenone, ASA or allopurinol. Incubation of platelets from saline-treated group with wortmannin
(100nM) and PP2 (5nM) reduced in 60% and 30%, respectively, the ADP-induced platelet aggregation, but did not affect the
ROS production. ADP-induced platelet aggregation was significantly reduced after LPS injection, which was not modified by
wortmannin or PP2 pre-incubation. However, the increased ROS production in non-stimulated or ADP-stimulated platelets of
LPS-treated rats at 6h was decreased in 2- and 2.5-fold by wortmannin and PP2, respectively.
Conclusions:
Our data indicate that NADPH-oxidase is the main source of ROS formation in ADP-stimulated platelets of LPS-treated rats, but
the cyclooxygenase and xanthine oxidase enzymes also contribute to this effect. Src kinase and PI3K are not involved in the
effect of LPS on platelet aggregation, but are important to modulate the increased ROS production in platelets of LPS-treated rats
Keywords: sepsis, reactive oxigen species, platelet, NADPH- oxidase
Resumo:17-031
THE PERIPHERAL ANTIEDEMATOGENIC EFFECT INDUCED BY INTRATHECAL MORPHINE IS
ASSOCIATED WITH POTASSIUM CHANNELS
Foletto, V. R. S. ; Martins, M. A. ; Tonussi, C. R.
Departamento de Farmacologia/UFSC, UFSC
Objectives:
The spinal cord has been considered as a potential modulation site of inflammation and not just a simple transmission line from
the periphery to the brain. The dorsal root reflex (RRD) characterized by antidromic activity in the primary afferents fibers can
modulate the peripheral inflammation. In this context, previous studies showed that administration of intrathecal morphine results
in anti-edematogenic effect with involvement of L-arginine / nitric oxide (NO) / cGMP. However, the mechanisms underlying
this action have not yet been fully elucidated. This study extends these findings to assess the hypothesis of the involvement of
potassium channels in this effect in carrageenan (CG)-induced paw edema.
The intrathecal drugs were injected in male Wistar rats (250-350 g) in a volume of 20 μL 30 min before stimulation with
carrageenan (150 μg/50 μl, subcutaneous) in the left hindpaw. The evaluation of edema was measured as increase in paw volume
(ml). The plasma extravasation was quantification by Evans blue staining. The migration of neutrophils was assessed indirectly
by testing the myeloperoxidase (MPO). The quantification of inflammatory infiltrate and vascular congestion was by histological
analysis. The administration of morphine (37 nmol) inhibited the edema, Evans blue dye leakage and vascular congestion.
However, had no effect on the inflammatory infiltrate and on the MPO activity in comparison to control group. The
coadministration of morphine (37 nmol) with 4-aminopyridine (10 nmol), glibenclamide (5 nmol) and Dequalinium (10 pmol)
reversed the effect of morphine.
Conclusions:
These results support the idea that morphine can act on spinal opioid receptors to produce antiedematogenic effect in periphery,
mediated by the opening of potassium channels by NO/cGMP pathway. Several types of K+ channels seem to be involved in the
mediation of the opiate effect, and this may help future pharmacological clinical approaches to control inflammation by acting in
the CNS. Several clinically available drugs that are K+ channel openers, as minoxidil and nicorandil could be associated to
opiates for a combined spinal therapy.
Keywords: edema, dorsal root reflex, morphine, potassium channel
Resumo:17-032
ANALGESIC POTENTIAL OF PHα1β IN A MODEL OF CANCER PAIN
Rosa, F. 2,2,2,2; Trevisan, G. 2,2,2,2; Rigo, F. 3,3,3,3; Ferreira, J. 2,2,2,2; Gomez, M. V. 3,3,3,3
2
Quimica, Ufsm
3
instituto de ensino e pesquisa da santa casa BH, Ufmg
Objectives:
Most cancer patients suffer pain sufficiently severe to require opioid, but sadly they develop tolerance after repeated treatment.
The N-type calcium channel blocker ziconotide (peptide ω-conotoxin MVIIA synthetic form) is efficacious to treat pain in cancer
patients refractory to other treatments, apart its adverse effects. Thus, the discovery of safer ziconotide-like drugs is relevant.
Thus, the aim was observe the analgesic potential a novel calcium channel blocker Phα1β (peptide purified from the spider
Phoneutria nigriventer venom) in a model of cancer pain.
Cancer pain was induced by the inoculation of melanoma B16–BL6 cells in the hind-paw of C57BL/6 mice (20-30 g). After 15
days, mice were intrathecally (i.t.) injected with Ph α1β (10-100 pmol/site) or MVIIA (10-100 pmol/site) and the painful
hypersensitivity (analyzed through von Frey filaments, expressed as paw withdrawal threshold - PWT in grams) and development
of side-effects were evaluated. In another experiment, the antinociceptive effect of Phα1β was also tested in morphine-tolerant
mice. After cancer induction, we observed significant painful hypersensitivity (the value of 50% PWT diminished from
2.068±0.220 to 0.048±0.007 g before and after inoculation of B16–BL6 cells), whereas PBS injection did not produce changes in
sensitivity (2.35±0.21 and 2.03±0.22 g before and after injection). The treatment with Phα1β (30 pmol/site) produced a longlasting (from 0.25 to 6 hours) reduction of cancer-induced allodynia, with maximum effect (Imax) of 57±13% and effective dose
50% (ED50) of 48 (32–72) pmol/site. The MVIIA (30 pmol/site) induced a similar effect (from 0.25 to 6 hours), with Imax of
71±17% and ED50 of 33 (21–53) pmol/site. Phα1β did not induce any detectable adverse effect. But, MVIIA induced doserelated side effects in mice in doses were it reduced pain. Finally, we had observed that Phα1β (30 pmol/site, i.t.) was capable to
control cancer pain in mice that become tolerant to morphine, reducing painful hypersensitivity from 0.25 to 6 hours (Imax of
72±16%).
Conclusions:
Phα1β is as effective as and as potent as MVIIA to induce analgesia in a cancer pain model without adverse effects or losing
efficacy in opioid-tolerant mice. This might point that Phα1β is a promising safe drug to treat cancer pain in patients.
Keywords: toxin, cancer, ziconotide, pain, allodynia
Financial Support: Capes, Cnpq, Fapergs, Famig
Resumo:17-033
THE CONTRIBUTION OF THE TRANSIENT RECEPTOR POTENCIAL A1 (TRPA1) FOR THE NOCICEPTION
MAINTENANCE IN A MICE NEUROPATHIC PAIN MODEL
Pinheiro, F. V. 1,3; Silva, C. R. 1; Oliveira, S. M. 1; Villarinho, J. G. 1; Cabreira, T. N. 1; André, E. 3;
Ferreira, J. 1
1
Programa de Pós-graduação em Farmacologia, UFSM
2
Programa de Pós-graduação em Bioquímica Toxicológica, UFSM
3
Departamento de Biofísica e Farmacologia, UFRN
Objectives:
Neuropathic pain is a debilitating condition that is poorly understood and often untreatable. A common complaint of neuropathic
pain patients is a painful hypersensitivity to noxious (hyperalgesia) or innocuous (allodynia) stimuli. Painful hypersensitivity to
norepinephrine (NE) has been reported in various neuropathic pain conditions that exhibit sympathetically maintained-pain,
particularly in traumatic lesion of peripheral nerves. It has been suggested that some reactive oxygen species of NE metabolism
are involved in neuropathic pain maintenance. TRPA1, a nonselective cation channel expressed by nociceptors, is involved in
acute pain, hyperalgesia and neurogenic inflammation and may be activated by several reactive species. Thus, the aim this study
was to investigate the involvement of TRPA1 receptor in painful hypersensitivity model of traumatic neuropathic pain in mice.
Adult male and female Swiss mice (25-30g; n=5-6) were used in this study. The animals were anesthetized (90 mg/Kg of
ketamine plus 3 mg/Kg of xylazine, intraperitoneal route, i.p.) and loosely constrictive ligatures were carried out around the right
sciatic nerve to induce neuropathy (chronic constriction injury-CCI). Sham surgery was done by exposing the sciatic nerve
without performing constriction. Before and 7 days after these procedures, the mechanical sensitivity was measured through
application of Von Frey filaments on mice right hind paw. The paw withdrawal threshold (PWT) response was evaluated to
verify the development of allodynia, characterized as a decrease on PWT. The mechanical allodynia were assessed 0.5-48 h after
the administration of the TRPA1 antagonist HC-030031 (100 mg/kg, i.p.) or vehicle (10 mg/kg, i.p.). To investigate the NEinduced hypersensitivity, distinct groups of animals were treated subcutaneously into the right hind paw with vehicle (20 µl/paw)
or NE (30 ng/paw) in presence or absence of the TRPA1 antagonist. Mice were observed for 5 min following NE injection, and
the time of paw licking was measured as nociceptive response. The injection of NE in sham-operated mice did not produce
nociceptive response when compared with its vehicle (5±6 and 4±2 s of response, respectively). On the other hand, injection of
NE in neuropathic mice produced a marked nociceptive response when compared with vehicle group (41±2 and 8±3 s of
response, respectively). As expected, CCI induced allodynia in mice when compared with sham-surgery (PWT of 0.35±0.06 and
2.89±0.17 g, respectively). The systemic injection of HC-030031 (100 mg/kg, i.p.) significantly reversed the decrease in PWT
produced by CCI surgery at 1, 2, 4 and 24 h after treatment (maximum inhibition of 76 ± 16% at 1h).
Conclusions:
These results indicate the participation of TRPA1 receptor in a mice model of neuropathic pain, especially in nociceptive
responses evoked by NE. Thus, TRPA1 receptor may be a new pharmacological target for the treatment of sympatheticallymaintained neuropathic pain.
Keywords: norepinephrine, TRPA1, hyperalgesia, neuropathic pain
Financial Support: CAPES, CNPq, CCNE/UFSM, CCS/UFSM
Resumo:17-034
PARTICIPATION OF GLIALS CELLS IN DORSAL ROOT GANGLION AFTER MUSCLE PAIN
Bonifacio, R. P. ; Freitas, M. F. D. ; Chacur, M.
Departamento de Anatomia - Universidade de São Paulo, USP
Objectives:
Pathological changes in skeletal muscle tissue during the inflammatory response manifest often in the presence of pain and
allodynia. These results are usually attributed to changes occurring in nociceptors caused by the release of pro-inflammatory
substances in the injured tissue. Glial cells have an important role for the transmission of nociceptive information in the central
nervous system. However, the participation of these cells in the dorsal root ganglion (DRG) of the spinal cord has been little
studied.
In this study, we evaluated the possible involvement of glial cells (astrocytes and microglia), through their expression, using
immunofluorescence assay in DRG after induction of musculoskeletal pain. We used Wistar male rats with 180-220g, supplied
by Institute of Biomedical Sciences, University of São Paulo (ICB-USP). For the inducing of painful process, we used
Carrageenan, at dose of 400μg/150μL through intramuscular injection. In order to determine the sensibility we used mechanical
hyperalgesia Von Frey electronic and thermal hyperalgesia Hargreaves test. Those tests were applied before (time zero-MI) and 2
hours after the induction of acute myositis. In other to study the involvement of glial cells, we used Fluorocitrate, a specific
inhibitor of those cells, by intrathecal injection. Two hours after carrageenan and fluorocitrate injections, a group of animals were
perfused and the DRG (L5) sections were incubated for GFAP staining. Our results showed an increase of glial cells in animals
with myositis compared to the control group and a decrease in painful process and glial cells in animals that received
Fluorocitrate injection. Regarding immunofluorescence assay, we observed an increase of GFAP- immunoreactivity after injury
and a decrease after glial inhibition administration.
Conclusions:
Our results suggest that glial cells are involved in muscle pain process, once the pain threshold was decreased after treatment with
glial inhibitor. These studies provide a basis for better understanding of the mechanisms involved in muscle pain, which are of
great clinical relevance.
Keywords: Hiperalgesia, spinal cord, glial cells, dorsal root ganglion, muscle pain
Financial Support: Financial support FAPESP (Research Support Foundation of São Paulo).
Resumo:17-035
PARTICIPATION OF THE SPINAL CORD TRANSIENT RECEPTOR POTENTIAL A1 (TRPA1) IN THE CFAINDUCED INFLAMMATORY HYPERALGESIA IN RATS
Klafke, J. Z. 1; Silva, M. A. 1; Rossato, M. F. 1; Guerra, G. P. 1; Ferreira, A. P. O. 1; Rigo, F. K. 3; Dalmolin,
G. D. 1; Mello, C. F. 2; Rubin, M. A. 1,2; Ferreira, J. 1,2,3
1
Programa de Pós Graduação em Bioquímica Toxicológica, UFSM
2
Programa de Pós-Graduação em Farmacologia, UFSM
3
Programa de Pós-Graduação em Farmacologia Bioquímica, UFMG
Objectives:
TRPA1 receptor is expressed in peripheral and spinal terminals of sensory neurons and is a cationic channel that is involved in
several physiological and pathological states. The tissue injury produces endogenous substances that directly activate or sensitize
TRPA1 to cause pain and inflammation, such as the lipid peroxidation product 4-hydroxy-2-nonenal (HNE). Recently, we
observed that lipid peroxidation may also occur in spinal cord in models of during inflammatory pain (such as that induced by
complete Freund's adjuvant-CFA), but the role of its products in activating TRPA1 is unknown. Thus, the aim of this study was
to evaluate the participation of the spinal cord TRPA1 receptor in the CFA-induced inflammatory hyperalgesia in rats.
Prior to injection with CFA, adult male Wistar rats (200-250 g, n = 4-8) were submitted to baseline measure of the paw
withdrawal latency to radiant heat through Hargreaves test. Then, rats were injected with 100 µL of 1 mg/mL CFA into the left
hind paw and after 24 h later, the development of thermal hyperalgesia was evaluated. Once thermal hypersensitivity had been
demonstrated, rats were counterbalanced in 2 groups: camphor, a TRPA1 antagonist, (3000 nmol/site) or its vehicle (5% DMSO,
5% Tween 80 on 10 mM PBS) were intrathecally administrated. Other group of rats was treated with camphor 15 min prior CFA
injection. Paw withdrawal latencies were then reassessed at 0.5, 1 and 2 h post-dosing. Immediately after the last test, the
ipsilateral and contralateral lumbar segments of rat‟s spinal cords were taken in order to determine the TRPA1 receptor
expression and 4-HNE levels. The expression of TRPA1 receptors was assessed by Western blot analysis and the relative
concentration of HNE adducts in proteins between groups was determined using slot blot immunoassay. The CFA clearly
decreased paw withdrawal latency after 24 h of its intraplantar injection. The intrathecal camphor administration significantly
reduced the CFA-induced hyperalgesia at 2 h post CFA injection. Intrathecal injection of camphor produced significantly a
reduction in about 39±5% of the CFA-induced hyperalgesia at sum of the 0.5, 1 and 2 h after of 24 h CFA injection. When the
rats were treated with camphor 15 min prior to CFA injection, no effect was observed compared with CFA group. Western blot
analysis showed that CFA injection significantly increased TRPA1 band density in 65±23% at the lumbar segment ipsilateral
when compared with the control values at 24 h post CFA injection. No change was observed in the contralateral side. Similarly,
CFA injection significantly increased the HNE levels in 83±23% at 24 h in the lumbar segment ipsilateral when compared with
the control values, whereas no change was observed in the contralateral.
Conclusions:
These results suggest that CFA possibly enhanced the amount of TRPA1 and HNE only at ipsilateral lumbar segment of spinal
cord. The results herein reported demonstrate that the production of HNE and the increase of the TRPA1 density seem to be an
important mechanism involved in the maintenance of the hyperalgesia induced by CFA injection in rats.
Keywords: 4-hydroxy-2-nonenal (HNE), camphor, complete Freund's adjuvant (CFA), inflammation, nociception
Financial Support: CAPES; CNPq; CCNE/UFSM.
Resumo:17-036
TESTOSTERONE ROLE IN THE ANTINOCICEPTIVE EFECT INDUCED BY ADRENOCEPTORS BLOCKADE IN
RATS.
Fávaro-moreira, N. C. 1; Okoti, L. W. 1; Furini, R. 1; Tambeli, C. H. 2,1
1
Ciências Fisiológicas/Faculdade de Odontologia de Piracicaba, FOP-UNICAMP
2
Anatomia, Biol Celular, Fisiologia Biofísica/IB-UNICAMP, IB-UNICAMP
Objectives:
We have previously demonstrated that testosterone protects males rats from developing articular pain and that β-adrenoceptors
contribute to formalin-induced temporomandibular joint (TMJ) nociception. Therefore, our aim was to investigate whether
testosterone modulates the antinociceptive effect induced by blockade of β1, β2 and β3 adrenoceptors in the rat´s TMJ.
Intact or gonadectomized with or without testosterone replacement Male Wistar rats (200-300g) were used. Saline (0.9% NaCl)
or 1.5% Formalin was co-administered with β1 (6, 18, 54, 162µg), β2 (0.1, 0.3, 0.9µg) or β3 (0.5, 1.5, 4.5, 13.5µg) adrenoceptors
antagonists, Atenolol, ICI 118.551 and SR59230A respectively, into the TMJ of rats. The nociceptive behavior was quantified for
45 minutes and used as a quantitative nociceptive behavior measure (Pain, 94: 185, 2001). The data were analyzed by One or
Two Way ANOVA and Tukey post-hoc test (p < 0.05). Beta-adrenoceptors antagonists significantly reduced formalin-induced
TMJ nociception in intact males (β1: 63.19%; β2: 66.14%; β3: 61.03%) and in gonadectomized males with (β1: 60,72%; β2:
57,39%; β3: 66,18%) or without (β1: 63.79%; β2: 66.26%; β3: 69,51%) testosterone replacement, confirming the βadrenoceptors contribuition to TMJ nociception. Furthermore, these findings indicate that endogenous testosterone does not affect
the antinociceptive effects Atenolol and ICI.118.551, but significantly reduces the antinociceptive effect of SR59230A.
Conclusions:
We conclude that local blockade of β1, β2 and β3 adrenoceptors significantly reduces TMJ nociception in males with or without
testosterone. Furthermore, since testosterone seems to be important to the antinociceptive effect of SR59230A, a higher dose of
β3-blockers may be necessary to induce TMJ antinociception in females.
Keywords: Adrenoceptors, Temporomandibular joint, Nociception, Testosterone
Resumo:17-037
OROFACIAL SENSORY CHANGES AFTER STREPTOZOTOCIN-INDUCED EXPERIMENTAL DIABETES IN
RATS
Nones, C. F. M. ; Cunha, J. M. ; Chichorro, J. G.
Farmacologia / Universidade Federal do Paraná, UFPR
Objectives:
Peripheral neuropathy is one of the most common complications of diabetes and is often accompanied by episodes of pain, which
can affect approximately 50% of patients (Diabetes Care 33:2285, 2010). There is growing evidence that diabetic neuropathy also
involves the trigeminal nerve, altering the transmission of orofacial sensory information (Brain Res. 865:112, 2000). This study
aimed to evaluate the time course of orofacial sensory changes in the face after streptozotocin-induced experimental diabetes in
rats.
After 12 h of food deprivation male Wistar rats (180–220 g), received vehicle (citrate buffer, 1mL/kg, i.p.; 10 mM, pH 4.5) or
streptozotocin (STZ, 50 mg/kg; i.p.). Three days later, the development of diabetes was confirmed by tail vein blood glucose
levels. Only rats with non-fast blood glucose levels higher than 250 mg/dL were included in the study. Thermal heat and cold
hyperalgesia were assessed before STZ injection and once a week up to 5 weeks after STZ. Cold stimulation consisted in the
application of a tetrafluoroethane spray to the center of the vibrissal pad and the duration of facial grooming behavior was
recorded over the first 2 min as an index of cold-induced nociception. Heat hyperalgesia was estimated as a decrease in the
latency to display head withdrawal or snout vigorous flicking after application of radiant heat to the face. All experimental
procedures were previously submitted to the Committee on the Ethical Use of Animals of the UFPR. The average baseline cold
responsiveness for the normoglycemic group (n = 6) was 9.1 ± 1.3 s and for the STZ group (n = 8) was 7.5 ± 1.4 s. One week
after vehicle or STZ treatment the facial grooming elicited by the cold stimulus was 10.4 ± 4.3 s and 20 ± 2.9 s, respectively (P
Conclusions:
Our results suggest that painful neuropathy associated with diabetes induces long lasting thermal hyperalgesia, to both heat and
cold, in the orofacial region. Further experiments are underway to assess the orofacial mechanical responsiveness of rats after
experimental diabetes.
Keywords: Diabetes, Orofacial pain, Rat, Streptozotocin
Resumo:17-038
EVALUATION OF ANTI-INFLAMMATORY ACTIVITY OF (+)-TRANS-4-HYDROXY-6-PROPYL-1OXOCICLOEXAN-2-ONE
Zardo, R. D. S. 1; Machado, L. D. S. 1; Miranda, L. S. D. M. E. 1; Vasconcellos, M. L. A. D. A. 2; Costa, J.
S. 1; Pereira, V. L. P. 1; Fernandes, P. D. 1; Marinho, B. G. 3
1
2
Departamento de Química, UFPB
3
Departamento de Ciências Fisiológicas, UFRRJ
Objectives:
Evaluate the anti-inflammatory properties of synthetic compound 4-hydroxy-6-propyl-1-one-2-oxocicloexan in air pouch model
in mice and nitric oxide production by RAW 264.7 cells.
Swiss mice, weighing 17–24 g were used in appropriate conditions for the implementation of research, obtaining approval of the
Ethics Committee in Animal Experimentation (ICBDFBC015). The animals were divided into 5 groups of 8 animals. The air
pouch model was performed as described in J. Neuro-endocrinol, 15: 93, 2003. A solution of carrageenan (1%) or saline was
administered in the air pouch. The mice received the compound (0.1; 1 and 10 mg/kg) and vehicle intraperitoneally 1 hour before
and 23 hours after injection of carrageenan. The animals were euthanized 24 hours after administration of carrageenan by
overdose of anesthetics ketamine and xylazine. The exudate was collected from the air pouch and then used for quantification of
total leukocytes and proteins. The total number of leukocytes obtained from the exudates was: Saline – 1.5 + 0.3 x 106 cells/ml;
Carrageenan + Vehicle - 16 + 2.4 x 106 cells/ml; Carrageenan + 0.1 mg/kg – 11.1 + 3.3 x 106 * cells/ml; Carrageenan + 1 mg/kg
– 9.3 + 2.2 x 106 * cells/ml; Carrageenan + 10 mg/kg – 9.2 + 2.2 x 106 * cells/ml. Protein values obtained from the exudate were:
Saline – 43.7 + 19 µg/ml; Carrageenan + Vehicle - 206.5 + 21.3 µg/ml; Carrageenan + 0.1 mg/kg – 166.6 + 23.1* µg/ml;
Carrageenan + 1 mg/kg – 127.1 + 31.1* µg/ml; Carrageenan + 10 mg/kg – 110.5 + 31.1* µg/ml. To evaluate the production of
nitric oxide, nitrite concentration in the supernatant of RAW 264.7 cells (macrophage-like cell line) was measured using the
Griess reaction according to Analytical Biochemistry, 126: 131, 1982. RAW 264.7 cells were activated with LPS
(lipopolysaccharide) for activation of nitric oxide production. RAW 264.7 cells activated with LPS were incubated with the
compound at concentrations of 10, 30 and 100 µM, obtaining the following results: Non-activated RAW 264.7 – 19 + 3.4 µg/ml;
Activated RAW 264.7 – 71.1 + 8.4 µg/ml; Activated RAW 264.7 + 10 µM – 49.3 + 9.2* µg/ml; Activated RAW 264.7 + 30 µM
– 51.4 + 1.9* µg/ml; Activated RAW 264.7 + 100 µM – 17.7 + 1.9* µg/ml. MTT assay was performed as described in Journal
Immunology, 89: 271, 1986 to assess cell viability. Incubation of cells with the compound was not able to alter cell viability at
the concentrations used (data not shown). The statistical significance between groups was performed by applying analysis of
variance (ANOVA) followed by Bonferroni test. A p value below 0.05 was used as significance level.
Conclusions:
The results show that the (+)-trans-4-hydroxy-6-propyl-1-oxocicloexan-2-one is able to reduce leukocyte migration and protein
extravasation, confirming its ability to reduce vascular permeability. Moreover, the compound was effective in inhibiting the
production of nitric oxide in vitro and did not develop cytotoxicity in RAW 264.7 cell line.
Keywords: Inflammation, nitric oxide, synthetic compound
Financial Support: FAPERJ and CNPQ
Resumo:17-039
ROLE OF CHEMOKINE RECEPTOR CCR4 AND REGULATORY T CELLS IN THE WOUND HEALING OF
SURGICALLY-INDUCED SKIN LESION IN MICE
Figueiredo, J. B. ; Borges, P. A. ; Brogliato, A. R. ; Benjamim, C. F.
Objectives:
Wounds of difficult healing affect 2% of population in developed countries. Despite of its clinical revelance, in Brazil there are
not statistical data yet. Several cell types and mediators are involved in the skin wound healing, among them we are interested in
the CCR4, CCL17 and CCL22 receptor, and T regulatory cells (Tregs). It is well known that Tregs express CCR4 in the cell
membrane, and this receptor is important for its activation and migration. Tregs main function is to modulate the immune system.
Tregs are found in the skin of humans and mice, however the role of this cell type in the healing process has not been described
yet. Assuming that the innate and acquired immune response is fundamental to the healing process, we believe that Tregs play an
important role in the healing of skin lesions. Our objective was to describe the role of CCR4-expressing Tregs in cutaneous
wound healing in normal and diabetic mice.
Diabetes was induced with a single dose of alloxan (75 mg/kg body weight) by intravenous injection (iv) in C57BL6 (WT) and
CCR4-/- (KO) mice. The wounds were induced surgically and followed for 14 days. Diabetic WT mice have a delayed healing
process compared to diabetics KO mice. Non-diabetic mice (WT and KO) did not present significantly difference in the wound
closure. Histological analysis was performed on sections stained with H&E obtained from lesions on day 7. It was observed that
the non-diabetic WT and KO mice have a similar histological profile. However, the diabetic WT mice have a greater number of
infiltrate cells and numerous new blood vessels compared to KO mice . In order to characterize the cellular infiltration in the
injury, we performed the quantification of neutrophils in the wound skin, but no differences were observed in non-diabetic and
diabetic KO and WT mice. Another parameter evaluated was the quantification of TNF-α, which was measured on day 2 after
lesion, by ELISA. In diabetic group it was observed that WT mice have a greater release of TNF-α than KO diabetics mice. In the
group of non-diabetic mice there was no statistical difference. Also, the production of growth factor TGF-β was evaluated, but it
did not show statistical difference between the groups.
Conclusions:
So far, we showed that WT and KO non-diabetics group have a very similar wound healing. However, diabetic KO mice close
the wound faster and produce less TNF- α , suggesting that the CCR4 and probably Tregs, recruited to local lesion in the skin by
this receptor, affect positively the skin wound healing.
Keywords: Wound healing, diabetic, Chemokine receptor CCR4
Financial Support: CNPq and Faperj
Resumo:17-040
ANIMAL MODEL FOR NEUROPATHIC PAIN STUDY AND DRUG DEVELOPMENT
Silva, A. M. S. 1; Trachez, M. M. 2; Antunes, F. 3; Zapata-sudo, G. 1; Sudo, R. T. 1
1
Programa de Desenvolvimento de Fármacos / ICB, UFRJ
2
3
Centro de Ciências Médicas, UFF
Centro de Ciências e Tecnologias Agropecuárias, UENF
Objectives:
Pharmacological treatment of chronic pain as neuropathic pain is still an unresolved issue in medical practice. Due to need of
animal model to investigate the efficacy of potentially new compounds designed for neuropathic pain control, the time course of
two different models based on chronic constriction injury (CCI) and spinal nerve ligation (SNL) was investigated in this study.
This study was performed in compliance with the Animal Care and Use Committee at Universidade Federal do Rio de Janeiro
(protocol # DFBCICB017). The CCI and the SNL were performed in male Wistar rats (180 – 200 g) under anesthesia with
ketamine (100 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.). Time course of behavioral tests were performed from day 2 after surgery
up to complete recovery of nociception to control level. Thermal and mechanical sensitivities were measured by plantar analgesia
meter (IITC mod 33) and modified digital von Frey filament (Insight Equipamentos) devices, respectively. Decreases of thermal
(thermal hyperalgesia) and mechanical (allodynia) sensitivities threshold were considered as success of nerve surgery induced
neuropathic pain. The data were expressed as mean ± S.E.M (n= 6). The latency to thermal stimulation decreased from 10.8±1.1 s
(control) to 6.2±0.4 s (P
Conclusions:
The thermal hyperalgesia and allodynia were successfully induced by CCI and SNL in rat. These models may be used for
effectiveness test of drug potentially used in the control of neuropathic pain.
Keywords: animal model, drug development, neuropathic pain
Financial Support: CNPq, FAPERJ, CAPES, INCT/INOFAR and CRISTÁLIA
Resumo:17-041
EFFECTS OF EZETIMIBE + SIMVASTATIN COMBINATION ON THE LIVER METABOLIC CHANGES OF
ARTHRITIC RATS
Bracht; L. 2; Barbosa, C. P. 3; Cuman, R. K. N. 3; Caparroz-assef, S. M. 3; Ishii-iwamoto, E. L. 3; Bersaniamado, C. A. 3
2
Universidade Tecnológica Federal do Paraná, UTFPR
3
Laboratório de Inflamação, Universidade Estadual de Maringá, UEM
Objectives:
In addition to their favorable effects on lipid levels, combined therapy with ezetimibe + simvastatin significantly reduces Creactive protein levels compared with statin monotherapy (Am. J. Cardiol. 99; 1706, 2007). Additionally, our group of
researchers has previously demonstrated that the ezetimibe + simvastatin combination was more effective in reducing the
inflammatory response in arthritic rats than atorvastatin, simvastatin or ezetimibe monotherapy. Despite their beneficial
advantages, statins can cause hepatotoxicity (Minerva Gastroenterol. Dietol. 38; 41, 1992), and combining simvastatin and
ezetimibe seems to increase plasmatic transaminase activity (Clin. Liver Dis. 11, 597, 2007). This fact may represent a major
problem, since the development of adjuvant arthritis in rats is often accompanied by several liver metabolic alterations, such as
the diminished gluconeogenesis from several substrates. Therefore, the aim of the present study was to investigate the effects of
ezetimibe + simvastatin combination on the plasma transaminases activity and on the liver metabolic changes in arthritic rats.
Arthritis was induced by the intradermal injection of a suspension of Mycobacterium tuberculosis (100 μg) in mineral oil (CFA)
into the plantar surface of the hind paws. Ezetimibe10mg/Kg + simvastatin40mg/Kg were given intragastrically and the treatment
began on the day of CFA injection and continued daily up to the 28th day after arthritis induction. At the end of the experimental
period, the rats were sacrificed and blood was collected to measure the activity of aspartate transaminase (AST) and alanine
trasaminase (ALT). Rats were also submitted to liver perfusion experiments. The perfusion fluid was Krebs/Henseleit bicarbonate
buffer (pH 7.4), saturated with O2 and CO2 (95:5) by means of a membrane oxygenator and simultaneously heated to 37o C.
Alanine was used as gluconeogenic substrate. The oxygen concentration in the perfusate was monitored continuously by means
of polarography. Metabolite production was measured by means of enzymatic assays. Treatment of arthritic animals with
ezetimibe + simvastatin combination markedly increased AST and ALT activity. Compared with control non-treated arthritic rats,
the activities were 366% and 450% higher, for ALT and AST, respectively. Liver metabolism was also affected by ezetimibe +
simvastatin treatment. Glucose production in normal or arthritic treated animals was considerably lower (-52%) in comparison
with their respective controls. Pyruvate and lactate production from L-alanine was comparably similar in all experimental
conditions. Treatment with ezetimibe + simvastatin led to a significant increase in basal oxygen uptake in normal treated (+15%)
or arthritic treated rats (+44%) when compared to normal or arthritic control animals. Ammonia and urea production was not
significantly altered.
Conclusions:
The ezetimibe + simvastatin combination clearly affect liver function, besides the augmentation in AST and ALT plasma
activities. This combination worsens the diminished gluconeogenesis seen in arthritic animals. Further experiments are needed to
better investigate these findings.
Keywords: adjuvant arthritis, liver metabolism, ezetimibe, simvastatin
Financial Support: CNpq/UEM, UTFPR
Resumo:17-042
TOPOGRAPHICAL -EVALUATION OF THE ANALGESIC EFFECT INDUCED BY TRANSDURAL ELECTRICAL
STIMULATION OF THE MOTOR CORTEX OF RATS: SOMATOTOPY OF BEHAVIORAL RESPONSE.
França, N. R. M. 1,2; Paes, R. A. 2; Fonoff, E. T. 2,4; Alves, A. D. S. 3; Britto, L. R. G. D. 3; Dale, C. S. 2,1
1
Departamento de Anatomia - ICB/USP, ICB-USP
2
Hospital Sírio Libanês, HSL
3
Departamento de Fisiologia e Biofísica - ICB/USP, ICB-USP
4
Departamento de Neurologia/Faculdade de Medicina-USP, IPq-HCFMUSP
Objectives:
Subliminal stimulation of the motor cortex has been used to treat patients with neuropathic pain resistant to other conventional
treatments (Nguyen et al., Pain, 82:245, 1999; Canavero e Bonicalzi., J Neurosurg, 94:688, 2001; Brow e Barbaro., Pain,
104:431, 2003; Nuti et al., Pain, 118:43, 2005). Data obtained by our group demonstrated that transdural electrical stimulation of
the motor cortex induces opioid-dependent analgesia in normal or neuropathic rats evaluated by the paw pressure test (Fonoff et
al., Behav Brain Res, 196:67, 2009a; Pagano et al, Eur J Pain, 15:268e1, 2011). This study investigates the possible topography
involved in this effect.
Male Wistar rats (200-250g) underwent surgery for implantation of transdural electrodes positioned on four distinct areas of the
corresponding motor cortex: whiskers, fore limb, hind limb and tail determined in accordance with cortical mapping carried out
by our group (Fonoff et al., Behav Brain Res, 202:138, 2009b). One week after surgery, animals were submitted to sessions of 15
min. of electrical stimulation (60Hz, 210ìs 1.25mA; Fonoff et al., Behav Brain Res, 196: 67, 2009a). At the end of 15 minutes,
and still under stimulation, animals were assessed in four behavioral tests to determine the pain threshold as follows: a) paw
pressure test of the hind paw (Randall e Selitto, Arch Intern Pharmacodyn 111: 209, 1957), b) mechanical hyperalgesia to fore
limbs with calibrated von Frey microfilaments (Takasaki et al. Pharmacol Exp There, 296:270, 2001), c) mechanical hyperalgesia
with electronic von Frey for wiskers (Denadai-Souza, Eur J Pain, 13: 812 , 2009) and d) tail pinch test (Nalwalk et al. Pharm
Biochem Behavior, 50: 421, 1995). Groups of animals implanted with electrodes but not electrically stimulated were evaluated as
control groups. Results demonstrate that transdural electrical stimulation induced antinociception correspondend to the cortex
implanted area for each experimental group: a) hind paw 98,33±4,594 for stimulated (ST) against 68.11±2,09 for control; b) fore
limbs 28.33±10 for ST against 20.83±7,04 for control; c) wiskers 74.26±6,65 for ST and 48.13±2,95 for control and d) tail
14.25±1,03 to ST and 10.33±0,88 to control.
Conclusions:
Data presented herein demonstrate that the analgesia induced by transdural electrical stimulation has topographic effect, specific
and relevant to the area of motor cortex stimulated. These data may help in understanding the mechanisms involved in analgesia
induced by cortical stimulation.
Keywords: Eletrical stimulation, Antinociception, Motor cortex, Analgesia
Financial Support: Hospital Sírio-Libanês, FAPESP and CAPES.
Resumo:17-043
ALPHA-7 NICOTINIC RECEPTOR STIMULATION MODULATES SOME ASPECTS OF ANTIGEN-SPECIFIC
RESPONSE
Pinheiro, M. L. ; Ribeiro, A. ; Ferraz-de-paula, V. ; Quinteiro-filho, W. M. ; Palermo-neto, J.
Faculdade de Medicina Veterinária - USP, FMVZ/USP
Objectives:
The inflammatory reflex is a neurophysiological mechanism that regulates the immune system. Recent studies indicate that the
vagus nerve modulates the immune response through a nicotinic anti-inflammatory pathway. The aim of this work was to
investigate the effects of an alpha7 nicotinic agonist (Anabasine) treatment on spleen dendritic cell phenotype, serum cytokines
production and on a Delayed Type Hypersensibility (DTH) model in ovalbumin (OVA)-sensitized mice.
The first experiment was done to evaluate dendritic cell phenotype and serum cytokines production: 20 C57BL/6 male mice were
used and divided in 2 groups: saline (S), anabasine 4mg/kg (A). Each mouse received one single dose (i.p.) and 30 minutes latter
mice were sensitized with OVA (50&mug/mouse, s.c.). After 24 hours, mice were euthanized and blood and spleens were
collected. Splenocytes were cultured with OVA for 12 hours, and dendritic cell phenotype was analyzed by flow cytometry.
Serum cytokines profile was rated also by flow cytometry using CBA inflammation kit. For the DTH: 24 C57BL / 6 mice were
divided in two groups and treated like the previous experiment. After 7 days, they were injected with aggregated-OVA (4%) on
the left paw to measure DTH by foot paw thickness. Anabasine decreased the MHC class II expression (S: 136.2±11.9;
A:117.5±8.5), increased CD80 (S: 90.3±9.0; A:101.6±8.7) and CD86 (S:75.1±5.1; A:83.6±8.3) expression, and increased IL-6 in
serum (S:28.7±8.9; A: 19.5±7.2) but had no effect on TNF, INF-gamma and MCP-1 (p&le0.05) serum levels. Furthermore
anabasine treatment, previously to the immunization, reduced DTH formation (p&le0.001), showing a reduction in the paw
thickness after 12, 24 and 48 hours (p&le0.05).
Conclusions:
We showed that the alpha-7 nicotinic receptor stimulation modifies dendritc cell phenotype in spleen and decrease IL-6
concentration in the serum. Moreover, alpha-7 nicotinic receptor stimulation decreases DTH response. Indeed, these finds suggest
that alpha-7 nicotinic receptor stimulation changes the immune sensitization development and regulates adaptive immunity.
Keywords: Cholinergic system, Dendritic cells, DTH, Neuroimmunomodulation
Resumo:17-044
ANTI-INFLAMMATORY PROPERTIES OF DIBENZALACETONE IN EXPERIMENTAL MODEL OF AIR POUCH
IN RATS.
Oliveira, K. C. M. . 1; Bobadilla, C. X. Ch. 1; Bastos, G. N. T. 1; Bitencourt, H. R. 2; Maués, L. A. L. 1; do
Nascimento, J. L. M. 1
1
Laboratório de Neuroquímica-ICB, UFPA
2
Laboratório de Sintese - ICE, UFPA
Objectives:
Inflammation is a response of the vascularizated tissues to an injury that is morphologically characterized by presence of exudate
and production of Reactive Oxygen Species (ROS), like Nitric Oxide (NO) and Superoxide (O2-). The presence of ROS is a
common characteristic in a oxidative stress of the cell during inflammation. Recent researches investigated the mechanisms
involved in the inflammatory process, to develop new medicines that are capable to treat the inflammatory process. Therefore,
our aim in this work was investigated the anti-inflammatory properties of Dibenzalacetone (DBZ), a synthetic drug.
The anti-inflammatory activity was analyzed in the air pouch model and the animals (Wistar rat´s weighting 150 - 200 g) were
randomly located in five groups: carrageenan (10 mg/Kg), indomethacin (10 mg/Kg i.p.) or DBZ (1mg/Kg; 5 mg/Kg and 10
mg/Kg i.p.). The exudates induced by carragenan were collected to analyze the cellular migration, vasodilatation, reactive oxygen
species and volume of exudates. Our results demonstrated a decrease of cellular migration of injury area in the group of
indomethacin or DBZ on following concentrations: 1 mg/Kg, 5 mg/Kg or 10 mg/Kg (164,5±40,9; 15±12,1; 24,2±5,7 and 7,4±6,8
cells; respectively). The synthetic drug also have decreased nitrite production (25,84±5,58; 17,03±2,86; 17,49±2,39; 17,08±2,0
and 16,03±1,79 μM; respectively). The qualitative decrease of vasodilatation and the depletion of SOD was observed in groups
treated with DBZ, but the depletion of this enzyme was not significant compared with carrageenan group. All procedures
involving animal care and experimentation were performed in accordance with guidelines of the Ethical Committee for Research
with Experimental Animals of the Universidade Federal do Pará (BIO001-09).
Conclusions:
These results suggest that DBZ was effective as anti-inflammatory drug and this was demonstrated by the decrease of cellular
migration, nitrite production (metabolite of nitric oxide) and vasodilatation.
Keywords: Anti-Inflammatory, Dibenzalacetone, Inflammation, Reactives Oxygen Species, Nitric Oxide
Financial Support: CNPq, FAPESPA, UFPA.
Resumo:17-045
EVALUATION OF THE MECHANISMS OF ACTION OF FATTY ACIDS FROM VEGETABLES OILS ON WOUND
HEALING
Brogliato, A. R. ; Borges, P. A. ; Figueiredo, J. D. B. ; Monte-alto-costa; Benjamim, C. F.
Objectives:
Wounds of difficult healing affect 2% of population in developed countries. Despite of its clinical relevance, in Brazil there are
not statistical data yet. Researchers have been showing that fatty acid from vegetal origin improves the wound healing.
Vegetables oils for wound treatment have been used in Brazil currently, but do not have scientific endorsement. Therefore, our
aim was to describe the mechanism of action of Curatec AGE® (commercial product rich with oleic acid, linoleic acid and
mediun chain triglycerides) in skin wound healing in mice.
The wound was surgically induced and treated daily with Curatec AGE®. Mineral oil was used as control. The Curatec AGE®
induced an increase of neutrophil accumulation in the wound at days 1, 4, 7 and 14 showing a peak at day 4. A high level of IL13 in wound homogenate was observed at day 7 but no difference was found in IL-10, IL-6, TNFα and TGFβ levels. Curatec
AGE® showed predominance of fibroblasts in newly formed tissue at day 7 after wound and also an increase of total collagen
deposit in the wound bed. However the wound closure in treated and control groups was similar throughout the 14 days of
evaluation.
Conclusions:
Taken together, Curatec AGE® promoted pro-inflammatory effects only in early days after injury. The presence of new blood
vessels and fibroblasts indicate granulation tissue formation that was not evident in control group. Data also suggest that fatty
acids from Curatec AGE® affect the wound microenvironments leading to an improvement of new tissue formation after
injury.We intend to evaluated the influence of Curatec AGE® on the formation of new blood vessels as well as parameters of
oxidative stress and deposit of matrix proteins in the wound bed.
Keywords: fatty acids, vegetables oils, wound healing
Financial Support: LM Farma Comércio e Indústria Ltda, and CAPES, CNPq and FAPERJ.
Resumo:17-046
ANTINOCICEPTIVE EFFECTS OF APOCYNIN
Castor, L. R. G. ; Hiruma, C. ; Rocha, N. P. ; Ximenes, V. F.
Depto de Farmacologia- Instituto de Biociências, UNESP
Objectives:
Aim: Apocynin, a compound originally extracted from the roots of the Hymalayan herb Picrorhiza Kurroa, has been used in
popular medicine for treatment of various diseases (Stefanka; 2010). Here we aimed to study mechanism of the antinociceptive
effects of apocynin
Methods and Results: Formalin-induced nociception: Animals (mice weighting 25 to 35 g) received 20 µL of 2.0 % formalin
(0.92 % formaldehyde in saline), injected intraplantar (i.pl) in the ventral surface of the right hindpaw. Animals were observed
from 0 to 5 min (neurogenic phase) and from 15 to 30 min (inflammatory phase), and the time spent licking the injected paw was
recorded considered as indicative of nociception. One hour before the administration of formalin, the animals received the
compounds (100 mg/Kg, v.o) and the control groups received vehicle (saline) or piroxicam (30 mg/Kg). To investigate the
oxidonitrergic system, L-arginine/65mg/kg i.p was administrated as a precursor for the synthesis of nitrite oxide (NO) and LName/40 mg/Kg i.p, as an analog of arginine that inhibits NO production. To the studied the serotonergic system the animals
were treated for 4 days with a tryptophan hydroxylase inhibitor (PCPA/100mg/Kg) previously the formalin test. This results were
in oxidonitrergic system in control (250±15.63),l-name(45.57±10.30), apocynin (157±28.61),l-arg(246±19.32),l-arg/lname(226±16.64), l-arg/apo (155±24.51. In serotonergic system the results were control (230±10.60), apocynin (149±14.40),
PCPA (206±14.31) apocynin/PCPA (200±10.86).
Conclusions:
We found that apocynin produces antinociceptive effect. However, ours results demonstrated that oxidonitrergic system was not
involved in antinoception provoked by apocynin. On the other hand, the antinociceptive effect of apocynin was reversed using
PCPA in the phase II of the formalin test. Apocynin has antinociceptive effect when mice are submitted to the formalin test.
Moreover, the serotonergic, but not the oxidonitrergic system, seems to be involved in this anti-inflammatory property of
apocynin.
Keywords: Apocynin, antinociceptive, formalin, test, mice
Financial Support: Capes
Resumo:17-047
PERIPHERAL MECHANISMS INVOLVED IN THE NOCICEPTION TRIGGED BY THE VENOM OF THE ARMED
SPIDER PHONEUTRIA NIGRIVENTER
Gewehr, C. 1; Oliveira, S. M. 2; Trevisan, G. 2; Rossato, M. F. 2; Rubin, M. A. 2; Ferreira, J. 2; Gomez, M.
V. 1
1
INSTITUTO DE ENSINO E PESQUISA DA SANTA CASA DE BH, IEP
2
UNIVERSIDADE FEDERAL DE SANTA MARIA, UFSM
Objectives:
Pain is the main local symptom presented by people inoculated with the Phoneutria nigriventer venom (PNV). However, the
mechaninsms involved in this nociception are poorly unknown. Therefore, the aim of the present study was to identify the
peripheral mechanisms involved in PNV-induced nociception in mice.
A volume of twenty ìl of PNV, diluted in phosphate-buffered saline (PBS), were injected subcutaneously (s.c.) under the surface
of the right hind paw. The amount of time spent licking the injected paw was timed with a chronometer and was considered as
indicative of nociception. This study was approved by the Committee on the Use and Care of Laboratory Animals of our
university (no. 23081.003193/200940). The s.c. injection of PNV (0.3-10 ìg/paw) produced an immediate and short lasting
spontaneous nociception. The effective dose to produce 50% effect (ED50) caused by PNV was 1.6 (0.8-2.4) µg/paw and the
maximal licking responses was 147.5±17.0 s. This effect was largely prevented by afferent C-fiber desensitization, but not mast
cells degranulation. Notably, the boiling or the dialysing of PNV reduced the spontaneous nociception induced by the venom,
with inhibition of 55±14% and 55±18%, respectively, indicating that termolabile and low molecular weight substances are
important by nociceptive action. We identified that serotonin (acting on 5-HT4 receptor), but not glutamate and histamine,
presented in PNV are important by nociceptive effect of the venom. Moreover, we detected that PNV contained a tissue
kallikrein-like activity that is also involved in venom-induced nociception, the kinetic curve demonstrated that the tissue
kallikrein-like enzyme in PNV (150 mg/ml) had a KM value of 1.47±0.09 mM and a maximal velocity Vmax of 16.13±0.62
nmol/mim/mg protein. Moreover, the kallikrein-activity of PNV (500 mg/ml) was inhibited by boiling PNV or by the tissue
kallikein inhibitor aprotinin (10 ìg/ml), but not by plasma kallikrein inhibitor SBTI (3 ìg/ml). The s.c. co-administration of the
inhibitor of tissue kallikrein aprotinin (100 µg/paw), but not of plasma kallikrein soybean trypsin inhibitor (3 µg/paw), was
capable to reduced the PNV-induced nociception, with a inhibition of 70±12% compared to control group. The s.c. coadministration of anti-ophidic serum (30 times dissolved in PBS) was capable to reduced the nociceptive response induced by
PNV (3 µg/paw), with inhibition of 54±8% compared to control group. The blockade of tetrodotoxin sensitive Na+ channel or the
antagonism of kinin B2 receptor, acid sensitive ion channel (ASIC) receptor or vanilloid receptor (TRPV1) reduced PNV-trigged
nociception. However, high concentrations of PNV (1.5-150 µg/ml) were not capable of altering the specific binding of
[3H]resiniferatoxin to TRPV1 receptor.
Conclusions:
Collectively, the results of the present study demonstrate that PNV produces spontaneous nociception by stimulation of receptors,
such as 5-HT4, present in C-fiber or by the activation of a kinin generating kallikrein-like enzyme.
Keywords: Phoneutria nigriventer, nociception, mice, vanilloid receptor, venom
Financial Support: Conselho Nacional de Desenvolvimento Científico
Resumo:17-048
LAPACHOL AS PROTECTIVE AGENT AGAINST INFLAMMATION INDUCED BY TOTAL AND SPLA2 FROM
CROTALUS DURISSUS CASCAVELLA VENOM.
Campos, S. C. D. 1; Bortoloti, L. V. 1; Fagundes, F. H. R. 1,2; de Paula, V. I. 2; Soares, V. C. G. 3,1; Filho, E.
B. S. D. 3; Minaya, M. A. B. 3; Marangoni, S. 3; Toyama, M. H. 3,4
1
2
ICS/Universidade Paulista, UNIP
Centro Universitário Padre Anchieta, Unianchieta
3
Depto. de Bioquímica, UNICAMP
4
Depto. de Bioquímica, UNESP
Objectives:
The search for new potentially active molecules such as anti-inflammatory ones is a wide field. New research strategies with
secretory fosfolipases A2 (sPLA2) purified from crotalic rattle snake venom used as pharmacologic responses inducers have
shown to be very useful to the discovery of new active principles. The aim of this study is to evaluate the protective activity of
Lapachol against acute inflammation induced by whole venom and sPLA2 from Crotalus durissus cascavella.
Paw edema was performed in groups of five female Swiss mice. The groups were divided in: A- subplantar injection of whole
venom (25μg) and after thirty minutes an intraperitoneum injection of Lapachol (25μg), B- subplantar injection of sPLA2 (25μg)
and after thirty minutes an intraperitoneum injection of Lapachol (25μg), C- Intraperitoneum injection of Lapachol (25μg) and
after thirty minutes a subplantar injection of whole venom (25μg), D- Intraperitoneum injection of Lapachol (25μg) and after
thirty minutes a subplantar injection of sPLA2 (25μg), E- subplantar injection of whole venom and Lapachol pre-incubated 1:1
(w/w)(25μg), F- Subplantar injection of whole venom (25μg), G-Subplantar injection of sPLA2 (25μg). The final paw volume
was measured with pletismometer. For Miotoxicity and hemorrhagic activity the inflammation inducers were injected
intramuscular and intradermal, respectively and the groups were divided the same way described above. The miotoxicity was
assessed by the release of Creatine-Kinase (CK) and the hemorrhagic activity by reducing hemorrhage in the mice skin. The
treatment that reduced faster the edema was injection of Lapachol 30 minutes after injection of whole venom with 9,4%
(p=0,003) and after injection of PLA2 with 26,5% (p=0,03)when compared withcontrol group(100%). After 4 hours, the
treatment that better reduced the edema was injection of Lapachol 30 minutes before injection of PLA2 with 29,1% (p=0,03) and
Lapachol after injection of whole venom with 19,2% (p=0,007)when compared with control group (100%). Miotoxicity was
reduced 16,1% (p=0,01) when Lapachol was injected after whole venom and was reduced 4,6%(p=0,004)when Lapachol was
injected after PLA2 and compared with control group (100%). Hemorrhage was reduced when Lapachol was injected after total
venom.
Conclusions:
Lapachol was effective for reducing inflammation caused by whole venom and rattle snake venom sPLA2, but their performance
depends on the form and via tested administration. Further studies are needed to determine the exact mechanism of action of
these substances so that it can be used as anti-inflammatory.
Keywords: Crotalus durissus cascavella, edema, Inflammation, Lapachol
Resumo:17-049
INVOLVEMENT OF MAST CELLS AND THEIR MEDIATORS IN A MOUSE MODEL OF POSTOPERATIVE PAIN.
Oliveira, S. M. ; Silva, C. R. ; Drewes, C. C. ; Trevisan, G. ; Ferreira, J.
QUÍMICA, UFSM
Objectives:
Recently, it was estimated that every year, 234.2 millions of surgeries are performed around the world, and only in Brazil, nearly
three million surgeries are performed annually. Studies have shown that surgical procedures are associated with mortality and
morbidity and that nearly half of surgical patients still experience postoperative pain moderate to severe and related inadequate
pain relief. Thus, it becomes important to understand the mechanisms involved in postoperative pain. We previously
demonstrated serotonin and histamine receptor antagonist post-operative nociception, suggesting a role of mast cell in this
process. Thus, the objective of the present study was to investigate the involvement of mast cells and their mediators in a mice
model of postoperative pain.
The experiments were conducted on adult male Swiss mice (25-35 g, n= 6-8). After anesthesia with halothane, a longitudinal
incision was made through the skin and fascia of the plantar foot. The underlying muscle was elevated with a curved forceps,
leaving the muscle origin and insertion intact and the skin was sutured. Before surgical procedure, the animals received by
intraplantar route sodium cromoglycate (200 µg/paw), a mast cell membrane stabilizer or vehicle (phosphate-buffered solutionPBS, 20 µl/paw). The mast cell degranulator compound 48/80 was administered daily at increasing doses (1, 3, 10 and 10
µg/paw). Histamine and serotonin levels were evaluated by fluorimetric method in the paw skin of animals that received or not
compound 48/48 or in the paw perfusate of animals that received cromoglycate. Histological procedure was carried out to
confirm mast cell degranulation. For the behavioral assessment of mechanical allodynia (pain in response to a non-nociceptive
stimulus verified as a threshold reduction) the animals were acclimated for behavioral accommodation. The mechanical threshold
of animals was determined before and after surgical procedure with flexible nylon von Frey filaments using the up-and-down
method. It was observed that plantar surgery induced mechanical allodynia (threshold reduction of 98±2%). The pre-treatment
with compound 48/80 significantly reduced the mechanical allodynia (reduction of 98±23%) after surgery and reduced histamine
and serotonin levels (88±4% and 68±10%, respectively) in operated tissue. Furthermore, plantar incision produced mast cell
degranulation, assessed by histology and confirmed by the increased levels of serotonin (three–fold higher) and histamine
(fifteen–fold higher) in the perfused tissue. The treatment with cromoglycate prevented the mechanical allodynia (inhibition of
96±21%) and the increase in histamine (44±10% of inhibition) and serotonin (73±5% of inhibition) levels induced by plantar
surgery.
Conclusions:
Taken together, our findings indicate the importance of mast cell activation as a peripheral mechanism that is involved in the
development and maintenance of postoperative nociception. Thus, mast cell activation mechanisms could be interesting targets
for the development of novel therapies to treat postoperative pain.
Keywords: ALLODYNIA, HISTAMINE, NOCICEPTION, SEROTONIN, SURGERY
Financial Support: CAPES, CNPq, PRONEX, FAPERGS
Resumo:17-050
CHRONIC TREATMENT WITH FLUOXETINE ENHANCES THE ELEVATED PLUS-MAZE-INDUCED
ANTINOCICEPTION IN MICE
Baptista, D. 1; Nunes-de-souza, R. L. 2; Canto-de-souza, L. 3; Canto-de-souza, A. L. M. 1
1
Dept Psychology-Psycobiology group, UFSCAR
2
Pharmacol, , FCF- UNESP
3
Faculty of Philosophy, Sciences and Letters of Ribeirão Pret, FFCLRP-USP
Objectives:
A single exposure in the elevated plus maze (EPM), an animal model of anxiety, induces antinociception in mice
(Psychopharmacol., 150; 300, 2000). Clinical and animal findings have suggested an analgesic role played by selective serotonin
reuptake inhibitors (SSRI, Brain Res., 915; 218, 2001). The present study investigated the effects of chronic treatment (21 days)
with the SSRI fluoxetine on nociceptive response in mice exposed to the (EPM).
Swiss-albino male mice (35-45g) (n= 7-9/group) received subcutaneous injection of fluoxetine (0, 5.0, 10 or 20 mg/kg) for 21
consecutive days. On 21th day, each mouse received an intraperitoneal injection of 0.6% acetic acid (0.1 ml/10g weight;
nociceptive stimulus) and then was confined to either open arm (OA) or enclosed arm (EA) of the EPM for 5 minutes. During
this period, the number of writhes was recorded. Two-way ANOVA (place of confinement x treatment) showed significant
effects for place of confinement factor [F(7.54) = 76.34, P < 0.05], and treatment factor [F(7.54) = 9.39, P < 0.05] but no effect
for place of confinement x treatment interaction [F(7.54) = 1.60, P > 0.05]. Posterior comparisons (Duncan‟s test) revealed that
OA-confined animals showed lower number of writhes when compared to EA-confined mice, an effect that was not changed by
fluoxetine treatment [saline (OA: 6.00 ± 0.58, EA: 12.71 ± 1.43, P < 0.05); fluoxetine 10 mg/kg (OA: 3.50 ± 0.82, EA: 11.13 ±
1.41, P < 0.05)] and fluoxetine 20 mg/kg (OA: 2.13 ± 0.48, EA: 6.63 ± 0.92, P < 0.05)]. OA-and EA-confined animals treated
with fluoxetine 20 mg/kg exhibited lower number of abdominal writhes when compared to the control group.
Conclusions:
Present results corroborate previous studies showing that OA confinement in the EPM elicits antinociception in mice
(Psychopharmacol., 150; 300, 2000). In addition, chronic treatment with fluoxetine (20 mg/kg) enhanced the OA-induced
antinociception and attenuated nociceptive response in EA-confined animals. These results confirm the previously reported
analgesic effect of SSRI drugs (Brain Res., 915; 218, 2001), suggesting an important role played by serotonin in the modulation
of nociception.
Keywords: Antinociception, Elevated Plus Maze, Fluoxetine, Mice
Financial Support: UFSCar, FAPESP (2009/17938-6).
Resumo:17-051
NO/CGMP/KATP PATHWAYÏ¿½S ACTIVATED BY NORADRENALINE TO INDUCE PERIPHERAL
ANTINOCICEPTION IN RAT.
Romero, T. R. L. ; Guzzo, L. S. ; Duarte, I. D. G.
Laboratório de Dor e Analgesia, Departamento de Farmacologia, ICB,UFMG
Objectives:
Recently studies showed that noradrenaline (NA) can induce peripheral pronociceptive and antinociceptive effect. In addition, the
participation of the NO/cGMP/KATP pathway in the peripheral antinociception has been established by our group as the
molecular mechanism of another adrenoceptor agonist xylazine. Thus the aim of this study was obtain pharmacological evidences
for the involvement of the NO/cGMP/KATP pathway in the peripheral antinociceptive effect induced by exogenous
noradrenaline.
The rat paw pressure test was used and hyperalgesia was induced by intraplantar injection of prostaglandin E2 (2 ï¿½g/paw). All
drugs were administered locally into the right hind paw of Wistar male rats. NA (05, 20 and 80 ï¿½g/paw) elicited a local
inhibition of hyperalgesia (35%, 55% and 85%, respectively). The non selective NO synthase (NOS) inhibitor L-NOarg (12, 18
and 24 ï¿½g/paw) antagonized the antinociception effect induced by NA, highest dose (60%, 80% and 100%, respectively). The
soluble guanylyl cyclase inhibitor ODQ (25, 50 and 100 ï¿½g/paw) antagonized the NA-induced effect (30%, 55% and 100%,
respectively); as well, cGMP-phosphodiesterase inhibitor zaprinast (50 ï¿½g/paw) potentiated the antinociceptive effect of a low
dose of NA from 60% to 90%. In additional, the local effect of NA was antagonized by a selective blocker of ATP-sensitive K+
channel glibenclamide (20, 40 and 80 ï¿½g/paw) (30%, 50% and 100%, respectively). On the other hand, the specifically voltagedependent K+ channel blocker tetraethylammonium (30 ï¿½g/paw), small and large conductance blockers of Ca2+-activated K+
channels dequalinium (50 ï¿½g/paw) and paxilline (20 ï¿½g/paw) were not able at blocking the local antinociceptive effect of
NA.
Conclusions:
The results provide evidences that NA probably induces peripheral antinociceptive effect by activation the NO/cGMP/KATP
pathway.
Keywords: Peripheral antinociception, NO/cGMP/KATP pathway, Noradrenaline
Financial Support: CNPq (473758/2007-5) and fellowships from CNPq, FAPEMIG
Resumo:17-052
ROLE OF 5-LIPOXYGENASE PRODUCTS IN ACUTE RESPIRATORY DISTRESS SYNDROME INDUCED BY
SEVERE SEPSIS
Monteiro, A. P. T. ; Pinheiro, C. D. S. ; Silva, E. S. ; Rocco, P. R. M. ; Benjamim, C. F. ; Canetti, C.
Inst de Biof. Carlos Chagas Filho/Univ Fed do Rio de Janeiro, IBCCF/UFRJ
Objectives:
Sepsis is the main cause of death in intensive care unit worldwide. A frequent outcome of sepsis is the acute respiratory distress
syndrome (ARDS), a pathological state which is characterized by overwhelming inflammation with increased microvascular
permeability that causes diffuse lung edema and mechanical dysfunction leading to respiratory failure. Leukotrienes are lipid
mediators of inflammation derived from arachidonic acid metabolism by 5-lipoxygenase (5-LO). We previously demonstrated
that 5-LO knockout mice (5-LO-/-) presented less mortality and less protein extravasation in peritoneum and kidney after sepsis.
The aim of this work is to evaluate the participation of 5-LO products in ARDS induced by sepsis.
5-LO-/- mice and their background lineage (SV129; WT) were submitted to cecal ligation and puncture (CLP; Ethical committee
# DFBCICB028). Briefly, mice were anesthetized, cecum was exposed, ligated, and punctured 2 times with an 18G needle. As
control, mice were submitted to sham surgery. After 16 h surgery, mice were sacrificed, perfused, and bronchoalveolar lavage
(BAL) performed and lung tissue collected. Histological analysis of lung tissue from WT mice submitted to CLP showed a
massive inflammatory cell infiltration, and loss of tissue architecture. In contrast, 5-LO-/- mice lungs were protected from sepsis
induced ARDS. MPO analysis also showed a decrease in the neutrophil influx in lung parenchyma of CLP-5-LO-/- mice when
compared with WT. Furthermore, morphometric analysis of lungs from CLP-5-LO-/- mice also revealed less collapsed alveolar
than CLP-WT mice. No difference in BAL cells was observed in any experimental group. LTB4 was found in BAL and in lung
parenchyma of WT mice subjected to clp. Pulmonar mechanics analysis was performed through occlusion at the end of
insufflations with constant influx as previously described. Pulmonar mechanics results also confirmed that 5-LO-/- mice are more
resistant, since the increase in elastance evoked by CLP was ameliorated in those animals compared to WT mice. Plasma
cytokine measurement from clp-mice did not show difference in IL-6 production between WT and 5-LO-/- mice, however IL1&beta was significantly lower in 5-LO-/- mice than in WT.
Conclusions:
These results suggest that 5-LO-/- mice are resistant to lung injury induced by sepsis. 5-LO-/- mice presented preservation of the
pulmonary components and less infiltration of inflammatory cells, observed in histological and MPO assays, and also in
mechanical performance.
Keywords: sepse, 5-lipoxigenase, ards, leucotrieno
Financial Support: CNPq, FAPERJ and FUJB
Resumo:17-053
CARVACROL ATTENUATES INFLAMMATORY RESPONSE IN MICE AND NITRIC OXIDE PRODUCTION BY
MURINE MACROPHAGES.
Xavier, M. A. 1; Guimarães, A. G. 1; Santana, M. T. 1; Cavalcanti, S. C. H. 1; Bonjardim, L. R. 1; Camargo,
E. A. 1; Brito, F. A. 7; Barreto, E. O. 7; Santos, M. R. V. 1; Quintans-júnior, L. J. 1
1
Departamento de Fisiologia/Universidade Federal de Sergipe, DFS/UFS
2
3
4
5
6
7
Universidade Federal de Alagoas , UFAL
8
Universidade Federal de Alagoas , UFAL
9
10
Objectives:
Carvacrol (CARV) is a phenolic monoterpene present in the essential oils of plants of the family Lamiaceae, as in Origanum and
Thymus genera. The purpose of the present study was to evaluate the anti-inflammatory activity of CARV by using tests in vivo
and in vitro.
The anti-inflammatory activity of CARV (25, 50 and 100 mg/kg; i.p.) was investigated in the models of carrageenan (CG)induced paw edema or pleurisy in male Swiss mice (n=8/group). Paw edema was induced by intraplantar injection of CG (300
ug/paw) and paw volume was measured before and at selected times (1-4 h) thereafter. Pleurisy was induced by intratoracic
injection of CG (300 ug/cavity) and after 4 h the total and differential counts were evaluated in pleural lavage, as well as TNF&alpha levels were measured in the supernatant by enzyme immunoassay. We also investigated the effect of CARV (1, 10 and
100 μg/mL) on LPS-induced nitrite production in isolated murine macrophages. The experimental protocols were approved by
the instituition‟s Ethic Committee (CEPA/UFS: 43/08). Data were evaluated by ANOVA followed by Tukey‟s test. Treatment of
mice with CARV significantly decreased (p
Conclusions:
It can be concluded from the present study that CARV possesses anti-inflammatory properties, by possible inhibition of TNF&alpha and NO production. We suggest that this molecule represents an important pharmacological tool for developing new
strategies to treat inflammation.
Keywords: Anti-inflammatory activity , Carvacrol, Paw edema, Pleurisy
Financial Support: CAPES, CNPq and FAPITEC-SE
Resumo:17-054
BDNF AND TNF AS MARKERS OF SEVERITY OF CHRONIC TENSION HEADACHE
Medeiros, L. F. 1,3; Deitos, A. 1,2; Souza, A. D. 1,2; Laste, G. 1,2; Dresch, F. 4; Silveira, J. 4; Souza, A. C. D.
1,2
; Kauffmann, C. 4; Fernandes, L. C. 4; Souza, I. C. C. 1,2; Hidalgo, M. P. L. 1,2; Torres, I. L. D. S. 1,2,3;
Caumo, W. 1,2
1
Hospital de Clínicas de Porto Alegre, HCPA
2
Programa de Pós-graduação Ciências Médicas - UFRGS, PPGCM/ UFRGS
3
PPG em Ciências Biológicas: Fisiologia/UFRGS, PPG Fisiologia/UFRGS
4
Centro Universitário Univates, Univates
Objectives:
The prevalence of chronic tension-type headache (CTTH) is about 2-5% in the general population. And in half of the cases of
CTTH is reported that headaches are related to impairment in work performance. The tumor necrosis factor (TNF) is a potent
cytokine that has essential roles in the activation and regulation of immune and inflammatory responses. The brain-derived
neurotrophic factor (BDNF) is critical for maintaining the survival and growth of many neuronal subtypes, and it may be a key
mediator of synaptic efficacy. The objective of this study was to evaluate the association between the impact of CTTH and BDNF
and TNF-alpha levels in a population of Taquari Valley.
This study was approved by the Ethics Committee of HCPA 08/087. The sample was composed by 11 cases (headache) and 11
controls (no headache) selected from a database of a population study. To assess the impact of headache on daily life of the
patient were used questionnaires: Short-Form Headache Impact Test (HIT-6) and the International Headache Society (IHS).
BDNF (pg/mL) and TNF (pg/mL) were determined by ELISA and presented by mean ± standard deviation. Statistical analysis
was performed by regression linear multiple analysis - stepwise backward (TNF and depressive symptoms) and regression
logistic analysis - stepwise backward (severity of headache and BDNF) and significant difference was considered P
Conclusions:
BDNF is a neurotrophin that has been implicated in the generation and modulation of pain, this study demonstrates that BDNF
may be a biomarker of the severity of the CTTH. It is important to note that this is a preliminary study and a new research with
larger sample is needed to confirm the findings.
Keywords: chronic tension headache, TNF-alpha, BDNF
Financial Support: GPPG / HCPA, Univates, CNPq, CAPES.
Resumo:17-055
LIGHT-EMITTING DIODE THERAPY REDUCES MECHANICAL HYPERNOCICEPTION INDUCED BY
PLANTAR INCISION VIA OPIOID MECHANISM
Cidral-filho, F. J. ; Mazzardo-martins, L. ; Martins, D. F. ; Moré, A. O. O. ; Santos, A. R. S.
Departamento de Ciências Fisiológicas, UFSC
Objectives:
Evaluate the antihypersensitivity effect of Light Emitting Diode Therapy (LEDT) in the plantar incision (PI) model in mice; as
well as investigate the possible involvement of the opioid system in this effect.
The experiments were approved by the institution ethics committee under protocol n. 23080.006492⁄2011-61. Male Swiss mice
were randomly divided in the following groups (n=8): naive, control (not treated), Off (LED device turned off), LEDT 1, 3, 5, 7
and 9 (treated with energy densities of 1 through 9 J⁄cm2). Control, Off and LEDT groups were submitted to a 5 mm longitudinal
PI (right hindpaw) under anesthesia (1-2% isoflurane). Mechanical hypersensitivity (MH) was assessed as withdrawal frequency
percentage to 10 presentations of a 0.4 g von Frey filament. Evaluations were conducted before and on day 1 through 5 after PI.
LEDT (MOLIMEDpen™ device; 950 nm wavelength, 80 mW⁄cm2 irradiance; 1 to 9 J⁄cm2 energy density) was applied directly
to the skin of the incision site. Results demonstrate that LEDT reduced MH in a dose-response manner with best results obtained
with 9 J⁄cm2 (inhibition of 55±10% and effect lasting for 1 hour). Treatment with 1 J⁄cm2 and with LED device turned off did not
reduce MH. Furthermore, LEDT as well as morphine (5 μg⁄site i.pl.) effects were blocked by intraperitoneal (i.p.), intraplantar
(i.pl.) or intrathecal pre-administration of naloxone (1 mg⁄kg i.p.; 5 μg⁄site i.pl. or 5 μg⁄site i.t.) 20 minutes prior to LEDT or
morphine treatment.
Conclusions:
LEDT reduced hypersensitivity induced by PI in mice via peripheral as well as central opioid mechanisms.
Keywords: Light-emitting diode therapy, Hypernociception, Plantar incision, Opioid, Mice
Financial Support: UFSC, Capes
Resumo:17-056
USE OF LOW-LEVEL LASER IN REPAIR PROCESS AFTER AVEOLAR INFERIOR INJURY: INVOLVEMENT OF
NERVE GROWTH FACTOR
Martins, D. O. ; Santos, F. M. ; Britto, L. R. G. ; Chacur, M.
Depto. de Anatomia-Instituto de Ciências Biomédica, ICB-USP
Objectives:
The removal of third molars is one of more frequent dental surgery practice, and is associated with a series of accidents and
complications, as well as, inferior alveolar nerve (NAI) lesion. The aim of this study is evaluate the expression of neurotrophic
factors after NAI injury. In addition, we will also evaluate the application of low power laser after the same injury and correlated
with behavior pain model in rats.
We used male Wistar rats in a model of NAI injury associated with treatment by laser therapy. The animals were divided into 4
groups: lesion + laser; lesion + without treatment; sham + laser and naive (used as control). The laser treatment was performed
every two days, during 10 sessions. To evaluate the nociception behavior we used the allodynia von Frey test. This behavior was
assessed after each session of laser therapy. Our results demonstrated an increase of nerve growth factor (NGF) expression (20%)
after NAI injury when compared with control group (naive and sham). On the other hand, we observed a higher increase of NGF
expression (30%) in animals treated with laser when compared with NAI injury group. Regarding nociceptive testes, we observed
an increase of pain sensitivity after nerve injury when compared with control animal and a decrease after treatment.
Conclusions:
NGF is an important neurotrophin and its presence is essential to the process of nerve regeneration. Our initial results allow us to
suggest that the laser therapy has a positive effect in nociceptive behavioral. And also induces NGF synthesis. This could be an
important therapeutic tool in treating patients with pain sensibility after oral surgeries.
Keywords: dor, nervo alveolar inferior, fator neurotrófico, reparação nervosa
Financial Support: FAPESP nº 2010/20026-6, IBRAMED
Resumo:17-057
SPINAL BRADYKININ NOCICEPTION IS MEDIATED BY GLUTAMATE RELEASED FROM VSOR CHANNEL
ACTIVATED BY REACTIVE OXYGEN SPECIES
Rossato, M. F. 1; Klafke, J. Z. 1; Gewehr, C. D. C. V. 2; Oliveira, S. M. D. 1; Trevisan, G. D. S. 1; Silva, M.
A. D. 1; Ferreira, J. 1
1
Química / Universidade Federal de Santa Maria, UFSM
2
Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizon, IEPSCBH
Objectives:
Bradykinin (BK) is a peptide produced in plasma and tissues via the kallikrein–kinin system in response to infection, tissue
trauma or inflammation, been responsible for increase in vascular permeability, edema formation and pain. Despite its peripheral
participation, BK also act as a excitatory neurotransmitter at central nervous system (CNS), were it contributes to pain
transmission as observed in several animal model, such as sciatic neuropathy and adjuvant-induced arthritis. Recently, it was
described BK promote glial activation in co-cultured glia/neurons through B2 receptor activation, with consequent production of
reactive oxygen species (ROS) by NADPH oxidase (NOX), activation the volume-sensitive outwardly rectifying anion channel
(VSOR), release of glutamate and stimulate NMDA receptor. Even though, the participation of VSOR channel and this pathway
(ROS-VSOR-glutamate) was not studied yet in the pain transmission. Thus, the main goal of this work was to investigate the
participation of VSOR channel in the nociception of spinal BK administration. This work was approved by the by the Ethics
Committee of the Federal University of Santa Maria (process number 23081.001086/2009-87).
For induction of spinal nociception, male Wistar rats (250 – 350g, n=6) were intrathecally (i.t.) injected with bradykinin (1 – 15
nmol/site) and the development of thermal hyperalgesia (increased response to noxious heat) and mechanical allodynia
(perception of innocuous mechanical stimulation as nocive) was evaluated 15, 30 and 60 minutes after. To investigate the
participation of B1 and B2 receptors, PKC, NOX, ROS, NMDA and VSOR channel, rats were co-administered with DALBK (B1
antagonist; 25 nmol/site), HOE-140 (B2 antagonist; 0.3 nmol/site), GF109203X (PKC inhibitor; 0.75 nmol/site), N-acetylcysteine
(NAC, ROS scavenger; 100 nmol/site), apocynin (NOx inhibitor; 300 nmol/site), MK801 (NMDA blocker 300 nmol/site) and
DCPIB (VSOR blocker; 1 - 100 nmol/site), respectively. To investigate the glial participation on BK nociception, we preadministered fluorocitrate (1 nmol/site, i.t.) 30 minutes prior to BK.Fifteen minutes after i.t. BK administration (5 nmol/site), it
was observed a decrease of 34.8±5.0 % in the paw withdrawal response, indicating the development of thermal hyperalgesia,
without alterations in the mechanical sensitivity. This nociceptive behavior was fully prevented by the co-administraion of HOE140, but not DALBK, indicating the dependence of B2 receptor. DCPIB, a blocker of VSOR channel was also able to prevent the
development of thermal hyperalgesia, with DE50 of 14.4 (7.1 - 29.1) nmol/site, i.t. To investigate the role of glia in this response,
we pre-administered fluorocitrate (1 nmol/site, i.t.) 30 minutes prior BK, an observed a reduction in 75.4±12.1% of nociception.
Similarlly, the co-administration of GF109203X, apocynin and NAC were able to reduce the hyperalgesia induced by BK in
99.7±13.7, 63.0±4.9 and 83.0±10.3%, respectively. To investigate the participation of glutamate in the BK hyperalgesia, we coadministered MK801, an antagonist of NMDA receptor. We observed that this treatment also reduced the hyperalgesia induced
by BK intrathecally in 116.4±5.4%.
Conclusions:
These results indicate the participation of VSOR channel at spinal pain transmission induced by BK, after glial activation,
showing a new pharmacological target for the treatment of pain.
Keywords: Bradykinin, VSOR, Pain, Spinal Cord, Glutamate
Financial Support: CNPq CAPES PPGBtox UFSM
Resumo:17-058
MOTOR AND NOCICEPTIVE CHANGES IN PARKINSON DISEASE RAT MODEL: DOPAMINERGIC
MODULATION OF MECHANICAL HYPERALGESIA.
Maciel, S. T. 1; Domenici, R. A. 1; Fonoff, E. T. 2,1; Pagano, R. L. 1
1
Hospital Sírio-Libanês, HSL
2
Dep. of Neurology, University of Sao Paulo Medical School, USP
Objectives:
Parkinson Disease (PD) is a neurodegenerative disorder characterized by progressive loss of nigrostriatal dopaminergic neurons,
resulting in chronic changes of neural circuits in basal ganglia. Before the motor and cognitive features of the disease, the
majority of PD patients experience persistent pain, which is commonly neglected and still poorly understood. Experimentally, it
was observed a decrease of the nociceptive threshold in PD rats, in different models of nociception; however, the mechanisms
involved remain unclear. The aims of the present study are the evaluation of the motor and nociceptive behavioral changes in 6OHDA PD rat model and to investigate the dopaminergic, opioidergic and GABAergic involvement in these changes.
Wistar male rats (200–250 g) were deeply anesthetized (tribromoethanol 2,5% i.p.) and under stereotaxic condition, were injected
with the neurotoxin 6-hidroxidopamine (6-OHDA; 12 &microg/2 &microl) in the left striatum. Animals injected with saline or
naive were used as control (n=5 animals per group). After 7, 14 or 21 days of neurotoxin injection, animals were submitted to the
evaluation of mechanical and thermal nociceptive response (paw pressure and tail flick tests, respectively).. Hemiparkinsonian
rats presented a 55%-decrease at the mechanical nociceptive threshold, with no interference in the thermal reflex, compared to
control animals. On the day 14 after 6-OHDA injections, animals received apomorphine, a dopaminergic agonist (0.5 mg/kg; s.c.)
and the number of total turns were calculated over a period of 30 min. Neurotoxin induced a asymmetric apomorphine-induced
rotational behavior characterizing the nigrostriatal lesion. Apomorphine also produced full reversal of mechanical hyperalgesia
induced by striatal 6-OHDA. On the day 21 after lesion, animals were submitted to catalepsy test and the lesioned rats showed an
exacerbated increase (244%) in the latency of removal both paws. The expression of enzyme glutamic acid decarboxylase
(GAD65/67), mu opioid receptor (MOR) and D1 dopaminergic receptor, in midbrain and striatum, was also evaluated.
Preliminary results indicate that D1 expression increased (97%), bilaterally, in midbrain of the lesioned rats, compared with the
expression pattern of control animals. GAD65/67 and MOR expression remained unaltered in midbrain and striatum in the same
animals.
Conclusions:
Hemiparkinsonian 6-OHDA rats showed marked mechanical hyperalgesia and it was fully reversed by apomorphine
administration, suggesting involvement of dopamine in pain modulation. Although, participation of midbrain D1 receptor during
descending inhibitory control of pain is well reported; the increase of midbrain D1 expression was first time observed in 6OHDA PD model. This fact possibly reflects the upregulation of D1 receptors in feedforward dopaminergic loops to pain
modulation node in midbrain. However, the exact mechanisms of this link remains unclear. A better understanding of the role of
nigrostriatal system in pain modulation can improve of therapeutic strategies for persistent pain in PD patients.
Keywords: 6-OHDA, Apomorphine, D1 dopaminergic receptor, Mechanical hyperalgesia, Parkinson disease
Financial Support: FAPESP and Hospital Sírio-Libanês
Resumo:17-059
NITRIC OXIDE (NO) MODULATES NOCICEPTION BY FORMALIN WITHOUT AFFECTING MAST CELL
DEGRANULATION
Mascarin, L. Z. ; Silva, E. S. ; Tonussi, C. R.
Universidade federal de santa catarina, UFSC
Objectives:
Mast cell degranulation is thought to be involved in the pain sensation induced by formalin, and nitric oxide (NO) was reported to
modulate its degranulation. In a previous study, we observed that N-Nitro-L-arginine (L-NA), a NOS inhibitor exert hypo or
hypernociceptive effects in the rat knee-joint formalin incapacitation. Thus, our aim was to evaluate whether L-NA can modify
mast cell degranulation in this nociceptive model.
After formalin (1.5%) injection into the right knee-joint, articular incapacitation was measured by counting the paw elevation
time (PET; s) during 1-min periods of forced walk on a revolving cylinder, each 5 min throughout a total test period of 20 min. LNA doses (0.07 and 70 µg/knee) were injected 20 min before formalin, and 20 min after formalin injection, the knee was
removed and fixed in paraformaldehyde (4%) for 24 hours. The anterior portion of the articular capsule (approx. 24 sq. mm) was
removed and kept in paraformaldehyde for another 24 hours. After the standard dehydration process in alcohol and xylene for
inclusion in paraffin, 4-µm slices were mounted in glass slides and stained by toluidine blue. The total number of degranulated
mast cells were counted under 40x optic magnification. This protocol was approved by the local ethical committee for animal use
(CEUA/PP00368/2009). The lower dose of L-NA decreased, and the higher dose increased the second phase of formalin induced
incapacitation (P
Conclusions:
These results suggest that L-NA effects in this model of articular nociception induced by formalin did not involve a change in the
mast cell degranulation dynamics.
Keywords: NITRIC OXIDE, MAST CELL DEGRANULATION, FORMALIN
Financial Support: Capes, CNPQ
Resumo:17-060
TRAMADOL AND MAPROTILINE WERE EFFECTIVE TO REDUCE HYPERALGESIA AND ALLODYNIA OF
NEUROPATHIC PAIN IN ANIMAL MODELS.
Monteiro, C. E. S. 1,1,1,1; Zapata-sudo, G. 1,1,1,1; Barreiro, E. J. 1,1,1,1; Sudo, R. T. 1,1,1,1
1
Programa de Desenvolvimento de Fármacos(Ciências Biomédicas), UFRJ
2
Faculdade de Farmácia (Centro de Ciências da Saúde), UFRJ
Objectives:
Treatment of neuropathic pain is an unsolved problem in medicine. Multi-target activation has been considered to reduce the
hyperalgesia and allodynia of this disease. Here, we investigated possible interaction of tramadol, an opioid receptor agonist, with
maprotiline, a selective neuronal norepinephrine uptake inhibitor to reduce hyperalgesia and allodynia of neuropathic pain in
animal models.
Protocols were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Neuropathic pain was
induced in male Wistar rats (180-220 g) by chronic constrictive injury (CCI) [1] and spinal nerve ligation (SNL) in L5 [2].
Thermal hyperalgesia and mechanical allodynia were evaluated by digital analgesymeter (Analgesia Meter IITC 336) and
modified von Frey filament (digital Analgesymeter EFF 302), respectively. Treatment was initiated 7 days after surgery, time in
which the neuropathic symptoms were established, by oral administration of tramadol (10 mg.kg-1) or maprotiline (0.25 mg.kg1) or by association at same doses, in CCI animals. The SNL animals received tramadol (20 mg.kg-1) or maprotiline (0.25
mg.kg-1) or association. The animals were treated for 7 days and thermal hyperalgesia and mechanical allodynia were evaluated
before surgery (control) and on days 1, 3, 7, 8, 10, 14 after surgery. No difference in the latency of thermal hyperalgesia or
mechanical allodynia was noted after treatment with tramadol or maprotiline and also by association in the sham groups. Latency
of thermal hyperalgesia was increased by tramadol (10 mg.kg-1 p.o., n= 5) from 5.6±0.2 s to 7.5±0.2 s (P
Conclusions:
The thermal hyperalgesia and mechanical allodynia induced by CCI and SNL were consistently reduced by combination of
tramadol with maprotiline.
Keywords: Allodynia, hyperalgesia, maprotiline, neuropathic pain, tramadol
Financial Support: INCT/INOFAR, CNPq, CRISTÁLIA, FAPERJ
Resumo:17-061
ANTI-HYPERNOCICEPTIVE EFFECT OF CITRONELLAL IS DEPENDENT OF NO-CGMP-ATP-SENSITIVE-K+
CHANNEL ACTIVATION.
Santana, M. T. 1,1; Guimarães, A. G. 1; Camargo, E. A. 1; Santana, D. G. 1; Oliveira, M. G. B. 1; Santana, M.
F. 1; Souza, D. P. 1; Almeida, J. R. G. S. 2; Lima, J. T. 2; Quintans-júnior. L. J. 1
1
Departamento de Fisiologia. Universidade Federal de Sergipe, UFS
2
Colegiado de C. Farmacêuticas, U. F. Vale do São Francisco, UNIVASF
Objectives:
The present study investigated the effect of citronellal (CTL) on inflammatory hypernociception induced by different stimuli and
examined the possible mechanism involved in this effect.
Male Swiss mice (28-32 g; n=6) were treated intraperitoneally with vehicle (saline + tween 80 0.2%), CTL (25, 50 or 100 mg/kg;
doses A, B and C respectively), 0.5 h after the subplantar injection of 20 μl of carrageenan (CG; 300 µg/paw), tumor necrosis
factor-α (TNF-α; 100 pg/paw), prostaglandine E2 (PGE2; 100 ng/paw) or dopamine (DA; 30 μg/paw). The hypernociception was
evaluated at time 1, 2, and 3 h after the injection of agents, using the digital analgesymeter (von Frey). CTL effects were
evaluated in the presence of L-NAME (30 mg/kg, i.p.), or glibenclamide (5 mg/kg, i.p.). The motor coordination was also
evaluated using Rota rod (7 rpm, 180 s). The data were evaluated by one-way analysis of variance (ANOVA) followed by
Tukey‟s test and the values represents the mean ± SEM, considering p
Conclusions:
The present data suggest that CTL presents anti-hypernociceptive property, and this effect is mediated, at least partially, by the
NO–cGMP–ATP-sensitive K+ channel pathway.
Keywords: Monoterpenes, Citronellal, Hypernociception, Pain, Inflammation
Financial Support: FAPITEC-SE and CNPq.
Resumo:17-062
ADENOSINE AND INOSINE ANTI-INFLAMMATORY EFFECTS IN PLEURISY-INDUCED INFLAMMATION:
EVIDENCE FOR THE PARTICIPATION OF ADENOSINE RECEPTORS AND ADENOSINE METABOLISM.
Lapa, F. R. 1; Cabrini, D. A. 1; Santos, A. R. S. 2
1
Depto de Farmacologia, UFPR
2
Depto. de ciências Fisiológicas, UFSC
Objectives:
Is increasingly evident that adenosine and inosine exert immunomodulatory effects and events such as inflammation account for
adenosine degradation to inosine, a process mediated by adenosine deaminase (Biochem Pharmacol. 65: 493, 2003). This study
aim to investigate adenosine and inosine anti-inflammatory effects, the participation of adenosine receptors and to evaluate
inosine contribution to adenosine effects on carrageenan (Cg)-induced pleurisy in mice.
Swiss female mice (18-25 g) were treated intraperitoneally with adenosine (100 mg/kg) or inosine (10 mg/kg), or pre-treated with
specific adenosine receptor antagonists A1 (DPCPX, 2.5 mg/kg), A2A (ZM241385, 1.5 mg/kg), A2B (Alloxazine 5 mg/kg),
unspecific adenosine receptor antagonist caffeine (1.5 mg/kg), or EHNA (5 mg/kg, i.p.) an adenosine deaminase (ADA) inhibitor,
30 min prior adenosine or inosine. To perform the protein vascular leakage measurement, mice were challenged with Evans blue
dye (25 mg/kg, i.p.), 30 min prior. The pleurisy was induced by intrapleural injection of Cg (1%) 30 min after treatments, and 4 h
later the exudates were collected to determinate total and differential leukocyte counting, determination of vascular leakage, IL1β and TNF-α level. To study the involvement of adenosine receptors, the doses of 100 mg/kg or 10 mg/kg of adenosine and
inosine, respectively were chosen taking account previous data from our group. In addition, a number of 6-8 animals were used
per group. Pre-treatment with ZM241385 and alloxazine, reversed completely (100 %) the effects of adenosine on total,
neutrophil cellular counts and vascular leakage. Adenosine treatment reduced 50 ± 2 % the TNF-α levels and the pre-treatment
with the antagonists did not reverted the adenosine effects. Moreover, the pre-treatment with DPCPX, ZM241385, alloxazine, or
caffeine reverted the inosine effects against total cell count in 77.5 ± 5.9 %, 80.1 ± 4.5 %, 82.9 ± 9.1 %, and 79.1 ± 12.5 % and
neutrophil count in 64.4 ± 6.1 %, 63.3 ± 6.3 %, 85 ± 13 %, and 70.6 ± 13 %, respectively, and reverted completely the inosine
effects against exudation. In addition, inosine reduced 51 ± 12 % the TNF-α levels without interfere with IL-1β levels. Only the
pre-treatment with ZM241385 and caffeine reverted 92.9 ± 7.4 % and 90.6 ± 10.3 %, respectively, inosine effects in TNF-α
assay. To study the adenosine metabolism and if inosine contributes with adenosine effects, mice were pre-treated with EHNA at
dose that inhibited ADA activity and hence, inosine generation. This treatment did not interfere with adenosine anti-inflammatory
effect.
Conclusions:
Our results suggest that adenosine effects probably involve the activation of A2A and A2B adenosine receptors, but all adenosine
receptors seems to be involved in the inosine effects. Interestingly, these receptors may not contribute with adenosine effects
against TNF-α release, however, our results suggests that A2A receptors mediate inosine effects against this cytokine. In addition,
this study suggests that the anti-inflammatory effects of adenosine seem not to be dependent on its switch on inosine.
Keywords: pleurisy, adenosine, inosine, adenosine deaminase
Financial Support: REUNI, CAPES, CNPq and FAPESC.
Resumo:17-063
ANTI-INFLAMMATORY ACTIVITY OF OMEGA-3: AN IN VIVO AND IN VITRO EVALUATION
Chakraborty, S. A. 1; Sampaio, T. M. A. 1; Correia, A. O. 1; Nobre, M. E. P. 1,2,4; Felipe, C. F. B. 1; Lucetti,
D. L. 1; Lopes, A. D. A. 3; Leal, L. K. A. M. 3; Viana, G. S. D. B. 1,3; Arida, R. M. 4
1
Faculdade de Medicina de Juazeiro do Norte, Estácio FMJ
2
Faculdade de Medicina do Cariri, UFC
3
4
Laboratório de Neurofisiologia , UNIFESP-EPM
Objectives:
Omega-3 fatty acids are known to decrease the production of inflammatory eicosanoids, cytokines and reactive oxygen species.
In the present work we evaluated the acute effects of omega-3 on the carrageenan-induced paw edema in rats and carrageenaninduced pertitonitis models. In addition, the Omega-3 activity on the myeloperoxidase (MPO, an inflammatory biomarker)
release from human neutrophils was also carried out.
For the carrageenan-induced paw edema, male Wistar rats (250 g) were treated with Omega-3 (50, 20, 10, 5, 2.5 e 1 mg/kg, p.o.)
or indomethacin (10 mg/kg, i.p.), 1 h before the intraplantar injection of 1% carrageenan (40 μL) in the right hind paw. The
control group received distilled water (10 mL/kg, p.o.). The paw edema volume (mL) was measured by a pletysmometer (Ugo
Basile, Italy) and results expressed as the difference between paw volume immediately before (initial volume) and at 1, 2, 3, 4
and 24 h (final volume) after carrageenan injection. In the carrageenan-induced pertitonitis model, male Wistar rats (250 g) were
distributed into 6 groups of 5 animals each: controls (pretreated with distilled water) and carrageenan in the absence and in the
presence of Omega-3 (2.5, 5 and 10 mg/kg, p.o., administered 1 h before carrageenan). Dexametasone (1 mg/kg, i.p.) was used as
the reference drug. Four hours after the intraperitoneal carrageenan administration (1 mL of a 1% solution), the animals were
sacrificed by decapitation, the abdominal cavity opened and the peritoneal exudate was collected for total leukocytes counting in
a Neubauer chamber. MPO assays were performed after PMA stimulation and in the absence and presence of Omega-3 (1, 10, 50
and 100 μg/mL). Indomethacin (35.7 μg/mL) was used as reference. Results were analyzed using one way of variance (ANOVA)
and expressed as means ± SEM. Data was further subjected to Student-Newman-Keuls as a post hoc test and differences between
means were regarded significant at p
Conclusions:
We showed that Omega 3 presented a significant anti-inflammatory effect as demonstrated by two experimental models in vivo.
Besides, at low concentrations Omega-3 inhibited MPO release from human neutrophils in vitro, what might be at least partly the
result of Omega-3 suppressing neutrophil adherence as shown by others.
Keywords: Omega-3 fatty acids, anti-inflammatory, carrageenan-induced paw edema, carrageenan-induced pertitonitis,
myeloperoxidase
Resumo:17-064
NEURAL MOBILIZATION IN NEUROPATHIC PAIN CONTROL: EVALUATION OF SATELLITE CELLS AND
NEURAL GROWTH FACTOR IN DORSAL ROOT GANGLION.
Silva, J. T. D. 1; Santos, F. M. D. 1; Giardini, A. C. 1; Silva, A. A. D. 2; Chacur, M. 1
1
Depto de Anatomia, ICB III, USP, ICB III - USP
2
Depto de Fisiologia e Biofísica, ICB I, USP, ICB I - USP
Objectives:
The technique of neural mobilization clinically is efficient in improvement of life quality of patients with neuropathic pain. Glial
cells and neurotrophins are directly involved with induction and maintenance of the painful processes, under chronic pain
conditions. However, the satellite cells and neurotrophins participation in dorsal root ganglion (DRG) of spinal cord is not fully
understood. In present project, we evaluated the glial fibrillary acidic protein (GFAP) of satellite cells and neural growth factor
(NGF) expressions by immunofluorescence and immunobloting methods in DRG after neuropathic pain and treatment with
neural mobilization.
Methods: Male Wistar rats (170-190g) were submitted to the neuropathic pain induction through nerve sciatic constriction (CCI).
The animals were anesthetized and the sciatic nerve was exposed and four ligatures (chromic catgut 4-0) were made around the
sciatic nerve trifurcation , a distance of approximately 1mm each ligature. The incision was sutured in layers using silk sutures
(number 4-0). Animals sham were submitted to the same incision, but without nerve ligation, and were used as controls. The
nociceptive threshold was determined before any procedure, after surgery and during treatment. The pain threshold was measured
by von Frey test (allodynia) and expressed in grams, by Hargreaves test (thermal hyperalgesia) and expressed in seconds and by
Randall & Selitto test (mechanical hyperalgesia) and expressed in grams. The results were analyzed by comparing the averages of
measurements obtained in base line and after treatments in all groups. After fourteenth day (14d) the animals received treatment
with neural mobilization during 10 sessions. After sedation, the rat was positioned in the left lateral position, then the right knee
joint (injured side) is positioned in extension, and ankle joint duty is handled in dorsi-flexion, and 20 oscillations per minute
during 02 minutes, with a pause of 25 seconds was made. The treatment lasts 10 minutes, and in the last minute is included
cervical flexion. At the end of the sessions, the DRG was performed to immunofluorescence or immunobloting using GFAP,
NGF and NeuroTrace antibodies. All procedures are in accordance with the protocol approved by the Ethical Principles of
Animal Experimentation adopted by Brazilian College of Animal Experimentation (COBEA) and the Ethics Committee on
Animal Experimentation (CEEA)of ICB, protocol nº 26 fl. Book 84 02.Results: The animals with CCI showed a decrease (90%
for allodynia, 75% for thermal and 47% for mechanical tests) of the nociceptive threshold, when compared with control. On the
other hand animals treated with neural mobilization induced effective improvement in allodynia (86%), thermal (66%) and
mechanical (63%) tests, when compared with animals without treatment. We also observed a decrease of GFAP (90%) and NGF
(98%) expressions in DRG of animals with neural mobilization treatment when compared with animals untreated, in both assays.
In addition, we also observed a colocalization of GFAP and NGF in DRG after injury and a decrease after neural mobilization
treatment.
Conclusions:
Ours results suggest the involvement of satellite cells and NGF in chronic pain model. We also emphasize the neural mobilization
technique importance and effectiveness as treatment for neuropathic pain.
Keywords: Hyperalgesia, Neural growth factor, Neural mobilization, Satellite cells, Sciatic
Financial Support: FAPESP 10/01730-4, 07/58136-4; CAPES, IASP.
Resumo:17-065
GENETIC BACKGROUND DETERMINES MOUSE STRAIN DIFFERENCES IN INFLAMMATORY
ANGIOGENESIS
Marques, S. M. ; Campos, P. P. ; Castro, P. R. ; Cardoso, C. C. ; Ferreira, M. A. N. D. ; Andrade, S. P.
Fisiologia/Instituto de Ciências Biológicas, UFMG
Objectives:
Inflammation and angiogenesis are key components of fibrovascular tissue growth, a biological event underlying both
physiological (wound healing) and pathological conditions (tumor development, chronic inflammation).We investigated these
components in three frequently used mouse strains (Swiss, Balb/c and C57BL/6J) to verify the influence of genetic background
on the kinetics of inflammatory cell recruitment/activation, neovascularization, extracellular matrix deposition, and cytokine
production in polyetherpolyurethane sponge implanted subcutaneously in male mice of these strains.
Male Swiss, Balb/c or C57BL/6J mice 7-8 weeks (20-30g body weight) were used in these experiments. The fibrovascular tissue
was induced by polyether polyurethane sponge discs implanted subcutaneously at the back of anesthetized mice. At days 4, 10
and 14 postimplantation the animals were killed, the implants removed and processed determination of the inflammatory,
angiogenic and fibrogenic components in the three mice strains. The kinetics of neutrophil recruitment/activation as assessed by
myeloperoxidase (MPO) activity was 2 and 3-fold higher in Balb/c implants at day 1 compared with Swiss and C57BL/6J
implants, respectively. Macrophage accumulation/activation as NAG (n-acetyl â-glucosaminidase) activity was higher in Swiss
implants. The levels the monocyte chemoattractant protein 1 (CCL2(MCP-1)) peaked at day 10 in the of implants of the three
strain but was produced more by C57BL/6J mice. Angiogenesis (hemoglobin, vascular endothelial growth factor-VEGF, and
number of vessels) differed among the strains. Swiss implants had the highest hemoglobin content at day 10 (1,3±0,18 µgHb;
n=14) but the lowest VEGF levels (0,37±0,05pg/mg wet tissue; n=8). In contrast, Balb/c implants had higher VEGF levels
(1,4±0,23pg/mg wet tissue; n=6) but lower hemoglobin (0,6±0,08µgHb; n=14). Collagen deposition and transforming growth
factor alpha-1; (TGFalpha-1) levels also varied among the groups. Swiss and Balb/c implants had progressive increase in
TGFalpha-1 from 4 to 14 days, while C57BL/6J implants achieved the peak at day 10 and fell at day 14.
Conclusions:
In conclusion, the sponge implant model elicited the formation of fibrovascular tissue which differed importantly among the
strains. Our experiments have quantified this difference in terms of inflammation, angiogenesis, cytokine production and tissue
remodeling. It is evident from our study that the genetic background influences quantitatively, temporally and qualitatively the
process of fibrovascular tissue growth. This information may be relevant to direct genetic manipulation to target gene/s involved
in fibroproliferative processes, as well as to direct the choice of the mouse strain for specific functional experiments.
Keywords: cytokines, sponge implant, C57BL/6J, Balb/c, Swiss mouse strain
Financial Support: CNPq, CAPEs and FAPHEMIG
Resumo:17-066
EFFECT OF DEXAMETHASONE ON OROFACIAL NEUROPATHIC THERMAL NOCICEPTION
Coelho, S. C. ; Chichorro, J. G. ; Zampronio, A. R.
Farmacologia / Universidade Federal do Paraná, UFPR
Objectives:
Trigeminal neuralgia (TN) is a form of neuropathic pain characterized by severe pain in orofacial regions innervated by the
trigeminal nerve. The cause of TN is not entirely known, but it is believed that sensory nerve root compression and subsequent
nerve fibers demyelization accounts for the pathology. It is known that in models of neuropathic pain involving other sites (e.g.
sciatic nerve) the nerve injury induces the recruitment of inflammatory cells to the damaged site which may release
proinflammatory cytokines such as tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) contributing to hyperalgesia
development and sensitization. However, it is not known if these cytokines contribute to the neuropathic nociception in the
trigeminal area. As an initial approach, in this study we aimed to evaluate the effect of dexamethasone (DEX), a steroidal antiinflammatory drug which blocks cytokine synthesis on the thermal (cold and heat) hyperalgesia caused the infraorbital nerve
constriction in rats.
All the methods used in this study were previously approved by Institution´s Ethics Committee for Animal Use under protocol #
499. Male Wistar rats (180g) had their basal threshold for heat (radiant heat source) and cold (tetrafluoroethane spray application,
1 s) measured before any procedure. On the following day, the animals were submitted to a chronic constriction of the infraorbital
nerve (CION) using two silk 4-0 ligatures under anesthesia. Sham-operated were submitted to the same procedure but no
ligatures were applied to the nerve. Nocifensive responses elicited by cold and heat stimulation applied to the snout of rats were
evaluated after 4 days. On the fourth day, CION induced reduction of the reaction time to heat from 9.7 ± 0.7s (basal) to 5.0 ± 1.1
s while sham-operated animals showed similar reaction time (10.1 ± 0.8 s). CION also induced an increase in the nocifensive
reaction to cold stimulation from 5.4 ± 1.3s (basal) to 14.1 ± 3.8 s while sham-operated animals showed similar response (6.4 ±
3.4s). The subcutaneous (s.c.) injection of DEX (0.5, 1.0 and 2.0 mg/kg) or saline (SAL) on days 2, 3 and 4 after surgery did not
modify cold or heat hyperalgesia induced by CION. Measurements were always done 1h after the last treatment with DEX.
However, when the treatment with the same doses of DEX was given 1h before and 1, 2, 3 and 4 days after the surgery, the heat
hyperalgesia was significantly reduced 3h (88% and 56%) after the last treatment. This reduction was maintained up to 6h.
Conclusions:
Our results suggest that the treatment with DEX after the installation of the nerve damage is not effective to reduce the thermal
(cold and heat) hyperalgesia induced by the CION. However, the first two days after the initiation of the process seem to be
crucial for the reversal of the thermal hyperalgesia by DEX and cytokine synthesis may be involved in the process.
Keywords: Neuropathic pain, Dexamethasone, Heat hyperalgesia, Cold hyperalgesia, Inflammation
Resumo:17-067
EFFECT OF APPLICATION OF ALTERNATING ELETRIC CURRENTS OF MEDIUM FREQUENCY (NEMEC),
EVALUATED BY SCAPULAR POSITION, ON THE CERVICOBRACHIALGY – CASE STUDY
Lemes, E. V. ; Beirith, A.
Department of Physiotherapy and Natural Sciences, FURB
Objectives:
Cervicobrachialgy is a pain on the cervical region that radiate to superior member that affects 12 a 32% of adult population (
The procedure was done with one female subject with cervicobrachialgy. Pain was evaluated with McGill test (n=2) and
measurement of scapular position (n=4) and the results were analyzed by t student test (significant values of p
Conclusions:
The application of NEMEC improves the posture of the patient with cervicobrachialgy by muscle relaxing, and this effect seems
to e associated to a reduction of pain.
Keywords: CERVICOBRACHIALGY, PAIN, NEMEC, SCAPULAR POSITION
Financial Support: FURB
Resumo:18-001
DOUBLE COLLECTION: AN IMPROVEMENT ON TUBERCULOSIS DIAGNOSIS
Laux, L. C. ; Michelon, C. T. ; Rosso, F. ; Schmid, K. B. ; Ribeiro, A. W. ; Verza, M. ; Dalla Costa, E. R. ;
Rossetti, M. L. R.
Centro de Desenvolvimento Científico e Tecnológico, FEPPS
Objectives:
The double collection of sputum for a correct diagnosis of tuberculosis is recommended by the World Health Organization
(WHO) to increase the chances of achieving positive results, since the amount of bacilli in each sputum sample is variable. The
aim of this study was to evaluate the importance of double collection of sputum samples in order to improve the rate of molecular
analysis in tuberculosis case-detection.
A total of 175 spontaneous sputum specimens, of which 47 had a single collection and were processed in duplicate, and 128 had
two collections on different days that were processed at a single time, were used on this study. All samples (500ml) were treated
with 2% N-acetyl-L-cysteine (500ml) / 1 M NaOH (500ml). Culture for mycobacteria and smear microscopy were carried out for
all clinical samples, the association of these results was chosen as gold standard. This study was approved by the Ethical
Committee of Fundação Estadual de Produção e Pesquisa em Saúde (FEPPS). DNA extraction, purification, DNA amplification
and Microwell hybridization assay was standardized based on a previously protocol described by Michelon et al. (2011). Among
the 175 spontaneous sputum samples analyzed, from the 128 samples, which had double collection, 1 (microscopy and culture
positive) showed inhibition in the PCR reaction, 14 had positive result for culture or microscopy (2 only microscopy positive, 7
only culture positive and 4 positive for both) and 13 showed positive results in the molecular test, while 114 were negative all
tests. From 47 samples, which had single collection and were processed in duplicate, 4 had positive results for culture or
microscopy (1 only culture positive and 3 positive for both), 3 showed positive results in molecular test while 1 (positive for
microscopy and culture) had negative readings (false-negative), 43 samples were negative for all tests. Sensitivity and specificity
were calculated separately for samples in double and single collection, being 100%, 100% and 75%, 100%, respectively.
Conclusions:
In conclusion, the double collection performs an improvement of sensitivity on the molecular analysis, in agreement with the
recommendations of WHO for conventional diagnosis methods for tuberculosis.
Keywords: double collection, molecular diagnosis, tuberculosis
Resumo:18-002
BLOCKADE OF THE CXCL-ELR+ CHEMOKINE / RECEPTOR CXCR2 AXIS DURING NEUTROPHILIC PHASE
ACCELERATES WOUND CLOSURE IN MICE
Castro, T. B. R. 1,2; Canesso, M. C. C. 1,2; Almeida, B. G. 1,2; Cisalpino, D. 1,4; Colotta, F. 5; Bertini, R. 5;
Proudfoot, A. 6; Andrade, S. P. 2; Teixeira, M. M. 1,3; Barcelos, L. S. 1,2
6
Merck Serono Geneva Research Centre, UNIGE
4
Microbiology, ICB-UFMG
5
Dompé Research and Development, Dompé S.p.A
1
Immunopharmacology group, ICB-UFMG
2
Department of Physiology and Biophysics, ICB-UFMG
3
Biochemistry and Immunology , ICB-UFMG
Objectives:
Chemokines are important regulatory molecules of inflammatory and angiogenic processes in the pathophysiology of wound
healing, representing, therefore, possible targets for new therapeutic strategies. CXC-ELR+ family members might regulate both
recruitment of polymorphonuclear leukocytes and angiogenesis. The receptor CXCR2 is considered the main receptor for the
CXC ELR+ chemokines CXCL1/KC and CXCL2/MIP-2. The aim of this study was to evaluate the kinetics of skin wound
healing in C57BL/6J mice and the effects of Evasin-3, a CXC-ELR+ binding protein isolated from salivary gland of the tick
Rhipicephalus sanguineus, and Meraxin (DF2156A), a non-competitive allosteric inhibitor of CXCR2, on skin wound healing in
mice.
Excisional wounds were created on the dorsum with the aid of a circular punch, removing the entire thickness of the skin. The
area of the wounds was measured with the aid of a digital caliper for monitoring the closure of wounds. The activity of the
enzymes myeloperoxidase and N-acetyl-glucosaminidase was used to indicate the accumulation of neutrophils and macrophages,
respectively. CXCR2 expression was evaluated by qRT-PCR. Chemokine levels were quantified by ELISA. Animals received
daily i.p. injection of Evasin-3 at doses of 40, 4 and 0.4 ug / kg or Meraxin (DF2156A) at doses 10, 1, 0.1 mg / kg and the control
group received PBS vehicle. All treatment schedule started just after wounding and last until day 3 post surgery. The animals
were sacrificed at different time points and their wounds collected for posterior analysis. All procedures described here had prior
approval from the local animal ethics committee (CETEA-UFMG, license 254/08). In control animals, we observed peak of
neutrophils at 12 hours after surgery, while the peak of macrophages occurred on day 3. These returned to baseline after days 3
and 7 respectively. CXCR2 mRNA expression peaked at day 3. Angiogenesis peaked at day 7. The groups treated with Evasin-3
showed faster closure of wounds in all doses evaluated when compared with the control group. We observed an increase of
macrophage content of treated animals. However, we did not observe any difference in neutrophil content or CXCL1 and CXCL2
levels after treatment. Evasin-3 had no effects on wound closure when treatment lasted till day 7 post injury. Likewise, animals
treated with Meraxin also displayed faster closure of wounds when compared to control group, although only at lower doses, and
showed an enhanced on macrophage content and no difference in neutrophil or CXCL1 and CXCL2 content.
Conclusions:
Our data suggest pharmacological blockade of the CXCL-ELR+ chemokine / receptor CXCR2 axis during neutrophilic, but not
angiogenic, phase accelerates wound closure in mice and may have therapeutical potential to treat non-healing wounds.
Keywords: CHEMOKINES, CXCR2, MACROPHAGES, NEUTROPHIL, WOUND HEALING
Financial Support: CNPq, FAPEMIG.
Resumo:18-003
THE ROLE OF EXTRACELLULAR ATP ON PROMASTIGOTES OF LEISHMANIA AMAZONENSIS
Uribe; G. D. C. S. ; Marques-da-silva; C. ; Coutinho-silva; R.
Instituto de Biofísica Carlos Chagas Filho/UFRJ, IBCCF
Objectives:
Leishmaniasis is one of the most important neglected tropical (World Health Organization), affecting approximately 88 countries.
The disease is caused by trypanosomatid protozoa Leishmania, that has two forms well described, the promastigote (flagelated
with free life, infective) and the amastigote form (Intracellular, replicative), that can infect virtually all cell with phagocytic
capacity. The Literature data indicate that the trypanossomatids are unable to perform de novo synthesis of purines, requiring
external sources for their metabolism. This study seeks to uncover the role of ATP in promastigotes of Leishmania amazonensis
and its role during the adhesion and infection of macrophages.
Metacyclic phase promastigotes were pre-incubated with crescent ATP concentrations or not, diluted in 199 medium for 30
minutes, centrifuged at 1500g for 2 minutes, washed and counted for subsequent infection on resident peritoneal macrophages
from Balb/c mice during 4, 24 and 48 hours. After exposure to 100μM ATP for 30 minutes, the promastigotes were centrifuged at
180g for 5 minutes, washed and counted. Then fixed with 4% paraformaldehyde for 1 hour at room temperature. The
concentration of parasites was adjusted to 5 x 105 in 200μL of PBS and performed cytospin. The samples were stained with Kit
Panótico Rápido. It was then taken to the confocal microscope to obtain images and the subsequent analysis.We observed an
increase in adhesion / infection of the parasites pretreated with ATP in a dose-dependent way until 24 hours, but not at 48 hours.
At 4 hours, 10, 50 and 100μM ATP pre-treatment induced an increase of (40.44% ± 17.08; 64.28% ± 12.26; 104.5 ± 24.55%);
respectively. After 24 hours, treatment with 10, 50 and 100 μM ATP augmented the infection in 61.87% ± 21.96; 50.14% ±
13.18; 93.59 ± 16.79% respectively; Conversely after 48 hours of infection with pre treated promastigotes the infection index did
not display alterations. We observed changes in the morphology of promastigotes when treated with ATP, as a more tapered, the
presence of more than one flagellum per cell and less clear kinetoplast.
Conclusions:
The molecule ATP increases adhesion and Leishmania amazonensis infection in macrophages through changes in their
morphology and metabolism.
Keywords: ATP, Extracellular, leishmania, promastigotes, role
Financial Support: CNPq, Faperj, PRONEX, INPeTAm.
Resumo:18-004
CELL NUMBER QUANTIFICATION AND IL-4 PRODUCTION OF MACROPHAGES IN YOUNG AND ADULT
RATS SUBMITTED TO NEONATAL MALNUTRITION
Galvao, A. M. 1; Melo, J. F. D. 1,2; Costa, T. B. D. 1; Chaves, M. E. C. 1; Nagel, M. 2; Castro, C. M. M. B.
D. 1,2
1
Laboratório de Imunopatologia Keizo Asami, LIKA
2
Université de Technologie de Compiègne, BMBI
Objectives:
To investigate the effects of a neonatal low-protein diet on the cell number and IL-4 production of macrophages in young and
adult rats.
Male Wistar rats (n=12) were suckled by mothers fed diets containing 17% protein (controls, C) or 8% protein (undernourished,
UN). All rats were fed a normal protein diet after weaning. Body weights were recorded every five days during lactation.
Bronchoalveolar lavage was collected of 42- (n=6) and 60-day-old rats (n=6). Alveolar macrophages were cultured (1.6 x 106
cells/35cm2 tissue culture wells) for 4 days in RPMI 1640 to assess cell number by measuring intracellular lactate dehydrogenase
(LDH) activity and IL-4 levels in culture supernatants by ELISA. After 3 days of culture, the cells were stimulated with LPS. The
IL-4 concentration was normalized to 108 cells using the LDH dosage. Offspring from mothers fed a low-protein diet showed a
lower body weight gain at 5th (C = 12.16 ± 0.4; UN = 10.38 ± 0.55), 10th (C = 24.11 ± 0.9; UN = 18.66 ± 0.8), 15th (C = 35.55 ±
0.6; UN = 24.38 ± 1.06) and 21st day of life (C = 55.55 ± 1.86; UN = 35.66 ± 1.34). Pups from undernourished mothers remain
with their lower body weight in comparison with control group at 42 d (C = 187.16 ± 9.16; UN = 161.33 ± 1.36) and 60 d (C =
275 ± 10.83; UN = 233.5 ± 5.25) p
Conclusions:
Dietary restriction during lactation reduces the number of macrophages in culture of young and adult rats. The higher production
of IL-4, an anti-inflammatory cytokine, in macrophages from UN adult rats may indicate an increase in the susceptibility to
infections.
Keywords: Interleukine, Macrophages, Malnutrition, Neonatal, Rats
Financial Support: CAPES-COFECUB (grant 584/07)
Resumo:18-005
EFFECT OF PALMITATE IN THE CELLULAR DYE REDUCTION OF 3-(4,5-DIMETHYLTHIAZOL-2-YL)-2,5DIPHENYLTETRAZOLIUM BROMIDE (MTT) AND PRODUCTION OF REACTIVE OXYGEN SPECIES IN
LEUKOCYTES FROM TYPE 2 DIABETIC PATIENTS
Volpe, C. M. O. 1; Fagundes-netto, F. S. 1; Veloso, C. A. 2; Fernandes, J. S. 1; Chaves, M. M. 2; Nogueiramachado, J. A. 1
1
Instituto de Ensino e Pesquisa da Santa Casa de BH, IEP - Santa Casa BH
2
Departamento de Bioquímica e Imunologia UFMG, ICB - UFMG
Objectives:
To investigate the effect of palmitate on the balance between oxidizing/reducing cellular responses in type 2 diabetic patients.
This study was approved by the ethics committee of Hospital Santa Casa of Belo Horizonte. Diabetic patients type 2 (T2DM) and
nondiabetic control (ND) were aged between 40 and 70 years. Leukocytes from T2DM (n=10) and ND (n=10) were purified
using the Ficoll-Hypaque gradient method. Either granulocytes (G) or peripheral blood mononuclear cells (PBMNC)
(1x10℘5/100μL in PBS) were incubated in the presence of palmitate (P) (100μM) and/or opsonized zymosan (ZC3b) (positive
control) with MTT (25μL; 5.0 mg/mL in PBS) for 3 hours at 37°C. The controls without palmitate or opsonized zymosan were
performed simultaneously. The reaction was stopped by addition of 1.5 mL of 0.04 M hydrochloric acid in isopropanol and the
absorbance measured at 570 nm. The phagocytosis assay was performed by evaluation of ROS production by granulocytes from
ND and T2DM. The ROS production was measured using a luminol-dependent chemiluminescence assay. Granulocytes
(1x10℘5/100μL in PBS) were stimulated by ZC3b or ZC3b+P (100μM). Cellular viability was performed by trypan blue
exclusion test. Statistical analysis was performed by Student t-test (p0.05). ROS production in the presence of palmitate was
greater in T2DM than ND. The results were expressed as mean measurements of relative light units/minute ± SE: G+ZC3b
4730.6±228.8 and 5446.5±180.4 (p>0.05); G+ZC3b+P 8691.4±204.6 and 10505.8±282.4 (p
Conclusions:
These results suggest that palmitate significantly affected the balance between oxidizing/reducing cellular responses in T2DM.
Keywords: MTT dye reduction, Palmitate, Reactive oxygen species, Type 2 diabetes mellitus
Financial Support: FAPEMIG, CAPES, CNPq.
Resumo:18-006
BRAIN MICROCIRCULATORY EFFECTS OF LOVASTATIN IN PLASMODIUM BERGHEI ANKA-INDUCED
EXPERIMENTAL CEREBRAL MALARIA.
Estato, V. 1; Reis, P. A. 2; Tibiriçá,1; Castro-faria-neto, 2
Laboratório de Investigação Cardiovascular, FIOCRUZ
2
Laboratório de Imuno-farmacologia, FIOCRUZ
1
Objectives:
Several studies have shown that statins, in addition to cholesterol-lowering properties, present antiinflammatory effects due to
several mechanisms, as well as alteration on geranylgeranylation of protein or enhance of heme-oxigenase-1 expression. In this
study we investigate the effects of lovastatin (LOVA, 20 mg/kg, per oral) treatment on brain microcirculatory alterations induced
by Plasmodium berghei ANKA-induced cerebral malaria (CM).
Methods: C57BL/6 mice were injected with 106 infected erythrocytes. On day 6 post infection they were treated with saline
solution (PbA, n=5) or LOVA (n=5). After 2 hours, intravital fluorescence videomicroscopy was performed to assess the pial
vasculature through a cranial window in anesthetized mice (PbA). Functional capillary density (FCD), considered as the number
of spontaneously perfused capillaries (vessels with diameters less than 10 µm) per mm², was determined in random microscopy
fields during 4 minutes. To observe leukocyte/endothelium interactions, leukocytes were labeled by i.v. administration of
rhodamine 6G (0.5 mg/kg) and rolling was defined as white cells moving at a velocity less than that of erythrocytes cells.
Adherent leukocytes were stationary at least 30 s to the venular endothelium. Results: Treatment with LOVA markedly reduced
the leukocyte rolling (25.9 ± 6 vs. 7.2 ± 2 cells/field; PbA vs LOVA, respectively; P
Conclusions:
Our results suggest that treatment with lovastatin is able to prevent the rolling and adhesion of leucocytes and may protect the
brain against microvascular alterations induced by cerebral malaria.
Keywords: CEREBRAL MALARIA, BRAIN MICROCIRCULATION, LOVASTATIN, INTRAVITAL MICROSCOPY,
LEUKOCYTE-ENDOTHELIUM INTERACTIONS
Financial Support: FAPERJ, CNPQ and PAPES-FIOCRUZ.
Resumo:18-007
MODULATION OF EXPERIMENTAL GLOMERULONEPHRITIS BY INVARIANT NATURAL KILLER T CELLS
AGONISTS.
Khaled, N. A. 2,3; Monteiro, A. P. F. S. 2,3; Reis, V. O. 2,3; Silva, J. C. 2,3; Trindade, G. 2,3; Savage, P. B. 4;
Keller, A. C. 2,3
3
Departamento de M.I.P./ Universidade Federal de São Paulo, UNIFESP
2
Laboratório de Imunopatologia Experimental, LIPE/UNIFESP
4
Department of Chemistry and Biochemistry, Brigham Young Univ, BYU
Objectives:
We have described that during the pathogenesis of the experimental anti-glomerular basement membrane glomerulonephritis
(anti-GBM GN) invariant Natural Killer T cells (NKT) migrate to inflammatory site and contributes to the modulation of the
disease (J Am Soc Nephrol 20; 1282, 2009). Because, the anti-GBM GN appears to be associated with an imbalance between Th1
and Th2 cytokines, we decided to study the effect of exogenous NKT cells agonists, with pro-Th1 or Th2 effect, on the
development of the disease. In this vein, we used GSL-1, a monoglycosilceramide from Sphingomonas ssp with pro-Th1 activity
and the pro-Th2 synthetic glycosphingolipids OCH, PBS-24 and PBS-120.
Passive anti-GBM GN was induced in C57Bl/6J mice by three consecutive intravenous administration of 200µL of sheep anti-rat
GBM serum (days 0, 1, and 2). The exogenous NKT cells agonists were injected i.v. at day 0 (10µg/mice). The experiment was
performed at day 14 and the renal function was determined by the proteinuria and creatininuria ratio (P/C). We found that the
levels of P/C were increased in the anti-GBM mice in comparison to the control group with two way ANOVA test, reflecting
renal failure (29,73 ± 7,14 versus 14,83 ± 0,93). In contrast, GSL-1 treated mice presented P/C levels comparable to control
animals (10,04 ± 1,00) whereas the pro-Th2 agonists OCH and PBS-120 increased P/C levels in comparison to anti-GBM group
(69,52 ± 3,01; 60,42 ± 5,01, respectively).
Conclusions:
Our data demonstrate that pro-Th1 polarization can exert a renoprotective effect on anti-GBM GN pathogenesis while the use of
pro-Th2 agonists resulted in a deleterious outcome. These results are in concordance with the notion that type-1 immune response
can be involved in the modulation of the anti-GBM GN (Faseb J 17, 860, 2003). Thus, our work suggests that NKT agonists can
be an alternative approach to anti-GBM GN management.
Keywords: NATURAL KILLER T, GLOMERULONEPHRITIS, AGONISTS
Financial Support: FAPESP and CNPq.
Resumo:18-008
INFLAMMATORY BIOMARKERS IN WORKERS EXPOSED TO SILICA
Mendonça, V. A. 1; Souto, M. F. O. 2; Carneiro, A. P. S. 2; Borges, V. O. 3; Mendonça, A. L. 2; Amorim, M.
R. 1; Teixeira, M. M. 3; Teixeira, A. L. 3
1
Laboratório de Imunologia/Departamento de Fisioterapia/FCBS, UFVJM
2
Centro de Referencia Estadual em Saúde do Trabalhador , CEREST
3
Laboratório de Imunofarmacologia – ICB , UFMG
Objectives:
Silicosis is an irreversible fibrotic and potentially fatal disease lung caused from inhalation of free crystalline silica particles. The
determination of biological markers associated with the pathophysiology (profibrotic and proinflammatory cytokines and
chemokines), has shown promise for early detection of disease and institution of control measures. Objectives: To evaluate the
plasma concentrations of TNF-α, CCL3, CCL11, CCL24 and CXCL10 of subjects exposed to silica (SES) and healthy controls.
TNF-α, CCL3, CCL11, CCL24 and CXCL10 were measured by ELISA in plasma of SES (n=34) from Corinto, Minas Gerais. In
this group, twenty subjects had the diagnosis of silicosis. Control group consisted of age and gender matched healthy subjects
(n=20). Results: Plasma concentrations of TNF-α and CCL11 were significantly increased in SES subjects when compared to
controls. Interestingly, the plasma concentration of CCL11 was higher in silicosis subjects (median value 534.1 pg/ml) in
comparison with SES without silicosis (278.1 pg/ml) and controls (100.0 pg/ml). There was no difference in the plasma
concentrations of CCL3, CCL24 and CXCL10 between SES and controls.
Conclusions:
This study demonstrated that CCL11 and TNF-α may play a role in silicosis pathogenesis. The role of these molecules as
biomarkers of silicosis development must be investigated.
Keywords: Chemokines, Cytokines, diagnosis, prognosis, Silicosis
Financial Support: FAPEMIG
Resumo:18-009
PRODUCTION OF IFN-γ, IL-12 AND IL-10 BY ALVEOLAR MACROPHAGES AND LYMPHOCYTES: STUDY OF
IN VITRO CELLULAR INFECTION BY METHICILLIN-SENSITIVE AND METHICILLIN-RESISTANT
STAPHYLOCOCCUS AUREUS IN RATS WITH NEONATAL MALNUTRITION
Costa, T. B. 1; Morais, N. G. 1; Almeida, T. M. D. 2; Ribas, K. H. S. 2; Araújo, F. R. G. 2; Castro, C. M. M.
B. 1
1
Department of Tropical Medicine, UFPE
2
Laboratory Immunopathology Keizo Asami, UFPE
Objectives:
Aim: To study the production of IFN-γ, IL-12 and IL-10 by alveolar macrophage (AM) and lymphocytes (LT) in vitro infected
with Methicillin-sensitive Staphylococcus aureus (MSSA) and Methicillin-resistant Staphylococcus aureus (MRSA) in groups of
rats submitted to neonatal malnutrition (NM) and a group of well-nourished rats (N).
Methods and Results: male Wistar rats (n=48) were breastfed by mothers that fed a diet containing 8% and 17% of protein during
lactation (MN and N groups, respectivelly). After weaning, normoproteic diet were administered to both groups. The AMs were
obtained after tracheostomy, through the collection of bronchoalveolar lavage. To obtain the LT, the surgical procedure for
cardiac puncture was initially performed in order to separate the mononuclear cells by Ficoll density gradient. After isolation of
different cell types in Falcon type plaques, four systems were formed: negative control, composed by AM or LT only (C-);
positive control, added with lipopolysaccharide (C+); and two test systems, MSSA and MRSA, stimulated by the strains. The
measurement was performed by ELISA, using the kit (Quantikine âm, R & D Systems), from samples collected from the culture
supernatants after 24 hours of incubation. For statistical analysis, Student-t test and Mann-Whitney were used, assuming a
significance level of p
Conclusions:
Conclusion: The model of neonatal malnutrition produced consequences on body weights and compromised the production of
proinflammatory cytokines (IFN-γ and IL-12), indicating that this model of malnutrition can affect the resolution of infectious
processes. The methicillin-resistant Staphylococcus aureus strain has stimulated a higher production of IFN-γ and IL-10 by
alveolar macrophages, suggesting more intense immune stimulation by this strain in this particular cell type.
Keywords: CYTOKINES, ALVEOLAR MACROPHAGES, LYMPHOCYTES, STAPHYLOCOCCUS AUREUS, NEONATAL
MALNUTRITION
Resumo:18-010
REDUCTION OF THE NUMBER AND REACTIVITY OF MAST CELLS INDUCED BY GLUCOCORTICOIDS
DEPENDS OF ADVANCED GLYCATION END PRODUCTS
Santoro, T. ; Torres, R. C. ; Cordeiro, R. S. B. ; Silva, P. M. R. ; Martins, M. A. ; Carvalho, V. F.
Laboratório de Inflamação/ Instituto Oswaldo Cruz, IOC/FIOCRUZ
Objectives:
Glucocorticoids are the most effective anti-inflammatory therapy for the treatment of many chronic inflammatory and immune
diseases, including asthma, rheumatoid arthritis and autoimmune diseases. There have been major advances in understanding the
molecular mechanisms whereby glucocorticoids suppress inflammatory response. One of these mechanisms is the activation of
genes encoding anti-inflammatory proteins, as annexin-1 and mitogen-activated protein kinase phosphatase-1. Moreover, chronic
corticosteroid therapy has shown direct inhibitory actions on several inflammatory cells, including a marked reduction in the
number and reactivity of mast cells by apoptosis induction. Mast cells play key roles in inflammatory responses as well as innate
and acquired immunity. Mast cells express two advanced glycation end products (AGE) cell surface receptors/binding proteins,
the receptor for AGE (RAGE) and galectin-3. Furthermore, AGE induces mast cell death by apoptosis through binding to RAGE.
More recently, it was observed that therapy with corticoids induces an increased expression of RAGE in the liver of patients with
atherosclerosis. In this study, we evaluated if the reduction in the number and reactivity of mast cells induced by glucocorticoids
is dependent on the AGE.
The animals were obtained from the Oswaldo Cruz Foundation breeding colony and used in accordance with the guidelines of the
Committee on Use of Laboratory Animals of the Oswaldo Cruz Foundation (CEUA-FIOCRUZ, license LW 23/10). Male Wistar
rats received dexamethasone or prednisolone (0.1 mg/kg, s.c.) for 21 days and were treated with aminoguanidine (250 mg/kg,
v.o.) after 3 days of glucocorticoid administration beginning, once daily for 18 consecutive days. The administration of
dexamethasone induced a reduction in the pleural mast cell numbers compared with controls (from 451.5 ± 28.89 to 308.17 ±
25.3 mast cells (x10,000)/cavity, respectively; mean ± SEM, n = 6). Treatment with aminoguanidine restored mast cell numbers
in the pleural cavity of rats were received dexamethasone (434.39 ± 19.17 mast cells (x10,000)/cavity, mean ± SEM, n = 6).
Aminoguanidine also significantly reversed the dexamethasone-induced reduction in histamine release, as measured by
fluorescence, following activation with antigen or compound 48/80 in vitro. Similar results were obtained using prednisolone
instead of dexamethasone.
Conclusions:
Our results showed that the inhibitory effect of glucocorticoids on number and reactivity of mast cells is dependent of AGE.
Keywords: Advanced glycation end products, Glucocorticoid, Mast cell
Financial Support: CNPq, FAPERJ and FIOCRUZ
Resumo:18-011
TRYPANOSOMA CRUZI INVADES HOST CELLS THROUGH THE ACTIVATION OF ENDOTHELIN AND KININ
RECEPTORS: A CONVERGING PATHWAY LEADING TO CHAGASIC VASCULOPATHY
Andrade, D. D. S. 1; Serra, R. R. 1; Svensjö, E. 1; Morandi, V. 2; Soeiro, ;. M. D. N. C. 3,3; Tanowitz, H. B. 4;
Scharfstein, J. 1
1
Instituto Biofísica Carlos Chagas Filho, IBCCF
2
Departamento de Biologia Celular, Universidade do Estado do, UERJ
3
4
Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fu, FIOCRUZ
Department of Pathology, Albert Einstein College of Medicine, Albert Einstein Coll
Objectives:
Background and purpose: Endothelin was previously implicated in the pathogenesis of Chagas heart disease. Since Trypanosoma
cruzi releases vasoactive kinins extravascularly, here we examined whether these parasites may infect host cells and evoke
interstitial edema through the activation of bradykinin (B2R) and endothelin receptors (ETRs).
Infection indexes were determined in CHO-cells overexpressing ETRs, mouse cardiomyocytes (MCMs), endothelial cells
(HUVECs), and human smooth muscle cells (HSMCs). Intravital microscopy in hamster cheek pouch (HCP) topically exposed to
T. cruzi was used to determine if accumulation of rhodamine-labeled leukocytes was reduced by HOE-140 (B2R antagonist),
BQ-123 or BQ-788 (ETAR and ETBR antagonists, respectively). Footpad swelling was used to measure parasite-evoked
inflammation in BALB/c mice. Although BQ-788 (but not BQ-123) partially protected HUVECs from infection, parasite uptake
was virtually abolished when BQ-788 was added to thapsigargin or wortmannin-treated-HUVECs. In contrast, parasite infectivity
was potentiated in HUVECs pretreated with the adenylyl cyclase inhibitor MDL12, irrespective of BQ-788. Assays with MCMs
or HSMCs showed that infection was similarly reduced by individual GPCR antagonists. Parasite internalization via the
ETAR/ETBR/B2R-dependent axis was abolished by MâCD, a cholesterol depleting drug. Studies of TCT-evoked inflammation
showed that BQ-123, BQ-788 and HOE-140 reduced leukocyte accumulation in HCP and blunted footpad swelling in mice, thus
linking development of interstitial edema to the activation of B2R, ETAR and ETBR.
Conclusions:
Our data indicate that trypomastigotes infect host cells, including cardiomyocytes, and evoke inflammatory edema through
intertwined activation of ETRs/B2R. These results substantiate the proposition that treatment with ETR/B2R antagonists might
alleviate infection-associated vasculopathy in Chagas heart disease.
Keywords: bradykinin, cardiomyopathy, Chagas disease, cruzipain, endothelin
Financial Support: FAPERJ, CNPq
Resumo:18-012
B7-CD28/CTLA-4 PATHWAY IN ALTERED IN ALZHEIMER AND FRONTOTEMPORAL DEMENTIA PATIENTS
Lima, G. S. F. ; Torres, K. C. L. ; Santos, R. R. D. ; Fiamoncini, C. ; Ferreira, R. O. S. ; Moraris, E. N. ;
Silva, M. A. R.
Departamento de Saúde Mental, Universidade Federal de Minas , UFMG
Objectives:
Frontotemporal dementia (FTD) is a devastating neurodegenerative disease characterized by behavioural and/or language
dysfunction and due to progressive atrophy and neuronal loss involving the frontal and/or temporal lobes. There is considerable
evidence to suggest that an inflammatory response may be involved in the neurodegenerative cascade in Alzheimer disease (AD)
and others dementias as well. Very little is known about the role of inflammatory process in FTD and most studies were derived
from cerebrospinal fluid, which is an invasive procedure. The ability to discriminate between self and nonself is perhaps the most
fundamentally important aspect of immune system. One mechanism designed to maintain the fidelity of the immune response is
the “two-signal” concept of lymphocyte activation: an antigen-specific signal via the T cell receptor (Signal 1) and a costimulatory signal (Signal 2) that is provided by soluble factors or cell-surface molecules on the antigen presenting cell (APC).
The main issue of this work was to study B7-CD28/CTLA-4 pathway in peripheral mononuclear cells from AD, FTD and control
patients (not demented not depressed patients).
Peripheral blood mononuclear cells (PBMC) from patients were separated cultivated with Lypopolysacaride (LPS). After 18h the
cells were harvested and stained with monoclonal antibodies (anti-CD80, anti-CD4, anti-CD28, anti-CD14, anti-CD19, antiCTLA-4). Afterwards they were fixed with phormadehyde 2% and acquired in flow cytometer (Guava/GE). The results were than
analysed in the Cytosoft software. The nonparametric test Kruskal-Wallis one-way analysis of variance was used for comparing
the distribution of the three unmatched group (FTD, AD and control group). The second signal can have a stimulatory or
inhibitory nature. Our results show that only inhibitory co-stimulatory molecule (CTLA-4) is altered. There is no difference in
CD80 and CD28 expression. CTLA-4 expression is reduced in CD4+ cells from FTD (0.457 ± 1.55%) when compared to AD
(0.999 ± 0.58 %) and control patients (3.669 ± 1.87 %) (p=0.006).
Conclusions:
By our knowledge this is the first study to point and alteration in B7-CD28/CTLA-4 pathway in dementia patients. FTD patient
usually progress more rapidly than AD and the reduction in inhibitory co-stimulatory CTLA-4 may be involved in this process.
Keywords: ALZHEIMER AND FRONTOTEMPORAL DEMENTIA , neurodegenerative disease , inflammatory response , antiCTLA-4 and anti-CD28, flow cytometer
Financial Support: INCT, CNPq, CAPES, AMBRIEX, John Simon Guggehnheim Foundation, FAPEMIG
Resumo:18-013
PERIPHERAL BIOMARKERS RELEASED BY HUMAN FIBROBLASTS AFTER STIMULATION WITH SOLUBLE
&BETA-AMYLOID PROTEIN.
Deus, J. L. D. 1; Rocha, N. P. 2; Santos, P. S. 3; Andrade, L. M. D. 2; Reis, H. . J. 2; Teixeira, A. L. 4;
Furtado, P. 1; Melo, G. E. . A. B. 1; Guimaraes, M. M. 1
1
UNIVERSIDADE FEDERAL DOS VALES DO JEQUITINHONHA E MUCURI, UFVJM
2
UNIVERSIDADE FEDERAL DE MINAS GERAIS, UFMG
3
4
UNIVERSIDADE FEDERAL DE MINAS GERAIS, UFMG
5
6
Objectives:
Examine potential participation of peripheral chemical mediators in the pathophysiologal mechanisms and developmental
prediction of Alzheimer's disease (AD).
In vitro conditions were generated, similar to those present during AD tissue changes derived from human gingival fibroblast
exposure to protein b-amyloid (bA). It is suggested that the latter was one of the key proteins involved in AD molecular
processes. Fibroblasts isolated from gingival tissue were stimulated with soluble protein at various concentrations of bA (10-4
mol/l, 10-6 mol/l, 10-8 mol/l, 10-10mol/l, 10-12mol/l), and at the following incubation periods: 12, 24, 48, 72, 96 and 120 hours.
The release of cytokines (IL-8, IL-10, IL-6, TNF-a) in supernatants of cells stimulated with bA protein was evaluated with
ELISA after various incubation periods. The control group went through the same procedures but without stimulation with
protein &betaA. Our preliminary results showed that bA peptides induced the increase of all cytokines analysed (IL-10, IL-8, IL6, TNF-a) in comparison with control group. The pattern of release was similar to both IL-8, IL-6 and TNF-a. It was observed a
peak of release of those cytokine release at 96 hours. The IL-10 pattern did not show a peak-like release as compared with the
others cytokines.
Conclusions:
Human fibroblast stimulation with different concentrations of &betaA soluble peptide is associated with IL-8, IL-6, TNF-a and
IL-10 release at different time of incubation with the peptide. The peak-like pattern of release verified at IL-8, IL-6 and TNF-a
cytokines could be associated with its pro-inflamatory function in contrast with distinct pattern presented by IL-10, un
uninflamatory interleukin. Our dada suggest that human fibroblast could be a model for the studies of inflammatory aspects
implicated at pathophysiology of AD.
Keywords: PERIPHERAL BIOMARKERS, HUMAN FIBROBLASTS, &Beta-AMYLOID PROTEIN, ALZHEIMER'S
DISEASE
Financial Support: CNPq, FAPEMIG and UFVJM
Resumo:18-014
HEPATIC AND SPLENIC HISTOLOGICAL EVALUATIONS IN MICE EXPOSED OF CIGARETTE SMOKE ON
GESTATIONAL PERIOD
Diniz, M. F. 1; Dourado, V. A. 1; Gonçalves, E. G. 1; Ferreira, B. S. B. 1; Silva, M. E. 2; Pedrosa, M. L. 1;
Bezerra, F. S. 1; Lima, W. G. 1
1
Departamento de Ciências Biológicas (DECBI), UFOP
2
Escola de Nutrição (ENUT), UFOP
Objectives:
Previous works of our group has been appointed the cigarette smoke ability of promotes tissue damage in organs of newborn
murine model. Thus to evaluate if exposition of cigarette smoke in the uterine life promotes changes in tissue architectural
organization we are studying the possible histological alterations in liver and spleen of newborn mice.
We promote the mate with eighteen C57BL6 mice that were disposes to in proportion of two females adults to each male. The
pregnancy was confirmed by endovaginal smear. Pregnant females were divided into two groups. The first group of eight females
was exposed for three times day to smoke of four cigarette during 21 days (IFC21) in chamber (30x60x40cm). The second one of
four females was exposed to ambient air receiving similar manipulation to IFC21 (control: CG). Six animals newborn of IFC21
and CG females were weighed and euthanized after 24 hours of life. In necropsy liver and spleen were collected and organ weight
was determined. Tissues slides of liver and spleen embedded paraffin were stained by Hematoxylin and Eosin and Masson´s
trichrome to histological analysis. Morphometric digital techniques were performed to quantify the hepatic inflammatory process
and the collagen tissue deposition. Moreover morphometric analyses were performed to evaluate the total area of the spleen,
white and red pulp total area and splenic capsule thickness. Liver and spleen weight was no different between IFC21 and CG
groups. Histological analyses showed no presence of hepatic tissue collagen deposition. However was observed differences (P5 x
9.8±2.0x105 µm2 , respectively), capsule thickness (32.8±2.3 x 38.6±4.0 µm, respectively), white pulp total area (3.2±1.2x10 4 x
4.6±2.7x104 µm2, respectively) and red pulp total area (9.3±1.4x10 5 x 9.4±2.0x105 µm2, respectively).
Conclusions:
Our results indicate cigarette smoke was not able to promote histological architectural change spleen of chosen murine model of
chronic exposure to cigarette smoke. We are accessing new histological and biochemical analyses in spleen, liver and other
organs trying to understand mechanism and effects of cigarette smoke over tissues and organic system.
Keywords: inflammation, cigarette smoke, liver, spleen, newborn
Financial Support: UFOP and FAPEMIG
Resumo:18-015
EFFECT OF DIPHENYLENEIODONIUM (DPI) ON PHAGOCYTOSIS OF OPSONIZED PARTICLES AND
PRODUCTION OF PROINFLAMMATORY CYTOKINES BY HUMAN LEUKOCITOSE FROM TYPE 2 DIABETIC
PATIENT.
Fagundes-netto, F. S. 1; Volpe, C. M. O. 1; Veloso, C. A. 2; Chaves, M. M. 2; Nogueira- Machado, J. A. 1
1
Instituto de Ensino e Pesquisa da Santa Casa de BH , IEP - SCMBH
2
Universidade Federal de Minas Gerais , ICB UFMG
Objectives:
Assess the phagocytic ability of leukocytes and pro inflammatory cytokine secretion by lymphocytes from diabetic and
nondiabetic patients. The possible association between the ability of phagocytic activity and NADPH oxidase are studied.
This study was approved by the ethics committee of SCMBH. Diabetic patients type 2 (T2DM) and nondiabetic control (ND)
were aged between 40 and 80 years, of both sexes. Mononuclear cells (PBMNC) of T2DM (n = 10) and ND (n = 10) were
purified and their number agreed to 1x105/100mL in PBS. Cell viability was performed by trypan blue exclusion test. Statistical
analysis was performed by X square and Student t-test (p
Conclusions:
The phagocytosis capacity of diabetic patients is reduced, with the exception of granulocytes. The mononuclear cells appear to
exert an inhibitory effect on granulocyte phagocytosis of non-diabetic patients. Possibly the MTT assay in diabetic patients
occurs through mitochondria, since when we inhibited NADPH oxidase did not alter phagocytosis.
Keywords: DIPHENYLENEIODONIUM (DPI), PHAGOCYTOSIS, PROINFLAMMATORY CYTOKINES
Financial Support: CAPES,CNPq,FAPEMIG
Resumo:18-016
EXPRESSION LEVELS OF ALPHA, BETA AND GAMMA INTERFERON GENES IN PATIENTS WITH
DIFFERENT CLINICAL FORMS OF DENGUE.
Silva, M. M. C. 1; Oliveira, A. G. 2; Bertani, G. R. 3; Gomes, A. L. V. 1; Calzavara-silva, C. E. 4; Gil, L. H.
V. G. 1
1
Depto. de Virologia e Terapia Experimental/CPqAM-FIOCRUZ, CPqAM-FIOCRUZ
2
Depto. de Genética, UFPE
3
Departamento de Bioquímica/UFPE, LIKA
4
Lab. Imunologia Celular e Molecular/CPqRR, CPqRR
Objectives:
Dengue virus (DENV) causes the most common arbovirosis worldwide. The most severe form of the disease, Dengue
Hemorrhagic Fever (DHF), presents the mortality rate usually between 1% and 10% and requires hospitalization and careful
hemodynamic management of patients. The type I IFNs (IFN-I), IFN-α and IFN-β, together with the type II IFN (IFN-II), IFN-γ,
are crucial in mediating antiviral response through blocking viral replication or through modulating immune responses to inhibit
viral spreading. In this study we investigated the expression levels of genes encoding for IFN-I and IFN-II, using cDNA obtained
from peripheral blood mononuclear cells (PBMC), and levels of IFN-I in serum samples from DENV-infected patients.
To study the gene expression of IFN-I and II, we used real-time quantitative PCR (qPCR), in which total RNA was extracted
from PBMC samples and reversibly transcribed to cDNA, and for IFN-I analysis in serum samples we used a sensitive nonviral
bioassay, based on a cell line that carries the luciferase reporter gene controlled by ISRE promoter (IFN-responsive region I). A
stable reporter cell line was established drawn from the transfection of BHK-21 cells (Baby hamster kidney cells) with pISRELuc-Hygro plasmid. These cells were then tested by the induction of ISRE promoter with human IFN-α which showed a high
expression of the reporter gene, demonstrating that cells are responsive to IFN-α human and therefore highly feasible detection
and measurement of IFN-I present in different types of samples. In serum samples of patients with dengue fever (DF) we detected
low levels of IFN-I, but more studies are being done to quantify IFN-I in patients with DHF. It was also observed, by qPCR, that
IFN-α levels increased in early infection (3 to 5 days of onset of fever), particularly in patients who develop DHF, suggesting that
these increased levels are associated with the development of severe disease. The IFN-β expression levels were similar in periods
between 3 to 10 days of onset of fever in DC patients, while IFN-γ were not significantly altered by DENV infection in all
periods analyzed in this study.
Conclusions:
In this study we observed high levels of IFN-I in acute disease, analyzed by qPCR, particularly in patients who develop DHF,
suggested association with the development of severe disease. However, more studies are being done to quantify IFN-I in patients
with DHF.
Keywords: Innate immune response, Interferon Expression, Dengue Hemorhagic Fever
Financial Support: CNPq, FIOCRUZ
Resumo:18-017
IN VIVO EFFECTS OF OUABAIN ON MICE LYMPHOCYTE POPULATIONS.
Sá, L. M. 1; Santos, L. F. C. 1; Pozzatti, R. R. 1; Silva, J. M. C. 1; Costa, K. M. 3,1; Do-canto, F. B. 2,1; Fucs,
R. 1; Rumjanek, V. M. 3; Paiva, L. S. 1
1
Instituto de Biologia/ Depto de Imunobiologia, UFF
2
Instituto de Microbiologia Prof. Paulo de Góes , UFRJ
3
Instituto de Bioquímica Médica/ Lab. de Imunologia Tumoral, UFRJ
Objectives:
Ouabain (OUA), previously known as a cardiotonic steroid capable of inhibiting Na+ K+-ATPase, has been identified as a
endogenous compound produced by the adrenals and hypothalamus and found circulating in mammalian plasma (Hypertension
30:886,1997). Additionally, it has been suggested that ouabain with other steroids such as glucocorticoids is also released by the
adrenal in stress situations. Besides, ouabain modulates several immunological functions.The aim of this study was to investigate
if pharmacological concentrations of ouabain regulate in vivo lymphocyte populations.
We used C57BL/6 or Balb/C mice (male or female 4-8 weeks old) injected intraperitoneally (i.p.) with 0.56 mg/kg ouabain for 3
days. In the fourth day the subpopulations of B and T lymphocytes were analyzed by flow cytometry, 24 hours after the last
injection. Ouabain had no effect in vivo on T cell subsets in the thymus, but the number of CD4+ Foxp3+ (10,6 x106 ± 1,6 CTR
vs 7,7 x106 ± 0,5 OUA) were significantly reduced in the spleen, whereas there was no significant modulation in the total number
of T CD8+ lymphocytes. The population of B cells in the bone marrow, spleen and peripheral blood was modulated in vivo by
ouabain. In the bone marrow it was possible to observe a decrease of total cellularity (22,5 x106 ± 1,1 CTR vs 17,3 x106 ± 0,6
OUA), affecting only mature B cells and preserving the Pro/PreB and immature B cells. Percentually, there was an increase in the
myeloid lineage in bone marrow (31,9 ± 8,0 CTR vs 59,0 ± 5,0 OUA). The decrease in bone marrow cell number, produced by
ouabain, was not observed in young animals until they were four weeks old. Numbers of mature B lymphocytes in the spleen
(55,4x106 ± 1,7 CTR vs 32,3x106 ± 2,8 OUA) and peripheral blood (228,0 x105 ± 117,3 CTR vs 78,8 x105 ± 26,4 OUA) were
also reduced by ouabain. In vitro, the B cell population of bone marrow was not affected.
Conclusions:
Ouabain, given in vivo, affects peripheral T and B subpopulations. Despite the absence of effect in the thymus of animals that
received ouabain, there was a reduction in the number and percentage of CD4+ CD25+ cells and regulatory T cells (CD4+
Foxp3+) in the spleen. Furthermore, these animals showed decreased numbers of mature B cells in the bone marrow, spleen and
peripheral blood. However, in vitro the B cell population was not affected by ouabain, suggesting that exogenous injected
ouabain is blocking maturation of B cells in the presence of endogenous glucocorticoids.The effects observed appeared to be
independent of strain and gender of animals, but age-dependent.
Keywords: B lymphocyte, development, ouabain, T lymphocyte
Financial Support: FAPERJ; CNPq and PROPPI/UFF
Resumo:18-018
IN VIVO MODULATION OF MULTIDRUG RESISTANCE RELATED PROTEINS BY OUABAIN
Lima, D. M. B. 1; Valente, R. C. 1; Lopes, A. G. 2; Capella, M. A. M. 1,2
1
Instituto de Bioquímica Médica, UFRJ
2
Objectives:
Besides the well documented role of kidneys in hypertension, several recent studies have shown an important role of the immune
system in the development of this patology. Ouabain, a hormone with immunomodulator properties, also acts as a prohypertensive agent. Recent works from our group suggest that ouabain can modulate the expression and activity of multidrug
resistance (MDR) related proteins, present in several tissues, including the immune system. In this way, we intend to investigate
if ouabain was capable of modulating, in vivo, the expression and/or activity of P-gp/ABCB1, MRP-1/ABCC1 and
BCRP/ABCG2 in cells of the immune system; and also whether this modulation correlates with an increase in the blood pressure.
To evaluate that, we used male Wistar rats with 12 weeks of age. These animals underwent acute treatment with ouabain in two
different concentrations (30ug/kg and 150ug/kg) by intraperitoneal injection. 24 hours later, these rats were sacrificed by
decapitation and their blood, mesenteric lymph nodes and thymus were collected for analyze. Blood pressure measurements
showed no significant change between controls and animals exposed to ouabain for 24 hours as well as the hemogram showed no
significant difference in blood parameters. The activity assays were performed with Rho 123 or CFDA, fluorescent substrates of
P-gp/ABCB1 and MRP-1/ABCC1. The obtained cells were seeded in 96-wells plate at a concentration of 3x10E5 cells per well
and incubated with the respective substrates for 30 minutes. After, the cells are incubated for 30 minutes in medium without the
fluorescent dye, for extrusion and the cellular fluorescence was measured by flow cytometry. Our results showed significant
difference in the amount of cells with P-gp/ABCB1 activity (75% of reduction, t-test, p
Conclusions:
The results suggest that ouabain is capable of modulating the activity and expression of P-gp/ABCB1 in cells of the immune
system without leading to increased blood pressure in rats. Since ouabain is a pro-hypertensive agent, these data suggest that
changes in expression and activity of the P-gp/ABCB1 transporter occurs before the development of essential hypertension, data
that may possibly contribute to the understanding of the genesis of hypertension.
Keywords: Ouabain, Multidrug Resistance, Hypertension, Immune System
Financial Support: CNPq, FAPERJ, PRONEX, FAF-Oncobio II.
Resumo:18-019
ECTONUCLEOTIDE PYROPHOSPHATASE/PHOSPHODIESTERASE (E-NPP) AND ADENOSINE DEAMINASE
(ADA) ACTIVITIES IN PLATELETS OF PATIENTS WITH CHAGAS' DISEASE
Souza, V. C. G. 1; Schlemmer, K. B. 1; Noal, C. B. 1; Pimentel, V. C. 3; Bagatini, M. D. 3; Jaques, J. A. S.
1,3
; Leal, C. A. M. 3; Fleck, J. 2; Casali, E. A. 4; Leal, D. B. R. 1,3
1
DEPARTAMENTO DE MICROBIOLOGIA E PARASITOLOGIA, UFSM
2
HOSPITAL UNIVERSITÁRIO DE SANTA MARIA, UFSM
3
DEPARTAMENTO DE QUÍMICA, UFSM
4
DEPARTAMENTO DE BIOQUÍMICA/ICBS, UFRGS
Objectives:
Chagas‟ disease caused by parasite Trypanosoma cruzi is characterized by microvascular disturbances that may contribute to the
pathogenesis of this disease. During the chronic phase, five anatomoclinical forms can be distinguished: indeterminate, cardiac,
digestive, mixed (cardiac and digestive) and nervous. After the acute phase the infection usually becomes chronic and clinically
silent, which is described as the indeterminate form. Cardiac and gastrointestinal disorders are the main forms developed during
the symptomatic chronic Chagas disease. Extracellular adenine nucleotides (ATP, ADP and AMP) and their metabolite,
adenosine, display many tissue functions including: development, blood flow, secretion, inflammation and thromboregulation
(Purinergic Signal. 2:409,2006). There are ecto-enzymes such as ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP)
and adenosine deaminase (ADA) that control the extracellular levels of circulating adenine nucleotides and adenosine, which are
important in the maintenance of hemostasis. The aim of this study was to investigate the E-NPP and ADA activities in platelets
from patients with indeterminate form of Chagas‟ disease.
The pheripheral blood platelets were collected from fifty human donors and separated as described in the literature (Platelets.
7:225,1996). The E-NPP activity was assayed as described by standardized tests (Platelets. 17:84,2006) and ADA activity was
determined by the direct production of ammonia when ADA acts in excess of adenosine. The control group was constituted by
twenty-five T. cruzi serum-negative subjects with an average age of 45 years old (SEM ± 2.354.) and twenty-five Chagas‟ disease
patients with an average age of 55 years old (SEM ± 1.572) were selected from the University Hospital of Santa Maria. The
protocol was approved by the Human Ethics Committee of the Health Science Center from the Federal University of Santa Maria
under the number 243/2009 and all the subjects gave written consent. Data were analyzed statistically by the T-test. Differences
were considered significant when the probability was PPP<0.001.
Conclusions:
These results suggest an increase in the E-NPP activity, which could be related to a compensatory physiological response to the
excessive platelet aggregation which occurs during the chronic inflammatory process observed in patients with indeterminate
form of Chagas‟ disease. The decrease in ADA activity found in this study could lead to an increase in adenosine concentration in
blood circulation. Adenosine, which is the product of the degradation of adenine nucleotides acts as a vasodilator and inhibits
platelet aggregation. This characteristic confers to the patients a cardioprotective effect.
Keywords: ADENOSINE, CHAGAS, E-NPP, PLATELETS
Financial Support: FIPE, CAPES
Resumo:18-020
IMMUNOGENICITY NANOPARTICLES OF BOVINE SERUM ALBUMIN ASSOCIATED FOUR STRAINS OF
DENGUE VIRUS INACTIVATED AND POTENCIALIZAÇÃO IGG PRODUCTION BY LAWSONIA INERMIS L
Silva, E. F. 2,1; Coelho, L. F. L. 1,1
1
Laboratório de Vacinas, Instituto de Ciências Biomédicas, UNIFAL
2
Nucleo de Pesquisas em Plantas Medicnais, UFPI
Objectives:
Dengue virus (DENV) is an enveloped virus belonging to the family Flaviviridae with icosahedral symmetry and a diameter of
40-50 nm. It has four DENV serotypes named 1,2,3 and 4. The World Health Organization estimates that between 50 and 100
million people become infected annually. The most effective way to reduce these illnesses and deaths from infectious diseases is
to vaccinate populations at risk. Unfortunately there are no vaccines for many diseases and in addition, some existing vaccines
are not completely protective. This study evaluated the immune response produced by nanoparticles (NPs) of biodegradable
bovine serum albumin associated with the four serotypes of DENV inactivated, as well as the adjuvant effect of aqueous extract
of Lawsonia inermis L. There are no reports on the activity of L. inermis BSA nanoparticles in the literature.
We evaluated whether the nanoparticles (NPs) with BSA as vehicle system of inactivated antigens of the four serotypes of DENV
(DENV 1, 2, 3 and 4). The statistical analysis of this study were performed using the software GraphPad Prism ® 5.03, the
Student t test and significance level was 5% (p < 0.05). The animals treated with inactivated antigen solution and Freund's
incomplete adjuvant showed anti-DENV immunoglobulin (p = 0.0466 for DENV-1, p = 0.0027 for DENV-2, p = 0.0043 for
DENV-3 and p = 0, 0018 for DENV-4). The hyper-immune serum showed a lot of anti-DENV IgG and was used as positive
control. BSA NPs in colloidal suspension had values of zeta potential of -35.8 mV. BSA NPs showed a controlled release of
antigens and inoculated into Swiss mice were able to activate the immune system and induce the production of anti-DENV IgG
antibodies (p = 0.0002) assessed by enzyme immunoassay (ELISA). The trials of immunization with adult Swiss mice showed
that the nanoparticles formulated with the aqueous suspension of L. inermis showed optical density (O.D.) 1.9-fold (p = 0.0111)
than that obtained for animals immunized with only NP + L (nanoparticles and L. inermis). The potentiation of the aqueous
extract of L. inermis in BSA NPs was confirmed by titration of anti-DENV IgG antibodies in serum from immunized mice, these
groups had a mean antibody titer 10 times higher than that observed for NPs + DENV.
Conclusions:
These data indicate that NPs BSA associated with the four serotypes of DENV inactivated, induced the production of IgG
antibodies to DENV and the addition of the aqueous extract of L. inermis enhanced the production of anti-DENV IgG, when
added to the preparation of BSA NPs.
Keywords: Dengue vírus, Nanoparticles, Bovine Serum Albumin, Lawsonia inermis L.
Financial Support: CNPq, FAPEMIG, UNIFAL.
Resumo:21-001
CONTROL OF SODIUM INTAKE BY OPIOID MECHANISMS INTERACTING WITH DIFFERENT
NEUROTRANSMITTERS IN THE LATERAL PARABRACHIAL NUCLEUS
Favero, M. T. ; Pavan, C. G. ; de Oliveira, L. B. ; Barbosa, S. P. ; de Luca, L. A. Jr ; Colombari, D. S. A. ;
Paula, P. M. ; Colombari, E. ; Menani, J. V.
Department of Physiology and Pathology, School of Dentistry, UNESP
Objectives:
Important inhibitory mechanisms modulated by serotonin (5-HT), cholecystokinin (CCK), opioids and GABA receptors in the
pontine lateral parabrachial nucleus (LPBN) are involved in the control of sodium and water intake. In the present study we
investigated the effects of the pre-treatment with naloxone (opioid antagonist) combined with muscimol (GABAA receptor
agonist), methysergide (5-HT receptor antagonist) or proglumide (CCK antagonist) injected into the LPBN on 1.8% NaCl and
water intake. In addition, it was also tested the effects of endomorphin (µ opioid receptor agonist) alone or combined with DOI
(5-HT2A/2C receptor agonist) injected into the LPBN on 1.8% NaCl and water intake.
Male Holtzman rats (290-310 g, n = 6/19 group) with bilateral stainless steel cannulas implanted into the LPBN were used. Water
and 1.8% NaCl intake was tested in satiated rats or in rats treated with the diuretic furosemide (FURO, 10 mg/kg of body weight)
combined with the angiotensin converting enzyme inhibitor captopril (CAP, 5 mg/kg) injected subcutaneously. Bilateral
injections of muscimol (0.5 nmol/200 nl) into the LPBN induced 1.8% NaCl intake (51.0 ± 10.6 ml, vs. vehicle: 3.5 ± 1.2 ml/240
min) and water intake (16.6 ± 4.4 ml, vs. vehicle: 4.7 ± 2.7 ml/240 min) in satiated rats. The pre-treatment with naloxone (40
µg/200 nl), bilaterally, into the LPBN reduced muscimol-induced 1.8% NaCl intake (25.9 ± 8.1 ml/240 min) and water intake
(7.0 ± 1.5 ml/240 min). Bilateral injections of methysergide (4 µg/200 nl) or proglumide (50 µg/0.2 µl) into the LPBN increased
1.8% NaCl intake induced by FURO + CAP (24.4 ± 2.9 and 25.3 ± 4 ml, respectively, vs. vehicle: 11.2 ± 1.9 ml/120 min).
Naloxone bilaterally injected into the LPBN also reduced the effects of methysergide or proglumide on 1.8% NaCl intake (17.3 ±
3 and 20 ± 3 ml/120 min, respectively). Methysergide or proglumide alone or combined with naloxone into the LPBN produced
no changes on FURO + CAP-induced water intake. Endomorphin (4 µg/200 nl) into the LPBN increased FURO + CAP-induced
1.8% NaCl intake (21.2 ± 3.6 ml vs. vehicle: 8.4 ± 2.4 ml/180 min) and the pre-treatment with DOI (5 µg/200 nl) reduced this
effect (14.9 ± 2.1 ml/180 min). Endomorphin into the LPBN alone or combined with DOI reduced FURO + CAP-induced water
intake in the first 30 min of test (vehicle + endomorphin: 3.8 ± 1.7 ml and DOI + endomorphin: 2.8 ± 1.2 ml vs. vehicle: 11.7 ±
1.6 ml).
Conclusions:
The effects of endomorphin alone or naloxone combined with methysergide, proglumide or muscimol into the LPBN suggest that
opioids deactivate LPBN inhibitory mechanism for sodium intake and that the release of opioids in the LPBN is reduced by
serotonergic or cholecystokinergic and enhanced by gabaergic activation. The effects of the combination of endomorphin and
DOI into the LPBN also suggest that serotonin directly activate the inhibitory mechanism independently on the action through
opioid mechanisms.
Keywords: lateral parabrachial nucleus , sodium appetite , serotonin, GABA, cholecystokinin
Financial Support: FAPESP, CNPq and CAPES
Resumo:21-002
ANTIOXIDANT CAPACITY ALTERATIONS IN SERUM OF PARKINSON’S DISEASE PATIENTS: A POSSIBLE
BIOMARKER?
Omura, K. M. 2; Kietzer, K. S. 1; Freitas, J. J. D. S. 1; Domingues, M. 1; Fujihara, S. 2; Yamada, E. 2
1
UNIVERSIDADE DO ESTADO DO PARÁ - DMCF, UEPA
2
Universidade Federal do Pará - ICB, UFPA
Objectives:
Parkinson‟s disease is the second most frequent neurodegenerative disease that afflicts elderly population. Diagnosis is based
mainly on clinical aspects, and by the time diagnosis is made, most of the neurons involved in disease have been lost. Therefore,
this study aimed to verify if measures of oxidative stress in serum of patients with Parkinson‟s disease could be used as
biomarkers for early diagnosis.
The study was conducted with 26 Parkinson's disease patients, of wich 9 were in early stage (ES), 9 in moderate stage (MG) and
8 in severe stage (SS) of diasease, and 33 control patients, all treated and followed at the Education and Assistance Unit of
Physiotherapy and Occupational Therapy of the Para State University (UEAFTO). Evaluation was performed through the
measurement of Trolox Equivalent Antioxidant Capacity (TEAC) and through thiobarbituric acid-reacting substances (TBARS)
by means of spectrophotometry.The results showed that TEAC was significantly reduced in patients with Parkinson‟s disease
(mean=1.69±0.008) compared to controls (mean=1.88±0.003). On the other hand, there was not a significant difference at TEAC
in different stages of the disease (ES=1,73; MD=1,64 ; SS=1,71 p>0,05). In the same form, there were no significant difference in
TBARS concentrations between patients and controls (PD patients=16.04±.4.22; Controls= 13.02±1.66) and in diferent stages of
disease (ES=14,45; MD=15,95; SS=17,74; p> 0,05).
Conclusions:
These results demonstrated that a reduction of the total antioxidant capacity could be detected in the serum of Parkinson‟s disease
patients even in early stages of the disease, and suggests that an evaluation of antioxidant capacity can be an adjuvant of early
diagnosis of PD.
Keywords: parkinson, antioxidant, oxidative stress
Financial Support: fapespa
Resumo:21-003
CHRONIC MILD AND UNPREDICTABLE STRESS AFFECTS DEPRESSION BEHAVIOR, BUT NOT ANXIETYLIKE BEHAVIOR
Costa, R. 2,1; Briet, L. D. S. 2; Tamascia, M. L. 1; Almeida, B. S. 2; Sanches, A. 1; Marcondes, F. K. 1
1
Piracicaba Dental School, University of Campinas, FOP/UNICAMP
2
Institute of Biology, University of Campinas, IB/UNICAMP
Objectives:
Chronic stress have been recognized as a risk factor for mood discords like anxiety and depression. The aim of this study was to
evaluate the depression and anxiety-like behaviors of male Sprague-Dawley rats submitted to chronic mild and unpredictable
stress (CMUS).
The animals were divided in two groups: control and CMUS (n=12). The CUMS protocol consisted of the application of different
stressors, 7 days/week during three consecutive weeks (from the 3rd to 5th weeks) of an experimental protocol lasting 7 weeks.
Each week the animals were exposed to repeated periods of immobilization twice a day, overnight illumination, water and food
deprivation for 20h, water deprivation for 18h followed by exposure to empty water bottle for 2h, wed bedding for 17h, and
reversed light/dark cycle throughout the week-end. Sucrose preference test was used to evaluate stress-induced ahnedonia and
was measured 3 times during the experimental protocol (2nd, 4th and 7th weeks). At week 6, the anxiety level was evaluated
using the elevated plus-maze (EPM) test and the depression-like behavior was evaluated using the forced swimming test (FST).
The behavioral responses were analyzed by Etho Vision XT™ 4.1 program. The preference sucrose test was determined by
ANOVA for Repeated Measures and behavioral measures were analyzed by Student´s t test. Values are presented as means ±
SEM (p
Conclusions:
These data indicate that CUMS induced depression-like behavior (anhedonia and hopelessness) in rats and increased the
locomotor activity, but did not influence the anxiety levels of rats.
Keywords: Anhedonia, Anxiety, Elevated plus-maze, Forced swimming test, Stress
Financial Support: FAPESP, CNPq, CAPES
Resumo:21-004
HEART RATE REACTIVITY TO HUMAN ATTACK PICTURES IS MODULATED BY TRAIT ANXIETY
Alves, R. C. S. 1; Lopes, T. 5; Andrade, J. R. 7; Mocaiber, I. 6; Souza, G. G. 2; Erthal, F. 3; Joffily, M. 4;
David, I. A. 1; Oliveira, L. 1; Pereira, M. G. 1
2
Departamento de Ciências Biológicas, UFOP
1
Departamento de Fisiologia e Farmacologia, UFF
3
Instituto de Biofísica, UFRJ
4
CCenter for Mind/Brain Research, Trento University
5
Instituto Alberto Luiz Coimbra - Coppe, UFRJ
6
Depto. Interdisciplinar, Puro/UFF
7
Instituto de Biologia, UFRJ
Objectives:
Emotional responses to appetitive and aversive stimuli motivate approach and avoidance behaviors essential for survival.
Previous studies have consistently shown a deceleration of heart rate response when participants viewed unpleasant pictures. The
aim of this study was to investigate the influence of trait anxiety over human defensive profiles. We studied how affective
predisposition (i.e. trait anxiety) could impact on defensive reactivity to threatening stimuli.
We employed a set of 16 pictures displaying scenes of human attack and a set of 16 matched neutral pictures for control. The
sequence of pictures was randomized and each picture remained on for 6s, with an inter-picture interval of 6 to 8s.
Electrocardiographic recordings were collected from 16 participants (15 women, with mean age of 22, 2 years; SD ± 3,75). The
neutral and unpleasant blocks of pictures were divided into 3 segments (early, middle and late) to estimate the time course of the
cardiac response. Mean heart rate during exposure to threatening pictures normalized by 1s pre-exposure correlated with anxiety
trait during the middle (ρ=0.60; p=0.01) and late segments (ρ=0.68; p=0.003). Participants with low anxiety slowed the heart rate
while those with high anxiety showed acceleration.
Conclusions:
The results suggest that increased predisposition to anxiety turn passive (i.e. freezing-like)into more active defensive reactions in
laboratory conditions.
Keywords: Anxiety, Emotion, Heart Rate
Financial Support: CAPES, PRONEX-FAPERJ, MCT-CNPq, IBN-Net, FAPERJ
Resumo:21-005
PROSTAGLANDINS MEDIATE SICKNESS BEHAVIOR INDUCED BY ZYMOSAN IN MICE
Lima, J. B. M. 1; Veloso, C. C. 3; Antunes-rodrigues, J. 2; Elias, L. L. K. 2; Vilela, F. C. 1; Soncini, R. 1;
Giusti-paiva, A. 1
1
Instituto de Ciências Biomédicas, UNIFAL
2
Departamento de Fisiologia, USP - Ribeirão Preto
3
Instituto de Ciências Biológias, UFMG
Objectives:
Zymosan (ZY) is a particle from the cell wall of yeast Saccharomyces cerevisiae, and previous studies have shown that ZY
induces sepsis in an experimental model. However, despite of the induction of sepsis, the sickness behavior induced by ZY is not
completely understood. This study was conducted to establish the possible participation of prostaglandins in ZY-induced sickness
behavior.
To evaluate the possible role of prostaglandins in sickness behaviour induced by ZY, male Swiss mice (n=10 per group) were
submitted to the forced swimming test (FST) and open field test (OFT) at 2, 6 and 24 hours after administration of ZY (10 and
100 mg/kg; ip). In addition, another group of animals were treated with vehicle (saline, 1 ml/100 g), indomethacin (10 mg/kg; ip)
or nimesulide (10 mg/kg, ip) and were submitted to the FST and OFT 2 hours after administration of ZY (10 mg/kg, ip). The
experiments were conducted with the approval of the Ethics Committee of the Federal University of Alfenas (protocol
#0269/2010). Zymosan (10 and 100 mg/kg) increased the time spent floating in the FST after 2 hours (106.9 ± 12.2 and 146.8 ±
10.7 s; respectively) and 6 hours (117.6 ± 14.5 and 120.6 ± 15.7 s, respectively) when compared with vehicle (46.7 ± 11.6 and
55.6 ± 6.4 s; after 2 and 6 hours, respectively). Zymosan (10 and 100 mg/kg) decreased the total number of line crossings in the
OFT after 2 hours (51.3 ± 6.4 and 41.6 ± 5.9 lines; respectively) when compared with vehicle (83. 7 ± 12.0 lines) evidencing an
attenuated exploratory and locomotor activity. After 6 hours, only ZY (100 mg/kg) produced effect in the total number of line
crossing (41.3 ± 8.8 vs. vehicle: 70.1 ± 4.1 lines; p
Conclusions:
The present results provide evidences that the synthesis of prostaglandins is necessary for changes in depressive-like and
exploratory behaviours in mice, which is supported by the fact that COX inhibitors also attenuate zymosan-induced behavioural
changes.
Keywords: prostaglandins, Saccharomyces cerevisiae, sickness behavior, stress, zymosan
Financial Support: FAPEMIG
Resumo:21-006
EFFECTS OF THE DIET KETOGENIC BASED ON TRIHEPTANOIN IN SEIZURES OF RATS WITH EPILEPSY
INDUCED BY PILOCARPINE.
Gomes, T. K. D. C. 1; Oliveira, S. L. D. 1; Ataíde, T. D. R. 1; Filho, E. M. T. 2; Rêgo, E. D. S. M. 1; Júnior,
C. R. C. 1; Machado, T. S. 1; Melo, I. T. 1; Galvão, J. A. 1; Barros, E. M. A. 1
1
Faculdade de Nutrição-Universidade Federal de Alagoas, UFAL
2
Universidade de Ciências da Saúde de Alagoas, UNCISAL
Objectives:
To investigate the effects of the ketogenic diet based on triheptanoin in frequency and duration of epileptic seizures in rats.
Male Wistar rats (n = 30) with thirty days old were submitted to step induction of Status Epilepticus by intraperitoneal
administration of pilocarpine(340mg/kg, ip). After the establishment of chronic epilepsy, the animals were subjected to one of
three experimental diets, Control (standard diet, n=7), CetoTAGsoya (ketogenic diet based on soya oil, n=8) or CetoTAGC7
(ketogenic diet based on triheptanoin TCM, n=6), and monitored by video, in order to record the frequency and duration of
spontaneous recurrent seizures. The variables were subjected to analysis of variance (ANOVA) with means compared by Tukey
test (P
Conclusions:
The ketogenic diet based on triheptanoin not reduced the frequency of spontaneous recurrent seizures, but reduced the average
duration of seizures at the end of the experiment. This result, albeit modest, could represent a potential benefit of triheptanoin for
outbreak control epileptic, hypothesis to be tested in future protocols, which could consider, a longer exposure to the ketogenic
treatment.
Keywords: experimental epilepsy, Status epilepticus, pilocarpine, ketogenic diet, triheptanoin
Resumo:21-007
AEROBIC EXERCISE EFFECTS UNDER BRAIN ELECTRICAL ACTIVITY IN STRESSED-DEPRESSED YOUNG
RATS.
Lins, J. C. P. S. ; Alves, A. R. A. ; Barros, M. A. ; Ximenes-da-silva, A.
Instituto de Ciências Biológicas e da Saúde, UFAL
Objectives:
Depression is one of the most prevalent diseases worldwide leading to a high morbidity. Studies of brain electrical activity
showed that in depressed patients prefrontal lobe exhibits particularly a reduced spontaneous brain activity. Cortical spreading
depression (CSD) is a depression of spontaneous brain electrical activity that can be triggered by stimulation of cortical surface.
Several studies have proposed stressful conditions as a causal pathogenic effect of depression and that physical training could act
ameliorating symptoms of depression. The aim of this study was to evaluate the effect of aerobic exercise on brain electrical
activity, through cortical spreading depression (CSD) recording in young rats (8-16 weeks), submitted to chronic unpredictable
mild stress (uCMS).
Fifty-nine Wistar rats were maintained under standard facilities conditions for two weeks, during which anhedonia tests were
conducted to evaluate preference to the consumption of 2% sucrose solution. Animals that presented normal sucrose consumption
were selected and were divided into four experimental groups: Sedentary Control (SC), Trained Control (TC), Control StressedSedentary (CS-S) and Control Trained-Stressed (CT-S). Animals were submitted to the uCMS protocol during 4 weeks and TC
and CT-S animals performed three times/60min week swimming sessions with a 2% load of their body weight. Oral glucose
tolerance and insulin resistance test were also performed. At the end of uCMS protocol, animals were submitted to
electrophysiological records of CSD for 4h. Data were analyzed using ANOVA one- or two-way. A significantly higher
preference for sucrose was observed in sedentary animals (SC) related to stressed-sedentary ones (CS-S) (P = 0.0088). Similarly,
animals of CS-S group had a more pronounced sucrose preference compared to trained-stressed control group (CS-T; P =
0.0328). Sedentary and stressed animals had an increased body weight compared to trained group (CT, P = 0.0290). Analysis of
oral glucose tolerance showed that CT-S animals had higher blood glucose levels compared to SC group (P = 0.0315). Cortical
spreading depression velocity of propagation was reduced after aerobic training in TC (1.82mm/min) vs SC (2.75mm/min,
P<0.05).
Conclusions:
Aerobic training changed brain electrical activity demonstrated here by slowing of cortical spreading depression. In addition,
stressed animals had longer latency to trigger slow potential change of CSD. Taken together, these conditions might suggest a
neuroprotective effect of aerobic training under electrophysiological brain changes.
Keywords: Spreading depression, Aerobic training, Depression, Brain electrical activity
Financial Support: Lins, J.C.P.S. was a fellowship from Fapeal (Alagoas)
Resumo:21-008
CHOLINERGIC STIMULATION OF THE MEDIAL SEPTAL AREA INCREASES VASOPRESSIN AND OXYTOCIN
MRNA EXPRESSION IN THE HYPOTHALAMUS.
Paulin, R. F. 1; Margatho, L. O. 1; Reis, W. L. 2; Elias, L. L. K. 1; Antunes-rodrigues, J. 1
1
Department of Physiology, School of Medicine , FMRP
2
Department of Physiology, Medical College of Georgia, MCG
Objectives:
Our previous data (not yet published) showed that carbachol into the medial septal area (MSA) increases plasma oxytocin (OT),
vasopressin (AVP), as well as the co-localization of c-Fos-OT and c-Fos-AVP in the magnocellular neurons of the hypothalamus.
In the present study we investigated the effects of the cholinergic antagonist atropine methyl, carbachol or vehicle injected into
the MSA on AVP and OT mRNA expression in the paraventricular nucleus (PVN) and supra-optic nucleus (SON) of the
hypothalamus by Real Time PCR.
Male Wistar rats (280-320 g, n=5-9) with cannulas unilaterally implanted into the MSA (0.96 in front of bregma, just at medline
and 0.40 in vertical) were used. After 5-7 days of the surgery, the rats were subjected to a first injection of vehicle or atropine
methyl (4 nmol/0.5 ul). Fifteen minutes later, a second injection was performed with carbachol (4 nmol/0.5 ul) or vehicle into the
MSA. All experimental groups of rats were decapitated after the second injection and, after the brain removal, a micropunch of
the PVN and SON was obtained and used to evaluate the relative AVP and OT mRNA expression by RT-PCR. Vehicle plus
carbachol into the MSA significantly increased AVP and OT mRNA expression (1.4 ± 0.07 vs. 0.8 ± 0.1 and 1.3 ± 0.1 vs 0.8 ±
0.1, respectively) in the PVN, while atropine methyl injected before carbachol produced opposite effects on AVP and OT mRNA
expression (0.8 ± 0.05 vs. 1.4 ± 0.07 and 0.8 ± 0.04 vs. 1.3 ± 0.1, respectively) in the same area. Similar responses were observed
in the SON when vehicle plus carbachol was injected into the MSA with an increase in AVP and OT mRNA (1.5 ± 0.08 vs. 1.0 ±
0.08 and 1.8 ± 0.2 vs 0.8 ± 0.2, respectively). Atropine methyl injected before carbachol also reduced AVP and OT mRNA (0.9 ±
0.06 vs. 1.5 ± 0.08 and 0.9 ± 0.06 vs 1.8 ± 0.2) in the SON.
Conclusions:
The blockade of cholinergic receptors with atropine methyl in the MSA produced a reduction in the genomic response (reducing
AVP and OT mRNA expression) in magnocellular neurons of the PVN and SON, which can be correlated with the reduction in
plasma OT and AVP levels observed previously, suggesting a tonic modulation of the cholinergic pathway of the MSA, since
carbachol produced stimulatory effects in these parameters.
Keywords: MEDIAL SEPTAL AREA, VASOPRESSIN, OXYTOCYN, SUPRAOPTIC NUCLEUS, PARAVENTRICULAR
NUCLEUS
Resumo:21-009
INFLUENCE OF FISH OIL SUPPLEMENTATION AND/OR PHYSICAL EXERCISE PROGRAM FROM
DEVELOPMENT TO MIDLIFE ON COGNITION IN RATS
Rachetti, A. L. F. 1; Cysneiros, R. M. 2; Patti, C. L. 3,4; Zanin, K. A. 3,4; Fernandes-santos, L. . 3,4; Frussafilho, R. . 3; Gomes da Silva, S. 5; Scorza, F. A. 1; Cavalheiro, E. A. 1; Arida, R. M. 5
1
Departamento de Neurologia/Neurocirurgia, Unifesp
2
Programa de Pós-Graduação em Distúrbios do Desenvolvimento, Mackenzie
3
Departamento de Farmacologia, Unifesp
4
Departamento de Psicobiologia, Unifesp
5
Departamento de Fisiologia, Unifesp
Objectives:
Fish oil supplementation (enriched in omega-3) or physical exercise can mprove cognition both in humans and animals. Recently,
it has been suggested that the association of these factors could potentiate such cognitive improvements. In this regard, the
present study was aimed to evaluate the cognitive effects of 1. prolonged 85 mg/Kg/day fish oil supplementation (rich in omega3) from prenatal period (1st day after conception) to midlife (300 day/old); 2. moderate physical exercise in treadmill initiated
from adolescent period (21-day-old) to midlife and 3. association of fish oil supplementation and physical exercise program.
Forty five 300-day-old male Wistar rats were allocated in one of the following groups: control/vehicle (CTL-VEH, n=11),
exercise/vehicle (EX-VEH, n=11), control/fish oil (CTL-FO, n=11) and exercise/fish oil (EX-FO, n=12). On 230 day/old,
animals were submitted to the open-field habituation test (OFH), on 240-day-old to object recognition (OR) test and on 250-dayold to plus-maze discriminative avoidance task (PM-DAT). Our results demonstrated that prolonged fish oil supplementation
facilitated both the persistence of a long-term OFH [since fish oil-treated animals displayed a decreased exploration in the 2nd
exposure (mean±SEM of frequency of locomotion from CTL-VEH: 22.64±4.85; CTL-FO: 16.36±3.05; EX-VEH: 37.64±8.92
and EX-FO: 26.5±5.45)] and OR (mean±SEM of the familiar or the novel objects time (s) exploration, respectively: CTL-VEH:
5.00±2.6 and 5.54±3.18; CTL-FO: 1.36±0.39 and 4.73±1.06; EX-VEH: 3.91±1.51 and 5.27±1.39; EX-FO: 2.42±0.6 and
7.33±2.1). Although the exercise program exerted no effects on OFH or OR tests, it was able to potentiate the persistence of the
discriminative avoidance memory [since trained animals discriminated the aversive from non-aversive enclosed arms
(mean±SEM of the non-aversive or aversive time (s) exploration, respectively: CTL-VEH: 69.82±23.25 and 75.82±22.27; CTLFO: 98.64±20.89 and 54.64±21.27; EX-VEH: 98.18±12.89 and 28.54±12.64; EX-FO: 114.42±14.6 and 36.25±13.59)]. Such
promnestic effects (induced both by FO supplementation and EX) were not accompanied by alterations in emotionality or
exploratory activity. No synergic cognitive effects induced by the association between FO supplementation and EX were
observed.
Conclusions:
Our data suggest that fish oil supplementation, initiated from prenatal period to midlife, and physical exercise program applied
throughout the life induced distinctly a better cognitive performance.
Keywords: Omega-3, Physical exercise, Memory, Desenvolviment, Rats
Financial Support: CAPES, FAPESP and CNPq.
Resumo:21-010
INVOLVEMENT OF THE NO-CGMP PATHWAY IN THE PILOCARPINE-INDUCED MODEL OF SEIZURES.
Rios, E. R. V. ; Rocha, N. F. M. ; Carvalho, A. M. R. ; Vasconcelos, L. F. ; Melo, F. H. C. ; Linhares, M. I.
; Lima, C. N. D. C. ; Lopes, K. S. ; Venâncio, E. T. ; Fonteles, M. M. F.
Depto. de Fisiologia e Farmacologia/Faculdade de Medicina, UFC
Objectives:
To investigate the involvement of NO pathway in the proconvulsant action of the pilocarpine though of behavioral alterations.
Mice (28-33g) were pretreated with L-NAME (10 mg/kg, ip), an inhibitor of the NOS, Aminoguanidine (25 mg/kg, ip), an
inhibitor of the iNOS, ODQ (10 mg/kg, ip), an inhibitor of soluble guanylate cyclase, or L-Arginine (150 mg/Kg, ip), donator of
NO, and 30 minutes after was induced the seizure with pilocarpine (400mg/kg, ip). A group received only vehicle (10mL/Kg, ip)
30 minutes before of the pilocarpine (400 mg/Kg, ip). In the behavioral assessment was observed following parameters by 60
minutes: Latency for a first seizure and latency to death (mortality). For statistical analysis, we used analysis of variance
(ANOVA) and Student-Newman-Keuls as post hoc. Our results show the attenuation of the behavioral parameters (increased
latency to first seizure and decreased time for death) in the L-NAME and Aminoguanidine groups (1035 ± 78.3 seconds/2382
seconds, the last death and 0% of survive) (1188 ± 194.1/1951 seconds, the last death and 37.5 % of survive) when compared
with control group (693.1 ± 23.7 seconds/1184 seconds, the last death and 0% of survive). The pretreatment with ODQ (912.1 ±
104.4/1133 seconds, the last death and 25% of survive) not altered time for first seizure but decreased the deathly and L-Arginine
(718.7 ± 32.64/991 seconds, the last death and 0% of survive) not has difference when compared with the control group.
Conclusions:
With these results we can suggest the involvement of the NO-cGMP pathway in the proconvulsant action of the pilocarpine.
Keywords: Pilocarpine, Neuroinflammation, Nitric Oxide, Seizures
Financial Support: CNPq and CAPES
Resumo:21-011
NUCLEUS RAPHE OBSCURUS PROJECTIONS TO MEDULLARY SITES IN BARODENERVATED RATS: A
C−FOS STUDY
Nunes, G. F. 1; Borges, A. P. R. 1; Meneguzzi, V. C. 1; Pires, J. G. P. 2,4; Futuro-neto, H. A. 1,2,3; Silva, N. F.
1
1
Depto. de Morfologia / Centro de Ciências da Saúde, UFES
2
Faculdade de Medicina, UNIVIX
3
Depto. Ciências Fisiológicas, EMESCAM
4
Faculdade de Medicina, UNESC
Objectives:
The participation of nucleus raphe obscurus (NRO) in autonomic regulation is well established. Its stimulation produces a variety
of different cardio −respiratory patterns. The present study aims to identify nuclei of the brainstem by activation of c −Fos, that
are involved in mediating a pattern of hypertension, bradycardia and hypopneia evoked by NRO stimulation.
Male Wistar rats of 300−350 g body weight were anesthetized with chloral hydrate (0.4 g⁄kg i.p.) barodenervated and had its right
jugular vein cannulated; 24 hrs later, anesthesia was induced and maintained with urethane (1.2 g⁄kg, i.v.) and the animals were
submitted to tracheotomy, had the right carotid artery cannulated and a craniotomy for NRO stimulation (11.8 mm caudal to the
Bregma and 7.8 mm from surface). After stabilization of the arterial pressure (BP, MBP), heart rate (HR) and respiratory
frequency (RF), electrical stimulation of NRO was started (SNRO, 1mV square waves, 1ms duration, 70μA, for 10s) and was
done every minute for one hour for the experimental group; control group had the electrode positioned but was not stimulated. At
the end of the experiment, phenylephrine was infused (0.6 μg⁄kg, 0.01mL⁄min iv), to test the efficacy of the barodenervation.
Following the animals were killed with an overdose of anesthetic, were perfused with paraformaldehyde (4%) and had the brain
removed and processed for c−Fos imunohystochemistry. The experimental protocol was approved by the Animal Use Committee
(No. 021⁄2007, CEUA EMESCAM, Vitória, ES). The results were presented as mean±SEM and statistical significance was
assessed with t−test, pIn situ expression of c−Fos in response to SNRO−induced hypertension were observed in neurons along the
rostral−caudal extent of the medulla, where they appeared as cell clusters. This micromapping study pointed c−Fos−expressing
neurons at sites coordinates of ~2.5 mm rostral to the calamus scriptorius, 1.5−2.0 mm lateral to the midline, and 1.0 mm dorsal
to the ventral surface of the medulla, in the rostral aspect of the rostral ventrolateral medulla. Also, c−Fos−expressing neurons
were observed in the caudal ventrolateral medulla, at level of obex, 1.5−2.0 mm lateral to the midline, and 1.0 mm dorsal to the
ventral surface of the medulla, and into the caudal pressor area ~1.0 mm caudal to the calamus scriptorius, 2.0 mm lateral to the
midline, and 1.7 mm ventral to the dorsal medullary surface, respectively. Other brain stem autonomic areas, including the NTS,
nucleus ambiguus also expressed c−Fos after SNRO.
Conclusions:
This study provides additional support to the idea that NRO influence upon autonomic functions is mainly mediated through
projections to ventral−medullary sites.
Keywords: Rafhe obscurus, Ventrolateral medulla, RVLM, CPA, c−Fos
Financial Support: Pronex Fapes, CNPq
Resumo:21-012
WHEN THE ATTRACTIVE BECOMES AVERSIVE: THE EFFECTIVENESS OF PICTORIAL WARNINGS ON
CIGARETTE PACKAGES DEPENDS ON THEIR EMOTIONAL IMPACT.
Tavares, G. 1; David, I. A. 1; Gleiser, S. 2; Pereira, M. G. 1; Oliveira, L. 1; Volchan, E. 2,3
1
Depto. Farmacologia e Fisiologia Inst. Biomédico, UFF
2
LINPES - Inst. de psiquiatria , UFRJ
3
Laboratório de Neurobiologia - IBCCF, UFRJ
Objectives:
Exposure to tobacco products is seriously harmful being considered the single most preventable cause of death in the world
today. Tobacco control actions involve a wide spectrum of interventions to help people stop smoking and to prevent them from
starting to smoke. Tobacco-related products in Brazil must carry pictorial warning labels on packages. Two successive sets of
pictorial warnings were implemented, one in 2002 and the other in 2004, contributing to the remarkable success in smoking
reduction in the country. Based on neuroscience grounds, the on-going third set of warning labels was elaborated on the
assumption that they should be highly aversive to impact on prevention and cessation of smoking. Our objective was to test this
assumption.
20 students (non-smoking, age: 21.4 ± 5.05 years old.) classified each of the 10 warnings labels that are exposed on cigarette
packages since 2008. They judged the emotional impact of the warnings on the packages using a standardized psychometric scale
(Self-Assessment Manikin - SAM) to assess the unpleasantness/pleasantness (valence) and emotional arousal associated with the
warnings. They also quantified in Likert-like scales how effectively each label would motivate smokers to quit, and how
effectively each label would prevent youth from start smoking. .Unpleasantness ratings were significantly associated with
motivation to quit (R = 0.84; p < 0.01) and with prevention from start smoking (R= 0.78; p < 0.01). Emotional arousal also
associated with motivation to quit (R= 0.75; p < 0.01) and with prevention from start smoking (R= 73; p < 0.01). The warning
labels considered more effective to induce quitting and to prevent smoke initiation were the most unpleasant and arousing ones.
Conclusions:
The present results strongly suggest that the emotional impact of the pictorial health warnings is an important factor to enhance
the effectiveness of warning labels for motivating smokers to quit and for preventing youth from start smoking. This is in line
with a vast body of research in the neurobiology of emotion demonstrating that visual stimuli affect attitude and behavior,
pleasurable ones prompting dispositions to approach and aversive ones, to withdrawal. The positive appeals of tobacco marketing
on packages can be undermined by warnings showing smoking risks. The present results also corroborates with one of the
tobacco control policies of the world‟s health treaty against the tobacco epidemic, which requests nations to employ pictorial
impacting warning labels on cigarette packages.
Keywords: CIGARETTE PACKAGES, EMOTION, PICTORIAL WARNINGS
Financial Support: Fundação do Câncer (Programa de Oncobiologia), CAPES; FAPERJ, FINEp, CNPq
Resumo:21-013
EFFECT OF ACUTE INTRAUTERINE ETHANOL EXPOSURE ON SODIUM APPETITE CONTROL IN WISTAR
RATS
Carvalho, A. A. V. D. 1,3; Galdino, P. M. 2,3; Costa, R. M. E. 3; Reis, L. C. 4
1
Depto de Ciências Fisiológicas/PMPGCF, UFRRJ
2
Dep. de Biologia/PPGF, UFSC
3
Depto. de Ciências Fisiológicas/ICB, UFG
4
Depto. de Ciências Fisiológicas/Instituto de Biologia , UFRRJ
Objectives:
The control of body fluids volume is a critical homeostatic mechanism. This regulation is coordinated by different neural,
endocrine and behavioral mechanisms. Salt-intake behavior plays an important role in this regulation. In this work we
investigated the alterations in sodium intake produced by the acute intrauterine ethanol exposure. The exposure was performed on
the fourteenth day of intrauterine life, period of the intensive Neurogenesis in midbrain of rats
Treatment procedures: Female Wistar rats (180-230 g) were housed overnight with male rats. Next morning, the presence of
sperm in vaginal fluid indicated the first day of gestation (E0). After pregnancy confirmation each female rat were housed
individually in cages (at 22 ± 0.4°C and 12 h light/dark cycle) with free access to food and water. On the 14th day of pregnancy
(E14), 5 rats received three intraperitoneal injections of a 20% ethanol solution at the dose of 3 g/kg body weight; at intervals of 8
h. Ethanol was administered intraperitonealy to ensure that all rats received equal volume and drug amount. In this same day and
conditions, the control pregnant rats received injections of 0.9 % saline solution, at the same intervals as the ethanol-treated rats.
With twenty one days of life, the pups were weaned and kept in separate boxes according to sex and experimental group until
they reach 90 days of life and/or the weight of 270 grams; to experimental protocols only the males were used. Sodium intake
assessment: The water and salt (0,3 M NaCl) intake were assayed by modified furocap method. The animals (n= 12 each group)
were submitted to a loss sodium diet during 5 days and water ad libitum. On the fifth day the animals were treated with a
combination of furosemide (10 mg/kg, s.c.) + captopril (5 mg/kg, s.c.). After these treatments, the animals were deprived of
access to water for 60 min and, then were placed in metabolic cages with access to distilled water and 0,3 M NaCl,
simultaneously. The fluid intake was measured every 60 minutes during the first five hours and 24 hours after the beginning of
the experiment. Results: Acute gestational treatment with ethanol produces significant increase in hypertonic saline intake at 300
min (8.4 ± 0.56 vs. 5.58 ± 0.49 ml/100g, ANOVA; p
Conclusions:
Influences produced by prenatal ethanol administration affected the offspring's adult life leading to a change in the satiety
response to sodium intake.
Keywords: ethanol neurotoxicity, hydroelectrolyte balance, Neurogenesis, Salt-intake behavior, sodium appetite
Financial Support: CAPES, CNPq and FAPERJ
Resumo:21-014
ACTIVATION OF THE CORTICOTROPIN-RELEASING FACTOR RECEPTORS FROM THE BASOLATERAL OR
CENTRAL AMYGDALA MODULATES THE NOCICEPTION IN GUINEA PIG.
1
Donatti, A. F. 1; Leite-panissi, C. R. A. 1,2
Programa de Pós-Graduação em Psicobiologia, FFCLRP-USP
2
Morfologia, Estomatologia e Fisiologia, FORP-USP
Objectives:
Corticotropin-releasing factor (CRF) is a peptide involved in the activation of the hypothalamic-pituitary-adrenal (HPA) axis.
However, evidence has suggested that the CRF acts as a neuromodulator outside the HPA axis, playing an important role in
anxiety, depression, fear and pain modulation. Electrophysiology, biochemical and behavioral studies indicate that the
amygdaloid complex is one of the sites of action of CRF in the nociception modulation. The aim of this study was to evaluate
whether the activation of the CRF receptors located in the basolateral (BLA) and central (CeA) nucleus of the amygdala promote
changes in nociception in guinea pigs evaluated in hot plate test (HP).
Adult male guinea pigs (± 450 g, anesthetized with ketamine 75 mg/kg and xilazine 10 mg/kg) were used to implantation of a
guide cannula located unilaterally into the BLA or CeA by stereotaxic surgery. After the recovery period (7 days) the animals
were evaluated in HP for determining the baseline, and then divided into different experimental groups, which received the
administration of 0.9% saline (control), CRF (CRF receptor agonist, 0.2 µg, 0.5 µg and 1.0 µg) or α-h-CRF9-41 (CRF receptor
antagonist, 0.2 µg, 0.5 µg or 1.0 µg). After different treatments, the animals were submitted again to the HP every 10 min for 1 h.
All experimental procedures were approved by the Ethics Committee for Animal Use of the Campus of Ribeirão Preto (Proc.
08.1.1368.53.1). The results showed that administration of CRF in the BLA and CeA 1.0 µg promoted an increase of the
antinociceptive index in the hot plate test (AIHP) (BLA, F6,237 = 56,32, P < 0.05, TWO-WAY ANOVA), (CeA, F3,174 = 21.67,
P < 0.05, TWO-WAY ANOVA) compared with groups control, CRF 0.2 µg and 0.5 µg CRF (P < 0.05, Newman Keuls). In
addition, control groups, CRF 0.2 µg and 0.5 µg CRF were not different among them. In groups that received different
concentrations of CRF receptor antagonist (α-h-CRF9-41) the statistical analysis showed no change in AIHP independent of the
concentration in all experimental time.
Conclusions:
Present results support that involvement of CRF in the modulation of nociceptive processes and include the participation of CRF
receptors located in the BLA and CeA in this mechanism. In addition, the CRF receptors of the BLA and CeA are involved in the
modulation of nociception in a phasic manner, since the blockade of CRF receptors per se do not alter the AIHP.
Keywords: CORTICOTROPIN-RELEASING FACTOR, BASOLATERAL OR CENTRAL AMYGDALA, NOCICEPTION,
GUINEA PIG
Financial Support: CAPES/PROEX, FAPESP, CNPq
Resumo:21-015
EXPRESSION OF TYROSINE HYDROXYLASE AND BRAIN-DERIVED NEUROTROPHIC FACTOR IN THE 6-
HYDROXYDOPAMINE RAT MODEL OF PARKINSON’S DISEASE AFTER DIFFERENT PROTOCOLS OF
TREADMILL EXERCISE.
Real, C. C. 1; Ferreira, A. F. B. 1; Chaves, G. P. 1; Torrão, A. S. 1; Pires, R. S. 2; Britto, L. R. G. 1
1
Fisiologia e Biofísica/ Universidade de São Paulo, ICB/ USP
2
PROGRAMA DE MESTRADO EM FISIOTERAPIA, UNICID
Objectives:
The neurodegenerative diseases represent a major challenge for current neuroscience research. As exercise has been shown to
have important effects on brain function, the aim of the present study was to investigate the exercise-induced
histological/neurochemical alterations in a rat model of unilateral Parkinson´s disease (PD) induced by striatal injection of 6hydroxydopamine (6-OHDA).
Adult male Wistar rats were divided into 4 groups: saline (SAL), sedentary (SED), exercised for 4 weeks after the induction of
PD (EXA), and exercised for 4 weeks before and 4 weeks after the induction of PD (EXB+EXA). The animals were exercised on
a treadmill 3 days/week at the speed of 10m/min during 40min. After the exercise protocol, the brains were collected for
immunohistochemistry and immunoblotting assays. We evaluated tyrosine hydroxylase (TH) and the brain-derived neurotrophic
factor (BDNF) in the striatum (CPu) and the substantia nigra (SN). Statistical analyses were performed using one-way ANOVA
with the Tukey pos hoc test. We observed that the exercise increased the levels of TH with both techniques, especially in the
CPu. The number of TH-positive cells in the SN decreased only 18% in the EXB+EXA (180±17 cells/mm2, n/s) when compared
to the SAL group (244±12 cells/mm2), whereas in the SED (145±24 cells/mm2, pimmunoblotting (BDNF/beta actin ratio), and
revealed a decrease in the SN in all groups in which we induced PD. No changes were seen in striatal levels. On the other hand,
in the EXB+EXA group (0.44±0.03, p
Conclusions:
Our data revealed that exercise may be capable of reducing the damage induced by 6-OHDA in dopaminergic neurons. The data
are indicative of a neuroprotective effect of exercise in the 6-OHDA PD model, leading to improved motor response. This
neuroprotective effect might be associated to the exercise-induced upregulation of BDNF.
Keywords: Parkinson´s Disease, tyrosine hydroxylase, brain-derived neurotrophic factor, treadmill exercise, neuroprotection
Financial Support: FAPESP and CNPq (Brazil)
Resumo:21-016
INHIBITION OF MICROGLIAL ACTIVATION, ASTROCYTOSIS AND NEURONAL DAMAGE IN THE ACUTE
FASE FOLLOWING STATUS EPILEPTICUS INDUCED BY PILOCARPINE INJECTION
Magno, E. N. 1; Pinto, A. I. S. 2; Lopes, R. T. S. 1; Cardoso, M. M. 1; Gomes-leal, W. 1
1
Institute of Biological Sciences, UFPA
2
ESCOLA SUPERIOR DA AMAZÔNIA, ESAMAZ
Objectives:
To investigate the patterns of microglial/macrophage activation, astrocytosis, neuronal damage, as well as the effects of
minocycline treatment, following status epilepticus induced by a single administration of pilocarpine.
Thirty male adult Wistar rats were used in the investigation. All procedures were approved by the Ethics Committee in Animal
Experimental Research of the Federal University of Pará (protocol nº BIO 025-2011). For induction of status epilepticus (SE),
animals were injected with 1mg/kg of scopolamine, follow by 400mg/kg of pilocarpine 30 minutes later. Diazepam was used to
interrupt seizures after SE onset. Epileptic animals were treated with sterile saline (Group 1, n=15) or minocycline (Group 2,
n=6). A third group consisted of non-SE sham animals without any treatment (Group 3, n =9). Minocycline treatment was
performed during 7 days (50 mg/kg, 2 times a day, in the first 2 days, following by single injections of 25mg/kg up to 7 days).
Animals were perfused at 1, 3 and 7 days after SE onset. Group 2 animals were perfused at 7 days only. Microglial activation
(ED-1), astrocytosis (GFAP) and neuronal loss (NeuN) were assessed by immunohistochemistry. After quantification,
comparisons were performed by analysis of variance followed by Bonferroni´s posthoc test (ANOVA-Bonferroni). There were
intense microglial/macrophage activation and astrocytosis in the hippocampus, thalamus motor nuclei, entorrinal and motor
cortices following pilocarpine-induced SE. Both microglial activation and astrocytosis were higher in later survival times in both
thalamus and hippocampus (p0.05). Minocycline treatment considerably reduced microglia/macrophage activation at 7 days postSE (p
Conclusions:
These results suggest that a strong inflammatory reaction is elicited, in different brain regions, in the first week after pilocarpineinduced SE in the absence of conspicuous neuronal loss. Minocycline treatment reduces microglial activation in this temporal
window with influencing neuronal loss. Future studies should investigate in chronic survival times the correlation between the
inflammatory response with neuronal loss and hippocampal neurogenesis following pilocarpine-induced SE.
Keywords: Status epilépticus, pilocarpine, inflammation, minocycline, hippocampus
Financial Support: Fundação de Apoio e Amparo à Pesquisa do Estado do Pará (FAPESPA)
Resumo:21-017
TRAFFIC OF LEUKOCYTES IN THE CENTRAL NERVOUS SYSTEM IS ASSOCIATED WITH CYTOKINE AND
CHEMOKINE UP-REGULATION IN A MODEL OF SEVERE SEPSIS
Comim, C. M. 1; Vilela, M. C. 2; Constantino, L. S. 1; Vuolo, F. 1; Miranda, A. S. 2; Lacerda-queiroz, N. 2;
Rodrigues, D. H. 2; Teixeira, A. L. 2; Quevedo, J. 1; Dal-pizzol, F. 1
1
2
Universidade Federal de Minas Gerais, UFMG
Objectives:
To evaluate the effects of sepsis on brain microvasculature leukocyte rolling and adherence, myeloperoxidase (MPO) activity,
cytokine and chemokine concentrations, and behavioral screening 6, 12, and 24 h after sepsis induction.
Methods: C57BL/6 mice or Wistar rats underwent cecal ligation and perforation (CLP) or sham operation. At 6, 12, and 24 h
after sepsis induction, intravital microscopy was performed in the mice brain microvasculature to evaluate leukocyte rolling and
adherence. Animals were killed and had the brain removed to determine MPO activity and the levels of cytokines and
chemokines. A behavioral screening (SHIRPA) was also performed in a separate cohort of animals. Blood–brain barrier (BBB)
permeability and cytokines and chemokines were determined in different brain regions in Wistar rats. Results: There was a
decrease in circulating leukocyte levels at 6, 12, and 24 h, an increase in rolling and adhesion of leukocytes in the brain
microvasculature, followed by an increase in brain MPO activity. In addition, there was an increase in both brain cytokines and
chemokines at different times. There was a decrease in the neuropsychiatric state muscle tone and strength only at 6h, and a
decrease in the autonomous function at 6 and 12h. The pattern of brain cytokines and chemokines, and BBB permeability
between the analyzed regions seemed to be similar with minor differences.
Conclusions:
Conclusions: During sepsis the brain‟s production of cytokines and chemokines is an early event and it seemed to participate both
in central nervous system (CNS) dysfunction and BBB permeability alterations, reinforcing the role of brain inflammatory
response in the acute CNS dysfunction associated with sepsis.
Keywords: brain, brain microvasculature, delirium, sepsis
Financial Support: UNESC, UFMG and CNPq
Resumo:21-018
EFFECTS OF ACUTE FORCED EXERCISE ON THE PRE-SYNAPTIC PROTEINS IN THE ADULT
HIPPOCAMPUS
Toscano-silva, M. 1; Novais, F. G. 1; Lee, K. S. 2; Gomes da Silva, S. 1; Blazechi, L. F. 1; Scorza, F. A. 3;
Cavalheiro, E. A. 3; Arida, R. M. 1
1
Department of Physiology, Universidade Federal de São Paulo, UNIFESP
2
Department of Biochemitry, Universidade Federal de São Paulo, UNIFESP
3
Department of Neurology, Universidade Federal de São Paulo, UNIFESP
Objectives:
Alterations of pre-synaptic proteins are closely related to synaptic plasticity, memory and learning. Synapsin I, Synaptophysin
and GAP-43 (Growth associated phosphoprotein-43) are involved in neurotransmitter release from adult nerve terminals,
synaptogenesis and axonal growth. Previous studies have demonstrated that the expression of these proteins in the adult
hippocampus is modulated by experimental models of neuronal injury as well as voluntary exercise. Here we sought to
investigate whether acute physical exercise alters expression of neuronal markers in two post-exercise periods.
Male wistar rats (250–300 g) were divided into two groups: exercise group (n=12) and control group (n=6). Animals from the
exercise group were submitted to 5 days of exercise in a treadmill at moderate intensity and further divided in two groups: 24h
(n=6) and 12 days (n=6) after the last session of exercise. Quantitative western blot analysis showed a significant increased of
synapsin I levels in the hippocampus 24 h post-exercise (p=0.02) but not 12 days post-exercise (p>0.05). Both synaptophysin and
GAP-43 did not change significantly 24 h and 12 days after the last physical exercise session (p>0.05).
Conclusions:
In conclusion, these results indicate that acute physical exercise can modulates hippocampal synapsin I in a time-dependent
manner, which may play an important role in the brain plasticity, learning and memory.
Keywords: physical exercise, synapsin, hippocampus, treadmill, synaptic plasticity
Financial Support: CAPES, CNPq, FAPESP
Resumo:21-019
FACILITATORY EFFECT OF AN AUDITORY WARNING STIMULUS IN VISUAL DISCRIMINATION TASKS
WITH THE SAME LEVEL AND DIFFERENT LEVELS OF PERCEPTUAL DEMAND.
Bueno, V. F. 1,2; Valle, L. E. R. D. 2,1
Department of Experimental Psychology, IP/USP
2
Department of Physiology and Biophysics, ICB/USP
1
Objectives:
We compared the influence of an auditory warning stimulus on reaction time to visual stimuli in discrimination tasks with
different levels of difficulty.
Forty-eight young adults (twenty-nine females), aged 18-30 (mean ± s.e.m.: 22,3 ± 0,3) were tested. In one experiment a location
identification task and a shape identification task with different difficulty levels were used. Each one of these tasks was
performed by a group of 12 participants. In half of the trials the target stimulus appeared without any warning and in the other
half, it occurred 50, 100, 200, 400, 800 or 1600 ms (depending on the block) after an auditory warning stimulus. The participants
should press one of two keys depending on the side (left or right) or shape (circle or ellipse) of the target stimulus. In the location
identification task, the warning stimulus produced a similar facilitatory effect for the 50, 100, 200, 400, 800, 1600 ms SOA (mean
± s.e.m.: 19,8 ± 3,9; 16,3 ± 3,5; 20,4 ± 5,5; 33,0 ± 6,3; 29,8 ± 4,0; 21,0 ± 7,0, respectively; p=0,001; p=0,006; p=0,001; p<
0,001). In another experiment, the level of difficulty of the location identification task was equated to that of the shape
identification task by requiring the differentiation of the vertical position of the target stimulus appearing on the left or right side.
As before, a group of 12 participants carried out one of the tasks and another group of 12 participants, the other task. In the
location identification task, the warning stimulus produced a similar facilitatory for the 100, 200, 400, 800 and 1600 ms SOA
(mean ± s.e.m.: 10,9 ± 7,4, 19,8 ± 5,8, 31,8 ± 5,6 24,3 ± 7,1 19,1 ± 4,9 and 20,2 ± 8,3 ms, respectively; p=0,017; p<0,001).
Conclusions:
The facilitatory influence of an auditory warning stimulus is more consistent across time when the location of a visual target
rather than its shape has to be identified. The relative level of difficulty of the location and the shape identification tasks does not
seem to make a difference.
Keywords: Auditory Warning Stimulus, Facilitatory Effect, Perceptual Demand, Reaction Time, Visual Discrimination Tasks
Resumo:21-020
ACUTE CARDIORESPIRATORY EFFECTS OF INTRACISTERNAL INJECTIONS OF MERCURIC CHLORIDE
Azevedo, B. F. 1; Futuro Neto, H. A1,2,3; Stefanon, I. 1; Vassallo, D. V. 1,2
1
Universiversidade Federal do Espírito Santo, UFES
2
Escola Superior de Ciências da Santa Casa de Misericórdia, EMESCAM
3
Faculdade de Medicina da Univix, Univix
Objectives:
The present study aims to evaluate changes in systolic arterial pressure (SAP), diastolic arterial pressure (DAP), respiratory rate
(RR) and heart rate (HR) following an intracisternal (i.c) injection of mercuric chloride (HgCl2) and the participation of the
autonomic nervous system in these responses.
Methods: Rats (58) were distributed randomly into 10 groups and receive 5 µl i.c. injection of 0.68 µg/kg HgCl2 (n =7), 1.2
µg/kg HgCl2 (n=7), 2.4 µg/kg HgCl2 (n=7), 60 µg/kg HgCl2 (n=7), 120 µg/kg HgCl2 (n=3), saline (control) (n=7), 60µg/kg
HgCl2 plus prazosin (n=6), saline plus prazosin (n=6), 60µg/kg HgCl2 plus methylatropine (n=4) or saline plus methylatropine
(n=4)HgCl2. Rats were anesthetized with halothane and maintained as needed with urethane (1.2 g/kg) administered
intravenously (i.v.). The left femoral artery was cannulated to record systolic arterial pressure (SAP), diastolic arterial pressure
(DAP) and heart rate (HR). A tracheotomy was performed to record respiratory rate. Animals were placed in a stereotaxic frame,
and the cisterna magna was exposed. After a stabilization period, solutions (saline or HgCl2) were injected and cardiopulmonary
responses were recorded for 50 minutes. Involvement of the autonomic nervous system was assessed through the i.v.
administration of hexamethonium (20 mg/Kg), prazosin (1 mg/kg) and methylatropine (1mg/kg) 10 minutes before the i.c.
injection of HgCl2 or saline. Results: Treatment with 0.68, 1.2, 2.4 µg/kg HgCl2 or saline did not modify basal cardiorespiratory
parameters, whereas the 120 µg/kg dose induced acute toxicity, producing respiratory arrest and death. The administration of 60
µg/kg HgCl2 , however, induced significant increases (p < 0.05) in SAP at the 30°, 40° and 50° min timepoints and DAP at the
5°, 10°, 20°, 30°, 40° and 50° timepoints. RR was significantly decreased at the 5°, 10°, 20°, 40° and 50°min timepoints;
however, there was no change in HR. Hexamethonium administration, which causes ganglionic blockade, abolished the observed
cardiorespiratory effects. Similarly, prazosin, a &alpha1-adrenoceptor blocker abolished HgCl2 induced increases in SAP and
DAP without affecting HR and RR. Methylatropine (1mg/Kg), a parasympathetic inhibitor, exacerbated the effects of HgCl2 and
caused slow-onset respiratory depression, culminating in respiratory arrest and death.
Conclusions:
Our results demonstrate that increases in SAP and DAP induced by the i.c. injection of mercuric chloride are mediated by
activation of the sympathetic nervous system.
Keywords: Mercury chloride, Central effects, Cardiopulmonary regulation.
Financial Support: FAPES/CAPES/CNPq
Resumo:21-021
MEMANTINE ALTERS THE SEIZURE PATTERN OF YOUNG RATS SUBMITTED EARLY IN LIFE TO LICLPILOCARPINE-INDUCED STATUS EPILEPTICUS
Zenki, K. C. ; Zimmer, E. R. ; Portela, L. V. ; Oliveira, D. L. D.
Departamento de Bioquímica, UFRGS
Objectives:
The status epilepticus (SE) is a prolonged seizure, or repeated brief seizures that affect the development of brain. Retrospective
studies indicate that up to 80% of patients with drug-refractory temporal lobe epilepsy had a history of SE early in life. The SEinduced brain damage is normally associated with overstimulation of glutamatergic system by excessive activation of NMDA
receptors. However, the high-affinity NMDA receptor antagonists are effective on blockade not only glutamate-induced
excitotoxicity but also glutamate-dependent normal brain processes. Thus, others low-affinity, uncompetitive, open-channel
NMDA receptor blockers, such as memantine, provide a new pharmacological tool for treatment of SE-induced brain damage
without disrupting physiological synaptic activity. The aim of this study was to investigate the effects of memantine on LiClpilocarpine-induced status epilepticus in young rats.
Rat pups 15 day old were injected i.p. with solution of LiCl (3mEq/Kg) 12-18 h prior to i.p pilocarpine hydrochloride
administration (60 mg/kg). Memantine (20mg/kg) was injected as a single dose in following times before SE induction: 0 (cotreatment), 3 and 6 hours. Control animals were handled and housed in the same manner as the treated animals and received an
equal volume of NaCl 0.9%. The number of animals per group were 7-14 rats. LiCl-pilocarpine-treated rats showed defecation,
salivation, body tremor, staring and scratching within a latency of 4.3+1.9 min after pilocarpine injection (stage 1). This
behavioral pattern progressed within 15.9+5.3 min to increased levels of motor activity and culminated in SE in all animals (stage
2). SE was characterized by sustained orofacial automatisms, salivation, chewing, forelimb clonus, loss of the righting reflex and
falling. All memantine-treated groups did not exhibit chewing and staring, and displayed a decreased body tremor. However,
others stage 1 seizure manifestations were observed with an increased latency to start in all groups. The administration of
memantine 6 h before pilocarpine injection increased latency for stage 1 (15+9.6 min; p
Conclusions:
In conclusion, this work showed that memantine-treatment alters the seizure pattern of LiCl-pilocarpine-induced status
epilepticus in young rats more efficiently when administered 3 hours after SE onset. However, further studies will be performed
in order to investigate the neuroprotective effects of memantine.
Keywords: Memantine, NMDA, status epilepticus
Financial Support: CNPq, INCTEN, CAPES, FAPERGS, UFRGS
Resumo:21-022
INFLUENCE OF SEROTONERGIC MODULATION IN THE CONTENT AND PROCESSING OF APP IN THE
SUPERIOR COLLICULUS OF RATS DURING POST-NATAL DEVELOPMENT AND IN A MODEL OF INDUCED
PLASTICITY.
Leo, L. M. ; Antonioli-santos, R. ; Vasques, J. F. ; Gonçalves, R. G. J. ; Campello-costa, P. ; Serfaty, C. A. ;
Faria-melibeu, A. C.
Dpto. Neurobiologia/Universidade Federal Fluminense, UFF
Objectives:
The Amyloid Precursor Protein (APP) is a transmembrane glycoprotein broadly presented in the Central Nervous System (CNS),
where it takes part in many cellular functions, as synaptogenesis and synaptic plasticity. Previous data from our group
demonstrated that the presence of APP in the visual system is chronologically related to the critical period of development of of
retinotectal pathways. Our group has also been demonstrating that mononuclear enucleation can alter the content and processing
of APP. Serotonin is a well-known neurotransmitter which has an important role in cognitive functions as well as in synaptic
plasticity. Serotonin is produced from the essential amino acid tryptophan. Previous studies have demonstrated that animals
depleted of serotonin through the intake of a tryptophan restriction diet have impaired plasticity; meanwhile the use of drugs that
enhance serotonergic function have the opposite effect. Furthermore, drugs that modulate the serotonergic system have been
shown to induce alterations in the metabolism of APP. Thus the objective of the present study is to evaluate and quantify through
Western blot analysis the content and processing of APP in rats submitted to tryptophan restriction diet or to daily intraperitoneal
injections of fluoxetine during the critical period of development and in response to mononuclear enucleation.
Lister Hooded rats were submitted to a tryptophan restriction diet from the day of birth. Another group received daily
intraperitoneal injections of fluoxetine, a selective serotonin reuptake inhibitor, also from the day of birth. These animals were
sacrificed at post-natal day (PND) 2, 7, 10, 14 and 21, in order to perform the ontogenesis of APP in the conditions described
above. Thereafter, the superficial visual layers of the superior colliculus (SC) wereremoved and prepared for evaluation of APP
content by Western blot analysis with the antibody anti-APP 22C11 Millipore (1:1000). Moreover, animals under both treatments
were submitted to monocular enucleation at PND10. After different survivals, the rats were sacrificed and the superficial layers of
the superior colliculus extracted and processed for western blotting analyses for APP (22C11 antibody). So far, we animals at
PND 10 submitted to the special diet have shown a 3-fold increase of a third lower molecular weight band of APP, while the two
higher molecular weight bands normally displayed remained unchanged (n=2) as compared to animals fed a control diet. The
treatment with fluoxetine had a similar result compared to non-injected animals (n=2).
Conclusions:
These results suggest that modulation of the serotonergic system, either by serotonin depletion or by increasing the levels of
serotonin in the synaptic cleft, is capable of altering the content and the processing of APP. Based on the literature we propose
that the band intensified by the treatments corresponds to the soluble form of APP.
Keywords: Alzheimer's disease, APP, Serotonin, Synaptic Plasticity, Visual System
Financial Support: Proppi-UFF, PRONEX-MCT
Resumo:21-023
GENE EXPRESSION OF ESTROGEN AND OXYTOCIN RECEPTORS IN LACTATING RATS AND THE
RELATIONSHIP WITH MATERNAL BEHAVIOR.
Moura, A. C. 1; Albiero, G. 1,1; Lazzari, V. M. 1,1; Becker, R. O. 1; Ruthschilling, C. A. 1; Almeida, S. D. 1;
Veiga, A. B. G. D. 1; Giovenardi, M. 1
1
PPG Pathology, UFCSPA
2
PPG Health Sciences, UFCSPA
Objectives:
In rats, there are changes in the circulating levels of oxytocin (OT) and estrogen, and also in the expression of their receptors
(OXTR and ER) in various brain regions during pregnancy and postpartum. Probably these changes can contribute to the
development and management of maternal behavior. The frequency that the mother licks her pups is a parameter used to evaluate
maternal care. Such behavior shows a pattern of normal distribution with two extremes, having mothers with low licking (LL)
and high licking (HL) frequencies. The main objective of this study was to analyze the expression of OXTR and ER in the
hippocampus (HC), prefrontal cortex (PFC), olfactory bulb (OB) and corpus striatum (CS) of lactating rats classified as LL and
HL.
Lactating rats (Wistar), from the animal care unity of UFCSPA, were divided according to the behavior of licking their pups: LL
(frequency of licking behavior below the bottom quartile) and HL (frequency of licking behavior above the upper quartile).
Maternal behavior was studied from the 1st to the 7th day postpartum. In the 8th day, they were sacrificed by decapitation and the
brain parts were removed and frozen at -80ºC. The number of samples of LL rats was: HC (n=8); PFC (n=8); OB (n=7); CS
(n=6); and of HL rats was: HC (n=7); PFC (n=7); OB (n=7); CS (n=6). After, RNA was extracted and used for cDNA synthesis
by RT-PCR to evaluate the expression of OXTR and ER. The levels of the constitutive β-actin gene expression were also
evaluated. Data were expressed by mean and standard media, and compared by the Mann-Whitney U test (p
Conclusions:
According to the results, we can conclude that the differences in the levels of OXTR gene expression in the OB and HP are
related to licking behavior in female rats, and that, in the studied brain areas, there is no relationship between the expression of
ER with the licking behavior.
Keywords: ESTROGEN RECEPTOR, LACTATING RATS , MATERNAL BEHAVIOR, OXYTOCIN RECEPTOR
Financial Support: PROAP/UFCSPA
Resumo:21-024
THE EFFECT OF ANGIOTENSIN II, OXYTOCIN AND SOMATOSTATIN MICROINJECTION IN POSTERIOR
DORSAL NUCLEUS OF MEDIAL AMYGDALA ON CONTROL OF HEMODYNAMICS RESPONSES IN RATS
1
Silva, J. B. 1,1; Quagliotto, E. 1,2; Dall'ago, P. 1; Rasia, A. 1,2
Universidade Federal de Ciências da Saúde de Porto Alegre, UFCSPA
2
Objectives:
The medial amygdala (MeA) modulates reproductive behaviors and is related with stress responses. The cardiovascular
adjustements for these responses could be, at least, in part due to the MeA activation. There is a high presence of angiotensin II,
somatostatin and oxytocin in the MeA. The aim of the present work was to determine the effects of these neuropeptides
microinjected in the posterior dorsal medial amygdala (MePD), a subnucleus of the medial amygdala (MeA), on the control of
cardiovascular responses in rats.
Adult Wistar male rats were anesthetized and submitted to both a stereotaxic implantation in the right MePD and catheter
implantation in the left femoral artery and vein. Afterwards, awake rats were microinjected with saline (0.3 µl, n= 6), angiotensin
II (50 pmol/ 0.3 µl or 50 fmol/ 0.3 µl, n= 6 in both cases), somatostatin (1 µM/ 0.3 µl or 15 fmol/ 0.3 µl, n= 6 and 4, respectively)
or oxytocin (10 ng/ 0.3 µl, n= 6). Heart rate (HR) and arterial pressure (AP) were recorded in basal conditions and after these
microinjections. Baroreceptor function was tested after phenylephrine (8 µg/ ml) and sodium nitroprusside (100 µg/ ml) systemic
injections. Chemoreflex was tested using potassium cyanide (from 60 to 180 µg/ ml). The ANOVA test was used to compare the
variables within and among groups. The significance level was set at á= 5%. No relevant statistical difference was found in the
HR, systolic AP, diastolic AP and medium AP following microinjections in the different groups (P > 0.05 in all cases). When
compared to the control group, oxytocin decreased the MAP50 variation in the point of the greatest slope of the pressure curve
and angiotensin II, at the dose of 50 pmol, reduced the gain (in bpm/ mmHg) variation of the average baroreflex sensitivity and
reduced the HR due to the chemoreflex response (P < 0.01 in all cases). Somatostatin did not promote statistically significant
effects compared to saline.
Conclusions:
The present study reinforces that the MePD is a relevant brain area for the central cardiovascular control and demonstrates that
locally microinjected oxytocin and angiotensin II can inhibit reflex responses mediated by the baroreflex and chemoreflex
stimulation in awake rats.
Keywords: MEDIAL AMYGDALA , HEMODYNAMICS RESPONSES, ANGIOTENSIN II, OXYTOCIN, SOMATOSTATIN
Financial Support: UFCSPA, UFRGS, CAPES and CNPq.
Resumo:21-025
EFFECT OF HYDROGEN PEROXIDE COMBINED WITH CARBACHOL INTO THE MEDIAL SEPTAL AREA ON
RENAL EXCRETION AND C-FOS EXPRESSION IN THE SUPRAOPTIC NUCLEUS.
Melo, M. R. . ; Zanella, R. C. ; Blanch, G. T. ; Menani, J. V. ; Colombari, E. ; Colombari, D. S. A.
Department of Physiology and Pathology, School of Dentistry, UNESP
Objectives:
In the present study, we investigated the effects of H2O2 injected into the MSA on the natriuresis, caliuresis and anti-diuresis
induced by the injection of carbachol into the same area. In addition, we also investigated the effects of carbachol alone or
combined with H2O2 into the MSA on c-Fos expression in the supra-optic nucleus of the hypothalamus (SON).
Male Holtzman rats (280-320 g) with stainless steel guide-cannulas implanted into the MSA were used. One group of rats (n=8)
received 2 gavages of water (10 ml/each) with 1 h interval between them. After the 2nd gavage, phosphate buffered saline (PBS,
0.5 µl) or H2O2 (5 µmol/0.5 µl) was injected into the MSA 1 min before the injection of carbachol (4 nmol/ 0.5 µl) or saline (0.5
µl) into the same site. Urine was collected for 2 hours after injection of saline or carbachol in MSA. Other groups of rats (n=3/4)
received H2O2 or PBS injected into MSA 1 minute before of the injection of carbachol in the same area. After 90 min, rats were
perfused and sections of SON underwent immunohistochemistry for c-Fos. The injection of carbachol into the MSA induced antidiuresis (PBS + carbachol: 7.9 ± 0.5, vs. PBS + saline: 11.8 ± 0.6 ml/2 h, p
Conclusions:
The results shows that H2O2 reduces the anti-diuresis, natriuresis, caliuresis and the increase in c-Fos expression in the SON
produced by cholinergic activation of the MSA. The changes in MSA cholinergic-induced renal responses produced by H2O2
into the MSA might be related to the changes in SON activity.
Keywords: Medial Septal Area, Reactive oxygen species, Supraoptic Nucleus
Financial Support: FAPESP, CNPq, CNPq-PIBIC
Resumo:21-026
CHARACTERIZATION OF CORTICAL SPREADING DEPRESSION IN YOUNG AND ADULT RATS TREATED
SYSTEMICALLY WITH L-ARGININE AND TOPICALLY WITH TIANEPTINE.
Maia, L. M. S. S. 1; Amâncio-dos-santos, Â. 2; Duda-de-oliveira, D. 3; Germano, P. C. P. 3; Falcão, A. C. S.
M. 3; Guedes, R. C. A. 3
2
Departamento de Fisiologia e Farmacologia, UFPE
1
Departamento de Histologia e Embriologia, UFPE
3
Departamento de Nutrição, UFPE
Objectives:
We have previously demonstrated that serotonin-enhancing drugs impair Cortical Spreading Depression (CSD; Exp.Neurol.
200:275, 2006), and L-Arginine treatment enhances it (Nutr. Neurosci. 12:73, 2009). Here we studied the CSD-effect of topic
administration of tianeptine, a serotonin antagonist. Also we investigated the interaction between this topic treatment and the
systemic treatment with L-Arginine.
Female Wistar rats (n=40) received L-Arginine (Arg; 300 mg/Kg/day; n=20) or water (H2O; n=20), daily by gavage, from
postnatal day 7 to 28. Half of each gavage group was submitted to a CSD recording session (recording in 2 points of the parietal
cortical surface), at 30-40 days of life (Young groups; n=10 for each treatment) and the rest was recorded at 90-120 days (Adult
groups). After CSD was elicited 3 times at 20-min intervals (baseline period), a tianeptine solution (TNP; 10 mg/ml) was
topically applied over the intact dura-mater. When compared with pre-TNP CSD recordings, topical application of tianeptine, as
well as the use of arginine, significantly increased CSD-velocities (p < 0.05), in both young and adult groups. The CSDenhancing effect of topical TNP was not modified by Arg treatment. In the H2O-treated groups the mean±sd velocities of CSD
(in mm/min) for the baseline, TNP and recovery periods were 3.85±0.07; 4.49±0.16; 3.90±0.16 in the young groups, and
3.46±0.18; 4.69±0.37; 3.88±0.25 in the adult groups. In the Arg-treated groups, the CSD velocities for the baseline, TNP and
recovery periods were 4.50±0.27; 5.50±0.56; 4.62±0.31 in the young groups, and 3.91±0.09; 4.94±0.48; 4.00±0.29 in the adult
groups.
Conclusions:
1)Data on early Arg-associated enhancement of CSD in adult life suggest a lasting effect of this amino acid; 2)the serotonin
antagonist TNP also enhances CSD, reinforcing previous findings regarding an antagonistic role of serotonergic activity on CSD;
3)the absence of interaction between the 2 treatments suggest that they probably act via two different pathways.
Keywords: Cortical spreading depression, L-Arginine, Tianeptine, Rats
Financial Support: FACEPE; CNPq; PROPESQ.
Resumo:21-027
NEONATAL BRAIN SEROTONIN DEPLETION INFLUENCES METABOLIC ASPECTS IN ADULT OFFSPRING
Lustrino, D. ; Mencalha, R. ; Castro, L. L. ; Almeida, N. A. S. ; Marassi, M. P. ; Mecawi, A. S. ; Reis, L. C.
Ciências Fisiológicas/ Instituto de Biologia, UFRRJ
Objectives:
Evidences have shown that serotonergic inputs modulate the feeding behavior in mammals, once that subset of basomedial and
lateral hypothalamic neurons are innerved by midbrain raphe serotonergic neurons. In addition, serotonergic system modulates
the ontogenetic development of several brain systems. Therefore, the aim of this study was to assess the influences of neonatal
brain serotonin (5-HT) depletion on the metabolic homeostasis in adult rats.
Male Wistar rats were treated daily with p-chlorophenylalanine (pCPA, 100mg. Kg-1, s.c.) or saline during 8th to 16th postnatal
day. Body weight and milk intake were evaluated at the same interval. Further, the animals were weighed weekly until adulthood
(63 postnatal day). In this period, the animals were individually housed in metabolic cages and cumulative food (g/100g bw) and
water (ml/100g bw) intakes through 24h and glycemia (mg/dl) were evaluated under basal conditions and after 24h food
deprivation. So, the rats were euthanized and blood and retroperitoneal adipose tissue were collected. Serum leptin (ng/ml), T3
(ng/dl) and T4 (µg/dl) were measured by radioimmunoassay under basal and food deprivation paradigms. The results are reported
as means ± SE. Differences were determined using either a Student “t” test, or a one-way or two-way ANOVA followed by
Bonferroni post hoc analysis (P
Conclusions:
Present results evidenced for the first time a clear influence of the serotonergic system on metabolic programming through
neonatal period. Data from body weight achieved by adult offspring raised the experimental evidence that neonatal brain
serotonin depletion possibly alters the plastic organization of hypothalamic system implied with body mass and energetic
metabolism adjustment throughout adulthood.
Keywords: Serotonin, Metabolic programming, food intake, Leptin, Thyroid hormones
Financial Support: CNPq; FAPERJ
Resumo:21-028
BLOCKADE OF THE HEME OXYGENASE-CARBON MONOXIDE-CGMP PATHWAY IN THE LOCUS
COERULEUS ALTERS BEHAVIORS EVALUATED IN THE ELEVATED PLUS MAZE IN RATS.
Carvalho, P. G. 1; Leite-panissi, C. R. A. 1,2
Psychobiology Graduation Program , FFCLRP-USP
2
Department of Morphology, Stomatology and Physiology , FORP-USP
1
Objectives:
The gaseous compound carbon monoxide (CO) is involved in modulating various physiological functions such as body
temperature and nociception. The participation of CO in physiological processes occurs through the activity of heme oxygenase
(HO) and CO forming intracellular cyclic guanosine monophosphate (cGMP) (Brain Res., 1385: 107-113, 2011). Emotional
stress situations promote increased of the release of noradrenaline in various brain regions, among them locus coeruleus (LC).
This structure which has a high expression of HO type 2 suggesting the involvement of CO in the modulation of the functions
performed by the LC (Pharmacol.Biochem.Behav., 16: 315-319, 1982). This study aims to evaluate the involvement of activation
of the HO-CO-cGMP pathway in the locus coeruleus modulation of anxiety, evaluated by elevated plus maze test (LCE) in rats.
Rats (250g; male Wistar, N = 7) anesthetized (ketamine 75mg/kg and xylazine 10 mg/kg, administered intramuscularly)
underwent surgery for implantation of unilateral guide cannula directed toward the LC. After the recovery period (7 days) the
animals received intra-LC administration of HO inhibitor (ZnDPBG/5 nmol/0.1 µL) or its vehicle (Na2CO3/50 mmol/0.1 µL) in
different experimental groups and were assessed 30 min after elevated plus maze test for 5 min. All experimental procedures
were approved the Animal Use and Ethics Committee of the Ribeirao Preto, University of Sao Paulo (Process no. 09.1.606.53.7).
These results show that the intra-LC administration of ZnDPBG changed the behavior of grooming (F(1,14)=9.58, P = 0.009, one
way ANOVA) and head-dipping (F(1,14) =7.37, P = 0.01, one way ANOVA) compared with the control group (P < 0.05,
Newman Keuls). Regarding the percentage of entry in the open arms and time spent in these arms, mean entry into the open and
closed arms and the different behavioral categories: end-arm-exploration, rearing, peeping out, scanning, stretched-attendposture, flat-back-approach and immobility was not observed statistical difference between the two groups on behavioral analysis
of the LCE.
Conclusions:
Our results suggest that the effect of intra-LC administration with an HO inhibitor (ZnDPBG) was able to block the behavior of
grooming and head-dipping, thus suggesting involvement of HO-CO pathway in the modulation of emotional behaviors in animal
models of anxiety.
Keywords: CARBON MONOXIDE, HEME OXYGENASE, LOCUS COERULEUS
Financial Support: CAPES / PROEX, FAPESP, CNPq
Resumo:21-029
CHEMICAL LESION IN THE MEDIAL NUCLEUS OF AMYGDALA CHANGED DEFENSIVE ANTINOCICEPTION
BUT NOT TONIC IMMOBILITY RESPONSE IN THE GUINEA PIGS.
Vieira-rasteli, E. B. 1; Leite-panissi, C. R. A. 1,2
Psychobiology Graduation Program, FFCLRP-USP/SP
2
Department of Morphology, Stomatology and Physiology , FORP-USP/SP
1
Objectives:
Tonic immobility behavior (IT) is an innate response, exhibited by the prey when the physical contact with the predator is
prolonged and the situation inescapable, characterized by profound motor inhibition. It can be suggested that triggering of the
defensive behavior involves the activation of a complex neural system responsible for detection of threatening stimuli, and
finally, the preparation of a planning engine suitable for the expression of defensive response chosen. Within this context, several
findings point to the medial hypothalamus, inferior colliculus, deep layers of superior colliculus, amygdala and periaqueductal
gray matter as fundamental structures in the modulation of emotional behavior. In addition, studies show that the simultaneous
activation of an antinociceptive system is essential for the animal to perform the response defense behavioral properly. The
objective of this study was to evaluate the effect of chemical lesions through the administration of ibotenic acid (0.4 µg/0.2 µL)
in the medial nucleus amygdala (MEA) of guinea pigs on the response of TI and on nociception by hot plate test.
Males guinea pigs (± 450 g) were induced manually to TI, followed by the hot plate test (52◦C) every 10 min for 1 h then before
and 7 days after the lesions. All experimental procedures were approved by the Ethics Committee for Animal Use of the Campus
of Ribeirão Preto (Proc. 06.1.220.53.9). Our data show that TI was able to activate an antinociceptive system, evidenced by the
hot plate test performed immediately after the response of TI in control sessions (before the lesion; N = 6; before the saline group,
N = 7). Moreover, the antinociceptive response triggered by the TI group with lesion in the MEA (N = 6) were decreased (TWOWAY ANOVA; F6,83 = 3.590; P = 0.004) when compared with control session (P < 0.05; Newman Keuls). The duration of the
TI behavior (seconds) not observed significant difference between the control session (130.1 ± 27.9 s) and after lesion (119.5 ±
16 s). Again, in the saline group did not demonstrated significant difference between the control session (131.7 ± 37.0 s) and after
the saline administration (133.0 ± 33.9 s).
Conclusions:
The results shown that although the chemical lesion of the MEA did not alter the TI duration, the defensive antinociception
induced by TI in guinea pigs was decreased. So, it is possible to suggest that MEA is particularly involved in the control
antinociceptive response associated with defensive behaviors.
Keywords: Antinociception, Medial nucleus of amigdala, Tonic immobility
Financial Support: FAPESP (07/06549-3), CAPES/PROEX, CNPq
Resumo:21-030
SEROTONINERGIC NEURONS RECRUITMENT IN THE DORSAL RAPHE NUCLEUS DID NOT ALTER THE
MECHANISMS OF THIRST SIGNALS IN WISTAR RATS.
Fonseca, F. V. 1; Mecawi, A. S. 2; Laureano-melo. R1; Almeida-pereira, G. 2; Araujo, I. G. D. 2; Menezes,
V. C. L. 1; Reis, L. C. 1
1
Department of Physiological Sciences, , UFRRJ
2
Department of Physiology - School of Medicine of Ribeirão Pr, FMRP/USP
Objectives:
Evidence from our laboratory revealed that the brain serotonin system is involved in regulating the balance hydroelectrolytic.
Electrolytic lesion in the DRN in rats given subcutaneous administration of isoproterenol had significant increase in water intake
compared to control animals (Braz. J. Med. Biol. Res. 36; 1709, 2003). We intend to examine the hypothesis of the existence of
mechanisms controlling serotoninergic activity specifically related to the integration of appetite and sodium satiety and thirst
signals.
Male Wistar rats (~ 320g, n = 5 to 10) were submitted to dipsogenic protocol by isoproterenol (a beta-adrenergic agonist)
administration. Intra-DRN guide cannula implant was stereotaxically completed in rats placed in a Kopf stereotaxic device for
microinjection of WAY100135, a 5-HT1A autoreceptor antagonist. Protocol 1: Acute intra-DRN administration of WAY100135
(2nmol/rat/0.2µl) in sodium-replete rats. Protocol 2: Acute intra-DRN administration WAY100135 (2nmol/rat/0.2µl) in betaadrenergic stimulated rats (DL-isoproterenol, 100µg/kg, S.C.). Protocol 3: Acute intra-DRN administration of WAY100135 in
beta-adrenergic stimulated rats (DL-isoproterenol, 100µg/kg, S.C) previously subjected to a low sodium diet during 5 days). For
the assessment of fluid intake, the animals were placed in metabolic cages equipped with graduated bottles for water and 0.3M
NaCl intake by 240min. Statistical analysis was performed by using one way ANOVA and Bonferroni test. Intra-DRN
Administration of WAY100135 did not alter the responses of ingestion of water in all protocols studied. In contrast, intra-DRN
WAY100135 administration in sodium-replete rats significantly decreased 0.3M NaCl ingestion (0.41 ± 0.06 ml vs 1.25 ±
0.16ml/100g bw, P
Conclusions:
We suggest that signaling dependent on 5-HT1A receptors within the DRN did not alter the thirst mechanisms related to
angiotensin II generation. Acute blockade of somatodendritic 5-HT1A autoreceptors at DRN clearly anticipates satiety signaling
in sodium deficient-rats probably by facilitated recruitment of serotoninergic neurons.
Keywords: Balance hydroelectrolytic, Intra-DRN , angiotensin II , Thirst, 5-HT1A autoreceptors
Financial Support: FAPERJ/CNPq
Resumo:21-031
INFLUENCE OF SEXUAL DIMORPHISM AND HEMISPHERIC LATERALIZATION IN HYPOXIC-ISCHEMIC
MATURE AND IMMATURE RATS IN BEHAVIORAL TASKS
Sanches, E. F. ; Arteni, N. S. ; Netto, C. A.
Bioquímica/ Universidade Federal do Rio Grande do Sul , UFRGS
Objectives:
The increasing of survival rates of preterm newborns (24-28 weeks) is associated to brain damage and several neurological
conditions, like those arising from neonatal hypo

XXVI Reunião Anual da FeSBE - FeSBE 2011 Resumo:01-001 Lima, A. M.

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