Programa de Pós-Graduação em Patologia Ambiental e

Transcription

COMPORTAMENTO DE CAMUNDONGOS
SOB INFECÇÃO SIMULADA DE LPS PERINATAL
TRATADOS POR ZINCUM METALLICUM
SILVIO LEITE MONTEIRO DA SILVA
COMPORTAMENTO DE CAMUNDONGOS SOB INFECÇÃO SIMULADA DE
LPS PERINATAL TRATADOS POR ZINCUM METALLICUM
Trabalho de conclusão de curso para
obtenção do título de doutor em Patologia
Ambiental e Experimental apresentado à
Universidade Paulista – UNIP.
Orientador: Prof.a Leoni Villano Bonamin
SÃO PAULO
2015
Silva, Silvio Leite Monteiro da.
Comportamento de camundongos sob infecção simulada de LPS
perinatal tratados por Zincum metallicum / Silvio Leite Monteiro da
Silva. - 2015.
84 f. : il. color. + CD-ROM.
Tese de Doutorado Apresentada ao Programa de Pós Graduação
em Patologia Ambiental e Experimental da Universidade Paulista,
São Paulo, 2015.
Área de Concentração: Patologia Ambiental.
Orientadora: Prof. Dr. Leoni Villano Bonamin.
1. LPS. 2. Perinatal. 3. Comportamento Zincum metallicum.
4. Altas diluições. 5. Homeopatia. 6. Depressão. 7. Infecção.
Bonamin, Leoni Villano (orientador). III. Título.
I.
COMPORTAMENTO DE CAMUNDONGOS SOB INFECÇÃO SIMULADA DE
LPS PERINATAL TRATADOS POR ZINCUM METALLICUM
Trabalho de conclusão de curso para
obtenção do título de doutor em Patologia
Ambiental e Experimental apresentado à
Universidade Paulista – UNIP.
Aprovado em: __________________
BANCA EXAMINADORA:
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AGRADECIMENTOS
Agradeço aos animais;
Aos meus pais Waldemar e Sara pelo apoio incondicional desta trajetória, a
Dora e a Jandira;
Aos amigos Adeline, Paulo de Poços, Rodrigo, Aline, Jefferson, Ricardo e
Andreia de São Paulo;
A Giang, Eduardo, Stefano, Ignazio, Maria Rosa, Tudor, Emanuele,
Alessandro, Filipo, Nico, amigos e colegas de Verona/estudantes da UNIVR;
Loris e colegas da ONAV - Organizzazione Nazionale Assaggiatori di Vino;
Aos colegas da UNIP Aloísio, Marlene, Chris, Toshie, Su, Cleide, Fabi, Thayná,
Tica, Paulo, Rodolfo, Osvaldo, Leandra, Kleber;
A Karina e Monique da equipe do pillattes em São Paulo, aos instrutores da
natação do SESC/SP; a Bulé e Donizete (Clube Caiçaras de Rio Pomba/MG) e
equipe multidisciplinar, e a equipe do Centro Federale di Alta Specializzazione
di Verona “Alberto Castagnetti”;
A Marta Marzotto, Clara Bonafini e Débora Olioso pelos momentos
inesquecíveis de convivência, pela recepção internacional, amizade, além da
oportunidade concedida por Paolo Belavitte da UNIVR;
Ao Tiago Berti Kirsten (USP/UNIP) pelo LPS; a Amarilys de Toledo Cesar
(HNCristiano) pelas potências; e a Carla Holandino (UFRJ) pela matriz do
Zincum metallicum;
A Maristela Dutra Correa e Vanessa Gallego Arias Pecorari da Odontologia da
UNIP que apoiaram incondicionalmente;
Ao Claudio Costa e Emiko Saito Arita da Faculdade de Odontologia da USP
pelos equipamentos de raios-X digital e MicroCT;
Ao Cassio Roberto Silva Noronha pelas valiosas sugestões;
As pessoas que fazem parte da equipe do GIRI e do grupo de estudo
internacional do Zincum metallicum;
A Capes pelo apoio com bolsa sanduíche no exterior (PDSE- 6225/14-4) e pela
taxa (PROSUP-1187970), a UNIP e o IF Sudeste MG pelo suporte geral;
A Maria Martha Bernardi por sua especial experiência na condução
organizacional do ambiente e admiração dos fenômenos e pessoas;
A Leoni Villano Bonamin pela sua atenção e referência ímpares como pessoa e
profissional com as quais pude desfrutar durante este período memoriável.
“Nós estamos diante da arte das verdadeiras histórias
Conectadas ao intrigante mistério da vida
Originais
Iluminadas pelo caminho que acontece
Mudam, se reinventam e se auto-organizam
Mas nunca terminam”
(Silvio Leite Monteiro da Silva)
RESUMO
Este é um estudo exploratório perinatal que aborda o comportamento de
camundongos BALB/C sobre efeito de infecção simulada por LPS durante o 9,5
dia de gestação, com tratamento de zinco diluído. Foram avaliados o
comportamento de depressão aparente e de doente através do teste de
suspensão pela cauda e campo aberto, além da massa corporal. A geração
parental foi cruzada com seleção polarizada pelo tempo de imobilidade ao teste
de suspensão pela cauda, considerado validado pelo uso, e com delineamento
em blocos inteiramente casualizados. O comportamento de depressão
aparente teve correlação inversa com o peso vivo à 2ª semana de gestação. A
prole apresentou redução significativa do comportamento de depressão
aparente em relação à geração parental de forma global. Os fatores de
interferência LPS e parto interagiram com o tratamento proposto e
demonstraram afetar evidentemente a geração parental. Dimorfismo sexual foi
observado na geração filial. Houve transmissão vertical deste efeito para a
geração filial fêmea das mães tratadas com o zinco diluído em 200cH. A
identificação de parâmetros de interferência potencialmente sensíveis neste
modelo apresenta seu potencial para estudo de depressão pós-parto.
Palavras-chave: LPS; perinatal; comportamento; Zincum metallicum; altas
diluições.
ABSTRACT
This is an exploratory perinatal study addressed to the behavior of BALB / C for
mice with simulated infection by LPS effect during pregnancy treated with
diluted zinc. The depression-like behavior and sickness behavior were
evaluated by the tail suspension test and open field arena, as well as live body
mass. The parental generation was mated with polarized selection by immobility
time from spontaneous tail suspension test, and randomized in blocks. The
depression-like behavior was inversely correlated with body weight at the
second week of gestation, when they received LPS. The offspring during
adulthood showed a significant reduction of depression-like behavior in relation
to parental generation globally. The interference factors LPS-delivery interacted
with the factor treatment and demonstrated clearly affect the parental
generation. Sexual dimorphism was observed in the filial generation. There was
vertical transmission of this effect to the female branch generation of mothers
treated with diluted zinc to 200cH. The identification of potentially interfering
sensitive parameters in this model has potential for the study of post-partum
depression.
Keywords: LPS; perinatal; behavior; Zincum metallicum; high dilutions.
SUMÁRIO
INTRODUÇÃO ................................................................................................... 9
CAPÍTULO 2 – Block randomization and tail suspension test as a method to
select parental mice mates to perinatal studies of depression-like behavior. ... 14
1.
2
Introduction ....................................................................................... 15
1.1
The depression-like behavior .......................................................... 15
1.2
Objectives ........................................................................................ 17
Material and methods ........................................................................... 17
2.1
Ethics............................................................................................... 17
2.2
General conditions........................................................................... 17
2.3
The behavior tail suspension test .................................................... 18
2.4
Statistical analysis ........................................................................... 19
3.
Results ............................................................................................... 20
4.
Discussion: ........................................................................................ 25
5.
Conclusion ......................................................................................... 26
CAPÍTULO 3 – Exploratory study with simulated infection to perinatal murine
model for depression-like and sickness behaviors treated with diluted zinc. .... 28
1.
Introduction ....................................................................................... 29
1.1.
The simulated infection by lipopolysaccharide (LPS) ...................... 29
1.2
The zinc and diluted zinc effects...................................................... 30
1.3
The depression-like and sickness behaviors ................................... 32
1.4
Grooming, defecation and stand up................................................. 33
1.5
Objectives ........................................................................................ 33
2.
Material and methods ....................................................................... 34
2.2
Experimental design ........................................................................ 34
2.3
The tail suspension tests and open field arena ............................... 36
2.3.1 Parental female generation (first experiment).................................. 37
2.3.2 Filial generation after adulthood (second experiment) ..................... 38
2.4
3.
Statistical analysis ........................................................................... 38
Results ............................................................................................... 39
3.1.
Comparison between maternal and filial immobility time ................ 39
3.2
Parental female generation ............................................................. 40
3.2.1 The parental female depression-like behavior ................................. 40
3.2.2 Suspension immobility time and weight ........................................... 44
3.2.3 The parental female sickness behavior ........................................... 46
3.2.4 The parental female grooming, stand up and defecation parameters..
........................................................................................................ 48
3.2.5 The delivery ..................................................................................... 50
3.3
The filial generation at adulthood..................................................... 51
3.3.1 The filial immobility time by the tail suspension test ........................ 51
3.3.2 The filial walking and immobility time............................................... 52
3.3.3 The filial grooming, stand up and defecation parameters ................ 55
4.
5.
Discussion: ........................................................................................ 56
4.1.
Parental females.............................................................................. 56
4.2
The filial generation at adulthood..................................................... 57
Conclusion ......................................................................................... 59
CAPÍTULO 4 - Considerações finais................................................................ 61
REFERÊNCIAS BIBLIOGRÁFICAS ................................................................ 62
APÊNDICE A – Apresentação de resultados parciais sobre comportamento no
GIRI 2014 (Romênia/Sighisoara). .................................................................... 73
APÊNDICE B – Apresentação de resultados parciais sobre densitometria
femoral no GIRI 2015 (Verona/Itália)................................................................ 74
APÊNDICE C – Apresentação de resultados parciais sobre citocinas
intraperitoneais na conferência do HRI 2015 (Roma/Itália). ............................. 75
APÊNDICE D – Publicação do resumo no 2nd HRI Research Conference ..... 76
APÊNDICE E – Imagens dos fêmures esquerdos aos raios-X. ....................... 77
APÊNDICE F – Tabela de dados da microtomografia computadorizada
(MicroCT) de fêmures. ..................................................................................... 78
APÊNDICE G – Microscopia eletrônica de varredura e espectroscopia de
energia dispersiva (MEV-EDS) de uma amostra de fêmur corte transversal. .. 79
ANEXO A – Certificado de aprovação pela (CEUA-UNIP) protocolo 156/13. .. 80
ANEXO B – Laudo de análise laboratorial externa da concentração de zinco. 81
ANEXO C – Carta com decodificação dos tratamentos em cego. ................... 82
ANEXO D – Parecer do Prof. Paolo Belavitte sobre o estágio de doutoramento
internacional na Università Degli Studio di Verona - Ospetale Policlinico. ....... 83
9
INTRODUÇÃO
O estado-da-arte atual das pesquisas científicas de homeopatia e altas
diluições encontra-se em um momento em que se observa ser de extrema
importância a coleção de diferentes dados experimentais. Dados sobre o uso
tradicional da homeopatia em humanos e animais são ferramentas que incitam
vários campos da pesquisa, pois seus fundamentos nascem da observação de
que muitas doenças e seus processos são passíveis de cura pela própria
resposta orgânica. Assim, tais princípios também podem ser estendidos do
reino animal para o vegetal, conforme demonstrado em diversos modelos
experimentais. Estudos experimentais com foco na compreensão dos
fenômenos naturais que cercam a ação biológica das altas diluições alimentam
a curiosidade científica exploratória. Tais estudos têm potencial particularmente
elevado e de impacto para oferecer novos tratamentos para doenças que ainda
não têm solução conhecida. A compilação de teorias construídas a partir da
abordagem fenomenológica de dados experimentais oferece pistas para o
desenvolvimento evolutivo esperado nesta intrigante área do conhecimento.
Neste estudo, o delineamento experimental escolhido foi da infecção
simulada por LPS em modelo perinatal em camundongos, tratados ou não com
diferentes potências (ou diluições) homeopáticas de zinco (Zincum metallicum
5cH, 30cH e 200cH), ou seja: tais tratamentos são o principal fator de
interferência para o modelo LPS-perinatal, cujas consequências foram
mensuradas em diferentes níveis e sistemas. Controle de tais medicamentos
foi feito pela administração do veículo dinamizado em via e posologia idênticas,
ou seja, preparado com os mesmos métodos farmacotécnicos utilizados para
os medicamentos. O controle da infecção simulada, per se, foi feito com a
adição de alguns animais em cada grupo que não receberam a injeção de LPS.
Os resultados obtidos a partir deste modelo são organizados em
diferentes artigos científicos, a serem submetidos para publicação em revistas
especializadas, de acordo com as variáveis estudadas. Assim, compõem o
volume desta tese de Doutorado, os capítulos concernentes à:
10
a)
Estratégias de parâmetros comportamentais e fatores de
interferência no modelo experimental;
b)
Efeitos comportamentais (parentais e filiais) do tratamento
com diferentes potências homeopáticas de Zincum metallicum.
A fim de refinar o uso de animais de laboratório, concorrendo a favor da
regra dos “3Rs”, outros parâmetros podem ser também avaliados amiúde a
partir de material colhido nas necropsias realizadas ao final dos experimentos
sem que, para isso, haja a necessidade de se utilizar mais animais. Tais
parâmetros, uma vez analisados e discutidos oportunamente, poderão compor
outros artigos derivados do mesmo modelo, além daqueles apresentados nesta
tese. São eles:
a)
Parâmetros referentes à avaliação da estrutura óssea nas
gerações prental e filial tratadas com Zincum metallicum, mensurados
por técnicas de raios-X digital para avaliação da densidade óssea,
microscopia eletrônica de varredura e espectroscopia de energia
dispersiva
(MEV-EDS),
micro-tomografia
e
dimensões
ósseas
macroscópicas.
b)
Parâmetros
referentes
à
relação
comportamento
x
inflamação, levando-se em conta a expressão de GFAP em áreas
específicas do SNC relacionadas aos comportamentos observados e a
mensuração de células inflamatórias e citocinas peritoniais (esta já
realizada, com a co-orientação do Prof. Paolo Bellavite da Universidade
de Verona, com auxílio do programa PDSE-CAPES).
Os resultados comportamentais que são apresentados neste volume
mostram como certas características individuais associadas ao histórico
perinatal interferem na eficácia do tratamento homeopático. Esse ponto de vista
enquadra exatamente no escopo da revista “Personality and Individual
Differences”, o qual valoriza o desenvolvimento de metodologia adequada ao
estudo dos fatores de interferência e de suas relações com as respostas
comportamentais individuais. Assim sendo, a organização desse volume
mereceu um formato exclusivo e completamente original, que permitisse
elucidar,
passo
a
passo,
como
tais
variáveis
podem
interferir
na
susceptibilidade a transtornos comportamentais e na capacidade de resposta
11
desses indivíduos ao tratamento homeopático. Por esse motivo, vê-se que o
discurso utilizado nos dois artigos apresentados a seguir inclui resultados e
discussão apresentados em conjunto e ordenados didaticamente ao longo da
narrativa. Para maior compreensão, o escopo da revista está apresentado no
quadro a seguir*:
Personality and Individual Differences is devoted to the
publication of articles (experimental, theoretical, review) which aim
to integrate as far as possible the major factors of personality
with empirical paradigms from experimental, physiological, animal,
clinical, educational, criminological or industrial psychology or to
seek an explanation for the causes and major determinants of
individual differences in concepts derived from these disciplines.
The editors are concerned with both genetic and environmental
causes, and they are particularly interested in possible interaction
effects. Ultimately they believe that human beings are bio-social
organisms and that work on individual differences can be most
fruitfully pursued by paying attention to both these aspects of our
nature. They believe that advances are more likely to be made by
the use of the hypothetical-deductive method, though empirical
data based on sound research and providing interesting new
findings, would of course not be rejected simply because they
might not have a good theoretical underpinning. All in all, the
traditional type of work on traits, abilities, attitudes, types and
other latent structures underlying consistencies in behavior has in
recent years been receiving rather short shrift in traditional
journals of personality; Personality and Individual Differences
aims to reinstate it to its proper place in psychology, equal in
importance with general experimental work, and interacting with it
to make up a unitary science of psychology.
*Escopo da revista Personality and Individual Differences. As partes sublinhadas no
texto
correspondem
aos
Durante
desenvolvimento
o
pontos
em
comum
das
com
o
trabalho
atividades
aqui
apresentado.
propostas,
após
a
apresentação no GIRI da Romênia (2014) – apêndice A, dos resultados
parciais de comportamento e nascimento de animais, a demanda de quantificar
a concentração de zinco evoluiu. Este micro elemento está presente nos
tecidos ósseos dos fêmures e deparamos com a oportunidade de utilizar uma
metodologia não destrutiva antes da quantificação. Com a hipótese de que
poderia haver alteração da disponibilidade de zinco nos tecidos ósseos do
fêmur, tanto pela demanda deste metal no pool metabólico na gestação quando
em situações de infecção, a demanda metabólica de macro elementos pode
12
alterar. Nesta linha, foram realizadas análises de raios-X dos fêmures
esquerdos de todos os camundongos.
Para surpresa, observamos alterações com tripla interação que inclui
efeitos do Zincum metallicum sobre a deposição mineral nos fêmures dos
mesmos camundongos utilizados nos experimentos de comportamento,
publicado no Simpósio do GIRI seguinte em Verona (2015), organizado pelo
Groupe Internacional de Recherche Sur L’infinitésimal, apêndice B. A relação
de imagens de raios-X realizadas em parceria com demais co autores está
apresentada no apêndice E. A relação dos parâmetros detectados pelas
análises de microtomografia computadorizada (microCT) em decorrência da
parceria anterior está apresentada no apêndice F. A análise da presença de
zinco presente na porção cortical do tecido ósseo em relação às demais
moléculas foi realizada pelo módulo de espectroscopia de energia dispersiva
presente no microscópio eletrônico de varredura (MEV-EDS) está ilustrada com
um exemplo da amostra 43 no apêndice G.
Os resultados parciais da determinação da concentração de citocinas
peritoneais realizada no período de estágio de doutoramento internacional sob
orientação do Prof. Paolo Belavitte com supervisão de Marta Marzotto foi
apresentada no 2nd. HRI International Homeopathy Research Conference,
organizado pelo Homeopathy Research Institute, appendices C e D.
Todos os procedimentos experimentais com animais foram realizados
após a obtenção do certificado de aprovação pelo comitê de ética em pesquisa
com animais sob o protocolo 156/13 (CEUA-UNIP) de acordo com a legislação
brasileira. A alimentação, a água e a medicação foram avaliadas pelo teor de
zinco em suas composições. Como a medicação é composta por zinco diluída,
é esperado apenas traços, assim como na água. Isso quer dizer que a ração
era a única fonte exclusiva de zinco, mas não a água e os medicamentos, o
que foi confirmado pelas análises externas. Como o limite de detecção do teste
é 0,3 mg/Kg, foi possível afirmar que a concentração das amostras onde eram
esperados traços era inferior a 0,3 mg/Kg. O nível de zinco mínimo garantido
na ração era de 70 mg/Kg e foi detectado 119 mg/Kg pelo método de absorção
atômica realizado no laboratório externo. Esta constatação foi de igual
influência entre todos os grupos e a influência deste aumento é considerada
limitada sobre os parâmetros avaliados.
13
O tratamento com Zincum metallicum aconteceu em cego, assim como
as análises estatísticas. A revelação da codificação entre o nome dos grupos
(A, B, C e D) e a relação das medicações utilizadas em cada frasco foi revelada
com a abertura da carta em posse de um funcionário sem vínculo direto ao
experimento, o conteúdo desta carta é apresentado no anexo C.
As atividades desenvolvidas durante o período do estágio de
doutoramento internacional foram relatadas e comentadas pelo professor
responsável e este documento é apresentado nos anexos D e E.
14
CAPÍTULO 2 – Block randomization and tail suspension test as a method to
select parental mice mates to perinatal studies of depression-like behavior.
Silvio Leite Monteiro da Silva, UNIP, IF Sudeste MG
Maria Martha Bernardi, UNIP
Leoni Villano Bonamin, UNIP
Abstract
Introduction: there are individual factors that are neglected in quantitative
analysis on depression studies developed on mice models. Parental generation
block randomization considers each subject and their inter-individual variations
towards a refined control over experimental variables. This method is useful to
evaluate the vertical transmission to maternal and offspring behavior in the
perinatal set up.
Methods: all 37 mice were previously accessed by spontaneous depressionlike behavior, observed by the immobilization time in Tail Suspension Test.
They were categorized in depressed-like; intermediate and healthy. The
females (n=28) were randomly blocked into four groups and the males selected
by complementary immobilization time to the mates. The aim of this procedure
is to control the individual parental influence over filial depression-like behavior.
Results/discussion: the parental generation was mostly in the intermediate
category which is good for depression studies with interference factors to reach
expected homogeneity of filial generation. The planned mating was performed
with more than one male option to be available in a 1:3 harem method, and
pregnancy was confirmed by the presence of vaginal plug. The mating plan
resulted in a significant inverse correlation between the female and the male
immobility time (p-value<0.01), in which the distribution of female and male
mice was similar in the four groups, considering behavior and body weight (pvalue>0.05). The block randomization method is useful to reduce the sample
size and increase the sensitivity of variables control of any proposed
treatments.
15
Conclusion: the mating plan performed under controlled randomness and
selection increased the accuracy of the spontaneous depression-like behavior
observation in parental mice to be used in transgenerational studies.
Highlights
•
Spontaneous depression-like behavior of parental selection mice may
affects the filial generation;
•
The block randomization design is a refinement method to control the
inter-individual variation of mates for perinatal studies;
•
The planned mates strategy reached an inverse correlation between
parental mates;
•
The parental and expected filial generation of further experiments are
more confident to study interference factors on depression-like behavior
Keywords:
Mice model; inter-individual; depression-like behavior;
Tail
Suspension Test; perinatal; block randomization
1. Introduction
1.1 The depression-like behavior
The depression is a world-wide disease that reduces the quality of life
and influence the death risk in humans. The treatment and exploration of
influent parameters on depression are currently relevant because of its social
demand thus, it is a hot topic (MACÍAS-CORTÉS et al., 2015; OSTERMANN et
al., 2015; VIKSVEEN & RELTON, 2014; ADLER et al., 2013; IJICHI et al 2013;
BAWDEN, 2012; LONGACRE et al., 2012; VAN DE VELDE et al., 2010).
The behavioral study using experimental models is a tool to evaluate how the
crosstalk complexes of neuronal and physiological functions respond over a
context (ROSENBLAT et al., 2012). Mice can be objectively assessed by
behavioral tests focused on correspondence effects with the human depression
16
(ENNACEUR, 2014; PINHEIRO et al., 2007; PRUT & BELZUNG, 2003). The
mice depression-like behavior model was validated 30 years ago, for
antidepressant drugs evaluation. The method measures the immobility time by
the tail suspension test and the paradigm is the continuous inescapable
situation during 5 minutes hanged: as more time the mouse take to give up and
keep immobile, more evident is the depression-like behavior.
In mice population, the spontaneous response in two equivalent tests, the tail
suspension test and the forced swimming test, classifies them in three
categories: depressed-like, intermediate and healthy subjects (REIS-SILVA,
2013). This parental and filial behavior are linked with the possibility of vertical
transmission as to humans as to mice (HAMMERTON, et al. 2015; MARS, et al.
2015; CRYAN et al 2005). REIS-SILVA (2013) showed that 70-75% of male and
female mice of three filial generations from depressed and non depressed-like
parents measured in tail suspension test presented the same behavior profile
than their parents. This author also revealed differential response to immune
activation by lipopolyssacharide in the offspring.
Due to these observations, in perinatal studies, a block randomization strategy
instead of simple sorting is needed to balance the individual parental effect
related to these parameters to expect the presence of controlled filial generation
(KAHAN et al., 2015). The proposed procedure reduce the risk of sorting artifact
to group treatments, for an example, the presence of more depressed-like
parental mice in one group than others and its potential impact over the filial
generation (KAHAN & MORRIS, 2012).
The strategy of individualizing the mates is a refinement technique of the simple
random mating arrangement. Such control increases the reliability of the results
and reduces the potential use of individuals. These concepts are adherent to
the 3R (Replace, Reduce and/or Refine) directives to works with participation of
laboratory animals (SCHIFFELERS et al., 2012).
Thus, in order to feed further experiments by the same author reported
elsewhere and to improve the quality of perinatal model studies we suggest the
matting plan one by one, according to each subject. The aim is to produce more
than a balanced group to evaluate the treatments: there is an individuality set
up of the parental generation and an expected reduction of bias from each
17
parental couple to their offspring. This way it is possible to evaluate carefully the
effects of different treatments to each individual and evaluate in detail choosen
interference factors.
1.2 Objectives
The objective of this work is to propose a method to individualize the mice
depression-like behavior to balance the mates in among experimental groups.
The specific objectives are: (1) to evaluate the immobility time from the tail
suspension test of all animals; (2) to randomize the female mice in blocks by the
immobility time among four groups; (3) to choose the male mice to mates by
polarization, according to the female immobility time.
2 Material and methods
2.1 Ethics
The participation of mice in this research was performed in accordance to
international protocols. The experiment was previously submitted and approved
by the CEUA-UNIP ethical committee in April, 17th 2013, under the protocol
156/13, appendix A.
2.2 General conditions
Thirty seven mice were enrolled to the experiment, 9 male mice and 28 female
mice from the lineage BALB/C ICB-USP originated from the Faculdade de
Medicina Veterinária e Zootecnia, Universidade de São Paulo (São Paulo-SPBrazil). All mice were kept at the Universidade Paulista bioterium (São PauloSP-Brazil) for at least 13 days to ambient.
The sanitary status was conventional: irradiated feed and bed (wood shaves),
filtered water and micro isolators under controlled temperature mean set to
24°C degrees, exclusive artificial illumination program set to 12-12h, turning on
at 7:00 am (local time).
The tests to access the individual immobility time were performed entirely in one
day at a separate room of the experimental cages; the animals were brought to
18
the new room and waited a minimum time of 30 minutes to adapt to the
ambient. The sequence order of the enrolled females also distributed the
animals in the four groups among the test queue.
2.3 The behavior tail suspension test
The tail suspension test (TST) was performed as described previously
(CASTAGNE et al., 2011; STERU et al., 1985). Briefly, mice were suspended
by the tail using a tape attached to a hook connected to a strain gauge.
Immobility time, defined as the mouse not struggling, was recorded in a single 6
min trial using a manual chronometer, registered after the end of each animal
assay.
The data collected by the immobility time in seconds was grouped in categories
to the set the random blocks by the depression-like behavior according to the
immobilization time: 1) from 1 to 100 seconds – mice are considered healthy; 2)
from 101 to 180 seconds – mice are considered intermediate and 3) higher than
180 – mice are classified as depressed-like behavior.
2.4. Experimental design
After the male and female were categorized, the animals were weighted and put
into individual micro isolator cages, where they received identification numbers
and an unique long-term color marks at hair coat back.
The females’ mice from each category were randomly sorted into four groups.
This way each group has the randomly homogeneous number of healthy,
intermediate and depressed-like females influence. The sorted numbers were
obtained by a validated live random numbers generator service (Random.org).
The second step was the choice of individuals to mate, intentionally done by the
opposite immobilization time of the male mice regarding to the female mice. For
example, the female with immobility time equal to 201 seconds (depressed-like)
mated with the male whose immobility time was 66 seconds (healthy). Thus, the
180 seconds-scored female mated with the 124 seconds-scored male (all
intermediate); the 154 seconds-scored female mated with the 147 secondsscored male; and the 105 seconds-scored female mated with the 161 secondsscored male. There was always a first and a second male options indication to
each female in case of recurrent absence of mating. The aim was to reach an
19
expected F1 balanced generation around a mean time of immobilization, thus
expected homogeneous depression-like behavior of F1 to control this parameter
for the evaluation of interference factors. After all, the body weight was
controlled by its distribution among the sorted groups.
To the mating, the female mice cycles were synchronized by the Lee-Boot and
Whitten effects (VAN DER LEE & BOOT, 1955; WHITTEN, 1956) using the
smell of the male urine, adsorbed by the bed wood shavings, put in the
respective female cage 3 days before the mating time. The controlled queue
mates were set by harem, setting by the maximum proportion of 1 male to 3
females. After mating, the females presented dried secreted liquids called
“vaginal plug” at early morning. The females presenting vaginal plug were put
into individual cage and this day was considered the gestational zero day (G0),
counting for the beginning of the following experiment. The procedure was
repeated after all mates finished. The male mice were submitted to euthanasia
after finishing their mating participation in the experiment.
In short, mice were mated with 93±10 days of age, the estrous cycle had 4 to 5
days and the gestational time was 21 days. The presence or absence of
delivery was recorded, the parental females were considered non-pregnant
(without delivery) 10 days after the expected 21th day of pregnancy. Fully
gestational mothers were considered by the presence of delivery. The result
from this procedure is inner model characteristic to next experiments and will be
mentioned further. After this experiment, all animals were enrolled to another
experiment presented elsewhere.
2.4 Statistical analysis
Validated random numbers were obtained by Random.org generator.
Descriptive analysis, Shapiro-Wilk normality test, Student’s T test, one-way
ANOVA, Kruskall-Wallis and Linear correlation had the calculations done by the
BioEstat 5.0 software (AYRES et al., 2007). Graphics were performed using
BioEstat 5.0 software (AYRES et al., 2007) and GraphPad Prism version 5.00
for
Windows,
Graphpad
Software,
San
Diego
California,
USA,
www.graphpad.com. The MS® Excel was used exclusively for data organization
and illustrative diagrams. The two tail p-value was considered to all tests and pvalues less than 0.05 were considered significant.
20
3. Results
The Tail Suspension Test showed 8 male mice out 9 classified as the
intermediate category; 1 out 9 as healthy category and 0 out 9 presented
occurrence of high immobility range to the depressed-like category; 23 out 28
female mice were classified by the intermediate category, 3 out 28 to the
depressed-like category and 2 out 28 to the healthy category. This means that
the female group was well distributed by the immobility time parameter (Graphic
1).
TST Immobility time (s)
250
200
150
100
50
0
Males
Females
n=9
n=28
Graphic 1. Tail Suspension Test immobility time. The dot-plot graphic shows the mean and
standard-deviation distribution of the depression-like behavior among the experimental mice.
Male and female mice were considered similar by the statistics (p-value>0.05; p-value=0.2870,
n=37, Student’s T test). Most of the animals were classified as the intermediate category (100180 seconds), the healthy category is set below 100 s., and the depressed-like behavior is set
above 180 s.
The animals of each category received a continuous number for auxiliary
identification. The randomization was performed and mice were distributed as
shown in the Figure 1. The block distribution of the previous tail suspension test
21
were homogeneous, without significant difference for both, female mice
(Kruskall-Wallis p-value>0.05; p-value=0.1829, H=4.8522) and male mice
(Kruskall-Wallis p-value>0.05; p-value=0.4386, H=2.7098) (Graphic 2). The
body weight of the animals were also checked and presented absence of
significant difference for its distribution (one-way ANOVA p-value>0.05; pvalue=0.2074, F=1.6318). A table containing the immobilization time with the
respective categories and the weight graphic are reported in the appendix B.
22
Figure 1. Schematic parental mice distribution to mates according to its time of immobility by the tail suspension test to randomized blocks set up. The aim of
this procedure is a refinement of the technique to control the vertical influence by the individual parental mates in a balance to expect as closest as possible
mean value homogeneity to F1 depression-like behavior born among the groups. (A) The mice had 13 days to adapt its physiology to the arriving bioterium
before to perform the test. (B) The mice were distributed by 3 categories according with each immobility time into Healthy; Intermediate; and Depressed
categories. (C) The animals of each category were randomized into 4 groups. The equivalent mean immobilization time among the treatment groups after all
were similar confirmed by statistical analysis with a p-value > 0.05.
23
Parental female depression-like behaviour
Immobilization time (s)
200
150
100
50
0
A
B
C
D
Groups
Parental male depression-like behaviour
Immobilization time (s)
200
150
100
50
0
A
B
C
D
Groups
Graphic 2. The distribution of immobility time among groups after the Tail Suspension Test
(female and male mice). The randomly sorted female groups were similar (p-value>0.05). The
ponderal male participation to groups was also similar (p-value>0.05).
The strategy of polarized mating is detailed by the Figure 2. The immobility time
from each gender by mating are highly correlated (p-value<0.01, pvalue=0.0021), as presented by the Graphic 3.
24
Crossed category sorting males mates for
each group
Males
Healthy
Intermediate
Females
Healthy
Intermediate
Depressed
Expected balanced distribution to
homogeneous F1 arround the mean
Mean±SD
142±28
Figure 2. The individual mates set up. The males from the healthy category mated with the
females from the depressed category and the Intermediate to each other. As no male mice were
found in the depressed category, the intermediate category was used instead. The mates
strategy objective was checked by correlation (p-value<0.05).
Graphic 3. Inverse correlation of male and female immobility time. Male mice were mated with
more than one female, according to the experimental queue. The choice of male to each female
was intentionally done to have inverse correlation, p-value<0.05; p-value=0.0021.
25
The female distribution by the immobility time was mainly of intermediate
depressed-like category. The randomization distributed all females in three
categories and four groups were performed. The mating selection method
reached the negative correlation by immobilization time. The correlation was
inversely proportional.
4. Discussion:
There is a continuous effort to discover human depression markers as
interference factors able to change treatment outputs, worldwide considered as
a social need. Some of the reported causes and markers are infection during
pregnancy, nutritional influence, social behavior of the couple, weight loss,
among others, but the high variability of real life situations implicate in
limitations of classical methodological solutions (HARRO, 2010), and this
suggests a strong clue towards individualized treatments. This work suggests a
refinement to the commonly used models and methodology, considering the
individualized control of the animals.
The technique refinement of random blocks is reported to reduce sorting bias,
reduce the width of confidence interval and the p-value in experiments
published in medical journals (KAHAN & MORRIS, 2012; KAHAN et al., 2015).
The refinement is part of the 3R acceptance model (Replace, Reduce and/or
Refine), is adherent to studies that refers to individual people and models,
leading to a possible reduction of the sample universe (N) (SCHIFFELERS et
al., 2012).
The overall immobility time of the enrolled animals was around the intermediate
category and this is a good sign of sample distribution to general experiments.
All mates were performed by 1:3 harems with the first and second male choice
for each female. The parental male mice distribution to mating was designed
according to the absence of a depressed-like category representative, but the
mating strategy done with the closest category reached significant negative
correlation.
This is important because the individual factor demonstrates high impact on the
depression subject (HARRO, 2010; DUCLOT et al. 2011). In this way, the
26
expected filial generation has the same inter-individual conditions among all
groups by their individually controlled parental mates. The observed effects to
the treatments are more reliable because considers the parental individuality
variations. The methodology of randomly blocks according to the depressionlike behavior can be extended not only to behavioral studies with pre- and/or
post-natal depression, but also to bio-molecular and immunity works.
5. Conclusion
The depression is currently in need of a solution worldwide. The random block
design applied individuality as a refinement method, increases the mice model
reliability and adheres to the 3R guidelines.
The individual immobilization time could be balanced in random blocks to four
groups of mating. This model set up was identified as potentially useful to
evaluate pre and post partum interference factors.
27
Appendix B
Auxiliary figure: immobility time by the TST for parental mating after female's
randomization.
Auxiliary graphic: at left is presented the homogeneous weight (g) for parental
distribution after females randomization by TSC, the difference was not
significant (one-way ANOVA p-value>0.05; p-value=0.2074, F=1.6318, n=28).
28
CAPÍTULO 3 – Exploratory study with simulated infection to perinatal murine
model for depression-like and sickness behaviors treated with diluted zinc.
Silvio Leite Monteiro da Silva, UNIP, IF Sudeste MG
Maria Martha Bernardi, UNIP
Leoni VillanoBonamin, UNIP
Abstract
Introduction: this is a perinatal exploratory study about behavior of BALB / C
mice under the effect of simulated infection by lippopolissacharide (LPS)
administered at the 9.5 days of gestation (100 µg/Kg i.p.) as a stress factor. The
treatment proposed is with diluted zinc (5cH, 30cH and 200cH dilutions).
Methods: the depression-like behavior and the sickness behavior were
assessed by tail suspension test (TST) and open field arena, respectively. The
body weight was registered weekly. All treatments were done in blind. The
parental generation was mated with polarized selection by immobility time in
TST for depression like behavior with completely randomized blocks.
Results/discussion: the depression like behavior was inversely correlated with
body weight at the second week of gestation. The offspring showed a significant
reduction of the TST immobility time comparing to the parental generation.
Parental LPS treatment and delivery interacted statistically with diluted zinc
treatment, as revealed by two-way ANOVA test. Sexual dimorphism was
observed in the filial generation. The mother’s treatment with Zincum metallicum
200cH changed the filial female sons. The identification of potentially interfering
sensitive parameters in this model has potential for the study of post-partum
depression homeopathic treatment.
Keywords: mice model; perinatal; simulated infection; lipopolysacharide (LPS);
depression-like behavior; sickness behavior; sex dimorphism; diluted zinc.
29
1. Introduction
This is a randomized blind exploratory perinatal study focused on mice behavior
after lipopolysacharide (LPS) exposition and high diluted zinc treatment during
pregnancy, with the possibility to affect the filial generation in the adulthood.
The perinatal setup is a classical model of human and animal interests. Some of
them are used in toxicology tests during pregnancy (AHMAD et al., 2013;
KIRSTEN et al., 2015), to mimetize mental diseases, like autism (KIRSTEN et
al., 2013). This model is important to other areas as veterinary because of
absence of drugs residues contamination of animal derived food such as eggs,
milk and meat (SATO et al., 2012; AMALCABURIO et al. 2009; ALMEIDA et al.,
2014; BONAMIN et al. 2012). The evaluation of the subjects of the parental
generation offers the opportunity to assess direct effects of the treatment, while
for the filial generation indirect vertical transmitted effects of their mother’s
treatments.
1.1. The simulated infection by lipopolysaccharide (LPS)
Inflammation triggered by a bacterial disease during the pregnancy is known to
perturb the mother-fetus complex (BIESMANS et al., 2013). Experimentally, the
simulation of an infection can be done with the injection of sterile purified
lipopolysaccharide (LPS) of Gram-negative bacteria wall, one of the main
components responsible for the classical inflammatory symptoms and
response. Lipopolysaccharide activates the immune system to release proinflammatory cytokines (interleukin-1, interleukin-6, tumor necrosis factoralpha, and others). LPS is known as agent able to stimulate monocytes,
neutrophils,
blood
platelets,
endothelial
cells,
and
first
activities
of
macrophages. A high dose of LPS can be lethal (PUJOL LOPEZ et al. 2015),
but a mild range dose affects central nervous system activity leading to
sickness behavior in many species (SALUK-JUSZCZAK & WACHOWICZ,
2005; ADEREM & ULEVITCH, 2000; AVITSUR et al., 1997). Systemic
inflammation or infection presents acute sickness symptoms, in which the
innate immune system is responsible for many of them (RIVEST, 2003;
MEDZHITOV & JANEWAY, 2000;). LPS-induced sickness behavior is generally
accompanied by a decrease in exploratory activity, social behavior, feeding
behavior, sexual behavior, induced anhedonia and poor learning and cognitive
functions (LARSON & DUNN, 2001).
30
The effects of maternal LPS exposure on the developing fetal brain have been
suggested to be mediated by the induction of pro-inflammatory cytokines within
the maternal circulation and placenta, reducing the born litter size (ASHDOWN
et al., 2006; CAI et al., 2000; URAKUBO et al., 2001). Cytokines can also
activate the hypothalamic–pituitary–adrenal (HPA) axis, leading to increased
glucocorticoid levels in peripheral blood, crossing the blood brain barrier and
influencing brain development processes (CRUDO et al. 2012). Some of those
effects can be strong enough to keep up to adulthood (KIRSTEN et al., 2012;
SCOTLAND & STABLES, 2011) and subject the offspring to mental disorders
(MEYER, 2013). Thus, LPS exposure in female mice can also be considered as
a source of stress.
On the other hand, cytokines produced after LPS exposure induces
metallothionein,
which
retains
zinc
and
induces
maternal
and
fetal
hypozincemia (COYLE et al., 2009). Coyle’s group reported that hypozincemia
induced by LPS leads to teratogenesis, and zinc supplementation prevented
some of the reproductive and offspring behavioral impairments (COYLE et al.,
2009; GALVÃO et al., 2005; CAREY et al. 2003). In a behavioral model of
autism, prenatal zinc administration appears to have a beneficial effect on the
prevention of autism (KIRSTEN et al., 2015). Indeed, in human studies have
previously investigated nutritional supplementation with zinc for autism
treatment (BILICI et al. 2004; AKHONDZADEH, 2004). However, according to
Theoharides and colleagues (THEOHARIDES, 2009), these studies are not
representative because they utilized a small number of subjects and did not
include appropriate controls, what keeps the subject controversial.
1.2 The zinc and diluted zinc effects
The zinc discovery as an essential trace element was convinced first in rodent
models than in humans, by previous empirical and observational studies.
Deprived
human
subjects
present
low
stature,
growth
retardation,
hypogonadism, mental lethargy and higher susceptibility to infectious diseases,
which will eventually cause death (PRASAD, 2001). In Life Sciences, zinc
functions are well known, as to be required in pregnancy and lactation, to
actiovation of the psycho-inflammatory hormonal response axes, neuronal and
reproductive development, embryogenesis, tissue growing and reparation,
some of them due to DNA translating by the zinc finger molecule
31
(DONANGELO & KING, 2012; SBARDELLA et al. 2012; CAULFIELD et al.,
1998). Excess of zinc due to over supplement or as environmental pollutant
intoxication change the metal distribution in the body and brain but low zinc
availability can be related to mental disorders (YASUDA et al., 2013;
CRADDOCK et al., 2012; WHITTLE, 2009; ANDREWS, 1990). The zinc is well
known by its anti-oxidant properties: as a supplement, it improves
immunological
response,
improves
the
depression
behavior
and
is
recommended to prevent deaths from certain diseases in both pregnant woman
and child presenting diarrhea (intestinal inflammation). Also, in nutrients
deprived populations, this fact made zinc as one of the 20 program
recommendations by WHO (LIBERATO et al., 2015; OSENDARP et al., 2001).
Strong observational reports are used by the empirical traditional knowledge to
link zinc to ultra dilution phenomena (MICB, 1895; ALLEN, 1880; HERING,
1872; HAHNEMANN, 1845). The treatment with high dilutions of zinc interferes
in inflammation of nervous system, depression and behavior (BOLOGNANI,
2011; SAELENS, 1997; BADULICI et al., 1994; JACK, 1984; GIBSON, 1967),
as do other high diluted compounds to behavior and psychiatry (BELLAVITE, et
al., 2012; MARZOTTO, et al., 2012; DAVIDSON, 1994), different from the
placebo effect (TEIXEIRA et al., 2010).
The reports from previous toxicology experiments about diluted zinc in human
subjects, repeated many times in the past, presented tiredness, prostration,
depression and inflammation effects, among others. The interference of this
medicine to the subject can increase or decrease the symptoms according to
the concentration and administration frequency over the time; the psychological
and somatic data collecting of such experiments with highly diluted medicines
are called proving (TEIXEIRA, 2009), they are systematically organized in
databases traditionally called material medica (VAN WIJK & ALBRECHT,
2007), and are an empirical paradigm of knowledge with pure natural
phenomena observations (SCHMIDT, 2014). The individuals registered effects
are used to counteract the same symptoms to be treated cancelling, reducing or
increasing. The diluted zinc preparation method for the release of medicinal
effects is described by the Farmacopéia Brasileira (BRASIL, 2011). Typical
dilutions are reported in the proper centesimal Hahnemannian (cH) scale of
5cH, 30cH and 200cH (BELAVITTE et al., 2014). Drugs are named in Latin
32
language and the original oxide zinc transformed by the high dilution process is
called Zincum metallicum (BRASIL, 2011).
1.3 The depression-like and sickness behaviors
The behavior is strategic to test because evaluates the psychopathology: it is
the result of neuronal and physiological crosstalk complex responses over a
context (ROSENBLAT et al., 2014). Mice can be objectively assessed by
behavioral tests and there are partial and limited animal to human
correspondence of effects (ENNACEUR, 2014;PRUT & BELZUNG, 2003).
The depression-like behavior is classically validated after 30 years of use with
antidepressants drugs. It measures the immobility time by the tail suspension
test in mice. The paradigm is the continuous inescapable situation during 6
minutes hanged, as more time the mouse give up and keeps immobile, as more
express the depression-like behavior. The spontaneous tail suspension test
exposition show results that occur three behavioral profiles: depressed-like,
intermediate and healthy subjects. These parental and filial behaviors are linked
to the possibility of vertical transmission in humans and mice (CRYAN et al
2005; HAMMERTON, et al. 2015; MARS, et al. 2015), and the live weight is
reported as strong predictor to humans depression (MARS et al., 2013).
Depression treatments with ultra dilutions are a hot topic and a social demand
(MACÍAS-CORTÉS et al., 2015; OSTERMANN et al., 2015; Gadassi, R.,
&Rafaeli, E. (2015); OLIOSO et al., 2014; VIKSVEEN & RELTON, 2014;
VILHENA, 2014; ADLER et al., 2013; IJICHI et al 2013; BAWDEN, 2012;
LONGACRE et al., 2012; ADLER et al. 2011; VAN DE VELDE et al., 2010).
The sickness behavior is evaluated by the walking space-temporal parameters
at the open field arena. The paradigm to explore the new ambient counts the
walking at the mapped squares on the device ground is interpreted with the
immobility time. As high the first counting as high the animal walked, it is
expected that tired animals walk less and stay more time immobile than the
counterparts, as reported to more ultra dilutions effects (MARZOTTO et al,
2012; BOUSTA, D. et al., 2001).
33
1.4 Grooming, defecation and stand up
The time spend grooming and the stand up position counting are markers to
their alertness to the ambient in the presence or absence of explore and the
danger. The full and relaxed grooming is a signal that shows absent of
perception of danger to the animal, but the incomplete and fast grooming
indicates anxiety, both processes are directly related to stress and GABA
(KALUEFF & TUOHIMAA, 2005). The stand up demonstrates their interest to
explore or alertness to the dangerous as hunted behavior. The defecation
shows the emotionality of the animal, as more bolus identified, as more
expression of emotionality presented (HONG et al. 2014; KALUEFF &
TUOHIMAA, 2005).
Herein, a treatment of pregnant mice is proposed to evaluate its effects to both
generations, using behavioral tests. The maternal treatment with diluted zinc
was evaluated as a possible transgenerational factor to reverse the effects of
prenatal treatment with 100 µg/Kg i.p. of LPS administered on between the 9th
and the 10th days of pregnancy. Thus, the open field behavior and the tail
suspension test were observed in the filial mice generation (F1), during the
adulthood. The maternal treatment with diluted zinc was performed from the
mating day up to 10 days after the delivery, total of 31 days.
1.5 Objectives
The objective of this work is to explore behavioral aspects of the perinatal
model under maternal simulated infectionby LPS and the possibility of influence
by the treatment with Zincum metallicum. The specific objectives are: to
simulate the infection by lipopolyssacharide (LPS) in pregnant parental female
mice; to treat the parental female with diluted zinc with the control, 200cH, 30cH
and 5cH potencies; to evaluate the animals by the tail suspension test, open
field arena and weight.
34
2. Material and methods
2.1Ethics
The participation of mice in this research was performed in accordance to
international protocols. The experiment was previously submitted and approved
by the CEUA-UNIP ethical committee in April, 17th 2013, under the protocol
156/13. All efforts were done to minimize animal suffering and discomfort.
2.2 Experimental design
Thirty seven mice were enrolled: 9 males and 28 female BALB/C mice lineage
ICB-USP originated from Faculdade de Medicina Veterinária e Zootecnia,
Universidade de São Paulo (São Paulo-SP-Brazil) were kept at the
Universidade Paulista experimental animal house (São Paulo-SP-Brazil). The
28 females were distributed into four balanced groups (n=7 each) by the
immobility time from the tail suspension test in a random block set up as
described elsewhere (MONTEIRO DA SILVA et al., [capítulo 2]). Each group
with the 7 parental females hat 5 to 6 animals with the LPS simulated infection
and 1 or 2 not. All animals of each group received one of the four diluted zinc
treatment: control, 200cH, 30cH or 5cH, see figure 1. The pups originated from
these experiment were enrolled to the next experiment with all born 35 filial
mice, 16 males and 19 females, see figure 2. The sanitary status is
conventional, it was used irradiated feed and bed (wood shaves), filtered water
and micro isolators under controlled temperature mean set to 24°C degrees,
exclusive artificial illumination program set to 12-12h, turning on at 7:00 am
(local time). All animals waited 13 days for to begin any experimental
procedures for the animals adapt and stabilize their physiology to the new
house.
35
Figure 1.Time line of the experimental steps and main procedures. Legend: Zm = Zincum metallicum; G# = gestational day; OF = open field arena; TST = tail
suspension test; F1 = filial; M = males; F = females.
Parental females
n=28
LPS +
n=20
Pf Control
n=4
Pf Zm 200cH
n=5
No LPS
n=8
Pf Zm 30cH
n=6
Pf Zm 5cH
n=5
Pf Zm Control
n=3
Pf Zm 200cH
n=2
Pf Zm 30cH
n=1
Pf Zm 5cH
n=2
F1m ♂
n=2
F1m ♂
n=5
F1m ♂
n=3
F1m ♂
n=0
F1m ♂
n=3
F1m ♂
n=3
F1m ♂
n=0
F1m ♂
n=0
F1f ♀
n=2
F1f ♀
n=4
F1f ♀
n=2
F1f ♀
n=2
F1f ♀
n=3
F1f ♀
n=4
F1f ♀
n=0
F1f ♀
n=2
Figure 2. Pups born according to the model characteristic. Sex and sample universe shown by parental female treatments. Legend: :Zm = Zincum
metallicum; Pf = parental females; LPS = simulated infection group by lippopolissacharide.
36
2.3 The tail suspension tests and open field arena
The depression-like behavior was evaluated by the tail suspension test
immobilization time parameter after the sickness behavior evaluation by time-space
walking parameters in open field arena. Complementary behavior parameters as
grooming, defecation, and stand up were controlled to all animals.
Open field arena
Tail suspension test
Walking area
Immobilization time
Immobilization time
Grooming time
Stand up position
Defecation bolus
Table 1. Behavior tests in order of performance and observational parameters.
The tail suspension test is done at an opaque box with a hook on the top and the test
begins when the mouse is hanged fixed by tail with a tape. The only opening side is
to the evaluator that measures the total time of immobilization in contrast to
movement when the mouse is trying to escape from the situation. The total time of 6
minutes hanged has the measure of the immobilization time during 5 minutes after
the first minute to pre-exposition to suspension without counting, both times were
controlled by manual chronometry. This test was performed with the parental female
mice, the first time to the previous experiment for the random blocks set up, then to
evaluate the treatments of the first and second experiments. Both previous and
current experiment data by treatment are shown here.
The open field arena performed with a device with delimited area by its ground and
begins with the mouse put in the middle. This case the arena had opaque walls and
measures 40cm of diameter. The walking area counting is considered after the last
footpad is removed from the delimited area. The stand position and defecation is
observed by events occurrence and fecal bolus eliminated. The time spent
grooming; the immobile time and the total time of 6 minutes are controlled by manual
individual chronographs. The evaluations of open field arena were video recorded.
After each animal test the devices were cleaned to remove the smell from the
previous animal.
The tests were performed one by each day in a separate room from the experimental
cages; the animals were brought to a new room and waited a minimum time of 30
minutes for to ambient. The parameters were directly observed by two persons with
37
the use of manual counters and chronometers, registered after the end of each
animal assay.
2.3.1 Parental female generation (first experiment)
All the animals came from the previous experiment (MONTEIRO DA SILVA et al.,
2015a) after the proof of mate by the presence of vaginal plug. The individual
treatment by the Zincum metallicumbegan at the day when the mate is detected,
keep during the 21 days of pregnancy and finished after 10 days of lactation, total of
31 days. The administration was continuous by the only drinking water source
exclusive to the mated females. Each labeled bottle received 250mL of drinking
water and then 100microL from the respective treatment flask with proper pipette
and disposable tips. Rigorous control of the water containers and tips is cared to
avoid cross-contamination between the groups. The 7 females from each group
received the respective treatment, total of 28 mated female, and one of these groups
were the placebo controlled.
The Zincum metallicum matrix was prepared from the zinc oxide according to the
Brazilian Pharmacopeia. The doses were prepared in liquid from the powder matrix
4cH up to the 5cH, 30cH and 200cH and the vehicle lactose at the 5cH as control.
The four identical flasks were relabeled in codes and the original identification was
kept with a staff personnel unrelated directly to the experiment; it was revealed only
after the statistical analysis, so the experiment and data analysis ran completely in
blind.
The LPS is single administrated by the intraperitoneal way at the dose of 100
micrograms/Kg between the 9th and 10th gestational day (G9.5). The LPS solution is
stored in -80°C freezer at special glass containers each one with 1.2 mL of to the
concentration of 50 microgram of LPS / mL of saline. The 5 to 6 animals received
LPS out 7 for each group.
Fully gestational mothers were considered by the presence of delivery. The absence
of delivery was evaluated together with the factor LPS/Delivery since LPS can affect
litter size and reproductive performance. The weaning after 21 lactating days
separated the mothers from their pups. Mice from the parental female generation
38
belonging to the same treatment group were put back together for recovering. A hard
paper card roll was put inside the cage for ambient enrichment at this time. The
behavior tests as described previously were performed from the 146 to the 149 days
of age. The evaluation of the depression-like behavior represented by the immobility
time during the post natal period was compared to the pre natal period, observed in
a previous experiment, considering the interaction factors. The weight was controlled
weekly and was evaluated by correlation with the immobility time. The number of
pups, abnormalities and their gender were recorded and the animals forwarded to
the next experiment by the same authors.
2.3.2 Filial generation after adulthood (second experiment)
The born puppies were feed during the first 10 days exclusively with the mother’s
milk, which supplies all requirements, including water source. The treated mother’s
drinking water was still available at the cage but unavailable to the pups up to the
10th day of life. This is possible because the pups barely walks and cannot reach the
mother’s treated drinking water hanged by the middle of the cage. They developed
since the uterine conception to the 10th day of age with their mother having the oral
treatments. The weaning happened by the 21 days of lactation, filial males and
females were uniquely marked to track their respective mother. Then, they were
divided by sex and treatment groups; a hard paper card roll was put into the cage for
ambient enrichment at this time. They were kept up to the age of 71±10 days for filial
males and 74±19 days for filial females, when the behavior tests were performed
with one week interval from each sex, as described previously. After the behavior
tests all filial mice were enrolled to another experiment, which is presented
elsewhere.
2.4 Statistical analysis
Descriptive analysis, Linear correlation, matrix correlation, Shapiro-Wilk normality
test, Student’s T-test, Mann-Whitney, one/two/three-way repeated measures by
ANOVA, Bonferroni’s or T-test post-tests, Kruskall-Wallis (K-W), and median test
were performed as described by the results, the calculations were done by the
39
BioEstat 5.0 software (AYRES et al., 2007). Graphics were performed using BioEstat
5.0 software (AYRES et al., 2007) and GraphPad Prism version 5.00 for Windows,
Graphpad Software, San Diego California, USA, www.graphpad.com. Two tail pvalue was considered to all tests and p-values less than 0.05 were considered
significant.
3. Results
3.1. Comparison between maternal and filial immobility time
Independent to the simulated infection by LPS or Zincum metallicum treatment
received, the depression-like behavior to the F1 generation was evaluated and
compared to the parental results, as shown elsewhere (MONTEIRO DA SILVA et al.,
2015a). The immobility time overall of the all filial generation was significantly lower
in relation to all parental generation time; the filial male was lower than the parental
male time; and the filial female was lower than parental female, all detected by oneway ANOVA, p-value<0.05; p-value<0.0001, F=13.5004, Figure 3.
Immobility time by the tail suspension test
with filial and parental generations by sex
TSC Immobility time (s)
250
Filial males and
females, n=35
Filial males, n=16
200
Filial females, n=19
Parental males
and females, n=37
150
Parental males, n=9
100
Parental females
before mates, n=28
50
Parental female
after weaning, n=25
0
F1 F1m F1f
Pm+f Pm Pf<m Pf>d
Generational groups
Mean±SD
one-way ANOVA, p-value<0.001;
p-value<0.0001, F=13.5004.
Figure 3.Immobilization time at tail suspension test. Values shown represents mean, SD and
max./min. range presented in box-plots for all the filial and generational sub-groups. The generational
data points were highly significantly different between them (one-way ANOVA, *** p-value<0.001, pvalue<0.00001).
40
All filial mice (males and females) presented lower values of immobilization time at
tail suspension test, regarding to the parental ones. All filial mice males were similar
to the filial females (p-value>0.05, p-value=ns, T test LSD post-test), the same result
occurred to the all parental male mice compared to the parental female. The filial
males group were significantly different with lower values in comparison to the male
parental group (p-value<0.05, p-value=0.0021, T test LSD post-test), as the F1
female group was different from the female parental group (p-value<0.05, pvalue<0.001, T test LSD post-test). This means that the F1 were healthier than the
parental group.
To perform the statistical analysis, the filial interference factor were: a) mother’s
simulated infection by lipopolyssacharide (LPS- or LPS+); b) the sex of the filial
generation; c) mother’s treatment of Zincum metallicum (Control, Zm 200cH, Zm
30cH, Zm 5cH).
Theoretical parameters
Parametertested
Depression-like behavior
TST immobility time (s)
Increased
Live weight (g)
Reduced
Table 2. Resume of the theoretical parameters from literature. The expression of depression-like
behavior is increased as the time of immobility by the tail suspension test increases. The reduction of
weight is a predictable parameter to depression in humans and was found to this mice model.
3.2 Parental female generation
3.2.1 The parental female depression-like behavior
The depression-like behavior is presented in details in the graphic 1 and in the table
3. The three-way ANOVA analysis did not identify differences among the factor
treatment, factor LPS/Delivery, and/or factor time of TSC. The three-way ANOVA
identified significant interaction between the factor Zincum metallicum treatments
and the factor LPS/delivery, p-value<0.05 (p-value=0.0124, F=5.079). The factor Zm
treatments and the factor previous/later immobility time did not interact p-value>0.05
(p-value=0.5137, F=0.8859). The factor LPS/delivery and the factor previous/later
immobility time did not interact p-value>0.05 (p-value=0.5741, F=0.7065).
41
Zincum metallicum treatments effect at parental female
Tail Suspension Test before and after to simulated infection
and/or delivery status to the control group
250
Control - 1st TSC n=7
Immobility time (s)
Control - 2nd TSC n=7
200
150
100
50
LPS-D-
LPS+D-
LPS-D+
LPS+D+
n=2
n=6
n=4
n=2
Groups
and/or delivery status to the control group
Immobility time (s)
250
Zm200C - 1st TSC n=7
Zm200C - 2nd TSC n=5
200
150
100
50
LPS-D-
LPS+D-
n=0
n=4
LPS-D+
n=4
Groups
LPS+D+
n=6
42
and/or delivery status to the Zm 30C group
250
Zm30C - 1st TSC n=7
Immobility time (s)
Zm30C - 2nd TSC n=5
200
150
100
50
LPS-D-
LPS+D-
LPS-D+
LPS+D+
n=2
n=7
n=0
n=3
Groups
and/or delivery status to the Zm 5C group
Immobility time (s)
250
Zm5C - 1st TSC n=7
Zm5C - 2nd TSC n=6
200
150
100
50
LPS-D-
LPS+D-
LPS-D+
LPS+D+
n=2
n=8
n=2
n=1
Groups
Graphic 1. The filled circles and full lines represents the parental female mice immobilization time effects of the
Zincum metallicum treatments influence (graphics A, B, C and D).The modelcharacteristic LPS and delivery are
represented by each X axis. The open circles and line represents the spontaneous immobility time prior to
mate.
43
The Control group represented in red in Graphic 1 shows the depression-like
behavior in pre and post-natal period and the effect of LPS. The Zincum metallicum
treatments factor interacts with the LPS/Delivery factor by the three-way ANOVA.
The pre and post natal factor does not interact significantly with the Zincum
metallicum treatments neither with the LPS/delivery factor.
1st TST
immobile
Control
Zm 200cH
Zm 30cH
Zm 5cH
Total
Mean±SD;
dot-plot (n=1)
LPS-D180, n=1
n=0
129, n=1
199, n=1
169±36, n=3
LPS+D156±44, n=3
169±0, n=2
124±31, n=4
146±9, n=3
145±29, n=13
LPS-D+
129±24, n=2
169±8, n=2
n=0
99, n=1
139±33, n=5
LPS+D+
154, n=1
141±36, n=3
163±26, n=2
128, n=1
147±27, n=7
Total
152±32, n=7
157±26, n=7
135±31, n=7
157±26, n=7
147±29, n=28
2nd TST
immobile
Mean±SD;
dot-plot (n=1)
LPS-D135, n=1
n=0
173, n=1
81, n=1
130±46, n=3
LPS+D108±25, n=3
141±30, n=2
99±37, n=3
113±15, n=4
113±26, n=12
LPS-D+
167±30, n=2
125±35, n=2
n=0
115, n=1
146±31, n=5
LPS+D+
115, n=1
180±38, n=3
117, n=1
n=0
154±44, n=5
Total
130±33, n=7
153±39, n=7
117±41, n=5
113±24, n=6
130±36, n=25
Table 3. Tail suspension tests immobility time (s) in two times (before mating and after weaning).
The ratio between the second and the first immobility time measured by the Tail
Suspension Test considers how much the value changed to the central value, thus,
ratio = 1 means no change, less than 1 means that the depressed-like behavior was
reduced, and more than 1 means that is increased.
nd
st
2 /1 TST Ratio
Control
Zm 200cH
Zm 30cH
Zm 5cH
LPS-D0.75
1.34
0.41
LPS+D0.69
0.83
0.80
0.77
LPS-D+
1.29
0.74
1.16
LPS+D+
0.75
1.27
0.72
Table 4. Tail Suspension test immobility time (s) ratio after/before to parental females under LPS
and/or Delivery (D) conditions among Zm treatments.
44
Qualitatively, the interpretation of the TST ratio indicates that the control group
shows that the LPS increased the immobility time while the delivery reduced, the
additive effect of LPS and delivery (LPS+D+) slightly reduced the immobility time,
which is curiously the same effect seen when both conditions were absent (LPS-D-).
Comparing the absent of LPS and Delivery conditions, the Zm30cH doubled the ratio
in comparison to the control and the Zm5cH keep it unchanged. The LPS reduced by
the half the time seen in mice submitted to all Zm treatments, in comparison to the
control. The delivery effect on the immobility time was neutralized by the Zm200cH
but increased, in non significant way, when the LPS was added.
3.2.2 Suspension immobility time and weight
The evaluation of the immobility time previous to mates was correlated to the body
weight. There were 5 weight (g) measurements: the first by the tail suspension test
day previous to mates, the second by the mating day, the others then weekly, up to
the 3rd week. The matrix correlation test identified significant correlations to the
immobility time and weight factors. The global data with all Zincum metallicum
groups show that the first immobility time was correlated with significance to the
mating day weight (p-value<0.05; p-value=0.0462, R2=0.1617, n=25) before the
treatments and correlated with highly significance to the 2nd week weight (pvalue<0.05; p-value=0.0041, R2=0.3055, n=25) as shown in graphic 2A-B. For both,
there were inverse correlations as reported to human markers: as high as the
depression-like behavior is as more the observed week weight was reduced (MARS
et al., 2013), what can be observed by the first weight evaluation. The LPS given to 5
or 6 out 7 females may expose their effect to reduce weight by the second week and
changed the significance level.
The first immobility time was not correlated to the second with border p-value (pvalue>0.05; p-value=0.0537; R2=1523; n=25). Three animals’ data had to be
censured due to missing values to be possible the matrix correlation.
The analysis by Zincum metallicum (Zm) groups in the 1st tail suspension test and
2nd week of pregnancy revealed that there were significant inverse correlation to the
control (p-value<0.05; p-value=0.0025, R2=0.8619, n=7) and Zm 200cH group (p-
45
value<0.05; p-value=0.0414, R2=0.5981, n=7) and the curve fit is not linear (graphic
2C-D). There is no significant correlation between Zm 30cH (p-value<0.05; pvalue=0.3737, R2=0.2011, n=6)
and Zm 5cH in the 2nd week (p-value>0.05; p-
value=0.0895, R2=0.4690, n=7), but there is significance in relation to the Zm 5cH 1st
week (p-value<0.05; p-value=0.0029, R2=0.8543, n=7). In this case, the curve fit is
not perfectly linear (graphic 2E-F-G).
2nd gestationalweek
Live weight (g)
Live weight (g)
Previousto mate week
TST Immobility time
(s)
A
TST Immobility time
(s)
TST Immobility time
(s)
D
G
Zm 30cH 2nd gestationalweek
Live weight (g)
Live weight (g)
F
Zm 200cH 2nd gestationalweek
Live weight (g)
Control 2nd gestationalweek
C
TST Immobility time
(s)
B
TST Immobility time
(s)
E
TST Immobility time
(s)
TST Immobility time
(s)
Graphic 2.Tail suspension test immobility time (s) and live weight (g) correlation as marker for the
depression-like behavior. (A) Inverse correlation to all parental female immobility time (s) previous to
2
the mates and the live weight (g) at the mating day, p-value<0.05; p-value=0.0462, R =0.1617, n=25.
46
Curves are best fit to linear correlation. (B) Inverse correlation to all parental female immobility time
(s) previous to the mates and the live weight (g) at the 2
nd
gestational week, p-value<0.05; p-
2
value=0.0041, R =0.3055, n=25. (C) Parental female from Control group inverse correlation at the 2
nd
2
gestational week p-value<0.05; p-value=0.0025, R =0.8619, n=7 (D) Parental female from Zm 200cH
group inverse correlation to 2
nd
2
gestational week p-value<0.05; p-value=0.0414, R =0.5981, n=7(E)
Parental female from Zm 30cH group with absent of correlation to 2
nd
gestational week p-value>0.05;
2
p-value=0.3737, R =0.2011, n=6 (F) Parental female from Zm 5cH group with absent of correlation to
nd
2
2
week p-value>0.05; p-value=0.0895, R =0.4690, n=7 (G) Parental female from Zm 5cH inverse
st
2
correlation to 1 week p-value<0.05; p-value=0.0029, R =0.8543, n=7.
All animals were assumed to be pregnant after the mating with the presence of
vaginal plug. The expected weight gain due to the beginning of pregnancy, from the
2nd week to the 3rd one, is remarkable. So, there is a direct impact of the depressionlike status on the weight in the 2nd week, what means that the depressed animals
gain less weight and may be not pregnant. It is noticeable that the weight may be
influenced due to method of mating: the 1 male/3 female harem, set up synchronized
by the Lee-Boot and Whitten effects (VAN DER LEE & BOOT, 1955; WHITTEN,
1956).
3.2.3 The parental female sickness behavior
The expression of sickness behavior is measured in open field arena. Few walking
squares and high time of immobility are the main marker of this sickness (Graphic 3
and table 5). The pregnancy with delivery or the simulated infection does not change
this pattern. The 3-way ANOVA could not detect significant difference to the open
field arena walking parameters (p-value>0.05) and the Mann-Whitney could not
detect significant differences to the open field arena immobility time (M-W, pvalue>0.05; p-value=0.0574, U=16.50).
Despite the statistical analysis accepted the nullity hypothesis, the reduced time of
immobility is compatible with the increase of pregnant mice exposed to LPS and
treated with Zincum metallicum in all tested dilutions. This fact shows the Zincum
metallicum as a reversion factor of the sickness behavior within this population. Such
effects are not identified in females without delivery, independent of the presence or
absence of LPS injection.
47
Zincum metallicum Treatments Effect at Parental Female Open
Field to LPS Simulated Infection and/or Delivery Status
250
Zm 5cH n=7
Zm 30cH n=7
Zm 200cH n=7
Control n=7
OF Walking
200
150
Mean±SD
and dot-plot
100
50
0
LPS-D-
LPS+D-
n=3
n=13
LPS-D+
n=5
LPS+D+
LPS=Simulated
infection
D=Delivery
"-" absent
"+" present
n=7
Model condition
Zincum metallicum Treatments Effect at Parental Female Open
Field to LPS Simulated Infection and/or Delivery Status
210
Zm 5cH n=7
Zm 30cH n=7
Zm 200cH n=7
Control n=7
OF Immobility Time (s)
205
20
Median
and dot-plot
15
LPS=Simulated
infection
D=Delivery
"-" absent
"+" present
10
5
0
LPS-D-
n=3
LPS+D-
n=13
LPS-D+
LPS+D+
n=5
n=7
Model condition
Graphic 3. The sickness behavior effects evaluated at the open field arena by space-temporal
parameters. The expression of sickness behavior is observed by walking reduction (A) and high time
of immobility (B). Legend: LPS- or LPS+ refers to the absent of presence respectively to LPS to
simulate an infection during the 9.5 days of pregnancy. The D- or D+ refers respectively to the
presence of delivery. The point plot represents the mean and median respectively or single value.
48
OF Walking
Mean±Std.
Deviation
dot-plot
LPS-DLPS+DLPS-D+
LPS+D+
Total
OF Immobile
time Median
(max.-min.)
dot-plot
LPS-D-
Control
126, n=1
97±66, n=3
97±80, n=2
80, n=1
110±56, n=7
Zm 200cH
Zm 30cH
n=0
113±4, n=2
127±3, n=2
169±40, n=4
141±36, n=7
133, n=1
106±21, n=4
n=0
124±74, n=2
115±36, n=7
Total
Zm 5cH
123, n=1
111±36, n=4
139, n=1
213, n=1
131±45, n=7
127±5, n=3
106±34, n=13
117±44, n=5
161±49, n=7
124±43, n=28
3 (3-55), n=3 3.5 (3-4), n=2
4.5 (0-6), n=4
1 (0-2), n=4
3 (2-4), n=3
16.5 (5-28),
4 (0-8), n=2
n=0
7, n=1 3 (0-55), n=13
LPS+Dn=2
LPS-D+
1, n=1
3 (1-4), n=4
0 (0-0), n=2
207, n=1
7 (0-28), n=5
LPS+D+
3 (3-55), n=3 3.5 (3-4), n=2
4.5 (0-6), n=4
1 (0-2), n=4 1 (0-207), n=7
Total
3(1-55), n=7
3(0-8), n=7
4(0-6), n=7
2(0-207), n=7 3(0-207), n=28
Table 5.The detailed data of the sickness behavior effects evaluated at the open field device by
space-temporal parameters.
3.2.4 The parental female grooming, stand up and defecation parameters
The data of the complementar paramenters from the open field arena are presented
by the graphic 4 and table 6.
The parental female stand up at Open Field under effects of
simulated infection and/or gestational status
influenced by the Zincum metallicum
20
Zm 5cH n=7
Zm 30cH n=7
Zm 200cH n=6
Control n=7
Stand up
15
10
Median
and dot-plot
5
0
LPS-D-
n=3
LPS+D-
LPS-D+
LPS+D+
n=5
n=7
n=12
Groups
LPS=Simulated
infection
D=Delivery
"-" absent
"+" present
49
The parental female grooming at Open Field under effects of
Grooming time (s)
15
Zm 5cH n=7
Zm 30cH n=7
Zm 200cH n=7
Control n=7
10
Mean±SD
and dot-plot
5
0
LPS-D-
n=3
LPS+D-
LPS-D+
LPS+D+
n=5
n=7
n=13
LPS=Simulated
infection
D=Delivery
"-" absent
"+" present
Groups
The parental female defecation at Open Field under effects of
8
Zm 5cH n=7
Zm 30cH n=7
Zm 200cH n=7
Control n=7
Defecation
6
4
Mean±SD
and dot-plot
2
0
LPS-D-
LPS+D-
n=3
n=13
LPS-D+
n=5
LPS+D+
LPS=Simulated
infection
D=Delivery
"-" absent
"+" present
n=7
Groups
Graphic 4. Grooming time, defecation, stand up for the parental females. The grooming time and
the standing up of the mothers that had simulates infection and had puppies under the treatment of
Zm 200cH is noticeable. Legend: LPS- or LPS+ = absent or presence of mother’s simulated infection
by LPS; D- or D+ = absence or presence of mother’s delivery; Zm = Zincum metallicum.
50
OF Grooming
Mean±SD/dotplot
LPS-D-
Control
Zm 200cH
Zm 30cH
Zm 5cH
3.0, n=1
4.3±3.5, n=3
n=0
2.5±0.7, n=2
0.0, n=1
6.0±4.5, n=4
2.0, n=1
5.5±2.4, n=4
2.5±3.5, n=2
11.0, n=1
2.0±2.8, n=2
4.7±1.5, n=3
n=0
3.5±4.9, n=2
4.0, n=1
0.0, n=1
4.6±3.9, n=7
3.3±2.0, n=7
4.4±4.4, n=7
4.0±2.8, n=7
4.1±3.2,
n=28
6, n=1
3±3, n=3
4±1, n=2
3, n=1
n=0
3±1, n=2
2±1, n=2
3±1, n=3
2, n=1
2±2, n=4
n=0
4±1, n=2
3, n=1
3±2, n=4
0, n=1
1, n=1
4±2, n=3
7±2, n=13
2±2, n=5
3±1, n=7
Total
4±2, n=7
2±1, n=7
2±1, n=7
2±2, n=7
3±2, n=28
OF Stand up
Median/dot-plot
LPS-DLPS+D-
11, n=1
5 (1-8), n=3
n=0
8 (6-10), n=2
5, n=1
6 (2-8), n=4
2, n=1
5 (0-18), n=4
LPS-D+
8 (3-13), n=2
3, n=1
n=0
9, n=1
LPS+D+
4, n=1
18 (9-30),
n=3
2 (1-3), n=2
1, n=1
5 (2-11), n=3
6 (0-18),
n=13
6, (3-13),
n=4
4 (1-30), n=7
5 (1-13), n=7
9 (3-30), n=6
5 (1-8), n=7
2 (0-18), n=7
LPS+DLPS-D+
LPS+D+
Total
OF Defecation
Mean±SD/dotplot
LPS-DLPS+DLPS-D+
LPS+D+
Total
Total
1.7±1.5, n=3
4.9±3.2,
n=13
2.6±2.4, n=5
4.6±4.0, n=7
5 (0-30),
n=27
Table 6. Detailed data of grooming time, defecation, stand up for the parental female.
3.2.5 The delivery
The puppies born were forwarded to the next experimentand are shown in the
figure 3. The birth was low particularly to parental females treated with the
Zincum metallicum dilutions of Zm 30cH and Zm 5cH. No male mice were born
to the parental female that received the LPS and had the Zm 5cH treatment, no
pups were born to the parental female that did not received LPS and had the
Zm 30cH treatment.
51
3.3 The filial generation at adulthood
3.3.1 The filial immobility time by the tail suspension test
Female mice presented decrease in TST immobility time, in comparison to the
control group, mainly those exposed to LPS during the intra-uterine time.
However, those born from mothers treated with Zm200cH had this effect
reverted, in order to present values comparable to the male ones (Median testFisher, p-value<0.05; p-value=0.0152; Median=94). The males born from
mothers treated with Zm 200cH had similar immobility time to the control group.
This is shown in Graphic 6 and detailed by Table 7.
The F1 male and female immobility time at the Tail Suspension Test
by parental simulated infection and sex under the effects of Zincum metallicum
TST Immobility time (s)
200
Control n=10
Zm 200cH n=16
150
*
100
50
Control x Zm200cH
2-way ANOVA,
p-value<0.05;
p-value=0.0319
0
F1m LPS-
n=6
F1m LPS+
n=7
F1f LPS-
n=7
F1f LPS+
n=6
Groups
Graphic 6. Filial immobility time observed by the Tail Suspension Test. The perinatal LPS
exposition during their development and thepup sex influenced the effect of Zincum metallicum
200cH mother treatment. Legend: *Median test-Fisher, p-value<0.05; p-value=0.0152; F1m =
Filial male; F1f = filial female; LPS- = absent of mother’s simulated infection by LPS; LPS+ =
presence of mother’s simulated infection by LPS; Zm = Zincum metallicum.
52
TST Immobile
time Mean±std.
deviation
dot-plot (for n=1)
F1m LPSF1m LPS+
F1f LPS-
Control
Zm 200cH
Zm 30cH
94±9, n=3
74, n=1
128±17, n=3
143±5, n=2
95±33, n=3
81±35, n=3
81±19, n=4
* 77±19,
n=4
83±25, n=16
n=0
122±14, n=3
n=0
235±48, n=2
Zm 5cH
total
n=0
n=0
64±4.5, n=2
103±20, n=2
94±21, n=6
92±38, n=10
93±31, n=9
107±36,
F1f LPS+
n=10
97±33,
110±35,
115±17, n=5
99±48, n=4
Total
n=35
n=10
Table 7.The immobility time at TST of filial generation by sex and maternal simulated infection
under the effect of all treatment groups. The Zincum metallicum 200cH reverted the immobility
shown by the control, Median test-Fisher, p-value<0.05; p-value=0.0152; Median=94).
3.3.2 The filial walking and immobility time
Sexual dimorphism was identified in relation to total locomotion in open field
arena, but not to the immobility time (Graphic 7-8 and table 8).
Total walking by the open field test
with filial generations by sex
OF Walking squares
400
Filial males and
females
300
Filial males
Filial females
200
Mean±SD
100
p-value<0.001,
p-value=0.0045,
Student's T-test
0
F1 (all)
F1 males
F1 females
182±57
n=29
145±60
n=11
204±43
n=18
Model groups
Graphic 7.
Open field arena walking squares of male and female filial generation
represented by mean, SD and max./min. range. Filial (F1) male mice presented reduction in
locomotion measurements, comparing to filial (F1) female mice (Student’s T-test, pvalue<0.001, p-value=0.0045, n=11 and 18), indicating the existence of sex dimorphism in the
general activity of filial generation.
53
Immobility time by the Open Field test
to filial and parental generations by sex
OF Immobility time (s)
200
F1 (all)
F1 males, n=11
F1 females, n=18
180
40
30
Median
20
Mann-Whitney,
p-value>0.05;
p-value=0.1009
10
0
F1 (all)
F1 males
F1 females
4
n=29
7
n=11
3
n=18
Model groups
Graphic 8. Open Field arena immobility time of male and female filial generation.Data is
median, quarters and max./min. range. The filial male group were identified as similar to the filial
female (Mann-Whitney, p-value>0.05, p-value=0.1009, n=11 and 18 respectivelly).
54
OF Walking
Mean±standard
deviation;
dot-plot (for n=1)
Control
Zm 200cH
Zm 30cH
Zm 5cH
F1m LPS-
151±26, n=3
229±82, n=2
n=0
n=0
F1m LPS+
127, n=1
121±61, n=3
95±18, n=2
n=0
F1f LPS-
178±28, n=3
216±33, n=4
n=0
188±88, n=2
F1f LPS+
228±11, n=2
225±27, n=3
235±48, n=2
152±43, n=2
Total
174±40, n=9
197±62,
n=12
166±85, n=4
170±60, n=4
182±57,
n=29
OF Immobile time
Median or
dot-plot (for n=1)
F1m LPSF1m LPS+
F1f LPSF1f LPS+
6, n=3
7, n=1
1, n=3
1.5, n=2
12, n=2
2, n=3
1, n=4
5, n=3
n=0
23.5, n=2
n=0
15.5, n=2
n=0
n=0
3.5, n=2
4.5, n=2
7±16, n=5
6.5±26, n=6
1±183, n=9
5±25, n=9
Total
182±62,
n=5
114±42,
n=6
197±43,
n=9
212±43,
n=9
2.5±16,
22.5±21,
3.5±5, n=4
4±183,
n=12
n=4
n=29
Table 8. Open field arena total walking and immobility time of male and female filial
Total
3±183, n=9
generation. Data showed by sex, mother’s simulated infection by LPS and Zincum metallicum
treatments. Legend: OF = Open Field test; Zm = Zincum metallicum.
55
3.3.3 The filial grooming, stand up and defecation parameters
OF Grooming
Median (max.min.);dot-plot (for
n=1)
Control
Zm 200cH
F1m LPS-
0(0-1), n=3
F1m LPS+
F1f LPS-
0, n=1
2(0-3), n=3
0(0-0), n=2
11(7-15),
n=2
0(0-5), n=3
0(0-6), n=4
0(0-6), n=3
F1f LPS+
Total
Zm 30cH
Zm 5cH
Total
n=0
n=0
1(0-15), n=5
0(0-0), n=2
n=0
1.5(0-3),
n=2
n=0
0(0-0), n=2
4.5(4-5), n=2
0(0-5), n=6
0(0-6), n=9
0(0-6), n=9
0(0-3), n=9
0(0-15),
n=12
0(0-3), n=4
2(0-5), n=4
0(0-15),
n=29
24(19-53),
n=3
0, n=1
10(8-12),
n=2
10(0-25),
n=3
15(3-24),
n=4
9(7-20), n=3
n=0
n=0
0(0-1), n=2
n=0
19(8-53),
n=5
0(0-25), n=6
n=0
9(6-13), n=2
10(10-11),
n=2
17(3-31), n=2
11(3-27),
n=9
13(3-31),
n=9
OF Stand up
Median (max.min.);
dot-plot (for n=1)
F1m LPSF1m LPS+
13(5-14),
n=3
17(14-21),
n=2
F1f LPSF1f LPS+
Total
OF Defecation
Mean±std.
deviation
dot-plot (for n=1)
F1m LPSF1m LPS+
F1f LPSF1f LPS+
14(0-53),
n=9
10(10-27),
n=12
3(0-13), n=4
10(3-31), n=4
11(0-53),
n=29
3±3, n=3
5, n=1
2.3±1, n=3
5±1, n=2
5±1, n=2
3±3, n=3
4±1, n=4
9±4, n=3
n=0
9±5, n=2
n=0
4±2, n=2
n=0
n=0
1±0, n=0
7±3, n=2
4±3, n=5
5±4, n=6
3±1, n=9
7±3, n=9
3±2, n=9
5±3, n=12
7±4, n=4
4±4, n=4
5±3, n=29
Total
Table 9. Open field arena grooming, stand up and defecation parameters of male and
female filial generation. Data showed by sex, mother’s simulated infection by LPS and Zincum
metallicum treatments. Legend: OF = Open Field arena; Zm = Zincum metallicum.
56
4. Discussion:
All parental female entered to the experiment after show the vaginal plug that is
composed by the male seminal liquids. All female mice survived after the
simulated infection by the lipopolyssacharide (LPS), injected at 9.5 days of
expected pregnancy; and all females without receiving LPS also survived. The
females that did the delivery from both control and the Zincum metallicum
200cH groups produced the highest number of pups, while the Zm 30cH and
Zm 5cH remarkably not. LPS has potential to reduce the number of pups to the
delivery and this is a factual observation in results. The experimental design to
evaluate the effects to reproductive performance is specific and the data
obtained should be considered as indicative of impairment to the reproductive
performance.
4.1. Parental females
The depression-like behavior had statistical interaction between the factor LPS
and/or delivery and the treatments by Zincum metallicum and the control; the
LPS interaction with depression-like behavior is in agreement to PITYCHOUTIS
et al. (2009). The control group presented all the typical features of this model
and it was reliable to compare with other treatments, in the previous and later
tail suspension test analysis of the same animals, but the difference among
treatments could not be detected by the three-way ANOVA. The absence of
change of depression in human parental females during pregnancy is reported
by VILHENA (2014), in which other high dilutions medicines were chosen by
individualized methodology treatments.
The mice body live weight corresponded inversely as predictor to depressionlike
behavior,
similarly
as
reported
to
depression
in
humans.
This
correspondence occurred during specific window time of the experiment, by the
day prior to the beginning of mates and strongly by the second week of all
animals, when they received LPS. Larson & Dunn (2001) report the reduction of
live weight by LPS and MARS et al. (2013) to depression. The correspondence
was also consistent to the control and Zincum metallicum 200cH groups, but not
to the Zm 30cH and Zm 5cH ones. Such modifications could be attributed to the
57
later observation of few pups delivery caused by the LPS. This was also
reported by Kirsten et al. (2013) in rats. The low pups delivery or absent of
delivery could be possibly due to interaction between the LPS with Zincum
metallicum 30cH and 5cH treatments.
The sickness behavior of mothers presenting LPS-simulated infection and
treated with Zincum metallicum 200cH was different in relation to the control,
but undetected by the statistical analysis, whose evaluation was done by the
walking and the immobility time parameters. Penteado et al. (2014) report no
change in open field arena parameters in rats under effect of LPS, which
partially corroborates the absence of effect in the immobility time, as seen
herein. Thus, the observed results regarding the walking count are divergent,
suggesting an effect of the treatment. The mechanism of action related to such
modifications can possibly occur by changes in the cytokine expression or in the
modulation of their effects, markedly IL-1beta, as reported by Larson & Dunn
(2001) and Kirsten et al. (2013). Indeed, the key symptom of a person that
would receive Zincum metallicum treatment in a homeopathic consultation is the
“tiredness” (HERING, 1872; HAHNEMANN, 1845). The present result show that
animal models can also be responsive to such treatments.
The authors observed an inverse effect between grooming and stand up
behavior to parental females submitted to simulated infection, that presented
delivery (LPS+D+) and were treated with Zincum metallicum 200cH, but it was
not statistically significant. While the stand up behavior increased, the grooming
reduced - in a no significant way - in relation to the control. We understand that
emotionality did not change in relation to the control. The mother’s grooming, in
control, had a central moderator role in relation to the effect of LPS and the
Zincum metallicum 200cH treatment. The structure of grooming should be
evaluated in further works, to check the presence of stress with impaired
grooming or not (HONG et al. 2014; KALUEFF & TUOHIMAA, 2005).
4.2 The filial generation at adulthood
The spontaneous immobility time in an overall comparison of all data (split just
by generation), from the previous and current experiments (MONTEIRO DA
SILVA et al. 2015ab), shows that the filial mice were significantly less
58
depressed-like than their parents. When exploring the differences by generation
and sex, one can observe significant reduction of the immobility time in filial
males regarding to all parental males; the same phenomenon occurs in the filial
female, if compared to all parental females. All filial males were significantly
similar to all filial females, as all parental males were significantly similar to all
parental females. These sex similarities in immobility time are in contrast with
the sex dimorphism, as shown by Dalla et al. (2009), in rats. The parental
female data from the first tail suspension test was significantly similar the
second one, with marginal p-value.
In short, the global filial time of immobilization by tail suspension test is reduced
in relation to the parental time. The immobility time is estimated as a complex
response with multiple causes. The results from the parental and filial
generations showed significantly lower values, most of them fit the healthy
category established for the depression-like behavior. The literature mentions
the phenomenon of selection as an important factor that can influence this
parameter (DALLA et al., 2009; CRYAN et al 2005; HAMMERTON, et al. 2015;
MARS, et al. 2015), what possibly happens to the current filial generation.
Observing deeply the influence of the treatment with Zincum metallicum on filial
mice behavior, the most noticeable effect was a decrease of immobility time in
tail suspension test exclusively in the filial females that were LPS injected (F1f
LPS+) and treated with Zincum metallicum 200cH. Similar findings were noticed
by KIRSTEN et al., 2015 using zinc as food supplement, however, this
parameter was neutral to males (no significant differences). The same increase
range was seen with the Zincum metallicum 5cH treatment in the filial females
without LPS inoculation (F1f LPS-). It means that the treatment with 200cH
diluted zinc followed the effects obtained by the zinc treatment reported in the
literature. The females demonstrate this effect by the immobility time, while the
males don’t. It was also reported by PITYCHOUTIS et al. (2009) using the same
generation of rats. This fact suggests that there is a sex dimorphism to the
female expression of the immobility time after the mother treatment with Zincum
metallicum 200cH, but it is not valid for males.
The males are known to present more clearly the effects of perinatal LPS
exposition and they walk significantly less than females, even if the immobility
time are similar (Kirsten et al., 2013). When the Zincum metallicum interference
59
is evaluated there is a much higher filial male grooming range, what can be
indicative of changes in grooming brain mechanisms such GABA (KALUEFF &
TUOHIMAA, 2005).
In short, the treatment of mothers with Zincum metallicum 200cH reverted the
depressive-like behavior of filial female mice, showing that the effects of Zincum
metallicum 200cH can assume a trans-generational feature and influence the
sexual dimorphism related to certain behavioral parameters. The absence of
effects of Zincum metallicum 200cH on the general activity, as observed in open
field arena, shows the effect-specificity on depressive-like behavior, without
relation to a possible interference of motor functions changes. This result is in
accordance to the literature, in which there is interference of individualized
maternal treatment by high diluted medicines during pregnancy on neonatal
parameters. In humans, the babies born from mothers treatedhomeopathically
presented increase of vitality, measured by the APGAR score, after the 5th
minute of life (VILHENA, 2014).
In this study, an experimental design is proposed to identify susceptible
individuals to post-delivery depression after simulated infection by LPS
exposition and their sensitivity to Zincum metallicum homeopathic treatment.
The statistical interaction among behavioral parameters using 3-way ANOVA,
correlation and non-parametric methods were crucial tools to reach this aim.
Pregnant mice exposed to LPS injection at the 9.5 day of gestation were more
responsible to the effects of Zincum metallicum treatment, especially at the
200cH dilution. Similar experimental designs could be useful to study other
biological aspects highly related to individuality.
5. Conclusion
The Zincum metallicum 200cH perinatal treatment can influence filial behavior,
with sexual dimorphism. Herein, the maternal treatment with Zincum metallicum
200cH reverted the filial female depressed-like behavior. The absence of effect
of the Zincum metallicum 200cH on the general mobility, observed by the open
field arena, rules out the hypothesis of unspecific action on motor functions and
reinforces the specificity on the depression-like behavior. The body weight is
identified as a potential marker to the depression-like behavior during the pre-
60
mating time. The body weight reduction was stronger after the simulated
infection with lipopoyssacharide (LPS) in the second week of pregnancy. The
effects of Zincum metallicum homeopathic dilutions (5cH, 30cH and 200cH)
have non-linear relationship to the observed behavior effects.
61
CAPÍTULO 4 - Considerações finais
O material apresentado para publicação referente aos aspectos
comportamentais foi dividido por apresentar maior coerência e compreensão
das atividades investigativas realizadas. Os demais dados adquiridos
referentes à produção intraperitoneal de citocinas e seu relacionamento com o
comportamento, e ainda com os aspectos de influência nos tecidos ósseos, e
possivelmente demais tecidos coletados para análise histopatológica estão em
fase de preparação para publicação futura.
62
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73
APÊNDICE A – Apresentação de resultados parciais sobre comportamento no
GIRI 2014 (Romênia/Sighisoara).
74
APÊNDICE B – Apresentação de resultados parciais sobre densitometria
femoral no GIRI 2015 (Verona/Itália).
75
APÊNDICE C – Apresentação de resultados parciais sobre citocinas
intraperitoneais na conferência do HRI 2015 (Roma/Itália).
76
APÊNDICE D – Publicação do resumo no 2nd HRI Research Conference
77
APÊNDICE E – Imagens dos fêmures esquerdos aos raios-X.
78
APÊNDICE F – Tabela de dados da microtomografia computadorizada (MicroCT) de fêmures.
neg
Quali/
BV
Res pix
8.71057 9.82257
Controle f1 femea
neg
8.71057 5.64846 49.77854 -68.99550 0.06879 7.23638 0.34860 2.65014 3,850.00000 0.07677 49.54487
5.62194
5.69872
Controle f1 macho
neg
8.71057 9.80527 61.43858 -48.12799 0.12333 4.98169 0.41961 2.74871 2,721.00000 0.11361 37.84953
6.04058
6.15420
2
geracao/
genero
Controle f1 femea
3
7
Amostras
Grupo
LPS
% BV
4.39780
TBPF
TBTH
TBN
TBSP
FD
-4.86075 0.24877 0.17678 2.13833 2.77320
PON
234.00000
% POop
POV op
POVtot
0.02242 95.59217 213.50697 213.52939
14
200ch
f1 macho
neg
8.71057 8.26947 48.47661 -42.57113 0.10091 4.80392 0.25597 2.67241
0.02221 51.39318
8.76700
8.78922
15
200ch
f1 macho
neg
8.71057 9.36854 65.21088 -52.79293 0.11953 5.45558 0.34715 2.76175 2,520.00000 0.11060 34.01930
4.88739
4.99799
18
200ch
f1 femea
neg
8.71057 8.01552 62.47161 -48.25101 0.13007 4.80300 0.46512 2.75135 2,016.00000 0.08643 36.85479
4.72872
4.81514
27
5ch
f1 femea
neg
8.71057 8.48359 61.56661 -50.96248 0.12328 4.99397 0.39826 2.74304 1,725.00000 0.06865 37.93646
5.22747
5.29594
28
5ch
f1 femea
neg
8.71057 9.83995 61.63749 -49.16015 0.12838 4.80100 0.48111 2.75610 2,562.00000 0.14275 37.46833
5.98153
38.36251
36
200ch
f1 macho positivo 8.71057 8.46703 61.92240 -43.28702 0.12933 4.78805 0.38628 2.73703 1,499.00000 0.06091 37.63213
5.14567
5.20658
37
200ch
f1 femea
neg
8.71057 7.31448 61.73267 -49.11240 0.12128 5.08997 0.44069 2.74550 2,078.00000 0.10917 37.34599
4.42499
4.53415
41
Controle f1 femea
neg
8.71057 8.20043 64.67124 -56.19065 0.11337 5.70461 0.34665 2.75466 2,465.00000 0.12878 34.31313
4.35097
4.47975
43
Controle f1 macho
neg
8.71057 8.18756 64.86262 -51.47879 0.11974 5.41715 0.34172 2.74593 2,015.00000 0.08415 34.47074
4.35121
4.43536
f1 macho animal2 8.71057 7.19116 61.99649 -55.55427 0.11708 5.29511 0.34447 2.73899 1,446.00000 0.06136 37.47455
4.34679
4.40814
9.65466
9.68630
63
71
200ch
8.71057 11.21545 53.65796 -34.56297 0.10094 5.31575 0.38254 2.63492
497.00000
POV cl
845.00000
0.03164 46.19066
79
APÊNDICE G – Microscopia eletrônica de varredura e espectroscopia de
energia dispersiva (MEV-EDS) de uma amostra de fêmur corte transversal.
80
ANEXO A – Certificado de aprovação pela (CEUA-UNIP) protocolo 156/13.
81
ANEXO B – Laudo de análise laboratorial externa da concentração de zinco.
82
ANEXO C – Carta com decodificação dos tratamentos em cego.
83
ANEXO D – Parecer do Prof. Paolo Belavitte sobre o estágio de doutoramento
internacional na Università Degli Studio di Verona - Ospetale Policlinico.
84
ANEXO D (Continuação) – Parecer do Prof. Paolo Belavitte sobre o estágio de
doutoramento internacional na Università Degli Studio di Verona - Ospetale
Policlinico.

Programa de Pós-Graduação em Patologia Ambiental e

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