Einfluss von Mikrogliazellen und retrospekt

Transcription

Tierärztliche Hochschule Hannover
Untersuchungen zu Rückenmarkstraumata beim Hund:
Einfluss von Mikrogliazellen und
retrospektive Untersuchung der MRT-Befunde
von Hunden mit thorakolumbalem Bandscheibenvorfall
INAUGURAL-DISSERTATION
zur Erlangung des Grades einer
Doktorin der Veterinärmedizin
- Doctor medicinae veterinariae (Dr. med. vet.)
vorgelegt von
Theda Marie Anne Boekhoff
Wilhelmshaven
Hannover 2010
Wissenschaftliche Betreuung: Prof. Dr. med. vet. Andrea Tipold
Klinik für Kleintiere
1. Gutachter: Prof. Dr. med. vet. Andrea Tipold
2. Gutachter: Prof. Dr. med. vet. Wolfgang Baumgärtner, PhD
Tag der mündlichen Prüfung: 12.05.2010
Diese
Arbeit
wurde
finanziell
unterstützt
durch
die
Deutsche
Forschungsgemeinschaft (DFG) (FOR 1103) und die Frauchiger Stiftung, Bern.
für meine Familie
Teile der vorliegenden Dissertation wurden bereits auf folgenden Tagungen
vorgestellt:
Posterpräsentation auf der 17. Jahrestagung der Fachgruppe
„Innere Medizin und Klinische Laboratoriumsdiagnostik“,
Deutsche Veterinärmedizinische Gesellschaft, e.V. (DVG)
31.01.2009 - 01.02.2009 in Berlin:
T.M. Boekhoff, E.M. Ensinger, R. Carlson, A. Tipold , V.M. Stein
„Funktionelle Untersuchungen von Mikrogliazellen aus dem Rückenmark des
Hundes.“
Posterpräsentation 22nd Annual Symposium of the European Society of Veterinary
Neurology (ESVN) and the European College of Veterinary Neurology (ECVN)
24th - 26th September 2009, Bologna, Italy:
T.M. Boekhoff, E.M. Ensinger, R. Carlson, A. Tipold , V.M. Stein
“Enhanced functional activity of canine microglial cells from the spinal cord.”
Posterpräsentation auf der 18. Jahrestagung der Fachgruppe
„Innere Medizin und Klinische Laboratoriumsdiagnostik“,
Deutsche Veterinärmedizinische Gesellschaft, e.V. (DVG)
06.02.2010 - 07.02.2010 in Hannover:
T.M. Boekhoff, E.M. Ensinger, R. Carlson, I. Spitzbarth, W. Baumgärtner,
A. Tipold , V.M. Stein
„Funktionelle Untersuchungen von Mikrogliazellen des Hundes bei nicht
entzündlichen Rückenmarkserkrankungen.“
Inhaltsverzeichnis
Inhaltsverzeichnis
I.
Einleitung........................................................................................................ 7
II.
Ergebnisse...................................................................................................... 9
A. Quantitative magnetic resonance imaging characteristics: evaluation of prognostic
value in the dog as a translational model for spinal cord injury ............................... 9
Abstract ................................................................................................................. 10
Introduction ........................................................................................................... 11
Materials and Methods .......................................................................................... 12
Results .................................................................................................................. 14
Discussion............................................................................................................. 21
Conclusion ............................................................................................................ 23
References............................................................................................................ 24
B. Upregulation of surface molecules and functional activity of canine microglia
following spinal cord trauma.................................................................................. 28
Abstract ................................................................................................................. 29
Introduction ........................................................................................................... 30
Materials and Methods .......................................................................................... 31
Results .................................................................................................................. 36
Discussion............................................................................................................. 42
References............................................................................................................ 47
III.
Zusammenfassung der Ergebnisse beider Studien.................................. 53
IV.
Übergreifende Diskussion........................................................................... 55
V.
Zusammenfassung (deutsch) ..................................................................... 59
VI.
Zusammenfassung (englisch)..................................................................... 61
VII.
Schrifttumsverzeichnis................................................................................ 63
VIII.
Anhang.......................................................................................................... 73
IX.
Danksagung ................................................................................................. 85
Einleitung
7
I. Einleitung
Bandscheibenvorfälle beim Hund stellen eine der häufigsten Ursachen für eine
traumatische Schädigung des Rückenmarkes dar. Vorgefallenes Bandscheibenmaterial
kann zu einer extraduralen Kompression von Rückenmark und Nervenwurzeln führen, in
deren Folge es zu einer mechanischen Schädigung sowie zur Minderdurchblutung der
betroffenen Gewebe (GÖDDE u. JAGGY, 1993) kommen kann.
Eine solche mechanische Schädigung des Rückenmarkes kann zu einer Zerstörung der
Zellmembranen von Gliazellen und Neuronen sowie zu einer Beeinträchtigung der lokalen
Durchblutung führen. Im weiteren Verlauf kommt es zu einer Kaskade von sekundären
pathophysiologischen Reaktionen wie zum Beispiel der Freisetzung von Neurotransmittern
sowie freien Radikalen, Ischämie, Bildung von Ödemen und Elektrolytimbalancen
(JEFFERY, 2009), welche in zellulärer Nekrose und Apoptose resultieren (PLATT u.
OLBY, 2004).
Das Ausmaß einer Rückenmarksschädigung kann mittels Magnetresonanztomographie
(MRT) zum Beispiel durch die Darstellung von Blutungen, Malazien des Myelons,
nekrotischen Bereichen sowie Ödemen als Hyperintensität in T2-gewichteten Sequenzen
evaluiert werden (SANDERS et al., 2002).
Bandscheibenvorfälle können neurologische Defizite zur Folge haben, welche von
Hyperästhesien bis hin zu einer vollständigen Lähmung der Gliedmaßen reichen. Eine
Behandlungsmöglichkeit stellt die chirurgische Dekompression dar (HOERLEIN, 1979),
welche bei bis zu 96% paraplegischer Hunde mit Tiefensensibilität (DAVIS u. BROWN,
2002; FERREIRA et al., 2002), sowie bis zu 58% paraplegischer Hunde ohne
Tiefensensibilität (OLBY et al., 2003) zu einer Behebung der klinischen Symptomatik führt.
Um eine Entscheidung für den Einsatz einer Therapie zu treffen, ist eine vorherige
Einschätzung
der
Prognose
unumgänglich.
Bisher
bekannte
prognostische
Einflussfaktoren sind unter anderem der Grad der neurologischen Ausfälle bei Vorstellung
des Patienten sowie die Beurteilung der Tiefensensibilität (SCOTT, 1997). Um die
Prognosefindung zu erleichtern und zu objektivieren, war das Ziel des ersten Teils dieser
Arbeit, qualitative sowie quantitative MRT-Befunde im Hinblick auf ihren prognostischen
Wert bei paraplegischen Hunden zu untersuchen.
Eine neuere Therapiestrategie für Rückenmarkstraumata stellt die Transplantation von
olfaktorischen Hüllzellen dar. Vorangegangene Studien haben aufgezeigt, dass diese
Zellen die Regeneration verletzter Neuronen sowie die Angiogenese unterstützen und
Einleitung
8
somit eine verbesserte Heilung traumatisierten Rückenmarkes bewirken können (RADTKE
et al., 2008; KOCSIS et al., 2009).
Eine
Zellpopulation
des
zentralen
Nervensystems
(ZNS),
die
sekundäre
Rückenmarksschäden positiv oder negativ beeinflussen kann, sind Mikrogliazellen. Diese
spielen als residente Immuneffektorzellen des ZNS eine wichtige Rolle in der Erhaltung
der Homöostase und der Infektionsabwehr (KREUTZBERG, 1996; ALOISI, 2001; STREIT,
2002). Bei Auftreten von pathogenen Stimuli sind sie zu Effektorfunktionen wie der
Phagozytose, der Bildung reaktiver Sauerstoffspezies (ROS), sowie der Sekretion von
Zytokinen befähigt (COLTON u. GILBERT, 1987; KREUTZBERG, 1996; STOLL u.
JANDER, 1999).
Durch die Sekretion von neuroprotektiven und neurotoxischen Substanzen können
Mikrogliazellen sowohl Degenerations- als auch Regenerationsprozesse im ZNS und
somit die Pathogenese von Rückenmarkserkrankungen entscheidend beeinflussen.
Eine Mikroglia-Aktivierung konnte bisher bei neurodegenerativen Erkrankungen wie zum
Beispiel Alzheimer, Parkinson, multipler Sklerose sowie amyotropher Lateralsklerose
nachgewiesen werden (THOMAS, 1992; WILLIAMS et al., 1994; YIANGOU et al., 2006).
Des Weiteren führten das kanine Staupevirus (STEIN et al., 2004b), die experimentelle
allergische Enzephalomyelitis (EAE; VASS u. LASSMANN, 1990; RUULS et al., 1995)
sowie experimentelle traumatisierende Läsionen des Rückenmarkes (SCHNELL et al.,
1999; SROGA et al., 2003; YANG et al., 2005; LONGBRAKE et al., 2007; YUNE et al.,
2009; Baloui et al., 2009) zu einer Aktivierung von Mikrogliazellen.
Ziel des zweiten Teils dieser Arbeit war die immunphänotypische und funktionelle
Untersuchung von Mikrogliazellen bei Hunden mit Rückenmarkstraumata. Anhand
erstellter Vergleichswerte aus einer im Vorfeld von uns durchgeführten Studie wurde
evaluiert, inwiefern es zu einer Aktivierung dieser Immuneffektorzellen kommt und
inwieweit die Pathogenese hierdurch beeinflusst wird beziehungsweise ob diese Zellen
eine erfolgreiche Zelltransplantation eventuell beeinflussen können.
Ergebnisse
9
II. Ergebnisse
A. Quantitative magnetic resonance imaging characteristics:
evaluation of prognostic value in the dog as a translational model for spinal cord
injury
Theda M. Boekhoffa, Cornelia Flieshardtb, Eva-Maria Ensingera, Melani Forka,
Sabine Kramera, Andrea Tipolda
a
Department of Small Animal Medicine and Surgery, University of Veterinary Medicine,
Hannover, Germany
b
LESIA Zentrum für Tiermedizin, Düsseldorf, Germany
Corresponding author:
Prof. Dr. Andrea Tipold
Department of Small Animal Medicine and Surgery
University of Veterinary Medicine Hannover
Bünteweg 9
D-30559 Hannover
Germany
Tel. 0049-511-953-6202
Fax 0049-511-953-6204
e-mail: [email protected]
Ergebnisse
10
Abstract
Thoracolumbar intervertebral disk herniations appear frequently in dogs and are a
common cause of neurological signs ranging from spinal hyperesthesia to paraplegia with
or without deep pain perception (DPP). For selection of the applied therapeutical approach
a prior assessment of prognosis is useful. The aim of this retrospective study was to
describe associations among the quantitative magnetic resonance imaging (MRI) signal
characteristics of the spinal cord in T2-weighted (T2W) sequences respectively degree of
spinal cord compression and clinical signs plus functional outcome in paraplegic dogs with
thoracolumbar disk herniation.
Medical records and MR images of 63 paraplegic dogs with intact or absent DPP referred
to and examined at the Department of Small Animal Medicine and Surgery, University of
Veterinary Medicine, Hannover, Germany between January 2005 and June 2009 were
reviewed and evaluated based on correlation of different clinical parameters.
A statistically significant correlation was seen between the neurological status before
surgery and both, presence and extent of the intramedullary hyperintensity adjacent to the
disk herniation in T2W sequences. Furthermore, in dogs with a longer duration of clinical
signs the degree of spinal cord compression was statistically significant higher. The extent
of hyperintensity and the degree of spinal cord compression presented a positive
correlation, whereas improvement in neurological score in one grade tended to advance
with absence of T2W hyperintensity respectively reduction of the extent of hyperintensity.
In conclusion, a direct correlation between neurological status and MRI signal intensity and
extent was proven. Moreover, presence and extent of T2W hyperintensity can assist in
determination of prognosis before surgery in respect to utilization of new therapeutical
strategies.
Key words: paraplegia, MRI, T2-weighted hyperintensity, prognosis, translational
model
Ergebnisse
11
1. Introduction
Displaying a common cause for spinal cord injury (SCI) in dogs thoracolumbar
intervertebral disk herniations can lead to neurological deficits ranging in severity from
spinal hyperaesthesia to paraplegia with loss of deep pain perception. Mechanisms of
injury in SCI in dogs are similar to those in human patients and the dog is considered to be
a valuable translational model for new treatment modalities (Jeffery et al., 2006). Surgical
decompression of the spinal cord is a common therapeutical approach in intervertebral
disk herniations (Hoerlein, 1979) and has been reported as successful in up to 96 % of
paraplegic dogs with intact DPP (Davis and Brown, 2002; Ferreira et al., 2002)
respectively 58 % of paraplegic dogs with loss of DPP (Olby et al., 2003). As the imaging
modality of choice (Ito et al., 2005) magnetic resonance imaging (MRI) gives important
insights in severity of SCI revealing information about presence of spinal cord edema,
hemorrhage and contusion (Kulkarny et al., 1987). There are several studies dealing with
the investigation of MRI signal characteristics and their prognostic value for functional
outcome after SCI in humans (Flanders et al., 1999; Selden et al., 1999; Shimada and
Tokioka, 1999; Yukawa et al., 2007; Miyanji et al., 2007) and in dogs (Ito et al., 2005;
Penning et al., 2006; Ryan et al., 2008; Levine et al., 2009). Nevertheless, there is a need
of further studies regarding quantitative characteristics of MRI findings (Miyanji et al.,
2007). Quantitative properties of signal characteristics of the myelon and spinal cord
compression in MRI images have been studied before (Ito et al., 2005; Levine et al.,
2009). However, to the best of our knowledge, there has been no study publishing results
of correlations between quantitative characteristics of T2W hyperintensity respectively
spinal cord compression and outcome after thoracolumbar disk herniation in a
homogenous dog population, exclusively in paraplegic dogs.
The aim of this study was to investigate correlations between clinical and qualitative
respectively quantitative imaging parameters and consequently evaluate potential
prognostic values of MRI findings. This prognostic information could give important
guidance in the choice of therapeutical strategies in respect to new approaches such as
implantation of olfactory ensheathing cells (OECs) additionally to surgery. There are
currently not enough data about preferable techniques and about the patient population
eligible for such transplantations (Jeffery et al, 2006). The current study should provide
information on in vivo imaging findings to provide prognostic values for severely injured
patients.
Ergebnisse
12
2. Materials and Methods
2.1 Case selection
Dogs referred to and examined at the Department of Small Animal Medicine and Surgery,
University of Veterinary Medicine, Hannover, Germany with a diagnosis of paraplegia
resulting from a thoracolumbar intervertebral disk herniation between January 2005 and
June 2009 were considered for inclusion in this study. The following inclusion criteria were
determined: body weight less than 20 kg in an effort to investigate a homogenous
population and exclude a worse prognosis given by a higher body weight, presence of
paraplegia with or without DPP, complete medical records of physical and neurological
examinations, MRI available for review, thoracolumbar disk herniation confirmed via MRI
and surgery.
2.2 Procedures
Information concerning breed, sex, age, body weight, interval between onset of
neurological
signs
and
performance
of
MRI/surgery,
pre-treatment
with
glucocorticosteroids before referral, neurological status before and after surgery and at
day of discharge, duration of hospitalization, and improvement in neurological score of one
grade was recorded for each case. For classification of neurological status the
neurological score by Sharp and Wheeler (2005) was used. This scoring system is defined
as spinal hyperesthesia only (grade 1), ambulatory paraparesis and ataxia (grade 2),
nonambulatory paraparesis (grade 3), paraplegia with intact DPP (grade 4), and
paraplegia with absent DPP (grade 5). Consequently, the dogs included in this study were
divided into two groups, paraplegic dogs with intact DPP (grade 4) respectively paraplegic
dogs with absent DPP (grade 5).
MRI was performed using a 1.0-Tesla scanner (Magnetom impact plus, 1.0 Tesla,
Siemens AG Medical Solutions Magnetic Resonance Imaging, Forchheim) under general
anaesthesia using propofol (Narcofol®, cp-Pharma, Burgdorf, dosis depending on effect)
and diazepam (diazepam-ratiopharm®, Ratiopharm GmbH, Ulm, 1 mg/kg, i.v.) for induction
respectively isoflurane (Isofluran-Baxter®, Baxter Deutschland GmbH, Unterschleißheim)
for maintenance of anaesthesia with each dog in dorsal recumbency. Sagittal (TR = 4,700,
TE = 112, slice thickness = 3mm) and transverse (TR = 3,458, TE = 96, slice
thickness = 3mm) T2W images were applied for evaluation. Afterwards all dogs underwent
hemilaminectomy to achieve a decompression of the damaged portion of the spinal cord.
Ergebnisse
13
MR images were reviewed by two board certified neurologists blinded to clinical
information by use of a computer workstation with appropriate software (dicomPACS
version 5.2, Oehm und Rehbein GmbH, Rostock, Germany). If an intramedullary
hyperintensity was present on T2W images, measurement of the length was performed in
sagittal T2W images and divided by the length of the L2 vertebra to create the T2W length
ratio described by Ito et al. (2005; Fig. 1a). Presence and degree of spinal cord
compression were evaluated by comparing the cross-sectional diameter of the spinal cord
at the site of disk herniation to the cross-sectional diameter of the spinal cord one vertebra
caudal to the herniation on transverse T2W images (Fig. 1b,c).
(a)
(b)
Figure 1
(c)
Determination of the extent of intramedullary hyperintensity in sagittal (a) and the
degree of spinal cord compression (b,c) in transverse T2W MR images.
(a) Measurement of the extent of the intramedullary T2W hyperintensity of the spinal cord
was performed (black line) in sagittal T2W images and divided by the length of the L2
vertebra (white line) to create the T2W length ratio described by Ito et al. (2005).
To determine the degree of spinal cord compression the cross-sectional diameter of the
spinal cord at the site of disk herniation (b) was compared to the cross-sectional diameter of
the spinal cord one vertebra caudal to the herniation (c) on transverse T2W images.
Ergebnisse
14
2.3 Statistics
A descriptive analysis was performed for all parameters. For analyzing parameters in
ordinal scale (interval between onset of neurological signs and performance of
MRI/surgery, neurological grade before surgery, improvement in neurological score in one
grade, degree of spinal cord compression, extent of hyperintensity) Spearman correlation
coefficients were used. Furthermore, to access correlations concerning presence of T2W
hyperintensity and pre-treatment with glucocorticosteroids a Chi-Square test was
performed. If sizes of analyzed groups were < 5, Fisher’s exact test was used additionally.
Regarding the experimentwise error rate, p values of < 0.05 were considered significant.
Analyses were carried out with the statistical software SAS®, version 9.2 (SAS Institute,
Cary, NC) in a Windows XP® environment. Microsoft® Office Excel® 2003 and 2007
(Microsoft Corporation, Redmond, Washington, USA) were used to display data in tables
and figures.
3. Results
Sixty-three dogs met the inclusion criteria for this study. Of these, 40 (63%) were
Dachshunds, 10 (16%) were mixed-breed dogs, 4 (6%) were Jack Russell Terriers, 2 (3%)
were Pekingese and there was 1 (2%) each of American Cocker Spaniel, Yorkshire
Terrier, Bulldog, Shi Tzu, Toy Poodle, Bolonka Zwetna, and Dachsbracke. Age at referral
ranged from 1 to 13 years (mean, 6.9 years). Thirty-nine dogs were male (7 castrated) and
24 were female (5 spayed). Mean body weight was 9.2 kg (range, 3.8 to 18.5 kg).
Intervals between onset of neurological signs and performance of MRI ranged from 1 to 32
days (mean = 4.5 days). Duration of clinical signs was further classified into the following 5
categories. Category 1 (duration 1 day) contained 25% of the dogs (n = 16), category 2
(duration 2 to 3 days) 35% (n = 22), category 3 (duration 4 to 7 days) included 27% of the
dogs (n = 17), into category 4 (duration 8 to 14 days) 10% of the dogs (n = 6) and category
5 (duration longer than 14 days) 3% of the dogs (n = 2) were classified.
Forty-nine dogs (78%) were presented with neurological signs classified into neurological
score 4, 14 (22%) dogs matched with neurological sore 5. Neurological status before, one
day after surgery and at the day of discharge were summarized (Table I). Improvement in
neurological score for one grade ranged from 1 day to several weeks and was grouped
Ergebnisse
15
into 5 categories (Table I). In one dog, improvement in neurological signs could not be
evaluated.
Table 1
Number and percentages of dogs concerning neurological status, improvement in
neurological score, and euthanasia respectively exitus letalis.
Of the 63 dogs, 28 had been treated with glucocorticosteroids, 23 dogs received no
glucocorticosteroids and in 12 dogs treatment before referral could not be evaluated.
Ergebnisse
16
Duration of hospitalization ranged from 4 to 26 days (mean = 9.4 days). Four dogs were
euthanized during hospitalization due to stasis or worsening of the neurological signs. Two
dogs died before discharge.
Review of MRI resulted in a confirmation of a thoracolumbar disk herniation in all 63
cases. The affected intervertebral disk was located at Th11-12 in 8 dogs (13%), Th12-13
in 22 dogs (35%), Th13-L1 in 19 dogs (30 %), L1-2 in 3 dogs (5%), L2-3 in 7 dogs (11%),
and L3-4 in 4 dogs (6%). A T2W hyperintensity was detected in 37 dogs (59% of all
investigated dogs). MRI findings concerning quantity of this hyperintensity and spinal cord
compression were each categorized into 4 groups and are displayed in figure 2.
Figure 2
MRI findings concerning presence and extent of a T2W hyperintensity and the degree
of spinal cord compression.
The extent of hyperintensity and the degree of spinal cord compression were each
categorized into 4 groups. For the extent of hyperintensity classification depended on the
length of the L2 vertebra (T2W length ratio): category 1 (≤ ½ L2); category 2 (≤ 1 L2);
category 3 (> 1 L2); category 4 (> 2 L2). The degree of spinal cord compression was
calculated in percent: category 1 (compression ≤ 25%); category 2 (compression > 25% to
50%); category 3 (compression > 50% to 75%); category 4 (compression > 75%).
The abscissa gives the qualitative and quantitative MRI characteristics in categories, the
ordinate represents the respective number of dogs.
Ergebnisse
17
Statistical analysis resulted in a significant correlation between the neurological status
before surgery and both, presence (p = 0.02) and extent (p = 0.02) of hyperintensity
(Fig. 3). A higher percentage of dogs with neurological grade 5 showed a T2W
hyperintensity compared to dogs with grade 4. Also the extent of hyperintensity enhanced
with increase in neurological grade.
Figure 3
Statistically significant () correlation between the neurological grade before surgery
and both, presence (p = 0.02) and extent (p = 0.02) of hyperintensity.
The abscissa gives the qualitative and quantitative characteristics of T2W hyperintensity, the
ordinate shows the respective percentage of dogs. Grey bars represent dogs with
neurological grade 4 respectively grade 5. 51% of the dogs with neurological grade 4 (dark
grey) showed a T2W hyperintensity in MRI, whereas in neurological grade 5 (grey)
percentage of dogs increases up to 86%. 58% of dogs with neurological grade 5 presenting
a T2W hyperintensity showed an extent of hyperintensity > 2 L2 vertebra (L2, category 4).
Extent of hyperintensity was classified into: category 1 (≤ ½ L2), category 2 (≤ 1 L2),
category 3 (> 1 L2), and category 4 (> 2 L2).
Furthermore, the degree of spinal cord compression was statistically significant higher in
dogs with a longer duration of clinical signs (p < 0.001, Fig. 4).
Ergebnisse
Figure 4
18
Correlation between the duration of clinical signs and degree of spinal cord
compression.
The abscissa displays the duration of clinical signs in categories, on the ordinate the
respective percentages of dogs are represented. Bars in different grey levels represent
categories describing the degree of spinal cord compression, which was classified into the
following categories: category 1 (compression ≤ 25%); category 2 (compression > 25% to
50%); category 3 (compression > 50% to 75%); category 4 (compression > 75%). For
duration of clinical signs the following classification was used: category 1 (duration 1 day),
category 2 (duration 2 to 3 days), category 3 (duration 4 to 7 days), category 4 (duration 8 to
14 days), and category 5 (duration longer than 14 days).
Degree of spinal cord compression was statistically significant higher () in dogs with a
longer duration of clinical signs (p < 0.001).
The extent of T2W hyperintensity and the degree of spinal cord compression were
positively correlated (statistically significant, p = 0.05, Fig. 5). Improvement in neurological
score for one grade was faster with absence of T2W intramedullary hyperintensity
respectively with a smaller extent of hyperintensity, if present (Fig. 6).
Comparison of pre-treatment with glucocorticosteroids did not reveal any statistically
significant correlation.
Ergebnisse
Figure 5
19
Correlation between the degree of spinal cord compression and extent of T2W
hyperintensity.
The abscissa displays the degree of spinal cord compression in categories: category 1
(compression ≤ 25%); category 2 (compression > 25% to 50%); category 3 (compression >
50% to 75%); category 4 (compression > 75%). On the ordinate the respective percentages
of dogs are represented. The extent of T2W hyperintensity classified into categories 1 – 4 is
represented by bars in different grey levels: category 1 (≤ ½ length of the L2 vertebra, L2);
category 2 (≤ 1 L2); category 3 (> 1 L2); category 4 (> 2 L2).
Expanding degrees of spinal cord compression lead to an increase in the extent of
hyperintensity, which was statistically significant (; p = 0.05).
Ergebnisse
20
(a)
(b)
Figure 6
Correlation between the improvement in neurological score in one grade and
presence (a) respectively extent (b) of T2W hyperintensity.
The abscissa shows improvement in neurological score in one grade in categories: category
1 (1 day), category 2 (2 to 3 days), category 3 (4 to 7 days), category 4 (8 to 14 days), and
category 5 (longer than 14 days). On the ordinate the respective percentages of dogs are
displayed. Bars in different grey levels represent presence (a) respectively categories
describing the extent (b) of hyperintensity: category 1 (≤ ½ length of the L2 vertebra, L2);
category 2 (≤ 1 L2); category 3 (> 1 L2); category 4 (> 2 L2). Some dogs were euthanized
because of stasis or worsening of neurological signs, shown as category “euthanasia” on the
abscissa.
Improvement in neurological score in one grade tended to prolong with presence of T2W
hyperintensity. In the category representing dogs with a duration longer than 14 days until
improvement in neurological score in one grade (category 5) all dogs showed a T2W
hyperintensity.
Ergebnisse
21
4. Discussion
Spinal cord injury in the dog is considered to be an ideal translational model between
rodent experiments and human clinical trials. To investigate the role of MRI findings on
prognostic values data of 63 paraplegic dogs with thoracolumbar disk herniations
confirmed via MRI and surgery were reviewed and correlations concerning clinical signs
and MRI characteristics were analyzed.
Results displayed a statistically significant correlation between neurological status before
surgery and presence as well as extent of a T2W hyperintensity in the myelon.
Consequently, dogs with a more severe pre-surgery neurological status more often
showed a T2W hyperintensity respectively displayed a more extended intramedullary
lesion. These findings are consistent with similar studies performed in a more
heterogenous dog population (Levine et al., 2009) and humans (Schaefer et al., 1989;
Miyanji et al., 2007; Miranda et al., 2008). Regarding the pathologic processes causing a
T2W hyperintensity (necrosis, myelomalacia, intramedullary hemorrhage, edema;
Ito et al., 2005) it becomes obvious that a higher neurological impairment is correlated with
a more severe spinal cord trauma.
The degree of spinal cord compression increased with duration of clinical signs. Previous
studies demonstrated that dynamic changes during a disk herniation have more distinct
influences on spinal cord damage than the duration of the compression (Bull, 2006).
Consequently, a sudden or explosive disk extrusion appears more harmful than a slowly
progressive disk herniation. Therefore, the detected correlation between duration of clinical
signs and spinal cord compression could be caused by the dynamic differences of the
individual disk herniation (Sharp and Wheeler, 2005). A slowly increasing amount of
extruded disk material seems to be leading to higher degree of spinal cord compression.
As described before no correlation between the degree of spinal cord compression and
initial neurological status (Levine et al., 2009) respectively outcome (Penning et al., 2006)
could be detected in our study.
A significantly higher extent of the T2W hyperintensity was seen in correlation with an
increased spinal cord compression. Consistent with this, Purdy et al. (2004) demonstrated
that compression injuries depend on the level of spinal cord occlusion. T2W hyperintense
signal changes of the spinal cord can be associated with hemorrhage, myelomalacia,
necrosis, edema, and fat (Sanders et al., 2002). It is known that disk herniations lead to a
spinal cord concussion respectively compression and induce a series of metabolic and
Ergebnisse
22
biochemical events (Jeffery, 2009) which may result in tissue necrosis and additional
damage to local vasculature (Griffiths, 1972; Platt and Olby, 2004). Thus, these
pathological insults can cause hemorrhage, malacia, edema, and necrosis detected by
T2W hyperintensities in MRI. In addition, an increased release of neurotoxic substances
such as reactive oxygen species (ROS) by microglia in case of spinal cord trauma
respectively disk extrusion can cause direct damage of neurons (Bruce-Keller, 1999)
leading consequently to necrosis and myelomalacia. Furthermore, the duration of clinical
signs was positively correlated with the degree of the spinal cord compression, which in
fact correlated with the extent of the T2W hyperintensity of the myelon. A longterm spinal
cord compression leads to a more severe spinal cord injury due to propagation of the
secondary injury process as shown before in histopathological studies (Carlson et al.,
2003). Secondary damage of the spinal cord is initiated by a cascade of secondary events
such as vascular dysfunction, edema, ischaemia, excitotoxicity, and delayed apoptotic cell
death and occurs over the following days and weeks, leading to a progressive tissue
disruption. This secondary injury can explain the lesion in the myelon detected by MRI.
Pre-treatment with glucocorticosteroids was evaluated to demonstrate a positive or
negative effect on intramedullary T2W MRI findings. This medication did not result in any
statistically significant influences on the other investigated parameters. These results
support studies by Davis and Brown (2002) and Bull et al. (2008) describing a missing
influence on the outcome using glucocorticosteroids as pre-treatment before surgical
spinal cord decompression.
In the present study, functional outcome respectively clinical improvement tended to
worsen with presence of T2W hyperintensity respectively increasing extent of this
hyperintensity in MR imaging. Several studies verified also a prognostic value of the
existence of T2W hyperintensity (Miyanji et al., 2007; Yukawa et al., 2007) and the extent
of hyperintensity (Flanders et al., 1999; Selden et al., 1999; Miyanji et al., 2007) in
humans. These findings highlight that the dog might be used for clinical trials of novel
therapeutic interventions and subpopulations of SCI affected dogs, in which recovery is
incomplete or does not occur at all, can be identified by MRI imaging. Ito et al (2005)
detected a poor prognosis for functional recovery in paraplegic dogs exhibiting an area of
T2W hyperintensity at least as long as the L2 vertebral body. Levine et al. (2009)
demonstrated a degraded long-term ambulatory outcome in association with occurrence of
T2W
hyperintensity
and
its
extent.
In
conclusion,
qualitative
and
quantitative
characteristics of T2W hyperintensity seem to have important prognostic value and serve
as meaningful addition to other prognostic factors such as initial neurological status (Scott,
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23
1997). The clinical examination can be supported by more objective measurements
especially in treatment studies.
Further prognostic information could be useful to choose the adequate therapeutical
approach such as implantation of OECs additionally to surgery. Previous studies revealed
an improved functional recovery due to the ability of OECs to provide trophic support for
injured neurons and angiogenesis (Radtke et al., 2008; Kocsis et al., 2009). Therefore, this
therapy displays a meaningful addition to surgery, especially in patients with poor
prognosis, detected by grade of neurological impairment in combination with the described
MRI findings.
Clinical SCI in dogs displays a model which can be compared to human SCI in terms of
mechanisms of injury, pathology, outcome, classification and functional monitoring (Jeffery
et al., 2006). Consistent with this, Purdy et al. (2004) described similarities between canine
and human spinal cord in terms of imaging. Thus, research in human spinal cord
pathology can benefit from this valuable translational model.
5. Conclusion
Qualitative and quantitative characteristics of T2W hyperintensity in spinal cord injury
seem to have important prognostic value in dogs. Findings are comparable to those in
human medicine. Consequently, these parameters can assist in the utilization of new
therapeutical strategies in veterinary and human medicine since the dog diplays a valuable
translational model for human spinal cord diseases. Choosing the correct subpopulation of
dogs with SCI using MR imaging might help in screening novel and diverse treatment
modalities.
Acknowledgements
The study was supported by the German Research Foundation (FOR 1103) and the
Frauchiger Stiftung, Bern, Switzerland.
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24
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Shimada K, Tokioka T. Sequential MR studies of cervical cord injury: correlation with
neurological damage and clinical outcome. Spinal Cord 1999;37:410-5.
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compression myelopathy: predictor of surgical outcomes. Spine 2007;32:1675-8.
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B. Upregulation of surface molecules and functional activity of canine microglia
following spinal cord trauma
Theda M. Boekhoffa,*, Eva-Maria Ensingera,*, Regina Carlsona, Andreas Beinekeb,
Wolfgang Baumgärtnerb, Karl Rohnc, Andrea Tipolda, Veronika M. Steina
a
Department of Small Animal Medicine and Surgery,
b
Department of Pathology,
c
Institute of Biometry, Epidemiology, and Information Processing,
University of Veterinary Medicine, Hannover, Germany
Corresponding author:
Veronika M. Stein
Department of Small Animal Medicine and Surgery
University of Veterinary Medicine Hannover
Bünteweg 9
D-30559 Hannover
Germany
Tel. 0049-511-953-6202
Fax 0049-511-953-6204
e-mail: [email protected]
*
the authors Boekhoff and Ensinger contributed equally to the manuscript
Ergebnisse
29
Abstract
Microglia cells represent the primary intrinsic immune effector elements of the central
nervous system (CNS) and show responses to many different pathological events. Spinal
cord trauma in dogs is a well recognized animal model to study pathogenesis and
treatment modalities. Therefore and to clarify the possible role in the pathology of spinal
cord trauma microglia from 15 dogs with spinal cord trauma confirmed by imaging, gross
and histopathological examination was isolated and characterized morphologically,
immunophenotypically, and functionally ex vivo by flow cytometry. Results were compared
to region-specific basic values.
Immunophenotypical characterization was performed using 12 different antibodies.
Surface markers responsible for co-stimulation of T-cells, leukocyte adhesion and
aggregation, and for lipid or glycolipid presentation showed an upregulation in traumatized
spinal cord. Statistically significant differences were found for the expression intensity of
B7-1, B7-2, MHC II, CD1c, ICAM-1, CD14, CD44 and CD45. Within the isolated microglia
from traumatized canine spinal cord a statistically significant higher expression intensity of
B7-1, MHC class I and class II was found in the cervical samples compared to the
thoracolumbar.
Functional investigation assessed by microglial phagocytosis of Staph. aureus together
with ROS generation after spinal cord trauma revealed a statistically significant decrease
in percentage of positive cells, whereas intensity of phagocytosis and ROS generation
appeared significantly increased.
In conclusion, due to their enhanced activation, microglia cells seem to play an important
role in the pathogenesis of spinal cord trauma. Consequently, an inhibition of this
activation has to be considered to encourage the success of new therapeutical strategies
such as transplantation of olfactoric ensheating cells (OECs).
Key words: spinal cord trauma, microglia, immunophenotyping, phagocytosis,
reactive oxygen species
Ergebnisse
30
1. Introduction
Microglial cells are known as the main immune effector cells of the central nervous system
(CNS; Kreutzberg, 1996; Aloisi, 2001; Liu et al., 2001; Streit, 2002; Streit, 2006). In case of
any pathologic stimuli they develop into an activated state.
For a considerable time the effect of microglial activation on neuronal repair respectively
damage are discussed controversly. A study by Rabchevsky and Streit (1997) resulted in a
creation of a pro-regenerative microenvironment evidenced by neuritic growth due to
transplantation of cultured microglia into injured, adult rat spinal cord. In agreement with
this, microglial activation after acute CNS injury reduces primary tissue damage and
promotes subsequent neuronal repair (Streit, 2002). In contrast to this, there are studies
underlining the harmful characteristics of microglia. Due to phagocytosis and the release of
potentially cytotoxic substances microglia play a key role in the initiation and mediation of
secondary autodestructive tissue damage (Banati et al., 1993) and contribute to further
axonal injury and cell death after spinal cord injury (SCI; Jeffery, 2009). Inhibition of
activated microglial cells lead to a decreased cell death and an improvement in functional
recovery after SCI in rats (Yune et al., 2009). Kempermann and Neumann (2003)
summarize microglial immune response to injury as a double-edged sword, simultaneously
beneficial and detrimental.
Microglial activation following pathological stimuli was described so far in human
neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, multiple
sclerosis, and amyotrophic lateral sclerosis (Thomas, 1992; Williams et al., 1994; Yiangou
et al., 2006), and in animals in canine distemper virus (Stein et al., 2004b), experimental
allergic encephalomyelitis (EAE; Vass and Lassmann, 1990; Ruuls et al., 1995), and
experimental traumatic lesions in mural brain (Schnell et al., 1999). Moreover, microglia in
an activated state was detected in the spinal cord of rodents in case of trauma (Schnell et
al., 1999; Sroga et al., 2003; Yang et al., 2005; Longbrake et al., 2007; Yune et al., 2009;
Baloui et al., 2009). The present study focuses on spinal microglial behaviour in respect to
trauma with the objective to evaluate influences on the pathogenesis. In an effort to reflect
microglial behaviour in their natural environment, these cells were characterized ex vivo.
Since spinal cord trauma in dogs provides an ideal translational model for new treatment
modalities (Jeffery et al., 2006), this species was investigated in the current study. Canine
spinal cord affected by a traumatic insult has not been investigated ex vivo so far. An
appropriate technique for examination of canine brain microglia via density gradient
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31
centrifugation has been established by Stein et al. (2004a) and modified for canine spinal
cord in our previous study. The aim of the present study was to accomplish a
morphological, immunophenotypical, and functional characterization of microglia in canine
spinal cord trauma to evaluate their pathogenetical role in degeneration and regeneration
of CNS tissue damage.
2. Materials and Methods
2.1 Animals
Fifteen three months to fifteen years old dogs of different breeds (6 mixed breeds, 3
Dachshunds, 2 Bernese Mountain Dogs and 1 each of German Shepherd Dog, Labrador,
Bavarian Mountain Dog, and Beagle) were presented at the Department of Small Animal
Medicine and Surgery, University of Veterinary Medicine, Hannover, Germany with spinal
cord traumata caused by intervertebral disk herniations or traffic accidents. Four dogs
(27%) were female, 11 were male (73%). Duration of neurological signs ranged from a few
hours to 42 days. In 5 dogs, the lesion was located in the cervical spinal cord, 10 dogs
were presented with a thoracolumbar lesion location. The dogs showed different severity
grades of neurological signs reflected by their status of ambulation, 7 dogs were paretic, 6
dogs were plegic. In the two remaining dogs ambulatory status could not be evaluated
reliably because of severe clinical signs. Of the 15 dogs included in the study, 6 had been
treated with glucocorticosteroids, 7 dogs had not received glucocorticosteroids, and in 2
dogs no information could be assessed about premedication. In 8 cases spinal cord
trauma was confirmed by magnetic resonance imaging (MRI). Euthanasia was elected by
the owners in all cases due to poor prognosis. Gross pathological and histopathological
examination confirmed spinal cord trauma in all cases. Ex vivo isolation and examination
of microglial cells from the lesion site was performed for each individual dog.
2.2 Monoclonal antibodies (mAbs) and membrane immunofluorescence
MAbs (Tab. 1) were used to detect the structures on the surface of the canine spinal
microglial cell as described before. The following antibodies were purchased: CD11b,
CD11c, CD45 (biotin linked), CD14 (conjugated with R-phycoerythrine), MHC class I,
CD44, CD3 (fluorescein-isothiocyanate-conjugated) and CD21 (conjugated with Rphycoerythrine). Monoclonal mouse antibodies against CD18, CD4, CD8α, B7-1 (CD80),
B7-2 (CD86), MHC class II, CD1c and ICAM-1 (CD54) were kindly provided by Professor
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32
Dr. Peter F. Moore (University of California, Davis, USA), prepared as tissue supernatants,
and used in a dilution of 1:5. Double stainings were performed for CD18 with CD45, and
CD3 with CD14.
Table 1
Monoclonal antibodies for immunophenotypical characterization of canine spinal
microglia.
The clone, isotype, dilution used in this study, and the company are given in the table.
CD = cluster of differentiation, MHC = major histocompatibility complex, ICAM-1 =
intracellular adhesion molecule-1.
Antibody
Host
Isotype
CD11b
CD11c
CD45
CD3
CD21
MHC I
CD14
CD44
mouse
mouse
rat
mouse
mouse
mouse
mouse
rat
IgG1
IgG1
IgG2b
IgG1
IgG1
IgG2a
IgG2a
IgG2a
CD18
mouse
IgG1
CD4
CD8α
B7-1 (CD80)
B7-2 (CD86)
MHC II
CD1c
ICAM-1
(CD54)
mouse
mouse
mouse
mouse
mouse
mouse
IgG1
IgG2a
IgG1
IgG1
IgG1
IgG1
mouse
IgG1
Dilution
Company
used
1:5
Serotec, Eching, Germany
1:5
1 : 16
1: 5
1:5
1 : 16
VMRD, Pullman, WA, USA
1:7
DAKO, Glostrup, Denmark
1 : 10
Leukocyte antigen biology
laboratory, Prof. Peter F.
1:5
Moore, University of
California, Davis, CA, USA
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
‫ײ‬
Human normal immunoglobuline G (in a dilution 1:16; Globuman Berna, Bern,
Switzerland) was added to the cell solution to block non-specific binding. Secondary,
fluorochrom-labled antibodies were given to bind the unconjugated primary antibodies
(Tab. 2). Goat anti mouse R-phycoerythrine (gαm-PE) was used for staining mAbs
originating from mouse tissues, the mAb of rat origin (CD44) was detected with rabbit anti
rat R-phycoerythrine (rαr-PE) and streptavidine-conjugated fluorescein-isothiocyanate (SAFITC) was used for the detection of CD45.
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Following staining procedure cells were measured ex vivo by flow cytometry with a
FACSCalibur® (BD Biosciences, Heidelberg, Germany). To identify microglial cells the
parameter size (FSC), complexity (SSC) and the relative expression of CD18, CD11b/c,
and expression intensity of CD45 were used as described before. Percentage of positive
cells (expression) and the mean fluorescence intensity (expression intensity) were
analyzed in two fluorescence channels (FL1, FL2) and results were evaluated with the
FACS Cell-Quest®-Software provided by BD Biosciences, Heidelberg, Germany.
Table 2
Secondary, fluorochrome-labeled antibodies for the detection of
primary antibodies.
The fragment, isotype, applied dilution, and the company are shown.
gαm-PE = goat anti mouse R-phycoerythrine, rαr-PE = rabbit anti rat Rphycoerythrine.
Antibody
Fragment
Isotype
Dilution
used
Company
gαm-PE
F(ab´)2
IgG
1 : 100
Dianova, Hamburg, Germany
rαr-PE
F(ab´)2
IgG
1 : 100
2.3 Isolation of spinal cord microglia
Isolation of microglia from the spinal cord was performed according to a protocol described
in our previous study. Briefly, an intravenous injection of pentobarbiturate (Narcoren®,
Merial GmbH, Hallbergmoos, Germany) in an overdose was used for euthanasia of dogs.
Spinal cord samples were removed immediately after death and contained the lesion site
and perilesional tissue from three to four spinal cord segments cranially and caudally.
Samples were immediately transferred to ice-cold Hanks´ solution with 3% fetal calf serum
and a pH of 7.36. Additionally, a segment of 1 cm size was taken from the epicenter of the
lesion and send to the Department of Pathology for histopathological examination. The
isolation protocol comprised a mechanical dissociation by mincing through a stainlesssteel sieve and enzymatical digestion of spinal cord tissue with collagenase (NB 8 from Cl.
histiolyticum, 5.7mg/g CNS tissue; SERVA, Heidelberg, Germany) and DNAse (DNAse
type IV: bovine pancreas, 500 U/g CNS tissue; Sigma-Aldrich Chemie GmbH, Steinheim,
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Germany) in dissociation buffer (89.4 g/l NaCl, 37.3 g/l KCl, 40.0 g/l MgCl2, 25.3 g/l CaCl2).
Microglial cells from the spinal cord were isolated using two consecutive density gradients.
For an initial gradient which was used to remove myelin and cell debris, cells were
resuspended in 45 ml of isotonic Percoll (GE healthcare, Uppsala, Sweden) at a density of
1.030 g/ml and underlayered with 5 ml of Percoll at a density of 1.124g/ml. After a
centrifugation step the cells were collected from the surface of the 1.124g/ml-layer. Cells
were washed and resuspended in 5 ml Hanks´ buffer, and added on top of a major
gradient consisting of 5 ml of Percoll at 1.124g/ml subsequently overlayered with Percoll
dilutions in Hanks’ buffer such as 12 ml of 1.077g/ml and 1.066g/ml Percoll each, followed
by 8 ml of the densities 1.050, and 1.030 g/ml Percoll each in a 50 ml-tube. Following a
centrifugation step the cells were gained from the surfaces of the 1.077 and 1.066g/mllayer.
The
collected
cells
were
used
immediately
for
the
morphological,
immunophenotypical and functional characterization.
2.4 Phagocytosis assay
The ability of spinal cord microglia to perform phagocytosis was evaluated as described
before by offering heat-killed and lyophilized FITC-labelled Staphylococcus aureus (Bio
Particles®, wood strain, without protein A, fluorescein conjugate, Molecular Probes Europe
B.V., Leiden, The Netherlands) for phagocytosis. The bacteria were adjusted to a
concentration of 8 x 108 bacteria/ml. 30 µl bacteria were treated with 30 µl pooled dog
serum diluted 1:5 with PBS for opsonization (opsonized bacteria) or 30 µl PBS (nonopsonized bacteria) were added and suspensions were incubated. Following incubation
180 µl PBS were added to each bacteria suspension resulting in a concentration of 108
bacteria/ml. 100 µl of microglial cells were mixed with 100 µl non-opsonized bacteria,
opsonized bacteria or PBS (negative control), the experiments were arranged in
duplicates. Following gentle suspension, assays were incubated as described before. To
stop phagocytosis reaction and to minimize adhesion of the cells on the surface of the
tubes, cells were put on ice for 15 minutes (min) after incubation.
FACSFlow® (BD Biosciences, Heidelberg, Germany) was added and the percentage of
microglia performing phagocytosis and the phagocytosis intensity (measured by
fluorescence intensity) of microglial cells were determined immediately ex vivo by flow
cytometry. To analyze the percentage of phagocytosis-positive microglia two steps were
used: a comparison of non-opsonized and opsonized bacteria with reference to the
negative control and phagocytosis of opsonized bacteria with reference to phagocytosis of
non-opsonized bacteria. Flow cytometry was accomplished in the fluorescence 1-height
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(FL1-H, green fluorescence) channel of a FACSCalibur® and the percentage of
phagocytoting
cells
respectively
the
mean
fluorescence
intensity
(intensity
of
phagocytosis) were analyzed using FACS Cell-Quest®-Software provided by BD
Biosciences, Heidelberg, Germany.
2.5 ROS generation test
The production of reactive oxygen species (ROS) by microglial cells was investigated as
described by Emmendörffer et al. (1990). In this method the non-fluorescent
dihydrorhodamine 123 (DHR 123, MoBiTec GmbH, Göttingen, Germany) is converted
during ROS generation by membrane-adapted myeloperoxidase into the green-fluorescent
rhodamine123. Microglial cells were triggered with Phorbol-myristate-acetate (PMA,
Sigma, Deisenhofen, Germany) which was dissolved in dimethylsulfoxide (DMSO, SigmaAldrich, Deisenhofen, Germany) and diluted with PBS to result in a concentration of 100
nmol/l cell suspension. Isolated microglial cells were pre-incubated at 37°C and 5 % CO2
for 15 min to achieve comparable and identical levels of activation. As a negative control
(evaluation of morphology and background fluorescence of the cells) one tube with
microglial cells only was applied. To compare non-stimulated and PMA-stimulated ROS
generation, either 10 µl PBS or 10 µl PMA were added to the microglial suspensions (90
µl). After a 15 min incubation time DHR 123 (20 µl) was added (except for the negative
control) and a further incubation step was performed. Samples were measured
immediately ex vivo by flow cytometry after adding of FACSFlow®. ROS-generation was
determined by analyzing both, the percentage of positive microglial cells, and the mean
fluorescence intensity as an indirect means for quantity of ROS-generation. The
percentage of microglial cells performing ROS-generation was evaluated by comparing
negative control with non-stimulated and PMA-stimulated microglia. In a further analyzing
step ROS generation of PMA-stimulated microglia was compared to that of non-stimulated
microglia. To ensure reproducibility of the results, approaches were performed in
duplicates and mean values were used for evaluation. Flow cytometric measurement and
evaluation of percentage of ROS generating cells and the mean fluorescence intensity
(intensity of ROS generation) was performed using a FACSCalibur® (BD Biosciences,
Heidelberg, Germany, FL-1 channel) and FACS Cell-Quest®-Software provided by BD
Biosciences, Heidelberg, Germany.
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2.6 Statistics
All data were included into a descriptive analysis. Shapiro-Wilk-Test and visual
assessment of normal probability plots were utilized for confirmation of normal distribution
of model-residuals. For the normal distributed parameter “percentage of positive cells”
arithmetic means () and standard deviation (S.D.) was calculated, for the right skewed
distributed parameter “fluorescence intensity” logarithmic transformation was performed
prior to analysis. Geometric mean and geometric standard deviation was calculated and
partly diagrammed on the original scale. For analyzing the effect of “localization” (cervical,
thoracolumbar) and “treatment of cells” (opsonization respectively concentration of PMA)
within localizations in dogs with spinal cord trauma two-way analysis of variance with
independent effect “localization” and “treatment of cells” as repeated measurements was
used, taking into account possible interactions between the two effects. A comparison of
“percentage of positive cells” and “fluorescence intensity” between dogs with spinal cord
trauma and healthy dogs, stratified by localization, “characterization” (mAbs, phagocytosis,
ROS generation) and “treatment of cells” was performed using unpaired two-sample ttests. Age, duration of clinical signs, pre-treatment, and ambulatory status were analyzed
by one-way analysis of variance with post-hoc tukey multiple pairwise comparisons.
Regarding the experimentwise error rate, values of p < 0.05 (), p < 0.01 (), and
p < 0.001 () were considered significant. Analyses were carried out with the
statistical software SAS®, version 9.2 (SAS Institute, Cary, NC) in a Windows
XP®environment. For the analysis of the linear model, the procedure mixed was used.
Data in tables and figures were presented using Microsoft® Office Excel® 2003 and 2007
(Microsoft Corporation, Redmond, Washington, USA) and GraphPad PRISM® (GraphPad
Software, La Jolla, California, USA).
3. Results
3.1 Identification and purity
Microglia was identified both, morphologically in a dot blot displaying size (FSC) versus
complexity (SSC) and immunophenotypically. Cells appeared as a population of relatively
small cells with a phenotype of CD18+, CD11b/c+ and CD45low. In five dogs with
traumatized spinal cord the microglial population showed a large diversity in morphology
with an increase in size and complexity (Fig. 1) compared to the healthy control dogs.
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Technical purification resulted in a microglial cell gate with sufficient purity (92.5%) for
further characterization. Only a very small proportion of the isolated cells showed an
expression of the lymphocyte markers CD4 ( = 4.1 %), CD8α ( = 3.4 %),
CD3 ( = 7.5 %), and CD21 ( = 2.2 %).
Figure 1
Microglial cell population isolated from healthy (a) and traumatized (b) cervical spinal
cord displayed in a Dot Plot.
The abscissa shows the size (FSC = forward scatter), complexity (SSC = side scatter) is
presented on the ordinate. In healthy spinal cord (a) microglia appears as a homogenous
population of relatively small cells (black line), whereas microglia from traumatized spinal
cord (b) showed a larger diversity in morphology with an increase in size and complexity
(black line) in five dogs.
3.2 Immunophenotypical characterization of canine microglia
Twelve different antibodies were used to characterize microglial immunophenotype.
Whereas no upregulation was found in the percentage of positive cells, microglia from
traumatized cervical and thoracolumbar spinal cord showed a significantly enhanced
expression intensity of B7-1 (p < 0.0001), B7-2 (p < 0.0001), MHC II (cervical: p < 0.0001,
thoracolumbar p = 0.0428), CD1c (p < 0.0001), ICAM-1 (cervical: p < 0.0001,
thoracolumbar p = 0.0007), CD45 (p < 0.0001), CD14 (p < 0.0001), and CD44 (cervical: p
< 0.0001, thoracolumbar p = 0.0006) compared to the region-specific values from healthy
dogs (Fig. 2). The highest enhancements in microglial expression intensities were found
for B7-1 and CD45 in the cervical spinal cord, which were about 11fold higher, and for
B7-1 in the thoracolumbar spinal cord which was 9.5fold higher than in the healthy control
dogs.
Comparison of the percentages of surface molecule expression from microglia originating
from traumatized cervical versus traumatized thoracolumbar spinal cord did not result in
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any differences. In contrast, the expression intensities of B7-1 (p = 0.0463), MHC class I
(p = 0.0173), and MHC class II (p = 0.0227) were significantly higher in the traumatized
cervical than in the thoracolumbar spinal cord (Fig. 3).
(a)
(b)
(c)
(d)
(e)
(f)
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Figure 2
39
Expression intensities of the surface antigens B7-1 (a), B7-2 (b), CD1c (c), MHC II (d),
ICAM-1 (e), CD45 (f) on microglia in dogs with spinal cord trauma compared to healthy
reference dogs.
Localizations of the trauma differentiated as cervical and thoracolumbar and their regionspecific references are shown on the abscissa and the fluorescence intensity on the
ordinate. Upper and lower limits of the box represent the 25% and 75% quartiles,
respectively. Horizontal bars give the median values, whiskers display minimum and
maximum values.
Microglia of dogs with cervical and thoracolumbar trauma (n = 5 and n = 10, respectively)
showed a significantly up-regulated expression intensity ( = p < 0.05, = p < 0.01,
= p < 0.001) measured by mean fluorescence intensity) of B7-1 (CD80) (p < 0.0001), B7-2
(CD86) (p < 0.0001), MHC II (cervical: p < 0.0001, thoracolumbar: p = 0.043), CD1c
(p < 0.0001), ICAM-1 (CD54) (cervical: p < 0.0001, thoracolumbar: p = 0.0007 ), CD45
(p < 0.0001), CD14 (p < 0.0001 ), and CD44 (cervical: p < 0.0001, thoracolumbar:
p = 0.0006) compared to the cervical and thoracolumbar values in healthy dogs (n = 22 and
n = 22, respectively). CD = cluster of differentiation, MHC = major histocompatibility complex,
ICAM-1 = intracellular adhesion molecule-1.
Figure 3
Comparison of microglial expression intensities of the surface antigens B7-1, MHC I,
and MHC II in traumatized cervical (grey box plots) and thoracolumbar (white box
plots) spinal cord.
The surface antigens are displayed on the abscissa, and mean fluorescence intensity is
shown on the ordinate. Upper and lower limits of the box represent the 25% and 75%
quartiles. Horizontal bars give the median values, whiskers display minimum and maximum
values.
Canine microglia from traumatized cervical spinal cord (grey boxes; n = 5) showed
significantly higher expression intensities () of B7-1 (p = 0.046), MHC I (p = 0.017), and
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40
MHC II (p = 0.023) compared to traumatized thoracolumbar spinal cord (white boxes; n =
10). CD = cluster of differentiation, MHC = major histocompatibility complex.
3.3 Phagocytosis assay
Microglia in dogs with spinal cord trauma showed a tendency for an increased
phagocytosis of opsonized bacteria when compared to the healthy controls, which was
very distinct in thoracolumbar spinal cord trauma (p = 0.05; Fig. 4a). Moreover, the mean
fluorescence intensity as a means for the microglial phagocytosis intensity revealed a
1.4fold and 1.5fold increase in cervical respectively thoracolumbar spinal cord trauma
compared to values from healthy control dogs. This finding was statistically significant for
microglia from traumatized thoracolumbar spinal cord and phagocytosis of opsonized
bacteria (p = 0.0366; Fig. 4b). A comparison of microglial phagocytosis activity in cervical
versus thoracolumbar spinal cord trauma did not reveal any statistically significant
differences.
Phagocytosis
(a)
Figure 4
(b)
Results of phagocytosis of opsonized Staphylococcus aureus in a) percentages of
phagocytosing microglia and b) phagocytosis intensity originating from traumatized
cervical and thoracolumbar spinal cord and region-specific reference values.
Localizations of the trauma differentiated as cervical (n = 5) and thoracolumbar (n = 10) and
the region-specific values of healthy control dogs (n = 22 each) are shown on the abscissa,
and a) the percentage of phagocytosing microglia or b) intensity of phagocytosis are
displayed on the ordinate. Upper and lower limits of the box represent the 25% and 75%
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41
quartiles. Horizontal bars display the median values, whiskers show minimum and maximum
values.
The percentage of microglia performing phagocytosis (a) as well as the phagocytosis
intensity (b) was higher in traumatized spinal cord compared to the healthy controls. This
enhanced percentage and intensity were statistically remarkable () respectively significant
() in traumatized thoracolumbar spinal cord compared to the healthy region-specific
reference (p = 0.05 and 0.04, respectively).
3.4 Generation of ROS
Whereas no upregulation could be found in the percentage of ROS generating microglia
the intensity of microglial ROS generation revealed a twofold increase after spinal cord
trauma compared to the region-specific values of healthy control dogs. This finding
reached the level of significance in thoracolumbar spinal cord trauma (p ≤ 0.03) whereas in
cervical spinal cord trauma only a distinct tendency for an enhanced ROS generation
intensity could be seen (Fig. 5) in comparison to the region-specific control. The increase
in ROS generation was very distinct in the five dogs showing the great diversity in
microglial morphology (data not shown). Furthermore, a comparison of microglial ROS
generation in spinal cord trauma did not result in any statistically significant differences
between the two regions - cervical and thoracolumbar - examined.
ROS generation
(a)
(b)
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Figure 5
42
Comparison of microglial non-stimulated (a) and PMA-stimulated (b) ROS-generation
intensity in traumatized cervical and thoracolumbar spinal cord, and their regionspecific reference values.
Cervical (n = 5) and thoracolumbar (n = 10) trauma and their region-specific references
(cervical ref. and thoracolumbar ref., n = 22 each) are shown on the abscissa, the ordinate
displays the ROS intensity. Upper and lower limits of the box represent the 25% and 75%
quartiles. Horizontal bars display the median values, whiskers show minimum and maximum
values.
Microglia in spinal cord trauma showed a higher ROS-generation intensity in respect to
healthy region-specific values in healthy control dogs which was statistically significant ()
in thoracolumbar samples both non-stimulated (a) (p = 0.003) and stimulated with PMA (b)
(p = 0.001). PMA = Phorbol-myristate-acetate, ref. = reference.
3.5 Age, duration of clinical signs, pre-treatment with glucocorticosteroids, and
ambulatory status
Results of microglial characterization were correlated to age, duration of clinical signs,
treatment of dogs with glucocorticosteroids, and their status of ambulation.
Older age of the dogs was correlated with an increase in the percentage of ROS
generating microglia, respectively CD1c+, and CD45+ cells, and with the expression
intensity of B7-1, B7-2, MHC I, ICAM-1, CD45, CD44, and CD14.
The percentage of microglia cells performing phagocytosis decreased with duration of
clinical signs. In contrast to this, expression intensity of CD11c, ICAM-1, and CD1c
increased with duration of clinical signs.
Dogs with spinal cord trauma treated with glucocorticosteroids prior to microglial
examination showed a lower percentage of ROS generating microglial cells. Moreover, a
higher expression intensity was seen for B7-1, MHC I and II, CD1c, ICAM-1, CD45, and
CD44 in these dogs.
Plegic dogs tended to have a higher percentage of ROS generating microglia after PMA
stimulation compared to paretic dogs.
4. Discussion
Spinal cord trauma in the dog is considered to be an ideal translational model between
rodent experiments and human clinical trials. To investigate the role of microglia in the
pathogenesis in respect to harmful or beneficial characteristics, these cell population was
isolated from dogs with spinal cord trauma using density gradient centrifugation, and
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43
characterized morphologically, immunophenotypically, and functionally by flow cytometry.
Only very few studies are published investigating microglial cells ex vivo in CNS injury
(Sedgwick et al., 1991; Stein et al., 2004b; Stirling and Yong, 2008), and this is the first
time canine microglia is examined and characterized ex vivo following spinal cord trauma.
There are many differences between naturally occurring and experimentally induced
injuries concerning the homogeneity in severity, type, and precise location of the lesion
(Jeffery et al., 2006) and therefore comparability of the results. However, the dog
represents an ideal model for isolating spinal cord microglia and offers the possibility to
investigate specific regions within the CNS as shown in our previous study. Due to
extensive homology of canine and human spinal cord trauma in terms of mechanisms of
injury, pathology, classification, functional monitoring, advanced imaging, and outcome
(Purdy et al., 2004; Jeffery et al., 2006), the dog represents a valuable translational animal
model for human spinal cord diseases and enables outstanding advances in human
pathology research. The amenability of spinal cord tissue for ex vivo examination
potentially closely reflecting conditions in vivo underlines the great advance of using
canine models compared to humans (Jeffery et al., 2006).
Density gradient centrifugation led to a convincing microglial purity and proved to be an
efficient method for the isolation of microglia from the canine spinal cord in our previous
study. Microglial purity achieved was slightly lower following trauma compared to our
previous study with spinal cord from healthy dogs. This might reflect a higher difficulty in
isolating microglial cells from traumatized canine spinal cord tissue due to their
hypertrophy and stout processes, following pathological insults leading to a bushy
appearance (Streit, 1995) and possibly resulting in entangled cells. Lymphocyte
contamination was extremely low.
Furthermore, isolated cells showed a higher expression intensity of CD14 and CD45 in
comparison to reference values of healthy dogs. This increased expression can have
several reasons (Popovich and Hickey, 2001). The phenotype could be ascribed to spinal
cord microglia up-regulating certain surface molecules in case of activation or tissue
macrophages adapted to the novel environment and tasks could have down-regulated
their surface antigens. Due to a lack of antigens uniquely expressed by microglia (Perry
and Gordon, 1991), it is difficult to make a distinction between these two cell types.
Recently, regional topographic differences were described in microglial expression of
surface molecules and function. These values were used to evaluate data in spinal cord
trauma
and
the
evaluation
revealed
an
increase
in
immunophenotypical, and functional characterization of microglia.
the
morphological,
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44
Consistent with this, several studies described an enhanced microglial activation following
spinal cord trauma in rats and mice (Schnell et al., 1999; Sroga et al., 2003; Yang et al.,
2005; Beck et al., 2010)
Immunophenotypic characterization revealed an upregulation of B7-1 and -2, CD1c,
MHC II, ICAM-1, CD14, CD44, and CD45 in dogs with spinal cord trauma compared to
healthy control dogs. This upregulation reflects microglial activation by transformation from
a resting phenotype into an activated state. It is well known that upon activation microglial
cells are capable of upregulation and de-novo expression of surface molecules (Thomas,
1992; Flaris et al., 1993; Streit, 1995; Stein et al., 2006). Microglial activation might also be
reflected by the morphological diversity seen in five dogs possibly representing different
functions of these cells.
Expression intensity of MHC II was significantly higher in the dogs with spinal cord trauma
compared to the healthy control dogs, which is consistent with immunohistochemical
studies investigating microglia from injured spinal cord in rats and mice (Popovich and
Hickey, 2001; Sroga et al., 2003). Furthermore, an upregulation of CD1c was seen on
canine spinal microglia after trauma. Upregulation of the MHC and CD1c underlines the
fact of microglia being an immunocompetent cell of the CNS, which is capable of
processing and presenting peptidic and non-pepidic lipid- and glycolipid antigen (Ulvestad
et al., 1994, Sedgwick et al., 1991; Gehrmann and Kreutzberg, 1995; Aloisi et al., 2000;
Bußhoff et al., 2001, Stein et al., 2004a, b). In the event of trauma, spinal cord microglia
cells seem to extend this ability to fulfill their tasks as repairing cells and gaining the
function of full blown macrophages.
According to their antigen presenting capacity by MHC or CD1c, for the activation of Tcells microglia require co-stimulating molecules on their surface such as B7-1 (CD80) and
B7-2 (CD86). Additionally, the expression of adhesion-molecules such as ICAM-1 (CD54)
is essential for interaction between the antigen presenting cell and the T-cell. In this study,
the expression intensity of B7-1, B7-2 and ICAM-1 was significantly higher compared to
the values in healthy control dogs. According to this, Rutkowski et al. (2004) also observed
an upregulation of B7-2 on spinal cord microglia following peripheral nerve injury. This
upregulation of B7-1, -2 and ICAM-1 might lead to facilitated cell adhesion and more
efficient co-stimulation of T-cells in the course of spinal cord injury.
Following activation, an upregulation of CD45 on microglia from rodents and canine brain
is described (Sedgwick et al. 1991; Stein et al. 2007). This upregulation of CD45 can be
confirmed for microglia from traumatized canine spinal cord. An increased expression
intensity of CD45 by microglial cells could lead to a more effective transduction of signals
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45
from molecules on the surface of cells, such as Fc-receptors, manifested in an elevated
phagocytosing
capacity.
Upregulation
of
CD44,
an
extracellular
matrix
phosphoglycoprotein, involved in cell adhesion and immunomodulation was found by
Moon et al. (2004) in rats with spinal cord injury after clip compression. This is in
accordance with the results of our study in naturally occurring spinal cord disease,
emphasizing the role of this surface molecule in response to trauma and repair of
damaged CNS tissue (Jones et al., 2000; Moon et al., 2004).
It was shown recently, that regional topographic immunophenotypic and functional
differences already exist in resting microglia in healthy spinal cord potentially reflecting
different states of microglial alertness. Interestingly, microglial immunophenotype in spinal
cord trauma also revealed an elevated microglial expression intensity of B7-1, MHC I and
II, in cervical compared to thoracolumbar spinal cord. In the healthy canine spinal cord
regional topographic differences occur in the state of alertness of microglia caused by
region specific requirements. Under pathological conditions this could lead to a higher
expression in regions with per se higher state of alertness.
In addition to an upregulation in the expression of surface molecules, canine microglia
performs macrophage effector functions such as phagocytosis and ROS generation during
the state of activation (Kreutzberg, 1996; Popovich, 2002). Functional characterization
revealed a distinct tendency of increased microglial phagocytosis in traumatized spinal
cord compared to findings in healthy dogs. Furthermore, the intensity of phagocytosis was
higher in traumatized spinal cord in relation to reference values of healthy dogs displaying
the higher activation status.
Enhanced phagocytosis is necessary to remove the high amount of damaged cells
respectively debris in an acute trauma. Therefore, an upregulation of phagocytosis in the
beginning of clinical signs might initialize subsequent healing processes of the spinal cord.
Consistent with this, the percentage of phagocytosis-positive microglia decreased with
duration of clinical signs.
According to our study, a microglial transformation into phagocytic cells was detected after
SCI in rats (Isaksson et al., 1999). Additionally, Taccola et al. (2010) described a
significant microglial activation associated with phagocytosis in segments below the lesion
after SCI in vitro in rat spinal cord.
A higher ROS generation was detected in traumatized spinal cord in comparison to
reference values of healthy dogs. This finding reached the level of significance in
thoracolumbar spinal cord whereas cervical spinal cord showed a distinct tendency for an
upregulation. Consistent with the present study, Yune et al. (2009) described a dramatical
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46
increase in microglial ROS generation following experimental SCI in rats. An elevated
ROS production following SCI in mice was also detected by Xu et al. (2005), who showed
a correlation between increased ROS generation and SCI-mediated motor neuron death
additionally. Furthermore, an in vitro spinal cord injury model performed by Luo et al.
(2002) revealed an increase of post-injury ROS signals detected by flow cytometry in
guinea pigs. Despite microglial ROS generation contributes to immune defence, this
function can lead to a direct damage of healthy neurons also (Bruce-Keller, 1999). Streit
(2002) described microglial activation after acute CNS injury as a reactive and adaptive
glial cell response (injury-induced activation; Streit, 2006) triggered by injured neurons to
promote subsequent repair. This injury-induced activation can be associated with an
irreversible microglial hyperactivation by which microglia is suspected to injure neurons
and to be involved in secondary damage after trauma (Nakamura, 2002).
Activated microglial cells are the primary source of the neurotoxic ROS (Qin et al., 2004).
Thus, a bystander damage in the traumatized spinal cord caused by microglial ROS
generation has to be taken into consideration. Indeed a higher percentage of ROS
generating microglia was seen in more severely compromised plegic dogs compared to
paretic dogs. This finding gives evidence for ROS generation as an important factor in
secondary damage (Jeffery, 2009) which could be manifested in more serious spinal cord
damage or in a functional damage causing the severe clinical signs. This is underlined by
an increased expression intensity of CD11c, ICAM-1, and CD1c correlated with a longer
duration of clinical signs.
In conclusion, the upregulation of certain surface molecules observed in this study is
indicative for an activation of microglia in spinal cord trauma. Furthermore, increased
microglial function seems to play a pivotal role in the pathogenesis of spinal cord trauma
and may have fundamental influences on recovery. Despite microglial potential of
destroying invading microorganisms, removing potentially deleterious debris and
promoting tissue repair (Kreutzberg, 1996) the potentially harmful characteristics of
microglial function have to be considered. This is of importance in the research concerning
new therapeutical strategies such as transplantation of olfactory ensheathing cells (OECs),
providing trophic support for injured neurons and angiogenesis which results in an
improved functional recovery (Radtke et al., 2008; Kocsis et al., 2009). A potentially
defense response directed against implanted cells such as OECs by activated microglia
has to be assumed. Therefore, a medication-induced repression of microglial activation
previous to implantation of OECs should be considered to ensure the success of this
approach (Ekdahl et al., 2003; Festoff et al., 2006).
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47
Acknowledgements
The study was supported by the German Research Foundation (FOR 1103) and the
Frauchiger Stiftung, Bern, Switzerland.
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Zusammenfassung der Ergebnisse beider Studien
53
III. Zusammenfassung der Ergebnisse beider Studien
Die erste Studie in dieser Arbeit beschäftigte sich mit der retrospektiven Evaluierung von
Korrelationen zwischen qualitativen beziehungsweise quantitativen MRT-Befunden und
Parametern des klinischen Verlaufs von paraplegischen Hunden, welche im Zeitraum von
Januar 2005 bis Juni 2009 in der Kleintierklinik der Tierärztlichen Hochschule Hannover
vorgestellt wurden.
Dreiundsechzig paraplegische Hunde erfüllten die Einschlusskriterien für diese Studie.
Darunter waren 63% Dackel, 16% Mischlinge, 6% Jack Russell Terrier, 3% Pekinesen und
jeweils ein Amerikanischer Cocker Spaniel, Yorkshire Terrier, Shi Tzu, Toy Pudel, Bolonka
Zwetna sowie eine Bulldogge und eine Dachsbracke. Das Alter der Tiere bei Vorstellung
reichte von einem Jahr bis zu 13 Jahren (Durchschnitt 6,9 Jahre). Neununddreißig Hunde
waren männlich, davon 7 kastriert, 24 Hunde waren weiblich und davon 5 kastriert. Das
durchschnittliche Körpergewicht betrug 9,2 kg. Bei Vorstellung in der Klinik zeigten 78%
der Hunde neurologische Ausfälle entsprechend Grad 4 nach Sharp und Wheeler (2005),
22% wurden mit Grad 5 präsentiert. Die Krankheitsdauer variierte von einem Tag bis zu 32
Tagen. Eine Vorbehandlung mit Glukokortikosteroiden erhielten 28 Hunde, bei 23 Hunden
konnte dieses ausgeschlossen werden und von 12 Hunden konnten keine Informationen
über etwaige Vorbehandlungen eingeholt werden. Der Zeitraum, nach dem eine
Besserung der neurologischen Symptomatik um eine Stufe des Schweregrades eintrat,
reichte von einem Tag bis hin zu mehreren Wochen. Die Auswertung der MRT-Befunde
zeigte das Auftreten einer intramedullären Hyperintensität bei 59% aller untersuchten
Hunde, von welchen 32% eine Ausdehnung entsprechend Kategorie 4 (> 2 x Länge des
zweiten Lendenwirbels (L2)), 38% entsprechend Kategorie 3 (> 1 x L2), 16%
entsprechend Kategorie 2 (≤ 1 x L2) und 14% eine Ausdehnung entsprechend Kategorie 1
(≤ ½ x L2) zeigten. Der Grad der Rückenmarkskompression konnte eingeteilt werden in
Kategorie 1: ≤ 25% (13% aller Hunde), Kategorie 2: ≤ 50% (46% aller Hunde), Kategorie
3: ≤ 75% (38% aller Hunde) sowie in Kategorie 4: > 75% (3% aller Hunde).
Statistisch signifikante Korrelationen konnten zwischen den Graden nach Sharp und
Wheeler (2005) bei Vorstellung und dem Vorliegen einer intramedullären Hyperintensität
sowie deren Ausdehnung in T2-Gewichtung festgestellt werden (p = 0,02). Hierbei zeigte
der
höhere
Grad
5
ein
wahrscheinlicheres
Auftreten
einer
Hyperintensität
beziehungsweise deren größere Ausdehnung. Außerdem wurde beobachtet, dass der
Grad der Rückenmarkskompression signifikant mit der Dauer des Krankheitsgeschehens
angestiegen ist (p < 0,001). Die Ausdehnung der Hyperintensität hing vom Grad der
Zusammenfassung der Ergebnisse beider Studien
54
Kompression des Rückenmarkes ab (p = 0,05). Der Zeitraum, in dem sich der
neurologische Zustand um einen Grad besserte, war tendenziell kürzer, wenn keine
beziehungsweise nur eine Hyperintensität geringer Ausdehnung vorlag.
Die zweite Studie in dieser Arbeit beschäftigte sich mit der immunphänotypischen und
funktionellen
Untersuchung
kaniner
Mikrogliazellen
aus
dem
zervikalen
und
thorakolumbalen traumatisierten Rückenmark. Die immunphänotypische Charakterisierung
wurde mittels 12 verschiedener Antikörper durchgeführt, in der funktionellen Untersuchung
wurde zum einen die Phagozytose von Staphylococcus aureus sowie zum anderen die
ROS-Bildung durch Mikroglia bei 15 Hunden mit Rückenmarkstraumata evaluiert.
Mikroglia, welche aus traumatisiertem Rückenmark isoliert wurde, zeigte eine signifikant
höhere
Expressionsintensität
von
B7-1
(p
<
0,0001),
B7-2
(p
<
0,0001),
Haupthistokompatibilitätskomplex II (major histocompatibility complex, MHC II; zervikal:
p < 0,0001, thorakolumbal: p = 0,0428), Cluster of Differentiation 1c (CD1c; p < 0,0001),
interzellulärem Adhäsionsmolekül 1 (intercellular adhesion molecule-1, ICAM-1; zervikal:
p < 0,0001, thorakolumbal: p = 0,0007), CD45 (p < 0,0001), CD14 (p < 0,0001) und CD44
(zervikal: p < 0,0001, thorakolumbal: p = 0,0006) verglichen mit gesundem Rückenmark.
Ferner stellte sich nach einem Trauma die Expressionsintensität von B7-1, MHC I und
MHC II im zervikalen Rückenmark signifikant höher dar als im thorakolumbalen
Rückenmark.
Die Phagozytoserate im traumatisierten Rückenmark war tendenziell erhöht im Vergleich
zu gesundem Rückenmark. Des Weiteren zeigte sich die Phagozytoseintensität
beziehungsweise die Menge der phagozytierten Bakterien deutlich erhöht nach
Rückenmarkstrauma,
was
durch
eine
statistische
Signifikanz
(p = 0,0366)
für
thorakolumbales Rückenmark unterstrichen wurde.
Die Untersuchung der ROS-Bildung resultierte in einem deutlichen Anstieg der Intensität
der ROS-Bildung nach Rückenmarkstrauma. Dies war statistisch signifikant für
thorakolumbale Proben (p ≤ 0,03), wobei zervikales Rückenmark eine deutliche Tendenz
zu erhöhten Werten zeigte.
Übergreifende Diskussion
55
IV. Übergreifende Diskussion
Die retrospektive Evaluierung von Korrelationen zwischen qualitativen beziehungsweise
quantitativen MRT-Befunden und Parametern des klinischen Verlaufs 63 paraplegischer
Hunde zeigte, dass Patienten mit hohem neurologischen Schweregrad vor der Operation
(OP) signifikant häufiger eine Hyperintensität sowie eine größere Ausdehnung der
Hyperintensität in T2-gewichteten MRT-Sequenzen aufweisen als Hunde mit einem
niedrigen neurologischen Schweregrad prä OP. Die Befunde wurden an einer annähernd
homogenen Hundepopulation erhoben. Die Hunde hatten ein Körpergewicht < 20 kg, um
die Prognose nicht durch unterschiedliche Körpergewichte zu beeinflussen. Diese
Ergebnisse stimmen mit ähnlichen Studien an einer heterogenen Hundepopulation
(LEVINE et al., 2009) sowie Menschen (SCHAEFER et al., 1989; MIYANJI et al., 2007;
MIRANDA et al., 2008) überein.
Eine Hyperintensität des Myelons in der T2-gewichteten MRT-Sequenz kann unter
anderem pathologische Prozesse wie Nekrosen, Myelomalazien, Blutungen sowie Ödeme
darstellen (ITO et al., 2005; SANDERS et al., 2002). Diese Veränderungen können den
höheren
neurologischen
Schweregrad
mit
einem
schwerwiegenderen
Rückenmarksschaden gut erklären. Eine solche Rückenmarksschädigung kann zum
Beispiel durch einen Bandscheibenvorfall verursacht werden.
Zum einen kann vorgefallenes Bandscheibenmaterial zu einer Erschütterung und
Kompression des Rückenmarkes führen und eine Reihe von metabolischen und
biochemischen Prozessen induzieren (JEFFERY, 2009), welche Gewebsnekrose sowie
eine Schädigung der Blutversorgung zur Folge haben können (GRIFFITHS, 1972; PLATT
u. OLBY, 2004).
So konnte in vorliegender Studie eine größere Ausdehnung der Hyperintensität des
Myelons in T2-gewichteten MRT-Aufnahmen bei Zunahme der Kompression des
Rückenmarkes durch Bandscheibenmaterial beobachtet werden, was im Einklang mit
einer Studie von PURDY et al. (2004) steht, in welcher eine Abhängigkeit der
Rückenmarksschädigung von dem Grad der Kompression aufgezeigt wurde.
Im zweiten Teil dieser Arbeit konnte nachgewiesen werden, dass ein Bandscheibenvorfall
zu einer Aktivierung der Mikroglia führt. Eine gesteigerte Produktion reaktiver
Sauerstoffspezies kann zu einer direkten Neuronenschädigung (BRUCE-KELLER, 1999)
und folglich Nekrose und Myelomalazie des Rückenmarkes führen, was wiederum die
MRT-Befunde gut erklären kann.
56
Der langfristige Therapieerfolg nach einem Bandscheibenvorfall fiel bei Vorliegen einer
Hyperintensität sowie einer größeren Ausdehnung in der T2-gewichteten MRT-Sequenz
tendenziell schlechter aus. Diese Tendenz wurde verifiziert durch andere Studien, welche
dem Vorhandensein einer intramedullären Hyperintensität (MIYANJI et al., 2007;
YUKAWA et al., 2007) beziehungsweise deren Ausdehnung (FLANDERS et al., 1999;
SELDEN et al., 1999; MIYANJI et al., 2007) beim Menschen einen prognostischen Wert
zusprachen.
Auch Studien mit Hunden zeigten Korrelation zwischen Therapieerfolg und Vorliegen
beziehungsweise Ausdehnung der Hyperintensität in der T2-gewichteten MRT-Sequenz.
ITO et al. (2005) wiesen eine schlechte Prognose für paraplegische Hunde nach, die eine
Hyperintensität ab einer Länge, die dem zweiten Lendenwirbel entsprach, aufwiesen. Dies
wird unterstützt durch eine Studie von LEVINE et al. (2009), in welcher ein herabgesetzter
langfristiger Therapieerfolg im Zusammenhang mit Vorhandensein und Ausdehnung einer
Hyperintensität in der T2-gewichteten MRT beschrieben wird.
So lässt sich also zusammenfassen, dass es deutliche Hinweise darauf gibt, dass den in
dieser
Studie
untersuchten
Hyperintensität
in
der
qualitativen
T2-gewichteten
und
MRT
quantitativen
ein
wichtiger
Eigenschaften
prognostischer
der
Wert
zugesprochen werden kann. Somit können sie bisherige prognostische Faktoren wie den
neurologischen Schweregrad prä OP und das Vorhandensein der Tiefensensibilität
(SCOTT, 1997) sinnvoll unterstützen.
Ergänzende Methoden zur Einschätzung der Prognose nach einem Bandscheibenvorfall
stellen wichtige Hilfsmittel für die Auswahl neuer Therapieansätze, wie zum Beispiel der
Implantation olfaktorischer Hüllzellen, dar.
Olfaktorische Hüllzellen haben in vorausgegangenen Studien durch die Unterstützung
geschädigter Neuronen und der Angiogenese gute Erfolge in der Regeneration des
Rückenmarkes erzielt (RADTKE et al., 2008; KOCSIS et al., 2009). Ihre Implantation in
traumatisiertes Rückenmark kann daher die Chancen auf einen größeren Therapieerfolg
erheblich steigern.
Fortschritte
in
der
Weiterentwicklung
dieser
Therapiemaßnahmen
von
Rückenmarkstraumata beim Hund sind auch auf den humanmedizinischen Bereich
übertragbar, da das kanine Rückenmarkstrauma aufgrund vergleichbarer Eigenschaften in
der Entstehung, Diagnostik und Behandlung ein wichtiges Translationsmodell für das des
Menschen darstellt (PURDY et al., 2004; JEFFERY et al., 2006).
Für
eine
57
erfolgreiche
Implantation
olfaktorischer
Zellen
ist
es
wichtig,
die
Abwehrmechanismen des Rückenmarkes, insbesondere die der Mikroglia, welche die
residenten Immuneffektorzellen in diesem Gewebe darstellen, einschätzen zu können.
Aus diesem Grund wurde in der zweiten Studie dieser Arbeit Mikroglia von Hunden mit
Rückenmarkstrauma
isoliert
und
mittels
Durchflusszytometrie
morphologisch,
immunphänotypisch sowie funktionell charakterisiert.
Vorausgegangene Studien beschäftigten sich mit der ex vivo-Untersuchung von
Mikrogliazellen
aus
pathologisch
verändertem
Gehirn
oder
Rückenmark
(SEDGWICK et al., 1991; STIRLING u. YONG, 2008), in der vorliegenden Studie wurde
zum ersten Mal kanines Rückenmark nach Rückenmarkstrauma ex vivo untersucht.
Klinische Rückenmarkstraumata weisen im Vergleich zu experimentellen Traumata in der
untersuchten Population eine größere Variabilität in Bezug auf den Schweregrad, die Art
sowie die genaue Lokalisation der Läsion auf (JEFFERY et al., 2006). Aus diesem Grund
können experimentelle Studien das klinische Geschehen oftmals nicht zuverlässig
widerspiegeln.
Die
in
dieser
Arbeit
beschriebene
ex
vivo-Untersuchung
von
traumatisiertem Rückenmark gibt im Gegensatz dazu einen besseren Aufschluss über
mikrogliale Funktionen in vivo.
Als Vergleichswerte dienten die Ergebnisse einer im Vorfeld durchgeführten Studie, in
welcher Mikroglia aus gesundem Rückenmark mit der gleichen Methodik charakterisiert
wurde.
Die morphologische Charakterisierung zeigte eine Diversität bezüglich Komplexität und
Größe der Mikroglia aus traumatisierten im Vergleich zu gesundem Rückenmark, was für
eine Aktivierung spinaler Mikrogliazellen nach einem Trauma spricht. Durch eine
gesteigerte ROS-Bildung und phagozytierte Partikel im Zellinneren kommt es zu einer
granulierten Struktur und folglich zu einem Anstieg in der Komplexität der Mikroglia. Eine
Zunahme der Zellgröße kann durch eine Hypertrophie der Mikroglia und eine starke
Ausprägung ihrer Zellfortsätze im Falle einer Aktivierung nach pathologischen Insulten
erklärt werden (STREIT, 1995).
Die immunphänotypische Untersuchung spinaler Mikroglia zeigte eine signifikante
Aufregulierung bestimmter Antikörper im Falle eines Traumas, welche eine Aktivierung
dieser Zellen widerspiegelt. So wurden MHC II und CD1c, welche für die Prozessierung
und Präsentation von Antigenen notwendig sind, höher exprimiert als im gesunden
Rückenmark. Des Weiteren zeigten B7-1, B7-2 und ICAM-1 eine Aufregulierung, was für
eine verstärkte Co-Stimulation beziehungsweise Zellinteraktionen mit T-Zellen spricht.
Eine höhere Expression von CD45 im traumatisierten Rückenmark erleichtert die
58
Signaltransduktion von Oberflächenmolekülen. Zudem wurde CD44 nach einem Trauma
stärker exprimiert, welches maßgeblich an der Zelladhäsion und Immunmodulation
beteiligt ist.
Die funktionelle Untersuchung zeigte, dass eine Traumatisierung des Rückenmarkes zu
einem Anstieg in der Phagozytoseintensität sowie zu einer erhöhten Bildung reaktiver
Sauerstoffspezies
führt.
durchflusszytometrischen
Bereits
vorausgegangene
Untersuchung
mikroglialer
Studien,
die
Phagozytose
sich
im
mit
der
pathologisch
veränderten ZNS beschäftigten, wiesen eine Steigerung der Phagozytoseintensität bei
kaniner Staupe und entzündlichen Infektionskrankheiten nach (STEIN et al., 2004b; STEIN
et al., 2006). Übereinstimmend mit der vorliegenden Studie wurde bei Ratten eine mit
Phagozytose
einhergehende
Aktivierung
von
Mikrogliazellen
nach
einem
Rückenmarkstrauma beobachtet (ISAKSSON et al., 1999; TACCOLA et al., 2010)
Eine gesteigerte ROS-Bildung durch Mikrogliazellen wurde bei kaniner Staupe (STEIN et
al., 2004b; STEIN et al., 2006), bei der experimentellen allergischen Enzephalomyelitis
(EAE; RUULS et al., 1995) sowie bei Alzheimer (LEFKOWITZ u. LEFKOWITZ, 2008)
berichtet. Zudem führten experimentelle Rückenmarkstraumata bei Ratten zu einem
deutlichen Anstieg der ROS Produktion durch Mikroglia (YUNE et al., 2009).
Des Weiteren beschreibt STREIT 2006 eine Aktivierung von Mikrogliazellen, die im Falle
eines Traumas induziert wird. Diese Aktivierung kann in eine irreversible Hyperaktivierung
übergehen, welche mit Schädigungen von Neuronen und Sekundärschäden nach einem
Trauma in Verbindung gebracht wird (NAKAMURA, 2002). Außerdem kann es bereits im
normal
aktivierten
Zustand
der
Mikroglia
durch
die
Bildung
von
reaktiven
Sauerstoffspezies zur direkten Neuronenschädigung kommen (BRUCE-KELLER, 1999).
Zusammenfassend lässt sich sagen, dass die Aktivierung der residenten Immunzellen des
Rückenmarkes und die damit verbundenen Effektorfunktionen eine entscheidende Rolle in
der Pathogenese von Rückenmarkstraumata spielen.
Angesichts der potentiell zellschädigenden Eigenschaften ihrer Funktionen sollte eine
Hemmung spinaler Mikrogliazellen vor der Implantation olfaktorischer Hüllzellen in
Betracht gezogen werden, um eine direkte Verletzung dieser Zellen einzuschränken. Eine
Hemmung der Mikroglia könnte beispielsweise mit der systemischen Verabreichung des
Breitbandantibiotikums Minocyclin erfolgen (EKDAHL et al., 2003).
Zusammenfassung (deutsch)
59
V. Zusammenfassung (deutsch)
Theda M. Boekhoff: Untersuchungen zu Rückenmarkstraumata beim Hund: Einfluss
von Mikrogliazellen und retrospektive Untersuchung der MRT-Befunde von Hunden
mit thorakolumbalem Bandscheibenvorfall
Thorakolumbale Bandscheibenvorfälle stellen eine häufige Ursache für Paraplegien bei
Hunden dar. Die chirurgische Dekompression des Rückenmarkes ist bei dieser
Symptomatik die Therapie der Wahl. Des Weiteren beschäftigen sich neue Ansätze bei
schwerster Schädigung des Rückenmarkes mit der Implantation von olfaktorischen
Hüllzellen, welche die Heilung durch Unterstützung von verletzten Neuronen sowie
Angiogenese positiv beeinflussen können.
Um die richtige Entscheidung treffen zu können, welcher Therapieversuch herangezogen
werden soll, ist eine vorherige Einschätzung der Prognose wichtig. Aus diesem Grund
gehörte es zu den Zielen der ersten Studie dieser Arbeit, Beziehungen zwischen den
qualitativen und quantitativen MRT-Befunden bezüglich intramedullärer Hyperintensität in
der T2-gewichteten Sequenz und Kompression des Rückenmarkes und klinischen
Parametern
sowie
Langzeiterfolg
nach
Dekompression
des
Rückenmarkes
zu
untersuchen. Hierfür wurden die Unterlagen und MRT-Befunde von 63 paraplegischen
Hunden mit einem Körpergewicht < 20 kg mit und ohne Tiefensensibilität, welche vom
Januar 2005 bis zum Juni 2009 in der Klinik für Kleintiere der Tierärztlichen Hochschule
vorgestellt wurden, ausgewertet und verschiedene Parameter miteinander korreliert.
Die Ergebnisse zeigten eine statistisch signifikante Korrelation zwischen dem Grad der
neurologischen Ausfälle prä OP und sowohl dem Vorliegen einer Hyperintensität in T2gewichteten MRT-Aufnahmen als auch deren Ausdehnung. Des Weiteren ergab sich bei
Hunden
mit
längerer
Krankheitsdauer
ein
signifikanter
Anstieg
im
Grad
der
Rückenmarkskompression. Die Hyperintensität war umso ausgedehnter, je grösser sich
der Kompressionsgrad des Rückenmarkes darstellte. Außerdem verlängerte sich der
Zeitraum, in dem es zur Besserung des jeweiligen neurologischen Grades um eine Stufe
kam,
bei
Vorliegen
einer
Hyperintensität
beziehungsweise
bei
deren
größerer
Ausdehnung. Somit konnte zusammenfassend eine direkte Korrelation zwischen
neurologischem Grad prä OP und Hyperintensität und deren Ausdehnung im MRT
nachgewiesen
werden.
Ein
Vorliegen
beziehungsweise
die
Ausdehnung
einer
Hyperintensität können außerdem zur Prognosefindung vor einem chirurgischen Eingriff
Zusammenfassung (deutsch)
60
im Hinblick auf den Einsatz verschiedener Therapiestrategien wie der Implantation
olfaktorischer Hüllzellen herangezogen werden.
Vor einer Implantation olfaktorischer Hüllzellen in das Rückenmark sollte das Verhalten
von Mikrogliazellen bei Rückenmarkstraumata berücksichtigt werden. In ihrem aktivierten
Zustand sind diese Immuneffektorzellen in der Lage, die implantierten Zellen zu schädigen
oder zu phagozytieren und könnten somit den Erfolg der Implantation gefährden. Um
diese Problematik einschätzen zu können, wurde in einer weiteren Studie Mikroglia aus
dem Rückenmark von 15 Hunden mit zervikalem oder thorakolumbalen Trauma mittels
Dichtegradientenzentrifugation gewonnen und immunphänotypisch sowie funktionell
anhand
ihrer
Phagozytoseaktivität
und
ROS-Bildung
charakterisiert.
Die
immunphänotypische Charakterisierung hatte eine Aufregulierung von B7-1, B7-2, MHC II,
CD1c, ICAM-1, CD45, CD14 sowie CD44 zum Ergebnis, welche eine mikrogliale
Aktivierung durch Rückenmarkstraumata widerspiegelte. Die funktionelle Untersuchung
zeigte einen signifikanten Anstieg sowohl in der Phagozytoseintensität als auch in der
Intensität der ROS-Bildung und somit eine Steigerung in der Ausübung mikroglialer
Abwehrmechanismen.
Aus diesem Grund sollte eine Hemmung aktivierter Mikrogliazellen vor einer Implantation
olfaktorischer
Hüllzellen
in
Betracht
Therapieansatzes zu gewährleisten.
gezogen
werden,
um
den
Erfolg
dieses
Zusammenfassung (englisch)
61
VI. Zusammenfassung (englisch)
Theda M. Boekhoff: Spinal cord trauma in dogs: characterization of microglia and
retrospective evaluation of MRI findings in dogs with thoracolumbar disk herniations
Thoracolumbar intervertebral disk herniations are a frequently found cause of paraplegia in
dogs. Surgical decompression of the spinal cord is the most common treatment modality,
when such clinical findings occur. Furthermore, new therapeutical strategies deal with the
implantation of olfactory ensheathing cells (OECs), which is known to promote functional
recovery after spinal cord trauma due to support of injured neurons and angiogenesis.
For selection of the best therapeutical approach a prior assessment of prognosis is useful.
Therefore, the aim of the first study was to describe associations between the qualitative
and quantitative magnetic resonance imaging (MRI) signal characteristics of T2-weighted
(T2W) hyperintensity respectively spinal cord compression and clinical signs and
functional outcome in paraplegic dogs with thoracolumbar disk herniation. Thus, medical
records and MR images of 63 paraplegic dogs with a body weight < 20 kg and intact or
absent deep pain perception (DPP) referred to and examined at the Department of Small
Animal Medicine and Surgery, University of Veterinary Medicine, Hannover, Germany
between January 2005 and June 2009 were reviewed and different clinical parameters
were correlated.
Statistically significant correlation was found between the neurological status before
surgery and both, presence and extent of T2W hyperintensity in MRI sagittal planes.
Moreover, dogs with a longer duration of clinical signs showed a significant increase in the
degree of spinal cord compression. Furthermore, the extent of T2W hyperintensity and the
degree of spinal cord compression presented a positive correlation. Improvement in the
neurological score for one grade was faster with absence of T2W hyperintensity
respectively with a smaller extent of this hyperintensity.
In conclusion, a direct correlation between neurological status and MRI signal intensity and
extent was shown. The presence and extent of T2W hyperintensity in the myelon may help
to determine the prognosis before surgery to decide, if new therapeutical strategies such
as implantation of OECs should be used in individual cases.
To prepare further studies on transplantation of OECs, the function of microglia in spinal
cord trauma has to be considered. In a potential activated state these immune effector
cells could defend or phagocytose the implanted cells and therefore diminish the support
Zusammenfassung (englisch)
62
of this therapeutical approach. In an effort to analyze this issue microglia of 15 dogs
suffering from cervical respectively thoracolumbar spinal cord trauma was isolated using
density gradient centrifugation and characterized using immunophenotyping and function
by examining phagocytosis and generation of reactive oxygen species (ROS).
Immunophenotypical characterization resulted in a significant upregulation of B7-1, B7-2,
MHC II, CD1c, ICAM-1, CD45, CD14, and CD44, reflecting an activation of microglial cells
due to trauma. Functional investigation revealed a significant increase in intensity of
phagocytosis and ROS generation in case of spinal cord trauma. This detection of
enhanced microglial defense mechanisms leads to the conclusion that repression of
microglial activation previous to implantation of OECs should be considered to ensure the
success of this approach.
Schrifttumsverzeichnis
VII.
63
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Anhang
73
VIII. Anhang
In Ergänzung zu Kapitel II. Ergebnisse
II A. Quantitative magnetic resonance imaging characteristics: evaluation of
prognostic value in the dog as a translational model for spinal cord injury
Tabelle 2 Daten zur Auswertung der Patienteninformationen
Legende: kg = Kilogramm; Geschlecht (M = männlich; W = weiblich; K = männlich kastriert; S = weiblich
kastriert); Dauer (Kategorie 1 = 1 Tag; Kategorie 2 = 2-3 Tage; Kategorie 3 = 4-7 Tage; Kategorie 4 = 8-14
Tage; Kategorie 5 = >14 Tage); Cortison (1 = Vorbehandlung mit Glukokortikosteroiden; 2 = keine
Glukokortikosteroide erhalten; 3 = Vorbehandlung unbekannt); Grad prä OP (4 = Grad 4 nach Sharp und
Wheeler, 2005; 5 = Grad 5 nach Sharp und Wheeler, 2005); Besserung (Kategorie 1 = 1 Tag; Kategorie 2 =
2-3 Tage; Kategorie 3 = 4-7 Tage; Kategorie 4 = 8-14 Tage; Kategorie 5 = >14 Tage).
Tiernummer Gewicht (kg)
1
9,0
2
4,0
3
9,5
4
5,8
5
9,1
6
8,5
7
6,2
8
5,2
9
5,0
10
7,7
11
6,1
12
12,8
13
9,5
14
11,8
15
10,0
16
17,8
17
6,9
18
4,7
19
9,4
20
5,8
21
8,4
22
14,5
23
14,5
24
7,1
25
4,3
26
10,5
27
5,0
28
13,0
29
8,2
Geschlecht
M
S
W
M
M
W
M
M
M
S
W
K
M
M
W
S
M
W
M
M
W
W
S
M
M
M
S
M
M
Patienteninformationen
Dauer Cortison Grad prä OP
4
1
3
3
3
1
2
1
2
2
4
3
1
2
1
3
1
2
3
1
3
1
1
2
1
2
4
1
1
2
2
2
4
1
2
1
2
2
3
1
2
3
1
1
2
2
2
1
2
1
3
2
2
1
3
2
2
3
Besserung
4
4
4
4
4
4
4
4
5
4
4
4
4
5
4
4
4
4
4
4
5
4
4
5
4
4
4
4
4
1
1
1
1
2
3
2
2
1
2
3
1
3
1
1
3
2
1
3
2
5
2
2
2
1
1
Anhang
74
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
4,0
12,8
5,3
12,3
7,4
3,8
15,0
8,7
12,4
10,2
5,5
13,9
15,5
7,9
12,0
7,0
11,3
6,6
10,0
16,4
9,5
10,9
9,9
8,0
5,3
6,3
8,0
9,0
9,0
13,3
9,9
18,5
6,2
8,6
W
M
M
W
W
W
M
W
M
K
M
W
M
M
M
K
M
W
K
S
W
M
W
S
M
W
M
M
W
M
M
K
M
W
3
3
4
1
2
3
1
2
3
4
1
2
1
1
3
2
1
2
3
2
2
1
5
3
1
2
3
2
2
3
3
5
1
2
1
3
1
1
2
3
2
1
1
1
2
1
2
2
1
1
2
2
3
2
2
3
1
3
2
2
3
1
1
1
1
1
2
3
5
4
4
5
5
5
4
5
5
4
4
4
4
4
4
4
4
4
5
4
4
4
4
4
4
5
4
5
4
4
4
4
4
5
1
2
4
2
1
2
1
3
5
3
4
3
2
1
1
2
1
2
2
1
2
1
1
1
5
1
1
2
1
2
Anhang
75
Tabelle 3 Daten zur Auswertung der Hyperintensität in der T2-gewichteten Sequenz
der Magnetresonanztomographie (MRT)
Legende: Hyperintensität = Hyperintensität des Myelons in T2-gewichteten MRT-Sequenzen; mm =
Millimeter; L2 = 2. Lendenwirbel; Kategorie Hyperintensität (Kategorie 1 ≤ ½ x Länge L2; Kategorie 2 ≤ 1 x
Länge L2; Kategorie 3 > 1 x Länge L2; Kategorie 4 > 2 x Länge L2); Kategorie Kompression
(Kategorie 1 ≤ 25%; Kategorie 2 > 25% bis 50%; Kategorie 3 > 50% bis 75%; Kategorie 4 > 75%).
Tiernummer
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
MRT-Befunde Hyperintensität
MRT Befunde Kompression
Länge
Länge
Quotient
Kategorie KomKategorie
Hyperintensität L2 in
HyperHyperpression KomHyperintensität in mm
mm
intensität/L2 intensität in %
pression Lokalisation
nein
75 3
Th12/13
nein
60 3
Th13/L1
ja
6
17
0,35
1
38 2
L3/4
nein
40 2
Th12/13
ja
33
16
2,06
4
60 3
Th12/13
nein
50 2
Th12/13
ja
6
13
0,46
1
40 2
L2/3
nein
50 2
Th13/L1
ja
36
13
2,77
4
67 3
L3/4
ja
20
14
1,43
3
67 3
Th13/L1
ja
33
13
2,54
4
75 3
Th12/13
nein
40 2
Th13/L1
ja
19
16
1,19
3
50 2
Th11/12
ja
44
16
2,75
4
80 4
Th11/12
nein
43 2
L2/3
nein
29 2
L2/3
ja
10
16
0,63
2
60 3
Th12/13
nein
75 3
Th12/13
ja
6
17
0,35
1
60 3
Th12/13
nein
75 3
Th12/13
ja
32
15
2,13
4
75 3
Th12/13
ja
9
18
0,50
1
20 1
Th12/13
ja
12
21
0,57
2
43 2
Th13/L1
ja
23
16
1,44
3
40 2
L1/2
ja
6
16
0,38
1
50 2
Th13/L1
nein
20 1
Th13/L1
nein
50 2
Th13/L1
nein
60 3
Th12/13
ja
26
14
1,86
3
40 2
L2/3
ja
10
11
0,91
2
50 2
Th11/12
nein
60 3
Th12/13
nein
50 2
Th13/L1
ja
9
17
0,53
2
20 1
Th13/L1
ja
36
15
2,40
4
25 1
Th12/13
nein
50 2
Th13/L1
ja
21
20
1,05
3
25 1
Th12/13
ja
86
16
5,38
4
60 3
Th11/12
nein
60 3
Th13/L1
nein
50 2
Th12/13
ja
12
14
0,86
2
50 2
Th13/L1
Anhang
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
76
ja
ja
ja
nein
ja
ja
nein
ja
nein
ja
nein
ja
nein
ja
ja
ja
ja
nein
ja
nein
ja
nein
ja
Figure 6c
16
23
17
17
19
14
0,94
1,21
1,21
2
3
3
30
16
13
15
2,31
1,07
4
3
33
15
2,20
4
29
16
1,81
3
37
15
2,47
4
14
30
31
23
11
15
14
14
1,27
2,00
2,21
1,64
3
3
4
3
21
17
1,24
3
24
19
1,26
3
64
17
3,76
4
50
50
25
75
25
33
60
60
43
50
25
75
80
40
60
40
60
67
40
75
60
50
50
2
2
1
3
1
2
3
3
2
2
1
3
4
2
3
2
3
3
2
3
3
2
2
Th13/L1
L2/3
L1/2
Th13/L1
Th11/12
L2/3
Th13/L1
Th12/13
L3/4
Th12/13
Th12/13
Th13/L1
L1/2
Th12/13
Th12/13
Th13/L1
Th13/L1
L3/4
Th11/12
Th11/12
L2/3
Th12/13
Th11/12
Correlation between the improvement in neurological score in one
grade and presence extent of T2W hyperintensity.
The abscissa shows improvement in neurological score in one grade in categories, on the
ordinate the respective percentages of dogs are displayed. Lines show dogs without
hyperintensity in comparison to dogs with a hyperintensity of category 3. Some dogs were
euthanized because of stasis or worsening of neurological signs, shown as category
“euthanasia” on the abscissa. Improvement in neurological score in one grade in categories:
category 1 (1 day), category 2 (2 to 3 days), category 3 (4 to 7 days), category 4 (8 to 14
days), and category 5 (longer than 14 days).
Anhang
77
II B. Upregulation of surface molecules and functional activity of canine microglia
following spinal cord trauma
Protokoll zur Isolierung kaniner spinaler Mikrogliazellen
Legende: min = Minuten; ml = Milliliter, g = Gravitationsbeschleunigung (9,81 m/sec²).
Vor Beginn der Arbeit die benötigten Materialien (Hanks`, Dichten,) aus dem Kühlschrank
nehmen. Wasserbad auf 37°C vorheizen. Mit Handschuhen arbeiten!
1. Mit Pinzetten die Meningen sorgfältig entfernen.
2. Mechanische Zerkleinerung erfolgt, indem das Material mit Hilfe eines
Spritzenkolbens durch ein Sieb aus rostfreiem Stahl in eine Petrischale gerieben
wird. Dabei mit Hanks` spülen. Auf Eis arbeiten.
3. Mit einer Pasteur-Pipette das gesiebte Material aufsaugen und in 50 ml-Röhrchen
überführen, mit Hanks`-Lösung auffüllen.
4. Zentrifugation bei 10°C 170 x g 10 min.
5. Überstand verwerfen, Gewebebrei mit ca. 8 ml Collagenase-DNAse-Puffer
versetzen und 60 min im Wasserbad bei 37 °C inkubieren. Zellsuspension nach
30 min resuspendieren.
6. Mit einer Pipette 10 ml Hanks`-Lösung hinzufügen und durch Auf- und Abpipettieren
verbliebene größere Gewebereste zerkleinern. Mit Hanks`-Lösung auf 50 ml
auffüllen. Zentrifugation bei 20°C 200 x g 10 min.
7. Überstand verwerfen und Schritt 6 wiederholen.
8. Zellsuspension in 50 ml-Röhrchen mit Percoll der Dichte 1,030 g/ml auf 45 ml
auffüllen und mit 5 ml Percoll der Dichte 1,124 g/ml unterschichten (=Vorgradient).
Zentrifugation bei 20°C 1250 x g 25 min, Bremse und Beschleunigung auf 1.
Anhang
78
9. Myelin und Zell-Debris befinden sich an der Oberfläche und werden verworfen. Die
Zellen auf der Dichte 1,124 g/ml Percoll werden mit einer 10 ml-Pipette geerntet
und in ein neues 50 ml-Röhrchen überführt.
Mit Hanks`-Lösung auf 50 ml auffüllen.
Zentrifugation bei 20°C 200 x g 10min.
Hauptgradienten gießen: Ein 50 ml-Röhrchen wird wie folgt befüllt:
1. 5 ml Percoll der Dichte 1,124 g/ml
10. Überstand verwerfen und das Pellet in 5 ml Hanks`-Lösung resuspendieren und
vorsichtig auf den Hauptgradienten pipettieren.
Zentrifugieren bei 20°C 1250 x g 25 min, Bremse und Beschleunigung auf 1.
11. Ernte der Zellen auf den Dichten 1,077 und 1,066 g/ml, mit Hanks` auf 50 ml
auffüllen und zentrifugieren bei 20°C 200xg 10 min.
Für alle weiteren Untersuchungen auf 2 ml mit Cell-Wash auffüllen.
Protokoll zur Durchführung der Membranimmunfluoreszenz
Legende: MIF = Membranimmunfluoreszenz; Ig = Immunglobulin; min = Minuten; CD = Cluster of
Differentiation; MHC = Major Histocompatiblity Complex (Haupthistokompatibilitätskomplex); ICAM =
intercellular adhesion molecule (Interzelluläres Adhäsionsmolekül); AK = Antikörper; gamPE = goat anti
mouse R-phycoerythrine; rarPE = rabbit anti rat R-phycoerythrine; SAFITC = streptavidine-conjugated
fluorescein-isothiocyanate; µl = Mikroliter.
1. 1000 µl der Zellsuspension 1/16 mit humanem IgG blocken:
Zugabe von 62,5 µl humanem IgG, dann 5 min bei 4°C inkubieren.
Antigen
CD1c
ICAM-1
Host +
Isotyp
Mouse
IgG1
Mouse
IgG1
Verdünnung
Zugabe
1/5
10 µl
1/5
10 µl
Anhang
79
Mouse
IgG1
Mouse
IgG1
Mouse
IgG1
Mouse
IgG1
Mouse
IgG2a
Mouse
IgG1
Mouse
IgG1
Mouse
IgG1
Mouse
IgG2a
Mouse
IgG1
Mouse
IgG2a
Mouse
IgG1
Rat IgG2a
Rat IgG2b
B7-1
B7-2
CD3
CD4
CD8α
CD21
CD11b
CD11c
CD14
CD18
MHC I
MHC II
CD44
CD45
1/5
10 µl
1/5
10 µl
1/5
10 µl
1/2
10µl
1/5
10 µl
1/5
10 µl
1/5
10 µl
1/5
10 µl
1/7
7 µl
1/5
10 µl
1/16
3 µl
1/5
10 µl
1/10
1/16
5 µl
3 µl
Doppelfärbungen mit
CD18+CD45
CD3+CD14
Kontrollen:
1) nur Cell-Wash
2) mouse IgG1 oder mouse IgG2a-Isotyp-Kontrolle + gamPE
3) rat IgG2b-Isotyp-Kontrolle + SAFITC
4) rat IgG2a-Isotyp-Kontrolle + rarPE
2. In jedes Röhrchen 50µl der geblockten Zellsuspension pipettieren und
entsprechend der Beschriftung und der vorgegebenen Verdünnung die PrimärAK dazupipettieren.
Durch Schwenken mischen, Röhrchen verschließen und 30 min bei 4°C
inkubieren.
Anhang
80
3. Zwei Waschschritte: Resuspension der Zellen und Zugabe von 200 µl CellWash,
Zentrifugieren bei Raumtemperatur 200 x g für 2 min
4. Zugabe der entsprechenden Sekundär-AK bzw. des SAFITC (Vorverdünnungen
in Cell-Wash). Bei direkt markierten Antikörpern (CD3, CD14, CD21) nur CellWash dazupipettieren.
gamPE Vorverdünnung 1:100
rarPE Vorverdünnung 1:100
SAFITC Vorverdünnung 1:100
Zugabe jeweils 50 µl
5. Inkubation 30 min bei 4°C
6. Zwei Waschschritte wie oben, danach Resuspension in 200 µl FACS-Flow,
Aufbewahrung bis zum Messen im Dunkeln bei 4°C.
Protokoll zum Phagozytose-Assay
Legende: ml = Milliliter; µl = Mikroliter; FITC = Fluoreszein-Isothiocyanat; min = Minuten.
FITC-markierte Staphylokokken, aliquotiert eingefroren.
Die Aliquots enthalten 30 µl Bakteriensuspension mit 8x108 Bakterien/ml.
Durchführung:
Die FITC-markierten Bakterien möglichst wenig dem Licht aussetzen, da sonst die
Leuchtkraft abnimmt.
2 Cups mit FITC-markierten Staphylokokken (je 30 µl) sowie ein Aliquot Hunde-Poolserum
(50 µl) auftauen.
Zu dem Hunde-Poolserum (HPS) 200 µl PBS pipettieren (Verdünnung 1/5).
Anhang
81
Cups markieren mit „O“ für opsonisiert und „N“ für nicht opsonisiert. In das Cup „O“ 30 µl
1/5 mit PBS verdünntes HPS geben, in das Cup „N“ 30 µl PBS, die Bakterien gründlich
resuspendieren und beide cups 60 min bei 37°C inkubieren.
Danach von der Bakterien-Suspension durch Zugabe von PBS eine 1:4-Verdünnung
herstellen (60 µl Bakteriensuspension +180 µl PBS).
5 FACS-Röhrchen beschriften und wie folgt vorbereiten:
1: Negativ-Kontrolle: nur Zellsuspension
2 und 3: 100 µl Zellsuspension + 100 µl Bakterien-Suspension „N“
4 und 5: 100 µl Zellsuspension + 100 µl Bakterien-Suspension „O”
Röhrchen abdecken, suspendieren, Inkubation über 60 min bei 37°C, nach der Hälfte der
Zeit noch einmal resuspendieren.
Röhrchen für 15 min auf Eis stellen, anschließend Zugabe von 100µl Facs-Flow je
Röhrchen und durchflusszytometrische Messung.
Protokoll zur Untersuchung der ROS-Bildung
Legende: mmol = milli mol; min = Minuten.
Phorbolmyristatacetat(PMA)-Stocklösung (10 mmol)
Dihydrorhodamin 123-working-solution (DHR) (1,5 mg/100 ml PBS)
Durchführung:
5 FACS-Röhrchen wie folgt beschriften:
1.: neg. Kontrolle: nur Zellsuspension
2. und 3.: Duplikate Zellsuspension mit PBS (=PMA 0) und DHR
4. und 5.: Duplikate Zellsuspension mit PMA (PMA 100) und DHR
In die markierten Röhrchen werden je 90 µl Zellsuspension pipettiert.
Anhang
82
Röhrchen 15 min bei 37°C im Brutschrank vorinkubieren.
PMA immer frisch verdünnen!!
In der Zwischenzeit das PMA 1/1000 vorverdünnen: Um auf eine 100 nmol Lösung zu
kommen, ist eine Verdünnung 1/10.000 erforderlich. Durch die Zugabe zur Zellsuspension
erfolgt die Weiterverdünnung 1/10.
Jeweils 10 µl der PMA-Verdünnungen (Röhrchen 4 und 5) bzw. PBS (Röhrchen 2 und 3)
zu den vorinkubierten Zellen pipettieren.
Röhrchen 15 min bei 37°C im Brutschrank inkubieren.
Zugabe von jeweils 20 µl DHR in Röhrchen 2-5.
Inkubation bei 15 min bei 37°C.
Nach der Inkubation Kühlung der Röhrchen für 15 min auf Eis, anschließend Zugabe von
je 100 µl FACS-Flow und durchflusszytometrische Messung.
Anhang
83
Table 3 Expression intensity of surface molecules on microglia from traumatized
spinal cord.
The mean fluorescence intensity, calculated as the geo mean of all results (cervical n = 5, thoracolumbar
n = 10) is presented. The upper and lower geometric standard deviations (geo S.D.) are shown.
CD = cluster of differentiation, MHC = major histocompatibility complex, ICAM-1 = intracellular adhesion
molecule-1.
Expression intensity
mean fluorescence intensity
Surface
antigen
cervical spinal
cord
thoracolumbar spinal
cord
geo mean
lower
geo S.D.
upper geo
S.D.
geo mean
lower
geo S.D.
upper geo
S.D.
CD18
1309.27
185.80
216.53
951.22
365.12
592.58
CD11b
2053.09
426.50
538.33
1592.79
514.75
760.53
CD11c
573.13
111.39
138.27
372.95
194.69
407.35
CD45
434.21
299.68
967.23
279.52
206.38
788.72
CD4
940.79
529.54
1211.42
386.78
261.60
808.27
CD8α
1181.67
722.31
1858.08
393.10
284.21
1026.05
CD3
343.50
140.62
238.08
292.61
218.87
868.42
CD21
355.18
78.50
100.78
300.89
195.40
557.37
MHC I
673.46
415.62
1085.60
271.60
165.48
423.52
MHC II
477.84
241.60
488.70
200.44
118.47
289.67
ICAM-1
267.25
117.24
208.88
163.88
91.58
207.57
CD1c
467.77
188.88
316.80
372.90
263.59
899.25
B7-1
890.38
481.34
1047.73
416.81
290.01
953.31
B7-2
422.93
227.27
491.28
241.65
154.18
425.93
CD14
365.01
137.65
220.99
284.14
164.11
388.51
CD44
200.53
34.42
41.56
158.81
84.91
182.45
Anhang
84
Table 4 Phagocytosis intensity of microglia from traumatized spinal cord.
n = 10), and the upper and lower geometric standard deviations (geo S.D.) are shown.
nops = non-opsonized bacteria, ops = opsonized bacteria.
negative
control
nops
ops
Phagocytosis intensity
cervical spinal cord
geo mean
lower geo
upper geo
S.D.
S.D.
67.3
36.4
79.3
1020.0
1186.3
437.2
355.2
765.1
507.1
thoracolumbar spinal cord
geo mean
lower geo
S.D.
43.6
25.9
821.6
1233.6
291.6
313.6
upper geo
S.D.
63.9
452.1
420.5
Table 5 Intensity of microglial ROS generation following spinal cord trauma.
n = 10), and the upper and lower geometric standard deviations (geo S.D.) are presented.
PMA = Phorbol-myristate-acetate.
negative
control
PMA 0
PMA 100
Intensity of ROS generation
cervical spinal cord
geo mean
lower geo
upper geo
S.D.
S.D.
48.1
28.7
71.1
802.3
781.5
513.5
472.0
1426.6
1192.0
thoracolumbar spinal cord
geo mean
lower geo
S.D.
38.1
20.7
605.8
754.5
299.5
440.8
upper geo
S.D.
45.5
592.2
1060.2
Danksagung
85
IX. Danksagung
Ganz herzlich möchte ich mich bei Frau Prof. Dr. Andrea Tipold für die Überlassung
meines Dissertationsthemas bedanken sowie für ihre kompetente und vor allem
warmherzige Unterstützung in den zwei Jahren. Neben meinem Thema habe ich viel über
die Neurologie im allgemeinen gelernt und bin froh darüber, ein Teil von Andreas
Arbeitsgruppe gewesen zu sein!
Bei Prof. Dr. Ingo Nolte bedanke ich mich für die Bereitstellung meines Arbeitsplatzes zur
Durchführung des praktischen Teils meiner Dissertation.
Dr. Veronika Stein danke ich für die Hilfe sowohl im praktischen als auch im
theoretischen Teil der Arbeit, für die hilfreichen Korrekturen der Veröffentlichungen, ihre
Einsatzbereitschaft und für die nette Zusammenarbeit.
Bei Dr. Cornelia Flieshardt möchte ich mich für ihre Unterstützung bei der Anfertigung
der MRT-Studie in dieser Arbeit bedanken.
Ganz herzlich bedanke ich mich bei Frau Regina Carlson für ihre liebe und auch
tatkräftige Unterstützung im Labor und dafür, dass sie zu jeder Tages- und auch Nachtzeit
ansprechbar war!
Mein besonderer Dank gilt auch Dr. Karl Rohn für seine geduldige Unterstützung in der
Erstellung des statistischen Teils dieser Arbeit.
Dem Institut für Pathologie danke ich für die Untersuchung der eingesendeten
Rückenmarksproben.
Danksagung
86
Bei meiner Arbeitsgruppe aus der Neurologie möchte ich mich für die nette
Zusammenarbeit und die lehrreiche Zeit in den letzten zwei Jahren bedanken.
Ein großes Danke auch an meine Kollegin und Freundin Eva-Maria Ensinger, mit der die
Zusammenarbeit einfach unheimlich viel Spaß gemacht hat! Mir werden die nächtlichen
Laborsessions, die witzigen Erlebnisse in und außerhalb der Klinik und die gesamte
Doktorandenzeit in sehr schöner Erinnerung bleiben!
Mädels! Euch danke ich für die schöne Zeit, auch schon im Studium. Ohne Euch wäre das
alles nicht dasselbe gewesen! Auch in den letzten zwei Jahren konnte ich mich immer auf
Euch verlassen, ob jetzt in kritischen Zeiten oder einfach zum Spaß haben. Danke!
Bei Friederike möchte ich mich für die Unterstützung, Ablenkung und Ratschläge in dieser
Zeit und im Besonderen für ihre Kritik und Anregungen bei der Korrektur dieser Arbeit
bedanken.
Meiner Familie danke ich dafür, dass sie immer für mich da ist und ich mich auf sie
verlassen kann. Ihre Liebe, Vertrauen und Glaube an mich waren eine wichtige
Unterstützung in dieser Zeit.

Einfluss von Mikrogliazellen und retrospekt

Transcription

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