elib.tiho-hannover.de fileelib.tiho-hannover.de

Small Animal Clinic

School of Veterinary Medicine Hannover

__________________________________________________________________

Ex vivo examination of canine microglia in different intracranial diseases:

stereotypic versus specific reaction profile

THESIS

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY – Ph.D. –

in the field of

Neurology

at the School of Veterinary Medicine Hannover

by

Dr. med. vet. Veronika Maria Stein Hollenstede/Fürstenau, Germany

Hannover, 2004

Supervisor: Prof. Dr. Andrea Tipold

Advisory Committee: Prof. Dr. Wolfgang Löscher

Prof. Dr. Dr. Gerhard Franz Walter

Prof. Dr. Andrea Tipold

First evaluation: 1. Univ.-Prof. Dr. Wolfgang Löscher, Department of

Pharmacology, Toxicology and Pharmacy, School of

Veterinary Medicine Hannover, Germany

2. Univ.-Prof. Dr. Dr. Gerhard Franz Walter, Rektorat,

Medical University Graz, Austria

3. Univ.-Prof. Dr. Andrea Tipold, Small Animal Clinic,

School of Veterinary Medicine Hannover, Germany

Second evaluation: Prof. Dr. Peter Schmidt, Institute of Pathology and

Forensic Veterinary Medicine, University of Veterinary

Medicine Vienna, Austria

Date of oral exam: June 3, 2004

This thesis was funded by the Deutsche Forschungsgemeinschaft (DFG)

Nature´s greatest miracles are manifested in the smallest things

Carl von Linnaeus

In den kleinsten Dingen zeigt die Natur ihre größten Wunder

Carl von Linné

Parts of this study have been published in the following form:

Publications: Stein, V.M.

Charakterisierung von Mikrogliazellen bei Hundestaupe Diss., Tierärztliche Hochschule Hannover, 2001 (Erich-Aehnelt-Gedächtnis-memorial award)

Stein, V. und Tipold, A.: Das zentrale Nervensystem und seine Privilegien: Sonderstellung bezüglich der Immunantwort hat Lücken! TiHo-Forschungsmagazin; Schwerpunkt: Neurowissenschaft, 16 – 20, 2002

Original Articles: Stein, V.M., Czub, M., Hansen, R., Leibold, W., Moore, P.F., Zurbriggen, A., Tipold, A. (2004):

Characterization of canine microglial cells isolated ex vivo Vet. Immunol. Immunopathol., 99, 73-85

Stein, V.M., Czub, M., Schreiner, N., Moore, P.F., Vandevelde, M., Zurbriggen, A., Tipold, A.:

Microglial cell activation in demyelinating canine distemper lesions J. Neuroimmunol., submittted 2003

Proceedings: Tipold, A., Stein, V.M., Moore, P.F., Zurbriggen, A., Vandevelde, M.:

Is the immune system responsible for acute canine distemper lesions? ACVIM-Proceedings on CD-Rom, Internet www.ACVIM.org; Charlotte, June 2003

Tipold, A., Stein, V.M., Moore, P.F., Zurbriggen, A., Vandevelde, M.:

Ex vivo examinations of microglial cells. ACVIM-Proceedings on CD-Rom, Internet www.ACVIM.org; Charlotte, June 2003

Presentations at scientific congresses: Posters Stein, V.M., Czub, M., Hansen, R., Moore, P.F., Vandevelde, M., Zurbriggen, A., Tipold, A.:

Microglial cells in canine distemper virus infection.

European Society and European College of Veterinary Neurology, 15th Annual Symposium „Inherited Neurological Diseases“, University of Pennsylvania, Philadelphia, USA, 26.-29.09. 2002

(Bayer Award)

In: European Society and European College of Veterinary Neurology, Proceedings of the 15th annual Symposium „Inherited Neurological Diseases“, University of Pennsylvania, Philadelphia, USA, 26.-29.09. 2002, p. 23

Stein, V.M., Carlson, R., Tipold A.:

Nitric oxide production in the canine brain – are microglial cells involved? European Society and European College of Veterinary Neurology, 16th Annual Symposium „Neurosurgery“, Prague, Czech Republic, 25.-27.09. 2003 In: European Society and European College of Veterinary Neurology Proceedings of the 16th annual Symposium „Neurosurgery“, Prague, Czech Republic, 25.-27.09. 2003, p. 56

Oral presentations Stein, V.M., Czub, M., Hansen, R., Zurbriggen, A., Tipold, A.:

Charakterisierung von Mikroglia bei Hundestaupe 10. Jahrestagung der Fachgruppe Innere Medizin und Klinische Laboratoriumsdiagnostik, Munich, February, 15.-17., 2001 In: Deutsche Veterinärmedizinische Gesellschaft e. V. (Hrsg.): Zusammenfassung der Vorträge zur 10. Jahrestagung der Fachgruppe Innere Medizin und Klinische Laboratoriumsdiagnostik, Munich, February, 15.-17., 2001, 126-127

Stein, V.M. und A. Tipold:

Canine microglial cells in dogs infected with CDV Vortrag im Rahmen des Infektionsbiologischen Seminars, Tierärztliche Hochschule Hannover, November, 4., 2002

Stein, V.M., Schröder, S., Carlson, R., Czub, M., Baumgärtner, W., Tipold, A.:

Mikroglia bei intrakraniellen Erkrankungen 12. Jahrestagung der Fachgruppe Innere Medizin und Klinische Laboratoriumsdiagnostik, Munich, February, 1.-2., 2003 In: Deutsche Veterinärmedizinische Gesellschaft e.V. (Hrsg.): Zusammenfassung der Vorträge zur 12. Jahrestagung der Fachgruppe Innere Medizin und Klinische Laboratoriumsdiagnostik, Munich, February, 1.-2., 2003, 132-133; Tierärztliche Praxis, 31, 73

Index

I. Introduction................................................................................................. 13 II. Literature Review........................................................................................ 15

II. 1. Microglia........................................................................................ 15 II. 1.1. Historical perspective......................................................................................15 II. 1.2. Morphology, developmental and functional states..........................................16 II. 1.3. Functional characteristics of microglia and their regulations ..........................18 II. 1.4. Microglia in different diseases ........................................................................22

II. 2. Surface molecules for immunophenotypic characterization.... 25 III. Aims of the study........................................................................................ 28 IV. Material and Methods ................................................................................. 30

IV. 1. Material .......................................................................................... 30 IV. 1. 1. Laboratory equipment.....................................................................................30

IV. 1.1.1. Technical equipment...............................................................................30 IV. 1.1.2. Equipment for sterile work ......................................................................31 IV. 1.1.3. Equipment for perfusion of brains ...........................................................31 IV. 1.1.4. Laboratory material .................................................................................32 IV. 1.1.5. Centrifuges..............................................................................................33

IV. 1.2. Reagents ........................................................................................................33 IV. 1.3. Solutions .........................................................................................................34 IV. 1.4. Reagents for separation und differentiation of cells........................................35 IV. 1.5. Enzymes .........................................................................................................36 IV. 1.7. Reagents for measurement of reactive oxygen intermediates .......................38 IV. 1.8. Material for measurement of phagocytic activity.............................................38 IV. 1.9. Material for measurement of nitric oxide (NO)................................................39 IV. 1.10. Material for flow cytometry..............................................................................39 IV. 1.11. Computer software .........................................................................................39 IV. 1.12. Virus................................................................................................................39 IV. 1.13. Animals ...........................................................................................................40

IV. 2. Methods.......................................................................................... 45 IV. 2.1. Cerebrospinal fluid (CSF) collection ...............................................................45 IV. 2.2. Isolation of canine microglial cells ..................................................................45 IV. 2.3. Histopathological and immunohistochemical examination..............................49 IV. 2.4. Light microscopic determination of cell yield ..................................................49 IV. 2.5. Indirect membrane immunofluorescence (MIF) ..............................................49 IV. 2.6. Functional examination of canine microglial cells ...........................................50

IV. 2.6.1. ROS generation test ...............................................................................50 IV. 2.6.2. Phagocytosis assay ................................................................................51

IV. 2.7. Cultivation of canine microglial cells ...............................................................52 IV. 2.8. Determination of NO2

-/NO3- (Griess reaction) .................................................52

IV. 2.9. Statistical evaluation .......................................................................................53

V. Results......................................................................................................... 55 V. 1. Cell yield, viability, and purity ...................................................... 55 V. 2. Immunophenotypical characterization of canine microglia ex vivo ............................................................................................ 56

V. 2.1. CD18, CD11b, and CD11c ............................................................................56 V. 2.2. CD45..............................................................................................................61 V. 2.3. CD1c, MHC I, and MHC II .............................................................................64 V. 2.4. ICAM-1 (CD54) ..............................................................................................69 V. 2.5. B7-1 (CD80) and B7-2 (CD86) ......................................................................70 V. 2.6. CD14..............................................................................................................72

V. 3. Functional characterization of canine microglial cells .............. 72 V. 3.1. ROS generation test ......................................................................................72 V. 3.2. Phagocytosis Assay.......................................................................................78 V. 3.3. Determination of nitric oxide (NO) in microglial culture supernatant and CSF.........................................................................................................88

VI. Discussion................................................................................................... 92 VII. Summary ................................................................................................... 106 VIII. Zusammenfassung ................................................................................... 108 IX. References ................................................................................................ 110 X. Acknowledgements .................................................................................. 130 XI. Appendix ................................................................................................... 132

Table XI. 1 Dogs of the vaccine challenge experiment ...............................................132

Table XI. 2 Dogs of the microglia study.......................................................................133

Table XI. 3 Dogs of the microglia study in which nitric oxide (NO) was determined in microglial culture supernatant and cerebrospinal fluid ..........................138

Table XI. 4 Dogs in which nitric oxide (NO) was determined in cerebrospinal fluid ....140

Table XI. 5. Statistical data for immunophenotypical characterization of canine microglial cells ex vivo in original and alternative examination groups .....144

Table XI. 6. Statistical data for functional characterization of canine microglial cells ex vivo - ROS generation test...........................................................149

Table XI. 7. Statistical data for functional characterization of canine microglial cells ex vivo - phagocytosis assay -..........................................................150

Table XI. 8. Boxplots for optical control of normality of the results obtained from the immunophenotypical characterizationof microglial cells ex vivo ...............152

Table XI. 9 Boxplots for optical control of normality of the results obtained from the ROS generation test of microglial cells ex vivo.........................................157

Table XI. 10 Boxplots for optical control of normality of the results obtained from the phagocytosis assay of microglial cells ex vivo ..........................................159

Figure XI. 1 Residualplots for the results of the immunophenotypical characterization of microglial cells ex vivo ................................................162 Figure XI. 2 Residualplots for the results of the ROS generation test of microglial cells ex vivo...............................................................................................170

Figure XI. 3 Residualplots for the results of the phagocytosis assay of microglial cells ex vivo...............................................................................................172

Abbreviations

AD Alzheimers Disease ADS AIDS dementia complex AIDS Acquired Immunodeficiency Syndrome ANOVA Analysis of Variance APC antigen presenting cell Aqua bidest. Aqua bidestillata Aqua tridest. Aqua tridestillata ATP Adenosintriphosphate bFGF basic fibroblast growth factor BSA bovine serum albumin c castrated ° C degree Celsius CD cluster of differentiation CDV canine distemper virus CO2 carbon dioxide cm centimeter CNS central nervous system CPSR-1/-3 controlled process serum replacement-1 or -3 CR3/4 complement receptor 3/4 CSF-1 colony stimulating factor -1 CSF cerebrospinal fluid CT computed tomography CTLA-4 cytolytic T-lymphocyte associated molecule-4 d days DHR 123 Dihydrorhodamine 123 DMSO Dimethylsulfoxide DNA deoxyribonucleic acid DNAse deoxyribonuclease EAE experimental autoimmune encephalomyelitis EEG electroencephalography e.g. exempli gratia, for example excl. exclusive FACS fluorescence-activated cell scanner, flow cytometer

Fc fragment cristalline (crystallizable part of an antibody, carboxy-terminal fragment of immunoglobulines following papain-splitting)

FCS fetal calf serum FITC Fluorescein-Isothiocyanate FL 1,2,3 Fluorescence 1,2,3 (1 = green/yellow, 2 = yellow/orange, 3

= red) FSC forward scatter g gravitation acceleration (9,81 m/sec2)

GABA γ-amino butyric acid GM-CSF granulocyte-monocyte colony stimulating factor GME granulomatous meningoencephalomyelitis gαm-PE goat-anti-mouse-Phycoerythrine (secundary antibody from

goat directed against mouse IgG bound to Phycoerythrine) h hour HE hematoxylin-eosin HEPES N-[2-hydroxyethyl] piperazine-N-2-Ethansulfonicacid HuIgG Human Immunoglobuline G ICAM-1 intercellular adhesion molecule-1 i.e. id est, that is

IFN-γ Interferon-γ IgG Immunoglobuline G IL Interleukin iNOS inducible nitric oxide synthase kDa kilo Dalton l Liter LBP Lipopolysaccharide-binding Protein LCA leukocyte common antigen = CD45 LFA-1 lymphocyte function-associated antigen-1 (= CD11a/CD18) log logarithmic value LPS Lipopolysaccharide m months Mac-1 membrane attack complex-1 mAb/s monoclonal antibody/ies M-CSF monocyte colony stimulating factor mg milligramm

MHC I/II major histocompatibility complex class I/II MIF membrane immunofluorescence min minute/s ml milliliter mmol molarity, milli mol/l MRI magnetic resonance imaging MS Multiple Sclerosis N. nervus NCV nerve conduction velocity NGF nerve growth factor NK cells natural killer cells, cytotoxic T-cells nm nanometer ( = 10-9 m) NMDA N-methyl-D-aspartate no. number NO nitric oxide NOI/s nitric oxide intermediate/s PBS phosphate buffered saline PE Phycoerythrine PGD2 Prostaglandine D2 PGE2 Prostaglandine E2 pH potentia Hydrogenii PI post infectionem PMA Phorbol-12-Myristate-13-Acetate PNS peripheral nervous system ROI/s reactive oxygen intermediate/s ROS reactive oxygen species RPE R-Phycoerythrine rpm revolutions per minute RPMI medium developed by Moore et al. at Roswell Park

Memorial Institute, hence the acronym RPMI RT room temperature s spayed SPF specific pathogen free SRMA steroid responsive meningitis arteritis SSC side scatter Tab. Table

TGF-β transforming growth factor-β

TNF-α tumor necrosis factor-α vs. versus y years µ micro (x 10-6) µl microliter µm micrometer arithmetic mean value

♀ female

♂ male

13

I. Introduction

Microglial cells are the main immune effector elements of the brain (Giulian 1987,

Streit 2002). Their cell numbers range from 5 to 20% of the entire central nervous

system glial cell population (Kreutzberg 1987, Streit 1995). They were first described

in 1932 by Del Rio-Hortega as a distinct cell type within the central nervous system

(CNS) with characteristic morphology and specialised staining characteristics.

Microglia respond to every kind of pathological event in the CNS (Giulian 1992a) and

rapidly progress from their resting ramified state to an activated state where they can

proliferate, migrate, and express surface molecules de-novo or at increased levels

(Streit et al. 1989). This cell population plays a critical role in host defense against

invading microorganisms and neoplastic cells or otherwise altered cells, and are

thought to determine the pattern and degree of central nervous system recovery or

disease (Giulian 1992b). Microglia share many properties with macrophages (Perry

and Gordon 1988), and upon activation can exert similar macrophage effector

functions such as phagocytosis, modulation of T-cell response, production, and

release of cytokines, chemokines, reactive oxygen (ROS), and nitrogen species

(Streit and Kreutzberg 1988, Kreutzberg 1996, Stoll and Jander 1999). Microglia are

also capable of processing and presenting antigen by expression of MHC class I and

MHC class II (Banati et al. 1993b; Gehrmann et al. 1995). These findings converge

into a concept that views microglia as the local immune system of the brain (Graeber

and Streit 1990, Streit 2002).

Microglial response to CNS injury has long been believed to be uniform and

stereotypic irrespective of the underlying insult (Gehrmann and Kreutzberg 1995,

Stoll and Jander 1999, Raivich et al. 1999, Graeber et al. 2002). This assumption is

now under intense investigation.

Isolation of canine microglia and purification of these cells without contamination by

other CNS macrophages or blood-derived cells is cumbersome. Difficulties include

the relatively small number of microglial cells and the absence of specific markers

differentiating microglia from other blood-derived mononuclear cells (Streit 1995,

González-Scarano and Baltuch 1999). The isolation protocol is complicated by the

fact that CNS tissue consists mostly of lipids. Furthermore, macrophages and

monocytes co-accumulate during density gradient centrifugation. Isolation protocols

_______________________________Introduction_________________________________

14

have already been established that provide good results for other species than dogs,

such as rats and mice (Sedgwick et al. 1991; Ford et al. 1995). We recently

developed an ex vivo microglial examination technique for the canine species (Stein

et al. 2004) in order to study the function of these cells immediately following their

isolation. Ex vivo examinations are rare (Hein et al. 1995) for all species and absent

for the dog. These have the great advantage over cell culture systems of more

closely reflecting conditions in-vivo. However, since microglial ex vivo examination

requires the isolation of several thousands of cells for which 10 to 20 g brain tissue is

needed, it is not possible to study single cells and cells in specific circumscribed

brain areas.

In a preliminary study we examined canine microglia in dogs from a vaccine

challenge experiment with canine distemper virus (CDV) infection. Dogs with

histopathologically confirmed demyelination had a marked upregulation of the

surface molecules CD18, CD11b, CD11c, CD1c, MHC class I, MHC class II, and a

tendency for increased expression intensity of ICAM-1 (CD54), B7-1 (CD80), and B7-

2 (CD86). Functionally, microglial cells isolated from demyelinating lesions due to

CDV infection developed a significantly enhanced phagocytosis and generation of

ROS.

In order to evaluate whether microglial reaction pattern is stereotypic or is specific for

the pathological condition in CDV infection the results from the vaccine challenge

experiment were compared to the results of dogs suffering from different intra- and

extracranial diseases. This study represents the first comparative examination of

microglial cells ex vivo in different canine diseases. The results are also respected to

contribute generally to our understanding of the pathogenesis of demyelination in the

CNS.

Furthermore, since NO production is part of the microglial immunological repertoire in

different species, this study examined microglial culture supernatant and CSF from

dogs with intracranial diseases in order to discover whether enhanced NO production

can be associated with a specific CNS disease, and if NO is produced by canine

microglial cells, as opposed to canine macrophages.

15

II. Literature Review II. 1. Microglia

II. 1.1. Historical perspective

As early as the nineteenth century, the famous neuropathologists and psychiatrists

Nissl and Alzheimer commented on the possibility that cells of non-neuroectodermal

origin invade the developing central nervous system (CNS). Ramon y Cajal (1913)

entitled these cells el tercer elemento, i.e. the third element of the central nervous

system, which were therefore morphologically distinct from the first and second

element, neurons and astrocytic neuroglia (summarized by Streit 1995). It was the

Spanish neuroanatomist Pio Del Rio-Hortega who with the help of his histochemical

technique of weak silver carbonate staining of CNS further differentiated Ramon´s

third element into oligodendrocytes and microglia. The discovery of microglial cells is

therefore ascribed to Del Rio-Hortega who performed the first systematic

investigations on microglial cells (1932).

Microglial cells are described in various ways because different developmental and

functional stages and anatomical localizations are used as criteria for their

descriptive names and attributes (Dickson et al. 1991, Streit 1995, Gehrmann and

Kreutzberg 1995). They are called “Hortega cells” which refers to their first

investigator. Following incorporation of detritus or fatty particles they are called “gitter

cells”, “lattice cells”, “fat granular cells”, “lipid phagocytes”, or “foamy cells”. Other

names in the literature include “rod cells”, “M-cells”, “rice cells”, “globoid and ameboid

cells”, “inflammation globules”, or – according to their presumed origin “mesoglia”

(Jordan and Thomas 1988, Leonhardt 1990, Gehrmann and Kreutzberg 1995, Nissl

1899 summarized by Streit 1995).

Microglial cell numbers range from 5 to 20% of the entire central nervous system glial

cell population (Kreutzberg 1987, Perry and Gordon 1988, Fedoroff 1995, Giulian

1995, Streit 1995, Rezaie and Male 1999, Stoll and Jander 1999). They reside in

brain, spinal cord and retinal ganglia and show an ubiquitous distribution (Perry and

Gordon 1988, Gehrmann and Kreutzberg 1995, Streit 1995). In grey matter they

occur adjacent to the neurons, whereas in white matter they are located between the

______________________________Literature review______________________________

16

nerve fibers (Lawson et al. 1993). It is estimated that the normal adult murine brain

contains 3.5 x 106 microglial cells (Lawson et al. 1993).

II. 1.2. Morphology, developmental and functional states

There are at least three clearly identifiable isoforms of microglia in the adult brain,

which correlate with different stages of activation and development (Davis et al.

1994, Streit 1995).

Resting or ramified microglial cells are present in the unaltered adult brain (Del

Rio-Hortega 1932, Giulian and Baker 1986, Perry and Gordon 1988). The cells are

between 5 and 10 µm in size, are highly branched with a small amount of perinuclear

cytoplasma, and have a small, dense, and heterochromatic nucleus (Streit 1995).

Depending on their localization in the CNS, the branching pattern of ramified

microglial cells is heterogenous: in white matter their branches spread in a bipolar

manner, whereas in grey matter their processes extend in all directions, resulting in a

stellate appearance (see. Fig 1; Streit 1995, Kettenmann and Ransom 1995,

Gehrmann and Kreutzberg 1995).

Figure 1. Heterogeneity of microglia according to functional and develop-mental states (modified according to Kettenmann and Ransom 1995)

A ameboid microglia B ramified microglia in grey matter C ramified microglia in white matter D activated or reactive, nonphagocytic

microglia E activated, phagocytic microglia


17

Activated or reactive microglia can be found in pathologically altered CNS tissue.

In this activation status microglia are nonphagocytic (Streit 1995, Gehrmann and

Kreutzberg 1995). During activation microglia become hypertrophic and retract their

branches (see Fig. 1; Jordan and Thomas 1988, Gehrmann and Kreutzberg 1995).

They are capable of migrating to the site of injury, where they proliferate (González-

Scarano and Baltuch 1999).

In the presence of neuronal degeneration, microglia may continue their

metamorphosis, and subsequently are transformed into the third isoform, phagocytic microglia (Streit and Kreutzberg 1988, Streit 1995). In this state, microglial cells are

round-shaped. A striking feature of these cells is their high content of lysosomes and

phagosomes (see Fig. 1; Leonhardt 1990, Schulz 1991).

Another isoform, the ameboid microglia, is found in fetal and early postnatal brain

(Del Rio-Hortega 1932, Giulian and Baker 1986). These cells have a large cell body,

a macrophage-like appearance, and only few and short processes (see Fig. 1; Perry

et al. 1985, Streit 1995). Ameboid microglial cells are not elicited by tissue damage;

they seem rather to be normal and wide-spread in the developing brain (Jordan and

Thomas 1988, Perry et al. 1995). Their function is believed to be the elimination of

cell debris caused by natural degeneration and reorganization processes in postnatal

development by phagocytosis (Streit et al. 1988, Perry and Gordon 1988, Perry et al.

1995).

In summary, microglia do not appear as a uniform cell population but show

pronounced phenotypic heterogeneity (DeGroot et al. 1992, Streit and Graeber 1993,

Davis et al. 1994). Ramified and activated microglia are convertible (Streit et al.

1988, Jordan and Thomas 1988, Gehrmann et al. 1991, Giulian 1995, Stoll and

Jander 1999), as has been demonstrated in vitro (Giulian and Baker 1986) and by

prolonged time-lapse video microscopy (Thomas 1992). This phenomenon is also

called morphological polymorphism (González-Scarano and Baltuch 1999), and since

these morphological changes are associated with different functions, the term

functional plasticity was introduced to emphasize the enormous variability of

microglial cells (Streit et al. 1988). The transformation of microglia in different

activation states according to the degree of tissue destruction is termed “graded

response” (Raivich et al. 1999)


18

II. 1.3. Functional characteristics of microglia and their regulations

As yet little is known about the function of ramified microglia and the factors that

keep the cells in this condition (Gehrmann et al. 1992a, Giulian 1992b, Thomas

1992). The ubiquity of these cells, their strategic spacing throughout the CNS, and

their morphology with numerous long branches predestine them for a function in

immune surveillance and protection of the cells of the CNS (Ulvestad et al. 1994a,

Streit 1995).

Ramified microglia are not capable of performing phagocytosis (Giulian and Baker

1986), but they are able to carry out pinocytosis effectively, as has been proven by

their uptake of horseradish peroxidase and specific dyes (Ransom and Thomas

1991). This feature and these cells´ high motility indicate a function in fluid cleansing

(Ransom and Thomas 1991). These cells can also remove potentially harmful agents

produced by impaired cells in addition to end-stage products of metabolism.

Therefore, microglial cells may play a fundamental role in the maintenance of

homeostasis and tissue integrity, the “steady state condition” in the CNS (Graeber

and Streit 1990).

Ramified microglia have a down-regulated expression of surface molecules (Perry

and Gordon 1991, Kreutzberg 1996, Rezaie and Male 1999). In the course of

disruption in homeostasis, microglia quickly respond and transform into an active

state.

It is still not clearly defined how the mechanism of activation is regulated in vivo and

which factors are needed (Gehrmann and Kreutzberg 1995). Various substances or

noxious stimuli can elicit microglial activation, for example neuronal debris, blood

clots, dead cells (Del Rio-Hortega 1932), cytokines, immunoactivators, and receptor

ligands (Giulian 1995). It has been shown that microglia can be triggered in vitro by

factors that can also influence characteristics of macrophages such as granulocyte-

monocyte colony stimulating factor (GM-CSF), monocyte colony stimulating factor

(M-CSF), and colony stimulating factor 1 (CSF-1) (Giulian and Baker 1986, Giulian

and Ingeman 1988, Sawada et al. 1990, Suzumura et al. 1990, Lee et al. 1992, Lee

et al. 1994, Liu et al. 1994, Raivich et al. 1994, Streit 1995). Furthermore, microglia

can be triggered by lipopolysaccharide (LPS) (Hetier et al. 1988) and interferon-γ

(IFN-γ) (Frei et al. 1987, Suzumura et al. 1990). Some of these substances are

produced by astrocytes, which is a sign of the close functional relation between these


19

two cell types (Fedoroff et al. 1993). Neurotransmitters such as γ-amino butyric acid

(GABA), glutamate, and acetylcholine, and also purines and adenosintriphosphate

(ATP) can activate microglial cells (Gehrmann and Kreutzberg 1995, Ferrari et al.

1996, Inoue 2002).

It is thought that microglial activation follows a rather stereotypical pattern

irrespective of the underlying pathological condition (Stoll and Jander 1999, Raivich

et al. 1999, Graeber et al. 2002). Microglial activation starts with proliferation even if

the distance to the insult is as much as several micrometers (Graeber et al. 1988b,

Moore and Thanos 1996). Subsequent to proliferation, the microglia are recruited to

the site of injury, where they undergo changes in their morphology, immuno-

phenotype, and functional properties (Frei et al. 1987, Gehrmann et al. 1992a,

Gehrmann and Kreutzberg 1995).

The immunophenotypical alterations include increased or de-novo expression of

surface molecules (Graeber et al. 1988a). These alterations can be immuno-

modulatory such as the upregulation of MHC class I, MHC class II, and complement

receptor 3 (CR3: CD11b/CD18) (Streit et al. 1988). Enhanced expression of MHC

molecules (Thomas 1992, Davis et al. 1994, Gehrmann and Kreutzberg 1995,

Raivich et al. 1999) enables microglial cells to process and present antigen and to

interact with T-cells (Hickey and Kimura 1988, summarized by Kreutzberg 1996). In

some species microglia also express CD4 (Perry and Gordon 1988), an accessory

molecule of T-helper cells (Janeway and Travers 1997). Microglia are known to be

able to present important co-stimulatory molecules such as B7-1 (CD80) and B7-2

(CD86) (Williams et al. 1994a and b), and ICAM-1, which can be upregulated

(Raivich et al. 1999), and induce T-cell proliferation in vitro (Frei et al. 1987).

The plasma membrane of microglial cells is very complex; it contains in addition to

enzymes a huge amount of receptors and adhesion molecules (Streit 1995). This

multiantigenicity makes it possible to use various antibodies for immunophenotypical

characterization of microglia (Dijkstra et al. 1985, Perry and Gordon 1988, Gehrmann

and Kreutzberg 1991, Flaris et al. 1993, Perry et al. 1995, Streit 1995). Differentiation

between ramified and activated microglia is possible in part because of the fact that

some surface molecules are upregulated or de-novo expressed on activated


20

microglia (Graeber et al. 1988a, Streit 1995). All surface molecules on microglia can

also be found on activated macrophages (Dijkstra et al. 1985), which is interpreted as

evidence of a close relationship to the myelo-monocytic cell lineage. Until today the

entirety of microglial surface molecules has not been identified. Moreover, no specific

antibody has yet been generated that allows the distinction between microglia and

macrophages in extracerebral tissue (Streit 1995, González-Scarano and Baltuch

1999).

Nevertheless, it is possible to distinguish between microglia and monocytes/macro-

phages in terms of the percentage of cells expressing a surface molecule or it´s

expression intensity (Stein et al. 2004). According to the literature, microglia are

identified by their expression of CD11b/c+ and CD45low, whereas monocytes show an

expression of CD 11b/c+ and CD45high (Ford et al. 1995).

Following activation, microglia can release several mediators. Numerous studies

describe the spectrum of secretory products belonging to cytokines and growth

factors (Frei et al. 1987, Streit et al. 1988, Banati et al. 1993a, Flaris et al. 1993, Lee

et al. 1994, Giulian et al. 1994, Liu et al. 1998). These include interleukin-1β (IL-1β),

IL-3, IL-6, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β),

nerve growth factor (NGF), basic fibroblast growth factor (bFGF), prostaglandine D2

(PGD2), and prostaglandine E2 (PGE2). Some of these growth factors are known to

be neuroprotective or neurotrophic (Cheng and Mattson 1991). Production of these

mediators demonstrates microglial effector function and immunomodulatory capacity

(Frei et al. 1987).

Continuous and repeated noxious stimuli acting upon neurons can lead to

neurodegeneration (Streit 2002). Activated microglial cells are capable of secreting

potentially detrimental factors, as described for macrophages (Nathan 1987). These

include reactive oxygen intermediates (ROIs), nitric oxide intermediates (NOIs),

proteolytic enzymes (e.g. Cathepsin B and L, lysozyme, acid hydrolases),

arachidonic metabolites, excitotoxines (e.g. quinolinic acid) (Espey et al. 1997),

complement proteins, and proinflammatory and potentially cytotoxic cytokines (e.g.

TNF-α and IL-1α) (Frei et al. 1987, Colton and Gilbert 1987, Giulian et al. 1990,

Banati et al. 1993b, Giulian 1995, Kreutzberg 1996, Chabot et al. 1997).


21

The generated reactive oxygen and nitrogen intermediates are short-lived cytotoxic

factors that can increase excitotoxic synaptic transmission and may be harmful to

healthy neurons (Giulian et al. 1995). ROIs and NOIs may trigger the release of large

amounts of glutamate (Piani et al. 1991), which may in turn contribute to NMDA-

receptor (N-methyl-D-aspartate) mediated neurotoxicity (Banati et al. 1993b).

Oligodendrocytes and myelin sheaths are important regulators for synaptic

transmission (Bruce-Keller 1999). They show a selective vulnerability to some

secretion products of microglia, for example cytokines (e.g. TNF-α) (Selmaj and

Raine 1988), ROI, and excitatory amino acids (Griot et al. 1990, Takahashi et al.

2003).

Therefore, microglial effector functions are viewed as a double-edged sword which

might play a pivotal role in the pathogenesis of different neurological disorders

including not only human Alzheimer´s Disease (AD), Multiple Sclerosis (MS), AIDS

dementia complex (ADC), but also stroke, neurodegeneration and tumors (Giulian

1995, González-Scarano and Baltuch 1999, Streit 2002).

In their third state of activation microglia develop the ability to perform phagocytosis

(Streit et al. 1988, Kreutzberg 1996). This transformation step is modified by the

presence of astrocytes and cytokines. Co-cultivation with astrocytes and incubation

with TGF-β and IL-4 inhibit phagocytosis (Von Zahn et al. 1997) whereas

preincubation with GM-CSF and TNF-α leads to enhanced phagocytosis (DeWitt et

al. 1988). In vitro phagocytic activity has been shown to be dependent on expression

of complement receptors (Ulvestad et al. 1994b) and Fc receptors for IgG (Fc:

fragment crystalline for immunoglobuline G) (Ulvestad et al. 1994c). The presence of

microglia in direct proximity to degenerating neurons (Akiyama et al. 1994) suggests

that microglia isolate dying neurons, show neuronophagia, and therefore limit

damage by removing these neurons before cell lysis and the release of toxic

cytosolic agents can harm neighboring cells (Bruce-Keller 1999). Elimination of cell

debris is facilitated by hydrolytic enzymes, which can be found in phagocytic

microglia (Ling and Wong 1993, Williams et al. 1994c).

A final step initiates the elimination of the activated microglia themselves, presumably

through a programmed mechanism such as apoptosis (Gehrmann et al. 1995).

Elimination of microglia is an important control mechanism to terminate local


22

inflammatory reactions and to avoid excessive “bystander lysis” of uninjured tissue

(Bruce-Keller 1999). Only recently has research begun on the underlying

mechanisms and factors initiating apoptosis of microglial cells.

II. 1.4. Microglia in different diseases

As microglial cells are the immune effector elements of the brain, their activation is

seen in virtually all kinds of pathologies of the CNS (Giulian 1992a). Animal models

of neurodegeneration have demonstrated reactive microgliosis as a time-dependent

graded response according to the severity of the lesion (Kreutzberg 1996, Raivich et

al. 1999) that starts at the epicenter of the lesion and spreads restrictively into

surrounding brain tissue (Gehrmann et al. 1995). Ebert et al. (1999) found microglial

activation in the ipsilateral piriform cortex and to a lesser extend in the contralateral

piriform cortex following amygdala kindling in rats. These findings point to regional

and time-dependent differences in microglial activation in brain pathology which need

to be considered, if several regions of brain tissue are pooled for ex vivo

examinations.

In human immunodeficiency virus (HIV) encephalitis, the cells from the

monocyte/macrophage lineage are a primary target, in addition to the CD4-positive

lymphocytes. Although weak, the microglial cells´ expression of CD4 is responsible

for being a target of the virus (Watkins et al. 1990, Jordan et al. 1991, Williams et al.

1992). The histopathologic hallmark of HIV encephalitis is the formation of

multinucleated giant cells (Sharer et al. 1985), which express microglial antigens and

HIV-coat proteins, and contain HIV nucleic acids. Activated microglial cells are

believed to play a central role in the pathogenesis of neuronal cell death through the

release of several cytotoxic substances such as ROS, proteases, quinolinic acid, and

excitatory amino acids (Graeber et al. 2002).

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the CNS.

Activated and lipid-laden microglia can be found abundantly at the borders of

demyelinating lesions (Li et al. 1993), and show an immunophenotype linking to

antigen presentation and to the mediation of T-cell response. Microglia are capable

of damaging antibody-coated or opsonized antigens, particularly oligodendrocytes

and the myelin sheaths which further stimulate their oxidative burst activity, and the


23

release of inflammatory cytokines (Graeber et al. 2002). The microglia are therefore

believed to play a key role in MS pathogenesis (Tan et al. 1999).

The role of activated microglia in Alzheimer´s Disease (AD) is not yet clear. They

are associated with senile plaque β-amyloid and extracellular neurofibrillary tangles

(Cras et al. 1991), which suggests both support of initial plaque formation or

phagocytosis and procession of amyloid (Graeber et al. 2002). More recently, a novel

view has been proposed as an alternative way of regarding microglial role in AD

pathogenesis: when microglia become senescent or dysfunctional with normal aging,

this consequently leads to impairment of neuron-supporting functions of the microglia

(Streit 2002). All of these theories consider the microglia to be a central actor in the

pathogenesis of AD.

Head injuries induce an immediate microglial activation followed by a release of high

levels of proinflammatory cytokines (Woodroofe et al. 1991). Microglial activation

may persist for years and can therefore serve as a sensitive indicator even for subtle

local brain damage (Graeber et al. 2002).

Brain tumors are infiltrated extensively by activated and partly phagocytic microglia.

However, this infiltration does not result in effective cytotoxic action in vivo, even

though microglial potential can be proven in vitro (Frei et al. 1987). This is explained

by the secretion of cytokines by tumor cells, e.g. TGF-β, which downregulates

microglial cytotoxicity (Kiefer et al. 1994).

Trauma, meningitis, and encephalitis have been associated with the onset of

seizures, but the mechanisms by which these pathologic conditions produce

aberrant electrical discharges are not known (Giulian 1995). The feature in common

to these CNS pathologies is the presence of brain inflammation. Exposure to

microglial secretion products induced a series of epileptiform discharges in

hippocampal slice preparations (Giulian et al. 1994).

Microglia become rapidly activated in cerebral ischemia, subsequently upregulate

the expression of several immune-related antigens, becomes phagocytic, and

synthesizes cytokines (Graeber et al. 2002). The functional consequences of

microglial activation in ischemia are not yet understood.

In addition to experimental autoimmune encephalomyelitis (EAE), canine distemper (CD) is a well established animal model for demyelinating diseases such

as MS (Appel et al. 1981). Histopathological characterization shows great

correspondence among these diseases, and it is consequently assumed that they


24

also have analogous pathogeneses. In comparison with EAE, canine distemper has

the advantage of being a naturally occurring disease, and it might therefore be

preferable as a model for MS.

In a vaccine challenge experiment with canine distemper virus infection, we found a

marked upregulation of the surface molecules CD18, CD11b, CD11c, CD1c, MHC

class I and MHC class II, and a tendency for increased expression intensity of

ICAM-1 (CD54), B7-1 (CD80), and B7-2 (CD86) in dogs with histopathologically

confirmed demyelination. Functionally, microglial cells in demyelination due to CDV

infection revealed a significantly enhanced phagocytosis and ROS generation (Stein

et al. 2003).

Results of various studies point to an important role of ROS generation in the

pathogenesis of demyelination (Griot et al. 1989, Griot et al. 1990, Ruuls et al. 1995,

Vladimirova et al. 1998). In vitro studies in humans suggest the central importance of

microglia as effectors and regulators of demyelination (Ulvestad et al. 1994a).

Therefore, the aim of the present study was to evaluate whether the generation of

ROS is specific to a demyelinating disease such as canine distemper or if ROS

generation also occurs in other diseases. This approach should give an answer to

the question if a stereotypical reaction pattern of microglia exists which is not

correlated to the underlying pathological condition or if microglia can react in a

specific way depending on the underlying cause of a disease in the CNS.


25

II. 2. Surface molecules for immunophenotypic characterization

Functionally distinct cell populations express distinct types of cell surface proteins.

These protein molecules play important roles in the development and function of

those cells. A considerable amount of cross-talk and interplay between cells is

mediated by cell surface molecules; therefore surface molecules play a fundamental

role in the function of the immune system. The cell surface molecules recognized by

monoclonal antibodies (mAbs) are called antigens or markers because they identify

and discriminate between different cell populations (Janeway and Travers 1997,

Abbas and Lichtman 2003). Initially, surface markers were named according to the

antibodies that reacted with them. A uniform international nomenclature system was

adopted in order to standardize their description. Surface proteins identifying

particular lineages or differentiation stages with defined structures of similar epitopes

that are recognized by a group (= “cluster”) of monoclonal antibodies belong to the

cluster of differentiation (CD) system. This nomenclature system was originally used

for human leukocyte antigens, but it is common nowadays to refer to homologous

markers in other species and other cells by the same CD designation (Roitt et al.

1991, Janeway and Travers 1997, Lai et al. 1996, Lai et al. 1998, Abbas and

Lichtman 2003).

Surface antigens with defined structures are given a CD designation, and 247

antigens (CD1-CD247) have now been identified (Abbas and Lichtman 2003). Most

mAbs for characterization of the canine immune system originated from human

species and showed cross-reactivity (Jacobsen et al. 1993). However, in recent

years a growing number of mAbs have also been generated against canine cells.

The characterization and specifity of many canine mAbs were established at the first

international workshop on canine leukocyte antigens in Cambridge in 1993

(summarized by Cobbold and Metcalfe 1994).

The relevant CD antigens used in this study are summarized in Table 1 below.


26

CD antigen Synonyms

Molecularweight (kDa)

Functions Cellular expression

CD18 + CD11b: Mac-1 or CR3 95

β2-subunit of the integrins; binds to CD11a, b and c (and

d), signaltransduction

Leukocytes, every cell expressing a β2-

integrin

CD11b + CD18: Mac-1 or CR3 170

αM-subunit of the integrin CR3; binds CD54,

complement fragment C3b and extracellular matrix

proteins

Myeloid cells and natural killer cells

(NK cells)

CD11c + CD18: CR4 or p150,95

150 αX-subunit of the integrin CR4; binds fibrinogen Myeloid cells

CD45 LCA

(leukocyte common antigen)

180-240 (multiple isoforms)

Tyrosine phosphatase; T and B-cell antigen receptor-

mediated signaling

Leukocytes, cells of hematopoetic origin

CD1c 43

Molecule similar to MHC class I, associated with β2-

microglobulin; presentation of nonpeptide (lipid and

glycolipid) antigens to some T-cells

Cortical thymocytes, Langerhans cells, dendritic cells, B-

cells, gut epithelium, smooth muscle

ICAM-1 CD54 75-115 Cell-cell adhesions;

ligand for LFA-1-integrin and Mac-1

Lymphocytes, monocytes, fibroblasts,

endothelial cells

B7-1 CD80 60 Costimulator for T lymphocyte

activation; ligand for CD28 and CTLA-4

B-cells, macrophages, dendritic cells

B7-2 CD86 80

Costimulator for T lymphocyte activation; ligand for CD28

and CTLA-4, signal enhancement (?)

Monocytes, B-cells, dendritic cells


27

CD antigen Synonyms

Molecularweight (kDa)

Functions Cellular expression

MHC I Antigen processing and

presentation, graft rejection, T-cell proliferation

Virtually all nucleated cells, except neurons

MHC II Antigen processing and

presentation, graft rejection, T-cell proliferation

B-cells, dendritic cells, macrophages, thymusepithelium, T-cells, microglia

CD14 LPS-receptor 53-55

Receptor for complex of LPS and LPS-binding protein (LBP), required for LPS-

induced macrophage activation

Monocytes, macrophages, granulocytes

CD3 CD3-complex

γ: 25-28 δ: 20 ε: 20 ξ: 16 η: 22

Required for cell surface expression of and signal transduction by the T-cell

antigen receptor; cytoplasmatic domains

contain specific sequences for binding tyrosinkinases

T-cells, thymocytes

Table 1. Overview of the relevant canine CD antigens used in this study Summarized according to Moore et al. 1992; Cobbold and Metcalfe 1994; June et al. 1994; Blumberg et al. 1995; Danilenko et al. 1995; Johnson et al. 1996; Lai et al. 1996; Janeway and Travers 1997; Alldinger et al. 2000; Tizard 2000, Busshoff et al. 2001, Abbas and Lichtman 2003. CD: cluster of differentiation; kDa: kilo Dalton; Mac-1: membrane attack complex-1; CR3/4: complement receptor 3/4; NK cells: natural killer cells; LCA: leukocyte common antigen; MHC class I and class II: major histocompatibility complex class I and II; ICAM-1: intercellular adhesion molecule-1; LFA-1: lymphocyte function-associated antigen-1; CTLA-4: cytolytic T lymphocyte associated molecule-4; LPS: Lipopolysaccharide; LBP: Lipopolysaccharide-binding protein

28

III. Aims of the study

Microglial cells represent the endogenous immune system of the central nervous

system (Graeber and Streit 1990). Their surveillance of the CNS aims at protection

and support of neurons (Streit 2002, Hanisch 2002). Upon disturbance in

homeostasis, microglial cells reveal their immunological potential by increased or de-

novo expression of surface molecules, modulation of T-cell response, phagocytosis,

production and release of cytokines, chemokines, ROI, and NOI (Graeber et al.

1988a, Gehrmann and Kreutzberg 1995, Williams et al. 1994a, Williams et al. 1994b,

Giulian et al. 1994, Frei et al. 1987, Streit et al. 1988, Banati et al. 1993a, Flaris et al.

1993, Streit and Kreutzberg 1988, Kreutzberg 1996, Stoll and Jander 1999). These

reactions have to be considered critically, as their effects may be ambivalent in the

vulnerable microenvironment of the CNS. Although reactions are aimed at the

beneficial removal of disturbing agents or nonviable cells, excessive or sustained

activation of microglia can promote harmful actions of the defense mechanism,

resulting in direct neurotoxicity and acute or chronic neuropathologies (Hanisch

2002).

Streit (2002) thinks that the challenge now is to determine what sort of pathological

scenarios could transform microglia into autoaggressive effector cells that attack

healthy neurons and cause neurodegeneration.

It has long been thought that microglial cells follow a uniform and unspecific reaction

pattern upon activation (Stoll and Jander 1999). Evidence has grown that this view

substantially underrates microglial competence in the immune surveillance of the

CNS.

The aim of our study was to evaluate whether canine microglial reaction profile is

stereotypic irrespective of the underlying pathological condition or if it follows a

specific scheme which can be classified according to the kind of causal insult.

Therefore, the results of a preliminary study with 20 dogs experimentally infected with

canine distemper virus (CDV) in the course of a vaccine challenge experiment were

compared to the results of the second part of the study of 25 dogs suffering from

different intra- and extracranial diseases. Examinations included immunopheno-

typical characterization and functional determination of phagocytosis activity and the

generation of reactive oxygen species (ROS).

______________________________Aims of the study______________________________

29

The second part of the study also examines another feature of the microglial

immunological repertoire, the production of nitric oxide (NO). NO is produced after

the oxidation of L-arginine by a family of nitric oxide synthases (NOS) (Howe and

Boothe 2001). One of these enzymes, the inducible NOS (iNOS), is not typically

expressed on resting cells (Howe and Boothe 2001). In astrocytes and activated

microglial cells, NO is synthesized from induction of iNOS (Chao et al. 1992,

Minghetti and Levi 1998), and this expression is stimulated by various substances

such as cytokines, endotoxins and microbial products. Neurons and oligodendrocytes

are particularly sensitive to NO-mediated toxicity (Merill et al. 1993, Wei et al. 2000,

Golde et al. 2002). Therefore, NO might be crucial in the development of specific

neurological signs.

The overall aim of the second part of the study was to evaluate whether production of

NO by microglial cells can be correlated with certain diseases of the CNS.

30

IV. Material and Methods

IV. 1. Material

IV. 1. 1. Laboratory equipment

IV. 1.1.1. Technical equipment

Aqua tridest. desalinator, Seralpur USF Serial, Ransbach- Delta UF Baumbach, Germany Flow cytometer FACSCalibur® with Becton Dickinson, Heidelberg, Apple Macintosh®-computer Germany Freezer, GS1583 Liebherr, Ochsenhausen,

Germany Ice machine, “Scotsman“, AF 10AS Tepa, Barsbüttel, Germany Laboratory balance Omnilab, OL 1500-P Nordlab, Bremen, Germany Magnetic mixer with hotplate, type MR 3001 Heidolph, Schwabach,

Germany Microscope, H 600 Helmut Hund GmbH, Wetzlar,

Germany Pipettes, adjustable (0.1-1000µl) Brand, Wertheim, Germany Pipette helper, Accu jet Brand, Wertheim, Germany Refrigerator, KT 1940 Liebherr, Ochsenhausen,

Germany Shaker for microtitreplate, Promax 1020 Heidolph, Schwabach,

Germany Shaker for test tubes, “Reax top“ Heidolph, Schwabach,

Germany Ultrasound treatment device “Sonorex“, Bandelin electronic GmbH, type RK 103H/13067/00 Berlin, Germany Water basin with thermostat Julabo Labortechnik GmbH, Seelbach, Germany

____________________________Material and Methods____________________________

31

Waterdesalination plant, DI 2800 TKA Wasseraufbereitungs- systeme GmbH, Niederelbert, Germany

IV. 1.1.2. Equipment for sterile work

Bunsen burner, „gasprofi1“ WLD-Tec, Göttingen, Germany CO2- incubator, type 36150180003100 WTB Binder Labortechnik

GmbH, Tuttlingen, Germany Fiberglass filter paper, no. 003048.083 Schleicher u. Schüll, Dassel,

Germany Forceps, BD 660 Aesculap, Tuttlingen, Germany Microbiological bench, CA/R Clean Air, Hilden, Germany

IV. 1.1.3. Equipment for perfusion of brains

Cannulae, Neolus, lot no. 8895B01F, Terumo, Leuven, Belgium 18 G x 11/2’’, 1.2 x 40 mm Catheter Vasofix®, Ch. 4268130B, Braun, Melsungen, Germany 0.9 x 25 mm Dressing scissor, BC 283R Aesculap, Tuttlingen, Germany Forceps, BD 27 Aesculap, Tuttlingen, Germany Forceps, rat tooth BD 557 Aesculap, Tuttlingen, Germany Gloves, Peha-soft Hartmann, Heidenheim,

Germany Infusion instruments, Perfudrop®-Air G Clinico, Bad Hersfeld, Germany Liston forceps, FO 626 Aesculap, Tuttlingen, Germany Mosquito forceps, BH 412R Aesculap, Tuttlingen, Germany Scalpel blades, BB510, lot no. H21312 Aesculap, Tuttlingen, Germany Scalpel handles, BB73 Aesculap, Tuttlingen, Germany Scissors, small, BC 111 Aesculap, Tuttlingen, Germany


32

Spinal needle, Yale, 0.7x40 mm Becton Dickinson, Heidelberg, Germany Syringes, 2 ml, Discardit II, lot no. 0002-086 Becton Dickinson, Fraga, Spain Tissue forceps, AE 160-12 Geomed Medizin-Technik,

Tuttlingen, Germany Tissue forceps, rat tooth, BD 512 Aesculap, Tuttlingen, Germany

IV. 1.1.4. Laboratory material

Cell count chamber TÜRK Brand, Wertheim, Germany Cup with lid, 1.5 ml, (order no. 7805 00) Brand, Wertheim, Germany Glass pipettes, sterile (1.5, 10 u. 20 ml) Brand, Wertheim, Germany Glass bottles, Plastibrand, various sizes Omnilab, Gehrden, Germany Microscope slides, Superfrost plus Menzel-Gläser, Braunschweig, Germany Microtitre plates, 96-well, round bottom, Brand, Wertheim, Germany non-sterile (order no. 7020147) Pasteur pipettes, 150 mm (order no. 7477 15) Brand, Wertheim, Germany Petri dish (order no. 450 2000) Brand, Wertheim, Germany Pipette tips Brand, Wertheim, Germany 0.1 - 20 µl (order no. 7023 12) 2 - 20 µl (order no. 7023 15) 50 - 1000 µl (order no. 7023 20) Refractometer, HRM-18, no. 10487 Krüss, Hamburg, Germany Steel sieve, diameter: 6 cm Teehaus Felber, Hannover,

Germany Tubes, 15 ml (order no. 1148 17) Brand, Wertheim, Germany Tubes, 50 ml (order no. 1148 22) Brand, Wertheim, Germany Tubes for flow cytometry, Becton Dickinson, Franklin Falcon, 12 x 75 mm (order no. 2008) Lakes, NJ, USA Ultracentrifugation tubes (order no. VS0101, Vivascience, Hannover, lot no. 03VS50018A) Germany


33

IV. 1.1.5. Centrifuges

Centrifuge “Rotina 35R“ Hettich, Tuttlingen, Germany Centrifuge “Rotanta RP“ Hettich, Tuttlingen, Germany

IV. 1.2. Reagents

Albumin, bovine, fraction V, 98% Roth, Karlsruhe, Germany pulverized (BSA, bovine serum albumin; order no. 8076.2) CPSR-1 (s. IV.1.3), (order no. C8905) Sigma-Aldrich, Schnelldorf,

Germany CPSR-3, (order no. C9155, lot no. Sigma-Aldrich, Schnelldorf, 071K8405), Hybri-Max® Germany Dihydrorhodamine 123 (s. IV.1.7) MoBiTec GmbH, Göttingen, Germany Dimethylsulfoxide (DMSO) Sigma-Aldrich, Deisenhofen, (order no. D-5879, lot no. 99H0020) Germany Hanks´ solution (s. 3.1.3) Sigma, Deisenhofen, Germany (order no. H4385) Heparin-Natrium-25000-ratiopharm® Ratiopharm GmbH, Ulm, (Ch.-B. A06837) Germany HEPES (= N- [2-hydroxyethyl] piperazine-N-2- Fluka, Buchs, Switzerland ethansulfonic acid, s.IV.1.3) Paraformaldehyde (order no. 1.04005.1000) Merck, Hannover, Germany PBS (s. IV.1.3) Penicillin (G-)Streptomycin (10.000U/ml Sigma, Deisenhofen, Germany + 10 mg/ml), CellCulture® (order no. PO781, s. IV.1.3) Eutha® 77 (400 mg pentobarbiturate/ml) Essex Tierarznei, München, Germany Percoll® (s. IV.1.4) Pharmacia, Freiburg, Germany PMA (s. IV.1.7) Sigma, Deisenhofen, Germany


34

RPMI 1640 with L-glutamine and 2g/l NaHCO3 Cytogen, Ober Mörlen, (order no. P04-16500, s. IV.1.3.) Germany Saponin (order no. 1.59665.0001) Merck, Hannover, Germany Sodium azide, (NaN3), > 99%, purest Merck, Darmstadt, Germany (molecular weight: 65.01 g/mol, order no. 6688) Sodium bicarbonate (Na2CO3) Sigma Chemical Co., St. Louis, (order no. S-8875) USA Sodium chloride (order no. 9265.2) Roth, Karlsruhe, Germany Softasept®N (74.1% ethanol, 10.0% Braun, Melsungen, Germany n-propanol; order no. 0388 7049) Trypan blue (order no. T-6146) Sigma Aldrich, Schnelldorf,

Germany Tutofusin solution® (s. IV.1.3) Baxter, Unterschleissheim, Ch.-B. 6309112 Germany

IV. 1.3. Solutions

Dissociation buffer: NaCl 89.4 g/l KCl 37.3 g/l MgCl2 40.0 g/l CaCl2 25.3 g/l Hanks’ solution (s. IV.1.2; Sigma, Deisenhofen, Germany): NaCl 8.0 g KCl 0.4 g Glucose 1.0 g KH2PO4 60.0 mg Na2PO4 47.5 mg Phenol red 17.0 mg Aqua bidest. ad 1000 ml + addition of: CPSR-1 20.0 ml Sodium bicarbonate (Na2CO3), 7.5% 4.7 ml

- for adjustment of pH-value on pH 7.3-7.4


35

Collagenase-DNAse buffer (Collagenase, s. IV.1.5, Serva, Heidelberg; DNAse I s.IV.1.5, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) (for 2 g brain material): DNAse 1.000 Units Aqua bidest. 2.0 ml Collagenase 11.4 mg Dissociation buffer 0.2 ml Culture Medium RPMI 1640 + CPSR-1 5% + HEPES 10 mmol + Penicillin (-G)-Streptomycin 100 U/ml + 100 µg/ml MIF (membrane immunofluorescence) buffer: PBS with 1% BSA and 0.01% NaN3 BSA 1.00 g NaN3, 10% 0.01 g PBS ad 100 ml PBS (phosphate-buffered saline) pH 7.4: NaCl 8.00 g KCl 0.20 g Na2HPO4 1.15 g KH2PO4 0.20 g Aqua bidest. ad 1000 ml Tutofusin®injection solution (s. 3.1.2, Baxter, Unterschleißheim, Germany): NaCl 8.182 g KCl 0.373 g CaCl2 x 2 H2O 0.368 g MgCl2 x 6 H2O 0.305 g Aqua ad inj. 1000 ml

IV. 1.4. Reagents for separation und differentiation of cells

Percoll® Pharmacia, Freiburg, Germany (order no. 17-089-01, lot nos. 278498 and 299354)


36

IV. 1.5. Enzymes

Collagenase of Clostridium histolyticum, Serva, Heidelberg, Germany EC 3.4.24.3, lyophilized (order nos. 17449 and 17456, contr. nos. 24029 and 15445) Deoxyribonuclease I (DNAse I, EC 3.1.21.1) Sigma-Aldrich Chemie GmbH, type IV: of bovine pancreas Steinheim, Germany (order no. D-5025, lot nos. 28H7015 and 31K7659) IV. 1.6. Reagents for indirect membrane immunofluorescence technique MIF buffer (s. IV.1.3) Primary antibodies Human normal immunoglobuline G Globuman Berna®, Serum- (HuIgG, lot no. 015 117.01); und Impfinstitut Berne, dilution used: 1:16 Switzerland Monoclonal antibodies (mAbs), unconjugated Class I Major Histocompatibility Antigen VMRD, Inc., Pullman, WA, USA (MHC I); cell line H58A; isotype: IgG2a (lot no. 0698-0899); dilution used: 1:16


37

Antibody against the

following surface molecules

Clone Isotype Dilution used

CD18 CA1.4E9 IgG1 1 : 5

CD11b CD16.3E10 IgG1 1 : 5

CD11c CA11.6A1 IgG1 1 : 5

CD1c CA13.9H11 IgG1 1 : 5

ICAM-1 (CD54) CL18-1D8 IgG1 1 : 5

B7-1 (CD80) CA24.5D4 IgG1 1 : 5

B7-2 (CD86) CA24.3E4 IgG1 1 : 5

MHC class II CA2.1C12 IgG1 1 : 5

CD3 CA17.2A12 IgG1 1 : 5 Table 2. Antibodies for immunophenotypic characterization of canine

microglial cells and their dilutions used, and which were provided by Prof. Peter F. Moore

Monoclonal mouse antibodies (mAbs) used for immunophenotypic analyses were

either specific for dog cell surface markers or cross-reacting with these markers.

Mabs directed against CD11b, CD11c, CD18, CD1c, ICAM-1 (CD54), B7-1 (CD80),

B7-2 (CD86), CD3, and MHC class II were provided by Prof. Peter F. Moore,

University of California, Davis, CA, USA (see Tab. 2). They were prepared as tissue

culture supernatant with addition of 0.1% sodium azide. MAbs against CD11b,

CD11c, CD18, and MHC class I and II were characterized at the First International

Canine Leukocyte Antigen Workshop (CLAW, Cobbold and Metcalfe, 1994). All

antibodies were used for flow cytometry (FACS analysis). Human normal

immunoglobuline G (Globuman Berna, Berne, Switzerland) was used to block non-

specific binding.

Monoclonal antibodies, conjugated Mouse anti-human CD14 conjugated with Dako, Glostrup, R-Phycoerythrine (RPE); purified IgG; Denmark clone TÜK4 (lot no. 091/101); dilution used: 1:7


38

Rat anti-dog CD45 conjugated with Biotin; Serotec, Eching, purified IgG; MCA 1042B; Germany clone no.: YKIX 716.13 (lot no. 0699); dilution used: 1:10 Secondary antibodies Purified R-Phycoerythrine-conjugated Dianova, Hamburg, F(ab´)2-fragment goat anti-mouse Germany (gαm-PE) IgG (code no. 115-116-072; lot no. 45974); dilution used: 1:100 Streptavidin conjugated fluorescein- Serotec, Eching, isothiocyanat (isomer 1; FITC); STAR2B Germany (lot no. 1199); dilution used: 1:100

IV. 1.7. Reagents for measurement of reactive oxygen intermediates

Dihydrorhodamine 123 (DHR 123) MoBiTec GmbH, (molecular weight: 346.38 g/mol; Göttingen, Germany order no. D632) PBS (s. IV.1.3) PMA, phorbol-12-myristate-13-acetate Sigma, Deisenhofen, (order no. P-8139; lot no. 071K2059) Germany Production of DHR working solution: Dried dihydrorhodamine 123 (DHR 123) was diluted in DMSO to a concentration of

1.5 µg/ml. Working solution was produced by further dilution with PBS to a

concentration of 15 µg/ml DHR 123.

IV. 1.8. Material for measurement of phagocytic activity

Staphylococcus aureus conjugated with Molecular Probes, fluorescein (wood strain without protein A) Leiden, The Netherlands BioParticels®; (order no. S 2851; lot no. 6301-2)


39

Serum for opsonization of bacteria: Blood was taken in the course of routine examinations from ten healthy beagles of

the Small Animal Clinic. After coagulation, blood samples were centrifuged for 15 min

and RT at 2,000 x g at room temperature (RT). To remove remaining corpuscles, the

supernatant was again centrifuged for 10 min at 3,500 x g at RT. Resulting sera from

the ten dogs were pooled and stored in aliquots at -20 °C. Serum was diluted 1:5 with

PBS for phagocytosis assay.

IV. 1.9. Material for measurement of nitric oxide (NO)

Nitric oxide (NO2

-/NO3-) assay R&D Systems GmbH,

(catalog no. DE1500) Wiesbaden, Germany

IV. 1.10. Material for flow cytometry

Cleaning solution: FACSRinse® Becton Dickinson, (order no. 340346; lot no. 28) Heidelberg, Germany Measuring solution: FACSFlow®; Becton Dickinson, (order no. 342003; lot no. 1294) Heidelberg, Germany

IV. 1.11. Computer software

Cell-Quest® software Becton Dickinson, (for Apple Macintosh® computer) Heidelberg, Germany

IV. 1.12. Virus

The virulent CDV strain A75/17 was provided by the Institute of Animal Neurology,

University of Berne, Switzerland. The virus was propagated in specific pathogen-free

(SPF) animals, and CDV containing lymphatic tissue had been homogenised, purified

and frozen at -70 °C in a previous experiment (Cherpillod et al. 2000). The 20


40

beagles of the vaccine challenge experiment were infected with 0.2 ml of virus

homogenisate (Schreiner 2001).

IV. 1.13. Animals

Microglia from 47 dogs were examined; 22 of the 47 dogs originated from a vaccine

challenge experiment while 25 dogs had been euthanized because of severe

neurological deficits on request of the owner, and subjected to histopathological

diagnosis (s. Appendix, Tab. XI. 2).

A DNA vaccine study was conducted with challenge infection with the virulent CDV

strain A75/17 according to Swiss national regulation 111/99/Berne/CH (Cherpillod et

al. 2000, Schreiner 2001). Microglia were examined ex vivo from 20 five- to six-

month old, experimentally CDV infected SPF Swiss Beagle dogs, 10 males and 10

females, from two litters. Thirteen animals developed mild transient systemic

symptoms and lymphopenia. Seven dogs developed a febrile condition, and four of

these dogs exhibited neurological signs. All dogs were euthanized between 20 and

26 days post infection (PI). In addition, two uninfected dogs of the same age and

breed (one male, one female) served as negative controls (see Appendix, Tab. XI. 1;

Stein et al. 2003).

The dogs were divided into three groups according to the histopathological findings

(see Appendix, Tab. XI. 1). Group I comprised the two uninfected, healthy negative

control dogs with entirely normal findings in CNS (dogs nos. 21 and 22). All dogs

displaying active lesions and CDV antigen in the CNS were grouped in group III,

while other dogs which had been challenged but had neither demyelinating lesions

nor CDV antigen in their CNS were assigned to group II (Stein et al. 2003). No

lesions nor CDV antigen were found in 10 animals (nos. 3, 4, 5, 6, 8, 9, 10, 11, 14,

16). Three dogs (nos. 12, 13, 18) had few scattered glial nodules in the CNS, but had

no inflammatory or demyelinating lesions (group II). These glial nodules consisted of

mononuclear cells, microglia and astrocytes. No CDV antigen was found in the CNS

of these animals. Seven dogs showed histopathological changes typical of acute

CDV infection (Vandevelde and Zurbriggen 1995). The brains of the dogs no. 2, 15

and 17 displayed small multifocal demyelinating lesions with vacuolation of the white

matter, astrocytic swelling, mild gliosis and occasional macrophages. Dogs no. 1, 7,


41

19 and 20 exhibited relatively large multifocal plaques with complete myelin loss,

marked gliosis and invasion with macrophages (group III). All demyelinating lesions

and additional periventricular and subpial areas contained many CDV infected cells

in this group.

Ex vivo microglial examination of the dogs of the vaccine challenge experiment was

performed from July 20, until August 9, 2000, in a high-security containment facility

(biosafety level 4) at the Institute of Virology and Immunoprophylaxis (IVI),

Mittelhäusern, Berne, Switzerland.

Results of microglial examination obtained from vaccine challenge experiment (22

dogs, Stein et al. 2003) were compared with results of microglial examination of 25

dogs with other diseases. These dogs had been euthanized because of severe

neurological deficits on request of the owners and submitted to histopathological

diagnosis in the years 2002 and 2003. Before euthanasia the dogs were examined

clinically and neurologically. Various techniques were used to confirm the clinical

diagnosis. These included digital radiography, electrodiagnostics (electroencephalo-

gram = EEG, electromyography = EMG, nerve conduction velocity = NCV), computed

tomography (CT), magnetic resonance imaging (MRI), and cerebrospinal fluid (CSF)

examination. Histopathological diagnosis was the basis for assigning these 25 dogs

to five additional different examination groups. Group IV consisted of five dogs with

intracranial tumors, group V consisted of five dogs with intracranial inflammation; one

of these animals suffered from CDV infection and showed classical demyelinating

lesions in CNS comparable to those of the dogs of group III of the vaccine challenge

experiment. Group VI comprised two dogs with idiopathic epilepsy. Group VII

comprised five dogs with other changes in CNS such as trauma, degeneration, and

malformations such as hydrocephalus internus and meningocele. Group VIII

contained eight dogs with extracranial diseases, five of which (group VIIIa) had a

manifestation of disease in the spinal cord or in the peripheral nervous system

(PNS); three dogs with no manifestation of disease in the nervous system were

assigned to group VIIIb. Findings in the CNS in these dogs were probably age-

related and must therefore be considered normal for senile dogs. See Appendix

(Tables XI. 1 and XI. 2) for further information about individual dogs.


42

Examination group

Number of dogs Histopathological diagnosis

I 2 vaccine challenge experiment: no changes, unchallenged

II 13 vaccine challenge experiment: no changes, challenged

III 7 vaccine challenge experiment: demyelinating lesions due to CDV infection

IV 5 intracranial tumors

V 5 intracranial inflammations

VI 2 no changes in CNS; idiopathic epilepsy

VII 5 other changes in CNS

VIIIa/b 8 extracranial diseases

VIIIa 5 disease of the spinal cord or PNS

VIIIb 3 no abnormalities in the nervous system

Table 3.: Definition of the eight examination groups according to histopathological findings in the central nervous system (CNS) and peripheral nervous system (PNS). CDV = canine distemper virus


43

For the determination of nitric oxide production by microglia, these cells were

cultured for 24 h in 5% CO2 and at 37 °C in a humid atmosphere; subsequently the

Griess reaction was performed on microglial culture supernatant and CSF (s. IV. 2.7.

and IV. 2.8.). To evaluate differences in NO production depending on occurrence of

seizures, the dogs of the second part of the study were divided into groups with and

without seizure activity.

The group of dogs with seizures comprised eight dogs, three of which had

intracranial tumors (dogs no. 1, 2, and 3): one dog had granulomatous meningo-

encephalitis (no. 4); two dogs had idiopathic epilepsy (nos. 5 and 6); one dog had

hydrocephalus internus and atrophy of the cortex cerebri (no. 7); and one dog had

cirrhosis of the liver and suspected hepatoencephalic syndrome in addition to

nephritis, endocarditis, and satellitosis in the lobus occipitalis, which was probably

age-related. See Appendix (Tab. XI. 3) for further information about individual dogs.

The group without seizures comprised 14 dogs, two of which had intracranial

inflammations (dogs no. 9 and 10); one dog had head trauma (no. 11); one,

hydrocephalus internus (no. 12); one, cerebellar abiotrophy (no. 13); four dogs had

no abnormalities in the CNS, two of these had polyradiculoneuritis (nos. 14 and 15);

one had retinal atrophy (no. 17); one had cervical disc herniation (no. 18); three dogs

had no abnormalities of the CNS except for some changes which were probably age-

related (nos. 16 and 22); one had thoracic disc herniation (no. 21); one had an

intracranial tumor (no. 19); and one dog had cerebellar malacia (no. 20). See

Appendix (Tab. XI. 3) for further information about individual dogs.

Additionally, NO was determined in the CSF of 56 dogs suffering from various CNS

diseases (see Appendix, Tab. XI. 4). Fourteen of these dogs showed seizure activity.

The clinical diagnosis of these dogs was confirmed by clinical and neurological

examinations and subsequent further examinations including complete blood cell

count and biochemical analysis, CSF diagnostics (cell count, differentiated cell count,

determination of protein and Immunoglobulin A [IgA] content) as well as with

advanced techniques such as electrodiagnostics, CT or MRI. The clinical diagnosis

was the basis of assigning the dogs to four examination groups, which were also

defined to differentiate NO production of dogs with and without seizures. The first

examination group contained 14 dogs with seizure activity and suffering either from


44

idiopathic epilepsy (n = 9 dogs, nos. 23 to 31), or from intracranial tumors (n = 5

dogs, nos. 32 to 36, see Appendix, Tab. XI. 4).

The second group comprised 15 dogs with extracranial diseases and normal CSF

but without seizures. Two of these dogs suffered from an intramedullary tumor in

the spinal cord (nos. 37 and 38); five dogs had disc herniations (dogs nos. 39-43),

and one dog had a compression of the cauda equina (no. 44); two dogs had

recovered from intracranial inflammations (nos. 45 and 46); two displayed peripheral

vestibular signs (nos. 47 and 48); one dog had thoracic vertebral fracture luxation

(no. 49), one had sudden blindness (no. 50), and one had facial palsy (no. 51).

The third group comprised 24 dogs with intracranial inflammation but without seizures. Among these were dogs suffering from granulomatous meningoencephalo-

myelitis (GME; n = 5, nos. 52 to 56), steroid responsive meningitis arteritis (SRMA; n

= 15, nos. 57 to 71), viral encephalitis (no. 72), encephalitis of unknown origin (nos.

73 and 74) and trauma and encephalitis (no. 75).

The fourth group comprised three dogs with intracranial tumors but without seizures. Two dogs had gliomas (nos. 76 and 77) and one dog had plexus

carcinoma (no. 78; s. Appendix, Tab. XI. 4).


45

IV. 2. Methods

IV. 2.1. Cerebrospinal fluid (CSF) collection

CSF was obtained by sterile puncture of the spatium suboccipitale with a spinal

cannula with the dog in lateral recumbency directly following euthanasia or in general

anesthesia. CSF was immediately frozen and stored at -70 °C.

IV. 2.2. Isolation of canine microglial cells

Isolation of microglia was performed according to the method described by Sedgwick

et al. (1991) and modified for the dog (Stein 2001; Stein et al. 2004). Animals were

given 12,000 units of heparin intravenously and killed by injection of an overdose of

pentobarbital. Immediately after death, the brains were perfused via the left ventricle

of the heart with between 1 and 2 l of cold electrolyte solution at an approximate

hydrostatic pressure of 1 m. The left auricula cordis was opened to ensure discharge

of blood while circulation in the body was prevented by compression of the vena cava

caudalis. Perfusion time was between 30 and 45 minutes for each dog. Following

perfusion, the brains were removed and cut sagitally along the midline, and one-half

was transferred to ice-cold Hanks´ buffer containing 3% fetal calf serum (FCS) at a

pH of 7.36.

After removal of the meninges, between 10 and 15 g of brain tissue around the forth

ventricle (for the dogs of the vaccine challenge experiment) or almost one half of the

brain, between 15 and 20 g of brain tissue, around the lesions identified with

advanced imaging techniques (for the dogs of the second part of the study) was

pureed by passing it through a stainless steel sieve (see Fig. 2). The mechanically

dissociated material was collected by centrifugation at 170 x g for 10 min at 4 °C in a

50 ml centrifugation tube. Supernatant was removed and the material from each

brain was then enzymatically digested by addition of Collagenase-DNAse buffer for

60 min at 37 °C. During the course of the digestion, the CNS preparation was gently

resuspended after 30 min. The digested brain material was washed twice with

Hanks´ buffer, pelleted and subjected to centrifugation in two consecutive density

gradients.


46

For production of the different densities Percoll was brought into an isotonic solution

with 1.5 M NaCl-solution at a ratio of 1:10. This solution has a density of 1.124 g/ml.

Other densities required – 1.077, 1.066, 1.050 and 1.030 g/ml Percoll – were made

by addition of Hanks´ solution according to the manufacturer´s formula:

1r0.9-r10.1-rVV

0

00 −

××=

V0 volume Percoll needed in ml

V end volume of working solution needed in ml

r densitiy of working solution needed in g/ml

r0 density of Percoll (in this case: 1.124 g/ml)

r10 density of Hanks’ solution (1.0046 g/ml)

The accuracy of the Percoll densities was monitored by use of a refractometer (s. IV.

1.1.).

For the initial density gradient, the cells were resuspended in 45 ml of isotonic

Percoll (Pharmacia, Freiburg, Germany) diluted in Hanks´ buffer resulting in a density

of 1,030 g/ml. This suspension was underlayered with 5 ml of isotonic Percoll at a

density of 1,124 g/ml. Centrifugation was performed at 1,250 x g for 25 min at 20 °C

(acceleration time of 1 min and deceleration without brake). Cells were collected from

the surface of the 1,124 g/ml layer after discarding the myelin debris on top of the

1,030 g/ml layer and washed in Hanks´ buffer.

The cells gained from the initial density gradient were resuspended in 5 ml of Hanks´

buffer and pipetted on top of the major gradient. For the major gradient, 5 ml of

Percoll at 1,124 g/ml was put into a 50-ml tube and subsequently overlayered with 12

ml of Percoll (1,077 g/ml and 1,066 g/ml) and with 8 ml Percoll (1,050 g/ml and 1,030

g/ml) diluted in Hanks´ buffer. Cells derived from up to 8 g of brain material can be

layered onto one major gradient. The gradient was centrifuged at 1,250 x g for 25

min at 20 °C (acceleration time of 1 min and deceleration without brake). As shown in

preliminary studies (Stein et al. 2004) canine microglia accumulate specifically on the

surfaces of the Percoll at densities of 1,077 and 1,066 g/ml Percoll. Cells were

collected and their viability was determined immediately by trypan blue exclusion.


47

Cell samples were counted using a hemocytometer and adjusted to the concentration

needed for further examination.

Sufficient perfusion of the CNS was indicated either by subsequent discharge of a

water-like fluid from the right auricula cordis in combination with the pale appearance

of the meninges and the brain and the absence of erythrocytes on top of the 1.124

g/ml interface, or in the pellet.


48

Figure 2. Isolation protocol for canine microglial cells For the dogs of the vaccine challenge experiment between 10 and 15 g

of brain tissue around the forth ventricle was used for microglial ex vivo examination (rhomb) whereas for the dogs of the second part of the study between 15 and 20 g of brain tissue was used from around the lesion (star) identified by advanced imaging techniques (ellipse).


49

IV. 2.3. Histopathological and immunohistochemical examination

Immediately after perfusion and removal of the part of the brain needed for microglia

isolation, the contralateral hemisphere of each dog was fixed in 4% buffered formalin

and processed for paraffin embedding. Sections of representative areas of brain and

spinal cord were mounted on positively charged slides (Superfrost plus, Menzel-

Gläser, Braunschweig, Germany) and stained with hematoxylin-eosin (HE).

Histopathological examination of dogs in the vaccine challenge experiment was

performed by Prof. Dr. Marc Vandevelde and Dr. Rosemarie Fatzer, Institute of

Animal Neurology, University of Berne, Switzerland. Histopathological evaluation of

the brains including parts of the lesions identified by imaging techniques (CT or MRI)

of the dogs of the comparative study (i.e. data from the examinations from the years

2002 and 2003) was performed by Prof. Dr. Wolfgang Baumgärtner, Institute of

Pathology, School of Veterinary Medicine, Hannover, Germany.

IV. 2.4. Light microscopic determination of cell yield

The cell yield was determined by suspension of 10 µl of the isolated cell suspension

in 90 µl of trypan blue. Ten µl of the suspension were placed into a TÜRK cell

counting chamber (s. IV.1.1) and the cell numbers determined. Only uncoloured,

therefore living, cells were taken into consideration. The cell sample was adjusted

with PBS to the concentration needed for further examination

IV. 2.5. Indirect membrane immunofluorescence (MIF)

After isolation of canine microglia, the cells were adjusted to a concentration of 2 x

105 cells in 50 µl PBS. Microglial cells were stained as previously described for

lymphocyte subsets (Tipold et al. 1999). Staining procedures were performed using

sterile reagents and equipment. All antibodies and solutions were used at 4 °C, and

incubation steps were performed on ice. After Fc receptor blockage with 10 mg/ml of

human IgG, primary antibodies were incubated for 30 min and washed twice with

Hanks´ buffered saline (Sigma, Deisenhofen, Germany).


50

Unconjugated mAbs were detected using the following secondary antibodies: F(ab´)2

goat anti-mouse R-phycoerythrine IgG (dilution 1:100; Dianova, Hamburg, Germany)

for the mouse mAbs, and streptavidine-conjugated fluorescein-isothiocyanate

(dilution of 1:100; Serotec, Eching, Germany) for the mAb detecting CD45. After

addition of the secondary antibodies incubation was continued for 30 min. Staining of

CD45 was performed as double-labeling with mAbs against CD18 and CD11b. After

two washing steps, cells were resuspended in between 200 and 400 µl FACS flow

solution and analyzed immediately after staining without fixation with a

FACSCalibur™ (for dogs of the second part of the study) or with FACScan™ (for

dogs of the vaccine challenge experiment) using the Cell-Quest software for Apple

Macintosh computer (Becton Dickinson, Heidelberg, Germany).

In general, a counting rate of 200 to 300 events per second was used, and the

sample and collection tube were cooled to 4 °C. Negative control samples were

stained with the secondary antibodies only. Analysis gates were adjusted not to

exceed 2% positive staining with negative controls. Between 5 and 103 live gated

events were analyzed for each sample. Data were stored and analyzed later.

In the immunophenotypic analysis, microglial cells were identified on the basis of the

parameters size (FSC), complexity (SSC), and relative expressions of CD18 and

CD11b/c+ (Stein et al. 2004), which resulted in a clearly detectable cell population in

flow cytometry. Cells were purified by technical means and re-analyzed by gating this

population and taking into consideration only cells inside the gate.

The results were evaluated on the basis of the two parameters, percentage of

positive cells for each surface marker and mean fluorescence intensity (measured by

means of fluorescent channel numbers, shown as log values) as a relative means of

the expression intensity of each surface marker.

IV. 2.6. Functional examination of canine microglial cells

IV. 2.6.1. ROS generation test

Analysis of the functional activity of canine microglial cells to produce reactive

oxygen species (ROS) was performed using the method described by Emmendörffer

et al. (1990). Briefly, the method is based on the fact that the non-fluorescent

derivative of rhodamine, dihydrorhodamine 123, is taken up by phagocytic cells such


51

as microglia and granulocytes, and is converted by membrane-adapted

myeloperoxidase during respiratory burst into a green-fluorescent compound,

rhodamine 123.

Dihydrorhodamine 123 (DHR 123) from MoBiTec GmbH (Göttingen, Germany) was

diluted in dimethylsulfoxide (DMSO, Sigma-Aldrich, Deisenhofen, Germany) to a

concentration of 150 µg/ml and further diluted with PBS to a concentration of 15

µg/ml.

To assess the production of ROS, cells were stimulated with a trigger to optimize

measurement of changes in fluorescence activity and intensity. Phorbol-myristate

acetate (PMA, Sigma, Deisenhofen, Germany) was diluted in DMSO and

consecutively diluted with PBS to a final concentration of 100 nmol PMA/l cell

suspension to optimize results.

Cell suspension was adjusted to a concentration of 2 x 105 cells in 100 µl. To achieve

identical and comparable levels of activation after the isolation process, the cells

were pre-incubated at 37 °C and in 5% CO2 for 15 min. The same incubation was

performed after adding the different triggers and PBS as a negative control, and after

adding DHR 123. The tubes were put on ice in dark surroundings to stop ROS

generation and minimize the amount of adherent cells.

Samples were measured immediately by flow cytometry in triplicates for each

approach; mean values were used for evaluation. The results were evaluated on the

basis of the two parameters, percentage of positive (ROS-producing) cells and mean

fluorescence intensity (measured by means of fluorescent channel numbers, shown

as log values) as a relative means of the intensity of ROS generation seen as a shift

in FL1. Results were evaluated using the multiple histogram analysis of the FACS

Cell-Quest software provided by Becton and Dickinson.

IV. 2.6.2. Phagocytosis assay

The ability of canine microglia to perform phagocytosis was measured using a flow

cytometry assay. Heat-killed and lyophilized Staphylococcus aureus (Molecular

Probes, Leiden, The Netherlands) were resuspended in PBS. This suspension was

sonicated for 4 min. After counting the bacteria, we adjusted the concentration to 108

bacteria/ml. Either PBS or 50 µl pooled serum from 10 dogs were added for


52

opsonization to each bacteria suspension. Incubation time was 60 min at 37 °C in 5%

CO2 and a humid atmosphere.

Canine microglia were mixed with opsonized and non-opsonized bacteria

suspensions, resulting in a ratio of 1:100, or with PBS (negative control). After gentle

suspension, incubation was performed for 60 min at 37 °C and in 5% CO2. After 30

min, cells were again gently resuspended to ensure optimal cell-to-cell contact. After

incubation cells were put on ice to stop the phagocytosis reaction and to minimize

adhesion of cells on the surface of the tubes. Percentage of cells performing

phagocytosis and intensity of phagocytosis (measured as the log value of the mean

fluorescent channel numbers) were determined by immediate flow cytometric

measurement. All phagocytosis experiments were performed in triplicates, and mean

values were used for evaluation.

IV. 2.7. Cultivation of canine microglial cells

Microglial cells were adjusted to a concentration of approximately 2 x 105 cells and

diluted in 100 µl medium. Medium and microglial cells were incubated in duplicates

for 24 h in 5% CO2 at 37 °C in a humid atmosphere. After incubation the culture

suspension was transferred into cups and centrifuged at 200 x g for 5 min. Culture

supernatant was immediately stored at -70 °C.

IV. 2.8. Determination of NO2-/NO3

- (Griess reaction)

Reagent preparation and assay procedure were performed according to the

manufacturer´s instructions (R&D Systems, Wiesbaden, Germany). The principle of

the assay is to determine the amount of nitric oxide (NO) present, based on the

enzymatic conversion of nitrate (NO3-) to nitrite (NO2

-) by nitrate reductase. The

reaction is followed by a colorimetric detection of nitrite as an azo dye product of the

Griess reaction. Therefore, with this method NO2- levels can be taken as an estimate

of the total NO generation. Griess reaction was performed on CSF and microglial

culture supernatant from 22 dogs suffering from various intra- and extracranial

diseases; the animals were divided into two groups those with seizures and those

without. We also examined CSF of 56 dogs with various diseases of the CNS; 14 of


53

these dogs had seizures. Pure cultured medium served as the negative control and

urine from dogs suffering from cystitis with elevated nitrite levels as the positive

control.

IV. 2.9. Statistical evaluation

Statistical evaluation was performed under the consulting of the Institute of Biometry,

Epidemiology, and Information Processing, School of Veterinary Medicine, Hannover,

using the SAS software (version 8.02.) in a Windows XP environment.

Data were analysed using the One-Way-ANOVA approach with grouping of dogs as

classification factor, i.e. a model of the form

ijiij ey ++= αµ , VIIIIIi ,...,= , inj ...,1=

was performed.

The null hypothesis tested was that the results of dogs with different intracranial

diseases differed generally.

Determination of correct association into specific examination groups was assessed

by an F-test which performs multiple comparisons of mean values of the examination

groups. Differences were assessed by ANOVA with single classification for the

examination groups. Multiple comparisons between the examination groups were

performed by tests following Scheffé for unbalanced models. In the multiple

comparisons, values for examination group I and II were combined in one group as

they both did not show alterations in the CNS (group I + II). In a further approach, all

dogs with demyelinating lesions were considered together as a composite group in

order to associate the results of the immunophenotypical and functional

characterization on the occurrence of demyelination. The results of this approach are

marked with the term “alternative”. An error probability of 0.05 (p) was used as the

significance level (see Appendix, Tabs. XI. 5, XI. 6, XI. 7) for all statistical tests and a

correction of test significance was not performed. Therefore, all test values are used

as exploratory values.


54

Values for R2 describe the variance of the parameters which can be explained by the

model used for evaluation. Therefore, R2 indicates the goodness of model fit (see

Appendix, Tabs. XI. 5, XI. 6, XI. 7). Analysis of residuals was performed to test

normality of the variance and to assess the model validity by comparing the model

residuals with the predicted values (see Hartung et al. 1999; see Appendix, Tabs. XI.

11, XI. 12, XI. 13).

Boxplots were used to visualize minimal and maximal values, lower and upper

quartile and median of the values obtained. The box contains the values of middle

50% of the sample values.

55

V. Results

V. 1. Cell yield, viability, and purity Microglial cells were identified as described before (Stein et al., 2004). Microglial cell

yields from the dogs of the vaccine challenge experiment ranged between 3 and 7 x

106 total viable cells per 10 to 15 g of CNS tissue, whereas cell yields from the 25

dogs of the second part of the study ranged between 2.6 x 105 and 1.7 x 106 per 15

to 20 g of CNS tissue.

The purity of the whole ex vivo cell yield was measured by the expression of CD18;

purity was found to be 62% ( ) for the dogs of the vaccine challenge experiment

(measured in July and August 2000), and 39.6% ( ) for the dogs of the second part

of the study. A separate evaluation of the purity was also performed with respect to

the dogs´ age, the time of year of the examination, and the underlying pathology. The

purity of the whole ex vivo cell yield was found to be 45.1% in dogs of the second

part of the study approximately the same age as the dogs of the vaccine challenge

experiment at the time of microglial examination (< 1 year of age, n = 5), whereas

purity was found to be 38.5% in dogs older than 1 year of age (n = 21). The purity

achieved in the summer was found to be 41.1% of the whole ex vivo cell yield (June

to August, n = 10); in the winter (November to March; n = 7) it was 47.5% ( ). Dogs

of the examination groups showed differences in the purity of the microglial

population depending on the underlying pathology. Thus, dogs of group VI had the

best results ( = 53.3%), followed by group VII ( = 51.1%), group V ( = 44.8%),

group IV ( = 39.0%) and group VIII ( = 25.9%). There was almost no difference in

the purity of the microglial cell population among the three examination groups of the

vaccine challenge experiment: group I: = 62.1%; group II: = 61.1%; group III: =

63.9%.

After technical purification, by which CD18-positive cells were gated, microglia with

purities ranging from 93.3% ( ) for the dogs of the vaccine challenge experiment and

73.8% ( ) for the dogs of the second part of the study could be used for further

examinations. The majority of the contaminating cells in these preparations were

damaged or dying cells that had not completely lost membrane integrity, but were

negative for CD18 and CD11b/c. There was a residual number of cells displaying an

expression of CD3 ( = 3.4% in dogs of the vaccine challenge experiment, = 6.4%

__________________________________Results__________________________________

56

in dogs of the second part of the study), which is indicative of T-lymphocytes. This

low percentage of cells expressing CD3 revealed low contamination with blood cells

which had left the circulatory system after stasis due to death.

V. 2. Immunophenotypical characterization of canine microglia ex vivo Eleven different dog-specific antibodies were used to characterize the

immunophenotype of microglia. Immunophenotypical characterization was performed

on the basis of two different parameters, the percentage of positive cells for the

expression of a special surface molecule, and the mean fluorescence intensity

(measured by mean fluorescent channel numbers, log values) as determined by flow

cytometry and used as a relative mean for determining the intensity of expression of

a certain molecule. Both characteristics were compared between the eight

examination groups.

V. 2.1. CD18, CD11b, and CD11c

CD18, CD11b, and CD11c were constitutively expressed by canine microglia ex vivo,

as was shown in the vaccine challenge experiment (Stein et al. 2003). CD18 was

expressed by 93.3% ( ) in the dogs of the vaccine challenge experiment (groups I –

III), and by 73.8% ( ) of microglia in the dogs of the second part of the study (groups

IV – VIII) (see V. 1., Fig. 3).

__________________________________Results__________________________________

57

Figure 3. Constitutive expression of CD18 on canine microglia. Dogs of the second part of the study showed a generally lower expression of CD18. Unanalyzable results were indicated with an x. The numbers I – VIII of the x axis refer to the eight examination groups. Group I (n = 2) comprised unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in the CNS, which were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated; group IV n = 5), dogs with intracranial tumors; group V (n = 5), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

The expression of CD11b and CD11c was between 84% and 99% of positive

microglial cells for the dogs of the vaccine challenge experiment (CD11b: = 92.0%;

CD11c: = 92.1%), it was 70.9% (CD11b, ) and 70.5% (CD11c, ) for the dogs of

the second part of the study.

In all eight examination groups, the expression intensity of the integrin CD18 was the

highest of the three integrins described here ( = 63.5 for the dogs of the vaccine

challenge experiment, groups I – III, and = 84.1 for the dogs of the second part of

the study, groups IV – VIII), followed by CD11b ( = 38.8 for groups I – III, and =

54.2 for groups IV – VIII), and CD11c ( = 29.5 for groups I – III, and = 37.7 for

groups IV – VIII). The ratios of these three integrins were almost identical for all

examination groups (see Fig. 4). The highest expression intensities were found in

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58

groups IV (intracranial tumors), V (intracranial inflammation), and VIII (extracranial

diseases) (see Fig. 4).

Expression intensities of CD18, CD11b, and CD11c

0

20

40

60

80

100

120

I II III IV V VI VII VIII

mea

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CD18 CD11b CD11c

Figure 4. Expression intensity of CD18, CD11b, and CD11c of the dogs of the vaccine challenge experiment (groups I – III) and the second part of the study (groups IV – VIII) shown as mean values of each group. The expression intensities were measured as mean fluorescent channel numbers (log values) in flow cytometry. Notice that expression intensity is highest for CD18, followed by CD11b and CD11c. The ratio of expression intensity of the three integrins was almost identical in all groups. Group I (n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in the CNS, and which were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experiment with demyelinating lesions in CNS, and which were infected and in part vaccinated; group IV (n = 5), dogs with intracranial tumors; group V (n = 5), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

There were moderate differences in the expression intensity of CD18, CD11b, and

CD11c between individual dogs of each examination group (see Fig. 5, which shows

as an example the expression intensity of CD11b; also see Fig. 6).

Dog no. 27 of group IV had the highest expression intensity for all three integrins.

This dog suffered from meningioma in the bulbus olfactorius and was presented in

status epilepticus (see Fig. 5, data for CD18 and CD11c not shown).

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59

The dog of the second part of the study with demyelination due to natural CDV

infection (no. 32) had the highest values for the intensity of expression of CD18 and

CD11c of the dogs with intracranial inflammations (group V, data not shown).

Figure 5. Expression intensity of CD11b of the examination groups. The expression intensities were measured as mean fluorescent channel numbers (log values) in flow cytometry. Unanalyzable results were indicated with an x. The numbers I – VIII on the x axis refer to the eight examination groups. Group I ( n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in the CNS, and which were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated; group IV (n = 5), dogs with intracranial tumors; group V (n = 5), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

The expression intensity of CD18, CD11b, CD11c in the eight examination groups

was highest in the dogs with demyelination due to experimental CDV infection (group

III), dogs with intracranial tumors (group IV), and dogs with intracranial inflammation

(group V). The expression intensity of CD11b was higher in dogs of group IV

(intracranial tumors) and of group V (intracranial inflammation) than in groups I + II

(both without changes in the CNS) (p = 0.0003 with R2 = 0.48). No upregulation was

found for CD18 and CD11c (see Tab. 4).

0

20

40

60

80

100

120

mea

n flu

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tens

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CD11b

VIII I II III IV V VI VII

x x x

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mea

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tens

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mea

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ores

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mea

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CD18 CD11b

0

30

60

90

120

150


0

20

40

60

80

100


CD11c

0

20

40

60


Figure 6. Expression intensity measured by mean fluorescence intensity of the surface antigens CD18, CD11b, and CD11c according to the examination groups. Examination groups are shown on the abscissa and mean fluorescence intensity measured by mean fluorescent numbers (log values) in flow cytometry on the ordinate. The boxplots display minimums and maximums, lower and upper quartiles, and medians. The box contains the middle 50% of the sample values. Group I (n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in CNS, and which were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated; group IV (n = 5), dogs with intracranial tumors; group V (n = 5), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

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61

mfi of variable R2 p

CD18 0.251872 0.0883

CD11b 0.477258 0.0003

CD11c 0.312922 0.0415

Table 4. Statistical data of the expression intensity (mfi = mean fluorescence intensity) of CD18, CD11b, and CD11c. R2 indicates the goodness of model fit, and an error probability of 0.05 (p) was used as the significance level.

V. 2.2. CD45

In the vaccine challenge experiment, only a low percentage of microglial cells

showed an expression of CD45 ( = 4.6%), regardless of the presence or absence of

pathological changes in the CNS. The expression intensity of CD45 was also low,

and there was no enhanced expression intensity found for the dogs with

demyelination (group III).

The expression profile of CD45 was not homogenous in the dogs of the second part

of the study. While 17 dogs had low percentages of positive cells ( = 10.1%, with a

minimum of 3.4% and a maximum of 16.6%), there were between 28.3% and 92.7%

positive microglial cells in seven dogs ( = 77.0%; see Fig. 7). Four of these seven

dogs were under one year of age, while the others were 3, 6, and 7 years old. The

underlying pathology of these dogs was as follows: two dogs had idiopathic epilepsy

(nos. 33 and 34, group VI); one dog had intraventricular plexuscarcinoma (no. 25,

group IV); one dog had CDV infection and deymelination (no. 32, group V); one dog

had head trauma (no. 35, group VII); one dog had meningocele (no. 37, group VII);

and one, hydrocephalus internus and atrophy of cortex cerebri (dog no. 39, group

VII).

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62

Figure 7. Expression of CD45 of the dogs of the vaccine challenge experiment (groups I – III) and the dogs of the second part of the study (groups IV – VIII). The expression of CD45 was high in seven dogs of groups IV – VIII, whereas CD45 expression was low in dogs of groups I – III. Dogs showing seizure activity are indicated with grey bars. Unanalyzable results are indicated with an x. The eight examination groups are displayed on the x axis. Group I (n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in CNS, and which were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated; group IV (n = 5), dogs with intracranial tumors; group V (n = 5), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

The expression intensity of CD45 in the 17 dogs of the second part of the study with

low percentages of positive microglial cells ( = 22.4) was comparable to that of the

dogs of the vaccine challenge experiment ( = 16.2).

Evaluation of the expression intensity of the seven dogs with high percentages of

CD45-positive cells permitted the identification of two different populations by flow

cytometrical analysis. A large subpopulation of cells expressed low levels of CD45

(CD45low) and a small subpopulation of cells expressed high levels of CD45

(CD45high, see Fig. 8). While the CD45low cells showed an expression intensity

comparable to that of microglia in the vaccine challenge experiment and that of the

Expression of CD45

0

20

40

60

80

100

%


x x x xxx

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63

other 17 dogs of the second part of the study ( = 18.7), the cells with an expression

of CD45high ( = 135.8) revealed an expression intensity comparable to that of

monocytes ( = 105.4) (see Fig. 8, Stein 2001).

CD45

CD45 CD45

Figure 8. Expression and expression intensity of CD45 in microglia (A+B) and monocytes (C) displayed as histograms in flow cytometry. Green line: FITC (fluorescein isothiocyanate, negative control); orange and turquoise lines: staining results with marker for CD45 in duplicates. Dogs of the vaccine challenge experiment and 17 dogs of the second part of the study with various diseases revealed a small percentage of microglial cells expressing CD45low (A). Seven dogs of this study had a high percentage of cells expressing CD45low (M2) and a small percentage of cells expressing CD45high (M3) (B). The fluorescence intensity measured by mean fluorescent channel numbers (log values) of the CD45high cells was comparable to that of monocytes (C). M1 comprises all cells expressing CD45; M2, the subpopulation of cells expressing CD45low; M3, the subpopulation of cells expressing CD45high. FL1-H: fluorescence channel 1 height, green fluorescence.

M1M2 M3

M1

A

B C

M1

microglia

microglia monocytes

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64

The percentage of CD45low and CD45high cells and their expression intensity were

assessed separately for dogs with the microglial profile shown in Figure 8B (M1

comprises all CD45-positive cells; M2, the CD45low, and M3, the CD45high cells). The

results for the dogs with high percentages of CD45-positive cells are displayed in

Table 5.

CD45low CD45high Dog no. Percentage of

positive cells Expression

intensity Percentage of positive cells

Expression intensity

25 51.6 27.2 28.2 135.3

32 67.2 27.6 19.1 123.8

33 82.9 12.0 9.8 125.0

34 80.3 10.7 1.8 111.5

35 88.0 17.7 4.0 147.4

37 28.0 13.7 0.7 109.4

39 75.8 22.3 6.3 169.4

Table 5. Differentiation of CD45-positive cells according to their expression intensity, CD45low and CD45high. The expression intensity was measured by mean fluorescent channel numbers (log values).

V. 2.3. CD1c, MHC I, and MHC II

The percentage of CD1c-positive cells of the dogs of the vaccine challenge

experiment was 75.6%, regardless of the group to which the dogs belonged (I, II or

III). The percentage of CD1c-positive cells in the second part of the study was

generally lower ( = 30.3%), with wide variation among the individual dogs of each

group. Both dogs with idiopathic epilepsy (group VI) showed comparably high

percentages of CD1c-positive cells ( = 65.4%), as was the case for dogs of the

vaccine challenge experiment.

__________________________________Results__________________________________

65

mea

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The expression intensity of the dogs of the second part of the study ( = 16.0) was

similar to that of the dogs of the vaccine challenge experiment ( = 14.1) but variation

between individual dogs of each group was notable (see Fig. 9). Dogs with the

highest expression intensity (> 40, n = 3) were one dog with meningioma and

seizures (no. 26, group IV); one dog with granulomatous meningoencephalitis with

seizure activity (no. 28, group V); and a dog of group VIII (no. 40) with disc herniation

and Alzheimer type II proliferation and satellitosis. No difference was found in the

expression of CD1c among the eight examination groups (p = 0.8155, see Fig. 9).

CD1c

0

20

40

60


Figure 9. Expression intensity of CD1c of the eight examination groups. Examination groups are shown on the abscissa and mean fluorescence intensity measured as mean fluorescent channel numbers (log values) in flow cytometry on the ordinate. Note the great individual variation between the dogs of the second part of the study and group III of the vaccine challenge experiment. The boxplots display minimums and maximums, lower and upper quartiles, and medians. The box contains the middle 50% of the sample values. Group I (n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in CNS, and which were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated; group IV (n = 5), dogs with intracranial tumors; group V (n = 5), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n= 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

The percentages of microglia expressing MHC I and MHC II were comparably high

for the dogs of the vaccine challenge experiment (MHC I: = 95.6%; MHC II: =

93.4%), whereas they were generally lower for the dogs of the second part of the

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66

study (MHC I: = 68.9%; MHC II: = 64.1%). Differentiation of the examination

groups revealed statistically higher expression of MHC I in groups I + II (both without

changes in the CNS) than in group IV (dogs with intracranial tumors) and in group

VIII (dogs with extracranial diseases). In addition group III (dogs with demyelination

due to experimental CDV infection) had statistically higher values than group IV

(dogs with intracranial tumors) (see Tab. 6 for the p-values attained).

The expression intensities of MHC I and MHC II in the dogs of the vaccine challenge

experiment were comparably high to those of the dogs of the second part of the

study (for example, for MHC I: = 43.3 for the dogs of the vaccine challenge

experiment and = 48.4 for the dogs of the second part of the study).

The expression intensity of MHC I was higher than that of MHC II (1.9 times, for the

dogs of the vaccine challenge experiment and 1.5 times, for the dogs of the second

part of the study). Furthermore, the expression intensity of MHC I and MHC II was

enhanced in dogs of group III as compared to that of groups I + II (see Fig. 10).

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67

Figure 10. Expression intensity of MHC I and MHC II of the eight examination groups. The expression intensities were measured as mean fluorescent channel numbers (log values) in flow cytometry. The expression intensity of MHC I was higher than that of MHC II. Unanalyzable results were indicated with an x. The eight examination groups are displayed at the x axis. Group I (n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in CNS, and which were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated; group IV (n = 5), dogs with intracranial tumors; group V (n = 5), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

The highest values for the expression intensity of MHC I was found in the dogs with

(experimental and natural) CDV infection, and in dogs with intracranial tumors (see

Figs. 10 and 11). Only group III, with p = 0.0033, differed from groups I + II (see Tab.

6 and Appendix, Tab. XI. 5). Statistical analysis revealed no other differences in

deviations in the other examination groups (see Appendix, Tab. XI. 5).

The expression intensity of MHC II was highest in the dogs with demyelination due to

experimental CDV infection (group III). Dogs with intracranial tumors (group IV) and

dogs with intracranial inflammation (group V) had comparably high values. Dog no.

28 of group V had the highest MHC II expression intensity. This dog suffered from

granulomatous meningoencephalitis with a focal demyelination in the cerebellum,

0

40

80

120

160m

ean

fluor

esce

nce

inte

nsity

MHC I MHC II


x x x

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and had seizures. Statistical testing revealed differences between the dogs of group

III and group V when compared with dogs of group I + II (p = 0.0004, for R2 attained

see Tab. 6). When all dogs with demyelinating lesions were considered together as a

composite group (named “alternative” in Tab. 6 and Appendix, Figs. XI. 1-3) the

upregulation of MHC II was significant in comparison to that of groups I + II (p =

0.0009, see Tab. 6), whereas there was no longer any difference between dogs with

intracranial inflammation (group V) and groups I + II (see Tab. 6).

MHC I MHC II

0

20

40

60

80

100

120

140


0

20

40

60

80


Figure 11. Expression intensity of MHC class I and MHC class II of the eight examination groups. Examination groups are shown on the abscissa and mean fluorescence intensity measured as mean fluorescent channel numbers (log values) in flow cytometry on the ordinate. The boxplots display minimums and maximums, lower and upper quartiles and medians. The box contains the middle 50% of the sample values. Group I (n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in CNS, and which were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated; group IV (n = 5), dogs with intracranial tumors; group V (n = 5), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

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69

Variable R2 p

% 0.482998 0.0003 MHC I

mfi 0.395919 0.0033

% 0.473586 0.0004 MHC II

mfi 0.473541 0.0004

% 0.463879 0.0005 MHC I alternative mfi 0.441790 0.0009

% 0.393029 0.0035 MHC II alternative mfi 0.530672 <.0001

Table 6. Statistical data of percentage and expression intensity (mfi = mean fluorescence intensity) of MHC I and MHC II. Values are shown for the eight examination groups. Statistical results are also shown for all dogs with demyelination taken together: MHC I alternative and MHC II alternative. R2 describes the goodness of model fit, the p-value displays the statistical level attained. An error probability of 0.05 (p) was used as the significance level for all statistical tests.

V. 2.4. ICAM-1 (CD54)

The expression of ICAM-1 in the dogs of the vaccine challenge experiment was

relatively high ( = 90.8%) compared to that of the dogs of the second part of the

study ( = 46.2%), whereas expression intensity was comparably low in both

experiments (dogs of the second part of the study: = 11.8; dogs of the vaccine

challenge experiment: = 10.1). There was wide variation in the expression of

ICAM-1 between individual dogs of each group.

There was no difference in the intensity of ICAM-1 expression between the

examination groups (p = 0.2991, Fig. 12, see Appendix, Tab. XI. 5).

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cenc

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tens

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ICAM-1

0

10

20

30

40


Figure 12. Expression intensity of ICAM-1 according to the eight examination groups. The examination groups are shown on the abscissa and mean fluorescence intensity measured as mean fluorescent channel numbers (log values) in flow cytometry on the ordinate. The boxplots display minimums and maximums, lower and upper quartiles and medians. The box contains the middle 50% of the sample values. Group I (n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in CNS, and which were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated; group IV (n = 5), dogs with intracranial tumors; group V (n = 5), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

V. 2.5. B7-1 (CD80) and B7-2 (CD86)

The expression of B7-1 and B7-2 differed markedly between the dogs of the two

studies. Dogs of the vaccine challenge experiment had a high percentage of positive

microglial cells (B7-1: = 33.0% and B7-2: = 77.0%), whereas dogs of the second

part of the study had only low percentages of positive cells for the two surface

markers (B7-1: = 3.1% and B7-2: = 3.0). Only two dogs of the second part of the

study had expression values of about 10% for both B7-1 and B7-2, one dog with

idiopathic epilepsy which was presented in status epilepticus (no. 33, group VI), and

one dog (no. 45) of group VIII suffering from osteosarcoma in the left humerus.

There also were differences in the expression intensity of B7-1 and B7-2 between the

dogs of the two parts of this study. Dogs of the vaccine challenge experiment had

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71

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relatively higher levels of expression intensities, which were enhanced in the dogs

with demyelinating lesions due to experimental CDV infection (B7-2: group I: = 3.1;

group II: = 4.1; group III: = 8.5; see Fig. 13). There also was great variation in the

expression intensity of B7-1 between individual dogs of the groups IV – VIII, whereas

B7-2 was expressed at very low levels except in one dog of group VIII with

osteosarcoma (see Fig. 13). Comparing the fluorescence intensity of a high

percentage of positive cells (dogs of the vaccine challenge experiment) with a small

percentage of positive cells (dogs of the second part of the study) we found

significantly greater upregulation of B7-1 and B7-2 in dogs with demyelination (group

III) than in all other examination groups (p < 0.0001; see Appendix, Tab. XI. 5).

B7-1 B7-2

0

10

20

30

40

I II III IV V VI VII VIII0

10

20

30

40


Figure 13. Expression intensity of B7-1 and B7-2 according to the examination groups. There was a marked difference between the two studies. Dogs of groups I – III had relatively high percentages of positive microglial cells, whereas dogs of groups IV – VIII had only low percentages of microglial cells expressing B7-2. Examination groups are shown on the abscissa and mean fluorescence intensity measured as mean fluorescent channel numbers (log values) in flow cytometry on the ordinate. The boxplots display minimums and maximums, lower and upper quartiles, and medians. The box contains the middle 50% of the sample values. Group I (n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in CNS, and which were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated; group IV (n = 5), dogs with intracranial tumors; group V (n = 5), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

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72

V. 2.6. CD14

The percentage of CD14-positive cells in dogs of the vaccine challenge experiment

had a mean value of 62.0% ( ), which differed from that of dogs of the second part of

the study, which had about 5.2% ( ) positive microglia.

The expression intensity was generally low in both studies ( = 3.7 for the vaccine

challenge experiment and = 4.0 for the dogs of the second part of the study). Three

dogs of the second part of the study had comparably higher levels of CD14

expression (17.6 minimum and 42.6 maximum). These were a dog suffering from

meningioma and seizures (no. 27, group IV), one with granulomatous

meningoencephalomyelitis (dog no. 29, group V), and one with hydrocephalus

internus and atrophy of cortex cerebri (dog no. 39, group VII). Statistical analysis

revealed no difference between the examination groups (p = 0.7119; see Appendix,

Tab. XI. 5).

V. 3. Functional characterization of canine microglial cells

V. 3.1. ROS generation test

Examination of triplicates in the ROS generation test revealed similar results (SD of

the mean ranged between 0.5 and 1.2). Therefore, the mean value of these

triplicates was used to evaluate the percentage of ROS-generating cells and the

intensity of ROS generation.

A relatively high percentage of ROS positive microglial cells (up to 24%) was found

only in dogs with demyelination due to experimental CDV infection (group III). Five

other dogs had similar percentages of ROS-positive microglial cells. These included

two dogs of group II of the vaccine challenge experiment with glial nodules as a sign

of recovery after CDV infection; one dog of group V with demyelination and GME (no.

28); one dog of group IV with meningioma with purulent encephalitis and malacia (no.

23); and one dog with hydrocephalus internus and atrophy of the cortex cerebri (no.

39). The dogs from groups IV, V, and VII showed seizure activity (see Appendix, Tab.

XI. 2). All dogs of group VIII with extracranial diseases had low percentages (below

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73

5%) of ROS-generating microglia (see Fig. 14). Statistical analysis revealed a

significant difference between dogs with demyelination (group III) and those without

changes in the CNS (groups I + II, group VIII) (p = 0.0033 and R2 = 0.379917; see

Appendix, Tab. XI. 6).

Dogs pretreated with prednisolone are indicated by crosshatching in the diagram

(Fig. 14). With one exception (dog no. 28 with demyelination and GME, group V),

these dogs had only low percentages (up to 5%) of ROS-generating microglial cells.

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74

Gen

erat

ion

of R

OS

of t

he d

ogs

of th

e va

ccin

e ch

alle

nge

expe

rimen

t (gr

oups

I –

III) c

ompa

red

with

dog

s of

the

seco

nd p

art o

f the

stu

dy (g

roup

s IV

– V

III).

PM

A 0

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ved

as n

egat

ive

cont

rol,

and

trigg

erin

g w

as p

erfo

rmed

usi

ng P

MA

100

. Dog

s pr

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with

pre

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e ar

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dica

ted

by c

ross

hatc

hing

. Una

naly

zabl

e re

sults

wer

e in

dica

ted

with

an

x. T

he e

ight

exa

min

atio

n gr

oups

are

dis

play

ed

on th

e x

axis

. Gro

up I

(n =

2) c

ompr

ises

unc

halle

nged

dog

s of

the

vacc

ine

chal

leng

e ex

perim

ent w

ith n

o ch

ange

s in

the

CN

S; g

roup

II (n

= 1

3), d

ogs

of th

e va

ccin

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alle

nge

expe

rimen

t with

no

chan

ges

in C

NS

, and

whi

ch w

ere

infe

cted

and

va

ccin

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; gro

up II

I (n

= 7)

, dog

s of

the

vacc

ine

chal

leng

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ith d

emye

linat

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in th

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whi

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nd i

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rt va

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; gr

oup

IV (

n =

5),

dogs

with

int

racr

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l tu

mor

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roup

V (

n =

5),

dogs

with

in

tracr

ania

l inf

lam

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gro

up V

I (n

= 2)

, dog

s w

ith id

iopa

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epi

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(n =

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dogs

with

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= 8

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Figu

re 1

4:

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75

The mean fluorescence intensity was used as a relative means for the amount of

ROS produced by microglia, which was generally low (see Fig. 15). The highest

values were found in dogs with demyelinating lesions in the CNS (group III and dogs

no. 28 and 32 of group V). High levels of ROS generation were present in dogs of

group V with demyelination despite pretreatment with prednisolone (see Fig. 15).

Dogs pretreated with prednisolone are indicated by crosshatching in the diagram.

No correlation was found between enhanced intensity of ROS generation and seizure

activity.

ROS generation without stimulation (PMA 0) was statistically significantly higher

(p < 0.0001) in dogs with demyelination due to CDV infection (group III) and in the

dogs with demyelination and inflammation in the CNS (group V) than in dogs with

virtually no changes in CNS (groups I + II). This difference was even more

pronounced after stimulation of the cells with PMA 100, and statistically significant

differences were obtained by comparing the dogs with demyelination (group III) with

all other examination groups except the dogs with intracranial inflammation (p <

0.0001; see Appendix, Tab. XI. 6).

__________________________________Results__________________________________

76

Figu

re 1

5.

RO

S ge

nera

tion

inte

nsity

of

the

dogs

of

the

vacc

ine

chal

leng

e ex

perim

ent

(gro

ups

I – II

I) as

com

pare

d to

th

at o

f dog

s of

the

seco

nd p

art o

f the

stu

dy (g

roup

s IV

– V

III).

PM

A 0

ser

ved

as th

e ne

gativ

e co

ntro

l, P

MA

100

was

use

d to

trig

ger m

icro

glia

. Dog

s pr

etre

ated

with

pre

dnis

olon

e ar

e in

dica

ted

by c

ross

hatc

hing

. A

rrow

s po

int

to d

ogs

of g

roup

V w

hose

his

topa

thol

ogic

al e

xam

inat

ion

reve

aled

de

mye

linat

ing

lesi

ons

in t

he C

NS

. U

nana

lyza

ble

resu

lts w

ere

indi

cate

d w

ith a

n x.

The

eig

ht e

xam

inat

ion

grou

ps

are

repr

esen

ted

on th

e x

axis

. Gro

up I

(n =

2)

com

pris

es u

ncha

lleng

ed d

ogs

of th

e va

ccin

e ch

alle

nge

expe

rimen

t w

ith n

o ch

ange

s in

the

CN

S; g

roup

II (n

= 1

3), d

ogs

of th

e va

ccin

e ch

alle

nge

expe

rimen

t with

no

chan

ges

in C

NS

, an

d w

hich

wer

e in

fect

ed a

nd v

acci

nate

d; g

roup

III

(n =

7),

dogs

of

the

vacc

ine

chal

leng

e ex

perim

ent

with

de

mye

linat

ing

lesi

ons

in t

he C

NS

, an

d w

hich

wer

e in

fect

ed a

nd in

par

t va

ccin

ated

; gr

oup

IV (

n =

5),

dogs

with

in

tracr

ania

l tum

ors;

gro

up V

(n

= 5)

, do

gs w

ith in

tracr

ania

l inf

lam

mat

ion;

gro

up V

I (n

= 2

), do

gs w

ith id

iopa

thic

ep

ileps

y; g

roup

VII

(n =

5),

dogs

with

oth

er c

hang

es in

the

CN

S; g

roup

VIII

(n =

8),

dogs

with

ext

racr

ania

l dis

ease

s.

__________________________________Results__________________________________

77

mea

n flu

ores

cenc

e in

tens

ity

%

In order to determine whether generation of ROS correlates specifically with the

occurrence of demyelination, all dogs with demyelination were combined into one

group for comparison with the results of the others. The results revealed a significant

difference between dogs with demyelination and all other examination groups for

PMA 100 (p < 0.0001) (see Fig. 16 and Appendix, Tab. XI. 6).

PMA 100 PMA 100

0

5

10

15

20

25


5

10

15

20

25


Figure 16. Generation of ROS displayed as percentage of ROS-generating microglial cells (on the left) and intensity of ROS generation (on the right). In these boxplots the results for all dogs suffering from demyelination are included in group III. Dogs with demyelination (group III) showed a significantly enhanced intensity of ROS generation (p < 0.0001, R2 = 0.719131). Examination groups are shown on the abscissa and the percentage of ROS-positive cells and mean fluorescence intensity measured as mean fluorescent channel numbers (log values) in flow cytometry on the ordinate. The boxplots display minimums and maximums, lower and upper quartiles, and medians. The box contains the middle 50% of the sample values. Group I (n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in CNS, and which were infected and vaccinated; group III (n = 9), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated plus dogs with demyelination and intracranial inflammation in the CNS; group IV ( n = 5), dogs with intracranial tumors; group V (n = 3), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

__________________________________Results__________________________________

78

V. 3.2. Phagocytosis Assay

Triplicates in the phagocytosis assay showed broad congruence (SD of the mean

ranged between 0.8 and 2.3). Therefore, the mean value of the triplicates was used

to evaluate the percentage of cells performing phagocytosis and the intensity of the

phagocytic reaction.

Generally, a distinct phagocytosis activity was found in all dogs, as indicated by the

high percentage of positive microglia after the addition of non-opsonized Staphylo-

coccus aureus (nops, see Fig. 17). PBS served as the negative control.

__________________________________Results__________________________________

79

Figure 17. Percentage of phagocytosis-positive m

icroglia ex vivo. D

ogs pretreated with prednisolone are indicated w

ith crosshatching. PBS

served as the negative control. Non-opsonized

(nops) and opsonized (ops) Staphylococcus aureus w

ere used as phagocytosis particles. Unanalyzable results w

ere indicated w

ith an x. There was distinct phagocytosis activity in all dogs of the study. The eight exam

ination groups are displayed on the x axis. G

roup I (n = 2) comprises unchallenged dogs of the vaccine challenge experim

ent with no

changes in the CN

S; group II (n = 13), dogs of the vaccine challenge experim

ent with no changes in C

NS

, and which

were infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experim

ent with dem

yelinating lesions in the C

NS

, and which w

ere infected and in part vaccinated; group IV (n = 5), dogs w

ith intracranial tumors; group V

(n = 5), dogs w

ith intracranial inflamm

ation; group VI (n = 2), dogs w

ith idiopathic epilepsy; group VII (n = 5), dogs w

ith other changes in the C

NS

; group VIII (n = 8), dogs w

ith extracranial diseases.

__________________________________Results__________________________________

80

Phagocytosis of opsonized Staphylococcus aureus led to results similar to those for

the non-opsonized bacteria. Enhancement of phagocytosis through opsonization was

assessed by an additional gating step. The cut-off for non-opsonized bacteria was

set not to exceed 2% positive cells. This cut-off point was used to evaluate

phagocytosis of opsonized bacteria, but the eight examination groups did not differ (p

= 0.1687, see Appendix, Tab. XI. 7). Opsonization of Staphylococcus aureus led to

generally enhanced phagocytosis by microglia.

Pretreatment with prednisolone did not seem to influence the percentage of microglia

performing phagocytosis (see Figs. 17 and 18).

The percentages of microglia performing phagocytosis in dogs with seizure activity

was not elevated in comparison to those of dogs without seizure activity.

__________________________________Results__________________________________

81

Figure 18. Percentage of phagocytosis-positive m

icroglia evaluated for phagocytosis of non-opsonized (nops) in com

parison with opsonized (ops) Staphylococcus aureus,

which served to docum

ent enhancement of phagocytosis through opsonization of bacteria. O

psonization of bacteria led to generally enhanced phagocytosis. D

ogs pretreated with prednisolone are indicated by crosshatching. The

eight examination groups

are displayed at the x axis. Group I (n = 2) com

prises unchallenged dogs of the vaccine challenge experim

ent with no changes in the C

NS

; group II (n = 13), dogs of the vaccine challenge experiment w

ith no changes in C

NS

, and which w

ere infected and vaccinated; group III (n = 7), dogs of the vaccine challenge experim

ent with dem

yelinating lesions in the CN

S, and w

hich were infected and in part vaccinated; group IV

(n = 5), dogs w


(n = 5), dogs with intracranial inflam

mation; group V

I (n = 2), dogs with

idiopathic epilepsy; group VII (n = 5), dogs w

ith other changes in the CN

S; group V

III (n = 8), dogs with extracranial

diseases.

__________________________________Results__________________________________

82

The intensity of phagocytosis was enhanced in all dogs of groups III to VIII in

comparison to groups I + II; values were lowest in the dogs of group VIII. The highest

values occurred in dogs of groups III, IV and V, which differed significantly from those

of dogs without changes in the CNS (groups I + II) (p < 0.0001; see Appendix, Tab.

XI. 7). Prednisolone pretreatment did not seem to alter the intensity of phagocytosis

(see Fig. 19).

__________________________________Results__________________________________

83

Figure 19. M

ean fluorescence intensity as a relative means for phagocytosis intensity of the exam

ination groups. P

BS

served as the negative control, and non-opsonized (nops) and opsonized (ops) Staphyloccoccus w

ere used as phagocytosis particles. D

ogs pretreated with prednisolone are indicated by crosshatching. U

nanalyzable results were

indicated with an x. The eight exam

ination groups are displayed on the x axis and fluorescence intentsity measured

by mean fluorescence channelnum

bers (log values) in flow cytom

etry. Group I (n = 2) com

prises unchallenged dogs of the vaccine challenge experim


NS

; group II (n = 13), dogs of the vaccine challenge experim


NS

, and which w


ent with dem


S, and w

hich were infected and in part vaccinated; group

IV (n = 5), dogs w


(n = 5), dogs with intracranial inflam

mation; group V

I (n = 2), dogs w


ith other changes in the CN

S; group V

III (n = 8), dogs with

extracranial diseases.

__________________________________Results__________________________________

84

Increased phagocytosis intensity due to opsonization of bacteria was assessed using

the same cut-off as described for the assessment of enhanced percentage of

phagocytosing microglia (see Fig. 20).

__________________________________Results__________________________________

85

Figure 20. M

ean fluorescence intensity as a relative means for m

easuring phagocytosis intensity of non-opsonized (nops) in com

parison with opsonized (ops) Staphylococcus aureus according to the exam

ination groups. This m

ethod proves the enhancement of the phagocytosis intensity through opsonization of bacteria. N

on-opsonized and opsonized S

taphylococcus aureus were used as phagocytosis particles. D

ogs pretreated with

prednisolone are indicated by crosshatching. The eight examination groups are displayed on the x axis

and fluorescence intentsity m

easured by mean fluorescence channel num

bers (log values) in flow cytom

etry.Group I

(n = 2) comprises unchallenged dogs of the vaccine challenge experim


NS

; group II (n = 13), dogs of the vaccine challenge experim


NS

, and which w


ent with dem


S,

and which w

ere infected and in part vaccinated; group IV (n = 5), dogs w


(n = 5), dogs w

ith intracranial inflamm

ation; group VI (n = 2), dogs w


ith other changes in the C

NS

; group VIII (n = 8), dogs w

ith extracranial diseases.

__________________________________Results__________________________________

86

Dogs of the groups III – VIII generally showed higher intensity of phagocytosis than

groups I + II (see Fig. 20). Levels were highest in dogs with demyelination due to

CDV infection (group III) and in dogs with intracranial inflammation (group V) as

compared to the other groups, comparison with dogs of groups I + II revealed

statistically significant enhancement in dogs of these two examination groups (p <

0.0001) (see Appendix, Tab. XI. 7).

The intensity of phagocytosis did not seem to be impaired by pretreatment of the

dogs with prednisolone.

In order to determine whether enhanced phagocytosis correlated with the occurrence

of demyelination in the CNS, all dogs with demyelination were subsumed in a

modified group III. The dogs with demyelinating lesions (group III) were then shown

to have slightly higher percentages of microglial cells performing phagocytosis (see

Fig. 21 A + B, and Appendix, Tab. XI. 7). Dogs with inflammation in the CNS (group

V) also displayed a high capacity for phagocytosis. Statistical analysis revealed

significant differences in the intensity of phagocytosis between dogs of group I + II

compared with dogs with demyelination (group III, p < 0.0001), and with dogs with

intracranial inflammation (group V, p < 0.0001, R2 = 0.511654; also see Appendix,

Tab. XI. 7, Fig. 21 C).

__________________________________Results__________________________________

87

%

%

mea

n flu

ores

cenc

e in

tens

ity

nops ops

0

30

60

90


30

60

90


nops : ops

0

500

1000

1500

2000

2500


Figure 21. Results of the phagocytosis assay with all dogs with demyelinating lesions in histopathology included in group III. Results are displayed as the percent of microglial cells performing phagocytosis of non-opsonized (nops, A) and opsonized (ops) Staphylococcus aureus (B) and in terms of mean fluorescence intensity measured by mean fluorescence channel numbers (log values) in flow cytometry as a relative measure of the intensity of phagocytosis of non-opsonized (nops) in comparison with opsonized (ops) Staphylococcus aureus (C). Dogs with demyelination showed slightly higher percentages of microglial cells performing phagocytosis and distinctly higher phagocytosis intensities. Examination groups are displayed on the abscissa and the percentage of phagocytosis-positive cells and the mean fluorescence intensity on the ordinate. The boxplots display minimums and maximums, lower and upper quartiles, and medians. The box contains the middle 50% of the sample values. Group I (n = 2) comprises unchallenged dogs of the vaccine challenge experiment with no changes in the CNS; group II (n = 13), dogs of the vaccine challenge experiment with no changes in CNS, and which were infected and vaccinated; group III (n = 9), dogs of the vaccine challenge experiment with demyelinating lesions in the CNS, and which were infected and in part vaccinated plus dogs with demyelination and inflammation in the CNS; group IV (n = 5), dogs with intracranial tumors; group V (n = 3), dogs with intracranial inflammation; group VI (n = 2), dogs with idiopathic epilepsy; group VII (n = 5), dogs with other changes in the CNS; group VIII (n = 8), dogs with extracranial diseases.

A B

C

__________________________________Results__________________________________

88

V. 3.3. Determination of nitric oxide (NO) in microglial culture supernatant and CSF

The NO content of microglial culture supernatant was assessed in 18 dogs (see

Appendix, Tab. XI. 3). Microglial culture supernatant of eight dogs showed enhanced

NO levels compared to pure cultivated medium, which served as the negative control

(see Fig. 22). These eight dogs belonged to five different examination groups.

Four of these dogs had seizures. The dogs with enhanced NO levels suffered from

meningioma (dogs no. 1 and 3), in one case with purulent encephalitis and malacia

(dog no. 1); one with suspected hepatoencephalic syndrome and satellitosis (dog no.

8), and one with idiopathic epilepsy (dog no. 6).

Four of the eight dogs with elevated NO levels did not have seizures. One of these

dogs suffered from granulomatous meningoencephalitis (dog no. 9), and one from

trauma and intracranial bleedings with degeneration of nervous tissue (dog no. 11).

One dog had no changes in the CNS (no. 15), and one had cervical disc herniation

(dog no. 18; see Appendix, Tab. XI. 3).

As a result elevated NO levels in microglial culture supernatant could not be

associated in this study with any specific CNS disease.

Nitrite measurement in microglial culture supernatant

1 3

2 4 5 7 10 12 13 14 16 17

9 11

1586

18

0

1

2

3

4

5

6

quot

ient

cul

ture

sup

erna

tant

: m

ediu

m

Figure 22. Nitrite measurement in microglial culture supernatant differentiated for dogs with seizures in comparison with dogs without seizures. Eight dogs showed enhanced NO levels, four dogs exhibited seizures and four did not. The numbers identify the dogs examined (see Appendix, Tab. XI. 3).

Dogs with seizures Dogs without seizures

__________________________________Results__________________________________

89

Figure 23 shows the results of the NO determination in CSF of the same dogs.

Values were assessed for 20 dogs. NO levels in CSF samples did not correspond in

all cases to the values obtained from culture supernatant from the same dogs.

Nitrite measurement in CSF

24

8

7

3 56

9

10

11

12

13

14

1516

17

19

20

21

220

2

4

6

8

10

12

Opt

ical

den

sity

Figure 23. Nitrite measurement in cerebrospinal fluid (CSF) differentiated for dogs with seizures as compared to dogs without seizures. The numbers identify the dogs examined (see Appendix, Tab. XI. 3). The results for dogs no. 1 and 18 were not analyzable.

The highest values in CSF were measured for four dogs without seizures (values >

4). These dogs suffered from granulomatous meningoencephalitis (dog no. 10);

hydrocephalus internus and atrophy of cortex cerebri (dog no. 12); intraventricular

plexuscarcinoma (dog no. 19); and thoracal disc herniation with proliferation of

Alzheimer type II cells and satellitosis (dog no 21; see Fig. 23).

Nine dogs showed moderate NO values in CSF, five of which had seizures. Three of

these five dogs also exhibited higher NO levels in culture supernatant. No NO or only

a very small amount of NO could be determined in seven dogs.

Additionally, CSF samples were examined from 56 dogs with different CNS diseases

(see Appendix, Tab. XI. 4); the results are displayed in Fig. 24. Every examination

group showed low, moderate, and high levels of NO in CSF. Therefore, no

association of NO levels in CSF samples with specific CNS diseases could be found

Dogs with seizures Dogs without seizures

__________________________________Results__________________________________

90

in the present study, nor were NO levels higher in dogs with seizures than in dogs

with extracranial diseases, intracranial inflammation, and intracranial tumor.

__________________________________Results__________________________________

91

Figure 24. D

etermination of N

O in cerebrospinal fluid.

CS

F of 56 additional dogs suffering from seizures of different origin (n = 14) in com

parison with dogs w

ithout seizures suffering from

extracranial diseases (n = 15), intracranial inflamm

ation (n = 24), and intracranial tumor (n = 3). The

numbers identify the dogs exam

ined (see Appendix, Tab. X

I. 4).

92

VI. Discussion

In the present study, microglia from 47 dogs were examined ex vivo. The data

originated from two studies differing in their experimental designs:

The first, a vaccine challenge experiment, was a well-defined study with 22 six-

month-old Beagles of two litters which were held under SPF conditions. Before

challenge infection with CDV, all dogs were healthy, and this study was intensively

monitored. The dogs lived in the same surroundings and received identical feed. The

dogs did not receive any medications. Microglial examination of all dogs was

performed during a very short period of time of three weeks in a high-security

containment facility. This means that the laboratory environment was held at defined

consistent conditions such as temperature and atmospheric pressure. Therefore, the

variation in the results among the individual dogs of the vaccine challenge

experiment was very low. Any influence on microglial immunophenotype or function

caused by different genetic background of the dogs or environmental interferences

could be excluded with high probability. Therefore, the results obtained could be

considered solely as the consequence of activation of microglia by CDV infection.

For the second part of the study, many factors have to be considered which might

have influenced the results. These dogs were recruited because they were to be

euthanized on request of the owner and had been released for histopathological

diagnosis, and thus represented a normal average of the dog population. Therefore,

the ages of the dogs ranged from very young (20 days) to very old (16 years). During

their lives the dogs may have had to cope with different disturbances in CNS

homeostasis such as a tremendous variety of antigens (viruses, bacteria, foreign

protein components) and other noxious stimuli. At the time of microglial isolation, the

dogs were suffering from various diseases at different stages of development. Prior

to euthanasia, some of the dogs were pretreated with prednisolone or phenobarbital.

Different medications might have influenced microglial activation or the functional and

immunophenotypic profiles (Kiefer and Kreutzberg 1991, Lieb et al. 2003). Dogs of

13 different breeds and 4 mixed-breed dogs were included in the study. Therefore,

genetic background may have had an unknown impact on the microglial evolvement

of the immunologic repertoires of these animals.

________________________________Discussion_________________________________

93

In sum, variation in the dogs of the second part of the study was very high. In any

statistical test, the ratio of the amount of data to variability determines significance

(Festing et al. 2002). Hence, the sample size in our study was often not great enough

to permit statements about the significance of the results obtained.

In the second part of the study, the purity of the microglial cell population isolated ex

vivo was found to be less than that of the vaccine challenge experiment. This purity

was therefore negatively correlated to the increased variation among the dogs in the

second part of the study. This might be explained in part by the sources of variation

indicated above. The ex vivo examination of the dogs of the second part of the study

was performed in the laboratory of the Small Animal Clinic, School of Veterinary

Medicine Hannover. The examinations took place over a period of two years (in 2002

and 2003). As a consequence of this long time period, different environmental

conditions such as seasonal differences in temperature, humidity or barometric

pressure, may have had an impact on the microglial isolation procedure. In the

second part of the study, purity was higher in younger dogs than in older dogs, a

phenomenon which can also be observed in rats (Czub 2004, personal communi-

cation).

Differences caused by seasonal variation were assessed through separate

evaluation of the dogs examined in winter and summer. Because of more constant

room temperatures, microglial cell yields and purity were higher in winter than in

summer.

The lower cell yield and purity made it necessary to use more brain tissue in the dogs

of the second part of the study as source material. For the dogs of the vaccine

challenge experiment brain material originating from around the forth ventricle was

used which is the predilection site of CDV induced lesions. For the dogs of the

second part of the study up to 20 g of brain tissue had to be used to perform the

same examinations. Therefore, not only tissue in direct proximity to the lesion was

used which might result in some kind of “dilution” of the results reflecting microglial

activation and enhanced functional properties.

However, the use of flow cytometry allows purity enhancement of the cells examined

by technical means. Therefore, a degree of purity of the isolated microglial cells was

achieved which allowed characterization of these cells without severe interference

________________________________Discussion_________________________________

94

with contaminating cells. Contamination, which may originate from blood cells or

other glial cells of the CNS, was ruled out by comparing size, complexity, and

staining characteristics of other cells with microglia. Contamination of the cell yield

with T-cells was low. This was assessed by determination of the percentage of CD3-

expressing cells ( = 6.4%), which are indicative of T-lymphocytes. Glial cells from

the CNS, such as astrocytes, oligodendrocytes and neurons, could be largely

excluded because these cells, unlike microglia, do not express CD18. Purity

enhancement and assessment of T-cell concentration made it possible to confirm the

assumption that contamination of the microglial cell yield was generally low, although

it was higher in the second part of the study than in the vaccine challenge experiment

(Stein et al. 2003). This implies that some results, especially those of the second part

of the study, might have been influenced to some extent by the decreased purity of

the microglial cell yield and their interference with contaminating cells.

In the present study, the results for 47 dogs were used to evaluate differences in

microglial reaction patterns in different diseases. Accordingly, the dogs were

assigned to eight examination groups and classified on the basis of the histopatho-

logical diagnosis of the CNS. In some cases histopathological diagnosis revealed

features seen in more than one classification group. For example, tumors were

associated with concomitant inflammatory reactions of the adjacent tissue.

Furthermore, some dogs without clinically manifest neurological signs had slight CNS

histopathological alterations. These changes were considered to be age-related to

some extent; they thus represent common findings in the geriatric canine but might

also have had an influence on the activation status of microglia (Perry et al. 1993,

Czasch 2001). As a consequence the dominant alterations in the CNS were decisive

for classification into the eight examination groups.

The protocol for treatment during isolation and the procedures for further examination

of canine microglial cells ex vivo themselves might have led to a certain degree of

microglial activation. However, since treatment was identical for all dogs, the

differences observed here probably reflect different states of microglial activation in

vivo.

________________________________Discussion_________________________________

95

In the vaccine challenge experiment, microglia of dogs with histopathologically

confirmed demyelination due to CDV infection showed marked upregulation of the

surface molecules CD18, CD11b, CD11c, CD1c, MHC class I and MHC class II, and

a tendency for increased expression intensity of ICAM-1 (CD54), B7-1 (CD80), and

B7-2 (CD86). No increased expression was found for CD44 and CD45 (Stein et al.

2003). Furthermore, microglial cells showed enhanced phagocytosis and generation

of ROS.

This thesis discusses in detail whether this reaction profile of microglial

immunophenotype and function is specific to demyelinating CDV infection or is

stereotypic of alterations of other etiology. This study is the first comparative

characterization of microglial cells ex vivo in different diseases.

Canine microglia constitutively express CD18, CD11b and CD11c (Stein et al. 2004),

a phenomenon which is also known for the human species (Akiyama and McGeer

1990). Akiyama and McGeer (1990) and other authors found an upregulation of

these integrins when microglia became reactive in the course of pathological

changes in the CNS (Graeber et al. 1988, Streit et al. 1989). In the present study, an

upregulated expression of CD18, CD11b, and CD11c was evident for every

examination group when compared to the combined control groups I + II. Dogs with

intracranial tumors (group IV) and intracranial inflammation (group V) showed

significant upregulation of CD11b compared to groups I + II (p < 0.05); the

expression intensity of CD11c was also highest in dogs with intracranial tumors.

The integrins are a family of cell adhesion molecules which promote cell-cell and cell-

matrix interactions (Janeway and Travers 1997). They play important roles in

immune function by providing chemotactic guidance of cells to their correct locations

and by supporting adherence to target structures (Akiyama and McGeer 1990).

CD11b and CD11c are receptors for complement proteins, e.g. C3b (Abbas and

Lichtman 2003). Complement proteins are key molecules which mark foreign or

damaged materials for disposal by phagocytes. Prominent staining of these receptors

on resting and activated microglia strongly suggests phagocytosis as one of the

primary functions of microglia (Akiyama and McGeer 1990).

In the vaccine challenge experiment, expression of CD45 was found to be very low or

even absent (Stein et al. 2003). This does not agree with descriptions in the literature

which claim that the immunophenotype of CD11b/c and CD45low specifically identifies

________________________________Discussion_________________________________

96

microglial cells in other species unlike monocytes, which are CD11b/c+ and CD45high

(Ford et al. 1995). Therefore, expression of CD45 was thought to be below the limit

of detection, and this has also been described for other species (Streit et al. 1989,

Perry et al. 1993) or as a species-dependent speciality of canine microglia (Stein et

al. 2004). For the second part of the study, the percentage of CD45-expressing

microglial cells was generally comparable to that of the vaccine challenge

experiment. However, seven dogs of the second part of the study showed higher

percentages of CD45-expressing cells. These CD45-positive cells are divided into

two subpopulations: a high percentage of cells expressing CD45low and a low

percentage of cells expressing CD45high. All seven dogs with relatively high

expression of CD45 suffered from intracranial diseases. Upregulation of the

expression intensity of CD45 was observed in microglia in response to inflammatory

alterations, with levels reaching that of the expression intensity of monocytes and

macrophages, which are CD11b/c+ and CD45high (Ford et al. 1995). However, no

upregulated expression intensity of CD45 was detected in demyelination due to

experimental CDV infection (Stein et al. 2003).

Two hypotheses must be taken into consideration to account for the CD45-

expressing cells in the second part of the study.

The first hypothesis would imply that the cells detected were leukocytes, which

accumulate on the same Percoll-density in density gradient centrifugation as

microglial cells (Ford et al. 1995). Therefore, a very important prerequisite for

avoiding contamination with blood-derived cells in this study was that brain tissue be

perfused immediately after death in order to prevent blood cell diffusion through

instabile vessel walls after stasis of blood flow. The effective perfusion of the brain

tissue in our study was proven by the pale appearance of the brain tissue and

meninges, and this was confirmed histopathologically. Leukocytes such as

monocytes could have invaded the brain in vivo via the disturbed blood brain barrier

in the course of the ongoing CNS disease. As they show a specific

immunophenotype of CD11b/c+ and CD45high, these leukocytes therefore cannot be

differentiated from activated microglial cells as they also express CD45 at a high

level.

As a second hypothesis it must be considered that the cells expressing CD45 in the

seven dogs of our study were microglia. In response to the disturbances in the CNS,

microglia progressed to an activated state, resulting in upregulated expression of

________________________________Discussion_________________________________

97

CD45. The expression intensity of CD45 in the other dogs was so low that the results

of indirect membrane immunofluorescence and flow cytometry were below the limit of

detection. Therefore, only a low percentage of microglial cells expressing CD45 ( =

9.5%) was found in these dogs.

Two distinct subpopulations of CD45-expressing cells can be differentiated: a high

percentage of cells expressing CD45low, and a small percentage of cells expressing

CD45high. According to the graded response in microglial activation described by

Raivich et al. (1999), these two subpopulations have to be seen as a result of graded

upregulation of CD45, which in turn might reflect different stages of microglial

activation.

With this hypothesis, it remains unclear why an expression of CD45 was found only

in these seven dogs, while CD45 expression was very low or even absent in other

dogs also suffering from intracranial diseases.

Microglial cells are considered to be the antigen presenting cells (APC) of the CNS

(Frei et al. 1987, Wekerle 1995). To meet the requirements of professional APCs,

microglial cells have to be capable of internalizing, processing and presenting

antigen via both MHC classes. Additionally, adhesion molecules are needed to bind

co-stimulatory molecules (B7-1 and B7-2) for the proliferation and differentiation of T-

cells.

Microglial cells are the most prominent MHC-expressing CNS cells in vivo (Wekerle

1995). Whereas MHC expression is generally minimal or absent in unaltered CNS

parenchyma and is restricted to microglia (Hayes et al. 1987), MHC II expression is

readily upregulated on activation (Kreutzberg 1996). This confirms our observation

that expression of MHC class I and MHC class II ex vivo was generally high for all

examination groups, and this suggests a constitutive expression of MHC I and II in

canine microglia. The expression intensity of the MHC molecules was different in the

two experimental designs: there was a significantly upregulated expression intensity

of MHC class I in the dogs with demyelination (group III, p < 0.05). But there also was

upregulated expression intensity in the dogs with intracranial tumors, inflammation,

and idiopathic epilepsy, as compared to group I + II. Upregulation of MHC class II

was significantly higher in the dogs of groups III and V than in group I + II. It has been

demonstrated that upregulation of MHC antigens occurs in alterations of the CNS of

various etiologies (McGeer et al. 1988, Streit et al. 1988, Sedgwick et al. 1991,

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98

Thomas 1992, Davis et al. 1994, Perry 1998). These disorders also include the

demyelinating diseases experimental autoimmune encephalomyelitis (EAE,

Gehrmann and Kreutzberg 1995) and Theiler´s murine encephalomyelitis virus

(TMEV) infection of mouse microglia in vitro (Olson et al. 2001). TMEV infection

induces a demyelinating disease and serves as a highly relevant virus-induced model

for human MS (Miller and Gerety 1990).

Generally speaking, it appears that many factors have an impact on MHC expression

of microglia.

The age of the animals is of special interest in regard to the expression of MHC II

(Nichols 1999). Perry et al. (1993) found that microglia in healthy, aged, but

pathogen-free rats expressed MHC class II antigens that are downregulated or

absent in juvenile rats. This is in agreement with immunohistochemical observations

of dogs in which there was a significant, age-related increase in activated microglia

expressing MHC II which are associated with plaques in the CNS (Czasch 2001).

Consequently, the possibility of an age-related influence of the immunophenotype

has to be taken into consideration for our study.

Furthermore, there seem to be environmental impact on the immunophenotype.

Studies of rodent microglia are usually performed under SPF conditions. Antigen

expression of rodents is unlike that in man, where microglia are commonly reported

to express MHC class II (McGeer et al. 1993). The results of experimentally

downregulated or absent antigen expressions may not reproduce real-life conditions.

Although humans do not live in SPF conditions, the expression of MHC class II in

man might indicate a comparable phenomenon in the dogs in our study. Our results

may also reflect the general conflict concerning knowledge gained from artificial or

clinical studies. Provided that adequate control groups are included, clinical studies

might in fact be more valuable because they more closely reflect conditions of real

life.

Enhanced levels of MHC class II expression have also been found in microglia from

animals with systemic infections (Perry 1998). Moreover, activation of microglia and

subsequent expression of MHC in the CNS was seen in experimental autoimmune

inflammation of peripheral nerves (Gehrmann et al. 1992b). These findings may

explain the higher values for the dogs with extracranial diseases (group VIII)

compared to dogs without changes in the CNS (group I + II).

________________________________Discussion_________________________________

99

The presence or absence of seizures may also influence the expression of MHC II. A

study with tetanus toxin-induced experimental epilepsy in the rat revealed that, in the

absence of degenerative changes, microglia upregulated MHC class II antigens in

response to abnormal electrical activity in the CNS (Shaw et al. 1994). Since

microglial activation following seizure activity is time-dependent the differences in

time span between seizure activity and microglial ex vivo examination in our study

might interfere with the results obtained. In our study we found no relation between

upregulated MHC class II expression and seizure activity in the dogs.

Originally, we thought that pretreatment of the dogs in our study would have an

impact on microglial expression profile, because the inhibitory effect of pretreatment

with prednisolone on MHC class I and class II expression has been demonstrated

(Kiefer and Kreutzberg 1991), and we thought this might also influence the

expression of other surface molecules. However, pretreatment with prednisolone did

not seem to have an intense influence on antigen expression in our study, as no

general downregulation of antigens was found on the microglia of the dogs which had

been medicated with prednisolone.

In addition to the classical molecules for antigen presentation (MHC class I and class

II), canine microglia also expressed CD1c, which is capable of presenting lipidic and

glycolipidic antigens to T-cells (Janeway and Travers 1997, Porcelli and Modlin 1999,

Busshoff et al. 2001). Upregulated expression of CD1c on microglia was found in the

dogs with demyelination (group III) in contrast to dogs with virtually no changes in the

CNS (group I + II). However, the intensity of CD1c expression was also higher in

dogs with intracranial tumors (group IV), inflammation (group V), and other changes

in the CNS (group VII). An upregulated expression of CD1c has also been found in

experimental autoimmune encephalomyelitis (EAE, Busshoff et al. 2001), which is

widely used as a model for CNS inflammation and demyelination, and is a well-

established model for multiple sclerosis (MS) (Wekerle et al. 1986). Upregulation of

CD1c demonstrates the presence of microglial activation and the potential to present

lipidic and glycolipidic substances (perhaps even auto-glycolipids) derived from brain

tissue during inflammation (Busshoff et al. 2001). The high percentage of CD1c-

positive microglia and upregulated intensity of expression in demyelination found in

the dogs of our study and in the observations of Busshoff et al. in EAE (2001)

suggest a model in which the expression of CD1c on microglia plays a central role in

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100

demyelinating diseases. However, it cannot be excluded that enhanced CD1c

expression is correlated with severe brain damage and general release of lipidic and

glycolipidic substances.

Microglial expression levels of B7-1 (CD80) and B7-2 (CD86) differed between the

vaccine challenge experiment and the second part of the study. When considering

the differences between the two groups of dogs in environmental conditions, it is of

note that expression levels of both antigens also varies from low to undetectable

levels among different mouse strains (Carson 2002). It is therefore likely that similar

variations exist in different dog breeds. The intensity of expression of B7-1 and B7-2

was significantly higher in dogs of group III than in all other examination groups,

which showed only low expression intensity of these surface molecules. This finding

is consistent with the in vitro observations made by Olson et al. (2001) with TMEV-

infected mouse microglia. In that study, examination of unstimulated mouse microglia

revealed low expression levels of B7-1 and almost no B7-2 and ICAM-1, although

there was upregulation of these surface molecules following TMEV infection or IFN-γ

stimulation (Olson et al. 2001). Expression and upregulation of B7-1 were also found

on human microglia in vitro after activation with IFN-γ and in MS lesions (Williams et

al. 1994a, De Simone et al. 1995, Windhagen et al. 1995). Attempts to co-cultivate

microglia and CD4+ T-cells resulted in clustering of T-cells around activated microglia

(Williams et al. 1994a, Ulvestad et al. 1994a). Similarly, a distinct diffuse clustering of

T-cells has also been observed in demyelinating CNS lesions due to CDV infection in

areas of microglial activation (Tipold et al. 1999). Therefore, the expression of B7-1

and B7-2 and the subsequent recruiting of T-cells might be a crucial characteristic of

the pathogenesis of demyelination in canine distemper. ICAM-1 (CD54) is described

as an adhesion molecule on APCs. It binds to the T-cell integrin LFA-1 (Janeway and

Travers 1997, Abbas and Lichtman 2003). An upregulated expression of ICAM-1 is

reported to occur in TMEV infection (Olson et al. 2001), in active MS lesions, and

around perivascular inflammatory infiltrates in EAE (Aloisi 1999). This is in contrast to

our study, where an upregulation was not found in statistical analysis. Only a certain

tendency to an upregulation of ICAM-1 was observed in demyelinating lesions. In

another study, transgenic mice that constitutively express B7-2 (CD86) on microglia

in CNS spontaneously developed an autoimmune demyelinating disease, whereas

transgenic animals lacking alpha-beta TCR+ T-cells were completely resistant to

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101

disease development (Zehntner et al. 2003). These data provide evidence that

interactions of co-stimulatory molecules with their counterparts on T-cells are critical

determinants in the pathogenesis of demyelinating diseases, and that such

interactions might be of crucial importance for disease progression.

Our results reflect an immunophenotype which strongly suggests the central role of

microglia in the mediation of cellular immune responses in demyelination due to CDV

infection, in intracranial tumors, and in intracranial inflammation due to the stimulation

of T-cell activation and proliferation by microglia.

Upon activation, microglial cells exert macrophage effector functions (Kreutzberg

1996, Stoll and Jander 1999). These include phagocytosis and synthesis of various

potentially harmful soluble factors (Nathan 1987, Banati et al. 1993b) such as

reactive oxygen species (ROS), which were found to be measured in this study.

Canine microglial cells were stimulated with PMA, which is known to be an effective

stimulator of ROS generation in monocytes/macrophages and in microglia of other

species (Smith et al. 1998, Possel et al. 2000). Triggering with PMA has also been

shown to result in ROS generation in canine microglia (Stein et al. 2004), and to be

indicative of quantitative and qualitative distinctions in dogs suffering from different

diseases, as well.

Accordingly, dogs with demyelination showed a statistically significantly higher

percentage of ROS-producing microglial cells and a statistically significantly higher

intensity of ROS generation than dogs of the other examination groups.

Correspondingly enhanced ROS generation was also found in studies with other

demyelinating diseases. For example, microglial cells of rats suffering from EAE

exhibited significantly elevated spontaneous and PMA-induced levels of ROS (Ruuls

et al. 1995); and a significant increase in DNA oxidation related to ROS was found in

MS plaques (Vladimirova et al. 1998).

Generation of ROS leads to peroxidation of proteins, DNA, and lipids and glycolipids,

which are components of the myelin sheath. Such peroxidation results in myelin

destruction and loss of myelin sheaths, and the brain is particularly vulnerable to

oxidative damage, due to its high oxygen consumption. In the CNS microenviron-

ment, oligodendrocytes which form the myelin sheath show selective vulnerability to

ROS (Griot et al. 1990). Therefore, microglial ROS generation could lead to altered

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102

synaptic transmission and direct damage of healthy neurons (Bruce-Keller 1999).

Although microglial ROS generation is distinctly enhanced in dogs with

demyelination, it must be said that the amount of ROS actually generated was only a

small proportion of that which macrophages, canine neutrophils respectively, could

potentially produce (data not shown; Stein 2001, Eickhoff 2002). This difference in

the potential for ROS generation might be considered as evidence for the presence

of a mechanism of protection for the vulnerable microenvironment of the CNS.

In our study, we first assumed that pretreatment with prednisolone would have an

impact on ROS generation, as it has been shown that glucocorticoids suppress the

production of pro-inflammatory cytokines (Kern et al. 1988), which might activate

microglia. Moreover, selective inhibition of microglial activation by dexamethasone

has been demonstrated in vivo in rats (Kiefer and Kreutzberg 1991). But in our study

we found no impact of pretreatment with prednisolone on ROS generation. Levels of

ROS in the dogs with demyelination in the second part of the study were comparable

to those of the dogs with demyelination of the vaccine challenge experiment, even

though both dogs of the second part of the study were pretreated with prednisolone.

The intensity of ROS generation might therefore even be enhanced if the dogs had

not been medicated.

Enhanced generation of ROS also has to be considered as a possible consequence

of ongoing seizure activity (Layton and Pazdernik 1999). Generation of ROS due to

seizure activity may play a key role in epilepsy-associated nerve cell damage

(Kovacs et al. 2002). Indeed, enhancement of the percentage of ROS-producing cells

was observed in dogs with seizures in our study.

The phagocytosis assay of dogs in all examination groups revealed a high

percentage of microglial cells performing phagocytosis. Opsonization of

Staphylococcus aureus resulted in an increase in the percentages of phagocytic

microglia without differences between the different examination groups. On the other

hand, it is possible to distinguish between examination groups in the intensity of

phagocytosis: dogs with demyelination (group III) and dogs with intracranial

inflammation (group V) exhibited statistically significantly enhanced phagocytosis of

non-opsonized and opsonized bacteria, and dogs with intracranial tumors and

secondary inflammatory reaction (group IV) showed statistically significantly

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103

enhanced phagocytosis of opsonized bacteria. This strongly suggests microglial

activation and execution of phagocytosis in vivo as a consequence of inflammation

and demyelination.

Stimulation of phagocytosis via opsonization is mediated via Fc- and complement

receptors (Ulvestad et al. 1994b, Ulvestad et al. 1994c), which specifically recognize

complement proteins bound to pathogens. Receptor binding of complement proteins

facilitates phagocytosis (Janeway and Travers 1997). Microglia are known highly to

express Fc-receptors (McGeer et al. 1993) and constitutively to express CR3 and

CR4 (CR3 = CD18/CD11b, CR4 = CD18/CD11c, Akiyama and McGeer 1990). CR3

and CR4 specifically bind inactive forms of the complement protein C3b, and thus

stimulate phagocytosis (Janeway and Travers 1997). In our study, an upregulated

expression of CD11b was found in dogs with intracranial tumors (group IV) and

intracranial inflammation (group V), which is in accordance with the significantly

enhanced intensity of phagocytosis in the dogs of these examination groups.

Phagocytosis capability can also be influenced by the age of the animals, as is the

case for alterations in immunophenotype. In humans 60 years of age or more without

evidence of clinical or pathological disease, Sheng and colleagues (1998) found

higher numbers of phagocytic microglia than in younger persons. It therefore seems

likely that phagocytosis in the dogs of our study might to a certain extent be due to

advanced age.

However, dogs with demyelination due to experimental CDV infection (group III)

showed the highest percentages of microglial cells performing phagocytosis and the

highest phagocytosis intensity, although these dogs were young. Dogs suffering from

intracranial inflammation (group V) also exhibited enhanced phagocytosis. Therefore,

the underlying disease seems to be the major trigger that elicits microglial

phagocytosis.

Another feature of the microglial immunological repertoire is the production of nitric oxide (NO). NO is produced after the oxidation of L-arginine by a family of nitric

oxide synthases (NOS) (Howe and Boothe 2001). In activated microglia, the

inducible NOS (iNOS) is responsible for the synthesis of NO (Chao et al. 1992,

Minghetti and Levi 1998). This radical can be transformed by superoxide to

peroxynitrite, which is an extremely aggressive oxidant that leads to lipid, protein,

and DNA damage (Rosen et al. 1995, Raivich et al. 1999).

________________________________Discussion_________________________________

104

Enhanced levels of NO can be found in various disease entities such as multiple

sclerosis and its animal model, EAE (Smith et al. 1999). NO is thought to be essential

in antitumoral and antimicrobial host defense. Although the production of NO may

represent a beneficial response in the host defense mechanisms against invading

microoorganisms, NO plays a critical role in neurodegeneration (Merill et al. 1993,

Wei et al. 2000, Golde et al. 2002). NO is presumed to play a distinct role in

demyelinating diseases (Smith et al. 1999).

Many influencing factors have to be taken into consideration in the induction of iNOS

and subsequent production of NO. For example, epileptic seizures are known to

elevate NO production significantly (Bashkatova et al. 2003). This contradicts the

results of our study, where there was no difference in NO levels between dogs with

and without seizure activity; nor did the levels of NO in CSF correlate with specific

CNS diseases.

However, many iNOS inhibitors have now been identified (Hays 1998). For example,

prednisolone is known to inhibit LPS-induced iNOS and NO synthesis (Lieb et al.

2003), and phenobarbital has been shown to prevent enhanced NO generation due

to seizure activity (Bashkatova et al. 2003). As the dogs of our “NO study” received

different medications, our results have to interpreted cautiously in regard to a

possible inhibition of NO generation.

Furthermore, the purity of the microglial yield in the second part of the study was

notably high, and this might have led to interference with NO production by the

contaminating cells. Although, all experiments were conducted with the same

methods, the results of dogs with different diseases could nevertheless be compared.

In our study, no correlation was found for NO levels in microglial culture supernatant

and CSF with a specific CNS disease. Our results therefore suggest a stereotypic

generation of NO in different disease entities.

Microglial activation has long been believed to follow a stereotypic reaction pattern

irrespective of the underlying pathological condition. In order to evaluate the question

whether this assumption underrates microglial immunological potential, we examined

microglia of dogs suffering from different intra- and extracranial diseases ex vivo, and

compared the result to those of a vaccine challenge experiment with CDV infection

and demyelination in the CNS.

________________________________Discussion_________________________________

105

Immunophenotypical characterization of canine microglia in various intracranial

diseases revealed a trend toward generalized upregulated expression of antigens.

Therefore it appears that immunophenotypical changes indicate a stereotypic

response by microglia, regardless of the underlying disease. Functionally,

phagocytosis was enhanced in dogs with demyelination and inflammation, which

suggests a trend toward specific enhancement in these disease entities. Significantly

enhanced ROS generation was found only in the dogs with demyelination and not in

the other groups examined here. This strongly suggests a specific response of

microglia to demyelination in regard to ROS generation and therefore a specific

immunological potential of canine microglial cells.

106

VII. Summary

Ex vivo examination of canine microglia in different intracranial diseases: stereotypic versus specific reaction profile

Veronika M. Stein, Hannover

Microglial cells are the main immune effector elements of the brain. They respond to

virtually every kind of disruption of homeostasis in the CNS and rapidly progress from

their resting, ramified state to an activated state where they can proliferate, migrate,

express surface molecules de novo or at increased levels, and exert functions similar

to those of macrophages such as phagocytosis and generation of ROS and NO.

It has long been believed that microglial cells follow an uniform unspecific reaction

pattern upon activation. Evidence has grown that this view seriously underrates

microglial competence in the immune surveillance of the CNS.

The purpose of this study was to elucidate whether the canine microglial reaction

pattern differs in response to different alterations in the CNS and whether the findings

of our preliminary study on demyelination due to CDV infection were specific to a

demyelinating disease or also apply to other disease entities. This would indicate that

the canine microglial reaction is stereotypic. To our best knowledge, this study is the

first comparative characterization of microglial cells ex vivo in different diseases.

Canine microglial cells from dogs suffering from different intracranial and extracranial

diseases were characterized immunophenotypically and functionally ex vivo by flow

cytometry. The dogs were divided into eight examination groups according to

histopathological diagnosis. The results of each group were evaluated and compared

to those of a preliminary study of the microglial ex vivo examination in dogs of a

vaccine challenge experiment with canine distemper virus infection and

demyelination in CNS.

In the vaccine challenge experiment, microglia of dogs with histopathologically

confirmed demyelination due to CDV infection showed marked upregulation of the

surface molecules CD18, CD11b, CD11c, CD1c, MHC class I, MHC class II, and a

_________________________________Summary_________________________________

107

tendency for increased expression intensity of ICAM-1 (CD54), B7-1 (CD80), and B7-

2 (CD86). Functionally, microglial cells in demyelination due to CDV were shown to

have significantly enhanced phagocytosis and generation of ROS.

A tendency for an upregulated expression was found for various surface molecules

on microglia in different CNS alterations. Statistical significance was found only in

certain surface molecules: there was an upregulated expression of CD11b in dogs

with intracranial tumors and inflammation; MHC II was also upregulated in dogs with

intracranial inflammation, whereas upregulation of B7-1 and B7-2 was found only in

dogs with demyelination.

Functionally, phagocytosis was significantly enhanced in dogs with demyelination

and in those with intracranial inflammation, but ROS generation was significantly

enhanced only in dogs with demyelination.

In conclusion, it appears that microglial response to various diseases can be both

stereotypic and specific. Our results point to a certain degree of conformity in the

immunophenotypical response of microglia, whereas the functional response of

canine microglia seems to differentiate between different underlying pathologies.

Phagocytosis was found to be enhanced for inflammation and demyelination in the

CNS, which therefore indicates a more specific microglial response. Significant

enhancement of ROS generation was found only in dogs with demyelination and not

in those with other diseases. This strongly suggests a specific response of microglia

to demyelination and underscores the pivotal role of microglia in the pathogenesis of

demyelination.

In contrast to findings regarding ROS generation, findings for the production of

another endogenous gas, NO, seems to be stereotypic. In our study there were no

enhanced levels of NO neither in microglial culture supernatant nor in CSF

corresponding to a specific disease entity.

108

VIII. Zusammenfassung

Ex vivo Untersuchung kaniner Mikrogliazellen bei verschiedenen intrakraniellen Erkrankungen: stereotypes versus spezifisches Reaktionsprofil

Veronika M. Stein, Hannover

Mikrogliazellen sind die Immuneffektorzellen des zentralen Nervensystems (ZNS).

Nahezu jede Störung der Homöostase im ZNS wie Infektion, Trauma, Ischämie,

Degeneration und Demyelinisierung führt zu ihrer Aktivierung und somit zur

Entfaltung ihres immunologischen Repertoires. Dieses umfasst eine Proliferation und

Migration, eine Aufregulation oder de-novo-Expression von Oberflächenantigenen

und die Ausübung typischer Makrophagen-Funktionen wie die Phagozytose und die

Generation von reaktiven Sauerstoffmolekülen (ROS) oder Stickstoffmonoxid (NO).

Die Aktivierung der Mikrogliazellen wurde lange Zeit als unspezifischer stereotyper

Prozeß angesehen, der ungeachtet seiner zugrunde liegenden Ätiologie einheitlich

abläuft. Ergebnisse unterschiedlicher Studien erhärten inzwischen den Verdacht,

dass diese Betrachtung die immunologische Kompetenz der Mikrogliazellen deutlich

unterschätzt.

Zur Klärung der Frage, ob das Reaktionsmuster der Mikroglia auf unterschiedliche

pathologische Stimuli tatsächlich einem stereotypen Schema folgt oder eine

spezifische Antwort der Mikroglia resultiert, wurde sie bei Hunden, die an

verschiedenen intrakraniellen Erkrankungen litten, untersucht. Unsere Studie stellt

die erste vergleichende Untersuchung der Mikroglia ex vivo bei verschiedenen

Erkrankungen des ZNS dar.

Die Hunde wurden auf der Grundlage der histopathologischen Diagnose in acht

verschiedene Untersuchungsgruppen eingeteilt. Die Ergebnisse der immunphäno-

typischen und funktionellen Charakterisierung der Mikroglia der einzelnen Gruppen

wurde bewertet und zudem mit den Resultaten einer vorangegangenen Studie mit

Hunden mit experimenteller Staupevirusinfektion verglichen.

______________________________Zusammenfassung____________________________

109

Hunde mit staupetypischen Läsionen und Demyelinisierung im ZNS wiesen eine

deutliche Aufregulation der Oberflächenmoleküle CD18, CD11b, CD11c, CD1c, MHC

Klasse I und MHC Klasse II, sowie eine Tendenz für eine aufregulierte Intensität von

ICAM-1 (CD54), B7-1 (CD80), und B7-2 (CD86) auf. Funktionell zeigte sich eine

statistisch signifikant aufregulierte Phagozytose und ROS-Bildung.

Eine Tendenz für eine aufregulierte Expression fand sich für mehrere Oberflächen-

antigene bei verschiedenen ZNS-Alterationen. Eine statistische Signifikanz fand sich

lediglich für die Aufregulation von CD11b bei Hunden mit intrakraniellen Tumoren

und Entzündungen, die zudem eine Aufregulation von MHC II aufwiesen. Eine

Aufregulation der Antigene B7-1 (CD80) und B7-1 (CD86) auf Mikroglia wurde

lediglich bei Demyelinisierungen verursacht durch Staupevirusinfektion gefunden.

Funktionell fiel eine statistisch signifikant verstärkte Phagozytose neben den Hunden

mit Demyelinisierung auch bei Hunden mit intrakraniellen Entzündungen auf,

wohingegen eine gesteigerte Bildung von ROS lediglich bei Hunden mit

Demyelinisierung gefunden wurde. Diese war signifikant höher als bei allen anderen

Untersuchungsgruppen.

Als Ergebnis scheint somit in Bezug auf den Immunphänotyp eine gewisse

Einheitlichkeit im Reaktionsmuster der Mikroglia auf unterschiedliche pathogene

Stimuli zu bestehen. Funktionell zeigt sich jedoch durch die signifikante Steigerung

der Phagozytose bei Hunden mit Demyelinisierung und intrakraniellen Entzündungen

eine Tendenz zu einer mehr spezifischen Antwort. Die signifikante Steigerung der

ROS-Bildung im Vergleich zu allen anderen Untersuchungsgruppen weist jedoch

sehr stark auf eine spezifische Reaktion der Mikroglia bei Demyelinisierungen hin,

welche zudem ihre zentrale Rolle in der Pathogenese der Demyelinisierung

unterstreicht.

Im Gegensatz zur spezifischen Bildung von ROS bei Läsionen im ZNS mit

Demyelinisierung konnte bei der Untersuchung eines weiteren endogenen Gases,

NO, keine Korrelation zu spezifischen intrakraniellen Erkrankungen gefunden

werden. Daher scheint die Bildung von NO eine eher unspezifische Reaktion der

Mikroglia bei verschiedenen Erkrankungen des zentralen Nervensystems zu sein.

110

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130

X. Acknowledgements I thank my supervisor Prof. Dr. Andrea Tipold for her unrestricted support, her enthusiastic motivation, academic guidance and confidence throughout every phase of my Ph.D. study. I would like to thank the members of my advisory committee, Prof. Dr. Wolfgang Löscher, Prof. Dr. Dr. Franz Walter, and Prof. Dr. Peter Schmidt for their intense interest in my work and their constructive discussions. Furthermore, I would like to thank Prof. Dr. Wolfgang Baumgärtner for the histopathological examination of the dogs of the second part of the study in the Institute of Pathology. Despite initial personal prejudices against statistics itself Prof. Dr. Lothar Kreienbrock´s friendly assistance resulted in understanding and appreciation of the statistical analysis. Whenever needed he was open for discussions even though questions arose out of working hours. Therefore, I would like to sincerely thank him. I thank Prof. Dr. Wolfgang Leibold for his helpful contribution in the development of the methods for microglial ex vivo isolation and functional characterisation. Many thanks to Prof. Dr. Marc Vandevelde and Prof. Dr. Andreas Zurbriggen for their friendly support with the vaccine challenge experiment and their critical and constructive discussions of the results obtained. I warmly thank Regina Carlson for her excellent technical and mental support. Her inexhaustible optimism was always a great help to beat the adversities during the time of the study. My grateful thanks are due to PD Dr. Markus Czub who was a valuable advisor for both scientific and personal questions. I gratefully thank Prof. Dr. Ingo Nolte for providing access to the laboratory equipment and infrastructure. I also like to thank PD Dr. M. Enss and S. Faber for their encouraging assistance during the last two years. I sincerely thank Judy McAlister-Hermann, PhD, for her proof-reading of my manuscript. There are many people from the stuff of the Small Animal Clinic whom I wish to warmly thank for their continuous support: Kai Rentmeister, Heike Schröder, Irene Böttcher, Thilo v. Klopmann, and Simone Schröder for her help with the microglial isolation procedure. I especially thank Henning Schenk for his unlimited help to battle against every arising computer problem. Schließlich möchte ich den Menschen danken, die mir durch ihr Interesse, ihr Vertrauen und ihre guten Wünsche eine unersetzliche Unterstützung sind: meinen Eltern und Geschwistern mit Familie und Frank.

_______________________________Acknowledgements___________________________

131

132

XI. Appendix

Table XI. 1 Dogs of the vaccine challenge experiment

Examination group

Histopathological findings in CNS CDV detection Dog no.

group I (negative control)

no lesions negative 21, 22

group II no lesions

few scattered glial

nodules

negative

negative

3, 4, 5, 6, 8, 9, 10, 11, 14, 16

12, 13, 18

group III demyelinating lesions in CNS positive 1, 2, 7, 15, 17, 19, 20

Table XI. 1. Dogs of the vaccine challenge experiment

were divided into three examination groups according to histopathological findings in the central nervous system (CNS): group I, group II and III. CDV: canine distemper virus

_________________________________Appendix_________________________________

133

Table XI. 2 Dogs of the microglia study

Vaccine challenge experiment

Ex gr Case no. Breed Age Seizures Histopathol. diagnosis

21 Beagle 5-6m - no abnormalities in CNS I

22 Beagle 5-6m - no abnormalities in CNS









12 Beagle 5-6m - no abnormalities in CNS, despite some glial nodules


II


_________________________________Appendix_________________________________

134

Ex gr Case no. Breed Age Seizures Histopathol. diagnosis



1 Beagle 5-6m - demyelinating lesions in CNS






III


Second part of the study

Ex gr Case no. Breed Sex Age Seizures Histopathol. diagnosis

23 Shih-Tzu ♂c 6y, 5m +++ (> 1 h)

fibroblastic meningioma with purulent encephalitis and

malacia

24 Rottweiler ♂ 7y, 6m + (> 2.5 d)

malignant blastoma with necrosis in occipital cortex

25 Eurasian ♀s 6y, 9m - intraventricular plexuscarcinoma

IV

26 Pitbull Terrier ♂c 5y, 6m + (> 3 d)

meningoepitheliomatous meningioma

_________________________________Appendix_________________________________

135


27 mixed breed ♀s 13y, 6m +++ (> 3 d)

meningioma in bulbus olfactorius + purulent necrotic inflammation,

satellitosis in cortex cerebri

28 Yorkshire -Terrier ♂ 3y, 3m +

(> 2 d)

granulomatous meningoencephalitis, focal

demyelination in cerebellum

29 Chihuahua ♂ 5y, 11m - granulomatous

meningoencephalomyelitis, pancreatitis

30 West Highland White Terrier ♀s 7y, 3m + (?)

(?)

granulomatous meningoencephalitis in cerebellum and cortical cerebellar abiotrophy

31 Newfoundland ♀ 3y, 5m - cerebellar malacia probably due to an infarct

V

32 Miniature Pinscher ♂ 7y, 9m +

(> 7 d)

demyelination with leukoencephalitis and

myelitis nonpurulenta due to CDV infection

33 Cocker Spaniel ♂ 2m +++

(> 5 d) no abnormalities in CNS,

granulomatous pneumonia VI

34 Jack Russell Terrier ♀ 5m +

(> 1 d) no abnormalities in CNS

and periphery

35 Miniature Poodle ♂ 4m -

head trauma with intracranial bleedings and degeneration of nervous

tissue

36 Jack Russell Terrier ♂ 10m - hydrocephalus internus,

atrophy of cortex cerebri

37 mixed breed ♀ 20 d - meningocele with

suppurative inflammation, low grade malacia in

cerebellum

38 Staffordshire Terrier ♂c 5y, 10m - cerebellar abiotrophy

VII

39 Yorkshire Terrier ♂ 3y, 11m +

(?) hydrocephalus internus, atrophy of cortex cerebri

_________________________________Appendix_________________________________

136


40 mixed breed ♀ 8y, 3m - disc herniation Th11/12, proliferation of Alzheimer

type II cells and satellitosis in cortex cerebri

41 Yorkshire Terrier ♀ 2y, 2m -

no abnormalities in CNS and periphery, clinical signs

of polyradiculoneuritis

42 Yorkshire Terrier ♂ 8y -

no intracranial abnormalities, cervical disc

herniation

43 Alaskan Malamute ♂ 1y, 10m - no abnormalities in CNS,

radiculoneuritis

VIIIa

44 mixed breed ♂c 16y + (> 1 d)

liver cirrhosis, suspected hepatoencephalic

syndrome, satellitosis in Lobus occipitalis, nephritis,

endocarditis

45 mixed breed ♂c 12y, 6m -

no abnormalities in CNS except low grade spongious

degeneration of white matter, osteosarcoma left

humerus

46 Yorkshire Terrier ♀s 12y, 7m -

no abnormalities in CNS except some vacuoles in white matter, pancreatitis,

peritonitis, hyperadrenocorticism

VIIIb

47 Labrador ♂ 7y, 11m - no abnormalities in CNS, retinal atrophy

Table XI. 2. Dogs of the microglia study.

Ex gr = examination group, ♀ = female, ♂ = male, s = spayed, c = castrated, y = years, m = months, d = days, h = hour, CDV = canine distemper virus, CNS = central nervous system, - absent, + present, +++ dog presented in status epilepticus. The time span from the last documented seizures to ex vivo examination of the microglia is mentionted below. According to histopathological findings in CNS the dogs were divided into examination groups: group I: dogs of the vaccine challenge experiment with no changes in CNS, non-infected and non-vaccinated group II: dogs of the vaccine challenge experiment with no changes in CNS, infected and vaccinated group III: dogs of the vaccine challenge experiment with demyelinating lesions in CNS, infected and partly vaccinated

_________________________________Appendix_________________________________

137

group IV: dogs with intracranial tumors, group V: dogs with intracranial inflammation, group VI: dogs with idiopathic epilepsy, group VII: dogs with other changes in CNS, group VIII: dogs with extracranial diseases, VIIIa: disease of the spinal cord or the peripheral nervous system (PNS), VIIIb: no abnormalities in the nervous system

_________________________________Appendix_________________________________

138

Table XI. 3 Dogs of the microglia study in which nitric oxide (NO) was determined in microglial culture supernatant and cerebrospinal fluid

Case no. Breed Sex Age Seizures Pathol. diagnosis

1 Shih-Tzu ♂c 6y, 5m +++ (> 1h)

fibroblastic meningioma with purulent encephalitis and

malacia

2 Rottweiler ♂ 7y, 6m + (> 2.5 d)

malignant blastoma with necrosis in occipital cortex

3 Pitbull Terrier ♂c 5y, 6m + (> 3 d)

meningoepitheliomatous meningioma

4 Yorkshire -Terrier ♂ 3y, 3m +

(> 2 d)

granulomatous meningoencephalitis, focal

demyelination in cerebellum

5 Cocker Spaniel ♂ 2m +++ (> 5 d)

no abnormalities in CNS, granulomatous pneumonia

6 Jack Russell Terrier ♀ 5m +

(> 1 d) no abnormalities in CNS and

periphery

7 Yorkshire Terrier ♂ 3y, 11m +

(?) hydrocephalus internus, atrophy of cortex cerebri

8 mixed breed ♂c 16y + (> 1 d)

liver cirrhosis, suspected hepatoencephalic syndrome,

satellitosis in Lobus occipitalis, nephritis, endocarditis

9 Chihuahua ♂ 5y, 11m - granulomatous

meningoencephalomyelitis, pancreatitis

10 mixed breed ♂ 7y, 2m - granulomatous encephalitis

11 Miniature Poodle ♂ 4m -

head trauma with intracranial bleedings and degeneration of

nervous tissue

12 Jack Russell Terrier ♂ 10m - hydrocephalus internus,

atrophy of cortex cerebri

_________________________________Appendix_________________________________

139

Case no. Breed Sex Age Seizures Pathol. diagnosis

13 Staffordshire Terrier ♂c 5y, 10m - cerebellar abiotrophy

14 Yorkshire Terrier ♀ 2y, 2m -

no abnormalities in CNS and periphery, clinical signs of

polyradiculoneuritis

15 Alaskan Malamute ♂ 1y, 10m - no abnormalities in CNS,

radiculoneuritis

16 mixed breed ♂c 12y, 6m - no abnormalities in CNS

except low grade spongious degeneration of white matter, osteosarcoma left humerus

17 Labrador ♂ 7y, 11m - no abnormalities in CNS, retinal atrophy

18 Yorkshire Terrier ♂ 8y - no intracranial abnormalities,

cervical disc herniation

19 Eurasian ♀s 6y, 9m - intraventricular plexuscarcinoma

20 Newfoundland ♀ 3y, 5m - cerebellar malacia probably due to an infarct

21 mixed breed ♀ 8y, 3m - disc herniation Th11/12,

proliferation of Alzheimer type II cells and satellitosis in

cortex cerebri

22 Yorkshire Terrier ♀s 12y, 7m -

no abnormalities in CNS except some vacuoles in white

matter, pancreatitis, peritonitis,

hyperadrenocorticism Table XI. 3. Dogs of the microglia study in which determination of nitric oxide

(NO) in microglial culture supernatant and cerebrospinal fluid (CSF) was performed. ♀ = female, ♂ = male, s = spayed, c = castrated, y = years, m = months, h = hours, CNS = central nervous system, - absent, + present, +++ dog presented in status epilepticus. The time span from the last documented seizures to ex vivo examination of the microglia is mentioned below.

_________________________________Appendix_________________________________

140

Table XI. 4 Dogs in which nitric oxide (NO) was determined in cerebrospinal fluid

Exam. group Case no. Breed Sex Age Seizures Pathol. diagnosis

S 23 mixed breed ♀ 9y + idiop. epilepsy

S 24 Airedale Terrier ♂ 2y + idiop. epilepsy

S 25 Siberian Husky ♂ 3y + idiop. epilepsy

S 26 Scotch Terrier ♂ 5m + idiop. epilepsy

S 27 mixed breed ♀ 9y + idiop. epilepsy

S 28 Great Dane ♂ 2y + idiop. epilepsy

S 29 Dachshund ♂ 5y + idiop. epilepsy

S 30 Golden Retriever ♂ 8y + idiop. epilepsy

S 31 Bordeaux Dogge ♂ 1y + idiop. epilepsy

S 32 Golden Retriever ♂c 12y + glioma in cerebrum

S 33 Great Dane ♂c 6y + oligodendroglioma

S 34 Golden Retriever ♀ 9y + lymphoma in cerebrum

S 35 Bernese Mountain Dog ♂ 7y + glioma in cerebrum

S 36 mixed breed ♂ 8y + tumor in brainstem

_________________________________Appendix_________________________________

141


ed 37 mixed breed ♂ 6y - intramedullary tumor in spinal cord

ed 38 Golden Retriever ♂ 9y - intramedullary tumor in spinal cord

ed 39 German Shepherd ♀ 6y - disc herniation

ed 40 Dachshund ♂ 9y - cervical disc herniation

ed 41 Greyhound ♂ 9y - cervical disc herniation

ed 42 mixed breed ♂ 9y - cervical disc herniation

ed 43 Bernese Mountain dog ♂ 5y - cervical disc

herniation

ed 44 German Shepherd ♂ 1y - Cauda equina compression

ed 45 Appenzeller ♂ 1y - SRMA, recovered

ed 46 Dachshund ♂ 7y - GME, recovered

ed 47 Briard ♀s 5y - periph. vestibular signs

ed 48 Boxer ♂ 9y - periph. vestibular signs

ed 49 Golden Retriever ♀ 2y - fracture luxation Th8

ed 50 Cairn Terrier ♂ 10y - sudden blindness

ed 51 Boxer ♂ 7y - facial palsy

_________________________________Appendix_________________________________

142


I 52 mixed breed ♀ 4y - GME

I 53 West Highland White Terrier ♂ 11y - GME

I 54 Bernese Mountain Dog ♂c 4y - GME

I 55 Scotch Terrier ♀ 5y - GME

I 56 Gordon Setter ♀ 9m - GME

I 57 Bernese MountainDog ? 5m - SRMA

I 58 Malinoise ♂ 1y - SRMA

I 59 Bernese Mountain Dog ♂ 8y - SRMA

I 60 Bernese Mountain Dog ♀ 8m - SRMA

I 61 Bernese Mountain Dog ♂ 9m - SRMA

I 62 Boxer ♂ 1y - SRMA

I 63 Springer Spaniel ♀s 5y - SRMA

I 64 Beagle ♀ 1y - SRMA

I 65 Bernese Mountain Dog ♂ 2y - SRMA

I 66 Dogue Argentine ♂ 6m - SRMA

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143


I 67 unknown - SRMA

I 68 Golden Retriever ♀ 1y - SRMA

I 69 Bernese Mountain Dog ? 1y - SRMA

I 70 Golden Retriever ♂ 2y - SRMA

I 71 Boxer ♂ 1y - SRMA

I 72 German Shepherd ♂ 2y - viral encephalitis

I 73 Springer Spaniel ♀ 5m - encephalitis of unknown origin

I 74 Beagle ♀ 4m - encephalitis of unknown origin

I 75 French Bulldog ♂ 3m - trauma and encephalitis

T 76 West Highland White Terrier ♂ 4y - glioma in midbrain

T 77 mixed breed ♀s 7y - central vestibular signs, glioma (?)

T 78 mixed breed ♂c 7y - plexus papilloma

Table XI. 4. Additional dogs in which nitric oxide (NO) was determined in

cerebrospinal fluid. S = dogs showing seizure activity, ed = dogs with extracranial diseases, I = dogs with intracranial inflammation without seizure activity, T = dogs with intracranial tumors without seizure activity, according to complete clinical diagnostic workup including blood diagnostics, examination of cerebrospinal fluid (CSF) and diagnostic imaging. ♀ = female, ♂ = male, s = spayed, c = castrated, m = months, y = years, GME = granulomatous meningoencephalomyelitis, SRMA = steroid responsive meningitis arteritis, Th = thoracal vertebra, periph. = peripheral

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144

Table XI. 5. Statistical data for immunophenotypical characterization of canine microglial cells ex vivo in original and alternative examination groups

Variable R2 p significant differences

% 0.608655 <.0001

I + II ─ IV I + II ─ VIII

III ─ IV III ─ VIII

CD18

mfi 0.251872 0.0883

% 0.537608 <.0001 I + II ─ IV I + II ─ VIII

III ─ IV CD11b

mfi 0.477258 0.0003 I + II ─ IV I + II ─ V

% 0.481998 0.0006 I + II ─ IV I + II ─ VIII CD11c

mfi 0.312922 0.0415

% 0.607838 <.0001

VI ─ I + II VI ─ III VI ─ IV VI ─ V

VI ─ VIII VII ─ I + II

CD45

mfi 0.497772 0.0004

% 0.532953 <.0001 I + II ─ IV I + II ─ VIII

III ─ IV CD1c

mfi 0.072860 0.8155

% 0.626596 <.0001

I + II ─ IV I + II ─ VIII

III ─ IV III ─ VIII

ICAM-1

mfi 0.173794 0.2991

_________________________________Appendix_________________________________

145


% 0.432304 0.0012 III ─ IV III ─ VII III ─ VIII

B7-1

mfi 0.535019 <.0001

I + II ─ III III ─ IV III ─ V III ─ VI III ─ VII III ─ VIII

% 0.981929 <.0001

I + II ─ IV I + II ─ V I + II ─ VI I + II ─ VII I + II ─ VIII

III ─ IV III ─ V III ─ VI III ─ VII III ─ VIII B7-2

mfi 0.790174 <.0001

I + II ─ III I + II ─ IV I + II ─ V

I + II ─ VII I + II ─ VIII

III ─ IV III ─ V III ─ VI III ─ VII III ─ VIII

% 0.482998 0.0003 I + II ─ IV I + II ─ VIII

III ─ IV MHC I

mfi 0.395919 0.0033 I + II ─ III

% 0.473586 0.0004 I + II ─ VIII

MHC II mfi 0.473541 0.0004 I + II ─ III

I + II ─ V

_________________________________Appendix_________________________________

146


% 0.803985 <.0001

I + II ─ III I + II ─ IV I + II ─ V I + II ─ VI I + II ─ VII I + II ─ VIII

CD14

mfi 0.101480 0.7119

% 0.620418 <.0001

I + II ─ IV I + II ─ V

I + II ─ VIII III ─ IV III ─ VIII

CD18 alternative

mfi 0.224596 0.1404

% 0.549182 <.0001 I + II ─ IV I + II ─ VIII

III ─ IV CD11b alternative

mfi 0.472433 0.0004 I + II ─ IV

% 0.495124 0.0004 I + II ─ IV I + II ─ VIII CD11c

alternative mfi 0.313790 0.0408

% 0.594932 <.0001

VI ─ I + II VI ─ III VI ─ IV VI ─ V

VI ─ VIII VII ─ I + II

VII ─ V

CD45 alternative

mfi 0.337490 0.0221

% 0.548353 <.0001

I + II ─ IV I + II ─ VII I + II ─ VIII

III ─ IV CD1c alternative

mfi 0.133829 0.4701

% 0.534705 <.0001 I + II ─ IV I + II ─ VIII ICAM-1

alternative mfi 0.188775 0.2429

_________________________________Appendix_________________________________

147


% 0.354128 0.0093 B7-1 alternative

mfi 0.433105 0.0012 I + II ─ III III ─ IV III ─ VII

% 0.909416 <.0001



B7-2 alternative

mfi 0.654395 <.0001


% 0.463879 0.0005 I + II ─ IV I + II ─ VIII

III ─ IV MHC I alternative

mfi 0.441790 0.0009 I + II ─ III

% 0.393029 0.0035 I + II ─ VIII MHC II alternative mfi 0.530672 <.0001 I + II ─ III

% 0.796712 <.0001


CD14 alternative

mfi 0.104965 0.6936

Table XI. 5. Statistical data obtained from immunophenotypical

characterization of microglial cells ex vivo. Variables without the term “alternative” represent the data obtained from statistical analysis of the eight examination groups. In order to evaluate whether results correlate specifically with the occurrence of demyelination all dogs with histopathologically confirmed demyelination

_________________________________Appendix_________________________________

148

were comprised in group III and analysed again. Results of this analysis are labelled with the word “alternative”. Dogs of group I and II without changes in CNS are comprised in group 1 for statistical analysis. R2 describes the goodness of model fit, the p-value displays the statistical level attained for the global F-test. An error probability of 0.05 (p) was used as the significance level for all statistical tests. mfi: mean fluorescence intensity

_________________________________Appendix_________________________________

149

Table XI. 6. Statistical data for functional characterization of canine microglial cells ex vivo - ROS generation test


% 0.393858 0.0023 I + II ─ VIII PMA 0

mfi 0.513512 <.0001 I + II ─ III I + II ─ V

% 0.379917 0.0033 I + II ─ III III ─ VIII

PMA 100 mfi 0.615580 <.0001

I + II ─ III III ─ IV III ─ VI III ─ VII III ─ VIII

% 0.393221 0.0023 I + II ─ VIII PMA 0 alternative

mfi 0.580604 <.0001

I + II ─ III III ─ IV III ─ VI

III ─ VIII

% 0.371949 0.0041 I + II ─ III

PMA 100 alternative mfi 0.719131 <.0001

I + II ─ III III ─ IV III ─ V III ─ VI III ─ VII III ─ VIII

Table XI. 6. Statistical data obtained from ROS generation test of microglial

cells ex vivo. PMA 0 and PMA 100 represent the data obtained from statistical analysis of the eight examination groups. In order to evaluate whether generation of ROS correlates specifically with the occurrence of demyelination, all dogs with histopathologically confirmed demyelination were comprised in group III and compared with the results of the other groups. Results of this analysis are labelled with the word “alternative”. Dogs of group I and II without changes in CNS are comprised in group 1 for statistical analysis. R2 describes the goodness of model fit, the p-value displays the statistical level attained for the global F-test. An error probability of 0.05 (p) was used as the significance level for all statistical tests.

_________________________________Appendix_________________________________

150

Table XI. 7. Statistical data for functional characterization of canine microglial cells ex vivo - phagocytosis assay -


% 0.116547 0.5344 PBS

mfi 0.218284 0.1319

% 0.334661 0.0106 nops

mfi 0.491131 0.0001 I + II ─ III I + II ─ V

% 0.281364 0.0355 ops

mfi 0.511201 <.0001 I + II ─ III I + II ─ IV

% 0.301482 0.0200 nops/ops n

mfi 0.481823 0.0001 I + II ─ III I + II ─ V

% 0.194838 0.1687 nops/ops o

mfi 0.514637 <.0001 I + II ─ III I + II ─ V

% 0.083689 0.7334 PBS alternative

mfi 0.296466 0.0293

% 0.260345 0.0548 nops alternative mfi 0.490891 0.0002 I + II ─ III

% 0.232079 0.0945 ops alternative mfi 0.484878 0.0002 I + II ─ III

I + II ─ IV

% 0.294155 0.0236 nops/ops n alternative mfi 0.493178 <.0001 I + II ─ III

I + II ─ V

% 0.213273 0.1223 nops/ops o alternative mfi 0.511654 <.0001 I + II ─ III

I + II ─ V

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151

Table XI. 7. Statistical data obtained from phagocytosis assay of microglial

cells ex vivo. Values for PBS, nops (non-opsonized) and ops (opsonized) Staphylococcus aureus represent the data obtained from statistical analysis of the eight examination groups. In order to evaluate whether enhanced phagocytosis correlated with the occurrence of demyelination all dogs with histopathologically confirmed demyelination were comprised in group III and compared with the results of the other groups. Results of this analysis are labelled with the word “alternative”. Dogs of group I and II without changes in CNS are comprised in group 1 for statistical analysis. R2 describes the goodness of model fit, the p-value displays the statistical level attained for the global F-test. An error probability of 0.05 (p) was used as the significance level for all statistical tests.

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Table XI. 8. Boxplots for optical control of normality of the results obtained from the immunophenotypical characterization

of microglial cells ex vivo

CD18 in % for 8 groups Boxplot

0 |

+-----+ *--+--* +-----+

| | 0 0

CD18 mfi for 8 groups Boxplot

0 | | | | |

+-----+ | + | *-----* | | +-----+

|

CD11b in % for 8 groups Boxplot

0 0 |

+-----+ *--+--* +-----+

| | *

*

CD11b mfi for 8 groups Boxplot

0 | | | |

+-----+ | + | *-----* +-----+

| | |

|

CD11c in % for 8 groups Boxplot

| | |

+-----+ *--+--* +-----+

| | | 0 0

0

CD11c mfi for 8 groups Boxplot

| | | |

+-----+ | + | *-----* +-----+

| |


* * |

+--+--+ *-----* +-----+

| 0 *

*


| | | | | |

+-----+ | | | | *--+--* | | | | +-----+

| | | | |

|

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153

CD1c in % for 8 groups Boxplot

| |

+-----+ | | | | | | *--+--* | | | | | | | | +-----+

| | | | | | | |

|

CD1c mfi for 8 groups Boxplot

0 0 | | | |

+-----+ | + | *-----* | | +-----+

| |

ICAM-1 in % for 8 groups Boxplot

0 0 0 | | |

+-----+ *--+--* | | +-----+

| | 0 0 0 *

*

ICAM-1 mfi for 8 groups Boxplot

0 0 | | | |

+-----+ *--+--* | | +-----+

| | | |

0

B7-1 in % for 8 groups Boxplot

* * 0 | |

+--+--+ *-----* +-----+

| 0

B7-1 mfi for 8 groups Boxplot

* * 0 0

+--+--+ | | +-----+

*

B7-2 in % for 8 groups Boxplot

| | | |

+-----+ | + | *-----* +-----+

| | | 0

0

B7-2 mfi for 8 groups Boxplot

* * * 0 | |

+--+--+ | | +-----+

| 0

* MHC I in % for 8 groups Boxplot

0 0 | |

+-----+ *--+--* +-----+

| | | *

*

MHC I mfi for 8 groups Boxplot

| | | |

+-----+ | | | + | *-----* +-----+

| | |

MHC II in % for 8 groups Boxplot

0 0 | |

+-----+ *--+--* | | +-----+

| | *

*

MHC II mfi for 8 groups Boxplot

0 | | |

+-----+ | + | *-----* +-----+

| |

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154


* 0 0 0 | |

+--+--+ *-----* +-----+

| | 0

*


* * 0

+--+--+ +-----+

|

CD18 in % for alternat grouping

Boxplot

0 |

+-----+ *--+--* | | +-----+

| |

0

CD18 mfi for alternat. grouping

Boxplot

0 | | | |

+-----+ | + | *-----* +-----+

| |

CD11b in % for alternat grouping Boxplot

0 0 |

+-----+ *--+--* +-----+

| | *

*

CD11b mfi for alternat grouping

Boxplot

0 0 | | |

+-----+ | + | *-----* +-----+

| | |

0

CD11c in % for alternat grouping Boxplot

0 0 |

+-----+ *--+--* +-----+

| | | 0 0 *

*

CD11c mfi for alternat grouping

Boxplot

| | | |

+-----+ | + | *-----* +-----+

| |

|

CD45 in % for alternat grouping

Boxplot

* * |

+--+--+ *-----* +-----+

| | 0

*

CD45 mfi for alternat grouping Boxplot

| | | | | |

+-----+ | | | | | + | *-----* | | | | +-----+

| | | |

|

_________________________________Appendix_________________________________

155

CD1c in % for alternat grouping

Boxplot

| | | |

+-----+ | | | | | | *--+--* | | | | | | | | +-----+

| | | | | | |

|

CD1c mfi for alternat grouping Boxplot

0 0 | | |

+-----+ | + | *-----* +-----+

| | |

|

ICAM-1 in % for alternat grouping

Boxplot

| |

+-----+ *--+--* +-----+

| | 0 0

*

ICAM-1 mfi for alternat grouping Boxplot

0 0 | | |

+-----+ | + | *-----* +-----+

| | | |

0

B7-1 in % for alternat grouping Boxplot

0 0 0 | |

+--+--+ *-----* +-----+

| |

|

B7-1 mfi for alternat grouping Boxplot

* * * * 0 |

+--+--+ | | +-----+

*

B7-2 in % for alternat grouping Boxplot

0 0 | |

+--+--+ *-----*

| |

*

B7-2 mfi for alternat grouping Boxplot

* * 0 |

+--+--+ +-----+

| 0

*

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MHC I in % for alternat grouping Boxplot

0 0 | |

+-----+ *--+--* +-----+

| | | 0 *

*

MHC I mfi for alternat grouping Boxplot

| | |

+-----+ | | | + | *-----* +-----+

| | |

|

MHC II in % for alternat grouping Boxplot

| | | |

+-----+ *--+--* | | +-----+

| | 0

*

MHC II mfi for alternat grouping Boxplot

0 | | |

+-----+ | + | *-----* +-----+

| |

CD14 in % for alternat grouping Boxplot

* 0 0 | |

+--+--+ *-----* +-----+

| | |

*

CD14 mfi for alternat grouping

Boxplot

* * 0

+--+--+ +-----+

|

Table XI. 8. Boxplots for optical control of normal distribution of the results

obtained from the immunophenotypical characterization of microglial cells ex vivo. Boxplots visualize minimal and maximal values, lower and upper quartile and median of the values obtained. The box shows the values of middle 50% of the sample values.

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Table XI. 9 Boxplots for optical control of normality of the results obtained from the ROS generation test of microglial cells ex vivo

PMA 0 in % for 8 groups Boxplot

| | | | | |

+-----+ | | *--+--* | | +-----+

| | | | 0

0

PMA 0 mfi for 8 groups Boxplot

0 0 |

+-----+ *--+--* +-----+

| |

*

PMA 100 in % for 8 groups Boxplot

0 0 0 | | |

+-----+ | + | | | *-----* +-----+

| | | |

0

PMA 100 mfi for 8 groups Boxplot

0 | | |

+-----+ | + | *-----* +-----+

| | |

0

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PMA 0 in % for alternat grouping Boxplot

| | | | | |

+-----+ | | *--+--* | | +-----+

| | | | 0

0

PMA 0 mfi for alternat grouping Boxplot

0 0 | | | |

+-----+ *--+--* | | +-----+

| | | |

0

PMA 100 in % for alternat grouping Boxplot

* 0 0 | |

+--+--+ *-----*

| | 0

0

PMA 100 mfi for alternat grouping Boxplot

| | | | | |

+-----+ | | *--+--* | | | | +-----+

| | | | | |

| Table XI. 9. Boxplots for optical control of normal distribution of the results

obtained from the ROS generation test of microglial cells ex vivo. Boxplots visualize minimal and maximal values, lower and upper quartile and median of the values obtained. The box shows the values of middle 50% of the sample values.

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159

Table XI. 10 Boxplots for optical control of normality of the results obtained from the phagocytosis assay

of microglial cells ex vivo

PBS in % for 8 groups Boxplot

* *

+--0--+ *-----*

*

PBS mfi for 8 groups Boxplot

* * * |

+--+--+ *-----*

| 0

nops in % for 8 groups Boxplot

| | | |

+-----+ | + | *-----* +-----+

| |

0

nops mfi for 8 groups Boxplot

* | | |

+-----+ | + | *-----* +-----+

| | | |

0

ops in % for 8 groups Boxplot

| |

+-----+ | | *--+--* | | | | +-----+

| |

ops mfi for 8 groups Boxplot

* 0 0 0 | | |

+-----+ | + | *-----* +-----+

| | | | 0

0

nops/ops nops in % for 8 groups Boxplot

0 | | | |

+-----+ | + | *-----* | | +-----+

| | | |

0

nops/ops nops mfi for 8 groups Boxplot

* 0 | |

+--+--+ *-----* +-----+

| 0

0

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160

nops/ops ops in % for 8 groups Boxplot

0 0 | | | |

+-----+ | | | + | | | *-----*

| | | |

nops/ops ops mfi for 8 groups Boxplot

* * 0 | |

+-----+ | + | *-----* +-----+

| | | 0

*

PBS in % for alternat grouping Boxplot

* *

+--0--+ *--0--* 0

PBS mfi for alternat grouping Boxplot

* * * 0

+--+--+ *-----*

| 0

nops in % for alternat grouping Boxplot

0 0 | | |

+-----+ | + | *-----* +-----+

| |

|

nops mfi for alternat grouping

Boxplot

0 | | |

+-----+ | + | *-----* +-----+

| | | | 0

0

_________________________________Appendix_________________________________

161

ops in % for alternat grouping

Boxplot

| |

+-----+ | | *--+--* | | +-----+

| |

|

ops mfi for alternat grouping

Boxplot

* 0 0 0 | | |

+-----+ | + | *-----* +-----+

| | | | 0 0

0

nops/ops nops in % for alternat grouping Boxplot

0 0 | | | | |

+-----+ | + | *-----* | | | | +-----+

| | |

0

nops/ops nops mfi for alternat grouping Boxplot

* 0 | | |

+--+--+ *-----* +-----+

| | | 0

0

nops/ops ops in % for alternat grouping Boxplot

0 0 | | | |

+-----+ | | | + | | | *-----*

| | | |

|

nops/ops ops mfi for alternat grouping

Boxplot

* 0 0 0 | |

+-----+ | + | *-----* +-----+

| 0 0 *

*

Table XI. 10. Boxplots for optical control of normal distribution of the results

obtained from the phagocytosis assay of microglial cells ex vivo. Boxplots visualize minimal and maximal values, lower and upper quartile and median of the values obtained. The box shows the values of middle 50% of the sample values.

_________________________________Appendix_________________________________

162

Figure XI. 1 Residualplots for the results of the immunophenotypical characterization of microglial cells ex vivo

_________________________________Appendix_________________________________

163

_________________________________Appendix_________________________________

164

_________________________________Appendix_________________________________

165

_________________________________Appendix_________________________________

166

_________________________________Appendix_________________________________

167

_________________________________Appendix_________________________________

168

_________________________________Appendix_________________________________

169

Figure XI. 1. Residualplots displaying the results of the analysis of the

residuals for the immunophenotypical characterization of the eight examination groups and for the analysis with all dogs with histopathologically confirmed demyelination comprised in group III (termed “with alternative grouping”). Dogs of group I and II without changes in CNS are comprised in group 1 for statistical analysis. Note that the scaling of the ordinates is different for various variables according to the signal size. mfi = mean fluorescence intensity, alternat = alternative groups with all dogs with demyelination in CNS comprised in group III.

_________________________________Appendix_________________________________

170

Figure XI. 2 Residualplots for the results of the ROS generation test of microglial cells ex vivo

_________________________________Appendix_________________________________

171

Figure XI.2. Residualplots displaying the results of the analysis of the

residuals for the ROS generation test for the eight examination groups and for the analysis with all dogs with histopathologically confirmed demyelination comprised in group III (termed “with alternative grouping”). Dogs of group I and II without changes in CNS are comprised in group 1 for statistical analysis. Note that the scaling of the ordinates is different for various variables according to the signal size. mfi = mean fluorescence intensity, alternat = alternative groups with all dogs with demyelination in CNS comprised in group III.

_________________________________Appendix_________________________________

172

Figure XI. 3 Residualplots for the results of the phagocytosis assay of microglial cells ex vivo

_________________________________Appendix_________________________________

173

_________________________________Appendix_________________________________

174

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175

Figure XI. 3. Residualplots displaying the results of the analysis of the

residuals for the phagocytosis assay for the eight examination groups and for the analysis with all dogs with histopathologically confirmed demyelination comprised in group III (termed “with alternative grouping”). Dogs of group I and II without changes in CNS are comprised in group 1 for statistical analysis. Note that the scaling of the ordinates is different for various variables according to the signal size. mfi = mean fluorescence intensity, alternat = alternative groups with all dogs with demyelination in CNS comprised in group III.

_________________________________Appendix_________________________________

176

Figure Index Figure 1. Heterogeneity of microglia according to functional and developmental states (modified according to Kettenmann and Ransom 1995) 16 Figure 2. Isolation protocol for canine microglial cells 48 Figure 3. Constitutive expression of CD18 on canine microglia. 57 Figure 4. Expression intensity (log values) of CD18, CD11b and CD11c

of the dogs of the vaccine challenge experiment (groups I – III) and the second part of the study (groups IV – VIII) shown as mean values of each group. 58

Figure 5. Expression intensity (log values) of CD11b of the examination groups. 59

Figure 6. Expression intensity (log values) measured by mean fluorescence intensity of the surface antigens CD18, CD11b, and CD11c according to the examination groups. 60

Figure 7. Expression of CD45 of the dogs of the vaccine challenge experiment (groups I – III) and the dogs of the second part of the study (groups IV – VIII). 62

Figure 8. Expression and expression intensity of CD45 in microglia (A+B) and monocytes (C) displayed as histograms in flow cytometry. 63

Figure 9. Expression intensity (log values) of CD1c of the eight examination groups. 65

Figure 10. Expression intensity (log values) of MHC I and MHC II of the eight examination groups. 67

Figure 11. Expression intensity (log values) of MHC class I and MHC class II in the eight examination groups. 68

Figure 12. Expression intensity (log values) of ICAM-1 according to the eight examination groups. 70

Figure 13. Expression intensity (log values) of B7-1 and B7-2 according to the examination groups. 71

Figure 14. Generation of ROS of the dogs of the vaccine challenge experiment (groups I – III) compared with dogs of the second part of the study (groups IV – VIII). 72

Figure 15. ROS generation intensity of the dogs of the vaccine challenge experiment (groups I – III) as compared to that of dogs of the second part of the study (groups IV – VIII). 74

Figure 16. Generation of ROS displayed as percentage of ROS-generating microglial cells (on the left) and intensity of ROS generation (on the right). 77

Figure 17. Percentage of phagocytosis-positive microglia ex vivo. 77 Figure 18. Percentage of phagocytosis-positive microglia evaluated for

phagocytosis of non-opsonized (nops) in comparison with opsonized (ops) Staphylococcus aureus. 79

Figure 19. Mean fluorescence intensity as a relative means for phagocytosis intensity (shown as log values) of the examination groups. 81

Figure 20. Mean fluorescence intensity as a relative means for measuring phagocytosis intensity (shown as log values) of non-opsonized (nops) in comparison with opsonized (ops) Staphylococcus aureus according to the examination groups. 83

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Figure 21. Results of the phagocytosis assay with all dogs with demyelinating lesions in histopathology included in group III. 87

Figure 22. Nitrite measurement in microglial culture supernatant differentiated for dogs with seizures in comparison with dogs without seizures. 88

Figure 23. Nitrite measurement in cerebrospinal fluid (CSF) differentiated for dogs with seizures as compared to dogs without seizures. 89

Figure 24. Determination of NO in cerebrospinal fluid. 89 Table Index Table 1. Overview of the relevant canine CD antigens used in this study 27 Table 2. Antibodies for immunophenotypic characterization of canine microglial cells and their dilutions used, and which were provided by Prof. Peter F. Moore 36 Table 3. Definition of the eight examination groups according to

histopathological findings in the central nervous system (CNS) and peripheral nervous system (PNS). 42

Table 4. Statistical data of the expression intensity (mfi = mean fluorescence intensity) of CD18, CD11b, and CD11c. 61

Table 5. Differentiation of CD45-positive cells according to their expression intensity, CD45low and CD45high. 64

Table 6. Statistical data of percentage and expression intensity (mfi = mean fluorescence intensity) of MHC I and MHC II. 69

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