UBC Theses and Dissertations

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UBC Theses and Dissertations

Major histocompatibility complex antigens in the rat CNS following herpes simplex virus type 1 infection Weinstein, Debra Lynn 1989

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I Major H i s t o c o m p a t i b i l i t y Complex A n t i g e n s i n t h e Rat CNS F o l l o w i n g Herpes Simplex V i r u s Type 1 I n f e c t i o n By Debra Lynn W e i n s t e i n B.A., The U n i v e r s i t y o f C a l i f o r n i a , Los A n g e l e s , 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES PROGRAM IN NEUROSCIENCE (Department of Psychiatry) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1989 (c) Debra Lynn W e i n s t e i n , 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Psychiatry The University of British Columbia Vancouver, Canada D a t e A p r i l 28, 1989 DE-6 (2/88) ABSTRACT The p r e s e n t s t u d y was d e s i g n e d t o d e t e r m i n e major h i s t o c o m p a t i b i l i t y complex (MHC) e x p r e s s i o n i n t h e r a t CNS f o l l o w i n g a v i r a l i n f e c t i o n . Male W i s t a r r a t s were a n e s t h e t i z e d w i t h e t h e r and t h e r i g h t c o r n e a was r e p e a t e d l y s c r a t c h e d w i t h a 23.5 gage n e e d l e . A 30 m i c r o l i t e r drop of Herpes Simplex V i r u s Type 1 (HSV1) (PFU 33,000/mL) was p l a c e d on t h e s c a r i f i e d c o r n e a . A n i m a l s were s a c r i f i c e d 3, 6, 8, 10, 12, and 30 days f o l l o w i n g i n f e c t i o n . F o l l o w i n g d e c a p i t a t i o n , t h e b r a i n s were removed and p l a c e d i n Zamboni f i x a t i v e f o r immersion f i x a t i o n . M o n o c l o n a l a n t i b o d i e s a g a i n s t r a t MHC c l a s s I / c l a s s I I a n t i g e n s and HSV1 as w e l l as p o l y c l o n a l g l i a l f i b r i l l a r y a c i d i c p r o t e i n (GFAP) were used i n b o t h s i n g l e and double s t a i n i n g p r o c e d u r e s . Neurons s t a i n i n g p o s i t i v e l y f o r HSV1 were o b s e r v e d i n t h e i p s i l a t e r a l p r i n c i p a l s e n s o r y n u c l e u s of t h e f i f t h n e r v e . F i f t h n e r ve axons were a l s o p o s i t i v e . A d d i t i o n a l i n f e c t e d a r e a s i n c l u d e d m i d l i n e b r a i n s t e m s t r u c t u r e s , hypothalamus, thalamus, c e r e b e l l u m and d i f f u s e c o r t i c a l r e g i o n s . I n f e c t i o n became i n c r e a s i n g l y pronounced t h r o u g h day 10. Two a n i m a l s who r e c o v e r e d from t h e v i r a l e n c e p h a l i t i s were s a c r i f i c e d on days 12 and 30; t h e y demonstrated much l e s s p o s i t i v e HSV1 s t a i n i n g . In t h e i n f e c t e d a n i m a l s , s e r i a l s e c t i o n s r e v e a l e d b o t h c l a s s I and c l a s s I I p o s i t i v e s t a i n i n g of non-neuronal c e l l s i n b r a i n a r e a s which were a l s o p o s i t i v e f o r HSV1. E n d o t h e l i a l c e l l s and c e l l s w i t h m i c r o g l i a - l i k e morphology were p o s i t i v e l y s t a i n e d f o r c l a s s I and e x p r e s s i o n i n c r e a s e d t h r o u g h day 10. The c l a s s I I p o s i t i v e c e l l s had t h e morphology of l e u k o c y t e s and m i c r o g l i a - l i k e c e l l s w i t h i i i i n c r e a s e d e x p r e s s i o n o c c u r r i n g t h r o u g h days 8 and 10 th r o u g h o u t t h e b r a i n . I n s e r i a l s e c t i o n s , dense a r e a s o f c l a s s I I p o s i t i v e m i c r o g l i a - l i k e c e l l s were l o c a t e d i n t h e same ar e a s as HSV1 p o s i t i v e c l u s t e r s . MHC e x p r e s s i o n i n t h e day 12 a n i m a l was q u i t e pronounced w i t h d e c r e a s i n g l e v e l s o b s e r v e d a t 30 days. T i s s u e d o u b l y s t a i n e d f o r c l a s s I I and GFAP demonstrated no o v e r l a p among p o s i t i v e l y s t a i n e d c e l l s s u g g e s t i n g t h a t a s t r o c y t e s were not e x p r e s s i n g MHC g l y c o p r o t e i n s . A s t r o c y t e s d i d , however, show m o r p h o l o g i c a l changes c o n s i s t e n t w i t h an acu t e CNS i n f e c t i o n . M i c r o g l i a a re an endogenous component o f t h e CNS which may be p h a g o c y t o t i c , but t h e f u l l range o f f u n c t i o n s which m i c r o g l i a p o s s e s s i s s t i l l u n c l e a r . The r e s u l t s o f t h e p r e s e n t s t u d y i n d i c a t e t h a t MHC e x p r e s s i o n o c c u r s i n t h e CNS as e a r l y as 6 days f o l l o w i n g v i r a l i n f e c t i o n w i t h i n c r e a s e d e x p r e s s i o n t h r o u g h day 10. C o n t i n u e d h i g h l e v e l s o f c l a s s I I e x p r e s s i o n a t 12 days, f o l l o w i n g s u b s t a n t i a l a c t i v e v i r u s c l e a r i n g , s t r o n g l y suggest ongoing immune system a c t i v i t y which appears t o be p r e s e n t a t l e a s t as l o n g as 30 days p o s t - i n f e c t i o n . P e r i p h e r a l l y , MHC e x p r e s s i o n i s i n v o l v e d i n t h e i n d u c t i o n o f an immune response, s u g g e s t i n g t h a t t h e r a t b r a i n i s c a p a b l e o f mounting an immune response t o v i r a l i n f e c t i o n . M i c r o g l i a e x p r e s s i n g c l a s s I I a n t i g e n s may p o s s e s s t h e a b i l i t y t o p r e s e n t a n t i g e n t o T c e l l s and t h e r e b y i n i t i a t e an immune response w i t h i n t h e CNS. The p r e s e n t s t u d y s u g g e s t s t h a t MHC a n t i g e n s may p l a y an a c t i v e r o l e i n v i r a l c l e a r a n c e from t h e CNS. i v T a b l e o f C o n t e n t s A b s t r a c t i i L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i Acknowledgement x I n t r o d u c t i o n 1 Model Systems 4 Normal T i s s u e 5 P a t h o l o g i c a l S t a t e s 6 T h e s i s R a t i o n a l e 10 P r e d i c t i o n s 15 M a t e r i a l s and Methods 17 V i r u s 17 V i r u s A p p l i c a t i o n 17 T i s s u e P r e p a r a t i o n 18 Immunohistochemistry 18 R e s u l t s 21 B e h a v i o r a l Changes 21 HSV1 S t a i n i n g 21 MHC C l a s s I E x p r e s s i o n 36 MHC C l a s s I I E x p r e s s i o n 45 GFAP S t a i n i n g 51 Double Immunostaining 57 Discussion 59 HSV1 59 MHC Antigens 64 GFAP 6 9 Microglia 69 Origin of Microglia 71 Summary 72 Direction of Future Studies 73 Bibliography 75 v i L i s t o f T a b l e s T a b l e 1 (Page 22) HSV1 i m m u n o r e a c t i v i t y w i t h i n 8 d i f f e r e n t b r a i n r e g i o n s t h r o u g h o u t a l l t i m e p e r i o d s examined. T a b l e r e c o r d s t i m e c o u r s e o f v i r a l i n f e c t i o n t h r o u g h o u t t h e b r a i n . v i i L i s t o f F i g u r e s F i g . l (Page 23) HSV1 i m m u n o r e a c t i v i t y i n t h e b r a i n s t e m 6 days a f t e r i n o c u l a t i o n . F i g . 2 (Page 24) HSV1 i m m u n o r e a c t i v i t y i n t h e b r a i n s t e m 8 days a f t e r i n o c u l a t i o n . F i g . 3 (Page 2 6) HSV1 immunoreactive axons w i t h i n t h e t r i g e m i n a l n e r v e . F i g . 4 (Page 27) HSV1 immunoreactive neuron w i t h i n t h e t r i g e m i n a l n u c l e u s . F i g . 5 (Page 28) HSV1 immunoreactive s m a l l , round c e l l s w i t h i n t h e t r i g e m i n a l n u c l e u s . F i g . 6 (Page 29) HSV1 immunoreactive G o l g i neuron w i t h i n t h e g r a n u l a r c e l l l a y e r o f t h e c e r e b e l l a r c o r t e x . F i g . 7 (Page 31) HSV1 immunoreactive f o c u s w i t h i n t h e v e n t r o p o s t e r i o r n u c l e u s o f t h e thalamus ( p a r v o c e l l u l a r r e g i o n ) . F i g . 8 (Page 32) HSV1 immunoreactive neurons w i t h i n t h e p a r a v e n t r i c u l a r h y p o t h a l a m i c n u c l e u s . F i g . 9 (Page 34) HSV1 immunoreactive p y r a m i d a l l y shaped neurons w i t h i n t h e f r o n t o p a r i e t a l c o r t e x . F i g . 1 0 (Page 35) HSV1 immunoreactive p y r a m i d a l l y shaped neurons w i t h i n t h e p r i m a r y o l f a c t o r y c o r t e x . F i g . 1 1 (Page 37) HSV1 i m m u n o r e a c t i v i t y i n t h e g r a n u l a r c e l l l a y e r o f t h e c e r e b e l l a r c o r t e x 12 days a f t e r i n o c u l a t i o n . v i i i Fig.12 (Page 38) MHC class I iramunoreactivity within the thalamus of a control animal. Fig.13 (Page 40) MHC class I immunoreactive microglia c e l l s within the trigeminal nucleus 6 days after inoculation. Fig.14 (Page 41) MHC class I immunoreactive small, round c e l l s within the trigeminal nucleus. Fig.15 (Page 43) MHC class I immunoreactive microglia c e l l s within the in t e r n a l capsule. Fig.16 (Page 44) MHC class I immunoreactive microglia c e l l s within the l a t e r a l septal nucleus. Fig.17 (Page 46) MHC class I immunoreactive focus of microglia c e l l s within the MGN of the thalamus. Fig.18 (Page 48) MHC class II immunoreactive microglia c e l l s within the trigeminal nucleus 6 days after inoculation. Fig.19 (Page 50) MHC class II immunoreactive focus of microglia c e l l s within the deep mesencephalic nucleus. Fig.20 (Page 52) GFAP immunoreactive astrocytes within the trigeminal nucleus 3 days after inoculation. Fig.21 (Page 54) GFAP immunoreactive astrocytes within the trigeminal nucleus 6 days aft e r inoculation. Fig.22 (Page 55) Cresyl v i o l e t s t a i n demonstrating swollen as t r o c y t i c nuclei within the trigeminal nucleus. Fig.23 (Page 56) Necrotic area within the cerebral peduncle immunostained with GFAP. i x F i g . 2 4 (Page 58) GFAP/MHC I I double i m m u n o s t a i n i n g d e m o n s t r a t i n g two d i s t i n c t c e l l groups w i t h i n t h e thalamus. F i g . 2 5 (Pages 60-61) E f f e r e n t p r o j e c t i o n s from t h e t r i g e m i n a l n u c l e u s . X Acknowledgement I would l i k e t o thank D r s . E d i t h and Pat McGeer f o r t h e i r i n f i n i t e a s s i s t a n c e and s u p p o r t i n c o m p l e t i n g t h i s t h e s i s . I would a l s o l i k e t o thank Dr. John O'Kusky f o r h i s c r i t i c a l comments and i n v a l u a b l e c o n t r i b u t i o n s t o t h e p r e p a r a t i o n o f t h i s m a n u s c r i p t . I am e s p e c i a l l y g r a t e f u l t o Dr. Doug Walker f o r p r e p a r a t i o n o f t h e v i r u s as w e l l as f o r h i s c o n t i n u e d a s s i s t a n c e and d i r e c t i o n t h r o u g h o u t t h e p r o j e c t . I would a l s o l i k e t o thank Dr. Haru Akiyama and Joanne Sunahara f o r t h e i r generous t i m e and e x p e r t i s e i n t h e f i e l d o f i m m u n o h i s t o c h e m i s t r y . F i n a l l y , t h e c o m p l e t i o n o f t h i s t h e s i s would not have been p o s s i b l e w i t h o u t t h e c e a s e l e s s s u p p o r t and l o v e from my p a r e n t s D a v i d and A n i t a , and my s i s t e r s L i s a and L i n d a . 1 INTRODUCTION Acute v i r a l i n f e c t i o n s o f t h e c e n t r a l nervous system (CNS) are r e s p o n s i b l e f o r a number o f n e u r o l o g i c a l d i s o r d e r s , such as e n c e p h a l i t i s , m e n i n g i t i s , p o l i o m y e l i t i s , and d e m y e l i n a t i n g d i s e a s e s (Johnson, 1982, K r i s t e n s s o n e t a l . , 1983). Some v i r u s e s , such as t h e h e r p e s v i r u s e s , have t h e c a p a c i t y t o produce l a t e n t i n f e c t i o n w i t h t h e p o s s i b i l i t y o f r e a c t i v a t i o n and subsequent i n f e c t i o n . R e a c t i v a t i o n o f l a t e n t v i r u s has been i m p l i c a t e d i n v a r i o u s n e u r o l o g i c a l and p s y c h i a t r i c d i s o r d e r s , such as A l z h e i m e r ' s d i s e a s e , s c h i z o p h r e n i a , and m u l t i p l e s c l e r o s i s ( K u r s t a k e t a l . , 1987, F l e m i n g e t a l . , 1983). D e s p i t e t h e s e v e r i t y o f t h e s e i n f e c t i o u s d i s o r d e r s , r e l a t i v e l y l i t t l e i s known o f t h e mechanisms o f t h e immune response t o v i r u s e s i n t h e CNS. V i r a l c a p s i d s and e n v e l o p e s c o n t a i n m u l t i p l e v i r u s - c o d e d ( a n t i g e n i c ) p r o t e i n s which a r e T c e l l dependent (Johnson, 1982). The s u r f a c e g l y c o p r o t e i n s o f a v i r u s w i l l a c t i v a t e T - h e l p e r c e l l s w hich w i l l i n t u r n s t i m u l a t e B c e l l a n t i b o d y p r o d u c t i o n . P r o d u c t i o n o f n e u t r a l i z i n g a n t i b o d y t o most v i r u s e s , i n c l u d i n g herpes s i m p l e x v i r u s , i s T - c e l l dependent. P h a g o c y t o s i s and p r o c e s s i n g o f b o t h s u r f a c e and i n t e r n a l v i r u s - s p e c i f i c p r o t e i n s w i l l r e s u l t i n t h e s y n t h e s i s of a n t i b o d i e s t o a l l o f t h e s e components. D u r i n g i n f e c t i o n , a n t i b o d y can be d i r e c t e d a g a i n s t b o t h c i r c u l a t i n g v i r a l p a r t i c l e s i n t h e e x t r a c e l l u l a r f l u i d o r plasma and v i r u s - c o d e d p r o t e i n s w i t h i n t h e h o s t - c e l l membrane. C i r c u l a t i n g v i r i o n s a re i n a c t i v a t e d o r n e u t r a l i z e d when a n t i b o d y 2 a t t a c h e s t o them and forms an immune complex. However, not a l l c i r c u l a t i n g v i r u s i s n e u t r a l i z e d and a s m a l l amount remains a c t i v e . A n t i b o d y n e u t r a l i z a t i o n i s c l e a r l y more e f f e c t i v e i n c l e a r i n g e x t r a c e l l u l a r v i r u s t h a n v i r u s which t r a v e l s i n t r a c e l l u l a r l y . Most a c u t e v i r a l i n f e c t i o n s a re a s s o c i a t e d w i t h an i n i t i a l i n f l a m m a t o r y response c h a r a c t e r i z e d by an i n f l u x o f p o l y m o r p h o n u c l e a r and mononuclear c e l l s (Johnson, 1982). The r o l e of t h e s e c e l l s i n c l e a r i n g v i r u s from t h e CNS i s p o o r l y u n d e r s t o o d . The i n f l a m m a t o r y response i s f o l l o w e d by a s p e c i f i c immune response known as c e l l - m e d i a t e d immunity (CMI). CMI i n v o l v e s s p e c i f i c T c e l l s u b s e t s as w e l l as B c e l l s and macrophages which work t o g e t h e r t o k i l l a v i r a l l y i n f e c t e d h o s t -c e l l . CMI i s d i r e c t e d a g a i n s t e i t h e r t h e v i r u s - c o d e d p r o t e i n i n c o r p o r a t e d i n t o t h e c y t o p l a s m i c membrane o r a c e l l c o n t a i n i n g an i n t e g r a t e d v i r a l genome e x p r e s s i n g c e l l s u r f a c e v i r u s - c o d e d a n t i g e n s . T - h e l p e r c e l l s t i m u l a t e d a n t i b o d y p r o d u c t i o n a i d s i n h o s t - c e l l d e s t r u c t i o n v i a antibody-dependent c e l l u l a r c y t o t o x i c i t y (ADCC). ADCC i s mediated t h r o u g h k i l l e r and p o l y m o r p h o n u c l e a r c e l l s as w e l l as macrophage p h a g o c y t o s i s . L i t t l e i s known about v i r a l c l e a r a n c e from t h e CNS o t h e r t h a n t h a t i t appears t o be h i g h l y dependent on immune responses ( G r i f f i n e t a l . , 1977). V i r u s appears t o be ph a g o c y t o s e d by mesenchymally d e r i v e d endogenous m i c r o g l i a l c e l l s (Johnson, 1982). That T c e l l r e s ponses may be more i m p o r t a n t t h a n B c e l l r e s p onses i n c l e a r i n g v i r u s i s i n d i c a t e d by f i n d i n g s on n a t u r a l i m m u n o d e f i c i e n c y s t a t e s i n man. P e o p l e w i t h CMI d e f e c t s may 3 d e v e l o p a CNS i n f e c t i o n t o a v i r u s which n o r m a l l y does not cause d i s e a s e (Johnson, 1982). R e c e n t l y , o t h e r f a c t o r s i n v o l v e d i n v i r a l c l e a r a n c e from t h e CNS have been uncovered. One i m p o r t a n t f a c t o r i n v o l v e s m o l e c u l e s o f t h e Major H i s t o c o m p a t i b i l i t y Complex (MHC). MHC m o l e c u l e s a r e known t o p l a y an i m p o r t a n t r o l e i n T c e l l - m e d i a t e d immune resp o n s e s ( S t i t e s e t a l . , 1987). The m o l e c u l e s which make up t h e MHC c o n s i s t o f c l a s s I a n t i g e n s (H-2 i n r o d e n t s , HLA-A,B,C i n humans) and c l a s s I I a n t i g e n s ( l a i n r o d e n t s , HLA-DR i n humans). C l a s s I a n t i g e n s a re p r e s e n t on most n u c l e a t e d c e l l s w h i l e c l a s s I I a n t i g e n s a re l a r g e l y r e s t r i c t e d t o immunocompetent c e l l t y p e s such as macrophages, B - c e l l s , and some T - c e l l s ( S t i t e s e t a l . , 1987). These c e l l s u r f a c e a n t i g e n s p l a y an i m p o r t a n t r o l e i n s e l f - r e c o g n i t i o n and a n t i g e n p r e s e n t a t i o n d u r i n g T c e l l - m e d i a t e d immune r e s p o n s e s . MHC m o l e c u l e s a re n e c e s s a r y f o r t h e i n i t i a t i o n o f CMI. CMI i n v o l v e s c l o n i n g o f s p e c i f i c T - h e l p e r c e l l s , s p e c i f i c T - c y t o t o x i c c e l l s , and s p e c i f i c a n t i b o d y p r o d u c i n g B c e l l s which f u n c t i o n t o g e t h e r t o k i l l v i r a l l y i n f e c t e d c e l l s . A l t h o u g h t h e importance o f MHC a n t i g e n s i n immune r e a c t i v i t y i s g e n e r a l l y a c c e p t e d , a few. s t u d i e s have c o n c l u d e d t h a t MHC m o l e c u l e s a r e l a r g e l y l a c k i n g i n CNS t i s s u e . I n a r e c e n t r e v i e w a r t i c l e (Lampson e t a l . , 1987), t h e d i s t r i b u t i o n o f c l a s s I and c l a s s I I immunohistochemical s t a i n i n g i n b o t h normal and p a t h o l o g i c a l t i s s u e (animal and human) was d e s c r i b e d . C l a s s I s t a i n i n g was d e t e c t e d on b l o o d v e s s e l w a l l s o f normal a n i m a l and human t i s s u e . Neurons, g l i a , and o t h e r parenchymal c e l l s were n e g a t i v e . I n s i m i l a r t i s s u e , r a r e c l a s s I I p o s i t i v e c e l l s and 4 v e s s e l w a l l s were seen, but most n e u r a l elements were n e g a t i v e . P a t h o l o g i c a l human t i s s u e ( l a r g e l y p h y s i c a l trauma) showed no i n c r e a s e i n c l a s s I e x p r e s s i o n . C l a s s I I r e a c t i v i t y was s l i g h t l y i n c r e a s e d i n m a l i g n a n c i e s and f o l l o w i n g v i r a l i n f e c t i o n . The a u t h o r c o n c l u d e d t h a t MHC e x p r e s s i o n i n b o t h normal and p a t h o l o g i c a l b r a i n t i s s u e was r a r e , s u g g e s t i n g l i t t l e o r no MHC a c t i v i t y i n t h e CNS. In c o n t r a s t t o t h i s c o n c l u s i o n , however, r e c e n t work i n t h e a r e a o f MHC e x p r e s s i o n s u g g e s t s an a c t i v e r o l e f o r MHC m o l e c u l e s i n CNS immunity. Both i n v i t r o and i n v i v o s t u d i e s p r o v i d e e v i d e n c e i n su p p o r t o f t h i s h y p o t h e s i s . Model Systems Under a p p r o p r i a t e c o n d i t i o n s , v a r i o u s c e l l t y p e s can be i n d u c e d t o e x p r e s s MHC a n t i g e n s on t h e i r s u r f a c e . C l a s s I e x p r e s s i o n can be i n d u c e d on a l l c e l l t y p e s i n t h e CNS i n c l u d i n g n e u r o b l a s t o m a and ependymoblastoma l i n e s f o l l o w i n g i n c u b a t i o n w i t h e i t h e r gamma-interferon (IFN-g) o r i n t e r l e u k i n - 2 (IL-2) (DuBois e t a l . , 1985, Lampson e t a l . , 1983, 1984, Male e t a l . , 1987, Suzumura e t a l . , 1986, 1987, T i n g e t a l . , 1987, Wong e t a l . , 1984, 1985). C l a s s I I e x p r e s s i o n has been demonstrated l a r g e l y on c u l t u r e d a s t r o c y t e s and b r a i n e n d o t h e l i u m (DuBois e t a l . , 1985, F i e r z e t a l . , 1985, H i r s c h e t a l . , 1983, Male e t a l . , 1987, Wong e t a l . , 1984). C u l t u r e d a s t r o c y t e s w i l l e x p r e s s c l a s s I I a n t i g e n s f o l l o w i n g i n c u b a t i o n w i t h IFN-g (Barna e t a l . , 1987, Male e t a l . , 1987), a c t i v a t e d T - c e l l s (Fontana e t a l . , 1984), o r c e r t a i n v i r u s e s (Massa e t a l . , 1986, 1987). B r a i n e n d o t h e l i u m w i l l e x p r e s s l a a n t i g e n s f o l l o w i n g i n c u b a t i o n w i t h IFN-g, p h y t o h e m a g g l u t i n i n (PHA) or c o n c a n a v a l i n A (conA) (Male e t a l . , 5 1987, W i l c o x e t a l . , 1987). F u r t h e r m o r e , c u l t u r e d a s t r o c y t e s , macrophages, and m i c r o g l i a can be s t i m u l a t e d t o produce i n t e r l e u k i n - 1 ( I L - 1 ) , a lymphokine n e c e s s a r y f o r CMI, under a p p r o p r i a t e c o n d i t i o n s ( A r t u r s s o n e t a l . , 1987, G i u l i a n e t a l . , 1986, M e r r i l l , 1987). Normal T i s s u e A n i m a l The h i g h e s t c o n c e n t r a t i o n o f c l a s s I e x p r e s s i o n i n normal r a t CNS t i s s u e was found on v a s c u l a r e n d o t h e l i a l c e l l s and on a few s m a l l g l i a l c e l l s (Akiyama e t a l . , 1988, Matsumoto e t a l . , 1987, Whelan e t a l . , 1986). C l a s s I I e x p r e s s i o n was r a r e , but has been found on a s m a l l number of m i c r o g l i a , mononuclear c e l l s , o l i g o d e n d r o c y t e s , and i n c h o r i o d t i s s u e (Akiyama e t a l . , 1988, Matsumoto e t a l . , 1986, T i n g e t a l . , 1981, Wang e t a l . , 1987) . I n a d d i t i o n , T - h e l p e r lymphocytes (CD4+) have been found i n s m a l l numbers i n mouse, r a t , and monkey b r a i n ( H i l l e t a l . , 1986, N e e l e y e t a l . , 1987, P e r r y e t a l . , 1987) w i t h I L -1 b i n d i n g n o t e d i n t h e r a t ( F a r r a r e t a l . , 1987). Human In normal human b r a i n t i s s u e , c l a s s I e x p r e s s i o n was l a r g e l y c o n f i n e d t o b l o o d v e s s e l w a l l s and a few parenchymal c e l l s . Neurons and g l i a were n e g a t i v e (Lampson e t a l . , 1986, S o b e l e t a l . , 1988). A l t h o u g h c l a s s I I e x p r e s s i o n was l a r g e l y absent from normal t i s s u e , a few HLA-DR p o s i t i v e a s t r o c y t e s , m i c r o g l i a , and e n d o t h e l i a l c e l l s were found (De T r i b o l e t e t a l . , 1984, Hauser e t a l . , 1983, Hayes e t a l . , 1987, Hofman e t a l . , 1986, Lampson e t a l . , 1986, McGeer e t a l . , 1987a, 1987b, 1988, N a t a l i e t a l . , 1981, S o b e l e t a l . , 1988). S m a l l numbers of T-6 h e l p e r c e l l s have been found i n t h e thalamus, c e r e b e l l u m , and pons ( H i l l e t a l . , 1986). P a t h o l o g i c a l S t a t e s A n i m a l A n i m a l models e m p l o y i n g a v a r i e t y o f i n s u l t s t o t h e CNS have demonstrated enhanced MHC e x p r e s s i o n i n b r a i n t i s s u e . MHC e x p r e s s i o n appears t o f o l l o w a f a i r l y p r e d i c t a b l e t i m e c o u r s e . 1) E x p e r i m e n t a l A l l e r g i c E n c e p h a l o m y e l i t i s (EAE) EAE i s an i m m u n o l o g i c a l l y - m e d i a t e d d e m y e l i n a t i n g d i s o r d e r o f t e n used i n s t u d y i n g MHC e x p r e s s i o n . C u r r e n t l y , i t i s c o n s i d e r e d t h e b e s t a n i m a l model f o r m u l t i p l e s c l e r o s i s (MS). S p i n a l c o r d homogenate from a l l o g e n i c r o d e n t s i s i n j e c t e d s u b c u t a n e o u s l y i n t o h o s t r a t s p r o d u c i n g r e l i a b l e motor d e f i c i t s as w e l l as h i s t o l o g i c a l e v i d e n c e o f d e m y e l i n a t i o n . The d i s e a s e o f t e n e x h i b i t s r e l a p s i n g and r e m i t t i n g phases. A l a r g e number o f EAE s t u d i e s have r e p o r t e d s i m i l a r f i n d i n g s . F o l l o w i n g t h e i n d u c t i o n o f EAE i n r a t s , c l a s s I s t a i n i n g was found l a r g e l y on d e n d r i t i c and e n d o t h e l i a l c e l l s w h i l e c l a s s I I s t a i n i n g was seen i n c e l l s q u e s t i o n a b l y i d e n t i f i e d as d e n d r i t i c c e l l s , a s t r o c y t e s (Matsumoto e t a l . , 1986a, 1986b, S a k a i e t . a l . , 1986), macrophages (Craggs e t a l . , 1985, S o b e l e t a l . , 1984, 1987), m i c r o g l i a ( A n t o n i o u e t a l . , 1987, H i c k e y e t a l . , 1988, Sedgwick e t a l . , 1987), and v a s c u l a r e n d o t h e l i a l c e l l s (Craggs e t a l . , 1985, H i c k e y e t a l . , 1985, Sobe l e t a l . , 1984, 1987, W i l c o x e t a l . , 1987). I n a d d i t i o n , T-c e l l s u b s e t s found p r e d o m i n a n t l y i n p e r i v a s c u l a r c u f f s i n c l u d e d T - h e l p e r (CD4+) and T - c y t o t o x i c / s u p p r e s s o r (CD8+) c e l l s ( E l l e r m a n e t a l . , 1988, M e r r i l l , 1987, Sedgwick e t a l . , 1987, Sob e l e t a l . , 1987). B - c e l l s a l s o appeared t o p l a y a r o l e i n EAE ( W i l l e n b e r g e t 7 a l . , 1983). In t h e p r e c e d i n g s t u d i e s , immunohistochemical a n a l y s i s o f CNS t i s s u e was performed u s i n g monoclonal a n t i b o d i e s a g a i n s t MHC a n t i g e n s , T - c e l l s ubset d e t e r m i n a n t s , m i c r o g l i a , monocyte/macrophages, and g l i a l f i b r i l l a r y a c i d i c p r o t e i n (GFAP) f o r a s t r o c y t e s . A l t h o u g h c l a s s I I e x p r e s s i o n on a s t r o c y t e s was r e p o r t e d , most s t u d i e s have found a s t r o c y t e s t o be n e g a t i v e f o r MHC g l y c o p r o t e i n s . The t i m e c o u r s e f o r t h e appearance o f MHC a n t i g e n s f o l l o w i n g t h e i n d u c t i o n o f EAE was as f o l l o w s . MHC a n t i g e n s appeared w i t h i n t h e f i r s t week o f t h e a c u t e phase and peaked a t a p p r o x i m a t e l y 10-18 days. E x p r e s s i o n began t o de c r e a s e by 1.5-2 months or d u r i n g t h e r e m i s s i o n phase. MHC e x p r e s s i o n was r a r e d u r i n g r e m i s s i o n (Antoniou e t a l . , 1987, Craggs e t a l . , 1985, S a k a i e t a l . , 1986, Sedgwick e t a l . , 1987). The r e s u l t s from t h e s e s t u d i e s demonstrate b o t h enhanced MHC e x p r e s s i o n and lymphocyte i n f i l t r a t i o n d u r i n g t h e a c u t e phase o f a d e m y e l i n a t i n g CNS d i s e a s e , s u g g e s t i n g t h a t MHC a n t i g e n s p l a y a s i g n i f i c a n t r o l e i n a n t i g e n p r e s e n t a t i o n and T - c e l l - m e d i a t e d immunity i n t h e r a t b r a i n . 2) K a i n i c A c i d (KA) L e s i o n s I n a r e c e n t s t u d y , (Akiyama e t a l . / 1988), e p i d u r a l a p p l i c a t i o n o f KA t o t h e c e r e b r a l c o r t e x was used t o examine MHC e x p r e s s i o n . T h i s p r o c e d u r e causes c o n s i d e r a b l e n e u r o n a l damage t o t h e u n d e r l y i n g t i s s u e , r e s u l t i n g i n f o c a l a r e a s o f enhanced MHC e x p r e s s i o n . Immunohistochemical a n a l y s i s was c a r r i e d out w i t h monoclonal a n t i b o d i e s a g a i n s t r a t MHC a n t i g e n s and l e u k o c y t e common a n t i g e n s (LCA). P o l y c l o n a l a n t i b o d y a g a i n s t GFAP was used t o d e t e c t a s t r o c y t e s . C e l l s 8 s t a i n i n g p o s i t i v e l y f o r c l a s s I i n c l u d e d round c e l l s ( p o s s i b l y macrophages) and m i c r o g l i a . Round c e l l s , "ameboid" c e l l s , and m i c r o g l i a were p o s i t i v e f o r c l a s s I I , w i t h b o t h round c e l l s and m i c r o g l i a a l s o p o s i t i v e f o r LCA. Neurons d i d not e x p r e s s d e t e c t a b l e l e v e l s o f c l a s s I , c l a s s I I , or LCA a t any t i m e . Double immunohistochemical s t a i n i n g r e v e a l e d most LCA p o s i t i v e c e l l s t o be c l a s s I p o s i t i v e w i t h o n l y a few c e l l s d o u b l y s t a i n e d f o r b o t h LCA and c l a s s I I . In a d d i t i o n , most c l a s s I I c e l l s s t a i n e d f o r c l a s s I , but t h e r e v e r s e was not t r u e . A s t r o c y t e s (GFAP+) were n e g a t i v e f o r b o t h c l a s s I and I I . The t i m e c o u r s e f o r MHC e x p r e s s i o n i n KA l e s i o n i n g was s i m i l a r t o t h a t seen i n EAE. MHC a n t i g e n s appeared w i t h i n t h e f i r s t week f o l l o w i n g KA a p p l i c a t i o n , peaked s h o r t l y a f t e r w a r d s , and d e c l i n e d o v er t h e f o l l o w i n g 16-20 weeks. C l a s s I I e x p r e s s i o n d e c l i n e d somewhat more r a p i d l y t h a n c l a s s I e x p r e s s i o n . As i n t h e EAE s t u d i e s , t h e s e r e s u l t s suggest MHC i n v o l v e m e n t i n t h e r a t CNS, as w e l l as a r o l e f o r m i c r o g l i a i n a n t i g e n p r e s e n t a t i o n . A s t r o c y t e s (GFAP+) d i d not d o u b l y s t a i n f o r c l a s s I I . 3) T r a n s p l a n t / G r a f t v s . Host CNS t i s s u e t r a n s p l a n t s , u n l i k e p e r i p h e r a l t r a n s p l a n t s , do not appear t o r e s u l t i n t o t a l t i s s u e r e j e c t i o n (Medawar, 1948). T h i s i s o f t e n used as e v i d e n c e f o r i m m u n o l o g i c a l p r i v i l e g e i n t h e CNS. However, a few s t u d i e s have demonstrated enhanced MHC ' e x p r e s s i o n , as w e l l as l y m p h o c y t i c i n f i l t r a t i o n , f o l l o w i n g t r a n s p l a n t a t i o n o f b r a i n t i s s u e i n t o t h e CNS. In one s t u d y , d e t e c t a b l e l e v e l s o f MHC e x p r e s s i o n were seen i n t h e b r a i n f o l l o w i n g t r a n s p l a n t a t i o n o f f o e t a l n e o c o r t i c a l 9 t i s s u e i n t o t h e l a t e r a l v e n t r i c l e o f a l l o g e n i c mice ( N i c h o l a s e t a l . / 1987). C l a s s I I p o s i t i v e e n d o t h e l i a l and parenchymal c e l l s , b o t h major T - c e l l s u b s e t s and macrophages were found. H i c k e y and Kimura (1987) , demonstrated a g r a f t v e r s u s h o s t immune response i n t h e r a t CNS which r e s u l t e d i n t h e appearance o f c l a s s I p o s i t i v e v a s c u l a r e n d o t h e l i a l c e l l s as w e l l as c l a s s I I p o s i t i v e e n d o t h e l i a l , parenchymal, and m i c r o g l i a c e l l s . I n a n o t h e r e x p e r i m e n t , b r a i n s t e m p i e c e s g r a f t e d i n t o t h e c e r e b e l l u m o f a l l o g e n i c mice r e s u l t e d i n t h e appearance o f b o t h T - h e l p e r and T-c y t o t o x i c / s u p p r e s s o r lymphocytes (Date e t . a l . , 1988). The above s t u d i e s employed immunohistochemical t e c h n i q u e s u s i n g monoclonal a n t i b o d i e s a g a i n s t mouse MHC a n t i g e n s , T - c e l l s u b s e t d e t e r m i n a n t s , and macrophages. A l t h o u g h CNS t i s s u e t r a n s p l a n t s do not r e s u l t i n a v i g o r o u s immune response, t h e r e s u l t s o f t h e s e t r a n s p l a n t s t u d i e s suggest t h a t some immune r e a c t i v i t y may s t i l l o c c u r . The reas o n s b e h i n d f a i l u r e o f a r o b u s t immune response and t r a n s p l a n t r e j e c t i o n f o l l o w i n g t i s s u e t r a n s p l a n t i n t h e CNS c u r r e n t l y remain u n c l e a r . Human MHC e x p r e s s i o n i n t h e CNS has been r e p o r t e d i n a few s t u d i e s e x a m i n i n g human p a t h o l o g i c a l t i s s u e . A number o f d i f f e r e n t c e l l t y p e s have been r e p o r t e d t o e x p r e s s MHC a n t i g e n s i n m u l t i p l e s c l e r o s i s (MS) p l a q u e s . C l a s s I p o s i t i v e a s t r o c y t e s have been found i n a c t i v e MS l e s i o n s ( T r a u g o t t , 1987), as w e l l as c l a s s I I p o s i t i v e m i c r o g l i a (Hayes e t a l . , 1987, Woodroofe e t a l . , 1986), macrophages ( E s i r i , 1987, T r a u g o t t e t a l . , 1984), a s t r o c y t e s (Hofman e t a l . , 1986, T r a u g o t t e t a l . , 1985, 1987), and e n d o t h e l i a l c e l l s ( T r a u g o t t e t a l . , 1985). Post-mortem b r a i n 10 t i s s u e from p a t i e n t s d i a g n o s e d w i t h P a r k i n s o n ' s o r A l z h e i m e r ' s d i s e a s e r e v e a l e d c l a s s I and I I p o s i t i v e m i c r o g l i a as w e l l as T-c e l l s . ( I t a g a k i e t a l . , 1987, McGeer e t a l . , 1987a, 1987b, 1988, Rogers e t a l . , 1988). A s t r o c y t e s d i d not show c l a s s I I s t a i n i n g . Tumor and g l i o m a b r a i n t i s s u e was examined f o r MHC e x p r e s s i o n . V e s s e l s s t a i n e d p o s i t i v e l y f o r c l a s s I w h i l e c l a s s I I r e a c t i v i t y was v i s u a l i z e d on a s t r o c y t e s , macrophages, and a few m i c r o g l i a -l i k e c e l l s (Bhondeley e t a l . , 1987, F r a n k s e t a l . , 1986, Lampson e t a l . , 1986, R o s s i e t a l . , 1987). A l t h o u g h a d d i t i o n a l p a t h o l o g i e s such as v i r a l i n f e c t i o n (Abbott e t a l . , 1987, B o u t i n e t a l . , 1987, Goudsmit e t a l . , 1987, M a n u e l i d i s e t a l . , 1987, P e t i t o e t a l . , 1986, R i c h e r t e t a l . , 1987, Zeman, 1978), s y s t e m i c l u p u s erythematosus (Ernerudh e t a l . , 1984), and s c h i z o p h r e n i a (Ahokas, 1986, R o b e r t s e t a l . , 1987, Steven s , 1982) have been examined f o r MHC e x p r e s s i o n , no c o n c l u s i v e e v i d e n c e has been r e p o r t e d a t t h i s t i m e . Purpose o f t h e P r e s e n t Study V i r a l i n f e c t i o n s have o n l y r e c e n t l y been u t i l i z e d t o examine MHC e x p r e s s i o n i n t h e CNS. A r e s e a r c h team i n Sweden r e p o r t e d a marked i n c r e a s e i n b o t h c l a s s I and c l a s s I I a n t i g e n e x p r e s s i o n (immunohistochemical a n a l y s i s ) i n t h e r a t b r a i n f o l l o w i n g t h e i n d u c t i o n o f measles v i r u s e n c e p h a l i t i s (Olsson e t a l . , 1988) . A l t h o u g h s p e c i f i c c e l l t y p e s were not d e s c r i b e d , d i s t r i b u t i o n o f t h e MHC m o l e c u l e s was more w i d e s p r e a d t h a n t h e o c c u r r e n c e o f v i r a l a n t i g e n , s u g g e s t i n g a s o l u b l e f a c t o r f o r t h e i r i n d u c t i o n . In s u r v i v i n g a n i m a l s , T - c e l l s u b s e t s were a l s o n o t e d p e r i v a s c u l a r l y and i n t h e b r a i n parenchyma. 11 The p r e s e n t s t u d y was d e s i g n e d t o d e t e r m i n e MHC e x p r e s s i o n i n t h e r a t CNS f o l l o w i n g c o r n e a l i n o c u l a t i o n w i t h herpes s i m p l e x v i r u s t y p e 1 (HSV-1). T h i s i s t h e f i r s t r e p o r t t o examine MHC e x p r e s s i o n and d e l i n e a t e a d e t a i l e d t i m e c o u r s e o f t h i s a n t i g e n e x p r e s s i o n i n t h e r a t CNS f o l l o w i n g HSV-1 i n f e c t i o n . HSV1 has been used t o produce CNS v i r a l i n f e c t i o n i n a n i m a l s v i a m u l t i p l e r o u t e s o f i n o c u l a t i o n . MHC e x p r e s s i o n , however, has not been c l e a r l y documented. Two s t u d i e s d e s c r i b e d an i n f l a m m a t o r y c e l l i n f i l t r a t i o n i n t h e CNS r e g i o n o f t h e t r i g e m i n a l nerve r o o t e n t r y zone (TREZ) f o l l o w i n g e i t h e r p e r i p h e r a l or snout i n o c u l a t i o n w i t h HSV-1 ( K a s t r u k o f f e t a l . , 1987, K r i s t e n s s o n e t a l . , 1983). The c e l l u l a r i n f i l t r a t e c o n s i s t e d o f lymphocytes, macrophages, and a few plasma c e l l s . In o t h e r l a b o r a t o r i e s , i n t r a c e r e b r a l , i n t r a v e n o u s , and s c i a t i c n e r v e i n o c u l a t i o n as w e l l as hippocampal m i c r o i n j e c t i o n l e d t o l y m p h o c y t i c m e n i n g i t i s w i t h p e r i v a s c u l a r l y m p h o c y t i c c u f f i n g (Anderson e t a l . , 1983, M c F a r l a n d e t a l . , 1986,1987). F o l l o w i n g c o r n e a l HSV-1 i n o c u l a t i o n , b o t h T - c e l l s and a few s c a t t e r e d B-c e l l s were found i n t h e CNS r e g i o n o f t h e TREZ (Townsend, 1985). In one a d d i t i o n a l s t u d y , r a b b i t s i n o c u l a t e d i n t r a t h e c a l l y o r v i a c o r n e a l s c a r i f i c a t i o n d e v e l o p e d CSF mononuclear p l e o c y t o s i s d u r i n g t h e a c u t e phase of i n f e c t i o n , f o l l o w e d by e l e v a t e d CSF i m m u noglobulin l e v e l s (IgG, IgA) d u r i n g t h e r e c o v e r y phase ( L a s k i n e t a l . , 1987). B e h a v i o r a l l y , t h e a c u t e e n c e p h a l i t i s was c h a r a c t e r i z e d by h y p o a c t i v i t y , d i m i n i s h e d f o o d and water i n t a k e , and hunched p o s t u r e , most o f t e n f o l l o w e d by d e a t h . A l t h o u g h l i t t l e work has been done t o date on MHC e x p r e s s i o n i n t h e CNS 12 f o l l o w i n g an HSV1 v i r a l i n f e c t i o n , i t i s an a r e a which s h o u l d p r o v i d e a c o n s i d e r a b l e c o n t r i b u t i o n t o t h e u n d e r s t a n d i n g o f CNS immunity. Herpes s i m p l e x i s f r e q u e n t l y a s s o c i a t e d w i t h human CNS d i s e a s e . I n i t i a l l y t h e cause o f a m i l d d i s o r d e r such as c o l d s o r e s , HSV i n f e c t i o n may become more s e r i o u s when t h e CNS i s i n v a d e d . HSV1 i s t h e cause o f a m a j o r i t y o f cases o f s p o r a d i c a l l y o c c u r r i n g a c u t e n e c r o t i z i n g e n c e p h a l i t i s i n t h e Western w o r l d w i t h an e s t i m a t e d 4,000 cases y e a r l y i n t h e U n i t e d S t a t e s (Viken e t a l . , 1978). A l t h o u g h o t h e r v i r u s e s more commonly produce an e n c e p h a l i t i c i l l n e s s , m o r b i d i t y and m o r t a l i t y r e s u l t i n g from herpes s i m p l e x e n c e p h a l i t i s exceeds t h a t o f o t h e r i n f e c t i o n s . The mechanism r e s p o n s i b l e f o r development o f t h e e n c e p h a l i t i s remains p o o r l y u n d e r s t o o d . C l i n i c a l l y , an i n d i v i d u a l s u f f e r i n g from herpes e n c e p h a l i t i s p r e s e n t s w i t h headaches and s e i z u r e s . These symptoms are f r e q u e n t l y a s s o c i a t e d w i t h p s y c h i a t r i c d i s t u r b a n c e s and s e l e c t i v e memory impairment (Viken e t a l . , 1978). The c l i n i c a l c o u r s e i s c h a r a c t e r i z e d by r a p i d changes over a p e r i o d o f a few days, r e s u l t i n g i n s t u p o r which o f t e n p r o g r e s s e s t o coma. Most cases o f HSV e n c e p h a l i t i s r e s u l t i n e i t h e r d e a t h , o r r e s o l u t i o n w i t h v a r i a b l e degrees o f i n t e l l e c t u a l , memory, or e m o t i o n a l d e f i c i t s . Few i n d i v i d u a l s r e c o v e r f u l l y . P a t h o l o g i c a l l y , l e s i o n s a re u s u a l l y l i m i t e d t o t h e s u b f r o n t a l and m e d i a l t e m p o r a l r e g i o n s o f t h e b r a i n (Viken e t a l , 1978). Hemorrhagic l e s i o n s and c o n g e s t i o n o f s m a l l v e s s e l s a re o f t e n g r o s s l y v i s i b l e . The i n f e c t i o n produces a n e c r o t i z i n g 13 p r o c e s s i n which neurons, o l i g o d e n d r o c y t e s , and a s t r o c y t e s a re a f f e c t e d . I n c l u s i o n s have been n o t e d i n a l l t h r e e c e l l t y p e s , but i n f e c t i o n o f e n d o t h e l i a l c e l l s has not been i d e n t i f i e d . P o s t -mortem t i s s u e from s u r v i v i n g i n d i v i d u a l s r e v e a l s c y s t i c n e c r o s i s o f t h e b r a i n . L a t e n t v i r u s p e r s i s t s i n t h e n a t u r a l h o s t i n a non-i n f e c t i o u s form and may remain l a t e n t f o r y e a r s (Johnson, 1982). The p r e c i s e n a t u r e and i n t r a c e l l u l a r s i t e o f t h e l a t e n t s t a g e a re unknown, but t h e v i r u s i s thought t o be s e q u e s t e r e d i n neurons o f s e n s o r y g a n g l i a (Stevens, 1978). R e a c t i v a t i o n may f o l l o w f e b r i l e i l l n e s s , s t r e s s , X-ray i r r a d i a t i o n , o r a v a r i e t y o f o t h e r non-s p e c i f i c e v e n t s , but t h e mechanisms i n v o l v e d i n r e a c t i v a t i o n a r e p o o r l y u n d e r s t o o d . S i n c e r e a c t i v a t i o n can o c c u r i n t h e p r e s e n c e of a n t i b o d y , c e l l - m e d i a t e d immune responses have been i m p l i c a t e d (Stevens, 1978). R e a c t i v a t i o n o f l a t e n t v i r u s i n aged i n d i v i d u a l s w i t h a compromised immune system has been h y p o t h e s i z e d as i m p l i c a t e d i n t h e e t i o l o g y o f c e r t a i n d e g e n e r a t i v e CNS d i s o r d e r s , such as P a r k i n s o n ' s and A l z h e i m e r ' s d i s e a s e ( K u r s t a k e t a l . , 1987, R y t e l , 1987) . P a t h o g e n e s i s o f HSV1 I n f e c t i o n s o f t h e CNS HSV a d s o r p t i o n t o a s t r o c y t e s and synaptosomes i s thought t o oc c u r t h r o u g h HSV t y p e - s p e c i f i c r e c e p t o r s (Johnson, 1982, Lycke e t a l . , 1984, Vahlne e t a l . , 1978). R e c e p t o r mediated attachment i s p r o b a b l y e s s e n t i a l f o r n e u r i t i c uptake o f v i r u s which i s t h e n f o l l o w e d by f u s i o n o f t h e v i r a l e nvelope w i t h t h e h o s t - c e l l membrane. A complex s e r i e s o f e v e n t s f o l l o w s , r e s u l t i n g i n t h e r e p l i c a t i o n o f t h e v i r u s and t h e e s t a b l i s h m e n t o f l a t e n c y 14 (Fleming e t a l . , 1983, Lycke e t a l . , 1984, Vahlne e t a l . , 1978). Once t h e v i r u s e n t e r s t h e CNS, l y s i s o f i n f e c t e d c e l l s appears t o be i m p o r t a n t i n a l l o w i n g e n v e l o p e d v i r u s t o e n t e r t h e e x t r a c e l l u l a r space where a d s o r p t i o n t o g l i a l c e l l s can o c c u r (Townsend, 1985). HSV has been shown t o t r a v e l t r a n s y n a p t i c a l l y i n b o t h a r e t r o g r a d e and a n t e r o g r a d e d i r e c t i o n (Fleming e t a l . , 1983, K u r s t a k e t a l . , 1987, Lycke e t a l . , 1984, U g o l i n i e t a l . , 1989, V a h l n e e t a l . , 1978, V i k e n e t a l . , 1978). I t has been su g g e s t e d t h a t HSV may s e r v e as a t r a c e r because r e p l i c a t i o n o f v i r u s i n t h e r e c i p i e n t neuron f o l l o w i n g t r a n s f e r a m p l i f i e s t h e " t r a c e r s i g n a l " ( U g o l i n i e t a l . , 1989). However, due t o t h e c o n c u r r e n t s p r e a d o f v i r u s w i t h i n g l i a l c e l l s , t h i s t e c h n i q u e i s l i m i t e d . I t i s thought t h a t b o t h t h e c e n t r i p e t a l and c e n t r i f u g a l n e u r a l t r a n s m i s s i o n o f HSV may be i m p o r t a n t i n t h e i n d u c t i o n o f l a t e n c y ( V iken e t a l . , 1978). L i t t l e i s u n d e r s t o o d about t h e p r o c e s s o f l a t e n t i n f e c t i o n o t h e r t h a n i t i s l i k e l y e s t a b l i s h e d i n neurons o f s e n s o r y g a n g l i a where v i r a l i n f o r m a t i o n i s c o n s e r v e d w i t h o u t n e c e s s i t a t i n g p r o d u c t i o n o f i n f e c t i o u s v i r u s ( Fleming e t a l . , 1983, K u r s t a k e t a l . , 1987, V i k e n e t a l . , 1978). HSV i s a p p a r e n t l y c a p a b l e o f i n f e c t i n g t h e f u l l range o f CNS c e l l t y p e s i n a number of d i f f e r e n t s p e c i e s (Viken e t a l . , 1978). The p r o d u c t i v e i n f e c t i o n i s c h a r a c t e r i z e d by an a l t e r a t i o n i n t h e n u c l e u s c o n s i s t i n g o f d i s p e r s i o n o f c h r o m a t i n t o t h e n u c l e a r margin and development o f e o s i n o p h i l i c i n c l u s i o n s w i t h i n t h e n u c l e u s . Some i n f e c t e d c e l l s may not show h i s t o l o g i c a l l y apparent 15 changes w h i l e o t h e r c e l l s may undergo d e g e n e r a t i o n even though t h e y may not be d i r e c t l y i n f e c t e d by t h e v i r u s . C o r n e a l i n o c u l a t i o n produces a r e l a t i v e l y s l ow and r e l i a b l e CNS i n f e c t i o n ( F l eming e t a l . , 1983, Tomlinson e t a l . , 1983, Townsend, 1985). I n t r a c e r e b r a l i n o c u l a t i o n , which i s commonly used t o model human HSV i n f e c t i o n , u s u a l l y r e s u l t s i n r a p i d d e a t h (1-6 days) (McFarland e t a l . , 1986). I n f e c t i o n i n i t i a t e d i n t h e co r n e a a l l o w s t h e s p r e a d o f v i r u s t o o c c u r r e l a t i v e l y more s l o w l y , t h e r e f o r e p r o v i d i n g an i n c r e a s e i n t h e t i m e span o f i n f e c t i o n (1-2 weeks). I n a d d i t i o n , more a n i m a l s are l i k e l y t o r e c o v e r from t h e e n c e p h a l i t i s f o l l o w i n g c o r n e a l i n o c u l a t i o n . The c o rnea i s i n n e r v a t e d by t h e o p h t h a l m i c b r a n c h o f t h e t r i g e m i n a l n erve and a number o f s t u d i e s have demonstrated a r e p l i c a b l e i n f e c t i o n i n t h e t r i g e m i n a l nerve f o l l o w i n g c o r n e a l i n o c u l a t i o n ( F l eming e t a l . , 1983, Tomlinson e t a l . , 1983, Townsend, 1985). Other i n f e c t e d a r e a s r e p o r t e d were t h e t r i g e m i n a l g a n g l i o n , thalamus, hypothalamus, and c e r e b e l l u m . V i r a l p a r t i c l e s were found i n neurons, o l i g o d e n d r o c y t e s , and a s t r o c y t e s . C o r n e a l i n o c u l a t i o n w i t h HSV1 a p p a r e n t l y r e s u l t s i n s e q u e s t r a t i o n o f v i r u s by t h e t r i g e m i n a l n e r v e , which t r a n s p o r t s t h e v i r u s back t o t h e CNS. Once i n t h e CNS, i n f e c t i o n c o n t i n u e s t o s p r e a d , t h u s e s t a b l i s h i n g a r e l i a b l e v i r a l i n f e c t i o n . P r e d i c t i o n s HSV1 w i l l be employed t o e s t a b l i s h an a c u t e CNS i n f e c t i o n i n the r a t v i a c o r n e a l s c a r i f i c a t i o n . Based on t h e i n f o r m a t i o n g l e a n e d from t h e l i t e r a t u r e , changes i n t h e f o l l o w i n g dependent v a r i a b l e s a re l i k e l y t o ensue. 1 6 1) U s i n g a n t i b o d i e s a g a i n s t HSV1, we are l i k e l y t o see i n f e c t e d neurons i n i t i a l l y i n t h e t r i g e m i n a l n u c l e u s . G l i a l c e l l s a r e a l s o l i k e l y t o be i n f e c t e d and t h e i n f e c t i o n may s p r e a d w i t h t i m e . 2) U s i n g a n t i b o d i e s a g a i n s t MHC c l a s s I and I I a n t i g e n s , c e l l s i n v a r i o u s b r a i n r e g i o n s w i l l p r o b a b l y s t a i n f o r t h e s e a n t i g e n s and i t i s p r o b a b l e t h a t t h e c e l l t y p e ( s ) e x p r e s s i n g t h e s e a n t i g e n s w i l l be n o n - n e u r o n a l . 3) The MHC i m m u n o r e a c t i v i t y i s l i k e l y t o be most i n t e n s e i n a r e a s s t a i n i n g f o r HSV1. 4) B r a i n s e c t i o n s d o u b l y s t a i n e d f o r MHC c l a s s I or I I and g l i a l f i b r i l l a r y a c i d i c p r o t e i n (GFAP) w i l l l i k e l y a i d i n i d e n t i f i c a t i o n o f MHC immunoreactive c e l l s . 5) E x a m i n i n g a number of d i f f e r e n t p o s t - i n f e c t i o n t i m e p e r i o d s w i l l l i k e l y c o n t r i b u t e t o a d e t a i l e d map o f HSV1 s p r e a d i n t h e CNS as w e l l as topography of MHC e x p r e s s i o n . I f MHC i m m u n o r e a c t i v i t y i s found i n t h e CNS, we may i n f e r t h e f o l l o w i n g . MHC m o l e c u l e s are l i k e l y t o be i n t e g r a l l y i n v o l v e d i n t h e p r o c e s s o f v i r a l c l e a r a n c e from t h e CNS. F u r t h e r m o r e , t h e c e l l t y p e s e x p r e s s i n g t h e s e a n t i g e n s are l i k e l y t o p l a y an e s s e n t i a l r o l e i n t h e immune res p o n s e . T h i s i s i m p o r t a n t because so l i t t l e i s known about v i r a l i n f e c t i o n s i n t h e CNS and t h e p r o c e s s e s i n v o l v e d i n t h e immune response f o l l o w i n g v i r a l i n f e c t i o n . I t i s t h e r e f o r e e s s e n t i a l t o examine c l o s e l y MHC e x p r e s s i o n i n t h e CNS f o l l o w i n g HSV1 i n f e c t i o n t o g a i n a b e t t e r u n d e r s t a n d i n g o f i t s r o l e i n CNS immunity. Such u n d e r s t a n d i n g may p o i n t t o improved t h e r a p e u t i c i n t e r v e n t i o n . 17 MATERIALS AND METHODS V i r u s : Herpes s i m p l e x v i r u s t y p e 1 ( s t r a i n F) was o b t a i n e d from t h e American Type C u l t u r e C o l l e c t i o n ( R o c k v i l l e , MD) ATCC VR-733. V i r u s s t o c k s were grown i n human f o r e s k i n d i p l o i d f i b r o b l a s t (HDF) c e l l s . The c e l l s were grown i n Dulbecco's m o d i f i e d E a g l e s Medium (DMEM) w i t h 10% f o e t a l b o v i n e serum (FBS) and 100 u n i t s / m l of P e n i c i l l i n and S t r e p t o m y c i n . The v i r u s was i n i t i a l l y p u r i f i e d by p l a q u e p u r i f i c a t i o n on HDF c e l l s and one p l a q u e was p i c k e d and a m p l i f i e d i n t h e s e c e l l s . The f i n a l s t o c k s were p r e p a r e d by i n f e c t i n g 20 x 100 mm p e t r i d i s h e s o f HDF c e l l s w i t h v i r u s a t a m u l t i p l i c i t y o f i n f e c t i o n o f 0.001. The v i r u s was h a r v e s t e d 24 hours a f t e r t h e c e l l s showed 100% c y t o p a t h i c e f f e c t (4 days p o s t -i n f e c t i o n ) . The s u p e r n a t a n t was h a r v e s t e d i n t o c e n t r i f u g e b o t t l e s and t h e c e l l d e b r i s removed by c e n t r i f u g a t i o n a t 5000 rpm f o r 10 min a t 4°C. The s u p e r n a t a n t was f r o z e n i n a l i q u o t s o f 1 ml and s t o r e d a t -70°C. The v i r u s was t i t e r e d i n HDF c e l l s u t i l i z i n g p l a q u e assay p r o c e d u r e s . S e r i a l d i l u t i o n s o f v i r u s were a p p l i e d t o d u p l i c a t e 60 mm p e t r i d i s h e s o f HDF c e l l s . The v i r u s was absorbed f o r 1 hour. The l i q u i d was t h e n removed and r e p l a c e d by an o v e r l a y o f DMEM w i t h 5% FBS and 0.5% agarose. P l a q u e s were v i s i b l e 2-3 days l a t e r (5.7 x 10 p f u / m l ) . The f i n a l p l a q u e assay of t h e s t o c k was performed on Vero c e l l s (3.3 x 10 p f u / m l ) . V i r u s A p p l i c a t i o n : Male W i s t a r r a t s (250-300 gm) were a n e s t h e t i z e d w i t h e t h e r and t h e r i g h t c o r n e a was r e p e a t e d l y s c r a t c h e d w i t h a s t e r i l e 23.5 gage n e e d l e . A 30 u,l drop o f Herpes Simplex V i r u s Type 1 (PFU 33,000/ml) was p l a c e d on t h e s c a r i f i e d 18 co r n e a and a n i m a l s were r e t u r n e d t o t h e i r cages w i t h ad l i b f o o d and water u n t i l t i m e o f s a c r i f i c e . C o n t r o l a n i m a l s r e c e i v e d c o r n e a l s c r a t c h i n g w i t h o u t a p p l i c a t i o n o f v i r u s . Rats were s a c r i f i c e d a f t e r p e r i o d s o f 3, 6, 8, 10, 12, and 30 days. Two t o f o u r r a t s were used f o r each o f t h e 3, 6, 8, and 10 day t i m e p e r i o d s and c o n t r o l a n i m a l s were s a c r i f i c e d on days 6 and 10. Days 12 and 30 had a s i n g l e r a t f o r each t i m e p e r i o d . T i s s u e P r e p a r a t i o n : Under deep sodium p e n t o b a r b i t a l a n e s t h e s i a , r a t s were d e c a p i t a t e d and t h e b r a i n , t r i g e m i n a l g a n g l i a w i t h t h e p r o x i m a l nerve r o o t s , s p l e e n , thymus, and l i v e r were immmediately removed. The b r a i n s were c u t i n t o 5 mm c o r o n a l s e c t i o n s and a l l t i s s u e was f i x e d i n a s o l u t i o n o f 2% p a r a f o r m a l d e h y d e and 1% p i c r i c a c i d i n 0.1 M phosphate b u f f e r (pH 7.4) f o r 48 h o u r s . T i s s u e was t r a n s f e r r e d t o p h o s p h a t e - b u f f e r e d 15% s u c r o s e s o l u t i o n f o r a t l e a s t 2 days, and t h e n s e c t i o n e d on a f r e e z i n g microtome a t a t h i c k n e s s o f 30 Jim. S e c t i o n s were c o l l e c t e d i n phosphate-b u f f e r e d s a l i n e c o n t a i n i n g 0.3% T r i t o n X-100 and s t o r e d as f r e e -f l o a t i n g s e c t i o n s a t 4°C u n t i l s t a i n e d . Immunohistochemistry: M o n o c l o n a l a n t i b o d i e s a g a i n s t r a t MHC a n t i g e n s were o b t a i n e d from S e r a - l a b (England) as s u p e r n a t a n t s o f c u l t u r e medium. 0X6 was used a g a i n s t a monomorphic d e t e r m i n a n t o f MHC c l a s s I I ( l a ) a n t i g e n (McMaster e t a l . , 1979) and 0X18 was used a g a i n s t a monomorphic d e t e r m i n a n t o f MHC c l a s s I a n t i g e n (Fukumoto e t a l . , 1982). D i l u t i o n s were 1:100 f o r 0X6 and 1:300 f o r 0X18. P o l y c l o n a l a n t i b o d i e s a g a i n s t HSV1 (Dako, Denmark) and r a b b i t a n t i - g l i a l f i b r i l l a r y a c i d i c p r o t e i n (GFAP) (Dako, 19 Denmark) were used a t d i l u t i o n s o f 1:1000 and 1:10,000 r e s p e c t i v e l y . GFAP was used t o i d e n t i f y a s t r o c y t e s . Immunostaining was c a r r i e d out u s i n g t h e ABC method (Hsu e t a l . , 1981) w i t h a few m o d i f i c a t i o n s (Kaneko e t a l . , 1987). F o l l o w i n g 30 minutes p r e - t r e a t m e n t w i t h 0.2% hydrogen p e r o x i d e i n PBS and 3 b u f f e r washes (x20 m i n ) , s e c t i o n s were i n c u b a t e d w i t h p r i m a r y a n t i b o d y f o r 24 hours a t room t e m p e r a t u r e . S e c t i o n s were t h e n i n c u b a t e d w i t h b i o t i n y l a t e d anti-mouse IgG (H&L) or a n t i -r a b b i t IgG ( V e c t o r , 3.75 (J.g/ml) s o l u t i o n c o n t a i n i n g 2% normal r a t serum f o r 2 hours a t room t e m p e r a t u r e . T h i s s t e p was f o l l o w e d by i n c u b a t i o n w i t h ABC s o l u t i o n (Vector) d i l u t e d 1:400 f o r 1 hour a t room t e m p e r a t u r e . The p e r o x i d a s e r e a c t i o n was c a r r i e d out w i t h a s o l u t i o n of 0.02% 3 , 3 ' - d i a m i n o b e n z i d i n e t e t r a h y d r o c h l o r i d e (DAB), 0.01% hydrogen p e r o x i d e and 25 mM n i c k e l ammonium s u l f a t e i n 0.05M T r i s - H C L b u f f e r pH 7.6. The r e a c t i o n p r o d u c t was p u r p l e i n c o l o r w i t h l i t t l e o r no background s t a i n i n g . Some s e c t i o n s were c o u n t e r s t a i n e d w i t h n e u t r a l r e d and a l l s e c t i o n s were mounted on g l a s s s l i d e s and c o v e r - s l i p p e d w i t h E n t e l l a n . MHC c l a s s I I and GFAP a n t i b o d i e s were used i n a double i m m u n o s t a i n i n g p r o c e d u r e . Double immunohistochemical s t a i n i n g was pe r f o r m e d i n two s t a i n i n g c y c l e s . D u r i n g t h e f i r s t c y c l e (GFAP), t h e s t a n d a r d p e r o x i d a s e - a n t i - p e r o x i d a s e (PAP) method o f S t e r n b e r g e r ( S t e r n b e r g e r , 1979) was used. S e c t i o n s were i n c u b a t e d w i t h GFAP as t h e p r i m a r y a n t i b o d y f o l l o w e d by goat a n t i - r a b b i t IgG (Cappel, USA) a t a d i l u t i o n o f 1:1000 o v e r n i g h t a t 4°C. T h i s s t e p was f o l l o w e d by i n c u b a t i o n w i t h r a b b i t PAP (Dako, Denmark) f o r 2 hours a t room t e m p e r a t u r e a t a d i l u t i o n o f 1:1000. The DAB 20 r e a c t i o n was c a r r i e d out minus t h e n i c k e l ammonium s u l f a t e t o produce a brown r e a c t i o n p r o d u c t . F o l l o w i n g a 30 minute t r e a t m e n t w i t h 0.5% hydrogen p e r o x i d e , 0X6 a n t i b o d y s t a i n i n g was p e r f o r m e d as d e s c r i b e f o r s i n g l e i m m u n o s t a i n i n g . P e r i p h e r a l t i s s u e s e c t i o n s from t h e thymus, s p l e e n , and l i v e r were s t a i n e d w i t h a n t i b o d y a g a i n s t HSV1 as d e s c r i b e d f o r b r a i n s e c t i o n s and c o n t r o l s t a i n i n g s e c t i o n s were t r e a t e d as d e s c r i b e d above w i t h o m i s s i o n o f t h e p r i m a r y a n t i b o d y from t h e p r o c e d u r e . S e r i a l s e c t i o n s were a l s o s t a i n e d w i t h c r e s y l v i o l e t . 21 RESULTS B e h a v i o r a l Changes A n i m a l s i n o c u l a t e d w i t h v i r u s appeared h e a l t h y f o r t h e f i r s t 6 t o 7 days and r a p i d l y became moribund d u r i n g days 7 t h r o u g h 10. The s e v e r e l y moribund a n i m a l s (day 10) were s t i l l a l i v e p r i o r t o s a c r i f i c e . A l l a n i m a l s e x h i b i t e d c l i n i c a l s i g n s c h a r a c t e r i s t i c o f an e n c e p h a l i t i s i n c l u d i n g h y p o a c t i v i t y , f a i l u r e t o groom, f a i l u r e t o consume f o o d and water, and hunched i m m o b i l i t y . Two a n i m a l s r e c o v e r e d and appeared h e a l t h y , r e g a i n i n g a c t i v i t y and f o o d consumption, when s a c r i f i c e d a t 12 and 30 days p o s t - i n f e c t i o n . Herpes Simplex V i r u s Type 1 (HSV1) S t a i n i n g (Table 1) At 3 days, a s m a l l amount of p o s i t i v e HSV1 s t a i n i n g was p r e s e n t i n t h e axons of t h e f i f t h nerve as w e l l as i n a few s c a t t e r e d c e l l s i n t h e t r i g e m i n a l n u c l e u s . V i r a l i n f e c t i o n was m i n i m a l at t h i s s t a g e . Days 6 t h r o u g h 10 were c h a r a c t e r i z e d by a s i g n i f i c a n t l y i n c r e a s e d number of HSV1 p o s i t i v e l y s t a i n e d c e l l s ( F i g s . 1 , 2 ) . S y s t e m i c i n f e c t i o n was r u l e d out based on n e g a t i v e s t a i n i n g o f p e r i p h e r a l o rgans. HSV1 i m m u n o r e a c t i v i t y was found i n i t i a l l y i n t r i g e m i n a l n erve axons, b r a i n s t e m n u c l e i and i n t h e c e r e b e l l u m . By t h e e i g h t h and t e n t h days, p o s i t i v e s t a i n i n g was f ound t h r o u g h o u t much of t h e b r a i n . I n f e c t e d a r e a s i n c l u d e d t h e hypothalamus, thalamus, and many d i f f u s e c o r t i c a l r e g i o n s . 22 Table 1 HSV1 i m m u n o r e a c t i v i t y w i t h i n v a r i o u s b r a i n r e g i o n s over t h e c o u r s e o f 30 days. I V Nu R + ++ ++++ +++ + + ( S p i n a l ) L + ++++ ++ - -C e r e b e l l a r R - ++ ++++ +++ C o r t e x L - + ++++ + Hypothalamus R - + ++++ ++ L - + ++++ ++ Thalamus R - + +++ + L - - ++ + I I I Nu R - + +++ +++ (Edinger-Westphal) L - - + + S e p t a l Nu R - ++ L - - + Caudate/ R - - ++ Putamen L - - ++ + C e r e b r a l R - - + + C o r t e x L - - + + 3 6 8 10 12 30 Days P o s t - I n o c u l a t i o n - = no v i s i b l e s t a i n i n g + = v e r y l i t t l e s t a i n i n g + = moderate s t a i n i n g (each a d d i t i o n a l + i n d i c a t i n g i n c r e a s e i n number o f p o s i t i v e l y l a b e l e d c e l l s ) R = r i g h t s i d e ( i p s i l a t e r a l t o i n o c u l a t i o n ) L = l e f t s i d e 23 F i g . l . S e c t i o n t h r o u g h t h e b r a i n s t e m o f an a n i m a l 6 days a f t e r c o r n e a l i n o c u l a t i o n . HSV1 immunoreactive c e l l s w i t h i n t h e s p i n a l t r i g e m i n a l n u c l e u s ( l a r g e a r r o w ) . Some HSV1 r e a c t i v i t y i s a l s o p r e s e n t i n t h e s u p r a f a c i a l n u c l e u s of t h e s e v e n t h nerve (arrowhead). V I I n., f a c i a l n u c l e u s . (Bregma (B)=-11.3 mm, a c c o r d i n g t o P a x i n o s and Watson, 1982) Bar=500 |lm. 24 F i g . 2 . S e c t i o n t h r o u g h t h e b r a i n s t e m o f an a n i m a l 8 days a f t e r i n o c u l a t i o n HSV1 immunoreactive c e l l s i n c r e a s i n g i n number from day 6. V n., s p i n a l t r i g e m i n a l n u c l e u s ; V I I n., f a c i a l n u c l e u s ; IV, f o u r t h v e n t r i c l e ; m, m i d l i n e . (B=-11.3 mm) Bar=500 U,m. 25 At 12 days, v e r y few p o s i t i v e l y s t a i n e d neurons remained and o n l y r a r e l y were HSV1 immunoreactive neurons n o t e d a t 30 days. HSV1 r e a c t i v i t y was i n i t i a l l y i p s i l a t e r a l a t 6 days, and became i n c r e a s i n g l y b i l a t e r a l t h r o u g h day 10. D i s t r i b u t i o n a t 12 days was i p s i l a t e r a l . Day 6 A t t h i s s t a g e , t h e i n f e c t i o n was l a r g e l y c o n f i n e d t o t h e t r i g e m i n a l n erve and c e r e b e l l a r c o r t e x . T r i g e m i n a l axons and l a r g e neurons w i t h i n t h e s p i n a l t r i g e m i n a l n u c l e u s were p o s i t i v e l y s t a i n e d f o r HSV1 ( F i g s . 3 , 4 ) . N e u r o n a l somas were a p p r o x i m a t e l y 20 (im i n d i a m e t e r , w i t h v i s i b l e d e n d r i t i c p r o c e s s e s , c h a r a c t e r i s t i c o f a t y p i c a l n e u r o n a l p r o f i l e . S m a l l e r , round c e l l s w i t h an approximate d i a m e t e r o f 5-10 |lm and no v i s i b l e p r o c e s s e s , were a l s o p o s i t i v e l y s t a i n e d , but were d i f f i c u l t t o i d e n t i f y from t h e s t a i n ( F i g . 5 ) . G i v e n t h e c h a r a c t e r i s t i c s o f t h e s e c e l l s , t h e y were most l i k e l y l e u k o c y t e s . A s m a l l f o c a l l e s i o n was p r e s e n t i n t h e E d i n g e r - W e s t p h a l n u c l e u s o f t h e t h i r d c r a n i a l n e r v e . A few HSVl p o s i t i v e neurons i n t h e g r a n u l a r l a y e r o f t h e c e r e b e l l u m were n o t e d . M o r p h o l o g i c a l l y , t h e s e neurons were a p p r o x i m a t e l y 25 u,m i n d i a m e t e r w i t h l o n g p r o c e s s e s and d e n d r i t i c branches e x t e n d i n g t h r o u g h o u t a d j a c e n t c e r e b e l l a r l a y e r s ( F i g . 6 ) . Based on morphology and l o c a t i o n o f t h e s e neurons, t h e y are most l i k e l y G o l g i neurons. 26 F i g . 3 . S e c t i o n t h r o u g h t h e t r i g e m i n a l nerve o f an a n i m a l 6 days a f t e r i n o c u l a t i o n . HSV1 p o s i t i v e l y s t a i n e d axons on t h e i p s i l a t e r a l s i d e , d e m o n s t r a t i n g a x o n a l l y t r a n s p o r t e d v i r u s . Bar=50 Lim. 27 F i g . 4 . S e c t i o n t h r o u g h t h e m e d i a l p o r t i o n of t h e f i f t h n u c l e u s of an a n i m a l 6 days a f t e r i n o c u l a t i o n . HSV1 p o s i t i v e neuron w i t h f i n e p r o c e s s e s . Note beaded appearance o f p r o c e s s e s i n n e u r o p i l . N e i g h b o u r i n g n e g a t i v e l y s t a i n e d neurons ( p o s s i b l y n o n - i n f e c t e d ) i n d i c a t e s i m i l a r i t y i n s i z e t o i n f e c t e d c e l l s . (B=-11.3 mm) Bar=50 |im. (See A-A' i n Fig.25) . 28 F i g . 5 . S e c t i o n t h r o u g h t h e m e d i a l p o r t i o n o f t h e f i f t h n u c l e u s of an a n i m a l 6 days a f t e r i n o c u l a t i o n . HSV1 r e a c t i v e s m a l l , round c e l l s , a p p r o x i m a t e l y 5-10 |im i n d i a m e t e r w i t h no v i s i b l e p r o c e s s e s . These c e l l s a r e most l i k e l y l e u k o c y t e s . (B=-11.3 mm) Bar=50 Lim. 29 F i g . 6 . S e c t i o n t h r o u g h t h e c e r e b e l l a r c o r t e x o f an a n i m a l 6 days a f t e r i n o c u l a t i o n . Large HSV1 r e a c t i v e neurons a p p r o x i m a t e l y 25 u,m i n d i a m e t e r a r e p r e s e n t w i t h i n t h e g r a n u l a r c e l l l a y e r . Note t h e f i n e d e n d r i t i c branches e x t e n d i n g i n t o a d j a c e n t l a y e r s . As d e s c r i b e d by E c c l e s e t a l . ( 1 9 6 7 ) , G o l g i neurons are l o c a t e d w i t h i n t h e g r a n u l a r l a y e r o f t h e c e r e b e l l a r c o r t e x and p o s s e s s d e n d r i t i c branches which o f t e n e x t e n d i n t o a d j a c e n t l a y e r s , g r , g r a n u l a r l a y e r ; mo, m o l e c u l a r l a y e r . (B=-10.3 mm) Bar=50 |im. 30 Day 8 By 8 days, t h e i n f e c t i o n had s p r e a d t o a l a r g e number o f b r a i n r e g i o n s . The number of HSV1 p o s i t i v e neurons w i t h i n t h e t r i g e m i n a l n u c l e u s had i n c r e a s e d s u b s t a n t i a l l y . L a y e r s 1-3 o f t h e c e r e b e l l a r c o r t e x and round c e l l s w i t h i n t h e i n f e r i o r c e r e b e l l a r p e d u n c l e (ICP) were a l s o i n f e c t e d . The c e l l s w i t h i n t h e ICP were s m a l l , round, and a p p r o x i m a t e l y 5-10 [im. i n d i a m e t e r w i t h no v i s i b l e p r o c e s s e s . S i m i l a r t o t h e c e l l s n o t e d i n t h e t r i g e m i n a l n e r v e , t h e s e c e l l s were most l i k e l y l e u k o c y t e s . P o s i t i v e l y s t a i n e d c e l l s i n t h e c e r e b e l l u m were l a r g e l y c o n f i n e d t o t h e g r a n u l a r l a y e r and were a l s o s m a l l and round i n shape. At t h i s s t a g e , b o t h t h e thalamus and t h e hypothalamus were i n f e c t e d . S u b s t a n t i a l i n f e c t i o n i n t h e thalamus was p r e s e n t m a i n l y i n t h e v e n t r a l p o s t e r o l a t e r a l (VPL), v e n t r a l p o s t e r o m e d i a l (VPM), and l a t e r a l g e n i c u l a t e n u c l e i (LGN). Large c e l l s w i t h i n t h e VPL and VPM were p o s i t i v e l y s t a i n e d and a l a r g e f o c u s o f i n f e c t i o n w i t h i n t h e v e n t r o p o s t e r i o r t h a l a m i c n u c l e u s ( p a r v o c e l l u l a r r e g i o n ) was a l s o HSV1 immunoreactive ( F i g . 7 ) . W i t h i n t h e hypothalamus, t h e p o s t e r i o r , d o r s a l , and l a t e r a l h y p o t h a l a m i c a r e a s were i n f e c t e d . Many l a r g e neurons were p o s i t i v e l y s t a i n e d ( F i g . 8 ) . A s m a l l number of l i g h t l y s t a i n e d c e l l s w i t h a t u f t e d appearance were e v i d e n t w i t h i n t h e a n t e r i o r o l f a c t o r y n u c l e u s . 31 F i g . 7 . S e c t i o n t h r o u g h t h e v e n t r o p o s t e r i o r n u c l e u s ( p a r v o c e l l u l a r r e g i o n ) o f t h e thalamus o f an a n i m a l 8 days a f t e r i n o c u l a t i o n . A l a r g e f o c u s c o n s i s t i n g o f HSV1 immunoreactive neurons, s m a l l round c e l l s , and n e u r o p i l i s l o c a t e d l a t e r a l t o t h e t h i r d v e n t r i c l e . (B=-3.8 mm) Bar=100 U,m. (See B-B' i n Fig.25) . 3 2 F i g . 8 . S e c t i o n t h r o u g h t h e p a r a v e n t r i c u l a r h y p o t h a l a m i c n u c l e u s of an a n i m a l 8 days a f t e r i n o c u l a t i o n . Note HSV1 r e a c t i v e neurons w i t h f i n e d e n d r i t i c p r o c e s s e s . (B=-1.8 mm) Bar=50 |im. 33 S m a l l f o c a l a r e a s o f p o s i t i v e s t a i n i n g were p r e s e n t i n a number o f d i f f e r e n t b r a i n r e g i o n s . Neurons and s m a l l e r , round c e l l s i n t h e caudate/putamen and l a t e r a l s e p t a l n u c l e i were p o s i t i v e f o r HSV1 and t h e number o f p o s i t i v e c e l l s w i t h i n t h e oculomotor n u c l e u s had i n c r e a s e d . The f r o n t o p a r i e t a l c o r t e x (Brodman's a r e a s 3,2,1) ( K r i e g , 1946) was a l s o i n f e c t e d . C e l l s w i t h p y r a m i d a l o r f u s i f o r m morphology were p o s i t i v e f o r HSV1 i n th e i n t e r n a l and e x t e r n a l p y r a m i d a l l a y e r s o f t h e f r o n t o p a r i e t a l c o r t e x ( F i g . 9 ) . A l t h o u g h t h e s e neurons p o s s e s s e d a p y r a m i d a l l y shaped soma and a p i c a l d e n d r i t e s , t h e b a s a l d e n d r i t e s were d i f f i c u l t t o i d e n t i f y . The s t r i a t e c o r t e x was n e g a t i v e . Day 10 At t h i s s t a g e , t h e t r i g e m i n a l and s e p t a l n u c l e i , thalamus, hypothalamus, ICP, and c e r e b e l l a r l a y e r s were a l l c h a r a c t e r i z e d by a s u b s t a n t i a l d e c r e a s e i n p o s i t i v e l y s t a i n e d c e l l s . W i t h i n t h e caudate/putamen, a s i n g l e s m a l l f o c u s o f p o s i t i v e l y s t a i n e d round c e l l s was v i s i b l e . The f r o n t o p a r i e t a l c o r t e x was n e g a t i v e f o r HSV1, but many l a r g e , p o s i t i v e p y r a m i d a l l y shaped neurons were p r e s e n t i n t h e p r i m a r y o l f a c t o r y c o r t e x ( F i g . 1 0 ) . A d d i t i o n a l l y , t h e o c ulomotor n u c l e u s showed an i n c r e a s e i n t h e number o f p o s i t i v e l y s t a i n e d e l e m e n t s . The elements were round i n shape, but m o r p h o l o g i c a l l y , d i f f i c u l t t o i d e n t i f y . 34 F i g . 9 . S e c t i o n t h r o u g h th e f r o n t o p a r i e t a l c o r t e x o f an a n i m a l 8 days a f t e r i n o c u l a t i o n . P y r a m i d a l / f u s i f o r m shaped neurons w i t h i n t h e e x t e r n a l p y r a m i d a l l a y e r are p o s i t i v e l y s t a i n e d f o r HSV1; however no b a s a l d e n d r i t e s are v i s i b l e . T h i s may be due t o i n c o m p l e t e i m p r e g n a t i o n o f v i r a l a n t i g e n . S e c t i o n i s l a t e r a l t o t h e p o s t e r i o r c i n g u l a t e g y r u s . (B=-3.8 mm) Bar=50 |im. (See C-C i n F i g . 2 5) . 35 F i g . 1 0 . S e c t i o n t h r o u g h t h e p r i m a r y o l f a c t o r y c o r t e x o f a a n i m a l 10 days a f t e r i n o c u l a t i o n . HSV1 immunoreactive p y r a m i d a l l y shaped neurons l a t e r a l t o t h e a n t e r i o r c o r t i c a l a m y g d a l i o d n u c l e u s . As i n F i g . 9 , b a s a l d e n d r i t e s are d i f f i c u l t t o i d e n t i f y . (B=-1.8 mm) Bar=50 U.m. 36 Day 12 By t h i s stage, very few areas remained v i s i b l y infected. The pyriform cortex was negative for HSV1, but a small focus of p o s i t i v e l y stained, small, dark, round c e l l s was present i n the cingulum. A few p o s i t i v e l y stained c e l l s remained within the granular layer of the cerebellum, most of which were small and round i n shape. What appeared to be a process and an elongated soma were also p o s i t i v e l y stained within the section (Fig.11). Day 30 One month after inoculation, a l l brain areas were e s s e n t i a l l y negative for HSV1, with an occasional spotting i n a few brain regions. MHC Class I Antigen Expression In control animals, MHC class I expression (0X18) was largely confined to vascular endothelial c e l l s and a few small g l i a l c e l l s throughout the brain (Fig.12). The immunohistochemical signal for the g l i a l c e l l s was stronger i n white matter than i n grey with morphology consistent with that of resting microglia. These c e l l s were multipolar with many fine spines and a s l i g h t l y elongated soma. Tissue examined at 3 days post-infection revealed no change i n class I staining from that seen i n control animals. By 6 days, however, a large number of c e l l s c h a r a c t e r i s t i c of reactive microglia were p o s i t i v e l y stained for MHC class I. These c e l l s were multipolar and star-shaped, with an elongated, 37 F i g . 1 1 . S e c t i o n t h r o u g h t h e c e r e b e l l a r c o r t e x ( g r a n u l a r c e l l l a y e r 1) o f an a n i m a l 12 days a f t e r i n o c u l a t i o n . HSV1 immunoreactive p r o c e s s ( l a r g e arrow) and s m a l l , round, c e l l s ( s m a l l arrow) are e v i d e n t . The round c e l l s a r e a p p r o x i m a t e l y 5-10 |im i n d i a m e t e r w i t h no v i s i b l e p r o c e s s e s . They a r e most l i k e l y l e u k o c y t e s . (B=-10.3 mm) Bar=100 urn. 38 F i g . 1 2 . S e c t i o n t h r o u g h t h e VPM n u c l e u s o f t h e thalamus o f a c o n t r o l a n i m a l 6 days a f t e r c o r n e a l s c a r i f i c a t i o n . MHC c l a s s I r e a c t i v e b l o o d v e s s e l s are e v i d e n t t h r o u g h o u t t h e t i s s u e . (B=-3.8 mm) Bar=100 Lim. 39 s l i g h t l y s w o l l e n soma. The soma was a p p r o x i m a t e l y 15-20 u,m i n l e n g t h , w i t h many s w o l l e n p r o c e s s e s . I n t e r n a l c e l l u l a r s t r u c t u r e s were d i f f i c u l t t o i d e n t i f y due t o t h e imm u n o s t a i n i n g . P o s i t i v e l y s t a i n e d m i c r o g l i a were p r e s e n t i n many b r a i n r e g i o n s s i m i l a r l y p o s i t i v e f o r HSV1. At 8 days, t h e number o f p o s i t i v e m i c r o g l i a i n c r e a s e d s u b s t a n t i a l l y and were l a r g e l y c o n f i n e d t o HSV1 p o s i t i v e b r a i n a r e a s , w i t h a few e x c e p t i o n s . Ten and 12 days p o s t - i n f e c t i o n were c h a r a c t e r i z e d by c l a s s I p o s i t i v e m i c r o g l i a t h r o u g h o u t t h e e n t i r e b r a i n . A t 30 days, however, t h e number o f p o s i t i v e c e l l s had d e c r e a s e d s u b s t a n t i a l l y , r e m a i n i n g o n l y i n r e g i o n s which had i n i t i a l l y been h e a v i l y s t a i n e d f o r HSV1. S i m i l a r t o s t a i n i n g p a t t e r n s v i s u a l i z e d f o r HSV1, c l a s s I s t a i n i n g was l a r g e l y i p s i l a t e r a l a t 6 days, p r o g r e s s i n g t o a b i l a t e r a l d i s t r i b u t i o n d u r i n g t h e 8-12 day p e r i o d , and r e t u r n i n g t o an i p s i l a t e r a l d i s t r i b u t i o n a t 30 days. Day 6 C l a s s I p o s i t i v e r e a c t i v e m i c r o g l i a and e n d o t h e l i a l c e l l s were p r e s e n t i n t h e a r e a o f t h e s p i n a l t r i g e m i n a l n u c l e u s ( F i g . 1 3 ) . P o s i t i v e m i c r o g l i a were a l s o p r e s e n t i n m o l e c u l a r l a y e r s 3-5 o f t h e c e r e b e l l u m as w e l l as i n t h e ICP. D a r k l y s t a i n e d , s m a l l , round c e l l s were a l s o e v i d e n t i n t h e ICP. ( F i g . 1 4 ) . P o s i t i v e l y s t a i n e d m i c r o g l i a were p r e s e n t w i t h i n t h e l a t e r a l h y p o t h a l a m i c a r e a as w e l l as i n t h e median eminence. 40 F i g . 1 3 . S e c t i o n t h r o u g h t h e m e d i a l p o r t i o n of t h e f i f t h n u c l e u s o f an a n i m a l 6 days a f t e r i n o c u l a t i o n . MHC c l a s s I p o s i t i v e l y s t a i n e d m i c r o g l i a c e l l s ( l a r g e arrowhead) w i t h t y p i c a l m i c r o g l i a morphology (see t e x t ) . A few MHC I immunoreactive b l o o d v e s s e l s a re a l s o e v i d e n t ( s m a l l arrowhead). (B=-10.3 mm) Bar=50 (i.m. 41 g.14. S e c t i o n t h r o u g h t h e i n f e r i o r c e r e b e l l a r p e d u n c l e o f an a n i m a l 6 days a f t e r i n o c u l a t i o n . MHC c l a s s I immunoreactive s m a l l , round c e l l s , w i t h no v i s i b l e p r o c e s s e s are p r e s e n t . They a r e a p p r o x i m a t e l y 5-10 u.m i n di a m e t e r and a r e most l i k e l y l e u k o c y t e s . (B=-10.3 mm) Bar=50 u,m. 42 Posterior and dorsal hypothalamic areas were negative. The medial geniculate nucleus (MGN) and superior c o l l i c u l u s both contained p o s i t i v e microglia, but no r e a c t i v i t y was noted i n either the LGN or VPL/VMP nu c l e i . A few reactive microglia were present i n the subiculum. P o s i t i v e l y stained microglia and endothelial c e l l s were noted i n the Edinger-Westphal nucleus, and, although the caudate/putamen was negative for class I, a few l i g h t l y stained microglia were present i n the inte r n a l capsule (Fig.15). C o r t i c a l and l a t e r a l septal areas were largely negative for class I. Day 8 A substantial increase i n the number of class I p o s i t i v e microglia was noted i n the aforementioned brain regions 8 days after inoculation. Additional brain areas containing p o s i t i v e l y stained microglia included posterior and dorsal hypothalamic areas, VPM, LGN, l a t e r a l habenular nuclei, and caudate/putamen. A large number of p o s i t i v e l y stained, small, round c e l l s were also present i n the posterior hypothalamic area. Class I po s i t i v e microglia were also noted i n the external pyramidal layer of the frontoparietal cortex (3,1,2) as well as i n the cingulum. Focal areas of r e a c t i v i t y were also present i n the l a t e r a l septal area (Fig. 16) . F i g . 1 5 . S e c t i o n t h r o u g h t h e i n t e r n a l c a p s u l e o f an a n i m a l 6 days a f t e r i n o c u l a t i o n . MHC c l a s s I immunoreactive m i c r o g l i a a re e v i d e n t . (B=-3.8 mm) Bar=50 u.m. 44 F i g . 1 6 . S e c t i o n t h r o u g h t h e l a t e r a l s e p t a l n u c l e i o f an a n i m a l 8 days a f t e r i n o c u l a t i o n . Note MHC c l a s s I immunoreactive m i c r o g l i a c e l l w i t h e l o n g a t e d soma and m u l t i p l e f i n e p r o c e s s e s . (B=1.7 mm) Bar=50 |im. 45 Davs 10 and 12 At 10 days, the entire brain was densely populated with class I p o s i t i v e microglia. A few focal patches of heavily stained microglia were present i n the MGN of the thalamus (Fig.17). By 12 days, a s l i g h t decrease i n the number of p o s i t i v e l y stained microglia was evident throughout the brain, but o v e r a l l , the brain remained densely populated. Day 30 A dramatic decrease i n the number of p o s i t i v e microglia was c l e a r l y evident at t h i s stage. Within the trigeminal nucleus and cerebellar layers, the l e v e l of r e a c t i v i t y was sim i l a r to that seen i n i t i a l l y at 6 days. Some microglia remained i n the ICP, but small, round c e l l s were no longer v i s i b l e . Decreased numbers of reactive microglia were also evident i n the l a t e r a l hypothalamus, Edinger-Westphal nucleus, superior c o l l i c u l u s , and in t e r n a l capsule. A few focal areas remained i n the VPL/VPM nuclei and a few p o s i t i v e c e l l s were present i n the caudate/putamen, cingulum and l a t e r a l septal n u c l e i . MHC Class II Antigen Expression Control tissue exhibited far less MHC class II (0X6) than class I staining. Only a few class II (la) po s i t i v e c e l l s with t y p i c a l resting microglia morphology were found. These c e l l s were largely confined to white matter. 46 F i g . 1 7 . S e c t i o n t h r o u g h t h e thalamus o f an a n i m a l 10 days a f t e r i n o c u l a t i o n . A l a r g e f o c a l a r e a o f MHC c l a s s I p o s i t i v e m i c r o g l i a are p r e s e n t w i t h i n t h e MGN, l a t e r a l t o t h e deep m e s e n c e p h a l i c n u c l e i . Note r e l a t i v e d e n s i t y compared t o background r e a c t i v i t y . (B=-6.3 mm) Bar=100 |lm. 47 Three days f o l l o w i n g i n o c u l a t i o n , b r a i n t i s s u e was n e g a t i v e f o r c l a s s I I a n t i g e n s . A t 6 days, c l a s s I I p o s i t i v e m i c r o g l i a were p r e s e n t i n b r a i n r e g i o n s s i m i l a r l y p o s i t i v e f o r c l a s s I . I a e x p r e s s i o n f o l l o w e d a p a t t e r n comparable t o t h a t seen i n c l a s s I e x p r e s s i o n d u r i n g t h e 8 t o 12 day p e r i o d . A d r a m a t i c d e c r e a s e i n I a e x p r e s s i o n was e v i d e n t a t 30 days. A l t h o u g h c l a s s I and c l a s s I I e x p r e s s i o n e x h i b i t e d l a r g e l y comparable d i s t r i b u t i o n s , some d i f f e r e n c e s were e v i d e n t . And s i m i l a r t o HSV1 and c l a s s I s t a i n i n g , c l a s s I I e x p r e s s i o n was i n i t i a l l y i p s i l a t e r a l , f o l l o w e d i n l a t e r s t a g e s by a b i l a t e r a l d i s t r i b u t i o n , and r e t u r n i n g t o an i p s i l a t e r a l p a t t e r n by 30 days. Day 6 P o s i t i v e l y s t a i n e d m i c r o g l i a and s m a l l , dark, round c e l l s were p r e s e n t i n t h e t r i g e m i n a l n u c l e u s ( F i g . 1 8 ) . These m i c r o g l i a were s l i g h t l y d i f f e r e n t m o r p h o l o g i c a l l y from c l a s s I p o s i t i v e m i c r o g l i a , i n t h a t t h e c l a s s I I p o s i t i v e m i c r o g l i a had fewer p r o c e s s e s and t h e s e p r o c e s s e s were l e s s s w o l l e n . The d i f f e r e n c e s n o t e d between t h e c l a s s I and I I p o s i t i v e m i c r o g l i a a re l i k e l y due t o d i f f e r e n c e s i n t h e q u a l i t y o f s t a i n i n g . The s m a l l , dark, round c e l l s were a p p r o x i m a t e l y 5-10 (i.m i n d i a m e t e r w i t h no v i s i b l e p r o c e s s e s . These c e l l s were most l i k e l y l e u k o c y t e s . 48 F i g . 1 8 . S e c t i o n t h r o u g h t h e m e d i a l p o r t i o n o f t h e f i f t h n u c l e u s of an a n i m a l 6 days a f t e r i n o c u l a t i o n . MHC c l a s s I I immunoreactive m i c r o g l i a . Note s l i g h t d i f f e r e n c e s i n morphology from MHC I p o s i t i v e m i c r o g l i a . These m i c r o g l i a p o s s e s s fewer p r o c e s s e s which a r e a l s o l e s s s w o l l e n r e l a t i v e t o c l a s s I m i c r o g l i a . The d i s p a r i t y i s l i k e l y due t o d i f f e r e n c e s i n q u a l i t y o f s t a i n i n g . (B=-11.3 mm) Bar=50 (im. 49 Day 8 Bo t h m i c r o g l i a and round c e l l s i n c r e a s e d i n number w i t h i n t h e t r i g e m i n a l n u c l e u s . The number of round c e l l s w i t h i n t h e ICP had a l s o i n c r e a s e d and s m a l l f o c i of I a p o s i t i v e m i c r o g l i a were e v i d e n t i n b o t h t h e g r a n u l a r and m o l e c u l a r l a y e r s (2-5) o f t h e c e r e b e l l a r c o r t e x . S m a l l f o c i o f c l a s s I I p o s i t i v e m i c r o g l i a were a l s o n o t e d i n t h e p o s t e r i o r h y p o t h a l a m i c a r e a , VPL/VPM n u c l e i , MGN, s u p e r i o r c o l l i c u l u s , and l a t e r a l s e p t a l n u c l e i . A few I a p o s i t i v e m i c r o g l i a and s m a l l , d ark, round c e l l s were p r e s e n t i n t h e i n t e r n a l c a p s u l e and a few p o s i t i v e l y s t a i n e d m i c r o g l i a were seen i n t h e caudate/putamen. I a p o s i t i v e round c e l l s were a l s o n o t e d i n t h e E d i n g e r - W e s t p h a l n u c l e u s . A few r e a c t i v e m i c r o g l i a were p r e s e n t i n t h e f r o n t o p a r i e t a l c o r t e x (3,1,2) and t h e c i n g u l u m . Davs 10 and 12 S i m i l a r t o t h e c l a s s I d i s t r i b u t i o n a t 10 days, c l a s s I I p o s i t i v e m i c r o g l i a were e v i d e n t t h r o u g h o u t t h e e n t i r e b r a i n . A few f o c a l a r e a s o f i n t e n s e s t a i n i n g were n o t e d i n s c a t t e r e d m i d b r a i n n u c l e i ( F i g . 1 9 ) . V e ry l i t t l e change i n t h e number o f I a p o s i t i v e m i c r o g l i a was e v i d e n t a t 12 days. A few a r e a s , n o t a b l y t h e p y r i f o r m c o r t e x , o l f a c t o r y t u b e r c l e , and hippocampus, c o n t a i n e d s l i g h t l y r e d u c e d numbers o f r e a c t i v e m i c r o g l i a . 50 F i g . 1 9 . S e c t i o n t h r o u g h t h e deep m e s e n c e p h a l i c n u c l e i o f an a n i m a l 10 days a f t e r i n o c u l a t i o n . Note h e a v i l y s t a i n e d f o c u s of MHC c l a s s I I immunoreactive m i c r o g l i a r e l a t i v e t o background s t a i n i n g . Focus i s m e d i a l t o t h e MGN o f the thalamus and l a t e r a l t o t h e t h i r d n u c l e u s . (B=-6.3 mm) Bar=50 |lm. 51 Day 30 A s u b s t a n t i a l d e c r e a s e i n t h e number of p o s i t i v e l y s t a i n e d m i c r o g l i a was e v i d e n t t h r o u g h o u t t h e b r a i n . A few r e a c t i v e m i c r o g l i a remained i n t h e t r i g e m i n a l n u c l e u s , ICP, g r a n u l a r and m o l e c u l a r c e r e b e l l a r l a y e r s 2-3, and i n t h e r o s t r a l p o r t i o n o f t h e c a u d a t e . A s i n g l e f o c u s o f l a p o s i t i v e s t a i n i n g was p r e s e n t i n t h e deep m e s e n c e p h a l i c n u c l e i and a s i n g l e p o s i t i v e m i c r o g l i a l c e l l was n o t e d i n t h e MGN o f t h e thalamus. U n l i k e t h e c l a s s I d i s t r i b u t i o n a t 30 days, a few l a p o s i t i v e m i c r o g l i a remained i n t h e o p t i c chiasm and t h e f o r c e p s major of t h e corpus c a l l o s u m . G l i a l F i b r i l l a r y A c i d i c P r o t e i n (GFAP) S t a i n i n g A s t r o c y t e s were e v i d e n t 3 days a f t e r i n o c u l a t i o n . These c e l l s were s t a r - s h a p e d w i t h an o v a l shaped n u c l e u s and w e l l d e v e l o p e d p r o c e s s e s emanating from a l l s i d e s ( F i g . 2 0 ) . The t i m e p e r i o d from 6 t o 10 days was c h a r a c t e r i z e d by a s t r o c y t i c r e a c t i v i t y w i t h i n HSV1 p o s i t i v e b r a i n r e g i o n s . At 12 days, t i s s u e n e c r o s i s was p r e s e n t i n t h e hypothalamus and c e r e b r a l p e d u n c l e . N e c r o t i c a r e a s were c h a r a c t e r i z e d by an absence o f c e l l s i n t h e c e n t e r , s u r r o u n d e d by a r i n g o f d a r k l y s t a i n i n g a s t r o c y t e s . A l t h o u g h some n e c r o s i s was e v i d e n t , t h e r e was no e v i d e n c e of n e u r o n a l d e g e n e r a t i o n i n e i t h e r c r e s y l v i o l e t or h e m a t o x y l i n and e o s i n s t a i n e d s e c t i o n s . T h i s may be due t o v a r i a b i l i t y i n e i t h e r s t a i n i n g or i n f e c t i o n , or i n b o t h . No n e c r o s i s or c y s t i c s c a r r i n g was v i s i b l e a t 30 days. 52 F i g . 2 0 . S e c t i o n t h r o u g h t h e m e d i a l p o r t i o n o f t h e f i f t h n u c l e u s of an a n i m a l 3 days a f t e r i n o c u l a t i o n . Note s t a r shaped GFAP p o s i t i v e r e s t i n g a s t r o c y t e s w i t h w e l l d e v e l o p e d p r o c e s s e s emanating from a l l s i d e s . (B=-11.3 mm) Bar=50 U.m. 53 Day 6 At t h i s s t a g e , changes c h a r a c t e r i s t i c o f a s t r o c y t i c r e a c t i v i t y were e v i d e n t w i t h i n t h o s e a r e a s which were s t a i n e d p o s i t i v e l y f o r HSV1. GFAP s t a i n i n g i n t e n s i t y was i n c r e a s e d , n u c l e i were s w o l l e n , and t h e number o f GFAP p o s i t i v e c e l l s had i n c r e a s e d o v er day 3. The somas were a p p r o x i m a t e l y 15-20 |im i n l e n g t h and a s t r o c y t i c p r o c e s s e s were s l i g h t l y more t u b u l a r and wispy i n appearance ( F i g . 2 1 ) . S e r i a l s e c t i o n s s t a i n e d w i t h c r e s y l v i o l e t r e v e a l e d s w o l l e n a s t r o c y t i c n u c l e i ( F i g . 2 2 ) . W i t h b o t h GFAP and c r e s y l v i o l e t s t a i n i n g , i n t e r n a l c e l l u l a r c h a r a c t e r i s t i c s were d i f f i c u l t t o i d e n t i f y . A s t r o c y t e s i n HSV1 n e g a t i v e b r a i n r e g i o n s were not r e a c t i v e . Days 8 and 10 HSV1 s t a i n i n g was p r e s e n t i n an i n c r e a s i n g number o f b r a i n r e g i o n s a t t h i s s t a g e , and a s t o c y t i c r e a c t i v i t y was n o t e d i n a l l of t h e s e a r e a s . S t a i n i n g i n t e n s i t y and t h e number o f GFAP p o s i t i v e c e l l s i n c r e a s e d . A few f o c a l a r e a s o f n e c r o s i s were p r e s e n t w i t h i n t h e l a t e r a l h y p o t h a l a m i c a r e a and t h e c e r e b r a l p e d u n c l e . The n e c r o t i c a r e a s were d e v o i d o f a l l parenchymal elements and were surrounded by a d a r k l y s t a i n e d a s t r o c y t i c margin ( F i g . 2 3 ) . Days 12 and 30 At 12 days, a d e c r e a s e i n t h e s t a i n i n g i n t e n s i t y and number of GFAP p o s i t i v e c e l l s was e v i d e n t i n a l l b r a i n r e g i o n s e x cept f o r t h e t r i g e m i n a l n u c l e u s , m o l e c u l a r c e r e b e l l a r l a y e r s 3-5, and th e ICP. These areas appeared as t h e y d i d a t 10 days. S m a l l 54 F i g . 2 1 . S e c t i o n t h r o u g h t h e m e d i a l p o r t i o n o f t h e f i f t h n u c l e u s of an an i m a l 6 days a f t e r i n o c u l a t i o n . GFAP p o s i t i v e r e a c t i v e a s t r o c y t e s p o s s e s s an e n l a r g e d and i r r e g u l a r l y shaped soma w i t h wispy p r o c e s s e s (arrowheads). These c h a r a c t e r i s t i c s a r e c o n s i s t e n t w i t h t h o s e d e s c r i b e d f o r r e a c t i v e a s t r o c y t e s (see t e x t , M o r i and L e b l o n d , 1969). (B=-11.3 mm) Bar=50 urn. 55 F i g . 2 2 . S e c t i o n t h r o u g h t h e m e d i a l p o r t i o n o f t h e f i f t h n u c l e u s of an a n i m a l 6 days a f t e r i n o c u l a t i o n . C r e s y l v i o l e t s t a i n r e v e a l s a s t r o c y t e s t o be s w o l l e n and g r a n u l a t e d (arrowheads). (B=-11.3 mm) Bar=50 |im. 5 6 F i g . 2 3 . S e c t i o n t h r o u g h t h e c e r e b r a l p e d u n c l e of an a n i m a l 12 days a f t e r i n o c u l a t i o n . A l a r g e n e c r o t i c a r e a (arrowheads) i s e v i d e n t w i t h an absence of c e l l s i n t h e c e n t e r , s u r r o u n d e d by a r i n g o f d a r k l y s t a i n e d a s t r o c y t e s . N e c r o t i c a r e a i s d o r s a l t o t h e p o n t i n e n u c l e i and v e n t r a l t o t h e m e d i a l l e m n i s c u s . (B=-6.8 mm) Bar=200 urn. n e c r o t i c a r e a s were v i s i b l e i n t h e cing u l u m , MGN, and caudate/putamen. A s u b s t a n t i a l d e c r e a s e i n s t a i n i n g i n t e n s i t y and c e l l number th r o u g h o u t t h e b r a i n was e v i d e n t a t 30 days. A few r e a c t i v e a s t r o c y t e s remained i n t h e VPL n u c l e u s o f t h e thalamus, but l i t t l e n e c r o s i s o r c y s t i c s c a r r i n g was v i s i b l e i n any a r e a o f th e b r a i n . Double Immunostaininq Many o f t h e c e l l s which s t a i n e d p o s i t i v e l y f o r c l a s s I and I a a n t i g e n s e x h i b i t e d c l a s s i c a l r e a c t i v e m i c r o g l i a morphology. Some c e l l s , however, were s t a r - l i k e i n appearance, c l o s e l y r e s e m b l i n g a s t r o c y t e s . I n o r d e r t o h e l p c l a r i f y t h e c e l l p o p u l a t i o n s e x p r e s s i n g MHC a n t i g e n s , t i s s u e was d o u b l y s t a i n e d w i t h GFAP f o r a s t r o c y t e s and MHC I or I I . I n a l l s e c t i o n s examined (from a l l t i m e p e r i o d s ) , double s t a i n i n g r e v e a l e d two s e p a r a t e c e l l p o p u l a t i o n s . There was no o v e r l a p between a s t r o c y t e s (GFAP+) and MHC c l a s s I or I I p o s i t i v e c e l l s ( F i g . 2 4 ) . 58 F i g . 2 4 . S e c t i o n t h r o u g h t h e VPM t h a l a m i c n u c l e u s o f an a n i m a l 8 days a f t e r i n o c u l a t i o n . GFAP (brown) and MHC c l a s s I I (purple) double s t a i n i n g r e v e a l s a s t r o c y t e s t o be n e g a t i v e f o r MHC g l y c o p r o t e i n s . (B=-4.3 mm) Bar=50 |im. 59 DISCUSSION In t h e p r e s e n t s t u d y , MHC e x p r e s s i o n was examined i n t h e r a t CNS f o l l o w i n g a herpes v i r a l i n f e c t i o n . To summarize b r i e f l y p r e d i c t i o n s based on t h e l i t e r a t u r e , i t seemed l i k e l y t h a t HSV1 r e a c t i v i t y would be n o t e d i n i t i a l l y i n t h e t r i g e m i n a l n u c l e u s , t h a t t h e i n f e c t i o n would s p r e a d t o o t h e r b r a i n r e g i o n s , and t h a t b o t h neurons and g l i a l c e l l s would be i n f e c t e d . A n t i b o d i e s a g a i n s t MHC c l a s s I and I I a n t i g e n s were used t o examine t h e b r a i n r e g i o n s , and c e l l t y p e s w i t h i n t h o s e r e g i o n s , which e x p r e s s MHC m o l e c u l e s . I t seemed l i k e l y t h a t MHC e x p r e s s i o n would be found w i t h i n t h e CNS and t h a t t h e c e l l t y p e ( s ) e x p r e s s i n g MHC a n t i g e n s would be n o n - n e u r o n a l . Double im m u n o s t a i n i n g c o u l d c l a r i f y t h e a n t i g e n p r e s e n t i n g c e l l t y p e s . To a l a r g e e x t e n t , t h e r e s u l t s o f t h e p r e s e n t s t u d y c l o s e l y f o l l o w e d t h e p r e d i c t i o n s . HSV1 B e h a v i o r a l changes c h a r a c t e r i s t i c o f an e n c e p h a l i t i s were e v i d e n t 6-7 days a f t e r i n o c u l a t i o n . The v i r u s was i n i t i a l l y found i n t h e f i b e r s and s e n s o r y n u c l e u s o f t h e f i f t h nerve and p r o g r e s s e d r a p i d l y t o o t h e r b r a i n r e g i o n s . These e a r l y s t a g e s o f i n f e c t i o n c l e a r l y demonstrated c e l l t o c e l l s p r e a d o f t h e v i r u s . E f f e r e n t p r o j e c t i o n s from t h e t r i g e m i n a l n u c l e u s have been documented ( F i g . 2 5 ) . Trigeminal Pathways 60 (From Haines, 1987) 61 Fig.25 Efferent Projections from the Trigeminal Nucleus Abbreviations AbdNu Abducens Nucleus ALS A n t e r o l a t e r a l System CC Crus C e r e b r i CSNu C h i e f Sensory Nucleus DTTr D o r s a l T r i g e m i n o t h a l a m i c T r a c t FacNu F a c i a l N u cleus HyNu H y p o g l o s s a l N ucleus IC I n t e r n a l C a p s u l e Man.V M a n d i b u l a r D i v i s i o n o f T r i g e m i n a l Nerve Max.V M a x i l l a r y D i v i s i o n o f T r i g e m i n a l Nerve OpTh.V Ophthalmic D i v i s i o n o f T r i g e m i n a l Nerve RB R e s t i f o r m Body RNu Red Nu c l e u s SN S u b s t a n t i a N i g r a SpTNu S p i n a l T r i g e m i n a l N ucleus SpTT S p i n a l T r i g e m i n a l T r a c t TriMoNu T r i g e m i n a l Motor Nucleus VPL V e n t r a l P o s t e r o l a t e r a l N ucleus o f Thalamus VPM V e n t r a l P o s t e r o m e d i a l N u c l e u s o f Thalamus VTTr V e n t r a l T r i g e m i n o t h a l a m i c T r a c t (From H a i n e s , 1987) Not Shown i n Diagram: C r o s s e d p r o j e c t i o n s t o t h e - Caudate/Putamen ( Y a s u i , 1987) - C e r e b e l l u m ( B r o d a l , 1981) - Cornea ( C a r p e n t e r e t al.j_ 1983) - D o r s a l Motor N u c l e u s o f X (C a r p e n t e r e t a l . , 1983) - Nucleus Ambiguus (C a r p e n t e r e t a l . , 1983) - Nucleus o f S o l i t a r y T r a c t (Menetrey e t a l . , 1987) - S u p e r i o r S a l i v a t o r y N u c l e u s ( C a r p e n t e r e t a l . , 1983) 62 HSV1 i m m u n o r e a c t i v i t y was c l e a r l y v i s i b l e w i t h i n a l l p r o j e c t i o n a r e a s of t h e s e n s o r y n u c l e u s of c r a n i a l n erve V by 8 days p o s t - i n o c u l a t i o n . S t a i n i n g was b i l a t e r a l a t t h i s s t a g e i n t h e s e b r a i n r e g i o n s e xcept i n t h e f r o n t o p a r i e t a l c o r t e x ( s omatosensory), where s t a i n i n g was s o l e l y c o n t r a l a t e r a l ( F i g . 9 ) . E f f e r e n t p r o j e c t i o n s from p o s i t i v e l y l a b e l e d c e l l s w i t h i n t h e VPM n u c l e u s of t h e thalamus t o t h i s c o r t i c a l r e g i o n , e x p l a i n t h e p r e s e n c e of HSV1 i m m u n o r e a c t i v i t y i n t h i s a r e a ( F i g . 7 ) . W i t h i n th e i n f e r i o r c e r e b e l l a r p e d u n c l e , s t a i n i n g was i p s i l a t e r a l a t 6 days but had become b i l a t e r a l a t 8 days, s u g g e s t i n g b i l a t e r a l p r o g r e s s i o n o f t h e v i r u s t o t h e c e r e b e l l u m from t h e t r i g e m i n a l n u c l e u s ( s p i n a l p o r t i o n ) . A f f e r e n t p r o j e c t i o n s t o t h e s p i n a l t r i g e m i n a l n u c l e u s o r i g i n a t e c o n t r a l a t e r a l l y from t h e m e d i a l and d e s c e n d i n g v e s t i b u l a r n u c l e i , p e r i h y p o g l o s s a l n u c l e i , and motor c o r t e x ( B r o d a l , 1981; Walberg e t a l . , 1985). HSV1 i m m u n o r e a c t i v i t y was c l e a r l y v i s i b l e i n t h e i p s i l a t e r a l t r i g e m i n a l axons as w e l l as b i l a t e r a l l y i n t h e s u p e r i o r , m e d i a l and d e s c e n d i n g v e s t i b u l a r n u c l e i , and p e r i h y p o g l o s s a l n u c l e i by 8 days p o s t - i n o c u l a t i o n . There was no v i s i b l e s t a i n i n g e v i d e n t w i t h i n t h e f r o n t o p a r i e t a l motor c o r t e x a t any s t a g e . Lack of s t a i n i n g i n t h i s r e g i o n may be t h e r e s u l t o f v a r i a b i l i t y i n e i t h e r s t a i n i n g or i n f e c t i o n , or i n b o t h . A number of b r a i n r e g i o n s were v i s i b l y i n f e c t e d w hich r e p o r t e d l y do not r e c e i v e e i t h e r p r o j e c t i o n s from or send a f f e r e n t s t o t h e s p i n a l t r i g e m i n a l n u c l e u s . E x t e n s i v e HSV1 i m m u n o r e a c t i v i t y was n o t e d i n t h e hypothalamus and moderate 63 l e v e l s were n o t e d i n t h e s e p t a l a r e a . A s m a l l number o f p o s i t i v e l y l a b e l e d c e l l s were e v i d e n t w i t h i n t h e a n t e r i o r o l f a c t o r y n u c l e u s which l i k e l y r e f l e c t s e n t r y o f t h e v i r u s i n t o t h e n a s a l pathway v i a t h e t e a r d u c t . Once i n s i d e t h e o l f a c t o r y pathway, t h e v i r u s most l i k e l y s p r e a d t o nearby l i m b i c s t r u c t u r e s , such as s e p t a l n u c l e i and p r i m a r y o l f a c t o r y c o r t e x ( F i g . 1 0 ) . These r e s u l t s suggest t h a t , a l t h o u g h herpes v i r u s t r a v e l s p r i m a r i l y v i a r e t r o g r a d e and t o a l e s s e r degree a n t e r o g r a d e a x o n a l t r a n s p o r t , o t h e r modes of t r a v e l must be c o n s i d e r e d . The v i r u s might bud i n t o t h e e x t r a c e l l u l a r space a l l o w i n g e n t r y i n t o t h e CSF; however l i t t l e e v i d e n c e o f HSV1 s t a i n i n g was p r e s e n t around v e n t r i c u l a r a r e a s . A b l o o d - b o r n e i n f e c t i o n from t h e p e r i p h e r y must a l s o be c o n s i d e r e d . P e r i p h e r a l organs ( s p l e e n , thymus, l i v e r ) were n e g a t i v e l y s t a i n e d f o r HSV1 but t h i s might i n d i c a t e e i t h e r an u n d e t e c t a b l e l e v e l o f v i r u s o r l a c k o f a b l o o d - b o r n e i n f e c t i o n . The w i d e s p r e a d d i s t r i b u t i o n o f HSV1 i m m u n o r e a c t i v i t y d u r i n g t h e l a t e r s t a g e s o f i n f e c t i o n s u g g e s t s t h a t t h e v i r u s was t r a v e l i n g v i a a number o f r o u t e s , i n c l u d i n g u n d i s c o v e r e d a f f e r e n t and e f f e r e n t s p i n a l t r i g e m i n a l c o n n e c t i o n s , as w e l l as secondary and t e r t i a r y c o n n e c t i o n s . In a number o f t i s s u e s e c t i o n s , neurons as w e l l as s m a l l , round c e l l s were p o s i t i v e l y s t a i n e d f o r HSV1. The s i z e , shape, and t i m e o f appearance o f t h e round c e l l s were c o n s i s t e n t w i t h a l e u k o c y t e p r o f i l e . L e u k o c y t e s are known t o e n t e r t h e t i s s u e d u r i n g e a r l y s t a g e s o f t h e i n f e c t i o n (Johnson, 1982) . A d d i t i o n a l l y , l a c k o f v i s i b l e p r o c e s s e s and t h e n o n - l i n e a r 64 d i s t r i b u t i o n o f t h e s e c e l l s ( o l i g o d e n d r o c y t e s o f t e n have a l i n e a r d i s t r i b u t i o n ) s u g g e s t e d t h a t t h e y were not g l i a l c e l l s . C o n s i s t e n t w i t h t h e s e r e s u l t s , o t h e r s t u d i e s have demonstrated i n f e c t e d neurons f o l l o w i n g HSV1 i n o c u l a t i o n (Lycke e t a l . , 1984, Vah l n e e t a l . , 1978, U g o l i n i e t a l . , 1989). In c o n t r a s t t o t h e p r e s e n t s t u d y , however, o t h e r i n v e s t i g a t o r s have r e p o r t e d t h e pr e s e n c e o f i n f e c t e d g l i a l c e l l s f o l l o w i n g i n o c u l a t i o n (Lycke e t a l . , 1984, Vahlne e t a l . , 1978). T h i s d i s c r e p a n c y may be due t o t h e i n a b i l i t y t o i d e n t i f y c l e a r l y c e l l t y p e s w i t h i m m u n o s t a i n i n g and t h e p o s s i b i l i t y o f f a l s e n e g a t i v e s f o l l o w i n g s t a i n i n g . L a t e r s t a g e s (days 12 and 30) w i t h l i t t l e o r no p o s i t i v e HSV1 s t a i n i n g , l i k e l y r e f l e c t e d e i t h e r t h e d i s a p p e a r a n c e o f a c t i v e v i r u s o r t h e p r e s e n c e of l a t e n t v i r u s which t h e a n t i b o d y was unable t o r e c o g n i z e . I t s h o u l d be n o t e d t h a t each a n i m a l i n t h e s e l a t t e r two t i m e p e r i o d s had r e c o v e r e d from t h e e n c e p h a l i t i s and appeared h e a l t h y p r i o r t o b e i n g s a c r i f i c e d . I t i s c o n c e i v a b l e , t h e r e f o r e , t h a t t h e s e a n i m a l s may not have s u f f e r e d as s e v e r e an i n f e c t i o n as t h e o t h e r a n i m a l s . A d d i t i o n a l l y , t h e r e was e v i d e n c e o f n e c r o s i s 12 days a f t e r i n o c u l a t i o n but no e v i d e n c e o f n e c r o s i s a t 30 days. I t i s u n l i k e l y t h a t n e c r o s i s would have d i s a p p e a r e d w i t h i n t h i s t i m e p e r i o d . MHC A n t i g e n s U n t i l r e c e n t l y , l a c k o f MHC e x p r e s s i o n i n t h e CNS was c o n s i d e r e d e v i d e n c e i n s u p p o r t o f i m m u n o l o g i c a l p r i v i l e g e . As r e v i e w e d i n t h e i n t r o d u c t i o n , however, i n v i t r o and i n v i v o s t u d i e s p r o v i d e e v i d e n c e i n d i c a t i n g t h a t MHC e x p r e s s i o n does 65 o c c u r under a p p r o p r i a t e c o n d i t i o n s . T h i s s t u d y c l e a r l y d e m onstrated such e x p r e s s i o n i n v i r a l l y i n f e c t e d r a t b r a i n . MHC p o s i t i v e r e a c t i v e m i c r o g l i a l c e l l s were m u l t i p o l a r and s t a r - s h a p e d , w i t h an e l o n g a t e d , s l i g h t l y s w o l l e n soma. They were a p p r o x i m a t e l y 15-20 |lm i n d i a m e t e r , w i t h many s w o l l e n p r o c e s s e s . T h i s p r o f i l e i s c o n s i s t e n t w i t h t h a t f o r r e a c t i v e m i c r o g l i a as o r i g i n a l l y d e s c r i b e d by d e l R i o H o r t e g a , (1919). U s i n g t h e s i l v e r c a r b o n a t e method, d e l R i o Hortega d e s c r i b e d t h e r e a c t i v e m i c r o g l i a as u n i - b i - , o r m u l t i p o l a r w i t h an e l o n g a t e d o r t r i a n g u l a r soma. T h i s c e l l t y p e p r o g r e s s e s t h r o u g h a number o f s t a g e s i n v o l v i n g s w e l l i n g o f t h e soma and p r o c e s s e s as w e l l as v e s i c u l a t i o n o f t h e c y t o p l a s m . E x t e n s i o n s a r e g r a d u a l l y r e t r a c t e d and numerous membrane-bound phagosomes become e v i d e n t . The c e l l e v e n t u a l l y becomes amoeboid or round i n shape w i t h a foamy c y t o p l a s m i c appearance. A l t h o u g h i n t e r n a l c e l l u l a r s t r u c t u r e s were not v i s i b l e w i t h i m m unostaining, t h e c e l l t y p e d e s c r i b e d i n th e p r e s e n t s t u d y i s more th a n l i k e l y a m i c r o g l i a l c e l l . R e p r o d u c i b i l i t y o f s t a i n i n g from r a t t o r a t w i t h i n groups s a c r i f i c e d a t t h e same t i m e p e r i o d was h i g h l y c o n s i s t e n t . MHC c l a s s I and I I p o s i t i v e l y s t a i n e d m i c r o g l i a l c e l l s were e v i d e n t i n a number o f b r a i n r e g i o n s s i m i l a r l y p o s i t i v e f o r HSV1. D i f f e r e n c e s i n s t a i n i n g between c l a s s I and I I were apparent 6 days a f t e r i n o c u l a t i o n . Thalamic and h y p o t h a l a m i c n u c l e i , as w e l l as t h e s u p e r i o r c o l l i c u l u s , s u b i c u l u m , and E d i n g e r - W e s t p h a l n u c l e u s c o n t a i n e d c l a s s I but not c l a s s I I p o s i t i v e l y l a b e l e d m i c r o g l i a a t 6 days. The reasons why c l a s s I I e x p r e s s i o n l a g g e d b e h i n d c l a s s I a t t h i s s t a g e , are not c l e a r l y u n d e r s t o o d . By day 66 8, however, b o t h MHC a n t i g e n s were e q u a l l y d i s t r i b u t e d t h r o u g h o u t i n f e c t e d b r a i n r e g i o n s . S m a l l , round, MHC p o s i t i v e c e l l s were note d , p a r t i c u l a r l y i n t h e a r e a o f t h e t r i g e m i n a l n u c l e u s , d u r i n g days 6 and 8. S e r i a l s e c t i o n s s t a i n e d w i t h c r e s y l v i o l e t r e v e a l e d a l a r g e number o f t h e s e c e l l s t o be p o l y m o r p h o n u c l e a r (PMN) c e l l s w i t h c h a r a c t e r i s t i c m u l t i - l o b e d n u c l e i . I n t e r e s t i n g l y , a l t h o u g h a l a r g e number o f b r a i n a r e a s were HSV1 p o s i t i v e a t 10 days, t h e e n t i r e b r a i n d i d not show such s t a i n i n g but i t d i d show c l a s s I and I I e x p r e s s i o n . A d d i t i o n a l l y , a f t e r p o s i t i v e HSV1 s t a i n i n g was no l o n g e r v i s i b l e (day 12), c l a s s I and I I p o s i t i v e m i c r o g l i a c o n t i n u e d t o p o p u l a t e t h e b r a i n . There were, however, a few b r a i n a r e a s a t t h i s s t a g e which had a s l i g h t d e c r e a s e i n t h e number o f p o s i t i v e l y s t a i n i n g m i c r o g l i a . B oth MHC a n t i g e n s were s t i l l v i s i b l e i n a few s e l e c t b r a i n r e g i o n s 30 days a f t e r i n o c u l a t i o n . N o t a b l y , c l a s s I I p o s i t i v e m i c r o g l i a were v i s i b l e w i t h i n t h e o p t i c chiasm, which was not p o s i t i v e f o r HSV1 a t any s t a g e . One p o s s i b l e e x p l a n a t i o n f o r t h i s d i s c r e p a n c y may be t h e l i m i t a t i o n s o f t h e t e c h n i q u e . Immunostaining can produce f a l s e n e g a t i v e s . And, as mentioned e a r l i e r , t h i s a n i m a l may have s u f f e r e d m i n i m a l i n f e c t i o n , which c o u l d a l s o l e a d t o d i s c r e p a n c i e s i n s t a i n i n g . These r e s u l t s suggest t h a t between 8 and 10 days, MHC e x p r e s s i o n was no l o n g e r c o n f i n e d t o v i s i b l y i n f e c t e d a r e a s , i n d i c a t i n g a s y s t e m i c CNS immune response was underway. T h i s v i g o r o u s response was s t i l l p r e s e n t a t 12 days, a l t h o u g h most o f th e v i r u s was a p p a r e n t l y no l o n g e r a c t i v e i n t h i s presumably r e c o v e r e d r a t . S i g n s o f immune r e a c t i v i t y were s t i l l v i s i b l e 30 67 days f o l l o w i n g i n o c u l a t i o n . The t i m e p a t t e r n f o r MHC e x p r e s s i o n i n t h e p r e s e n t s t u d y was s i m i l a r t o o t h e r CNS i n s u l t s (Akiyama e t a l . , 1988) . A l t h o u g h p r e v i o u s s t u d i e s have not examined MHC e x p r e s s i o n i n t h e CNS f o l l o w i n g HSV1 i n f e c t i o n , a few s t u d i e s have r e p o r t e d MHC p o s i t i v e r e a c t i v e m i c r o g l i a f o l l o w i n g v a r i o u s i n s u l t s (Akiyama e t a l . , 1988, A n t o n i o u e t a l . , 1987, H i c k e y e t a l . , 1987). F u n c t i o n a l l y , p e r i p h e r a l MHC c l a s s I e x p r e s s i o n i s known t o be i n v o l v e d i n v i r a l immunity (Lampson, 1987). C e l l - m e d i a t e d c y t o l y s i s o f v i r a l l y i n f e c t e d c e l l s i s thought t o o c c u r t h r o u g h t h r e e d i s t i n c t mechanisms: c y t o t o x i c T c e l l s , n a t u r a l c y t o t o x i c i t y , and a n t i b o d y dependent c e l l - m e d i a t e d c y t o t o x i c i t y ( S t i t e s e t a l . , 1987). D u r i n g a v i r a l i n f e c t i o n , v i r a l a n t i g e n s a r e " p r e s e n t e d " on t h e c e l l s u r f a c e o f t h e i n f e c t e d c e l l i n c o m b i n a t i o n w i t h a MHC c l a s s I a n t i g e n ( S t i t e s e t a l . , 1987) T h i s c o m b i n a t i o n i s t h e n r e c o g n i z e d by c y t o t o x i c T-lymphocytes p o s s e s s i n g r e c e p t o r s s p e c i f i c t o t h i s c o m b i n a t i o n . R e c o g n i t i o n t h e n i n i t i a t e s c e l l - m e d i a t e d l y s i s (CML) of t h e i n f e c t e d c e l l . T h i s a l l o w s f o r t h e r e l e a s e o f v i r a l progeny i n t o t h e e x t r a c e l l u l a r space which i n f e c t g l i a l c e l l s and i n i t i a t e f u r t h e r i m m u n o l o g i c a l r e s p o n s e s . T - h e l p e r c e l l s and a n t i b o d y p r o d u c i n g plasma c e l l s p l a y an i m p o r t a n t r o l e i n e l i m i n a t i n g t h e v i r u s d u r i n g t h i s s t a g e . C l a s s I e x p r e s s i o n i s c l e a r l y i n s t r u m e n t a l i n t h e i m m u n o l o g i c a l d e f ense p r o c e s s d u r i n g p e r i p h e r a l HSV1 i n f e c t i o n . C l a s s I e x p r e s s i o n i n t h e CNS may be i n d i c a t i v e o f s i m i l a r immune r e a c t i v i t y . 68 The functional significance of peripheral class II expression during HSV1 i n f e c t i o n d i f f e r s from that of class I expression (Lampson, 1987) . Class II antigen presentation i s also integral.to the recognition process, but recognition i s followed by a series of events functionally d i f f e r e n t from class I antigen presentation. Antigen presentation occurs when a foreign antigen i s consumed by an antigen presenting c e l l (APC), often a macrophage, r e s u l t i n g i n presentation of pieces of the foreign p a r t i c l e on the c e l l surface i n combination with a class II molecule. This combination i s then recognized by T-helper lymphocytes possessing receptors s p e c i f i c for t h i s combination. Once recognition occurs, an immune cascade begins. T-helper c e l l s i n i t i a t e cloning of additional T-helper and T-cytotoxic c e l l s , as well as stimulating B - c e l l s to d i f f e r e n t i a t e into antibody-producing plasma c e l l s . Large amounts of antibody s p e c i f i c to the foreign antigen are produced i n an attempt to eliminate the invading antigen. Humoral antibody production i s demonstratable in both peripheral and central HSV1 inf e c t i o n , but offers very l i t t l e protection (Johnson, 1982, Lampson, 1987). Due to i t s a b i l i t y to tr a v e l transynaptically, HSV1 i s largely protected from antibody in the surrounding e x t r a c e l l u l a r f l u i d . However, i t i s apparent that MHC class II expression i s an active component during v i r a l i n f e c t i o n . Based on the known functions of both class I and II MHC antigens in the peripheral immune response, expression of these molecules i n the CNS suggests a similar pattern of immune r e a c t i v i t y . 69 GFAP GFAP s t a i n i n g r e v e a l e d p a t h o l o g i c a l changes i n a s t r o c y t e s c h a r a c t e r i s t i c o f a v i r a l i n f e c t i o n (Johnson, 1982, V i k e n e t a l . , 1978). S t a i n i n g i n t e n s i t y was i n c r e a s e d , n u c l e i were s w o l l e n , and th e number o f GFAP p o s i t i v e c e l l s had i n c r e a s e d . The somas were a p p r o x i m a t e l y 15-20 |im i n d i a m e t e r and a s t r o c y t i c p r o c e s s e s were s l i g h t l y wispy i n appearance. R e a c t i v e a s t r o c y t e s , as d e s c r i b e d by M o r i and L e b l o n d , show an enlargement o f t h e soma w i t h a s l i g h t l y e n l a r g e d , e c c e n t r i c a l l y l o c a t e d n u c l e u s (Mori and L e b l o n d , 1969) . The p r o c e s s e s l o s e t h e i r s h e e t - l i k e e x p a n s i o n s and become t u b u l a r and wispy i n appearance. G l i a l f i b r i l l a r y a c i d i c p r o t e i n i s a l s o g r e a t l y i n c r e a s e d . A l t h o u g h i n t e r n a l c e l l u l a r changes were not c l e a r l y v i s i b l e i n t h e c e l l s i d e n t i f i e d i n t h e p r e s e n t s t u d y , t h e i r morphology i s c o n s i s t e n t w i t h t h a t o f r e a c t i v e a s t r o c y t e s . These changes i n a s t r o c y t e morphology were e v i d e n t i n areas of HSV1 r e a c t i v i t y and p e r s i s t e d t h r o u g h day 12. At 10 days, s m a l l n e c r o t i c - l i k e a r e a s c o u l d be seen i n a few b r a i n r e g i o n s and some n e c r o s i s was e v i d e n t a t 12 days. No n e c r o s i s o r c y s t i c s c a r r i n g was v i s i b l e a t 30 days, but MHC r e a c t i v e m i c r o g l i a were p r e s e n t , s u g g e s t i n g some l e v e l o f immune a c t i v i t y . The l a c k o f n e c r o s i s and c y s t i c s c a r r i n g a t t h i s s t a g e , however, s u g g e s t s t h a t t h e i n f e c t i o n i n t h i s a n i m a l may have been m i n i m a l . M i c r o g l i a In t h e p r e s e n t s t u d y , t h e main CNS c e l l t y p e s t a i n i n g p o s i t i v e l y f o r b o t h MHC a n t i g e n s was t h e r e a c t i v e m i c r o g l i a - l i k e c e l l . T h i s was m o r p h o l o g i c a l l y d e t e r m i n e d based on c l a s s i c a l 70 d e s c r i p t i o n s o f t h i s c e l l t y p e by d e l R i o Hortega and P e n f i e l d ( d e l R i o Hort e g a , 1919, P e n f i e l d , 1925). The two main i s s u e s s u r r o u n d i n g t h i s c o n t r o v e r s i a l c e l l t y p e a re a n t i g e n p r e s e n t a t i o n and t h e o r i g i n o f t h e s e c e l l s . As mentioned e a r l i e r , a n t i g e n p r e s e n t a t i o n i s e s s e n t i a l f o r i n i t i a t i o n o f t h e immune cascade, o r CMI. P e r i p h e r a l l y , APCs c o n s i s t l a r g e l y o f macrophages/monocytes and a few T and B lymphocytes. The q u e s t i o n a r i s e s as t o which c e l l o r c e l l s i n t h e CNS a r e p e r f o r m i n g t h i s f u n c t i o n . S t u d i e s i n v o l v i n g i n v i t r o work w i t h c u l t u r e d a s t r o c y t e s (Barna e t a l . , 1987, Fontana e t a l . , 1984, Male e t a l . , 1987, Massa e t a l . , 1986, 1987) and a n a l y s i s o f post-mortem MS b r a i n t i s s u e (Hofman e t a l . , 1986, T r a u g o t t , 1985, 1987) have r e p o r t e d c l a s s I I p o s i t i v e l y s t a i n e d a s t r o c y t e s . The a u t h o r s s t a t e t h a t t h e s e r e s u l t s suggest t h a t a s t r o c y t e s a r e s e r v i n g t h e f u n c t i o n o f a n t i g e n p r e s e n t a t i o n i n t h e CNS. Double s t a i n i n g t e c h n i q u e s were used i n t h e p r e s e n t s t u d y i n an attempt t o c l a r i f y t h i s i s s u e . A n t i b o d i e s a g a i n s t b o t h GFAP ( a s t r o c y t e s ) and MHC c l a s s I or I I d e t e r m i n e n t s (0X18,0X6) were used i n a double s t a i n i n g p r o c e d u r e t o examine t h e q u e s t i o n o f a n t i g e n p r e s e n t a t i o n . M u l t i p l e s e c t i o n s r e v e a l e d no o v e r l a p i n p o s i t i v e r e a c t i v i t y f o r GFAP and 0X18 or 0X6. Two d i s t i n c t c e l l p o p u l a t i o n s were p r e s e n t , i n d i c a t i n g a s t r o c y t e s were not e x p r e s s i n g c l a s s I o r I I a n t i g e n s on t h e i r s u r f a c e and t h e r e f o r e d i d not p o s s e s s a n t i g e n p r e s e n t a t i o n c a p a b i l i t i e s . These r e s u l t s have been r e p l i c a t e d i n our l a b o r a t o r y w i t h b o t h MS and A l z h e i m e r ' s b r a i n t i s s u e , and a f t e r KA l e s i o n i n g i n t h e r a t CNS (Akiyama e t a l . , 1988, McGeer e t a l . , 1987a, 1987b). The c e l l s 71 which did s t a i n p o s i t i v e l y for MHC class II were morphologically i d e n t i f i e d as reactive microglia. Many studies have demonstrated that MHC class I and II expression i s a function of immune c e l l s and not a function of ectodermally derived g l i a l c e l l s . Although currently a controversial c e l l type, microglia possess phagocytotic c a p a b i l i t i e s and are thought to be the brain macrophage (Akiyama et a l . , 1988, Jordan et a l . , 1988). Peripheral macrophage antigen presentation together with evidence of class II expression on microglia i n the CNS suggests functional s i m i l a r i t i e s between these two c e l l types. However, the o r i g i n of these brain macrophages remains elusive. Origin of Microglia Microglia are thought to be either part of the g l i a l family (neuroectodermal o r i g i n ; F u j i t a , 1981, Matsumoto et a l . , 1987, Schelper et a l . , 1986) or part of the mononuclear phagocyte system (hematogenous o r i g i n ; Akiyama et a l . , 1988, Dolman, 1985, E s i r i et a l . , 1986, Giulian, 1987, Hickey et a l . , 1988, Imamoto et a l . , 1978, Kitamura et a l . , 1972, 1980, Murabe et a l . , 1981, Oehmichen, 1980, Wood et a l . , 1979). Although during disease processes involving compromise to the blood-brain b a r r i e r , monocytes may enter the tissue and d i f f e r e n t i a t e into macrophages, i t i s not clear whether an endogenous g l i a l macrophage performing the same function also e x i s t s . A number of studies attempting to answer t h i s question have met with inconclusive r e s u l t s . One study, however, provides some important evidence i n support of a hematogenous o r i g i n (Akiyama et a l . , 1988). In t h i s study, CNS tissue from KA lesioned rats was 72 s t a i n e d w i t h a n t i b o d i e s a g a i n s t b o t h MHC c l a s s I I (0X6) and l e u k o c y t e common a n t i g e n (LCA). The r e s u l t s showed t h a t m i c r o g l i a and i n f i l t r a t i n g macrophages were p o s i t i v e l y s t a i n e d f o r b o t h 0X6 and LCA. G l i a l c e l l s o f n e u r o e c t o d e r m a l o r i g i n ( a s t r o c y t e s , o l i g o d e n d r o c y t e s ) d i d not s t a i n f o r e i t h e r o f t h e two a n t i b o d i e s i n a l l s e c t i o n s examined. The a u t h o r s suggest t h a t t h e r e s u l t s i n d i c a t e d i f f e r i n g gene e x p r e s s i o n between m i c r o g l i a and n e u r o g l i a and s i m i l a r i t i e s i n gene e x p r e s s i o n between m i c r o g l i a and macrophages. A l t h o u g h t h e s e r e s u l t s l e n d s u p p o r t f o r t h e hematogenous o r i g i n o f m i c r o g l i a , t h e c o n t r o v e r s y s t i l l r emains. Summary The p r e s e n t s t u d y demonstrated MHC e x p r e s s i o n i n t h e r a t CNS f o l l o w i n g a herpes v i r u s i n f e c t i o n . A d e t a i l e d map o f i n f e c t e d b r a i n a r e a s as w e l l as a comprehensive t i m e c o u r s e o f MHC e x p r e s s i o n were o u t l i n e d . An i n i t i a l PMN i n f i l t r a t e was f o l l o w e d by e l e v a t e d MHC a n t i g e n e x p r e s s i o n on m i c r o g l i a c e l l s i n and around HSV1 p o s i t i v e b r a i n a r e a s . D u r i n g l a t e r s t a g e s o f i n f e c t i o n , t h e e n t i r e b r a i n was d e n s e l y p o p u l a t e d w i t h r e a c t i v e m i c r o g l i a , even i n areas n e g a t i v e f o r HSV1. A s t r o c y t e s showed t y p i c a l p a t h o l o g i c a l changes, but d i d not e x p r e s s MHC a n t i g e n s a t any t i m e . A few s m a l l f o c i o f n e c r o s i s were e v i d e n t a t 10 and 12 days, but no e v i d e n c e o f n e c r o s i s was v i s i b l e a t 30 days p o s t -i n o c u l a t i o n , s u g g e s t i n g m i n i m a l i n f e c t i o n i n t h i s r a t . T h i s sequence o f e v e n t s c o n c e i v a b l y r e f l e c t s CMI and a s y s t e m i c CNS immune response, r e s u l t i n g i n p h a g o c y t o s i s o f t h e v i r u s w i t h l a t e n t v i r u s r e m a i n i n g i n t h e b r a i n . The immune system was 73 a p p a r e n t l y u n able t o overcome t h e v i r a l i n f e c t i o n i n a n i m a l s which d i d not r e c o v e r from t h e e n c e p h a l i t i s . The r e s u l t s from t h e p r e s e n t s t u d y are i m p o r t a n t f o r two main r e a s o n s . Most i m p o r t a n t l y , MHC e x p r e s s i o n on endogenous CNS elements f o l l o w i n g v i r a l i n f e c t i o n s u g g e s t s t h e c a p a c i t y f o r a n t i g e n p r e s e n t a t i o n and t h e i n i t i a t i o n o f CMI. These r e s u l t s i n d i c a t e t h a t MHC m o l e c u l e s p l a y an e s s e n t i a l r o l e i n CNS v i r a l c l e a r a n c e i n a manner s i m i l a r t o t h e p e r i p h e r y . S e c o n d l y , i t i s i m p o r t a n t t o u n d e r s t a n d f u r t h e r how t h e CNS responds t o v i r a l i n f e c t i o n . A c l e a r u n d e r s t a n d i n g o f t h e CNS response t o a v i r a l i n f e c t i o n may p r o v i d e i n f o r m a t i o n n e c e s s a r y f o r t h e r a p e u t i c i n t e r v e n t i o n , as w e l l as p r o v i d i n g a d d i t i o n a l i n f o r m a t i o n r e g a r d i n g t h e e t i o l o g y o f c e r t a i n CNS d i s e a s e s . The F u t u r e The r e s u l t s g l e a n e d from t h e p r e s e n t s t u d y suggest f u r t h e r e x a m i n a t i o n and m a n i p u l a t i o n o f t h e r e l e v a n t i s s u e s . Based on t h e l i m i t a t i o n s o f im m u n o h i s t o c h e m i s t r y , such as f a l s e n e g a t i v e s and d i f f i c u l t y i n d i s t i n g u i s h i n g i n t r a c e l l u l a r elements o r s t a i n e d s t r u c t u r e s , e l e c t r o n m i c r o s c o p y (EM) would p r o v i d e d e f i n i t i v e e v i d e n c e o f t h e f i n d i n g s put f o r t h i n t h e p r e s e n t s t u d y . A l t h o u g h a l s o somewhat l i m i t e d , EM would a i d i n c l a r i f i c a t i o n o f c e l l t y p e s e x p r e s s i n g MHC a n t i g e n s as w e l l as i n i d e n t i f i c a t i o n o f v i r a l p a r t i c l e s t h r o u g h o u t t h e b r a i n . To complete t h e p i c t u r e , e x a m i n a t i o n o f o t h e r i m p o r t a n t i m m u n o l o g i c a l markers may prove i n f o r m a t i v e . R e c e p t o r s f o r lymphokines, such as i n t e r l e u k i n - 2 , and f o r t h e v a r i o u s lymphocytes, such as T c e l l s u b s e t s and B c e l l s , can be 74 i m m u n o h i s t o c h e m i c a l l y l a b e l e d and examined f o r t h e i r r o l e i n t h e immune response t o v i r a l i n f e c t i o n . In t h i s same v e i n , human post-mortem t i s s u e from P a r k i n s o n ' s and A l z h e i m e r ' s p a t i e n t s has been examined f o r t h e s e i m m u n o l o g i c a l markers i n t h i s l a b o r a t o r y ( I t a g a k i e t a l . , 1987, 1988, McGeer e t a l . , 1988). The r e s u l t s suggest some form o f immunologic d i s t u r b a n c e p r i o r t o dea t h , y e t e v i d e n c e f o r v i r a l i n f e c t i o n i s s t i l l b e i n g sought. Post-mortem e x a m i n a t i o n o f herpes e n c e p h a l i t i s b r a i n t i s s u e may p r o v i d e i m p o r t a n t i n f o r m a t i o n r e g a r d i n g t h e i m m u n o l o g i c a l p r o c e s s e s i n v o l v e d i n a t t e m p t i n g t o c l e a r herpes v i r u s from t h e CNS. F i n a l l y , m a n i p u l a t i o n s d u r i n g t h e acu t e i n f e c t i o n may p r o v i d e u s e f u l i n f o r m a t i o n f o r t h e r a p e u t i c i n t e r v e n t i o n . Such m a n i p u l a t i o n s might i n v o l v e t h e use o f i n t e r l e u k i n s o r i n t e r f e r o n s t o a c c e l e r a t e o r a t t e n u a t e t h e immune re s p o n s e . A d d i t i o n a l l y , p h a r m a c o l o g i c a l m a n i p u l a t i o n o f t h e b l o o d - b r a i n b a r r i e r may a l s o have an e f f e c t on t h e c o u r s e o f t h e i n f e c t i o n ( M o k h t a r i a n e t a l . , 1984). I t i s i m p o r t a n t t o examine b o t h b a s i c s c i e n t i f i c q u e s t i o n s as w e l l as c l i n i c a l l y r e l a t e d q u e s t i o n s i n o r d e r t o u n d e r s t a n d f u l l y t h e p r o c e s s e s i n v o l v e d i n v i r a l i n f e c t i o n s o f t h e CNS and t h e i r p o s s i b l e r e l a t i o n t o n e u r o l o g i c a l d i s o r d e r s . 75 B i b l i o g r a p h y A b b o t t , R., B o l d e r s o n , I . , Gruer, P., and P e a t f i e l d , R. (1987) . Immunoreactive IFN-gamma i n CSF i n n e u r o l o g i c a l d i s o r d e r s , j N e u r o l N e u r o s u r a P s y c h i a t r y , 50:882-885. Ahokas, A. (1986). Immunoglobulin and C3 a b n o r m a l i t i e s i n ac u t e p s y c h i a t r i c d i s o r d e r s . A c t a P s v c h i a t r s c a n d , 74:542-547. Akiyama, H., I t a g a k i , S., and McGeer, P. ( 1 9 8 8 ) . 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MHC a n t i g e n e x p r e s s i o n on b u l k i s o l a t e d m a c r o p h a g e - m i c r o g l i a from newborn mouse b r a i n : i n d u c t i o n o f I a a n t i g e n e x p r e s s i o n by gamma-interferon. J Neuroimmunol, 15:262—278. T i n g , J . , Shigekawa, B., L i n t h i c u m , D., Weiner, L., and F r e l i n g e r , J . . (1981). E x p r e s s i o n and s y n t h e s i s o f murine immune r e s p o n s e - a s s o c i a t e d (Ia) a n t i g e n s by b r a i n c e l l s . Immunol, 78:3170-3174. T i n g , J . , T a k i g u c h i , M., M a c c h i , M., and F r e l i n g e r , J . (1987) . The e x p r e s s i o n and d e t e c t i o n o f MHC c l a s s I a n t i g e n s on murine n e u r o b l a s t o m a and ependymoblastoma l i n e s . J Neuroimmunol, 14:87-98. Tomlinson, A. and E s i r i , M. (1983). Herpes s i m p l e x e n c e p h a l i t i s i m m u n o h i s t o l o g i c a l d e m o n s t r a t i o n o f s p r e a d o f v i r u s v i a o l f a c t o r y pathways i n mice. J N e u r o l S c i , 60:473-484. Townsend, J . (1985). Macrophage response t o herpes s i m p l e x e n c e p h a l i t i s i n immune competent and T c e l l - d e f i c i e n t mice. J Neuroimmunol, 7:195-206. 85 Traugott, U. and Raine, C. (1984). Further lymphocyte characterization i n the central nervous system i n multiple s c l e r o s i s . Ann NY Acad S c i , 436:163-180. Traugott, U. and Raine, C. (1985). Multiple s c l e r o s i s . Evidence for antigen presentation i n s i t u by endothelial c e l l s and astrocytes, j N e u r o l S c i , 69:365-370. Traugott, U. (1987). Multiple s c l e r o s i s : Relevance of class I and class II MHC-expressing c e l l s to l e s i o n development. J Neuroimmunoi, 16:283-302. Ugolini, G., Kuypers, H., and Strick, P. (1989). 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( 1 9 7 9 ) . The f a i l u r e of microglia i n normal brain to exhibit mononuclear phagocyte markers. J N e u r o p a t h o i Exp  N e u r o l , 3 8 : 3 6 9 - 3 7 6 . Woodroofe, M., Bellamy, A., Feldman, M., Davison, A., and Cuzner, M. (1986) . Immunocytochemical c h a r a c t e r i s t i c s of the immune reaction i n the central nervous system i n multiple s c l e r o s i s . Possible role for microglia i n lesion growth, j N e u r o l S c i , 7 4 : 1 3 5 - 1 5 2 . Yasui, Y., Itoh, K., and Mizuno, N. ( 1 9 8 7 ) . Direct projections from the caudal spinal trigeminal nucleus to the striatum in the cat. Brain Res, 4 0 8 : 3 3 4 - 3 3 8 . Zeman, W. (1978) . Subacute sclerosisng panecephalitis and paramyxovirus i n f e c t i o n s . In: Handbook o f c l i n i c a l  N e urology, I n f e c t i o n s i n t h e Nervous System, part II, Vol. 34, North-Holland Publishing. Amsterdam. Honors/Awards/Publications 1982-1987 1982-1987 1987 1987 1986- 1987 1986 1985-1986 1987- 1988 1988 Honors Psychology Undergraduate Dean's Honor L i s t Magna Cum Laude Phi Beta Kappa Sigma Xi President's Undergraduate Award for Research Undergraduate Research Summer Stipend University Graduate Fellowship Health Sciences Research Day Microbiology/Immunology Award Publications 1) D.L. Weinstein, G.D. E l l i s o n , R. See, and E. Levin (1986). A Model For Tardive Dyskinesia: The Effects of Chronic Fluphenazine Deconate on Guinea Pigs. Proc. West. Pharmacol. S o c , 29:405-407. 2) D.L. Weinstein, G.D. E l l i s o n , E. Levin, and R. See (1986). Dyskinetic Oral Movements in Guinea Pigs Following Chronic Neuroleptic Treatment. Soc.  Neurosci. Ann. Meet. Abst., 8.:1203. 3) D.L. Weinstein, D.R. Walker, and P.L. McGeer (1988). Major Histocompatibility Antigens i n The CNS of The V i r a l l y Infected Rat. Soc. Neurosci. Ann.  Meet. Abst., 14:1301. 4) D.L. Weinstein, R.E. See, and G. E l l i s o n (1989). Delayed Appearance of F a c i a l Tics Following Chronic Fluphenazine Administration to Guinea Pigs. Pharmacol, Biochem, & Behavior, A p r i l , In Press. 

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