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The effect of electrolytic lesion and neural implants on glial fibrillary acidic protein expression in… Falconer, Robert J. 1989

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THE EFFECT OF ELECTROLYTIC LESION AND NEURAL IMPLANTS ON GLIAL FIBRILLARY ACIDIC PROTEIN EXPRESSION IN THE RAT SPINAL CORD By Robert J . Falconer B.A. , B . S c , The U n i v e r s i t y of B r i t i s h Columbia A THESIS IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF i n THE FACULTY OF GRADUATE STUDIES (Physiology) We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October 1989 ( c ) Robert J . Falconer, 1989 MASTER OF SCIENCE 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 The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT T h i s t h e s i s a s s e s s e d t h e s u i t a b i l i t y o f u n i l a t e r a l , e l e c t r o l y t i c l e s i o n s as a model o f s p i n a l c o r d damage and r e p a i r i n t h e a d u l t r a t . T h i s t y p e o f l e s i o n r e s u l t e d a c u t e l y i n l o c a l i z e d damage i n t h e upper motor neuron a t t h e L2-L3 l e v e l o f t h e s p i n a l c o r d . M i n i m a l a c u t e damage t o a s c e n d i n g s e n s o r y pathways was i n d i c a t e d by p r e s e r v e d somatosensory evoked p o t e n t i a l s e l i c i t e d by s t i m u l a t i o n o f t h e t i b i a l n e r v e . I m m e d i a t e l y a f t e r l e s i o n g e n e r a t i o n one o f s e v e r a l s u b s t r a t e s was i n j e c t e d i n t o t h e l e s i o n c a v i t y . These s u b s t r a t e s were s a l i n e b u f f e r , l i q u i d c o l l a g e n s o l u t i o n , f o e t a l s p i n a l c o r d c e l l s from 14 day o l d r a t embryos, and a m i x t u r e o f c o l l a g e n and E 14 f o e t a l s p i n a l c o r d c e l l s . The 4 groups were compared f o r f u n c t i o n a l r e c o v e r y o v e r 3 months u s i n g t h e i n c l i n e d p l a n e t e s t and a T a r l o v movement s c a l e . A f t e r s a c r i f i c e , t h e t i b i a l i s a n t e r i o r m u s c l e s were d i s s e c t e d and weighed t o a s s e s s a t r o p h y due t o l o w e r motor neuron i n j u r y . A f t e r removing and embedding t h e s p i n a l c o r d s i n p a r a f f i n , t r a n s v e r s e and l o n g i t u d i n a l s e c t i o n s were t a k e n f o r c y t o a r c h i t e c t u r a l i n v e s t i g a t i o n . C r e s y l v i o l e t was u s e d t o i n d i c a t e N i s s l s u b s t a n c e , L u x o l f a s t b l u e s t a i n e d f o r m y e l i n and 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) a n t i b o d y r e v e a l e d t h e e x p r e s s i o n o f GFAP i n t h e c o r d s e c t i o n s . C h r o n i c e l e c t r o l y t i c l e s i o n s were c h a r a c t e r i z e d by t h e h i g h l y v a r i a b l e degree o f c a v i t a t i o n , d e m y e l i n a t i o n and macrophage i n f i l t r a t i o n t h a t was p r e s e n t . There was no s i g n i f i c a n t performance d e f i c i t on t h e i n c l i n e d p l a n e t e s t i n any o f t h e l e s i o n e d groups when compared t o u n o p e r a t e d a n i m a l s . The t i b i a l i s m u s c l e s from a l l groups were o f normal w e i g h t , i n d i c a t i n g t h a t t h e l o w e r motor neurons were n o t s i g n i f i c a n t l y damaged by t h e l e s i o n s used. There was, however, a marked d e c r e a s e i n t h e number o f GFAP r e a c t i v e a s t r o c y t e s i n t h e l e s i o n e d a n i m a l s when compared t o u n l e s i o n e d c o n t r o l s (P < 0.01, W i l c o x o n t e s t ) . Moreover, t h i s r e d u c t i o n o f GFAP - l i k e i m m u n o r e a c t i v i t y was n o t p r e v e n t e d by i m p l a n t s o f f o e t a l neurons, c o l l a g e n o r f o e t a l neurons suspended i n c o l l a g e n . P o s s i b l e e x p l a n a t i o n s f o r t h e re d u c e d GFAP - l i k e i m m u n o r e a c t i v i t y seen i n a l l e l e c t r o l y t i c a l l y l e s i o n e d c o r d s a r e d i s c u s s e d . i i 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 T a b l e o f C o n t e n t s i i i L i s t o f T a b l e s i v L i s t o f F i g u r e s v Acknowledgements v i I n t r o d u c t i o n 1 -Mammalian S p i n a l Cord Anatomy 2 - C l i n i c a l E f f e c t s o f S p i n a l C o r d L e s i o n s 4 - S u r g i c a l P r o c e d u r e s f o r t h e R e p a i r o f S p i n a l C o r d L e s i o n s 6 - R e p a i r o f S p i n a l C o r d F u n c t i o n U s i n g C o l l a g e n G e l s 7 - R e p a i r o f S p i n a l C o r d F u n c t i o n U s i n g N e u r a l I m p l a n t s .... 8 - E x p e r i m e n t a l Models o f S p i n a l Cord Damage 9 -The E x p e r i m e n t a l R a t i o n a l e 11 Methods and M a t e r i a l s 12 - S u r g e r y 12 - L e s i o n G e n e r a t i o n 13 -Somatosensory Evoked P o t e n t i a l R e c o r d i n g 14 - C o l l a g e n I m p l a n t s 15 - S p i n a l C o r d C e l l / C o l l a g e n I m p l a n t s 16 - I n j e c t i n g t h e Implant 16 - B e h a v i o u r a l T e s t i n g o f S p i n a l Cord F u n c t i o n 17 - H i s t o l o g y 21 -Immunocytochemistry 22 -GFAP C o n t r o l s 23 - A n a l y s i s o f a n t i - GFAP R e a c t i v e C e l l s 23 - T i b i a l i s M u s c l e Weights 24 -Acute L e s i o n Measurement 24 -The E x p e r i m e n t a l Groups 25 R e s u l t s 25 -Normal Anatomy 26 -Acute E l e c t r o l y t i c L e s i o n s 26 -Somatosensory Evoked P o t e n t i a l s 35 - H i s t o l o g i c a l Appearance o f C h r o n i c E l e c t r o l y t i c L e s i o n s 44 - C o l l a g e n I m p l a n t 51 - F o e t a l C e l l I m p l a nt 51 -Assessment o f V o l u n t a r y Motor Performance 58 - I n f l u e n c e o f C h r o n i c L e s i o n s on T i b i a l i s M u s c l e Weights 63 - D i s t r i b u t i o n o f GFAP i n Normal S p i n a l C o r d S e c t i o n s .... 67 - D i s t r i b u t i o n o f GFAP i n L e s i o n e d S p i n a l C o r d S e c t i o n s .. 73 D i s c u s s i o n 76 R e f e r e n c e s 87 i i i L i s t of Tables 1. E l e c t r o l y t i c l e s i o n diameter f o r 5 sec and 30 sec l e s i o n s . 36 2. Assessment of l e g f u n c t i o n u s i n g a m o d i f i e d T a r l o v s c a l e .. 64 3. A s t r o c y t e counts f o r normals and f o u r implant p r o t o c o l s ... 70 i v L i s t of Figures 1. Inclined plane apparatus 19 2. Transverse section of normal rat spinal cord 28 3. Transverse section of rat spinal cord - 30 sec lesion . . . . 30 4. Longitudinal section of rat spinal cord - 30 sec lesion . . 32 5. Longitudinal section of rat spinal cord - 5 sec lesion . . . 34 6. Somatosensory evoked potential - control recording 39 7. Somatosensory evoked potential - pre lesion recording . . . . 41 8. Somatosensory evoked potential - post lesion recording . . . 43 9. Transverse section of rat spinal cord - demyelination . . . . 46 10. Transverse section of rat spinal cord - macrophages 48 11. Transverse section of rat spinal cord - cavitation 50 12. Transverse section of rat spinal cord - collagen+cells . . . 53 13. Longitudinal sect, of rat spinal cord - spherical implant 55 14. Longitudinal sect, of rat spinal cord - e l l i p t i c a l implant 57 15. Longitudinal sect, of rat spinal cord host-donor interface 60 16. Bar graph of maximum angles achieved on incl ined plane . . . 62 17. Bar graph of weights of t i b i a l i s anterior muscles 66 18. Fluorescent labelled GFAP in normal rat spinal cord . . . . . . 69 19. Peroxidase labelled GFAP in normal rat spinal cord 72 20. Peroxidase labelled GFAP in lesioned rat spinal cord 75 v I wish to thank Dr. David A. Mathers for his guidance and assistance i n the preparation of t h i s t h e s i s . Thanks to Dr. J . Anthony Pearson for providing rats used i n t h i s research. In addition, I would l i k e to thank the MRC Peptide Group for the advice and material assistance with immunostaining procedures. v i i INTRODUCTION There a r e a p p r o x i m a t e l y 11,500 s p i n a l c o r d i n j u r i e s p e r y e a r i n t h e U n i t e d S t a t e s r e s u l t i n g p r i n c i p a l l y from a u t o m o b i l e a c c i d e n t s , s p o r t s i n j u r i e s and m i s s i l e wounds. Of t h e s e , about 6,500 d i e and 5,000 have some degree o f r e s i d u a l n e u r o l o g i c a l damage [Mahoney, 1988]. I n 1945 t h e p o s t - i n j u r y l i f e e x p e c t a n c y o f t h e q u a d r i p l e g i c and p a r a p l e g i c was 3 y e a r s ; t o d a y i t i s 90 % o f normal b u t b o t h t h e i n i t i a l and c h r o n i c c a r e o f t h e s e p a t i e n t s a r e r e s o u r c e i n t e n s i v e and c o s t many m i l l i o n s o f d o l l a r s each y e a r [Guttman, 1985]. F o r t h e p a r a p l e g i c and q u a d r i p l e g i c t h e q u a l i t y o f l i f e i s g r e a t l y r e d u c e d , and t h e a l t r u i s t i c m o t i v a t i o n o f w i s h i n g t o a l l e v i a t e s u f f e r i n g , as w e l l as t h e economic m o t i v a t i o n o f t r y i n g t o r e duce t h e c o s t o f c a r e , have been p o t e n t s t i m u l i t o s p i n a l c o r d r e s e a r c h . T r a u m a t i c s p i n a l c o r d i n j u r i e s f a l l i n t o two groups. One k i n d o f i n j u r y i s s u s t a i n e d when t h e c o r d i s deformed as a r e s u l t o f a r a p i d change i n a c c e l e r a t i o n o f t h e body. T h i s group i n c l u d e s s p o r t s and a u t o m o b i l e i n j u r i e s where t h e r e i s b l u n t trauma t o t h e c o r d . A n o t h e r k i n d o f s p i n a l c o r d i n j u r y o c c u r s when a m i s s i l e e n c o u n t e r s t h e c o r d a t a speed r e l a t i v e l y much g r e a t e r t h a n t h a t o f t h e c o r d such as o c c u r s i n a k n i f e o r b u l l e t wound. 1 Depending upon t h e e x t e n t o f t h e damage done and t h e p a r t s o f t h e c o r d w h i c h a r e a f f e c t e d , permanent i n j u r y c a n be m i n o r , w i t h s m a l l a r e a s o f numbness and s l i g h t m uscle weakness o r major, w i t h t o t a l a n a e s t h e s i a and l a c k o f movement below t h e l e s i o n [Wolman, 1964; P u c h a l a & W i n d l e , 1977]. Mammalian S p i n a l C o r d Anatomy Motor a c t i v i t y c a n be b e s t e x p l a i n e d u s i n g t h e c o n c e p t s o f t h e upper and l o w e r motor neuron. The c e l l body o f t h e upper motor neuron r e s i d e s i n t h e motor c o r t e x and has an axon w h i c h t r a v e l s down t h e s p i n a l c o r d t o i n f l u e n c e t h e l o w e r motor neuron i n t h e v e n t r a l h o r n . The l o w e r motor neuron i n n e r v a t e s t h e s k e l e t a l m u s c l e . The c o r t i c o s p i n a l t r a c t i s an e f f e r e n t p a t h w h i c h s t a r t s i n t h e p y r a m i d a l c e l l s o f t h e motor c o r t e x i n t h e r e g i o n o f t h e p r e c e n t r a l g y r u s . Axons from t h e s e c e l l s t r a v e l 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 and i n t o t h e m i d b r a i n . C o l l a t e r a l s a r e g i v e n o f f t o o t h e r a r e a s such as t h e b a s a l g a n g l i a , r e d n u c l e u s and i n f e r i o r o l i v a r y n u c l e u s . A f t e r d e s c e n d i n g t o t h e m e d u l l a , t h e axons d e c u s s a t e i n t h e py r a m i d s . I n t h e r a t t h e s e motor axons r u n c a u d a l l y i n t h e m e d i a l a r e a o f t h e normal s p i n a l c o r d . The t e r m i n a t i o n o f t h e s e upper motor neuron axons i s on t h e c e l l s o f t h e v e n t r a l h o r n o f t h e s p i n a l c o r d an on i n t e r n e u r o n s w h i c h i n f l u e n c e t h e v e n t r a l h o r n c e l l s . The v e n t r a l h o r n c e l l s a r e t h e 2 l o w e r motor neurons and i t i s t h e s e w h i c h a r e r e s p o n s i b l e f o r t h e d i r e c t i n n e r v a t i o n o f t h e muscle f i b r e [ A f i f i & Bergman, 1986]. The s p i n o t h a l a m i c pathway i s an a f f e r e n t system and i s made up o f axons from c e l l s i n t h e c o n t r a l a t e r a l g r e y m a t t e r o f t h e s p i n a l c o r d . Most o f t h e c e l l s a r e i n l a m i n a V w i t h a few i n l a m i n a e I , V I I and V I I I . Axons from t h e s e c e l l s d e c u s s a t e i n t h e v e n t r a l w h i t e commissure t o e n t e r t h e v e n t r o l a t e r a l and v e n t r a l f u n i c u l i and e v e n t u a l l y t e r m i n a t e i n t h e tha l a m u s . P a i n and t e m p e r a t u r e i n f o r m a t i o n from t h i s pathway i s e v e n t u a l l y t r a n s m i t t e d t o t h e v e n t r a l p o s t e r o l a t e r a l thalamus and t h e n t o t h e somatosensory c o r t e x . A d d i t i o n a l s e n s o r y i n f o r m a t i o n such as p r o p r i o c e p t i o n and l i g h t t o u c h a r e c a r r i e d i n t h e axons o f t h e d o r s a l columns. Axons i n t h e s e columns a r i s e from c e l l s i n t h e d o r s a l r o o t g a n g l i a and ascend t o synapse on p o s t e r i o r column n u c l e i and t h e n c e t o t h e thalamus [ A f i f i & Bergman, 1986]. Neurons and g l i a a r e t h e two main t y p e s o f c e l l s i n t h e s p i n a l c o r d . O l i g o d e n d r o c y t e s g r e a t l y outnumber neurons and a r e r e s p o n s i b l e f o r t h e p r o d u c t i o n o f m y e l i n and n u t r i e n t r e g u l a t i o n [Clemente, 1955; R e i e r & Hou l e , 1988; V a r o n , 1988]. Damage t o neurons i n t h e s p i n a l c o r d w i l l c ause d e g e n e r a t i o n t o t a k e p l a c e i n two d i r e c t i o n s . I n t h e r o s t r a l d i r e c t i o n 3 d e g e n e r a t i o n w i l l o c c u r o v e r s e v e r a l weeks, e v e n t u a l l y r e a c h i n g t h e c e l l b o d i e s i n t h e c e r e b r a l c o r t e x [ K a n d e l & S c h w a r t z , 1985]. I n t h e c a u d a l d i r e c t i o n , a x o n a l d e g e n e r a t i o n w i l l a l s o o c c u r up t o t h e axon t e r m i n a l . There i s l i t t l e o r no d e g e n e r a t i o n a t t h e axon t e r m i n a l u n t i l some t i m e a f t e r t h e axon has been l e s i o n e d . I t i s l i k e l y t h a t t h e axon t e r m i n a l f u n c t i o n s u n t i l t h e s u p p l y o f p r o t e i n s s u p p l i e d v i a a x o p l a s m i c t r a n s p o r t from t h e c e l l body c e a s e s [ K a n d e l & S c h w a r t z , 1985]. Once t h i s has o c c u r r e d , t h e r e w i l l be d e g e n e r a t i o n o f t h e p o s t - s y n a p t i c membrane as a r e s u l t o f d e c r e a s e d s y n a p t i c a c t i v i t y . C l i n i c a l E f f e c t s o f S p i n a l C o r d L e s i o n s M a j o r f u n c t i o n a l d e f i c i t s r e s u l t from l e s i o n s i n t h e c o r t i c o s p i n a l and s p i n o t h a l a m i c t r a c t s . C o r t i c o s p i n a l damage r e s u l t s i n i n t e r r u p t i o n o f s k i l l e d motor movements and i n f i v e m a j or upper motor neuron symptoms. Clon u s i s a r h y t h m i c c o n t r a c t i o n o f t h e muscle upon f a s t e x t e n s i o n . S p a s t i c i t y o f t h e m u scle i s a s t a t e o f s u s t a i n e d i n v o l u n t a r y c o n t r a c t i o n . H y p e r r e f l e x i a o c c u r s when an a t t empt i s made t o e l i c i t a normal s t r e t c h r e f l e x . Some a t r o p h y o f t h e muscle e v e n t u a l l y o c c u r s from l a c k o f normal use [ K a n d e l & S c h w a r t z , 1985]. The B a b i n s k i s i g n c a n be e l i c i t e d upon m i l d s t r o k i n g o f t h e s o l e o f t h e f o o t . I t i s g e n e r a l l y r e c o g n i z e d t h a t t h i s f l e x i o n r e s p o n s e i s a r e f l e x l o c a t e d i n t h e s p i n a l c o r d . T h i s r e s p o n s e 4 i s n e c e s s a r y t o w i t h d r a w t h e l e g from a n o x i o u s s t i m u l u s such as b u r n i n g . However, an i n t a c t p y r a m i d a l t r a c t i n h i b i t s t h i s f l e x o r r e s p o n s e i n t h e c a s e o f m i l d s t i m u l a t i o n . T h e r e f o r e t h e d i s p l a y o f t h e B a b i n s k i s i g n i s an i n d i c a t i o n o f damage somewhere i n t h e upper motor neuron [ A f i f i & Bergman, 1986]. Lower motor neuron s i g n s a r e f l a c c i d i t y and r a p i d , s u b s t a n t i a l m uscle a t r o p h y due t o l a c k o f spontaneous a c t i v i t y from t h e l o w e r motor neuron [ K a n d e l & S c h w a r t z , 1985]. A d e f i c i t i n s p i n o t h a l a m i c pathway t r a n s m i s s i o n g i v e s r i s e t o s e v e r a l c o n d i t i o n s t y p i c a l o f t h e s p i n a l c o r d i n j u r e d p a t i e n t . F i r s t l y , one may m e n t i o n t h e f o r m a t i o n o f s e v e r e p r e s s u r e s o r e s . These a r e due t o t h e c o n t i n u o u s p r e s s u r e on one a r e a o f t h e s k i n because t h e v i c t i m can not move. A d d i t i o n a l l y , t h e r e i s a l a c k o f normal vasomotor t o n e i n t h e a f f e c t e d dermatomes. T h i s r e s u l t s i n a c c e l e r a t e d c o l l a p s e o f t h e s e v e s s e l s when under p r e s s u r e and i n subsequent n e c r o t i c changes t o t h e u n d e r l y i n g d e r m i s . These s o r e s c a n be s e v e r e enough t o expose bone t i s s u e and h a r b o u r l i f e - t h r e a t e n i n g i n f e c t i o n s [Guttman, 1985]. A f u r t h e r d e f i c i t i s s u s t a i n e d i n t h e form o f i m p a i r e d u r o g e n i t a l r e f l e x e s . T h i s r e s u l t s i n poor v o l u n t a r y b l a d d e r c o n t r o l . I f l e f t u n t r e a t e d t h i s c o n d i t i o n w i l l l e a d t o t h e f o r m a t i o n o f a h y p e r e x t e n d e d b l a d d e r s i n c e u r i n e volume w i l l i n c r e a s e u n t i l t h e b l a d d e r w a l l s c o n t r a c t r e f l e x i v e l y . There a r e 5 two main consequences of the prolonged retention of urine i n the bladder. There w i l l be an increased frequency of bladder i n f e c t i o n since organisms are not flushed through with normal frequency. Also, prolonged retention of urine i n the bladder has been correlated with the formation of bladder neoplasms [Guttman, 1985]. Since the reflexogenic bladder w i l l void unpredictably, i t becomes convenient for the spinal cord injured patient to catheterize himself. This represents another opportunity for i n f e c t i o n and consequent l i f e - t h r e a t e n i n g damage. Surgical Procedures for the Repair of Spinal Cord Lesions At present, there are no c l i n i c a l l y useful procedures for the r e p a i r of neuronal damage following sp i n a l cord damage. Direct suturing of the cut ends of the cord does not r e s u l t i n the detectable recovery of nerve function i n mammalian species [Das, 1983a]. Segments of peripheral nerve have been grafted across the plane of spinal transections i n r a t s . In these experiments, the CNS axons were found to grow into the peripheral nerve bridge, but stopped immediately upon re-entering the host s p i n a l cord [David & Aguayo, 1981; Sceats et a l , 1985]. A major reason for the f a i l u r e of these procedures i s the formation of a prominent g l i a l scar at the s i t e of sp i n a l cord 6 damage [ P u c h a l a & W i n d l e , 1977; K i e r n a n , 1979; Das, 1983b;Reier e t a l . , 1983]. I n t h e normal c e n t r a l nervous system, g l i a a r e g e n e r a l l y a t t r i b u t e d w i t h r e g u l a t o r y f u n c t i o n s and s e r v e t o s e p a r a t e i n d i v i d u a l axons. However, i n t h e t r a u m a t i z e d c o r d , a s u b - c l a s s o f g l i a , t h e r e a c t i v e a s t r o c y t e s , p h a g o c y t i z e d e b r i s i n t h e l e s i o n a r e a [Das, 1983b]. The p r e s e n c e o f r e a c t i v e a s t r o c y t e s c a n be v i s u a l i z e d by t h e use o f f l u o r e s c e n t l y l a b e l l e d a n t i b o d i e s d i r e c t e d a g a i n s t 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) a component o f t h e i n t r a c e l l u l a r g l i o f i l a m e n t s [ B a r r & K i e r n a n , 1983]. R e p a i r o f S p i n a l C o r d F u n c t i o n U s i n g C o l l a g e n G e l s A number o f s t u d i e s have demonstrated t h e a b i l i t y o f c e r t a i n porous g e l s t o s u p p o r t n e u r o n a l growth b o t h i n v i v o and i n v i t r o . Grega [ 1 9 8 4 ] , J a y and B a r a l d [1984] and C o a t e s , [1986] showed t h a t s p i n a l c o r d e x p l a n t s i n V i t r o g e n c o l l a g e n g e l e x h i b i t enhanced n e u r i t i c growth when compared t o o t h e r s u b s t r a t e s . C o l l a g e n o f t h i s t y p e has been t r a n s p l a n t e d i n t o t h e i n j u r e d s p i n a l c o r d , where i t s u p p o r t s t h e growth o f n e u r i t e s , f i b r o b l a s t s and b l o o d v e s s e l s . The c o l l a g e n i s n o r m a l l y m a i n t a i n e d i n a f l u i d s t a t e by c o o l i n g o v e r i c e . A f t e r i n j e c t i o n i n t o t h e h o s t c o r d , t h e g e l i s h e a t e d t o 37° C by c o n t a c t w i t h t h e r a t t i s s u e . I t t h e n s o l i d i f i e s i n t o a s t a b l e , t h r e e -d i m e n s i o n a l s u b s t r a t e f o r n e u r o n a l growth. 7 P a r t i a l r e c o v e r y o f t h e somatosensory evoked p o t e n t i a l ( S E P ) has been c l a i m e d t o o c c u r i n r a t s r e c e i v i n g a c o l l a g e n i m p l a n t w h i c h p r o v i d e s t i g h t p h y s i c a l c o n t i n u i t y between t h e s e v e r e d ends o f t h e c o r d [de l a T o r r e e t a l , 1984]. T h i s r e c o v e r y would i n d i c a t e r e s t o r e d t r a n s m i s s i o n i n t h e a s c e n d i n g s e n s o r y pathways o f t h e c o r d . R e p a i r o f S p i n a l C o r d F u n c t i o n U s i n g N e u r a l I m p l a n t s T r a n s p l a n t s i n t o t h e CNS have been e s t a b l i s h e d i n a number o f e x p e r i m e n t s [Thompson, 1890; Ranson, 1914; C l a r k , 1940; B j o r k l u n d & S t e n e v i , 1971; Schmidt e t a l , 1981; I s a c s o n e t a l , 1985; M i c k l e y e t a l , 1987; A quino e t a l , 1988]. S e v e r a l s t u d i e s have d e m o n s t r a t e d t h e l o n g t e r m s u r v i v a l and growth o f embryonic s p i n a l c o r d neurons t r a n s p l a n t e d t o a l e s i o n e d c o r d i n t h e a d u l t r a t [ R e i e r e t a l , 1986; Houle & R e i e r , 1988; T e s s i e r e t a l , 1988]. A x o n - t r a c i n g s t u d i e s have shown t h a t i m p l a n t e d neurons e x t e n d p r o c e s s e s 5-20 mm i n t o t h e h o s t c o r d . These o b s e r v a t i o n s have p r o v i d e d r e a s o n t o hope t h a t a degree o f f u n c t i o n a l r e c o v e r y may be p o s s i b l e , f o l l o w i n g i m p l a n t a t i o n o f f o e t a l c e l l s . A r e c e n t s t u d y has l e n t s u p p o r t t o t h i s v i e w . C a t e c h o l a m i n e s e c r e t i n g neurons i n t h e l o c u s c e r u l e u s o f t h e r a t c a n be s e l e c t i v e l y d e s t r o y e d u s i n g a n e u r o t o x i n . T h i s l e s i o n i m p a i r s normal e x p r e s s i o n o f t h e f l e x o r w i t h d r a w a l r e f l e x i n t h e s e a n i m a l s . I m p l a n t a t i o n o f embryonic l o c u s c e r u l e u s c e l l s d i r e c t l y 8 into the cord of lesioned rats restores the strength of the flexor reflex [Buchanan & Homes, 1986]. Experimental Models of Spinal Cord Damage Several methods for creating spinal cord lesions have been investigated by various researchers. These include knife edge section of the cord [Reier et a l , 1983; Reier et a l , 1986,Reier & Houle, 1988], cooling [Carlstedt, Dalsgaard & Molander, 1986], and weight-drop injury [Black et a l , 1988]. There are problems associated with each of these techniques. The use of a scalpel to par t ia l l y section the cord represents a crude approach to a delicate s ituation. Severe bleeding occurs as a result of ruptured arteries and the stumps of the cord are affected by this loss of perfusion. Addit ional ly , some traction of the cord is inevitable as a result of compression of the cord by the knife. Local freezing of the cord has been tr i ed in an attempt to model spinal cord lesion [Carlstedt et a l , 1986]. However, this results in a large, diffuse zone of destruction with no tissue removal. Any injected ce l l s would have to displace dead ce l l s in the frozen area. 9 The weight-drop paradigm i s widely considered to represent the most accurate model of spinal cord injury [Black et a l , 1988]. In t h i s model, a measured weight i s dropped onto the exposed cord from a measured height. The damage sustained by the cord may then be correlated with the force applied. Although complete transection of the cord would y i e l d the most qua n t i f i a b l e r e s u l t s , f u l l y paraplegic animals present s p e c i a l problems. These include the absence of any sensation from the affected limbs. In the rat t h i s r e s u l t s i n the phenomenon of autocannibalism which i s possibly r e l a t e d to the lack of normal feedback from grooming a c t i v i t y . Also noted i s a lack of normal grooming of the head and eyes, r e s u l t i n g i n a buildup of exudate around the eyelids. In addition, the animal i s unable to move around his cage and thus requires constant cleaning and moving by the experimenter i f infected pressure sores are to be avoided. The animal vigorously opposes t h i s interference and quickly becomes unmanageable. In addition, there are reports of s u s c e p t i b i l i t y to urinary i n f e c t i o n s which are d i f f i c u l t to control [ R i v l i n & Tator, 1977]. E l e c t r o l y t i c l e s i o n i n g has been widely employed i n the i n v e s t i g a t i o n of neuronal function i n the brains of mammalian species. The technique involves l o c a l destruction of c e l l s by the a p p l i c a t i o n of a high current through a metal electrode inserted at known coordinates i n the brain t i s s u e [Huang & 10 Rottenberg, 1971; Olds & Olds, 1969]. In contrast, at present there are no reports of the use of e l e c t r o l y t i c lesions i n the study of spi n a l cord damage and i t s repair. The Experimental Rationale In view of these considerations, i t was decided to investigate the s u i t a b i l i t y of the e l e c t r o l y t i c l e s i o n as a model system for studying the damage and repair of the spinal cord i n adult r a t s . The experiments were conducted according to the following protocol. U n i l a t e r a l e l e c t r o l y t i c lesions of various sizes were generated i n the dorsomedial area of the cord and the acute damage done by the procedure was assessed h i s t o l o g i c a l l y . The stimulus i n t e n s i t y required to produce a c l e a r motor d e f i c i t on the l e s i o n side of the animal was determined. The extent to which the stimulus disturbed ascending sensory t r a f f i c i n the cord was assessed by the somatosensory evoked p o t e n t i a l (SEP) evoked by stimulation of a d i s t a l leg nerve. The chronic h i s t o l o g i c a l changes and g l i a l i n f i l t r a t i o n produced by the lesions were determined i n rats s a c r i f i c e d up to 3 months a f t e r l e s i o n generation. The influence of transplanted spi n a l cord neurons on the development of these lesions was determined by i n j e c t i n g a suspension of embryonic cord c e l l s into the l e s i o n s i t e , at the time of the i n s u l t to the host cord. The implanted c e l l s were injected i n eit h e r saline medium or i n a collagen 11 medium, t o d e t e r m i n e i f c o l l a g e n g e l s were a b l e t o i n f l u e n c e t h e appearance o f c h r o n i c e l e c t r o l y t i c l e s i o n s . METHODS AND MATERIALS S u r g e r y The W i s t a r r a t s w e i g h i n g 200 - 250 gm were a n a e s t h e t i z e d w i t h p e n t o b a r b i t a l sodium (65 mg/kg body w e i g h t ) . To p e r f o r m t h e laminectomy, t h e s k i n o v e r l y i n g t h e s p i n a l column was c u t and r e t r a c t e d . The o v e r l y i n g f a s c i a were c u t on e i t h e r s i d e o f t h e s p i n a l column t o a d i s t a n c e o f 2 v e r t e b r a e r o s t r a l and c a u d a l t o t h e t a r g e t v e r t e b r a ( t e n t h t h o r a c i c ) . Cuts i n t h e f a s c i a were t h e n made above and below t h e t a r g e t v e r t e b r a so as t o i s o l a t e i t from n e i g h b o u r i n g ones. U s i n g P e a r s o n r o n g e u r s , t h e s p i n o u s p r o c e s s was removed from t h e t a r g e t v e r t e b r a . The v e r t e b r a i m m e d i a t e l y r o s t r a l t o t h e t a r g e t v e r t e b r a was g r a s p e d w i t h t i s s u e f o r c e p s and p u l l e d d o r s a l l y . The t i p o f t h e r o n g e u r s was i n s e r t e d under t h e r o s t r a l edge o f t h e t a r g e t v e r t e b r a and s m a l l p i e c e s removed u n t i l a 2 mm2 a r e a o f s p i n a l c o r d was exposed. Care was t a k e n n o t t o d i s t u r b t h e s u r f a c e o f t h e c o r d w h i l e t h e d o r s a l bone was b e i n g removed. The c o r d was k e p t m o i s t w i t h s a l i n e soaked c o t t o n b a l l s . P r i o r t o l e s i o n g e n e r a t i o n , t h e a n i m a l was p o s i t i o n e d i n a s t e r e o t a x i c frame. T h i s e n a b l e d t h e s p i n a l column t o be 12 immobilized with clamps placed r o s t r a l and caudal to the exposed area of sp i n a l cord. This was necessary to minimize displacement of the r a t both during positioning of the l e s i o n electrode and the microsyringe and during the application of current. Upon i n i t i a t i o n of current flow through the l e s i o n electrode a strong spasm was induced i n the muscles i p s i l a t e r a l to the l e s i o n . The use of the sp i n a l clamps greatly reduced cord displacement due to t h i s e f f e c t . Lesion Generation The e l e c t r o l y t i c l e s i o n i n the cord was made with a Grass Instruments l e s i o n maker (Model D.C. LM5A) d r i v i n g a needle which was insulated to within 0.1 mm of the end. The needle was positioned approximately 0.5 mm to the r i g h t of the dorsal spinal artery and lowered approximately 1.5 mm into the cord. To f a c i l i t a t e entry of the needle into the nervous t i s s u e , a small hole was made i n the dura with a 26 ga. i . v . needle. T y p i c a l l y , a 0.1 mA current at 150 V was applied for 30 sec a f t e r which the needle was withdrawn from the cord. To investigate the acute e f f e c t s of the e l e c t r o l y t i c l e s i o n , some lesions were made using a 0.1 mA current at 150 V for 5 sec. The l e s i o n i n the spinal cord was made i n the dorsal-medial region i n an attempt to interrupt the major portion of the c o r t i c o s p i n a l t r a c t without greatly a f f e c t i n g surrounding 13 s t r u c t u r e s s u c h as t h e d o r s a l columns and t h e d o r s a l s p i n o c e r e b e l l a r t r a c t . Somatosensory Evoked P o t e n t i a l R e c o r d i n g Somatosensory evoked p o t e n t i a l s were r e c o r d e d by s t i m u l a t i n g t h e exposed t i b i a l n e r v e w i t h a 1.5 V, 0.02 msec d i r e c t c u r r e n t p u l s e t h r o u g h double-hook t y p e e l e c t r o d e s . These were made by b e n d i n g 0.5 mm s t a i n l e s s s t e e l w i r e t h r o u g h 90° 4 mm from t h e end o f t h e w i r e . The two w i r e s were p l a c e d 5 mm a p a r t and s l i p p e d under t h e t i b i a l n e rve o f t h e upper t h i g h r e g i o n , r a i s i n g i t f r e e o f t h e s u r r o u n d i n g t i s s u e . The immediate a r e a was f l o o d e d w i t h p a r a f f i n o i l t o s t o p t h e n e r v e d r y i n g o u t . The evoked p o t e n t i a l s were r e c o r d e d from a p l a t i n u m w i r e e l e c t r o d e l o w e r e d t h r o u g h a b u r r h o l e i n t h e s k u l l . The h o l e was d r i l l e d a p p r o x i m a t e l y 3 mm a n t e r i o r o f bregma and 1.2 mm l a t e r a l t o t h e m i d l i n e . The ground c o n s i s t e d o f a Ag/AgCl b u t t o n t y p e e l e c t r o - c a r d i o g r a p h i c e l e c t r o d e p l a c e d i n t h e r a t ' s mouth. The s i g n a l was a m p l i f i e d 2 x 10 4 by a T e k t r o n i x d i f f e r e n t i a l a m p l i f i e r (model AM502) f i l t e r e d below 60 Hz and above 600 Hz and d i g i t i z e d a t 200 usee/point by a Data P r e c i s i o n D6000 d i g i t a l s t o r a g e o s c i l l o s c o p e . The low a m p l i t u d e o f t h e evoked p o t e n t i a l s n e c e s s i t a t e d a v e r a g i n g t h e r e s u l t s o f 1024 s t i m u l u s - r e s p o n s e e v e n t s t o make a r e c o r d i n g . 14 C o l l a g e n I m p l a n t s The c o l l a g e n s o l u t i o n ( V i t r o g e n C o r p o r a t i o n , P a l o A l t o , CA.) w h i c h was t o be i n j e c t e d i n t o t h e l e s i o n c a v i t y e i t h e r a l o n e o r w i t h f o e t a l neurons i n s u s p e n s i o n was k e p t r e f r i g e r a t e d i n 1:1000 a c e t i c a c i d . C o l l a g e n g e l m a t r i x was p r e p a r e d by a d d i n g 10 u l o f c o o l e d medium 199 o f 10 X c o n c e n t r a t i o n and 10 u l o f c o o l e d 1 N NaOH t o 80 u l o f c o l l a g e n s o l u t i o n . The amount o f NaOH was a d j u s t e d t o b r i n g t h e f i n a l pH o f t h e m i x t u r e t o 7.4. T h i s r e q u i r e d t i t r a t i o n o f t h e p a r t i c u l a r b a t c h o f c o l l a g e n used s i n c e each b a t c h has a s l i g h t l y d i f f e r e n t c o n c e n t r a t i o n o f a c e t i c a c i d . The c o l l a g e n s o l u t i o n was t h e n k e p t on i c e f o r as s h o r t a t i m e as p o s s i b l e . A p p r o x i m a t e l y 4 u l o f t h e p r e p a r e d c o l l a g e n were i n j e c t e d i n t o t h e l e s i o n s i t e s o f s e l e c t e d a n i m a l s u s i n g an 5 u l H a m i l t o n m i c r o s y r i n g e . The c o l l a g e n p r e p a r a t i o n formed a g e l i n 5-10 min a t 37° C a t a pH o f 7.3 - 7.4. t h i s g e l i s a non-immunogenic, t h r e e - d i m e n s i o n a l o r g a n i c complex w h i c h a l l o w s d i f f u s i o n o f n u t r i e n t s and gas e s . I n a d d i t i o n , i t s g e l - l i k e c o n s i s t e n c y a l l o w s i t t o f l e x w i t h t h e a n i m a l and t o p o s s i b l y s l o w down m i g r a t i o n o f d u r a and g l i a i n t o t h e l e s i o n a r e a . 15 Spinal Cord C e l l / Collagen Implants Fourteen day o l d f o e t a l spinal cord neurons were obtained by removing the u t e r i from a pregnant Wistar r a t which had been s a c r i f i c e d by carbon dioxide inhalation. The i n d i v i d u a l embryos were dissected out of the u t e r i and away from the placenta. Under a d i s s e c t i n g microscope the spinal cords were removed from the fetuses using iridectomy s c i s s o r s . Six to nine spinal cords were dissected free of any adherent tiss u e and placed together i n a s t e r i l e culture dish. The spi n a l cords were minced with iridectomy scissors and transferred to a tube containing 5 ml of s t e r i l e Medium 199. Using a succession of pasteur pipettes with flamed t i p s of decreasing diameter, the chopped spinal cords were t r i t u r a t e d into a f i n e suspension. This suspension was centrifuged for 1 min i n a benchtop centrifuge to p e l l e t the large pieces. The supernatant was transferred to another tube and centrifuged for 4 min at a higher speed. The supernatant from t h i s treatment was discarded and the p e l l e t of neurons kept at 4° C u n t i l required. Injecting the Neural Implant P r i o r to t h i s i n j e c t i o n , spinal cord c e l l s were resuspended i n collagen matrix. Approximately 4 ul of collagen matrix containing sp i n a l cord c e l l s was introduced into the l e s i o n 16 cavity in the spinal cord over a period of 10 min through a 5 ul Hamilton microsyringe coupled to a manually operated micrometer drive. The density of the ce l l s in the implant matrix was checked with a haemocytometer and was in the range of 60,000 -95,000 c e l l s / u l . After withdrawing the microsyringe, the exposed spinal cord was covered with a 2 mm2 piece of bone wax and each layer of the overlying fascia was sutured. Approximately 3 mg of p e n i c i l l i n powder was dusted onto the wound area and the skin was closed. The animals were returned to their cages and allowed to recover overnight. Behavioural Testing of Spinal Cord Function The principal objective test of the animal's voluntary motor function was the incl ined plane test [Rivl in & Tator, 1977]. This apparatus comprised two boards (45 cm x 21 cm) joined at the short edge by a hinge. The top of the board had a rough surface glued to i t . This surface had ridges 3 mm high, spaced 3 mm apart, running across the board (Fig. 1). A testing session consisted of placing the animal on the ridges with i t s right limbs nearest the hinge and slowly raising the top board. When the animal was no longer able to maintain his posit ion on the board for 5 sec, the angle of the inclined 17 F i g . 1. Drawing o f i n c l i n e d p l a n e a p p a r a t u s . The t i l t i n g b o a r d i s 30 cm wide and 45 cm h i g h . The r i d g e s r u n n i n g a c r o s s t h e b o a r d a r e 3 mm h i g h , spaced 3 mm a p a r t . T e s t i n g c o n s i s t e d o f p l a c i n g t h e a n i m a l f a c i n g a c r o s s t h e b o a r d so t h a t t h e a n i m a l ' s r i g h t s i d e l i m b s were n e a r e s t t h e h i n g e . The b o a r d was s l o w l y r a i s e d from t h e h o r i z o n t a l u n t i l t h e a n i m a l was u n a b l e t o m a i n t a i n c o n t r o l o f i t s p o s i t i o n f o r 5 seconds. 18 19 b o a r d was n o t e d . F i v e t r i a l s were averaged t o form t h e v a l u e f o r one s e s s i o n o f t e s t i n g . T e s t i n g o c c u r r e d e v e r y f o u r days and was t e r m i n a t e d a f t e r 3 months o r when no improvement was n o t e d f o r 1 month. The m o b i l i t y o f t h e h i n d l i m b s was a s s e s s e d f o r t h e l e f t and r i g h t s i d e s i n d e p e n d e n t l y , u s i n g t h e T a r l o v s c a l e o f measurement [ R i v l i n & T a t o r , 1977; T a t o r , 1975; T a t o r , 1973]. T h i s s c a l e r e c o g n i z e s t h e f o l l o w i n g performance grades : grade 0 = complete p a r a l y s i s o f l e g grade 1 = t r a c e o f movement grade 2 = good movement a t a l l j o i n t s b u t no w a l k i n g o r w e i g h t b e a r i n g grade 3 = w a l k i n g and w e i g h t b e a r i n g b u t n o t n o r m a l l y grade 4 = normal T h i s assessment was con d u c t e d under s i n g l e - b l i n d c o n d i t i o n s . Measurements made a f t e r 3 months were compared a c r o s s t h e v a r i o u s e x p e r i m e n t a l groups u s i n g t h e W i l c o x o n t e s t . The W i l c o x o n t e s t i s a n o n - p a r a m e t r i c s u b s t i t u t e f o r t h e t - t e s t and i s c o n s i d e r e d t o have a h i g h degree o f a c c u r a c y [ S c h e f l e r , 1980]. 20 H i s t o l o g y The a n i m a l s were s a c r i f i c e d a f t e r 3 months w i t h an overdose o f sodium p e n t o b a r b i t a l . P e r f u s i o n f o r i m m u n o h i s t o c h e m i s t r y and g e n e r a l c y t o l o g y c o n s i s t e d o f 200 ml 0.9% NaCl l e f t v e n t r i c u l a r p e r f u s i o n f o l l o w e d by 20 min o f 4 % p a r a f o r m a l d e h y d e (PFA) i n 0.1 M Sorenson's phosphate b u f f e r pH 7.4. V a s c u l a r e f f l u e n t was a l l o w e d t o d r a i n from t h e r i g h t a t r i u m . The s p i n a l c o r d s were removed and were k e p t o v e r n i g h t i n t h e same s o l u t i o n . A 1 cm s e c t i o n o f c o r d , c e n t r e d around t h e l e s i o n s i t e , was d i s s e c t e d from t h e removed s p i n a l c o r d . These p i e c e s were d e h y d r a t e d i n 70% e t h a n o l o v e r n i g h t f o l l o w e d by 90 min each o f 95% e t h a n o l , a b s o l u t e e t h a n o l and x y l e n e . The p i e c e s were embedded i n P a r a p l a s t s e c t i o n i n g p a r a f f i n ( F i s h e r S c i e n t i f i c ) f o r a f u r t h e r 75 min. S e r i a l t e n m i c r o n t h i c k c r o s s - s e c t i o n s were t a k e n u n t i l t h e l e s i o n s i t e was r e a c h e d . Mounted p a r a f f i n s e c t i o n s 5 m i c r o n s t h i c k were r e h y d r a t e d by s u c c e s s i v e e x p o s u r e t o 5 min each o f 95% e t h a n o l , a b s o l u t e e t h a n o l , x y l e n e , a b s o l u t e e t h a n o l , 95% e t h a n o l , 70% e t h a n o l and d i s t i l l e d w a t e r . S t a i n i n g was done w i t h c r e s y l v i o l e t f o r N i s s l s u b s t a n c e and L u x o l f a s t b l u e f o r m y e l i n . S e c t i o n s were mounted on s l i d e s and c o v e r e d w i t h g l a s s c o v e r s l i p s and Permount. Axon and c e l l 21 photographs were made under t h e 20 X o r 40 X o b j e c t i v e o f a s t a n d a r d compound m i c r o s c o p e . Immunocytoc h e m i s t r y To q u a n t i t a t i v e l y a s s e s s a s t r o c y t e i n f i l t r a t i o n , s t a i n i n g f o r 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) i n a s t r o c y t e s was done u s i n g an a n t i - G F A P a n t i b o d y . T h i s a n t i b o d y i s v e r y s p e c i f i c t o t h e i n t r a c e l l u l a r p r o t e i n a s s o c i a t e d w i t h l o n g f i b r e s p a r t i c u l a r t o g l i a . A f t e r b l o c k i n g endogenous p e r o x i d a s e a c t i v i t y f o r 1/2 hour w i t h 0.5 % H2O2 i n methanol and n o n s p e c i f i c b i n d i n g w i t h 50 % goat serum f o r 1/2 hour, t h e s e c t i o n s were i n c u b a t e d f o r 48 hours a t 4° C w i t h a n t i - G F A P (ICN B i o c h e m i c a l s ) d i l u t e d 1:1000. A f t e r 3 x 5 min wash i n p h o s p h a t e - b u f f e r e d s a l i n e , t h e goat anti-mouse b i o t i n second l a y e r ( J a c k s o n ) was d i l u t e d 1:1000 and a p p l i e d t o t h e s e c t i o n f o r 1 hour a t 20° C. A f t e r a n o t h e r 3 x 5 min PBS wash, t h e t h i r d l a y e r , a p e r o x i d a s e - l a b e l l e d a v i d i n m o l e c u l e ( V e c t a s t a i n ) , was a p p l i e d a t 1:1000 d i l u t i o n f o r 1 hour a t 20° C. U n l e s i o n e d s e c t i o n s were p r o c e s s e d w i t h s e c t i o n s from l e s i o n e d groups t o c o n f i r m a n t i b o d y a c t i v i t y . The r e s u l t s o b t a i n e d w i t h 15 min p e r o x i d a s e development a r e s u i t a b l e f o r c o u n t i n g b u t t h e s u b t l e shades o f brown r e s u l t i n l o w - c o n t r a s t p h o t o g r a p h s . T h e r e f o r e , f l u o r e s c e n t F I T C - a v i d i n ( J a c k s o n ) t h i r d l a y e r s t a i n i n g was done on some s e c t i o n s t o produce h i g h c o n t r a s t a s t r o c y t e p h o t o g r a p h s . GFAP Controls Control experiments for the antibody l a b e l l i n g experiments consisted of processing, incubating, and developing sections of unlesioned Wistar r a t spin a l cord t i s s u e . Once the antibody s t a i n i n g technique was established, the experimental sections were s i m i l a r l y processed. In addition, to t e s t the s p e c i f i c i t y of the second and t h i r d layer antibodies, a negative control was performed using an i r r e l e v a n t (anti - actin) primary antibody. Further t e s t s t a i n i n g was done on normal spinal cord sections a f t e r s t a i n i n g the lesioned cords to ensure that the antibody had not degraded during the course of processing the lesioned sections. Analysis of a n t i - GFAP Reactive C e l l s The numbers of a n t i - GFAP l a b e l l e d astrocytes i n the grey matter were determined for a l l experimental groups as well as for control (unlesioned) s p i n a l cords. A c e l l was counted as an astrocyte i f i t possessed a r a d i a l pattern of a n t i - GFAP l a b e l l e d c e l l u l a r dendrites and an associated unlabelled, smooth c e l l body. Pieces of a n t i - GFAP l a b e l l e d c e l l u l a r material were not counted as an astrocyte unless the c e l l body was v i s i b l e . The Wilcoxon t e s t for non-parametric data was performed on these data. 23 T i b i a l i s M u s c l e Weights E a r l y o b s e r v a t i o n s i n d i c a t e d t h a t t h e major d e f i c i t c a u s ed by t h e e l e c t r o l y t i c l e s i o n s was an i n a b i l i t y t o f l e x t h e f o o t . A d e f i c i t o f t h i s k i n d would a r i s e i f t h e l o w e r motoneurons s u p p l y i n g t h e t i b i a l i s a n t e r i o r muscle were d e s t r o y e d by t h e l e s i o n p r o c e d u r e . I f t h i s was t h e c a s e , t h e n marked a t r o p h y o f t h e t i b i a l i s a n t e r i o r muscle would be e x p e c t e d t o o c c u r w i t h i n t h e t i m e p e r i o d o f t h e e x p e r i m e n t [Guttman, 1985]. To t e s t f o r t h i s p o s s i b i l i t y , t h e t i b i a l i s a n t e r i o r muscles o f b o t h h i n d l e g s were removed i m m e d i a t e l y a f t e r t h e s p i n a l c o r d was e x t r a c t e d . The s k i n o v e r l y i n g t h e muscle was c u t away and t h e l a t e r a l and m e d i a l b o r d e r s o f t h e muscle were s e p a r a t e d from t h e a p p o s i n g f a s c i a . The p r o x i m a l and d i s t a l tendons were c u t a t t h e i r n a r r o w e s t p o i n t and t h e muscle p l a c e d i n t o 4% f o r m a l - s a l i n e f o r s t o r a g e . The m u s c l e s were d r i e d o f e x c e s s m o i s t u r e and weighed 24 hours a f t e r r emoval from t h e a n i m a l . A c u t e L e s i o n Measurement To d e t e r m i n e t h e r e l a t i o n s h i p between l e s i o n s i z e and d u r a t i o n o f a p p l i e d c u r r e n t , s i x 250 gram W i s t a r r a t s underwent b i l a t e r a l e l e c t r o l y t i c l e s i o n g e n e r a t i o n a t t h e l e v e l o f t h e 10th t h o r a c i c v e r t e b r a . Two c u r r e n t a p p l i c a t i o n t i m e s were us e d , 5 s and 30 s, b o t h a t 150 V and 0.1 mA. The s p i n a l c o r d s were removed i m m e d i a t e l y a f t e r l e s i o n g e n e r a t i o n and p r o c e s s e d as 24 above. A f t e r s e c t i o n i n g and s t a i n i n g , t h e r a d i u s o f damage f o r each c u r r e n t d u r a t i o n was measured. The E x p e r i m e n t a l Groups F i f t y - o n e female W i s t a r a d u l t r a t s were d i v i d e d i n t o f o u r e x p e r i m e n t a l g r o u p s . Group 1 c o n s i s t e d o f f i f t e e n a n i m a l s w h i c h underwent t h e l e s i o n p r o c e d u r e o n l y . Group 2 c o n s i s t e d o f s i x a n i m a l s i n w h i c h t h e l e s i o n was made and a s u s p e n s i o n o f E 14 f o e t a l c e l l s i n b u f f e r was i n j e c t e d a t t h e l e s i o n s i t e . F i f t e e n group 3 a n i m a l s had a l e s i o n made w h i c h was s u b s e q u e n t l y f i l l e d w i t h l i q u i d c o l l a g e n - g e l m a t r i x c o n t a i n i n g no c e l l s . The f i f t e e n a n i m a l s i n group 4 were t r e a t e d t h e same as t h o s e i n group 3 e x c e p t t h a t 14 d a y - o l d embryonic (E14) s p i n a l c o r d c e l l s were added t o t h e c o l l a g e n m a t r i x p r i o r t o i n j e c t i o n . A l l i n j e c t i o n s were made w i t h i n 30 mi n u t e s o f l e s i o n g e n e r a t i o n . RESULTS The a n i m a l s t o l e r a t e d t h e s u r g i c a l p r o c e d u r e w e l l . E i g h t a n i m a l s d i e d s h o r t l y a f t e r t h e s u r g e r y a l t h o u g h t h i s may have been due t o t h e use o f a n a e s t h e t i c w h i c h was o l d e r t h a n was recommended f o r s a f e usage by t h e m a n u f a c t u r e r . One a n i m a l d e v e l o p e d c o m p l e t e p a r a p l e g i a and was t h e r e f o r e o m i t t e d from t h e perf o r m a n c e a n a l y s i s . I n s p i t e o f t h e t o t a l motor p a r a l y s i s i n t h i s a n i m a l , s e n s o r y c a p a b i l i t y was a p p a r e n t l y p r e s e r v e d s i n c e 25 t h e r e was no a u t o c a n n i b a l i s m and t h e a n i m a l p e r f o r m e d normal grooming f u n c t i o n s . Normal Anatomy F o r c o m p a r i s o n p u r p o s e s , F i g . 2 shows a t r a n s v e r s e s e c t i o n o f a n o r m a l , u n o p e r a t e d s p i n a l c o r d . The s e c t i o n was t a k e n a t t h e l e v e l o f t h e 1 0 t h t h o r a c i c v e r t e b r a and was s t a i n e d w i t h c r e s y l v i o l e t and L u x o l f a s t b l u e . The c r e s y l v i o l e t s t a i n s t h e N i s s l s u b s t a n c e , e s s e n t i a l l y RNA, and i s r e s p o n s i b l e f o r t h e d a r k e r g r e y ' b u t t e r f l y ' shape a r e a i n t h e c e n t r a l t w o - t h i r d s o f t h e c o r d . L u x o l f a s t b l u e s t a i n s t h e m y e l i n w h i t e m a t t e r and r e s u l t s i n t h e l i g h t e r g r e y around t h e p e r i p h e r y o f t h e c o r d . A c u t e E l e c t r o l y t i c L e s i o n s The a c u t e e f f e c t s o f t h e e l e c t r o l y t i c l e s i o n p r o c e d u r e were e s t a b l i s h e d by s a c r i f i c i n g t h e e x p e r i m e n t a l a n i m a l s w i t h i n 15 min o f t h e l e s i o n p r o c e s s . The r e s u l t s o f a t y p i c a l l e s i o n o f 30 s d u r a t i o n a r e shown i n F i g . 3. A s i m i l a r 30 s l e s i o n i s shown i n l o n g i t u d i n a l s e c t i o n i n F i g . 4. To a s s e s s t h e r e l a t i o n s h i p between t h e degree o f damage and t h e d u r a t i o n o f c u r r e n t , 3 s p i n a l c o r d s were s u b j e c t e d t o b i l a t e r a l 5 s l e s i o n s and 3 t o b i l a t e r a l 30 s l e s i o n s . A r e p r e s e n t a t i v e 5 s l e s i o n i s shown i n F i g . 5. 26 F i g . 2. A 5 um p a r a f f i n embedded transverse section of normal, unlesioned spinal cord stained with c r e s y l v i o l e t and Luxol f a s t blue. The section was taken at the l e v e l of the tenth thoracic vertebra. 35 x 27 C O R T I C O S P I N A L T R . V E N T R A L 28 F i g . 3. A 5 um p a r a f f i n embedded transverse section stained with c r e s y l v i o l e t and Luxol fast blue. Low power view of acute e l e c t r o l y t i c l e s i o n . Lesion parameters : 150 V, 0.1 mA, 30 s. Note intensely stained c i r c u l a r area and cracking of section due to high carbon content. The l e s i o n i s l a t e r a l to the c o r t i c o s p i n a l t r a c t because the midline positioning required for a c o r t i c o s p i n a l l e s i o n r e s u l t s i n possible rupture of the dorsal s p i n a l artery and death of the animal. L a t e r a l placement resulted i n zero mortality. 40 x 29 N E E D L E T R A C K Fig . 4 . A 5 um paraffin embedded longitudinal section stained with cresyl v io let and luxol fast blue. Low power view of a 30 sec e lectrolyt ic lesion at the level of the 10th thoracic vertebra. Note spherical shape of lesion. Together with F ig . 3 this confirms the symmetry of the lesion area and localized extent of damage. 6 0 x 31 L E S I O N A R E A 3 2 F i g . 5. A 5 um p a r a f f i n embedded longitudinal section stained with c r e s y l v i o l e t and Luxol fast blue. Low power view of acute e l e c t r o l y t i c l e s i o n at tenth thoracic l e v e l . Lesion parameters : 150 V, 0.1 mA, 5 s. Note much smaller area of intense s t a i n i n g compared to F i g . 4. 40 x 33 L E S I O N A R E A 34 The e l e c t r o l y t i c l e s i o n s i z e was d e t e r m i n e d by t h e d u r a t i o n o f a known v o l t a g e and c u r r e n t . I n t h i s e x p e r i m e n t , t h e s e p a r a m e t e r s were 150 V and 0.1 mA. The d i a m e t e r o f t h e l e s i o n was found t o be r e l a t e d t o t h e d u r a t i o n o f t h e c u r r e n t f l o w . The l e s i o n a r e a was s p h e r i c a l l y s y m m e t r i c a l about t h e t i p o f t h e e l e c t r o d e and i t was found t h a t t h e d i a m e t e r o f t h e l e s i o n was i n c r e a s e d by a p p r o x i m a t e l y 11 um f o r each a d d i t i o n a l 5 s o f c u r r e n t passage ( T a b l e 1 ) . There was no d i s c e r n a b l e d i f f e r e n c e between t h e e x t e n t o f t h e l e s i o n i n w h i t e o r g r e y m a t t e r . L e s i o n s i t e s t y p i c a l l y showed c a v i t a t i o n i n j u r y s u r r o u n d e d by c a r b o n i z e d d e b r i s w h i c h appeared b l a c k on t h e s e s e c t i o n s ( F i g . 4 ) . U n l e s s o t h e r w i s e s t a t e d , a l l t h e l e s i o n s d i s c u s s e d i n t h e re m a i n d e r o f t h i s t h e s i s were made w i t h a 150 V, 0.1 mA c u r r e n t a p p l i e d t o t h e r i g h t s i d e o f t h e s p i n a l c o r d f o r 30 s. A l l c h r o n i c l e s i o n s were made i n t h e d o r s o m e d i a l r e g i o n o f t h e s p i n a l c o r d a t t h e t e n t h t h o r a c i c l e v e l . L e s i o n s were made as c l o s e t o t h e m i d l i n e as t h e d o r s a l s p i n a l a r t e r y would p e r m i t , t y p i c a l l y 1.5 mm from t h e m i d l i n e . The E f f e c t o f A c u t e E l e c t r o l y t i c L e s i o n on t h e Somatosensory  Evoked P o t e n t i a l To a s s e s s t h e a c u t e e f f e c t o f t h e 30 s e l e c t r o l y t i c l e s i o n on s t r u c t u r e s n e a r t h e s i t e o f t h e l e s i o n , a somatosensory evoked p o t e n t i a l (SEP) t e s t was c o n d u c t e d l e s s t h a n 1 hour a f t e r l e s i o n 35 30 second l e s i o n 5 second l e s i o n s i z e (mm) s i z e (mm) 0.25 0.28 0.28 0.30 0.30 0.05 0 . 0 5 0.06 0.06 0.07 mean ± S.D 0.28±0.02 0.06±0.02 T a b l e 1. Di a m e t e r s o f e l e c t r o l y t i c l e s i o n s p roduced i n t h e s p i n a l c o r d by a p p l i c a t i o n o f a 150 V, 0.1 mA c u r r e n t f o r e i t h e r 5 o r 30 s e c . C u r r e n t was a p p l i e d t o t h e c o r d t h r o u g h a s t a i n l e s s s t e e l l e s i o n n e e d l e i n s u l a t e d t o w i t h i n 0.1 mm o f t h e end. The l e s i o n e l e c t r o d e i n t h e s e a c u t e a n i m a l s was p o s i t i o n e d s l i g h t l y more l a t e r a l t h a n i n t h e c h r o n i c a n i m a l s t o l e s s e n t h e chance o f f a t a l l y r u p t u r i n g t h e d o r s a l s p i n a l a r t e r y . 36 g e n e r a t i o n . Damage t o t h e a s c e n d i n g s e n s o r y pathways o f t h e c o r d i s e x p e c t e d t o r e s u l t i n i n c r e a s e d l a t e n c i e s i n t h e SEP peaks [C h i a p p a & Ropper, 1982]. F i g . 6 shows a c o n t r o l SEP r e c o r d i n g made w i t h t h e e n t i r e a p p a r a t u s , i n c l u d i n g t h e s t i m u l u s e l e c t r o d e s , i n p l a c e b u t w i t h t h e s t i m u l a t o r s w i t c h e d o f f . F i g . 7 shows t h e SEP w h i c h r e s u l t e d from t h e average o f t h e evoked p o t e n t i a l s o f 1024 s t i m u l u s - r e s p o n s e p a i r s r e c o r d e d b e f o r e an e l e c t r o l y t i c l e s i o n was made i n t h e c o r d . F i g . 8 shows t h e average o f 1024 s t i m u l i a p p l i e d a f t e r t h e a p p l i c a t i o n o f 30 s o f l e s i o n i n g c u r r e n t (0.1 mA) a t t h e L2-L3 l e v e l o f t h e c o r d as p r e v i o u s l y d e s c r i b e d . Note t h e s i m i l a r i t y i n t h e shape, s p e c i f i c a l l y t h e l a t e n c i e s o f t h e peaks, o f t h e p r e - and p o s t - l e s i o n r e c o r d i n g s . There i s a s l i g h t upward d r i f t i n t h e p o s t - l e s i o n r e c o r d i n g b u t t h i s i s an a r t i f a c t r e s u l t i n g from t h e c h o i c e o f t h e v e r t i c a l r e c o r d i n g s c a l e . U n l i k e t h e a u d i t o r y evoked p o t e n t i a l , t h e r e has been no d e t e r m i n a t i o n o f t h e n e u r o n a l s t r u c t u r e s r e p r e s e n t e d by t h e v e r t i c a l components o f t h e waveforms i n t h e somatosensory evoked p o t e n t i a l . The d i a g n o s t i c parameter i s t h e s i m i l a r i t y i n t h e l a t e n c i e s o f t h e v e r t i c a l components o f t h e r e c o r d i n g . T h i s e x p e r i m e n t was r e p e a t e d i n a f u r t h e r 5 a n i m a l s each o f w h i c h y i e l d e d a s i m i l a r r e s u l t . These o b s e r v a t i o n s s u g g e s t t h a t t h e e l e c t r o l y t i c l e s i o n u sed i n t h i s s t u d y d i d n o t a c u t e l y cause major l o s s o f a s c e n d i n g s e n s o r y t r a f f i c i n t h e s p i n a l c o r d . 37 F i g . 6. Typical control somatosensory evoked p o t e n t i a l recording taken with the ent i r e apparatus connected but the stimulator output switched o f f . Time 0 represents the t r i g g e r pulse which st a r t s both the stimulator and the averager. Note e s s e n t i a l l y random noise i n t r i n s i c to the recording system and the absence of any time-locked waveforms. Sweeps averaged : 1024. Recording electrode l o c a t i o n : 7.6 mm anterior of bregma; 1.2 mm l a t e r a l . 38 0 50 100 Time (ms) 39 F i g . 7. Same animal and stimulus parameters as i n F i g . 6. Stimulator switched on, no l e s i o n . Stimulating electrode on deep peroneal nerve. Stimulation rate = 1 Hz. Sweeps averaged = 1024. 40 1 1 1 1 1 1 1 1 1 1 0 50 100 Time (ms) 41 F i g . 8. Same animal as Figs. 6, 7. U n i l a t e r a l r i g h t - s i d e l e s i o n , stimulator switched on. Sweeps averaged = 1024. Note preservation of most features seen i n the i n t a c t wave of F i g . 7 and s i m i l a r latencies of the spikes i n d i c a t i n g that afferent transmission i s not abolished. 42 5 0 Time (ms) 1 0 0 43 H i s t o l o g i c a l Appearance o f C h r o n i c E l e c t r o l y t i c L e s i o n s The appearance o f e l e c t r o l y t i c l e s i o n s was examined i n 43 r a t s m a i n t a i n e d f o r 3 months a f t e r l e s i o n g e n e r a t i o n . The m a j o r i t y (26/43) o f t h e s e l e s i o n s were c o n f i n e d t o t h e m e d i a l p a r t o f t h e s p i n a l c o r d . However, i n (17/43) c a s e s t h e l e s i o n s i n v o l v e d more l a t e r a l s t r u c t u r e s . The c h r o n i c l e s i o n d i f f e r e d from a c u t e i n j u r y i n t h e g r e a t e r degree o f c a v i t a t i o n and d e m y e l i n a t i o n t h a t was o b s e r v e d . D e m y e l i n a t i o n was seen i n a l l c h r o n i c l e s i o n s examined b u t was h i g h l y v a r i a b l e i n e x t e n t r a n g i n g from moderate ( F i g . 9) t o e x t e n s i v e i n w h i c h t h e c r o s s -s e c t i o n a l a r e a o f t h e c o r d was r e d u c e d by up t o t w o - t h i r d s . I n 28 l e s i o n s t h e p r e s e n c e o f i n t e n s e l y c r e s y l v i o l e t p o s i t i v e c e l l s was n o t e d ( F i g . 1 0 ) . These c e l l s t y p i c a l l y i n f i l t r a t e d t h e l e s i o n s i t e and p r o b a b l y r e p r e s e n t macrophages, known t o accompany t h e i n f l a m m a t o r y r e s p o n s e t r i g g e r e d by s p i n a l c o r d damage [ R e i e r e t a l , 1986]. These o b s e r v a t i o n s s u g g e s t t h a t e l e c t r o l y t i c l e s i o n s t y p i c a l l y t r i g g e r d e l a y e d c e l l u l a r damage i n t h e s p i n a l c o r d and t h a t t h e e x t e n t o f t h e s e s e c o n d a r y p a t h o l o g i c a l changes was h i g h l y v a r i a b l e from l e s i o n t o l e s i o n . The c a v i t a t i o n p r e s e n t a t t h e i n j u r y f o c u s ranged from n e g l i g i b l e t o e x t e n s i v e ( g r e a t e r t h a n 1 mm2 ). These c a v i t i e s c o u l d be o f s p h e r i c a l o r i r r e g u l a r shape ( F i g . 1 1 ) . 44 F i g . 9. A 5 um p a r a f f i n embedded t r a n s v e r s e s e c t i o n o f l e s i o n e d s p i n a l c o r d s t a i n e d 3 months a f t e r l e s i o n i n g w i t h c r e s y l v i o l e t and L u x o l f a s t b l u e . Note d e m y e l i n a t i o n and l o s s o f g r e y m a t t e r on r i g h t ( l e s i o n e d ) s i d e o f t h e c o r d . 60 x 45 F i g . 10. A 5 um p a r a f f i n embedded transverse section taken 3 months post l e s i o n and stained with c r e s y l v i o l e t and Luxol f a s t blue. Note the intensely stained, granular material present on the r i g h t (lesioned) side t y p i c a l of macrophage i n f i l t r a t i o n . 40 x. 47 M A C R O P H A G E S C Y S T I C A R E A 48 F i g . 11. A 5 um p a r a f f i n embedded transverse section of lesioned s p i n a l cord stained 3 months post l e s i o n with c r e s y l v i o l e t and Luxol fa s t blue. Note large cyst i n the area of the l e s i o n . 40 x 49 C Y S T I C A R E A C E N T R A L C A N A L 50 H i s t o l o g y o f C h r o n i c L e s i o n s T r e a t e d w i t h C e l l - F r e e C o l l a g e n  I m p l a n t I n 11/43 r a t s , t h e e l e c t r o l y t i c l e s i o n s i t e was i n j e c t e d w i t h a c e l l - f r e e c o l l a g e n g e l a t t h e t i m e o f l e s i o n f o r m a t i o n . When examined 3 months l a t e r , t h e s e l e s i o n s d i d n o t appear t o d i f f e r q u a l i t a t i v e l y o r q u a n t i t a t i v e l y from i n j u r y s i t e s l a c k i n g c o l l a g e n i m p l a n t s . D e m y e l i n a t i o n , c a v i t a t i o n and macrophage i n f i l t r a t i o n were a l l d e t e c t e d i n t h e c o l l a g e n t r e a t e d l e s i o n and d i d n o t appear t o be l e s s e x t e n s i v e o r l e s s v a r i a b l e t h a n damage seen i n u n t r e a t e d l e s i o n s . H i s t o l o g y o f C h r o n i c L e s i o n s T r e a t e d w i t h C e l l I m p l a n t s The s u r v i v a l o f i m p l a n t e d embryonic s p i n a l c o r d c e l l s was d e t e c t e d i n 4/6 3 month l e s i o n s i n j e c t e d w i t h t h e c e l l s suspended i n s a l i n e and i n 7/15 l e s i o n s t r e a t e d w i t h embryonic c e l l s suspended i n c o l l a g e n g e l . T h e r e f o r e i t appears t h a t t h e p r e s e n c e o r absence o f t h e c o l l a g e n g e l d i d n o t g r e a t l y i n f l u e n c e t h e s u c c e s s o f n e u r a l t r a n s p l a n t a t i o n i n t h i s e x p e r i m e n t . I m p l a n t e d s p i n a l c o r d c e l l s c o u l d be d i s t i n g u i s h e d from h o s t t i s s u e i n t h e s e s e c t i o n s by t h e i r d i s t i n c t morphology and dense p a c k i n g i n t h e l e s i o n a r e a , as shown i n F i g s . 12, 13 and 14. The i m p l a n t s were o f s p h e r i c a l o r e l l i p s o i d a l form, as shown 51 F i g . 12. A 5 um p a r a f f i n embedded t r a n s v e r s e s e c t i o n o b t a i n e d 3 months p o s t l e s i o n g e n e r a t i o n f o l l o w e d by i m p l a n t a t i o n o f f o e t a l s p i n a l c o r d c e l l s i n c o l l a g e n m a t r i x . The s e c t i o n was s t a i n e d w i t h c r e s y l v i o l e t and L u x o l f a s t b l u e . Note t r a n s p l a n t e d c e l l s i n v e n t r o - m e d i a l a r e a o f c o r d . The t r a n s p l a n t i s more l a t e r a l t h a n w o u l d be e x p e c t e d because t h e l a t e r a l d i s p l a c e m e n t o f t h e d o r s a l s p i n a l a r t e r y p r e v e n t e d a c c e s s t o t h e optimum a r e a o f t h e c o r d . Some macrophage i n f i l t r a t i o n i s a l s o seen i n d o r s o - m e d i a l r e g i o n . 60 x. 52 I M P L A N T M A C R O P H A G E S 53 F i g . 13. A 5 una. p a r a f f i n embedded l o n g i t u d i n a l s e c t i o n o f r a t s p i n a l c o r d s t a i n e d 3 months p o s t i m p l a n t a t i o n . Note s p h e r i c a l shape o f i m p l a n t r e g i o n and c y s t i c c a v i t y f o r m a t i o n a l o n g one edge. 35 x 54 C A V I T A T I O N T R A N S P L A N T C E N T R A L C A N A L 55 Figure 14. A 5 um p a r a f f i n embedded longitudinal section of r a t spina l cord stained 3 months post implantation. Note e l l i p s o i d a l shape of implant region and c y s t i c c a v i t y development. 35 x 56 C Y S T I C A R E A I M P L A N T H O S T W H I T E M A T T E R 5 7 i n F i g . 13 and F i g . 14 r e s p e c t i v e l y . I n a l l c a s e s where t r a n s p l a n t e d m a t e r i a l c o u l d be seen, some degree o f c a v i t a t i o n p e r s i s t e d a t t h e margins o f t h e l e s i o n c a v i t y and macrophage i n f i l t r a t i o n was p r e s e n t i n t h e s e s e c t i o n s ( F i g . 1 2 ) . However, t h e r e were a l s o e x t e n s i v e a r e a s o f h o s t - d o n o r i n t e r f a c e where t h e r e appeared t o be no c a v i t a t i o n o r macrophage i n f i l t r a t i o n , as shown i n F i g . 15. Assessment o f V o l u n t a r y Motor Performance i n L e s i o n e d Rats The i n c l i n e d p l a n e t e s t was used as an i n d e x o f v o l u n t a r y motor a b i l i t y i n r a t s s u b j e c t e d t o a c h r o n i c e l e c t r o l y t i c l e s i o n . F i g . 16 compares t h e maximum a n g l e s o b t a i n e d by an u n l e s i o n e d c o n t r o l group, a l e s i o n e d group i n j e c t e d w i t h f o e t a l c e l l s o n l y , a group i n j e c t e d w i t h c o l l a g e n s o l u t i o n o n l y , a group w h i c h was i n j e c t e d w i t h c e l l s suspended i n c o l l a g e n and a group w h i c h underwent o n l y t h e l e s i o n p r o c e d u r e . The maximum a n g l e s o b t a i n e d by any o f t h e l e s i o n e d groups were n o t s i g n i f i c a n t l y d i f f e r e n t from t h e performance seen i n i n t a c t c o n t r o l r a t s (P < 0.05, W i l c o x o n t e s t ) . F o r c o m p a r i s o n , F i g . 16 a l s o i n d i c a t e s t h e perf o r m a n c e o f a f u r t h e r e x p e r i m e n t a l group s u b j e c t e d t o a co m p l e t e s p i n a l c o r d t r a n s e c t i o n a t t h e L2-L3 l e v e l , 70 days p r i o r t o t e s t i n g . T h i s p r o c e d u r e d i d r e s u l t i n a l a r g e , s i g n i f i c a n t (P < 0.01, W i l c o x o n t e s t ) r e d u c t i o n i n t h e maximum a n g l e s o b t a i n e d , i n agreement w i t h p r e v i o u s s t u d i e s o f t h i s k i n d [ R i v l i n & T a t o r , 1977; T a t o r , 1975; T a t o r , 1973]. 58 F i g . 15. A 5 ura p a r a f f i n embedded l o n g i t u d i n a l s e c t i o n s t a i n e d w i t h c r e s y l v i o l e t and L u x o l f a s t b l u e . H i g h power v i e w o f h o s t w h i t e m a t t e r (bottom) -donor ( t o p ) i n t e r f a c e . Note good a p p o s i t i o n and l a c k o f c e l l u l a r a c c u m u l a t i o n a t i n t e r f a c e . 300 x. 59 I M P L A N T Fig . 16. Histogram of inclined plane scores attained by rats treated with various experimental protocols. Numbers of rats in each group : lesion only : 15; c e l l implant : 6; collagen implant : 15; collagen + ce l l s implant : 15. No significant difference exists between any of the e l ec tro ly t i ca l ly lesioned groups and the control , unlesioned rats . No significant differences exists between any of the e lectrolyt ic lesion groups (P > 0.05, Wilcoxon test) . Note dramatic performance def ic i t in the group subject to complete (bilateral) transection at the L2-L3 level (P < 0.01, Wilcoxon test) when compared to control group. Error bars represent the 95 % confidence interval . 61 Inclined P lane P e r f o r m a n c e oo 100-1 P 9 0 -No Surgery Lesion Only Lesion it Lesion & Lesion & Spinal Cord Foetal Cells Collagen Collagen Transection & Cells 62 The motor performance of the experimental groups was also graded i n a b l i n d assessment using the Tarlov scale (Table 2). The data obtained from one lesioned animal were not used i n s t a t i s t i c a l analysis because of the onset of t o t a l b i l a t e r a l paraplegia. In a l l remaining lesioned animals, the l e f t , unoperated legs were graded at l e v e l 4, i n d i c a t i n g that they showed no detectable functional d e f i c i t . In a l l lesioned groups, the mean Tarlov score for the r i g h t limb was s i g n i f i c a n t l y lower than 4 (P < 0.05, Wilcoxon t e s t ) . This r e s u l t indicated that a s i g n i f i c a n t degree of motor d e f i c i t had indeed been produced by the l e s i o n procedure. A s t a t i s t i c a l comparison was made between l e s i o n only rats and animals receiving collagen, c e l l s or collagen plus c e l l implants. I t was found that none of the treated l e s i o n groups d i f f e r e d s i g n i f i c a n t l y i n t h e i r mean Tarlov scores when compared to l e s i o n only rats (Table 2). The Influence of Chronic E l e c t r o l y t i c Lesions on the Weight  of the T i b i a l i s Anterior Muscle To detect possible damage to lower motor neurons 3 months post l e s i o n , the t i b i a l i s anterior muscle was dissected out and weighed. F i g . 17 shows that the weights of the t i b i a l i s muscles removed from lesioned rats were not s i g n i f i c a n t l y d i f f e r e n t from muscles of control animals (P < 0.05, Wilcoxon t e s t ) . This P r o t o c o l l e s i o n + c o l l a g e n Performance S c o r e R i g h t L e f t mean ± S.D. l e s i o n + c o l l a g e n + c e l l s mean ± S.D. 1 2 2 2 3 3 1 1 2 3 3 ,1±0.8 3 3 1 3 3 2 3 2 2 2 2 2.4±0.6 4 4 4 4 4 4 4 4 4 4 4 ,0±0.0 4 4 4 4 4 4 4 4 4 4 4 4.0+0.0 P r o t o c o l l e s i o n + c e l l s P erformance Score mean ± S.D l e s i o n + b u f f e r mean ± S.D. R i g h t 2 2 2 3 0 1 1.7±1.( 2 0 1 0 3 2 3 3 2 2 3 3 2 2 3 2.1±1.0 L e f t 4 4 4 4 4 4 4.0±0, 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 0±0 T a b l e 2. Performance s c o r e s o f i n d i v i d u a l r i g h t and l e f t r a t h i n d l i m b s . The assessments were made 3 months a f t e r r i g h t s i d e l e s i o n g e n e r a t i o n and i m p l a n t , c o l l a g e n o r b u f f e r i n j e c t i o n . S c o r e s a r e d e r i v e d from a T a r l o v s c a l e [ R i v l i n , 1977,1973] where complete p a r a l y s i s i s a s s i g n e d a s c o r e o f 0 and normal movement a s c o r e o f 4. W i t h i n a l l f o u r g r o u p s , t h e r i g h t and l e f t l i m b s c o r e s a r e s i g n i f i c a n t l y d i f f e r e n t from each o t h e r (P <0.05, W i l c o x o n t e s t ) . I n a l l a n i m a l s t h e l e f t ( u n l e s i o n e d ) s i d e showed normal f u n c t i o n ( s c o r e 4 ) . Comparison o f r i g h t l i m b s c o r e s between any two groups f a i l e d t o show a s t a t i s t i c a l d i f f e r e n c e (P > 0.05, W i l c o x o n t e s t ) . 64 F i g . 17. Histogram of muscle weights of dissected, f i x e d t i b i a l i s anterior muscles. In each group, damage to the lower motor neuron would r e s u l t i n atrophy of the muscle. Note lack of s i g n i f i c a n t difference e i t h e r between l e f t and r i g h t muscles i n any group or between any groups (P > 0.05, Wilcoxon t e s t ) . Different body weights were taken into account by adjusting the muscle weight up or down to match a body weight of 275 gm. For example : the muscle weight of a 300 gm r a t would be m u l t i p l i e d by 275/300 to a r r i v e at the adjusted muscle weight. 65 cn ^ 1.0 -+-' c < 0 .8 o 0.6-I o • Left TD CD CO 0.4 0.2 o c 0.0 o -•—' CO Cont ro l Buf fer Co l lagen Cel ls Col lag + Cell e*' 66 r e s u l t i n d i c a t e s t h a t , i n t h e g r e a t m a j o r i t y o f l e s i o n e d r a t s , e x t e n s i v e l o s s o f l o w e r motor neurons s u p p l y i n g t h e t i b i a l i s a n t e r i o r m u s c l e s d i d not t a k e p l a c e . An e x c e p t i o n t o t h i s o c c u r r e d i n t h e c a s e o f t h e one r a t w h i c h d e v e l o p e d t o t a l p a r a p l e g i a and consequent w a s t i n g o f t h e t i b i a l i s m u s c l e s ( w e i g h t s : l e f t 0.35 g, r i g h t 0.17 g ) . H i s t o l o g i c a l e x a m i n a t i o n showed t h a t t h e s p i n a l c o r d l e s i o n had expanded 1 t o 2 c o r d segments beyond t h e i n i t i a l l e s i o n a r e a and l i t t l e i d e n t i f i a b l e v e n t r a l g r e y m a t t e r remained i n t h i s a r e a . The D i s t r i b u t i o n o f 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 m m u n o r e a c t i v i t Y i n Normal S p i n a l Cord S e c t i o n s The d i s t r i b u t i o n o f l a b e l l e d GFAP - l i k e i m m u n o r e a c t i v i t y i n s e c t i o n s o f u n l e s i o n e d s p i n a l c o r d i s i l l u s t r a t e d i n F i g 18. I n t h e s e t y p i c a l s e c t i o n s , t h e average number o f GFAP r e a c t i v e p r o f i l e s , as d e f i n e d i n Methods and M a t e r i a l s , was 27 ( T a b l e 3 ) . I t was n o t e d t h a t GFAP p o s i t i v e p r o f i l e s near t h e margins o f t h e s e c t i o n s t e n d e d t o p o s s e s s a f i b r o u s appearance and t o be r a d i a l l y a r r a n g e d . I n c o n t r a s t , GFAP p r o f i l e s i n t h e g r e y m a t t e r o f t h e c o r d were t y p i c a l l y s t e l l a t e i n appearance ( F i g . 1 9 ) . 67 F i g 18. A 5 um p a r a f f i n embedded transverse section at L2-L3 l e v e l of unlesioned r a t spinal cord l a b e l l e d with fluorochrome-conjugated a n t i - GFAP antibody. Note l a b e l l e d c e l l s near periphery of cord c h a r a c t e r i s t i c of fibrous g l i a i n contrast to l a b e l l e d stellate-shaped c e l l s i n the grey matter. 135 x. 68 F I B R O U S G L I A 69 normals l e s i o n collagen c e l l s collagen-t-cells Mean±S.D. n 27±4* 9 3±1 9 3±1 9 4±1 9 4±1 9 * s i g n i f i c a n t l y d i f f e r e n t (P < 0.01, Wilcoxon test) from l e s i o n , collagen, c e l l s and collagen + c e l l s groups, n = number of sections counted. Table 3. Astrocyte counts taken from 5 um p a r a f f i n embedded sections of spinal cord. An astrocyte was counted i f a r a d i a l pattern of processes was seen i n association with an unlabelled c e l l body. The negative control sections were incubated with an i r r e l e v a n t (anti -actin) antibody. Astrocyte counts for these negative controls revealed no l a b e l l e d c e l l s per section. Normal sections were taken from animals not subjected to any experimental protocol. Other groups were inje c t e d with buffer, collagen, c e l l s , and a mixture of collagen and c e l l s . No s t a t i s t i c a l differences were detected between the 4 groups of rats receiving e l e c t r o l y t i c lesions and subsequent implants. 70 F i g . 19. A 5 urn p a r a f f i n embedded transverse section of normal spin a l cord grey matter immunostained for GFAP using a double l a b e l l i n g process and developed with peroxidase. Note the presence of many ast r o c y t i c c e l l s . The white arrow shows an astrocyte q u a l i f y i n g for counting since the soma can be seen. Note GFAP r e a c t i v i t y around blood vessel t y p i c a l of g l i a l connectivity with blood supply. 430 x 71 B L O O D V E S S E L A S T R O C Y T E 72 GFAP - l i k e I m m u n o r e a c t i v i t y i n S e c t i o n s Taken from L e s i o n e d S p i n a l Cords H i s t o l o g i c a l s e c t i o n s t a k e n from l e s i o n e d s p i n a l c o r d s e x h i b i t e d d e c r e a s e d numbers o f a s t r o c y t i c , GFAP r e a c t i v e c e l l s when compared t o s e c t i o n s from a n i m a l s w h i c h had n o t been l e s i o n e d ( F i g . 2 0 ) . T h i s change i n t h e d e n s i t y o f GFAP i m m u n o r e a c t i v i t y c o u l d be d e t e c t e d i n s e c t i o n s t a k e n d i r e c t l y t h r o u g h t h e p l a n e o f t h e l e s i o n . But i t was a l s o p r e s e n t i n s e c t i o n s w h i c h appeared m o r p h o l o g i c a l l y normal i n a l l o t h e r r e s p e c t s and w h i c h were t a k e n from an a r e a o f t h e s p i n a l c o r d up t o 5 mm d i s t a n t from t h e l e s i o n p l a n e . F o r t h e sake o f comparison t h e n , c o u n t s o f GFAP p r o f i l e s were av e r a g e d from s e c t i o n s w h i c h l a y w i t h i n 1 mm o f t h e l e s i o n p l a n e f o r a l l l e s i o n b e a r i n g groups i n t h i s s t u d y . Counts o f GFAP p o s i t i v e p r o f i l e s p e r s e c t i o n a r e compared f o r t h e v a r i o u s e x p e r i m e n t a l groups i n T a b l e 3. The W i l c o x o n t e s t f o r u n p a i r e d d a t a showed t h a t s i g n i f i c a n t (P < 0.01) d i f f e r e n c e s e x i s t e d i n t h e c o u n t s o f b o t h t h e i r r e l e v a n t a n t i b o d y c o n t r o l group and t h e n o n - l e s i o n c o n t r o l group, when compared t o any o f t h e l e s i o n e d groups. There was no s i g n i f i c a n t d i f f e r e n c e i n t h e numbers o f GFAP p o s i t i v e p r o f i l e s p e r s e c t i o n when compared between l e s i o n o n l y a n i m a l s and a n i m a l s r e c e i v i n g any o f t h e c o l l a g e n o r c e l l i m p l a n t s ( T a b l e 3, P > 0.05, W i l c o x o n t e s t ) . 73 F i g . 20. A 5 um p a r a f f i n embedded transverse section of lesioned spin a l cord grey matter immunostained for GFAP using a double l a b e l l i n g process and developed with peroxidase. Note v i r t u a l absence of astrocytes s i m i l a r to those i n F i g . 18. Note blood vessel with attached fragment of an as t r o c y t i c process. 430 x 74 DISCUSSION The present experiments have u t i l i z e d the e l e c t r o l y t i c l e s i o n generation method to produce u n i l a t e r a l , l o c a l i z e d damage at the L2-L3 l e v e l of the r a t spinal cord. The Tarlov motor performance assessment showed that a s i g n i f i c a n t motor d e f i c i t was created as a r e s u l t of these lesions and that t h i s d e f i c i t affected only the r i g h t hindlimb, as intended. In contrast, the i n c l i n e d plane t e s t of voluntary motor function f a i l e d to discriminate between lesioned rats and normal controls. The u t i l i t y of t h i s t e s t therefore appears to be l i m i t e d to b i l a t e r a l lesions of the cord, such as those produced by complete transection ( R i v l i n & Tator, 1977). In the present experiment, the animals were uniplegic and were able to cope with the challenge of the i n c l i n e d plane well enough to mask any differences i n performance. However, the uniplegic animal was s i g n i f i c a n t l y easier to care for than was the paraplegic one since bladder and grooming functions are not affected enough to warrant constant care of the animal. When examined 3 months post-injury, e l e c t r o l y t i c l e s i o n s i t e s showed considerable v a r i a b i l i t y with respect to the degree of c a v i t a t i o n , demyelination and macrophage i n f i l t r a t i o n that was present. The following factors may have been responsible for 76 some of t h i s v a r i a b i l i t y . I t was d i f f i c u l t to determine exactly when the l e s i o n i n g electrode was i n contact with the surface of the cord and t h i s affected the ultimate depth of the l e s i o n . Some e l e c t r o l y t i c destruction of the electrode occurred during a p p l i c a t i o n of the current and t h i s tended to cause v a r i a t i o n i n the current density over the borders of the l e s i o n . At the voltages used (150 V) some e l e c t r o l y t i c chemical breakdown was occurring at the electrode. This resulted i n the formation of bubbles of gas near the electrode, which e f f e c t i v e l y insulated that part of the electrode from the surrounding t i s s u e since gas i s a poor e l e c t r i c a l conductor. Perhaps the largest problem with the e l e c t r o l y t i c technique was the muscular spasm which occurred at the onset of the current. Since the current was applied near the c o r t i c o s p i n a l t r a c t , there was extensive recruitment of motor units i n the muscles i n f e r i o r to the s i t e of current a p p l i c a t i o n . This caused a l a t e r a l curvature of the spinal column while the current remained on. There was a further twitch when the current was switched o f f . Both of the muscle spasms were very strong, r e s u l t i n g i n spin a l column movement, and could not be eliminated completely with the spinal clamps used. The r e s u l t of t h i s movement was that the spinal cord could s h i f t p o s i t i o n during the generation of the l e s i o n , r e s u l t i n g i n additional damage to both grey and white matter. 77 The variable histology of the lesions formed by the e l e c t r o l y t i c process meant that a large number of animals would be required i n studies aimed at assessing the e f f e c t s of c e l l u l a r implants on the appearance of these lesions. Measurement of the t i b i a l i s anterior muscle weights showed that the e l e c t r o l y t i c lesions caused l i t t l e d i r e c t damage to the lower motor neurons innervating t h i s muscle. This suggests that these lesions were successfully l o c a l i z e d to the dorsal part of the cord, since the lower motor neurons for the t i b i a l i s anterior muscle are i n the L2-L3 region of the cord which l i e s i n the area of the T10 vertebra. If the lower motor neurons had been disrupted by the l e s i o n i n g procedure, atrophy and wasting of the muscle should have been observed [Guttman,1985] as well as f l a c c i d p a r a l y s i s . Moreover, since lesioned animals did show a functional d e f i c i t and spastic paralysis i n the t i b i a l i s anterior muscle, the d e f i c i t must have resulted l a r g e l y from interruption of the descending c o r t i c o s p i n a l or other motor t r a c t s . In the rats subjected to u n i l a t e r a l e l e c t r o l y t i c lesions there was l i t t l e evidence of autocannibalism and of the consequent u l c e r a t i o n seen i n denervated grooming rats [ R i v l i n & Tator, 1977]. The somatosensory evoked po t e n t i a l data from the acutely lesioned animals also showed that such an e l e c t r o l y t i c l e s i o n d i d not block impulse transmission i n the ascending columns. I t i s generally accepted that impulses evoked by the 78 t e s t s t i m u l u s ascend i n t h e s p i n o t h a l a m i c t r a c t a f t e r e n t e r i n g t h e c o r d i n f e r i o r t o t h e l e s i o n , i n t h e a r e a o f t h e 3 r d lumbar, by way o f t h e p o s t e r i o r b r a n c h o f t h e s a c r a l p l e x u s and deep p e r o n e a l n e r v e and t h e common p e r o n e a l n e r v e [Greene, 1959]. I t has been shown i n t h e p r e s e n t e x p e r i m e n t s t h a t f o e t a l s p i n a l c o r d neurons can be s u c c e s s f u l l y t r a n s p l a n t e d i n t o a r e a s o f s p i n a l c o r d s s u b j e c t e d t o e l e c t r o l y t i c l e s i o n s . These g r a f t s appear t o r e m a i n v i a b l e f o r months and a l s o appear t o undergo d i f f e r e n t i a t i o n t y p i c a l o f normal f o e t a l c e l l s . More s o p h i s t i c a t e d n e u r o a n a t o m i c a l and immunocytochemical i n v e s t i g a t i o n i s , however, r e q u i r e d t o demonstrate p o s s i b l e a x o n a l c o n n e c t i v i t y between t h e h o s t and t h e g r a f t . I t would appear from t h e s e d a t a t h a t embryonic s p i n a l c o r d c e l l s have t h e p o t e n t i a l t o r e p l a c e h o s t neurons a b l a t e d from t h e c o r d by means o f e l e c t r o l y t i c l e s i o n i n g . I n t h e p r e s e n t e x p e r i m e n t s , however, t h e r e was no e v i d e n c e o f r e s t o r a t i o n o f v o l u n t a r y motor performance i n a n i m a l s r e c e i v i n g any o f t h e c e l l u l a r o r c o l l a g e n i m p l a n t s , as judged by t h e T a r l o v s c a l e o f motor f u n c t i o n . T h i s r e s u l t i s i n agreement w i t h p r e v i o u s s t u d i e s c o n d u c t e d on s p i n a l s e c t i o n e d r a t s , w h i c h showed t h a t n e i t h e r i m p l a n t s o f c o l l a g e n g e l s [ S c h r e y e r & J o n e s , 1987] n o r embryonic c e l l s [ B e r n s t e i n , H o o v l e r & T u r t i l , 1985; Das, 1983b] s i g n i f i c a n t l y r e s t o r e d v o l u n t a r y motor performance i n t e s t a n i m a l s . 79 Further studies would benefit from using i n t r a c e l l u l a r axon-tracing techniques such as wheat germ agglutinated horseradish peroxidase to allow determination of the extent of transplanted neural processes [Carlstedt, Dalsgaard & Molander, 1987; Schreyer & Jones, 1987]. In addition, t r i t i a t e d thymidine fed to pregnant donor rats would y i e l d embryonic c e l l s i n which the neuronal DNA was r a d i o a c t i v e l y l a b e l l e d . This would allow c l e a r discrimination between host and transplanted neurons. Electron microscopic i n v e s t i g a t i o n of the donor-host interface might y i e l d further information about the nature of the g l i a l r e action i n t h i s area. Ultimately, i t would be valuable to know i f the surviving transplanted neurons had any detectable e l e c t r o p h y s i o l o g i c a l a c t i v i t y and i f any functional connectivity to the res t of the cord occurred. The f a i l u r e of some researchers to obtain f o e t a l spinal cord gr a f t s u r v i v a l [Das, 1983a] may have been due to the excessive age of the f o e t a l t i s s u e at the time of gr a f t i n g . Older (16-18 day old) foetuses were used i n these experiments, i n contrast to the 14 day o l d foetuses used i n the present experiment. Autoradiographic evidence from the study of developing neurons [Altman and Bayer, 1984] has shown that most neurons originate between day 11 and 16 of gestation. Since g l i a have a s i m i l a r developmental period [Gilmore, 1971] a normal r a t i o of neurons to g l i a should be preserved during growth of the gr a f t . A 80 t r a n s p l a n t o b t a i n e d from E14 embryos s h o u l d c o n t a i n l a r g e numbers o f r e l a t i v e l y u n d i f f e r e n t i a t e d neurons. I n p r e v i o u s s t u d i e s , i t has been r e p o r t e d t h a t s u r g i c a l i n t e r v e n t i o n i n t h e c o r d n o r m a l l y r e s u l t s i n t h e appearance o f i n c r e a s e d GFAP - l i k e i m m u n o r e a c t i v i t y i n t h i s t i s s u e . T h i s phenomenon has been seen a f t e r c o r d t r a n s e c t i o n [ P r e d y & M a l h o t r a , 1989], a s p i r a t i o n o f a wound c a v i t y [ B e r n s t e i n & G o l d b e r g , 1986] and s e c t i o n o f a c r a n i a l n e r v e [ H a l l e t a l , 1989]. I n t h e p r e s e n t s t u d y , a l l s p i n a l c o r d s u b j e c t e d t o e l e c t r o l y t i c l e s i o n s were found t o e x h i b i t r e d u c e d numbers o f GFAP p o s i t i v e p r o f i l e s when examined 3 months p o s t l e s i o n . T h i s u n e x p e c t e d r e s u l t c o u l d have o c c u r r e d f o r t h e f o l l o w i n g r e a s o n s . I t i s p o s s i b l e t h a t l e s i o n i n d u c e d damage t o t h e l o c a l c i r c u l a t o r y system o f t h e c o r d s e r v e d t o i s o l a t e t h e l e s i o n a r e a d u r i n g subsequent p e r f u s i o n o f t h e t i s s u e . Inadequate f i x a t i o n c o u l d t h e n r e s u l t i n d e c r e a s e d s e n s i t i v i t y t o t h e GFAP immunoassay. T h i s p o s s i b i l i t y seems u n l i k e l y s i n c e a l l l e s i o n e d c o r d s were removed from t h e h o s t i m m e d i a t e l y f o l l o w i n g p e r f u s i o n and p l a c e d i n f i x a t i v e s o l u t i o n o v e r n i g h t p r i o r t o f u r t h e r p r o c e s s i n g . I n a d d i t i o n , t h e o v e r a l l h i s t o l o g i c a l q u a l i t y o f s e c t i o n s t a k e n from t h e l e s i o n e d a n i m a l s was n o t n o t i c e a b l y i n f e r i o r t o t h a t seen i n s e c t i o n s t a k e n from u n o p e r a t e d c o n t r o l a n i m a l s . I t s h o u l d be n o t e d t h a t s u r g i c a l damage t o t h e c o r d s h o u l d a l s o produce compromised b l o o d f l o w t o t h e a r e a o f damage, 81 yet in this case, enhanced, not reduced, GFAP - l ike immunoreactivity is invariably seen [Reier, 1986; Reier et a l , 1983]. Possible differences in technique and antibody degradation were accounted for by control experiments. The most l i k e l y reason for the disappearance of GFAP react iv i ty in the present experiment is that the e lectrolyt ic lesion resulted in some alteration of the character or expression of GFAP so that this molecule became unrecognizable to the anti - GFAP antibody. Commercial GFAP antibodies are mouse monoclonal and are very specific in their ac t iv i ty . These monoclonal antibodies are prepared by rais ing antibodies against purif ied GFAP, resulting in an antibody specific to only that one GFAP molecule. A polyclonal antibody is prepared by rais ing antibodies to a re la t ive ly unpurified neuronal extract. This results in antibodies to many different neural c e l l molecules. Because of the spec i f ic i ty of anti - GFAP, i t is unlikely that the antibodies would label an altered state of the molecule. In addition, there is evidence for the transient expression and deletion of ce l lu lar antigenic determinants within the injured spinal cord [Aquino et a l . , 1988; Maehlen et a l . , 1988]. It has been shown that g l i a move and proliferate at the s ite of a lesion so i t is l i k e l y that, given the spec i f ic i ty of this anti -82 GFAP a n t i b o d y u s e d , a s l i g h t change i n t h e c h a r a c t e r o f GFAP i s r e s u l t i n g i n a l a c k o f r e c o g n i t i o n by t h e a p p l i e d a n t i b o d y . T h i s p o s s i b i l i t y s h o u l d be t e s t e d by a more e x t e n s i v e l o n g i t u d i n a l s t u d y o f GFAP e x p r e s s i o n i n s p i n a l c o r d s r e c e i v i n g e l e c t r o l y t i c damage. I t i s a l s o i m p o r t a n t t o d e t e r m i n e t h e volume o f t h e c o r d w h i c h shows t h i s change i n GFAP e x p r e s s i o n , as a f u n c t i o n o f t h e s e v e r i t y o f t h e a p p l i e d l e s i o n . Whatever t h e cause o f t h e d e c r e a s e d GFAP - l i k e i m m u n o r e a c t i v i t y i n l e s i o n e d s p i n a l c o r d s , i t was found t h a t t h i s change c o u l d n o t be p r e v e n t e d by i n j e c t i o n o f l e s i o n s i t e s w i t h any o f t h e c o l l a g e n o r n e u r o n a l i m p l a n t s used i n t h i s s t u d y . T h i s r e s u l t c o r r e l a t e s w e l l w i t h an analogous s t u d y p e r f o r m e d on s u r g i c a l l y l e s i o n e d s p i n a l c o r d s . Here i t was shown t h a t i m p l a n t a t i o n o f embryonic c e l l s c o u l d n o t p r e v e n t t h e i n c r e a s e i n GFAP i m m u n o r e a c t i v i t y w h i c h accompanies m e c h a n i c a l damage t o t h e c o r d . I t seems l i k e l y , t h e r e f o r e , t h a t GFAP e x p r e s s i o n i n damaged c o r d s i s c o n t r o l l e d by f a c t o r s w h i c h cannot be p r o v i d e d e i t h e r by c o l l a g e n i n j e c t i o n s o r by c e l l u l a r i m p l a n t s . The n a t u r e o f t h e s e f a c t o r s i s a t p r e s e n t u n c l e a r . However i t i s known t h a t GFAP e x p r e s s i o n i n c u l t u r e d a s t r o c y t e s i s up r e g u l a t e d i n t h e p r e s e n c e o f h i g h K + i n t h e c u l t u r e medium [Canady e t a l , 1987], down r e g u l a t e d i n t h e p r e s e n c e o f h y p e r o s m o t i c media [Canady e t a l , 1988] and i n v e r s e l y r e l a t e d t o 83 the level of c irculat ing corticosterone [O'Callaghan et a l , 1988]. It i s possible that the unique environment generated by the e lectro lyt ic lesion is in some way affecting expression of the GFAP. In the studies of the effect of potassium, osmolarity and corticosterone, which represent f a i r l y typical physiological manipulations, alteration of GFAP expression may be secondary to an alteration in the functions of the astrocytes rather than being an indication of the actual number of astrocytes in the t issue. For example, one of the functions attributed to g l i a is the regulation of extracellular potassium. An increase in this parameter might result in greater act iv i ty of existing g l i a , the production of new g l i a or the activation of a population of g l i a l precursor. A l l of these situations would add to the pool of GFAP antigen s i tes . The e lectrolyt ic lesion technique may have resulted in moderated GFAP - l ike immunoreactivity by fa i l ing to stimulate astrocytes as is often seen in surgical manipulations. In addition, a reaction product unique to the e lectrolyt ic lesion procedure may have affected the g l ia to suppress GFAP immunoreactivity. Such a premise is perhaps tenable to explain GFAP label l ing for a period of days after lesion generation. 84 However, the reason f o r the persistence of the reduced GFAP -l i k e immunoreactivity up to 3 months post-lesion i s unclear. On the basis of t h i s study and others l i k e i t , i t i s doubtful whether simple transplantation of f o e t a l s p i n a l cord neurons can lead to s i g n i f i c a n t functional recovery of the voluntary motor performance i n mammals. Indeed there e x i s t several reasons to regard such transplants as being p o t e n t i a l l y hazardous to the r e c i p i e n t . For example, crossed connections i n sensory pathways could lead to confusion, since sensation a r i s i n g i n one dermatome might f e e l as though i t arose somewhere else. Analogous problems might occur with the control of muscle. There i s also a p o t e n t i a l problem regarding the long-term s u r v i v a b i l i t y of embryonic implants. It w i l l be c r i t i c a l to ensure that enough c e l l s are transplanted to make the appropriate connections since further transplants would increase the damage done to the s p i n a l cord. It w i l l also be necessary to monitor the growth of the transplanted c e l l s over a long period of time to e s t a b l i s h the ultimate degree of p r o l i f e r a t i o n of a l l transplanted c e l l s . If g l i a continued to p r o l i f e r a t e , for example, pressure on the healthy ends of the cord could develop and cause further d i s a b i l i t y to the patient. 85 I t i s also possible that an imbalance i n the innervation of feedback and control c i r c u i t s would r e s u l t i n an inoperative network. For example, i f presynaptic i n h i b i t o r y inputs were enhanced by the implant, the target neuron might not depolarize appropriately on e x c i t a t i o n of the upper motoneuron. These possible problems lead to the question of why spontaneous regeneration i s apparently so l i m i t e d i n the adult mammalian c e n t r a l nervous system. Given that sprouting of CNS axons does occur, i t may be agreed that functional r e p a i r , i f not c e l l u l a r r e p a i r , can occur. This regeneration on a small scale depends upon the existence of i n t a c t target neurons upon which the sprouts can synapse. The capacity for CNS mitosis and c e l l u l a r d i f f e r e n t i a t i o n may have been counterproductive due to the complexity of the connections required to r e b u i l d a large area of damage. There would be no way that regrowing neurons could be directed to synapse i n the pre-injury pattern. The r e s u l t i n g confusion of neuronal t r a f f i c might have resulted i n no functional benefit to the injured organism. 86 R e f e r e n c e s A f i f i , A.K., Bergman, R.A. (1986) B a s i c N e u r o s c i e n c e . 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