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The effect of denervation on the contractile properties of skeletal muscle Webster, Deirdre M. S. 1982

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THE EFFECT OF DENERVATION ON THE CONTRACTILE PROPERTIES OF SKELETAL MUSCLE by DEIRDRE M. S. WEBSTER B.S.R.(PT), The University of B r i t i s h Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Anatomy We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA MAY 1982 0 DEIRDRE M. S. WEBSTER, 1982 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Anatomy  The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 D a t e May 5, 1982 ABSTRACT The l i t e r a t u r e has provided ample evidence that neural i n f l u e n c e i s res p o n s i b l e f o r the r e g u l a t i o n and maintenance o f muscle p r o p e r t i e s . This study, conducted on approximately 100 mice of the C57 BL/6J+/+ species i n v e s t i g a t e d the d i f f e r e n t i a l e f f e c t s of denervation on the i s o m e t r i c c o n t r a c t i l e p r o p e r t i e s of a f a s t - t w i t c h (extensor digitorum longus) and a slow-twitoh (soleus) muscle. Adult male animals were studied at 1, 28, 84 and 210 days f o l l o w i n g u n i l a t e r a l s e c t i o n o f the s c i a t i c nerve. The muscles were stimulated i n v i t r o at 37°C at optimal l e n g t h by supramaximal square puls e s . The data f o r a l l muscles i n each experimental group were pooled and compared to age-matched c o n t r o l s . In both the denervated soleus (SOL) and extensor digitorum longus (EDL) the time-to-peak t w i t c h tension and the h a l f r e l a x a t i o n were prolonged by 28 days post-denervation and t h i s trend continued to the o l d e s t age groups s t u d i e d . The weight of the denervated muscles was l e s s than t h a t of the c o n t r o l s . Consequently, although the force that could be generated per u n i t mass by the EDL was i n i t i a l l y w e l l maintained, a l l muscles showed reduced peak t e t a n i c tension i n the long term, f o l l o w i n g denervation. Even when developed tension was expressed on a per wet weight b a s i s , soleus became weaker with increased time post-denervation. A s u r p r i s i n g and unexpected-result was the f i n d i n g that 28 days a f t e r denervation both the f a s t and slow-twitch muscles developed increased t e n s i o n . The denervated SOL - i i i -showed a marked decrease i n r e s i s t a n c e to f a t i g u e at 1 and 28 days, whereas the EDL showed an increase i n r e s i s t a n c e to f a t i g u e at 28 days and beyond. I t was concluded that denervation a f f e c t e d the tension generat-i n g a b i l i t y and the c o n t r a c t i o n time of the SOL more than the EDL. The f a t i g u e response i n d i c a t e d that conversion of f i b r e types may have occurred i n the EDL and to a l e s s e r extent i n the SOL. The r e s u l t s support the hypothesis that slow muscle may be more dependant upon neural i n f l u e n c e than f a s t muscle f o r the maintenance of i t s c o n t r a c t i l e p r o p e r t i e s . Further experiments to t e s t t h i s hypothesis are o u t l i n e d . TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i GLOSSARY v i i i ACKNOWLEDGEMENTS i x I INTRODUCTION 1 Co n t r a c t i l e Properties 2 Neural Influence on Muscle 2 Purpose of Study 3 I I REVIEW OF LITERATURE 5 Cross Innervation 6 Neurotrophic Factor 8 Nerve Impulse A c t i v i t y 11 Denervation I1* (a) Morphology I 1* (b) Biochemical Changes 16 (c) C o n t r a c t i l e Properties 17 (d) Membrane Properties 20 Summary 21 I I I METHODS AND PROCEDURES 22 Denervation 23 Functional Denervation Test 24 Muscle Dissection 25 Experimental Apparatus 26 Experimental Procedures 27 Histology 28 Analysis of Data 28 IV RESULTS 30 Cont r a c t i l e Properties of Denervated Muscle 31 (a) Twitch Contraction Time 31 (b) Isometric Tension 37 - i v -- V -Page (c) Fatigue C h a r a c t e r i s t i c s 42 Morphology .... 45 Reinnervated Muscle 55 V DISCUSSION 61 Con t r a c t i l e Properties of Denervated Muscle 62 (a) Twitch Contraction Time 62 (b) Isometric Tension 65 (c) Fatigue C h a r a c t e r i s t i c s 69 Ef f e c t of Time on Post-denervation Changes 71 Slow and Fast Muscle Response to Denervation 75 VI CONCLUSION 77 LIST OF REFERENCES 79 APPENDICES 87 1. E f f e c t of Double-Denervation Procedure 87 2. Comparison of Normalised Tension Data 91 3. Twitch Contraction Times of Normal and Denervated SOL and EDL Muscles 96 4. Experimental Variables 97 - v i -LIST OF TABLES Page I. Denervated SOL muscle data 32 I I . Denervated EDL muscle data 33 I I I . Tension c h a r a c t e r i s t i c s of SOL muscle i n normal and denervated mice 40 IV. Tension c h a r a c t e r i s t i c s of EDL muscle i n normal and denervated mice 41 V. Cross s e c t i o n a l areas of SOL and EDL muscles from normal and denervated mice 48 VI. Isometric c o n t r a c t i l e properties of reinnervated SOL and EDL muscles 60 VII. Comparison of isometric c o n t r a c t i l e properties of 1 X denervated and 2 X denervated SOL and EDL muscles... 88 VIII. Normalised tetanus tension of SOL muscles 94 IX. Normalised tetanus tension of EDL muscles 95 X. Contraction times of SOL and EDL muscles 96 XI. Time taken to di s s e c t SOL and EDL muscles 98 XII. pH of Krebs s o l u t i o n i n SOL and EDL experiments 99 - v i i -LIST OF FIGURES Page 1. Percentage change i n wet weight of denervated SOL and EDL. 3^ 2. Isometric twitch myogram of f a s t and slow muscle 36 3. Time to peak twitch tension of SOL and EDL i n normal and denervated mice 38 4. Half relaxation time of SOL and EDL i n normal and denervated mice 39 5. Fatigue myogram of fast and slow muscle 44 6. Fatigue pattern of normal and denervated SOL muscles 46 7. Fatigue pattern of normal and denervated EDL muscles 47 8. Transverse sections of SOL and EDL 28 day post-denervation 50 9. Transverse sections of SOL and EDL 210 days post-denervation 52 10. Transverse sections of normal and denervated SOL 1 day post-denervation 54 11. Transverse sections of normal and denervated SOL 210 days post-denervation 57 12. Transverse sections of normal and denervated EDL 210 days post-denervation 59 13. SOL and EDL tetanus tension normalised with respect to dry weight and cross s e c t i o n a l area 93 - v i i i -GLOSSARY Po - Maximum isometric tetanus tension Pt - Maximum isometric twitch tension TTP - Time from f i r s t stimulus to maximum height of twitch 1/2RT - Time for maximum twitch tension to decay to half twitch tension lo - Length at which the maximum isometric twitch tension was recorded EDL - Extensor digitorum longus FDL - Flexor digitorum longus FHL - Flexor hallucis longus SOL - Soleus RMP - Resting membrane potential SR - Sarcoplasmic reticulum SO - Slow-oxidative FG - Fast-glycolytic FOG - Fast-oxidative-glycolytic ACKNOWLEDGEMENTS The author wishes to express appreciation to Dr. B. H. Bressler f o r h i s guidance and encouragement throughout t h i s p r o ject. Grateful thanks are extended to Hildegarde Erber and Edie Goble f o r excellent technical assistance; to the members of the Committee, Dr. M. Menard, Dr. L. Jasch and Dr. P. Vaughan f o r t h e i r advice and assistance; to Dr. W. Ovalle f o r help with the photography and to Mary Raphael f o r the preparation of the manuscript. F i n a l l y , thanks are due to my colleagues i n the School of R e h a b i l i t a t i o n Medicine, for t h e i r willingness to cope with my i r r e g u l a r schedule. - 1 -I INTRODUCTION - 2 -Contractile Properties Three types of muscle fibre have been identified according to their histochemistry, speed of contraction and resistance to fatigue and named slow oxidative (SO), fast oxidative glycolytic (FOG), and fast glycolytic (FG) according to the classification of Peter et a l . (1972). According to histochemical c r i t e r i a , based solely on their ATPase activity, they are also termed Type I, IIA and IIB respectively (Brook and Kaiser, 1970). Differentiation of muscle fibres into these various physiologically and histochemically distinct types seems to be largely determined by neural influence. The functional role of the muscle may also influence differentiation, as the proportion of d i f -ferent fibre types in a particular muscle has been found to vary in different species. Since the distribution of fibre types in a muscle determines i t s overall contractile characteristics, fast and slow mus-cles not only differ in their speed of contraction and resistance to fatigue, but are also reported to dif f e r in the amount of tension per unit area they are able to generate. Neural Influence on Muscle Both nerve impulse activity and a neurotrophic factor, of unknown chemical composition, seem to play roles, possibly in some way comple-mentary, in the regulation and maintenance of muscle properties (Guth, 1968; Drachman, 1972*; Gutmann, 1976). Although the specific effects of each of these influences i s s t i l l unclear, their combined effects have been studied by means of denervation. On the whole, loss of neural influence appears to result in an increase in contraction time and a decrease in weight with a concomitant decrease in tension. Since denervated muscle has been shown to exhibit an increase in weight, - 3 -length and contraction time i n response to passive s t r e t c h (Melchina and Gutmann, 1974), i t i s apparent that despite i t s prominent role the neural influence does not exert exclusive control over muscle properties. Purpose of Study Previous studies have shown some inconsistencies i n t h e i r f i n d -ings with regard to the contraction time and tension generating a b i l -i t y of denervated muscle. It appears that the onset and extent of denervation changes d i f f e r not only i n animals of d i f f e r e n t species, but also i n young and old animals of the same species. It also ap-pears that changes exhibited may vary according to the length of time that has elapsed since denervation (Gutmann et a l . , 1972). Since many investigations have been li m i t e d i n both scope and duration i t seems appropriate to conduct a study i n animals of precise age for an ex-tended period of time post-denervation. In t h i s study, the e f f e c t of denervation on the c o n t r a c t i l e properties of slow and f a s t twitch-muscles of the mouse i s i n v e s t i -gated. A review of the l i t e r a t u r e on the e f f e c t s of denervation i s presented. It shows that there i s evidence that there i s a neural i n -fluence on muscle, which involves both a trophic factor and nerve impulse a c t i v i t y , and i s responsible for the maintenance and regula-t i o n of the c o n t r a c t i l e properties of- the muscle. The known e f f e c t s of denervation on fast and slow-twitch muscle are summarized. Then the r e s u l t s of measurements of the e f f e c t of denervation on the con-t r a c t i l e properties of c e r t a i n muscles from approximately 100 mice are presented for i n t e r v a l s varying from 1 to 210 days post-denervation. The c o n t r a c t i l e properties examined include the maximum isometric twitch tension (Pt), time to peak twitch tension (TTP), h a l f r e l a x -ation time ( 1 / 2 R T ) , the maximum tetanus tension (Po) and the r e s i s t -ance to fatigue. The responses of the slow-twitch soleus (SOL) and the fast-twitch extensor digitorum longus (EDL) muscles are compared. It i s shown that the two muscles respond d i f f e r e n t l y to denervation. - 5 -II REVIEW OF LITERATURE - 6 -M o d e r n p h y s i o l o g i c a l a n d h i s t o c h e m i c a l t e c h n i q u e s h a v e e n a b l e d c l a s s i f i c a t i o n o f m u s c l e f i b r e s t h a t r e c o g n i s e s a t l e a s t t h r e e t y p e s ; c o r r e l a t i o n c a n b e made b e t w e e n c o n t r a c t i o n s p e e d a n d r e s i s t a n c e t o f a t i g u e o n t h e o n e h a n d a n d m e t a b o l i c p r o f i l e a n d e n z y m e a c t i v i t i e s o n t h e o t h e r . B u l l e r ( I 9 6 0 ) p r o v i d e d some o f t h e f i r s t s u b s t a n t i v e e v i d e n c e t h a t f i b r e - t y p e d i f f e r e n t i a t i o n may b e u n d e r n e u r a l c o n t r o l , i n h i s e x e c u t i o n o f c r o s s - i n n e r v a t i o n e x p e r i m e n t s . C r o s s - i n n e r v a t i o n B u l l e r , E c c l e s a n d E c c l e s ( 1 9 6 0 b ) d e s i g n e d t h e f o l l o w i n g e x -p e r i m e n t w h i c h e s t a b l i s h e d , f o r t h e f i r s t t i m e , t h a t t h e n e r v e s u p p l y t o f a s t a n d s l o w m u s c l e s i n f l u e n c e d t h e i r s p e e d s o f c o n t r a c t i o n . T h e n e r v e s t o f l e x o r d i g i t o r u m l o n g u s ( F D L ) a n d SOL m u s c l e s w e r e d i v i d e d a n d c r o s s - u n i t e d , s o t h a t u p o n r e g e n e r a t i o n t h e p r e d o m i n a n t l y f a s t t w i t c h F D L was i n n e r v a t e d b y t h e s o l e u s n e r v e ( X - F D L ) a n d v i s e v e r s a , ( X - S O L ) . I n h a l f o f t h e e x p e r i m e n t s t h e n e r v e s w e r e s i m i l a r l y d i -v i d e d , b u t e a c h p r o x i m a l s t u m p was r e u n i t e d t o i t s own d i s t a l s t u m p t o f o r m s e l f - i n n e r v a t e d c o n t r o l s ( S - F D L , S - S O L ) . U s i n g t h i s p r o c e d u r e , i n k i t t e n s a p p r o x i m a t e l y t h r e e w e e k s o l d , t h e y f o u n d a r e v e r s a l i n t h e s p e e d s o f c o n t r a c t i o n o f t h e c r o s s -i n n e r v a t e d m u s c l e s , a s s h o w n b y t h e p a r a m e t e r s T T P , 1 / 2 R T a n d t h e f r e q u e n c y o f s t i m u l a t i o n r e q u i r e d t o p r o d u c e a f u s e d t e t a n u s . T h r e e w e e k s p o s t o p e r a t i v e l y t h e T T P a n d 1 / 2 R T o f t h e X - S O L w e r e s h o r t e n e d f r o m 60 t o 42 ms a n d 62 t o 45 ms r e s p e c t i v e l y , c o m p a r e d t o t h e S - S O L . I n c o n t r a s t t h e T T P a n d 1 / 2 R T o f t h e F D L w e r e l e n g t h e n e d f r o m 33 t o 5 9 ms a n d 32 t o 67 ms c o m p a r e d t o t h e S - F D L . I n a d d i t i o n t h e y f o u n d t h e t w i t c h t e n s i o n ( P t ) o f b o t h c r o s s e d m u s c l e s was 50% l e s s t h a n t h e c o n t r o l s . Post-operative tests were c a r r i e d out from periods ranging from 23-200 days and a s i m i l a r trend was noted throughout. These findings were subsequently confirmed i n the cat ( B u l l e r and Lewis, 1965), and the rat (Close, 1969). I t was also demonstrated that the speed of i s o t o n i c contraction (Close, 1969), and character-i s t i c s such as potentiation of the twitch immediately following a tetanus or i n reponse to cooling (Hoh, 1974) were altered by cross-innervation. Bara'ny (1967) was the f i r s t to demonstrate that the myosin ATPase a c t i v i t y of fast and slow-twitch muscles correlated with t h e i r speeds of contraction. B u l l e r et a l . (1969) demonstrated s i m i l a r findings i n the cat following cross-innervation. In 1970 Guth et a l . investigated the d i s t r i b u t i o n of a c i d -l a b i l e , intermediate, and a l k a l i - l a b i l e actomyosin ATPases i n the f a s t and slow-twitch muscles of the cat and the rat following cross-innervation. As t h e i r r e s u l t s showed that there was a q u a l i t a t i v e change i n the type of ATPase, i n a small proportion of the f i b r e s , they concluded that the nerve influenced the type of myosin ATPase, as well as i t s s p e c i f i c a c t i v i t y . In extensions of these observations, electrophoretograms were used to determine the type of myosin i n the cross-innervated muscles i n the c a t . I t was shown that the X-SOL myosin l o s t i t s character-i s t i c sub-units of low molecular weight proteins, and acquired those of fast muscle myosin, while the reverse changes occurred i n the X-FHL (Samaha et a l . , 1970). Further studies on the e f f e c t s of cross-innervation i n the rat were undertaken by Bardny and Close (1971). They found that isometric - 8 -c o n t r a c t i l e c h a r a c t e r i s t i c s , such as the TTP, 1/2RT, Pt and Po and the structure of myosin as well as i t s ATPase a c t i v i t y were alt e r e d to a greater extent i n the X-EDL than the X-SOL. The r e s u l t s of these experiments (Buller et a l . , 1969; Guth et a l . , 1970; Samaha et a l . , 1970; Barany and Close, 1971) strongly sup-port the hypothesis that the nerve supply of a muscle a c t u a l l y i n f l u -ences the gene expression of the muscle c e l l and thus the nature of the protein the muscle c e l l synthesizes. In the early experiments a small sample and f a i l u r e to prevent inadvertent s e l f reinnervation of the X-SOL and X-EDL may have accoun-ted f or the incomplete e f f e c t of cross-innervation. However even i n cross-innervation experiments where s e l f - r e i n n e r v a t i o n was prevented complete conversion of muscle types was not obtained. Since the controls were self-innervated muscles the f a i l u r e of conversion to be complete was probably not due to the procedure i t s e l f . Therefore there must be some other influence which regulates the c h a r a c t e r i s t i c s of the muscle. Perhaps the functional role of the muscle also has to be changed before complete conversion can be expected. The preceding experiments have c l e a r l y demonstrated the nerve's a b i l i t y to influence the c o n t r a c t i l e properties of muscle. How that influence i s mediated has been the subject of considerable i n v e s t i -gation. B u l l e r and colleagues (1960a) hypothesised'that the e f f e c t of cross-innervation was due to the difference i n nerve impulse a c t i v i t y between the f a s t and slow nerves or to the presence of a trophic factor, Neurotrophic Factor A neurotrophic factor has been defined as a substance respons-i b l e for long-term maintenance of muscle properties, which i s not - 9 -mediated by nerve impulses (Gutmann, 1976). The existence of such a f a c t o r has been supported by empirical observations such as those made by Denny-Brown and Brenner (1944) who noted that "atrophy of the par-alysed muscle appears to be prevented by anatomical connection with the motor horn c e l l i n the absence of conducted nerve impulses". Sub-sequent evidence, that the length of the nerve stump had an e f f e c t on the development of denervation changes, led to further speculation about the nature and function of an 'axonal substance'. Blockade of axonal transport by means of c o l c h i c i n e and v i n b l a s t i n e has shown that denervation-like changes i n membrane properties, such as depolarization, extra-junctional acetylcholine (ACh) s e n s i t i v i t y and tetrodotoxin r e s i s t a n t action p o t e n t i a l s , can be induced without loss of muscle a c t i v i t y (Albuquerque et a l . , 1972; Hoffman and Thesleff, 1972). Tissue culture studies have provided the most conclusive e v i -dence that a d i f f u s a b l e and non-impulse regulated agent i s e f f e c t i v e i n nerve-muscle r e l a t i o n s . Crain and Peterson (1974) demonstrated that rodent f o e t a l muscle explants atrophied unless they were co-cultured with a s p i n a l cord dorsal root ganglion. Furthermore, i f the i s o l a t e d explant was f i r s t allowed to atrophy, recovery ensued when the cord-ganglion complex was added. In other experiments, i n the chicken, soluble proteins were extracted from normal nerves and added to myotube cu l t u r e s . It was found that, i n the presence of the extract, these cultures were main-tained f o r several weeks, whereas control cultures degenerated a f t e r 3-4 days (Oh et a l . , 1980). - 10 -Tissue c u l t u r e has a l s o been used to analyse the mechanisms u n d e r l y i n g denervation atrophy i n mature muscle. In these experiments i t was shown that when adu l t mouse muscle was co-cultured with a f o e t a l mouse s p i n a l cord explant, and subsequently denervated by ex-t i r p a t i o n of the explant, the mature muscle atrophied w i t h i n a month, whereas immature muscle atrophied f a r more r a p i d l y . This suggested that the mature muscle may have developed a n e u r a l l y induced, endogen-ous r e g u l a t o r y system, which allowed the muscle f i b r e s t o maintain t h e i r i n t e g r i t y a f t e r denervation. Thus i t was concluded that the n e u r a l f a c t o r s i n v o l v e d may be d i f f e r e n t from those enhancing e i t h e r muscle development before i n n e r v a t i o n , or maintenance of membrane pro-p e r t i e s a f t e r denervation (Peterson and C r a i n , 1979). Davis and Kiernan (1980) have tested the e f f e c t i v e n e s s of a ne u r a l e x t r a c t i n v i v o i n the r a t . Their experiments co n s i s t e d of d a i l y i n j e c t i o n s of s c i a t i c nerve e x t r a c t , or heat i n a c t i v a t e d ex-t r a c t used as c o n t r o l , i n t o the EDL muscle immediately f o l l o w i n g denervation. Measurement, 7 days l a t e r , demonstrated that the ex-perimental muscles had s i g n i f i c a n t l y l e s s l o s s of weight, p r o t e i n and AChE a c t i v i t y , than the c o n t r o l s . Furthermore the a m e l i o r a t i o n o f atrophy i n the type IIB f i b r e s was h i g h l y s i g n i f i c a n t , which suggested that these f i b r e s were most dependent on neurotrophic f a c t o r s . The preceding experiments i n d i c a t e that a t r o p h i c f a c t o r does play a r o l e i n the maintenance of the muscle's s t r u c t u r a l i n t e g r i t y . However i t has not yet been demonstrated that t h i s t r o p h i c f a c t o r i n -fluences the c o n t r a c t i l e p r o p e r t i e s of muscle. The other mode by which the nerve can i n f l u e n c e muscle i s v i a the nerve impulse a c t i v i t y . - 11 -Nerve Impulse A c t i v i t y The e f f e c t o f h y p o a c t i v i t y of muscle on i t s various p r o p e r t i e s has been studied by a v a r i e t y of experimental procedures. These i n c l u d e i s o l a t i o n o f a s p i n a l segment of the cord, tenotomy, i m m o b i l i s a t i o n , a p p l i c a t i o n of pharmacological agents, and e l e c t r i c a l s t i m u l a t i o n . In some of these s t u d i e s the a c t i v i t y of the muscle was monitored by electromyography (EMG) which provided i n d i r e c t evidence o f reduced nerve impulse a c t i v i t y (Fischbach and Robbins, 1969; Bagust, 1979). S p i n a l cord i s o l a t i o n , a procedure f i r s t described by Tower (1937), c o n s i s t e d of t r a n s e c t i o n of the s p i n a l cord above and below the lumbosacral enlargement. This together w i t h b i l a t e r a l deaf-f e r e n t a t i o n , by severance of the d o r s a l r o o t s , r e s u l t e d i n v i r t u a l c e s s a t i o n o f impulse a c t i v i t y . Using a s i m i l a r technique i n the c a t , B u l l e r et a l . (1960a) showed that the TTP and 1/2RT of f a s t muscles remained unchanged, whereas the same parameters i n slow muscles were decreased to about h a l f t h e i r normal value. Steinbach et a l . (1980) a l s o working i n the cat found that cord i s o l a t i o n f o r a pe r i o d of 2.5 years r e s u l t e d i n the conversion of the soleus to a f a s t t w i t c h muscle, w i t h q u a l i t a t i v e changes i n both the myosin s t r u c t u r e and the ATPase a c t i v i t y whereas the f a s t - t w i t c h FHL remained unchanged, and showed a normal array of f a s t p r o t e i n s . They concluded that the f a i l u r e of the slow nerve to maintain the slow t w i t c h muscle proper-t i e s was due to i n a c t i v i t y , or to the i n t e r r u p t i o n of t r o p h i c s i g n a l s emmanating from s u p r a - s p i n a l o r sensory neurones. Tenotomy has a l s o been shown to r e s u l t i n an increased speed of - 12 -contraction of a slow muscle, in these experiments the tendons of a l l muscles around the ankle joint were severed and isometric contractions were elicit e d by indirect stimulation. Using this procedure in the rabbit Vrbova (1963) demonstrated that the TTP and 1/2RT in the SOL decreased from 69 to 17 ms, while those of the plantaris remained virtually the same with a change from 23 to 24 ms. As these changes were partially reversed when the SOL tendon was resutured, she con-cluded that the change in the speed of contraction of the SOL was due to the decrease in impulse activity. Similar results were obtained in the cat, although the increase in speed of the contraction of the SOL was not as great as in the rabbit (Vrbova, 1963). Bagust (1979), working in the rabbit, also demonstrated that the SOL had an increased speed of contraction following tenotomy. He found a greater decline in the 1/2RT than the TTP, and also a greater reduction in the iso-metric tetanus tension than the twitch tension. Immobilisation, by means of splinting or joint fixation, has been used to demonstrate that reduced activity results in alterations in muscle properties. Fischbach and Robbins (1969) pinned the ankle and knee joints in the rat, recorded the EMG activity, and 4 weeks later measured the mechanical properties of the SOL muscle, in vitro at 23-26°C. They found that the EMG activity in the immobilised muscles was 5-15% of the controls and that the TTP, tetanus and twitch tensions were reduced to an intermediate level between the values of normal fast and slow-twitch muscles. In addition they found that although the frequency of the nerve impulses remained the same, the number and pattern of the impulses changed towards those of a fast nerve. They concluded that the speed of the contractile mechanism was not dependent upon some factor unique to a particular class of - 13 -motoneurones, but i f a neurotrophic influence was at work, i t was modifiable and p a r t i a l l y dependent on nerve impulse a c t i v i t y . The most s t r i k i n g e f f e c t s of induced a c t i v i t y have been obtain-ed by means of long-term e l e c t r i c a l stimulation. Salmons and Vrbova (1969) used implanted electrodes, i n normal freely-moving cats, to d e l i v e r continuous stimulation (10 shocks/s with pulses of 0.5 ms dur-ation) to the t i b i a l nerve f o r 7 to 13 days. By t h i s means they showed that the speed of contraction of the f l e x o r h a l l u c i s longus (FHL) was prolonged about 50% as judged by the TTP and 1/2RT. At the same time the tetanus and twitch tensions were markedly reduced to about a t h i r d of the controls, but no explanation was given with, regard to a possible cause. I t would appear that there must have been some concomitant nerve damage. In s i m i l a r experiments i n the rabbit i t was shown that stimula-t i o n of the peroneal nerve at a frequency of 10 shocks/s f o r 6 weeks resulted i n marked lengthening of the TTP and 1/2RT of the EDL and t i b i a l i s a n t e r i o r (TA) muscles. The slowing e f f e c t of the stimulation was appreciable within 9-12 days and increased progressively. In ad-d i t i o n i t was noted that whereas the twitch tension was maintained i n the stimulated muscles there was a progressive decline i n the tetanus tension. I t was concluded that the low frequency stimulation had changed the c o n t r a c t i l e properties of the f a s t muscle, and indicated an adaptive response by the muscle to a more continuous pattern of a c t i v i t y (Salmons and Vrbova, 1969). In summary, i t appears from the experiments described that nerve impulse a c t i v i t y plays a major role i n the maintenance of speed of contraction, p a r t i c u l a r l y of slow twitch muscles. Reduced a c t i v -i t y , on the whole, tends to r e s u l t i n an increased speed of - 14 -contraction of a slow muscle, regardless of whether the disuse i s caused by cord isolation (Buller et a l . , 1960a; Steinbach et a l . , 1980), tenotomy (Vrbova-, 1963; Bagust, 1979), or joint fixation (Fischbach and Robbins, 1969). Both trophic factor and impulse activity have been shown to regulate muscle properties. To determine the total influence of the nerve both these effects have to be eliminated. This may be achieved by means of denervation. Denervation Denervation results in widespread changes in the morphological, contractile, biochemical and membrane properties of the muscle (Gutmann, 1962). Many of these induced changes are reversed once the nerve supply is restored, thus verifying that the loss of neuronal influence i s the cause of the changes in the denervated muscle. (a) Morphology Atrophy, a predominant feature of denervated muscle, was f i r s t noted in clinical-pathological studies and was observed to consist of generalised thinning of muscle fibres with disintegration and absorption of tissue so that only sarcolemmalotubes containing nuclei remained. Later experimental studies demonstrated additional features such as early degeneration of the motor end plate, persistence of the •muscle spindles-and long term fibrosis. Gutmann (1962) in reviewing the early work, noted that there were considerable differences between the species with regard to onset, extent, and reversibility of the degenerative changes. In 1963 Pellegrino and Franzini working in the rat, used - 15 -e l e c t r o n microscopy to demonstrate that there were two phases to the atrophic process. The f i r s t , which consisted of a u t o l y t i c degenera-t i o n of the m y o f i b r i l s , accounted for a 50% los s of weight within 7-14 days. The second phase consisted of detachment of m y o f i b r i l s from the periphery which resulted i n a gradual reduction i n the f i b r e diameter. Other u l t r a s t r u c t u r a l changes which have been reported i n -clude accumulation of free ribosomes at the sarcolemma (Gauthier and Dunn, 1973); nuclear and mitochondrial changes; and d i l a t i o n and r e l -a t i v e increase of the sarcoplasmic reticulum (Engel and Stonnington, 1974). The degree of atrophy i n f a s t and slow-twitch muscles i n res-ponse to denervation has been reported to be v a r i a b l e . In the f i r s t few days of denervation the SOL has been found to atrophy more than the EDL i n the rabbit (Syrovy et a l . , 1971) and the rat (L0mo and Rosenthal, 1972). In contrast Engel and Stonnington (1974) found an equal reduction i n the f i b r e area i n fast and slow-twitch muscles a f t e r 3 weeks denervation. The response of s p e c i f i c f i b r e types to denervation has pro-duced l e s s contradictory r e s u l t s . Karparti and Engel (1968), working i n the guinea pig, found more atrophy i n Type II (F0G,FG) f i b r e s than i n Type I (SO) f i b r e s i n the gastrocnemius muscle, 4 weeks post-denervation. Similar r e s u l t s have been reported i n the vastus l a t e r a l i s i n the guinea pig (Tomanek and Lund, 1973), i n the rat p l a n t a r i s (Jaweed et a l . , 1975), semitendinosus (Gauthier and Dunn, 1973) and EDL (Niederle and Mayr, 1978). In contrast, Herbison et a l . (1979) found equal reduction of Type I and Type II (FOG, FG) f i b r e s i n the rat p l a n t a r i s . In the rat SOL an equal amount of atrophy has been - 16 -found i n the SO and FOG f i b r e s at 1 and 2 weeks post-denervation (Jaweed et a l . , 1975; Herbison et a l . , 1979). However the SO f i b r e s i n the SOL have been observed to be more atrophic than SO f i b r e s i n a f a s t muscle (Tomanek and Lund, 1973). Fibre type d i s t r i b u t i o n appears to play a major role i n these d i f f e r e n c e s , as the predominant f i b r e type usually appears to atrophy the most. (b) Biochemical Changes Denervation has been shown to have a pronounced e f f e c t on protein metabolism. Pearlstein and Kohn (1966), working i n the r a t , investigated the turnover of myosin and t o t a l protein content f o l -lowing denervation and found that loss of muscle substance was due to a c c e l e r a t i o n of catabolic processes and not to a defect i n synthesis. Goldspink (1976) systematically studied the rates of protein turnover f o r the f i r s t 10 days of denervation and found an increase i n the rate of protein breakdown i n the rat EDL and SOL a f t e r 24 hours. In ad-d i t i o n there was a transient decrease i n protein synthesis, which was followed by an increase at 7-10 days. This changing pattern seems to account for the contradictory reports that claimed synthesis to be both increased and decreased following denervation. Another area of study has been the e f f e c t of denervation on the types of c o n t r a c t i l e proteins synthesized. Carrara et a l . (1981) working i n the rat soleus muscle 6 months post-denervation, demon-strated by use of two-dimensional gel electrophoresis a decrease i n the heavy and l i g h t chains of slow myosin. As the function of the myosin l i g h t chains has not yet been determined any suggestion that such a decrease may be related to a change i n the speed of contraction remains purely speculative. - 17 -(c) C o n t r a c t i l e P r o p e r t i e s Numerous i n v e s t i g a t i o n s have shown that denervation has a pro-found e f f e c t upon the speed of c o n t r a c t i o n o f f a s t and slow muscles. The m a j o r i t y o f s t u d i e s followed the same procedure. The nerve was sectioned c l o s e to the muscle. A f t e r a c e r t a i n time the muscle was s t i m u l a t e d supramaximally and the c o n t r a c t i l e p r o p e r t i e s were measured i n v i t r o or i n v i v o , while the muscle was held at optimal l e n g t h . The r e s u l t s were compared to those of the c o n t r a l a t e r a l limb which was l e f t i n t a c t . The e f f e c t of denervation on the c o n t r a c t i o n time of f a s t muscles has produced c o n s i s t e n t r e s u l t s . Lewis (1962, 1972) working i n the c a t , i n v i v o , found that the TTP of the FDL was increased from 27 to 50 ms f o l l o w i n g 4 weeks denervation. This prolongation of the TTP which was approximately 180% of the c o n t r o l has been confirmed i n other s t u d i e s i n the cat (Eccles et a l . , 1962; Syrovy et a l . , 1972; Kean et a l . , 1974; Lewis et a l , 1978) and to a l e s s e r extent i n the r a b b i t (Syrovy et a l . , 1972). Drachman and Johnston (1975) working i n the r a t , found a progressive p r o l o n g a t i o n of the TTP from 10 to 20 ms i n the denervated EDL. S i m i l a r r e s u l t s have been reported by others working i n the r a t (Gutmann et a l . , 1972; F i n o l and Lewis, 1975; Ranatunga, 1977), and i n the mouse (Zeman and Sandow, 1979) Studies of the e f f e c t of denervation on the speed of contrac-t i o n of slow-twitch muscles have produced v a r i a b l e r e s u l t s . Lewis (1962, 1972) found that the TTP of the soleus muscle i n the cat was increased from 80 to 120 ms, whereas Syrovy, Melchina and Gutmanm (1972) found a minimal p r o l o n g a t i o n . In the r a b b i t the TTP was shortened by 40% (Syrovy et a l . , 1972). The m a j o r i t y of researchers working i n the r a t have found the TTP to be prolonged from 30 to 40 ms - 18 -following 4 weeks denervation (Drachman and Johnston, 1975; F i n o l and Lewis, 1975; Ranatunga, 1977). At variance with t h e i r r e s u l t s are those of Gutmann, Melchina and Syrovy (1972) who found a shortened TTP i n the soleus of both 1 month and 6 month old rats a f t e r 30 days of denervation, athough 1 month old animals showed an i n i t i a l prolonga-t i o n of the TTP. The duration of the experimental period cannot account for the inconsistent r e s u l t s as some studies were f o r a s i m i l a r time period of 64 or 74 days (Gutmann et a l . , 1972; F i n o l and Lewis, 1975). In addition to the e f f e c t on the TTP denervation has been shown to r e s u l t i n the prolongation of the 1/2RT (Eccles et a l . , 1962; Lewis, 1972; Drachman and Johnston, 1975; Zeman and Sandow, 1979). In a l l cases t h i s parameter was increased more than the TTP and to a greater extent i n the EDL than the SOL muscle. The time of onset of the prolonged time of contraction has varied iri d i f f e r e n t species. In the cat, slowing developed at 9 days to 3 weeks following denervation (Lewis, 1962, 1972; Lewis et a l . , 1978) whereas i n the rat the TTP became prolonged within 3 days of denervation (Gutmann et a l . , 1972; F i n o l and Lewis, 1975). In contrast to the number of studies dealing with the speed of contraction, only a few of the investigators have studied the e f f e c t of denervation on the tension generating a b i l i t y of muscle. Kean, Lewis and McGarrick (1974) working i n vivo i n the cat, found that the tetanus tension of the denervated EDL f e l l to nearly 50$ of the c o n t r a l a t e r a l control limb within 35 days. Drachman and Johnston (1975) reported an even greater decline i n the tetanus tension of the EDL i n the rat, i n s i m i l a r experimental conditions. In both of these studies there was a smaller, but s i g n i f i c a n t , decrease i n the tension - 19 -of the soleus muscle. In contrast, Ranatunga (1977) found the isometric tetanus tension of the SOL i n the rat to have diminished more than that of the EDL a f t e r IH days denervation. The e f f e c t of denervation on the twitch tension has produced a s i m i l a r trend i n several i n v e s t i g a t i o n s . The twitch tension has been shown to decline, but to a l e s s e r extent than the tetanus, so that the twitch to tetanus r a t i o i s increased. Zemen and Sandow (1979), working on i s o l a t e d mouse muscle at 15°C, found that the twitch tension increased to 180% of the controls within 3 weeks of dener-vation. Although no figures were c i t e d , t h i s potentiation of the twitch has been reported i n both slow and fast-twitch muscles i n the rat (Finol and Lewis, 1975; Kotias, 1975). The experiments which have been described demonstrate that the twitch contraction time of the fast muscle i s prolonged following denervation. However there i s some c o n f l i c t with regard to the reaction of the slow-twitch SOL. The tension generating a b i l i t y of both f a s t and slow muscles i s generally impaired but on occasion the twitch i s found to be potentiated. Moreover d i f f e r e n t responses have been obtained according to age, species, and time since denervation, i n addition to the v a r i a t i o n s i n experimental design. As species vary i n t h e i r speed of contraction (Close, 1972) absolute data between species cannot be compared. However even i n the same species, studies which involve d i f f e r e n t experimental procedures make comparisons of dubious value. Although a l l studies s p e c i f i e d that stimulation was effected at optimal length and by a supramaximal stimulus, there was some v a r i a t i o n i n temperature. The choice of s i t e of the nerve transection provided the greatest source of v a r i a -t i o n . The l e v e l of transection not only a f f e c t s the length of the - 20 -nerve stump, but also the degree to which the antagonist and synergist muscles are involved. Lack of tone i n other muscles can a f f e c t the degree of s t r e t c h applied to the denervated muscles, and has been shown to act as a variable i n the a l t e r a t i o n of c o n t r a c t i l e properties (Goldspink, 1978). F i n a l l y the majority of studies were r e l a t i v e l y short term, and therefore did not elaborate on the precautions used to prevent reinnervation. However only one stated the method used to v e r i f y that denervation was i n e f f e c t at the time of the experimental procedures (Zeman and Sandow, 1979). Despite these objections there i s , nevertheless, a c l e a r picture of the changes induced by denervation. However the mechanisms responsible for the a l t e r a t i o n s i n the speed of contraction remain speculative. Factors which have been suggested include a decrease i n ATPase a c t i v i t y (Gutmann et a l . , 1972) and changes i n the membrane properties ( F i n o l and Lewis, 1975). (d) Membrane Properties Denervation has been shown to r e s u l t i n a decrease i n the restin g membrane pot e n t i a l (Albuquerque and Mclsaac, 1970), decrease i n the rate of r i s e of the action p o t e n t i a l ( S e l l i n and Thesleff, 1980), cessation of spontaneous transmitter release, and appearance of a c e t y l c h o l i n e (ACh) s e n s i t i v i t y (Albuquerque and Thesleff, 1968; Lomo and Rosenthal, 1972), and decrease i n junctional AChE a c t i v i t y (Guth et a l . , 1981). I t i s beyond the scope of t h i s thesis to review these changes i n d e t a i l . However, i t i s important to acknowledge that the length of the nerve stump has been implicated i n determining the time of onset of these changes (Stanley and Drachman, 1980). Thus the conclusion has been reached that changes i n membrane properties - 21 -induced by denervation r e s u l t p r i m a r i l y from loss of axonally trans-ported neurotrophic influences but may be modulated by loss of a c t i v i t y (Guth and Albuquerque, 1979; Desphande et a l . , 1980). Summary A large body of research supports the concept that both neuro-trophic and nerve impulse a c t i v i t y contribute to the maintenance of the properties of normal muscle. Deprivation of t h i s neural influence r e s u l t s i n morphological, p h y s i o l o g i c a l and biochemical changes, which are associated with changes i n the c o n t r a c t i l e properties of the muscle. The changes have been found to vary i n fa s t and slow-twitch muscles and i n addition vary according to species, age of animal and the time since denervation. There i s a lack of longterm studies i n which a l l the isometric c o n t r a c t i l e properties are considered. In addition, there are va r i a t i o n s i n experimental design such as length of nerve stump and the e f f e c t of st r e t c h which makes the r e s u l t s d i f f i c u l t to compare. The present study was undertaken i n an attempt to circumvent some of these discrepancies. - 22 -III METHODS AND PROCEDURES - 23 -A l l experiments were ca r r i e d out on adult male mice of the C57-BL/6J s t r a i n , bred and raised i n our own colony. The o r i g i n a l breeding pairs were obtained from Jackson Laboratories. Adult male mice were selected as i t has been reported that the number and growth of muscle f i b r e s d i f f e r s between the sexes and f u l l y d i f f e r e n t i a t e d patterns become established at about 15 weeks of age (Goldspink and Ward, 1979). In the major s e r i e s of animals used, the r i g h t hindlimb was denervated at 12 weeks of age and isometric c o n t r a c t i l e properties of the fa s t - t w i t c h EDL and the slow-twitch SOL muscles were studied at 28, 84, and 210 days post-denervation. In addition, another group of animals was denervated at 16 weeks of age and studied at 1 day post-denervation. This allowed 16 week unoperated muscles to be used as controls f o r both the 1 day and 28 day post-denervation groups. In a l l other instances age-matched unoperated animals were used as c o n t r o l s . Despite the known v a r i a b i l i t y between animals with regard to the contraction times (Lewis and Parry, 1979), control animals were preferred to the use of the c o n t r a l a t e r a l limb as i t was anticipated that work induced hypertrophy would occur i n the unoperated l e g . Denervation The mice were anaesthetized by i n t r a p e r i t o n e a l i n j e c t i o n of c h l o r a l hydrate (600 mg/kg). With the leg positioned i n 90° of hip and knee f l e x i o n , the skin over the r i g h t thigh was swabbed with alchohol and a 10 mm i n c i s i o n made along the posterior border of the femur. Straight s t a i n l e s s s t e e l forceps were used to s p l i t the overlying f a s c i a and separate the f i b r e s of the gluteus maximus and so expose the s c i a t i c nerve. The nerve was then elevated with curved forceps and a 5 mm segment i s o l a t e d with two s u r g i c a l s i l k l i g a t u r e s . - 24 -This segment was then removed by cuts placed beyond the l i g a t u r e s . No a d d i t i o n a l precaution was taken to prevent the p o s s i b i l i t y of r e i n n e r v a t i o n . The s k i n f l a p was then closed w i t h one or two s u r g i c a l c l i p s and the animal placed on i t s s i d e to recover. In order to circumvent the p o s s i b i l i t y that r e i n n e r v a t i o n might occur, an a d d i t i o n a l group of animals was denervated a second time s i x weeks a f t e r the i n i t i a l operation was performed. The second procedure con-s i s t e d of c u t t i n g any v i s i b l e branches o f the proximal stump which may have appeared and removal of an a d d i t i o n a l 1 mm segment from t h i s stump. At t h i s point a s i l k l i g a t u r e was placed on the cut d i s t a l stump so that i t could be i d e n t i f i e d l a t e r . This group was studied only at 12 weeks post-denervation (see Appendix 1 ) . Fun c t i o n a l Denervation Test P r i o r to removal of the muscles f o r study, the denervated limb was assessed to determine i f f u n c t i o n a l r e i n n e r v a t i o n had occurred. As a c l i n i c a l assessment of the denervated limb, the mouse was sus-pended by i t s t a i l and the absence of a r e f l e x extension of the foot w i t h spreading toes was used as an i n i t i a l i n d i c a t i o n that the limb was s t i l l denervated. Fol l o w i n g t h i s , the mouse was anaesthetized and the body weight of each animal was recorded. The denervated limb was p a r t i a l l y skinned and the cut end of the s c i a t i c nerve exposed and kept moist w i t h warm Krebs s o l u t i o n . E l e c t r i c a l s t i m u l a t i o n was then c a r r i e d out us i n g symmetrical b i p h a s i c square pulses of 1 ms d u r a t i o n , varying i n stren g t h from 1-10 v o l t s and a p p l i e d by means of two n i c k e l chromium wire e l e c t r o d e s . In order to prevent the stimulus from spreading to the u n d e rlying muscle, a small piece of p a r a f i l m was placed underneath the nerve stump. F i r s t , d i r e c t s t i m u l a t i o n of the t i b i a l i s a n t e r i o r - 25 -muscle was used to e l i c i t a ctive d o r s i f l e x i o n , then a threshold stimulus was applied to the cut proximal stump of the s c i a t i c nerve. The lower l e g was considered to be f u n c t i o n a l l y denervated i f t h i s i n d i r e c t stimulation f a i l e d to produce a v i s i b l e contraction of the t i b i a l i s a n t e r i o r or gastrocnemius muscle or any movement of the toes or ankle j o i n t . A t o t a l of 10 animals f a i l e d the functional test and showed evidence of reinnervation i n response to i n d i r e c t stimulation. Direct scrutiny of the EDL and SOL was rejected on the grounds that i t would require d i s s e c t i o n of the overlying muscles and possible damage to any reinnervating nerve f i b r e s . In addition, the gastrocnemius and t i b i a l i s a n t e r i o r muscles have the same nerve supply, and s i m i l a r l o c a t i o n of motor point as the underlying SOL and EDL muscles as well as a l a r g e r muscle mass. They thus serve as s u i t a b l e i n d i c a t o r s for signs of functional reinnervation. However as synapses may be s i l e n t and i t i s known that there i s a delay between establishment of motor end plate and onset of transmission, lack of f u n c t i o n a l reinnervation does not imply lack of morphological r e -innervation. A l l c o n t r o l animals were assessed f o r the presence of normal toe spread b i l a t e r a l l y , but were not subjected to e l e c t r i c a l s t i mulation. F i n a l l y , the 10 animals which exhibited some evidence of re-innervation were retained for study as a separate group. Muscle Dissection Following functional t e s t i n g , the animal was k i l l e d by c e r v i c a l d i s l o c a t i o n . The r i g h t leg was removed from the body, skinned and bathed i n a pool'of Krebs s o l u t i o n . The t i b i a l i s a n t e r i o r was removed to expose the underlying EDL. The proximal and d i s t a l tendons were - 26 -tied with surgical silk (3.0) close to the musculotendinous junction to avoid any stray series compliance due to excessive tendinous material. The SOL was similarly exposed underneath the gastrocnemius, tied and dissected away from the surrounding connective tissue. Either the SOL or the EDL was used from a single animal for the physiological experiments. Once the f i r s t muscle was set up in the experimental apparatus the alternate muscle was dissected for subsequent histological study. Experimental Apparatus The muscle was placed on a perspex block containing two platinum wire multielectrode arrays, one on each side of the muscle and parallel to, but not touching, i t s fl a t surface. The distal tendon was tied to the anode pin of an RCA 5734 tension transducer (resonant frequency, 1.2 KHz) and the proximal tendon was secured to a straight annealed stainless steel wire. The wire passed through a short brass tube and was rigidly connected to a light aluminium rod extending from the centre of a Ling shaker. The shaker was part of a closed loop length servo-system which was extremely s t i f f and allowed examination of the' isometric contractile properties of the muscle. The muscle in i t s electrode assembly was immersed in a bath of Krebs solution for stimulation and recording. The composition of the bath was, in millimoles per l i t r e : NaCl, 121; NaHCO^ , 2.5; KH2P0i), 0.5; KCL, 4.75; CaCl 2, 1.5; and MgCl 2 > 0.23; and glucose, 2 g/l i t r e and the pH approximated 7.7. Throughout the experiment the Krebs solution was continuously bubbled with 95% 0^  and 5% CO^. The temperature of the bath was held constant at 37°C by circulating water from the thermostatically controlled pump (Haake FE) through a water jacket surrounding the glass chamber housing the - 27 -muscle. The temperature of the bathing s o l u t i o n was constantly monitored with a c a l i b r a t e d thermistor probe. Experimental Procedures A routine procedure was used to set the length and voltage to ensure accuracy and consistency. The muscle length was i n i t i a l l y set at 12 mm which approximated the mean r e s t i n g length. Further adjust-ments were made i n increments of 0.5 mm u n t i l maximum twitch height was obtained. The maximum twitch height was s p e c i f i c a l l y chosen to es t a b l i s h the resting length, since most of the data used to compare normal and denervated muscles originated from the twitch. Stimulation was achieved by supermaximal square pulses of 1 ms duration. The fusion frequency f o r a smooth isometric tetanus at 37°C was determined to be 80-100 shocks/s f o r the SOL and 120-160 shocks/s f o r the EDL. A stimulus pattern of three twitches followed by a tetanus with a contraction once every 90 s was used throughout the experiment. This pattern was maintained to prevent fatigue of the muscles due to repeated t e t a n i . Although none was observed, the f i r s t twitch following each tetanus was not recorded to avoid any p o s s i b i l i t y of post-tetanic potentiation. For each muscle a minimum of 9 twitches and 4 tetani were recorded. At the end of an experimental run, the fatigue c h a r a c t e r i s t i c s of the muscle were measured. The fatigue procedure consisted of gi v i n g the muscle a 1 s tetanus, once every 5 s and recording the re-sponses on an oscilloscope sweep speed of 5 s / d i v i s i o n . The decline i n maximum tension with time was compared to the average maximum tetanic tension recorded during the f i r s t phase of the experiment. At the conclusion of each experiment, the length of the muscle between the t i e s was recorded with f i n e c a l i p e r s . The muscle was - 28 -blotted between two layers of paper towel by a l i g h t pinch and the wet weight was recorded. Then the muscle was placed i n a holding chamber and allowed to a i r dry for 15-24 hours before the dry weight was de-termined . Histology The alternate muscle to the one used i n the phy s i o l o g i c a l experiment was usually dissected and prepared f o r h i s t o l o g i c a l exami-nation (33 SOL and 34 EDL specimens). The muscle was pinned at just greater than slack length i n Karnovsky's f i x a t i v e f o r a minimum of 30 minutes, then divided at the midpoint of the b e l l y . The proximal portion remained i n Karnovsky's f i x a t i v e and was kept f o r future study by electron microscopy, while the d i s t a l h a l f of the muscle was trans-ferred to 10% formalin f o r 24 hours and then stored i n 70% a l c o h o l . A f t e r dehydration through ascending alcohols, the specimen was embed-ded i n p a r a f f i n wax. S e r i a l transverse sections (7p thick) were cut through the mid b e l l y region, then mounted, and stained with hemotoxylin and eosin (H. and E.). Analysis of Data An image, magnified 100 times, was obtained of each i n t a c t histology specimen by the use of a drawing attachment f i t t e d to a microscope. Then the circumference was traced and a Zeiss MOP 3 image analyzer was used to compute the cross-sectional area of each muscle. The c o n t r a c t i l e responses were photographed from the o s c i l l o -scope. In addition the signals were d i g i t i z e d at the rate of 0.5 ms/address and subsequently printed out on a Decwriter. A 12 ms delay between the t r i g g e r i n g of the an a l o g - d i g i t a l converter and the stimulator provided a baseline on the records. From these records the maximum isometric twitch and tetanus tension, time-to-peak twitch - 29 -tension (TTP), and half-relaxation time (1/2RT) were measured. As this was a new procedure the data was compared with a sample measure-ment from each film. Fatigue responses were measured from enlarge-ment-projections of filmed tetanus records. The absolute values for each parameter studied were pooled. This amounted to approximately 36 tetani and 72 twitches for each muscle in each age group. In addition both the tetanus tension and twitch tension were normalised with respect to wet weight. Wet weight was chosen as a means to normalise tension as myofibrillar proteins form the largest component of muscle tissue. Other methods of normalising tension are with respect to dry muscle weight or estimated cross-sectional area. A comparison of the results determined by d i f -ferent methods of normalisation may be seen in Appendix 2. A Student's t-test for unmatched pairs was used throughout this study to compare the differences in the various parameters examined. The difference between the means was considered to be significant i f p 5 0.05. - 30 -IV RESULTS - 31 -Denervation of soleus or extensor digitorum longus muscles had no e f f e c t on whole animal weight except i n those mice that had gone 210 days post EDL-denervation. Here the weight loss was s t a t i s t i c a l l y s i g n i f i c a n t (p = .05)' and almost 12% of control body weight. This delayed loss was unexplained and not seen i n animals with denervated SOL. Denervated EDL muscles themselves showed rapid weight l o s s : within 28 days they had l o s t almost 50% of the weight of control muscles and by 210 days they weighed only one t h i r d of control muscles. A s i m i l a r rapid decline appeared to occur i n denervated SOL, but whereas the within-group variance for EDL was small, that f or SOL was large, and while the means f o r the control and denervated SOL seemed quite d i f f e r e n t (11.70 and 8.57 mg, respectively) at 28 days the diffe r e n c e was not supported s t a t i s t i c a l l y . However, at 210 days post-denervation the denervated muscles weighed only a quarter of the control muscles. Muscle length was not affected by denervation. These data are c o l l e c t e d i n Tables I and II and Figure 1. C o n t r a c t i l e Properties of Denervated Muscle (a) Twitch Contraction Time Denervation resulted i n a marked slowing of the twitch of both the SOL and the EDL. O r i g i n a l records of the time course of the isometric twitch of denervated muscles and t h e i r respective controls at 28 days post-denervation can be seen i n Figure 2. The changes i n TTP twitch tension and 1/2RT of the denervated SOL and EDL are shown i n Figures 3 and 4 respectively (see also Appendix 3). At 1 day post-denervation the TTP of the SOL showed no change, whereas i n the EDL the TTP was s i g n i f i c a n t l y shorter than normal. In both muscles the greatest proportion of change occurred within 28 days. TABLE I Age, Weight and Length Data f o r Soleus Muscles from Normal and Denervated M i c e a . T i m e b A S e C Body Wt M. Length Wet M.W. Dry M.W. (days) (g) (mm) (mg) (mg) ( , ) d Norm. Den. Norm. Den. Norm. Den. Norm. Den. Norm. Den. 1 (6,6) P 28 (6,6) P 84 (6,6) P 210 (6,3) P 115.83 118.67 ±1.36 ±0.19 N.S. 115.83 117.50 ±1.36 ±2.23 N.S. 170.67 174.50 ±1.07 ±1.43 N.S. 296.50 292.33 ±0.84 ±1.96 N.S. 29.003 27.76 ±1.38 ±0.63 N.S. 29.00 28.96 ±1.38 ±0.77 N.S. 29-85 27.20 ±0.69 ±0.86 N.S. 34.57 36.23 ±0.79 ±0.14 N.S. 13.08 13.08 ±0.42 ±0.22 N.S. 13.08 13.00 ±0.42 ±0.43 N.S. 12.83 12.42 ±0.19 ±0.27 N.S. 13-33 11.67 ±0.47 ±0.14 N.S. 11.70 12.50 ±0.85 ±0.63 N.S. 11.70 8.57 ±0.85 ±1.07 N.S. 10.90 6.18 ±0.36 ±0.38 ;.001 14.02 3-60 ±0.59 ±0.37 ?.001 3-45 3.62 ±0.21 ±0.20 N.S. 3.45 2.42 ±0.21 ±0.47 N.S. 3.48 1.85 ±0.29 ±0.22 5.01 3.63 0.93 ±0.29 ±0.19 5.001 a Values are means ± S.E. b Time, i n days post-denervation c Age i n days d Number of normal and denervated muscles tested r e s p e c t i v e l y TABLE II Age, Weight and Length Data for Extensor Digitorum Longus Muscles from Normal and Denervated Mice a. Time b ( , ) d Age c (days) Body Wt (g) M.Length (mm) Wet M.W. (mg) Dry M.W. (mg) Norm. Den. Norm. Den. Norm. Den. Norm. Den. Norm. Den. 1 1 1 4 . 0 0 1 1 7 . 1 7 3 0 . 0 2 2 7 . 1 0 1 2 . 6 7 1 2 . 6 7 1 2 . 1 7 1 1 . 3 8 2 . 9 3 3 .17 ( 6 , 6 ) ± 0 . 8 2 ± 0 . 9 6 ± 1 . 3 8 ± 0 . 9 5 ± 0 . 3 0 ± 0 . 1 9 ± 0 . 2 2 ± 0 . 6 2 ± 0 . 2 8 ± 0 . 2 7 P 5 . 0 5 N.S. N.S. N.S. N.S. 28 1 1 4 . 0 0 1 1 7 . 2 9 3 0 . 0 2 2 9 . 5 8 1 2 . 6 7 1 2 . 7 1 1 2 . 1 7 6 .86 2 . 9 3 1.92 ( 6 , 7 ) ± 0 . 8 2 ± 1 . 9 1 ± 1 . 3 8 ± 0 . 8 5 ± 0 . 3 0 ± 0 . 2 8 ± 0 . 2 2 ± 0 . 2 4 ± 0 . 2 8 ± 0 . 2 6 P N.S. N.S. N.S. ? . o o i 5 . 0 5 84 1 6 8 . 5 0 1 7 1 . 7 5 31.75 3 4 . 2 5 13.00 1 2 . 6 3 13-32 5 . 5 3 3 .67 1 .60 ( 6 , 4 ) ± 0 . 4 6 ± 2 . 8 2 ± 0 . 9 8 ± 0 . 9 5 ± 0 . 2 4 ± 0 . 3 3 ± 0 . 7 4 ± 0 . 9 3 ± 0 . 1 7 ± 0 . 1 5 P N.S. N.S. N.S. 5 . 0 0 1 5 . 0 0 1 210 287 .00 2 9 4 . 7 5 3 8 . 5 0 33-96 1 2 . 8 8 1 2 . 6 7 1 2 . 2 0 3 .77 3 . 0 0 1.07 ( 4 , 3 ) ± 0 . 0 0 ± 0 . 2 2 ± 0 . 8 2 ± 0 . 9 4 ± 0 . 1 1 ± 0 . 2 7 ± 0 . 4 7 ± 0 . 6 2 ± 0 . 2 6 ± 0 . 2 4 P ;.05 5.05 N.S. 5 . 0 0 1 51.01 a Values are means ± S.E. b Age, i n days post-denervation c Wet muscle weight in milligrams d Number of normal and denervated muscles tested respectively - 3h -W e t m u s c l e w e i g h t 8 0 " -* SOL • EDL 1 2 8 8 4 2 1 0 P o s t - d e n e r v a t i o n ( d a y s ) Figure 1. Histogram summarizing the percentage change from control i n wet muscle weight of SOL arid EDL muscles a f t e r denervation. - 35 -Figure 2. Isometric t w i t c h myograms of normal muscles (a,b) and muscles denervated f o r 28 days (c,d) i n mice 16 weeks of age. Records from SOL are on the l e f t and EDL on the r i g h t . The i n t e r v a l between time markers i s 1 ms. C a l i b r a t i o n bars equal 1.6 ( a ) , 2.0 (b) and 3.0 (c,d) gram weights. o - 37 -o f d e n e r v a t i o n a n d a p p r o x i m a t e d a n i n c r e a s e o f 82% i n t h e SOL a n d M l ? i n t h e E D L . O v e r t h e s u c c e e d i n g p o s t - d e n e r v a t i o n t i m e p e r i o d s t h e r e was a m o r e g r a d u a l i n c r e a s e i n t h e T T P o f b o t h t h e SOL a n d E D L . A t a l l t i m e s t h e d i f f e r e n c e b e t w e e n t h e s l o w a n d f a s t m u s c l e was m a i n t a i n e d a s t h e T T P o f t h e d e n e r v a t e d EDL r e m a i n e d f a s t e r t h a n t h e T T P o f t h e SOL ( F i g u r e 3). T h e 1 / 2 R T i n t h e d e n e r v a t e d SOL s h o w e d a s i g n i f i c a n t p r o l o n g -a t i o n a t 1 d a y p o s t - d e n e r v a t i o n . A f u r t h e r s l o w i n g was s e e n a t 84 d a y s , w i t h n o a p p a r e n t i n c r e a s e b e y o n d t h a t t i m e . T h e d e n e r v a t e d EDL s h o w e d n o c h a n g e i n t h e 1 / 2 R T a t 1 d a y p o s t - d e n e r v a t i o n b u t d e v e l o p e d a p r o g r e s s i v e s l o w i n g o f t h e 1 / 2 R T t h e r e a f t e r . B y 2 1 0 d a y s p o s t -d e n e r v a t i o n t h e 1 / 2 R T o f t h e SOL a n d t h e EDL w e r e o f n e a r l y s i m i l a r d u r a t i o n ( F i g u r e 4 ) . ( b ) I s o m e t r i c T e n s i o n T h e i s o m e t r i c t e n s i o n d a t a f o r t h e SOL i s s u m m a r i z e d i n T a b l e I I I . T h e d e n e r v a t e d SOL s h o w e d a m a r k e d d e c r e a s e i n t h e a b s o l u t e max imum i s o m e t r i c t e t a n u s t e n s i o n w i t h i n 24 h o u r s . A s t h e r e was no a c c o m p a n y i n g l o s s o f w e i g h t , t h e t e t a n u s t e n s i o n n o r m a l i s e d w i t h r e s p e c t t o w e t w e i g h t s h o w e d a s i m i l a r r e d u c t i o n ( T a b l e I I I ) . A t 28 d a y s p o s t - d e n e r v a t i o n , a l t h o u g h t h e a b s o l u t e t e t a n u s t e n s i o n was d e c r e a s e d t h e r e was n o s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e d e n e r v a t e d a n d n o r m a l SOL when t e n s i o n was e x p r e s s e d r e l a t i v e t o w e t w e i g h t . H o w e v e r , b y 84 d a y s a d r a m a t i c d r o p i n t e t a n u s t e n s i o n was o b s e r v e d b o t h i n a b s o l u t e a n d n o r m a l i s e d t e r m s . T h i s t r e n d c o n t i n u e d t o t h e o l d e s t a g e g r o u p s t u d i e d . I n a d d i t i o n t h e n o r m a l SOL m u s c l e s h o w e d a s i g n i f i c a n t d e c l i n e i n t e t a n u s t e n s i o n w i t h a g e . T h e d e n e r v a t e d E D L e x h i b i t e d a d i f f e r e n t r e s p o n s e f r o m t h e S O L a t n e a r l y a l l t i m e p e r i o d s a s may b e s e e n i n T a b l e I V . No d e c r e a s e o f S O L i E D L L 1 0 N O R D E N £ 5 -I, I « '0 1 2 8 8 4 2 1 0 0 1 2 8 8 4 2 1 0 T i m e P o s t - d e n e r v a t i o n ( d a y s ) Figure 3. Time to peak twitch tension of soleus and EDL muscles of denervated and normal mice. Each point i s the mean in ms ± standard error of the mean. The asterisks indicate values which are stati s t i c a l l y significant at P ; 0.001. u CD CO E CD E c o 4 5 r 4 0 3 5 3 0 2 5 S O L £ / / J ~ 2 0 h */ CO X jO CD CC I to X 1 5 1 0 5 • N O R • D E N 0 1 2 8 8 4 J E D L / / / ± 2 1 0 0 1 2 8 8 4 2 1 0 T i m e P o s t - d e n e r v a t i o n ( d a y s ) Figure 1 . Half relaxation time of the isometric twitch of the soleus and EDL muscles of denervated and normal mice. Each point i s the mean in ms ± standard error of the mean. Asterisks indicate values which are s t a t i s t i c a l l y significant at P ; 0.001. TABLE III Tension Chara c t e r i s t i c s of Soleus Muscles from Normal and Denervated M i c e a . fime b Po (g) Po/M.W.c Pt (g) Pt/M.W.c Norm. Den. Norm. Den. Norm. Den. Norm. Den. 1 (6,6)d P 34.82 19.11 ±1.35 ±1.00 5.001 3.05 1.54 ±0.14 ±0.08 5.001 7.71 4.74 ±0.28 ±0.16 5.001 0.67 0.38 ±0.02 ±0.01 5.001 28 (6,6) P 34.82 21.62 ±1.35 ±0.52 5.001 3.05 2.71 ±0.14 ±0.11 N.S. 7.71 10.16 ±0.28 ±0.22 5.001 0.67 1.28 ±0.02 ±0.04 5.001 84 (6,6) P 31.16 7.54 ±1.40 ±0.43 5 .001 2.93 1.25 ±0.17 ±0.08 5.001 8.44 3 . 5 8 ±0.46 ±0.15 5.001 0.80 0.60 ±0.05 ±0.03 5.01 210 (6,3) P 18.39 2.27 ±0.76 ±0.10 5 .001 1.31 0.63 ±0.05 ±0.01 5.001 4.80 1.38 ±0.14 ±0.05 5.001 0.35 0.38 ±0.01 ±0.01 5.02 a Values are means ± S.E. b Time, i n days post-denervation. c Tension per unit of wet muscle weight d Number of normal and denervated muscles tested respectively TABLE IV Tension Characteristics of Extensor Digitorum Longus Muscles from Normal and Denervated M i c e a . Time b Po (g) Po/M.W.c Pt (g) Pt/M.W.c ( , ) d Norm. Den. Norm. Den. Norm. Den. Norm. Den. 1 36.62 38.51 3.02 3.50 10.78 6.68 0.89 0.60 (6,6) ±1.09 ±2.24 ±0.10 ±0.24 ±0.38 ±0.19 ±0.03 ±0.02 P N.S N.S. 5.001 5.001 28 36.62 24.28 3.02 3-57 10.78 9.19 0.89 1.36 (6,7) ±1.09 ±0.10 ±0.10 ±0.16 ±0.38 ±0.28 ±0.03 ±0.04 P 5-001 5.01 5.001 5.001 84 30.28 14.62 2.33 2.66 8.65 5.77 0.66 1.13 (6,4) ±1.37 ±1.29 ±0.13 ±0.10 ±0.29 ±0.38 ±0.03 ±0.04 P 5.001 N.S. 5.001 5.001 210 51.68 ' 9.62 4.21 2.66 11.93 4.04 0.99 1.21 (4,3) ±2.55 ±0.67 ±0.25 ±0.20 ±0.47 ±0.20 ±0.05 ±0.06 P 5-001 = .001 5.001 5.01 a Values are means ± S.E. b Time, i n days post-denervation c Tension per unit of wet muscle • weight d Number of normal and denervated muscles tested respectively - 42 -tetanus tension was observed in either absolute or normalised terms at 24 hours post-denervation. However at 28 days, although the absolute tetanus tension had declined, the denervated EDL was capable of generating more tension per unit of wet weight than control muscles. This trend was reversed at the later time periods, as the normalised tetanus tension of the denervated EDL showed no difference at 84 days but a significant decrease at 210 days post-denervation, when compared to the controls. With regard to twitch tension the denervated SOL generated less tension in both absolute and normalised terms at 1 day post-denervation. At 28 days post-denervation the SOL showed a maximum twitch tension which was significantly above normal both in absolute terms and when normalized with respect to wet weight. By 84 days both absolute and normalised tension was again decreased when compared to control muscles. However at 210 days post-denervation although absolute twitch tension was decreased, tension expressed relative to wet weight was greater than the controls. The denervated EDL also generated less twitch tension than the control muscles in both absolute and normalised terms at 1 day post-denervation. However at the subsequent time periods although the absolute tension of the denervated muscles was significantly reduced, tension expressed relative to wet weight was significantly increased (Table IV). (c) Fatigue Characteristics The normal response of the slow-twitch SOL and the fast-twitch EDL to a fatiguing pattern of stimulation i s seen in Figure 5. The fatigue patterns of normal and denervated SOL at 1, 28 and 84 days - 4 3 -Figure 5. Isometric tetanus tension myograms of normal SOL (a) and EDL (b) muscles ill u s t r a t e different responses to a fatiguing pattern of stimulation. The intervals between the time-markers i s 1 second. The calibration bar is equal to 8.2 and 4.7 gram weights for the SOL and EDL respectively. - 4 5 -post-denervation are compared and shown in Figure 6. At 1 day post-denervation, the SOL demonstrated a marked decrease in i t s resistance to fatigue but a relatively normal response at subsequent time periods. The denervated EDL, on the other hand, exhibited the same lack of resistance to fatigue as normal muscles at 1 day post-denervation. However at 28 days and 84 days post-denervation, the fast-twitch muscle appeared to be resistant to fatigue when compared to the normal counterpart (Figure 7). Morphology Intact cross-sections were obtained from 30 of the original sample of 67 SOL and EDL muscles. The cross-sectional areas for the individual muscles are listed in Table V. Despite the small sample there appeared to be an obvious difference between the cross-sectional areas of normal and denervated muscles at 28 days post-denervation (Figure 8). This diminution in cross-sectional area progressed with time post-denervation, so that i t was extremely marked by 210 day period (Figure 9). The percentage change observed in the cross-sectional area of the denervated SOL and EDL muscles were comparable to the changes in wet weight (refer to Figure 1). In the denervated SOL, morphological abnormalities were evident at 1 day post-denervation where the presence of several hypertrophic c e l l s was observed (Figure 10). Although there appeared to be some enlargement of some of the muscle fibres of the denervated EDL at 1 day post-denervation, they were neither as large nor as rounded as those seen in the denervated SOL. Signs of decreased angularity of c e l l s , central nucleation and variation in fibre diameter were apparent in both SOL and EDL at 28 days post-denervation and became - 46 -T I M E (sec) Figure 6. The change i n maximum isometric tetanus tension, with time during rapid stimulation of soleus muscles from normal and denervated mice at various times post-denervation. A l l tension values are normalised with respect to mean tetanus tension ( P o a ) . - 4 7 -E D L 1 DAY 0 30 60 90 120 150 180 210 240 270 300 TIME (see) Figure 7 . The change i n maximum isometric tetanus tension, with time during rapid stimulation of EDL muscles from normal and denervated mice at various times post-denervation. A l l tension values are normalised with respect to mean tetanus tension (Po a). - 48 -TABLE V Cross Sectional Area of Individual SOL and EDL Muscles from Normal and Denervated Mice. 3 SOL EDL; Time 0 Norm Den Norm Den 1 0.892 0.757 1.207 1.0991 0.761 0.568 1.292 1.328 0.845 28 0.892 0.753 1.207 0.719 0.761 0.808 1.292 0.402 0.554 84 0.788 0.408 1.057 0.389 1.478 0.899 210 1.198 0.372 1.049 0.486 1.131 0.477 1.079 0.485 1.214 0.406 1.041 0.235 1.257 0.336 1.288 a Values are millimeters squared, b Time post-denervation i n days. - 49 -Figure 8 Transverse p a r a f f i n sections of normal and denervated muscles stained with H and E i n mice 16 weeks of age i l l u s t r a t i n g decrease i n c r o s s - s e c t i o n a l area. The c a l i b r a t i o n bar i n t h i s and subsequent photographs equals 100 u. (a) normal EDL (b) SOL 28 days post-denervation (c) EDL 28 days post-denervation - 51 -Figure 9 Transverse p a r a f f i n sections of normal and denervated muscles stained with H and E i n mice 42 weeks of age i l l u s t r a t i n g decrease i n cros s - s e c t i o n a l area. (a) normal SOL (b) SOL 210 days post-denervation (c) EDL 210 days post-denervation - 53 -Figure 10 Transverse p a r a f f i n sections of normal and denervated muscles stained with H and E i n mice 16 weeks of age. (a) normal SOL, c e l l s are regular i n s i z e and angular i n shape (b) SOL 1 day post-denervation. Asterisks indicate hypertrophied c e l l s . - 55 -more prevalent at l a t e r periods post-denervation. At 210 days post-denervation an a d d i t i o n a l feature of marked connective ti s s u e i n f i l t r a t i o n was observed i n the denervated SOL (Figure 1 1 ) , whereas the denervated EDL at t h i s stage showed no evidence of increased connective tissue (Figure 1 2 ) . Reinnervated Muscles No signs of functional reinnervation were evident i n the muscles of animals denervated for 28 days. A muscle twitch i n response to nerve stimulation was observed i n a t o t a l of 10 (38%) of the muscles examined 84 and 210 days post-denervation. No animal demonstrated a p o s i t i v e toe spread reaction to being suspended by the t a i l . Due to the small sample the c o n t r a c t i l e properties of the re-innervated muscles have not been subjected to s t a t i s t i c a l analysis and the r e s u l t s of i n d i v i d u a l muscles are presented i n Table VI. Three SOL (#3i 8 , 9) and 2 EDL (#4, 5) muscles showed obvious signs of recovery i n the development of absolute tetanus tension. However, 2 of these muscles f a i l e d to develop a concomitant increase i n weight compared to t h e i r respective c o n t r o l s . The 3 SOL muscles showed an increase i n the TTP whereas the 2 EDL muscles showed no change. A l l the other muscles showed values which were i n between those of normal and f u n c t i o n a l l y denervated muscles with a decrease i n tetanus tension and a prolonged contraction time compared to the normal muscles. - 56 -Figure 11 Transverse p a r a f f i n sections of normal and denervated muscles stained with H and E i n mice 42 weeks of age. (a) normal SOL. (b) SOL 210 days post-denervation. Compared to the normal muscle the c e l l s here are rounded and i r r e g u l a r i n siz e with c e n t r a l n u c l e i . There i s a considerable amount of connective t i s s u e . - 58 -Figure 12 Transverse p a r a f f i n sections of normal and denervated muscles stained with H and E i n mice 42 weeks of age. (a) normal EDL. (b) EDL 210 days post-denervation. Compared to the normal muscle the c e l l s here are rounded and i r r e g u l a r i n s i z e with central n u c l e i . The empty spaces indicate shrinkage during preparation but there i s no evidence of an increase i n connective t i s s u e . - 60 -TABLE VI Isometric Contractile Properties of Reinnervated Soleus and EDL Muscles 3 Muscle 3 (n) Wet Wt Po Pt TTP 1/2RT (mg) (s) (g) (ms) (ms) SOL 84 N (6) 10.90 31.16 8.44 17.77 23.19 ±0.36 31.40 30.46 30.21 30.48 SOL 84 R (# 1) 4.9 17.08 8.34 38.08 49.83 SOL 84 R (# 2) 6.8 11.76 2.92 13.17 22.5 SOL 84 R (# 3) 15.0 26.77 7.04 12.9 20.15 EDL 84 N (6) 13.32 30.26 8.70 9.02 9.76 zo.74 3:1.37 3:0.29 3:0.17 30.17 EDL 84 R (# 4) 8.8 47.36 10.67 11.23 12.42 EDL 84 R (# 5) 8.6 22.69 6.51 9.35 18.00 EDL 84 R (# 6) 4.3 14.17 5.57 12.18 18.50 EDL 84 R (# 7) 5.8 15.66 6.42 14.25 21.71 SOL 210 N (6) 14.02 18.39 4.80 19.09 28.15 30.59 30.76 30.14 30.29 30.52 SOL 210 R (# 8) 14.8 24.02 6.21 13.27 20.93 SOL 210 R (# 9) 6.4 .28.02 6.35 14.42 19.08 EDL 210 N (4) 12.20 51.68 11.93 8.60 7.52 30.47 32.55 30.47 30.19 30.18 EDL 210 R (#10) 4.2 19.44 7.83 17.73 15.73 a Normal (N) and reinnervated (R) SOL and EDL muscles b Values f o r normal muscles are means 3: S.E. c Values f o r reinnervated muscles are means for i n d i v i d u a l muscles - 61 -V DISCUSSION - 62 -This study has shown that the denervated SOL and EDL muscles developed, with time, a prolonged contraction time, a reduced a b i l i t y to generate tension, and an al t e r e d resistance to fatigue. The changes observed i n the denervated SOL were, on the whole, more marked than those i n the EDL and occurred over a d i f f e r e n t time course. However the denervated EDL tended to exhibit a more pronounced change i n i t s response to fatigue. C o n t r a c t i l e Properties of Denervated Muscle (a) Twitch Contraction Time The denervated SOL and the EDL showed a progressive prolonga-t i o n of the TTP and the 1/2RT (refer to Figures 3 & 4). However, there are some i n t e r e s t i n g v a r i a t i o n s i n the behaviour of the two muscles. With regard to the TTP, at a l l times the difference between the slow and fast-twitch muscle was maintained as the TTP of the de-nervated EDL remained f a s t e r than the TTP of the SOL. This suggests that a common mechanism underlies the changes i n the TTP seen i n the two muscles. With only two exceptions (Gutmann et a l . , 1972; Syrovy et a l . , 1972) , previous studies have found that the TTP i s prolonged a f t e r denervation (Eccles et a l . , 1962; Lewis, 1962, 1972; Kean et a l . , 1974; Drachman and Johnston, 1975; F i n o l and Lewis, 1975; Ranatunga, 1977; Zeman and Sandow, 1979). It i s i n t e r e s t i n g to note that the onset of the prolongation of the TTP has been correlated with changes i n the e l e c t r i c a l properties of the membrane. A decline i n the re s t i n g membrane p o t e n t i a l (RMP) i s the f i r s t change to be observed following denervation. The extent of the depolarization i n the rat has been reported to be 10 and 24 mV a f t e r 2 and 45 days denervation - 63 -res p e c t i v e l y (Albuquerque and Mclsaac, 1970). S e l l i n and Thesleff (1980) reported a s i m i l a r loss of 16-20 mV i n the mouse 3 to 6 days post-denervation. The depolarization has been at t r i b u t e d to an increase i n membrane permeability ( S e l l i n et a l . , 198l) and r e d i s t r i b u t i o n of K + ions (Albuquerque and Mclsaac, 1970). In turn the membrane action p o t e n t i a l becomes prolonged from 40-60% with a decrease i n the amplitude and a decrease i n the rate of r i s e of voltage (dV/dt) (Albuquerque and Thesleff, 1968). This decrease i n dV/dt occurred, i n the rat, 3 days following s c i a t i c section (Redfern and Thesleff, 1971) and coincided with prolongation of the TTP ( F i n o l and Lewis, 1975). Lewis, Webb and Pardoe (1978) have also shown i n the cat that prolongation of the TTP and the ac t i o n p o t e n t i a l occur together at 9 days post-denervation. Even though i t i s hard to reconcile a change of 50% i n the action p o t e n t i a l with a change of up to 100% which i s seen i n the TTP, i t appears that a r e l a t i o n may e x i s t between the two events. This i s further sup-ported by the work of Taylor et a l . (1972) who demonstrated i n the frog that the use of a twitch potentiator, such as zinc, resulted i n an increase i n the duration of the action p o t e n t i a l and a slowing of the C a + + pulse with a corresponding increase i n the TTP. Other membrane changes are known to occur following denervation which may also a f f e c t the exci t a t i o n - c o n t r a c t i o n mechanism. A decrease i n T-tubule membrane protein phosphorylation has -been ob-served, i n the r a t EDL, 10 days post-denervation. I t has been sug-gested that such a membrane change may disturb the t r i a d junction (Blaise Smith and Appel, 1977). In addition s t r u c t u r a l changes have been observed, such as p r o l i f e r a t i o n and disorganisation of the - 64 -T-tubule system, the formation of pentads, and swelling and vesicu-l a t i o n of the sarcoplasmic reticulum (SR) (Pellegrino and F r a n z i n i , 1963; M i l e d i and S l a t e r , 1969; Tomaneck and Lund, 1973). Although there i s no evidence that t h i s delays the inward spread of a c t i v a t i o n , i t has been suggested that calcium release may be impaired from the i s o l a t e d v e s i c l e s (Martinosi, 1972). In terms of 1/2RT a s i g n i f i c a n t slowing of the SOL was seen within 24 hours post-denervation, whereas no change was recorded f o r the EDL. The presence of hypertrophic f i b r e s i n the SOL seen i n t h i s study at 1 day post-denervation i s i n d i c a t i v e of some early s t r u c t u r a l abnormality, but the r e l a t i o n s h i p between t h i s observation and the relaxation process remains speculative. At subsequent time periods there was a progressive slowing of the 1/2 RT i n both muscles, which reached an apparent plateau i n the denervated SOL at 84 days, but continued to develop i n the EDL. The rate of C a + + transport i n normal fast muscle has been estimated to be double that of slow muscle, based on y i e l d of SR membranes, concentration of C a + + s i t e s (Harigaya et a l . , 1968, 1969) and volume assessed by electron microscopy (Luff and Atwood, 1971). Aft e r denervation, i t has been shown that there i s an increase i n the amount of SR in both slow and fast muscle, as the m y o f i b r i l s degenerate more than the SR (Engel and Stonnington, 1974). Although the r e l a t i v e volume suggests that the capacity of the SR to accumulate C a + + i s increased, at the same time the rate of C a + + uptake has been shown to decrease following denervation (Sreter, 1970). Further-more the rate of uptake decreases as the Ca concentration i n the v e s i c l e s increases (Martinosi, 1972). As the decrease i n uptake has - 65 -been noted to be greater i n the f a s t gastrocnemius than the SOL (Sreter, 1970), t h i s would explain the progressive increase seen i n 1/2RT the EDL i n t h i s study. (b) Isometric Tension Muscle tension i s l a r g e l y dependant upon the number of cross-bridges which are formed on a c t i v a t i o n , and thus, at optimal length, upon the number of myo f i b r i l s acting i n p a r a l l e l . However, tension i s not s o l e l y a function of cross-sectional area. There are differences i n the maximum tetanus tension generated per unit cross-section i n slow and fast-twitch muscles (Buller, 1977). Nevertheless, any dimunition i n the number of m y o f i b r i l s , as indicated by a loss of weight, would be expected to r e s u l t i n a proportional loss of absolute tension. This i n fac t was the response obtained i n the EDL at a l l time periods except 210 days post-denervation, at which time tension with respect to wet weight was decreased. Wet weight has been used as an i n d i c a t o r to normalise tension values as loss of weight following denervation has been found to be proportional to loss of m y o f i b r i l l a r proteins (Goldberg, 1969; Engel and Stonnington, 1974; Goldspink, 1976). U l t r a s t r u c t u r a l studies have indicated that early changes following denervation include m u l t i f o c a l disruption of several contiguous m y o f i b r i l s f o r a distance of 1 sarcomere (Cullen and Puskal, 1977) or more (Pellegrino and Fr a n z i n i , 1965). I f these m y o f i b r i l s are not connected i n serie s they should not contribute to the development of tension or at le a s t only to a limited extent. Nevertheless, the degenerative areas con s i s t i n g of undifferentiated sarcoplasm (Cullen and Pluskal, 1977) would s t i l l contribute to muscle weight u n t i l the absorption process was completed. - 66 -In a d d i t i o n Goldspink (1980), working i n the rat, has demon-strated a 40? decrease i n the rate of synthesis and a 20% increase In the rate of degradation of m y o f i b r i l l a r proteins within 2 days of de-nervation. Other studies have shown that there i s also a decrease i n the rate of amino acid transport (Goldberg et a l . , 1974) and an increase i n acid proteinase a c t i v i t y (Pluskal and Pennington, 1976) following denervation. Whether these factors contribute to the f i n d i n g i n the SOL that l o s s of weight was preceded by l o s s of tension, within hours of dener-vation, remains e n t i r e l y speculative. However, the s t r i k i n g d i f -ference between the SOL and EDL i n the a b i l i t y to generate tension during the early phase of denervation would seem to indicate a u t o l y s i s of e a r l i e r onset i n the slow than the fast muscle. This, i n f a c t , has been observed both i n rate of protein degradation (Goldspink, 1976) and m y o f i b r i l l a r disruption (Cullen and Pluskal, 1977). This f i n d i n g of early impairment of tension generating a b i l i t y i n the denervated muscle was s u r p r i s i n g , but not without precedent (Gutmann and Sandow, 1965; Takamori et a l . , 1978). However i n both of these studies the f a s t muscle was reported to be s i m i l a r l y a f -fected. An attempt was made to i d e n t i f y any possible experimental error which would account for the differences seen i n the acute phase . between the SOL and the EDL. Both the time taken to d i s s e c t the muscle preparation and also the pH of the bathing solution were examined (see Appendix 4). The pH of the s o l u t i o n bathing the SOL at 1 day post-denervation was s i g n i f i c a n t l y higher than that of the c o n t r o l muscles (Table XII). Although a r i s e i n pH i s usually asso-ciated with a r i s e i n tension (Roos and Boron, 1981) i t has also been - 67 -found to cause a diminution (Pannier et a l . , 1970). So whether this factor can account for the loss of tension in the denervated SOL remains undecided. At 28 days post-denervation both the SOL and EDL showed a similar response in which loss of tension was largely accompanied by a proportional loss of weight. This indicated that the denervated muscles were able to generate as much or even greater tension per unit of wet weight as the normal controls at this time period. By 84 days post-denervation the SOL again showed loss of tension in absolute and normalised terms. If the change in the SOL at 24 hours i s explained by increased autolysis as an acute reaction to denervation, the same reaction occurring weeks later i s inconsistent with that theory. An alternative explanation might be that the SOL muscle has been reported to develop a greater amount of absolute collagen after denervation (Tomanek and Lund, 1973). If more collagen i s present in the SOL than the EDL i t would present an artifact in the weight tension ratio. On subjective examination of the EDL and SOL histological sections there appeared to be no difference in the amount of connective tissue between the two muscles at 84 days although there was a marked increase in the denervated SOL at 210 days (see Figure 11). Unfortunately a quantitative assessment of collagen to muscle content has not been performed. However sections are available for further study by electronmicroscopy. At 210 days post-denervation both SOL and EDL showed decreased tension with respect to wet weight. It is feasible to assume that at this late stage when muscle weight is only to 20% of normal, and i t is known that myofibrillar proteins degenerate more rapidly than non-contractile elements (Engel and Stonnington, 1974), the weight/tension - 68 -r a t i o has f i n a l l y been disrupted. Further explanation for the decrease i n tension/unit weight i s provided by the connective tissue i n f i l t r a t i o n which was a prominent feature i n the 210 day SOL but not i n the EDL. However with regard to the EDL i t should be noted that the normal EDL animals at 210 days were not only s i g n i f i c a n t l y larger i n body weight but were from the same l i t t e r and therefore do not represent a normal sample. With regard to twitch tension the denervated SOL and EDL generated l e s s tension i n both absolute and normalised terms at 1 day post-denervation. In contrast to t h i s immediate response to dener-vation both muscles developed more tension/unit weight at 28 days post-denervation. This response at 1 day i s i n t e r e s t i n g as i t serves to h i g h l i g h t the fact that the development of twitch tension may be dependent upon the duration of the contraction as well as crossbridge formation. I t has been shown that the greater the time for a c t i v a t i o n the greater i s the c o n t r a c t i l e response. Thus i n the EDL, although the tetanus tension was normal at 1 day post-denervation, the TTP was shortened and the twitch tension was correspondingly reduced. The decrease i n twitch tension i n the denervated SOL, however, corres-ponded to a s i m i l a r 50% decrease i n the tetanus tension. At 28 days post-denervation the twitch, when normalised with respect to wet weight, was observed to be potentiated 91% and 52% above normal i n the SOL and EDL r e s p e c t i v e l y . This approximates an increase i n the TTP of 82% and k l % i n the corresponding muscles. The potentiation of the twitch has been reported i n previous studies. Zeman and Sandow (1979), working i n the mouse found the twitch tension to be increased by 8 0 % i n the EDL 13-16 days post-denervation. F i n o l and Lewis (1975) whilst g i v i n g no figures, reported the twitch tension - 69 -to be increased 3 days following denervation. As the TTP i s a s s o c i -ated with membrane changes which include prolongation of the action p o t e n t i a l (Lewis et a l . , 1978; S e l l i n and Thesleff, 1980) i t i s reasonable to speculate that these membrane changes could invoke action s i m i l a r to known potentiators (Taylor et a l . , 1972) or else herald concomitant changes i n the membranes of the T-tubule system. Further evidence that the potentiated twitch tension i n denervated muscle may be associated with the slowing of the TTP i s provided by the twitch to tetanus tension r a t i o (Pt/Po). Drachman and Johnston (1973), working i n the rat have shown that, during the f i r s t 20 days of post-natal development, the Pt/Po decreased from 0 . 6 to 0.2 with a concomitant decrease i n the TTP. In t h i s study the Pt/Po increased from approximately 0.2 to 0.5 i n both the SOL and the EDL and was accompanied by an increase i n the TTP. Similar figures have been reported i n othe denervation studies (Kean et a l . , 1974; Drachman and Johnson, 1975; Kotias, 1975; Ranatunga, 1977). (c) Fatigue C h a r a c t e r i s t i c s The response of the muscle to fatigue i s a c h a r a c t e r i s t i c of the general metabolic properties of that muscle, that i s , oxidative or g l y c o l y t i c . The SOL showed a marked decrease i n i t s resistance to fatigue at 1 day post-denervation but a r e l a t i v e l y normal response at the l a t e r time periods. Thus the denervated SOL muscle appeared to regain i t s primarily oxidative c h a r a c t e r i s t i c s a f t e r 28 days of dener-vat i o n . In contrast, the EDL showed an increased resistance to fatigue at and beyond 28 days post-denervation. Again, a d i f f e r e n t response i s seen i n the SOL and EDL which involves d i r e c t i o n and extent of change, as well as time of onset. The d i s t r i b u t i o n of f i b r e types i n the normal C57 SOL has been - T O -reported to change during post-natal development so that the percen-tage of SO fibres increases from 40% at 4 weeks to 60% at 32 weeks of age. There i s at the same time a corresponding decrease in the percen-tage of FOG fibres (Ovalle et a l . , 1981). During the time course of this study the normal muscles would be expected to undergo similar changes. The slight increase in TTP of the normal SOL, which developed with age, marks the physiological expression of this change (refer to Table III). However i t i s not known how much lack of normal function would affect this process in the denervated muscles. Denervation has been shown to result in a marked reduction of enzyme activity, particularly in fibres in which activity i s high, with l i t t l e change in the activity of enzymes which are low (Romanul and Hogan, 1972). Thus oxidative enzymes should decrease in the de-nervated SOL. This would result in the observed decrease in resis-tance to fatigue. The fact that this change in the response to fatigue was only transitory requires some explanation. There are two po s s i b i l i t i e s . There may have been preferential atrophy of the FOG fibres although other studies indicate that Type I (SO) and Type IIA (FOG) fibres', in the rat SOL atrophied at a similar rate (Jaweed et a l . , 1975). Alternatively, induced activity in the form of f i b r i l -lation may have prevented the depletion of oxidative , enzymes (Silverman and Atwood, 1980). The same rationale may be used to explain the changes which were demonstrated in the denervated EDL. A change in the fibre type distribution of the EDL muscle has also been shown to occur during development (Goldspink and Ward, 1970). Although the exact propor-tions have not yet been documented in the mouse, the FG fibres have - 71 -been reported to increase while the FOG f i b r e s decrease (Ovalle and Bressler, 1981). P r e f e r e n t i a l l o s s of the f a s t f i b r e s i n a f a s t muscle following denervation has been well documented (Karparti and Engel, 1968; Gauthier and Dunn, 1973; Jaweed et a l . , 1975). Although e a r l i e r studies did not d i s t i n g u i s h between type IIA and IIB f i b r e s , more recently Niederle and Mayr (1978) and Davis and Kiernan (1980) working i n the rat EDL have observed that greater atrophy occured i n the f a s t -g l y c o l y t i c (FG) than the f a s t - o x i d a t i v e - g l y c o l y t i c (FOG) f i b r e s . The combination of these factors, together with the e f f e c t of f i b r i l l a t i o n would tend to widen the d i s p a r i t y between the normal and denervated EDL muscles. I f the normal muscle becomes more g l y c o l y t i c while the denervated muscle becomes more oxidative, i t would explain the marked increase i n resistance to fatigue shown by the denervated muscle. The fatigue responses of the denervated muscles indicate a pos-s i b l e trend i n changes i n the d i s t r i b u t i o n of f i b r e types. Of course c o r r e l a t i v e histochemical studies are required before t h i s can be sub-stantiated . E f f e c t of Time on Post-denervation Changes To determine underlying mechanism behind the changes which are exhibited by denervated muscle, a l l the a v a i l a b l e evidence has to be integrated and any anomalies have to be recognised and reconciled. Thus i t i s important to view the spectrum of changes which occur at a s i n g l e point i n time following denervation, as well as the pattern of events which occur over a prolonged period. The early phase of denervation i s of p a r t i c u l a r i n t e r e s t as i t serves to provide some clue as to the t r i g g e r responsible f o r - 7 2 -i n i t i a t i o n of the induced changes. At 1 day post-denervation although nerve impulse a c t i v i t y has ceased, the e f f e c t of a neurotrophic f a c t o r may s t i l l be at work. As degeneration of the nerve i s not complete fo r a few days, neurotrophic products would s t i l l be present i n the d i s t a l stump at t h i s time. The length of the nerve stump has been observed to a f f e c t the onset of membrane changes following denervation. Drachman and Johnston (1980) found that although the stump length did a f f e c t membrane changes, there was an i n i t i a l delay of 18 hours when the RMP was measured i n vivo i n the rat with a 2 mm stump. However, others also working i n vivo i n the rat found a decrease i n the res t i n g membrane po t e n t i a l (RMP) a f t e r 6 or 18 hours, i n the soleus, depending on whether the nerve was sectioned 2 mm or 65 mm from the muscle (Despande et a l . , 1980). Thus the influence of the stump length was 5 mm/hr, a f t e r accounting f o r the i n i t i a l delay, which presumably was due to the length of nerve within the muscle. This e f f e c t of stump length suggests that the mechanism responsible f o r the membrane changes i s associated with f a s t axonal transport, which has a rate of 410 mm/day (Ochs, 1974). In t h i s study the length of the d i s t a l stump was approximately 15 mm. There was no increase i n the TTP within 24 hours which suggests that changes i n the contraction time may be dependent upon the loss of a trophic factor or that loss of nerve impulse a c t i v i t y requires more than 24 hours to produce an e f f e c t . A loss of tension occurred i n the SOL at 1 day post-denervation, which suggests that loss of nerve impulse a c t i v i t y may have i n i t i a t e d t h i s response. Loss of tension appears to be associated with an increase i n p r o t e o l y t i c a c t i v i t y . Boegman and - 73 -Scarfe (1981) have demonstrated that an increase i n a u t o l y t i c enzyme a c t i v i t y i n the rat soleus occurs 3 days post-denervation. Furthermore, they found that the increase was associated with loss of a c t i v i t y and l o s s of trophic factor as batrachotoxin, which blocks both nerve impulse a c t i v i t y and axonal transport, had a greater e f f e c t than c o l c h i c i n e , which only blocks the l a t t e r . Loss of tension has been reported to occur within 24 hours i n the r a t , following peroneal nerve section (Gutmann and Sandow, 1965), and i n the rabbit following s c i a t i c section (Takamori et a l . , 1978). This would support the contention that loss of impulse a c t i v i t y may be p a r t l y responsible for t r i g g e r i n g changes i n tension generating pro p e r t i e s . At 28 days post-denervation the potentiation of the tetanus as well as the twitch tensions indicated the p o s s i b i l i t y of a recupera-t i v e a ction i n both the SOL and the EDL. Whatever the mechanism t h i s appears to have been abortive as i t was not present at 84 days post-denervation. However, i t raises the p o s s i b i l i t y that reinnervation had occurred by t h i s time. The functional test indicated there was no reinnervation i n these muscles. Although EMG would have been a more rigorous means of detection, a previous study has shown that miniature excit a t o r y end plate p o t e n t i a l s , detected by intramuscular electrodes, were accompanied by a v i s i b l e weak contraction ( S e l l i n et a l . , 1980). Nevertheless morphological reinnervation could have been established without synaptic transmission. An increase i n weight i s a general i n d i c a t i o n that reinnervation i s i n progress. In f a c t , i n t h i s study there was a progressive decrease i n weight with time post-denervation. At 84 days post-denervation the changes i n both the SOL and the EDL appeared progressive and can be a t t r i b u t e d to progressive loss of - 74 -myofibrils. The percentage loss in wet weight continued to be less in the SOL than the EDL. As atrophy has been shown to be retarded by the mechanism of passive stretch i t is appropriate to explore the pos-s i b i l i t y that stretch could have an influence on the difference between the muscles at this period. Previous studies in the rat have indicated that the animal was observed to drag i t s leg so that the EDL which spanned 2 joints was subjected to more stretch than the 1-joint SOL. This could explain why in some instances SOL has been reported to be more atrophied than the EDL. In this study the mouse was observed to walk with an abducted gait with the hip and knee in flexion. Therefore stretch of the SOL, even though i t is a 1-joint muscle, could have been inflicted by body weight and lack of muscle tone in the antagonistic groups. Although the EDL would be subjected to stretch by the toes curling, the solid floor of the cage would reduce this effect. As stretch has been shown to decrease the rate of protein degradation in denervated muscle, the smaller loss of weight in the SOL may indicate a contribution of stretch to this effect. On the other hand there was no significant difference in length between the denervated and control muscles which suggests that i f stretch was a factor i t was intermittant and insufficient to produce a change in length. Nevertheless as loss of tension in the denervated SOL at this time was far greater than the loss of weight, factors other than myofibrilar loss must be at work to explain the effects seen in the denervated SOL 84 days post-denervation. At the 84 and 210 days post-denervation the marked loss of tension in the denervated SOL is accompanied by a diminution in the tension generating a b i l i t y of the normal muscles. Normal muscles in - 75 -the rat have been shown to develop .an ..impairment of _ strength with advanced age, as a. r e s u l t . o f neuronal atrophy. As...the. l i f e span of the C57 mouse i s i n excess of 2 years, i t would appear that age i s not the contributing factor to the decline i n normal tension. Further experiments are required to c l a r i f y t h i s issue. Slow and Fast Muscle Response to Denervation. The response of the denervated SOL showed a marked d i f f e r e n c e from the response of the EDL both i n the extent and early i n i t i a t i o n of change. This suggests that the slow muscle i s more dependant on neural influence than the f a s t muscle. The l i t e r a t u r e provides evidence to support t h i s viewpoint (Buchthal and Schmalbruch, 1980). Studies of cross-innervation have shown that the slow nerve converts f a s t muscle more rea d i l y and more completely than the f a s t nerve transforms slow muscle (Buller et a l . , 1960a, 1960b; Close, 1969; B u l l e r et a l . , 1969; Samaha et a l . , 1970; Barany and Close, 1971). E l e c t r i c a l stimulation with a frequency of 10-20 shocks/s i s able to convert a fast muscle to a slow muscle (Salmons and Vrbova, 1969; Pette et a l . , 1976) whereas a frequency of 40-60 shocks/s has been found to convert the properties of the slow muscle only i f i t i s de-nervated (Lcmo et a l . , 1974). F i n a l l y i t has been shown that neonatal neurectomy prevents the d i f f e r e n t i a t i o n of the slow muscle properties so that the slow muscle f a i l s to develop slow myosin l i g h t chains (Ishiura et a l . , 1981), retains a high l e v e l of myosin ATPase a c t i v i t y (Shafiq et a l . , 1972), and a f a s t speed of contraction ( B u l l e r and Lewis, 1965, Drachman and Johnston, 1973). There are, however, a few proponents f o r the suggestion that f a s t muscle i s more dependent on neural influence, mainly on the grounds that the f a s t muscle i s seen to atrophy more than the slow - 76 -muscle (Bajusz, 1964). In addi t i o n i t has been suggested that the slowing e f f e c t of denervation indicates that a l l muscles revert to an immature stage which i s characterized by a slow speed of contraction. The opposing view i s that the d i f f e r e n t i a t i o n of the slow muscle i s i n i t i a t e d by slow nerve influence upon elimination of polyneural innervation. Therefore the e f f e c t s of denervation repre-sent a derepression of fa s t muscle c h a r a c t e r i s i c s and r e s u l t i n changes such as an increase i n the amount of sarcoplasmic reticulum. The observation that a slow muscle acquires a f a s t speed of contrac-t i o n when subjected to a vari e t y of methods of disuse further supports t h i s argument. The apparent c o n t r a d i c t i o n that disuse r e s u l t s i n an increase i n speed while t o t a l disuse by denervation r e s u l t s i n a decrease i n speed of contraction may be explained by the onset of membrane changes which supersede a l l other influences. - 77 -VI -CONCLUSION - 78 -This study has shown that denervation has a profound effect on the contractile properties of both slow and fast muscle in the mouse. The denervated muscles exhibited a prolonged contraction time and a decreased a b i l i t y to generate tension. These changes occurred earlier in the slow-twitch SOL than in the fast-twitch EDL. In the EDL an increased resistance to fatigue became evident at 28 days post-denervation. Since the SOL was affected to a greater extent than the EDL, on the whole, this suggests that the slow-twitch muscle may be more dependant on i t s nerve supply than the fast-twich muscle for the maintenance of i t s contractile properties. 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R., Preiser, H. and Sandow, A. (1972). Action p o t e n t i a l parameters a f f e c t i n g excitation-contraction coupling. J. Gen.  Physiol. 59, 421-436. Tomanek, R. J . and Lund, D. D. (1973). Degeneration of d i f f e r e n t types of s k e l e t a l muscle f i b r e s . I Denervation. J . Anat. 116, 395-407. Tower, S. S. (1937). Trophic control of non-nervous tissue by the nervous system: a study of muscle and bone innervated from an i s o l a t e d and quiescent region of s p i n a l cord. J . Comp. Neurol. 67, 241-267-Vrbova, G. (1963). The e f f e c t of motoneurone a c t i v i t y on the speed of contraction of s t r i a t e d muscle. J . Physiol. 169, 513-526. Zeman, R. J . and Sandow, A. (1979). Denervation e f f e c t s on dystrophic and normal muscles and the etiology of dystrophy. Ann. N.Y. Acad. S c i . 317, 171-184. 87 -APPENDIX 1: The e f f e c t of a second denervation procedure upon the con-t r a c t i l e properties of SOL and EDL muscles i n normal and denervated mice. Introduction To circumvent the possible occurrence of reinnervation a second operative procedure was performed i n animals i n the 84 day post-denervation group. Known rates of nerve regeneration vary from 3 to 4.5 mm/day. This occurs a f t e r 3 days i n i t i a l delay following a crush but when the nerve trunk i s transected delay i n crossing the gap can be several weeks. In t h i s study, the distance from the s c i a t i c sec-t i o n to the motor point of the SOL and EDL muscles was approximately 15 mm. Therefore, there was l i t t l e chance of reinnervation in the 28-day animals but considerable p o s s i b i l i t y i n the 84 day animals. Method Six weeks a f t e r the i n i t i a l denervation the r i g h t s c i a t i c nerve was exposed i n a manner s i m i l a r to that previously described (see Methods). The stump was located and the presence of any branches was noted. They were then severed and an a d d i t i o n a l 1mm was removed from the proximal end of the nerve. The end of the nerve was then l i g a t e d to a id i d e n t i f i c a t i o n l a t e r . The f i n a l experimental procedures were c a r r i e d out 6 weeks l a t e r which was 84 days following the i n i t i a l de-nervation. Results With respect to the tension generating a b i l i t y the 2XD SOL showed an increase i n tetanus and twitch tensions both i n absolute TABLE VII Contractile Properties of 2 X Denervated and 1 X Denervated Soleus and EDL Muscles at 84 days post-denervation a. Muscle b Po (g) Pt (g) TTP (ms) 1/2RT (ms) 2XD 1XD 2XD 1XD 2XD 1XD 2XD 1XD SOL (6,6)c 12.63 7.54 4.69 3.58 26.94 34.82 34.94 42.20 ±1.01 ±0.43 ±0.47 ±1.48 ±0.45 ±0.27 ±1.29 ±0.88 P ?.001 5.05 5.001 5.001 EDL (5,4) 17.00 14.62 6.24 5.77 14.50 13-00 18.88 19-67 ±0.54 ±1.29 ±0.18 ±0.38 ±0.17 ±0.56 ±0.02 ±0.83 P NS NS ;=.01 NS a Values are means ± S.E. b Denervated muscle 84 days a f t e r f i r s t or only denervation procedure at 12 weeks of age. c Number of 2 X denervated and 1 X denervated muscles tested r e s p e c t i v e l y . - 89 -terms and when expressed r e l a t i v e to wet weight, compared to the 1XD SOL. In contrast, there was no difference between the 1XD and the 2XD EDL muscles (Table VII). There was a s i m i l a r discrepancy with regard to twitch con-t r a c t i o n time between the e f f e c t of a second denervation procedure on the EDL and the SOL muscles. The TTP and the 1/2RT of the 2XD SOL had not slowed to the same extent as the 1XD. However, the TTP of the 2XD EDL was s i g n i f i c a n t l y slower than the 1XD and there was no difference i n the 1/2RT. Discussion There was a s i g n i f i c a n t d i f f e r e n c e between the 1XD and the 2XD SOL muscles but not the EDL muscles. This would suggest that i f r e -innervation were the cause of the dif f e r e n c e s seen between the 1XD and 2XD muscles, i t had occurred to a greater extent i n the SOL than the EDL. Three possible explanations which may account for the changes seen i n the 1XD and the 2XD muscles are: (1) Both 1XD and 2XD muscles are reinnervated. This i s not supported by the f a c t that there was no increase i n weight i n the f u n c t i o n a l l y denervated muscles. (2) Only 2XD are reinnervated. Again there i s no functional evidence. However, i f regeneration had occurred within s i x weeks, a second i n s u l t to the s c i a t i c nerve could r e s u l t i n a spurt of the growth cone r e s u l t i n g i n greater outgrowth during the second s i x week period. I f t h i s were the case the 2XD muscles should show a decrease i n weight and tension compared to the 1XD. (3) Neither reinnervated. There i s no apparent cause f o r the discrepancy between the 1XD and 2XD muscles. - 90 -Conclusion As the 1XD and 2XD experimental procedures produced d i f f e r e n t r e s u l t s of unknown cause i t was deemed i n a d v i s a b l e to use the 2XD group f o r comparison w i t h 1XD animals of other ages. The reason why the 2XD procedure appeared to a f f e c t the SOL and not the EDL i s not known. - 91 -APPENDIX 2: Tetanus Tension Normalised with Respect to Wet Weight, Dry Weight and Estimated Cross-sectional Area. Introduction As denervated muscle i s subject to a considerable loss of muscle weight, tension i s usually normalised by some means, so that the tension generating a b i l i t y of denervated and control muscles can be compared. Some investigators have normalised tetanus tension with respect to wet weight (Kean et a l . , 1974; Zeman and Sandow, 1979). An a l t e r n a t i v e method used i s the estimated cr o s s - s e c t i o n a l area of the muscle, which i s obtained by Po (kg)/wet muscle weight (g)/muscle length (cm) (Sabbadini and Baskin, 1979). In addition i t has been suggested that dry weight could be used as an estimate of the m y o f i b r i l l a r protein content. Results The percentage changes i n the tetanus tension of the denervated SOL and EDL compared to the control muscles are i l l u s t r a t e d i n Figure 13- There i s some v a r i a t i o n i n the r e s u l t s obtained from the use of wet weight as opposed to dry weight (Tables VIII and IX). However the only a l t e r a t i o n to a s i g n i f i c a n t difference between the denervated and control muscles i s seen i n the EDL at 28 days post-denervation. In that muscle, tension with respect to wet weight i s s i g n i f i c a n t l y above the control muscles, whereas when expressed i n r e l a t i o n to dry weight, there i s no s i g n i f i c a n t difference between the denervated muscles and t h e i r c o n t r o l s . There i s also some v a r i a t i o n when tension normalised with respect to wet weight i s compared to tension expressed i n r e l a t i o n to the estimated cross-sectional area (mass/length). Again the only - 92 -alteration to a significant difference between denervated and control muscles occurred at 28 days post-denervation in the SOL. At 1 day post-denervation tension of the denervated SOL showed less loss when normalised with respect to cross-sectional area than when normalised with respect to wet weight. Discussion The variations in percentage change seen in tension expressed in relation to dry weight may be accounted for by the the d i f f i c u l t y experienced in obtaining accurate measurements. In addition there was some variation in the percentage change from wet weight to dry weight which ranged from 68%-7k% in SOL and 71%-7&% in EDL. This suggests that there was also an atmospheric influence but the study was reaching completion before i t was realised that this variable was uncontrolled. There was no significant difference between the muscle lengths of the denervated and control muscles to account for the difference in tension expressed relative to estimated cross-sectional area. In order to determine whether the wet weight or the cross-sectional estimate provides the most accurate means of estimating tension/fibre, actual tension/fibre needs to be studied. Although ideal, this method was considered to be beyond the scope of this study. G3 d Post-denervation days Figure 13« Percentage change from normal of tetanus tension of denervated SOL and EDL'muscles normalised with respect to dry weight (d), wet weight (w) and estimated cross-sectional area (cm2). - 9k -TABLE VIII Normalised Tetanus Tension of Soleus Muscles from Normal and Denervated Mice 3. Time b ( , ) d Po/cm^ Po/W.W. Po/D.W.c Norm. Den. Norm. Den. Norm. Den. 1 2.75 2.00 3.05 1.54 10.08 5.42 (6,6) ±0.11 ±0.10 ±0.14 ±0.08 ±0.34 ±0.35 P ?.001 5.001 5.001 28 2.75 3.46 3-05 2.71 10.08 10.62 (6,6) ±0.11 ±0.12 ±0.14 ±0.11 ±0.34 ±0.86 P 5.001 N.S. N.S. 84 3-71 1.57 2.93 1.25 9.24 4.69 (6,6) ±0.22 ±0.10 ±0.17 ±0.08 ±0.44 ±0.48 P 5.001 5.001 5.001 210 1.76 0.74 1.31 0.63 5.16 2.68 (6,3) ±0.08 ±0.01 ±0.05 ±0.01 ±0.22 ±0.17 P 5.001 5.001 5.001 a Values are means ± S.E. b Time, i n days post-denervation c Tension per unit of mass/length (cm 2), wet weight (W.W.), dry weight (D.W.) d Number of normal and denervated muscles tested respectively - 95 -TABLE IX Normalised Tetanus Tension of Extensor Digitorum Longus Muscles from Normal and Denervated Mice 3. Time b Po/cm2 Po/W.W. Po/D.W.c ( , ) d Norm. Den. Norm. Den. Norm. Den. 1 (6,6) P 3.82 4.44 ±0.13 ±0.31 N.S 3.02 3.50 ±0.10 ±0.24 N.S. 13-00 14.41 ±0.63 ±1.83 N.S. 28 (6,7) P 3.82 4.50 ±0.13 ±0.19 ?.01 3-02 3.57 ±1.00 ±0.16 ?.01 13.00 14.60 ±0.63 ±1.67 N.S. 84 (6,4) P 3.01 3.40 ±0.15 ±0.14 N.S. 2.33 2.66 ±0.13 ±0.10 N.S. 8.19 9-30 ±0.30 ±0.97 N.S. 210 (4,3) P 5.40 3.34 ±0.32 ±0.21 5.001 4.21 2.66 ±0.25 ±0.20 5.001 17.37 9.84 ±1.28 ±0.72 5.001 a Values are means ± S.E. b Time, i n days post-denervation c Tension per unit of mass/length (cm 2), W e t weight (W.W.), di weight (D.W.) d Number of normal and denervated muscles tested respectively APPENDIX 3 TABLE X Contraction times of Soleus and Extensor Digitorum Longus Muscles from Normal and Denervated M i c e a SOL EDL Time 0 ( , ) c TTP (ms) 1/2RT (ms) ( , ) TTP (ms) 1/2RT (ms) Norm. Den. Norm. Den. Norm. Den. Norm. Den. 1 (6,6) 16.82 16.01 20.75 24.93 (6,6) 9.10 7.54 8.89 8.43 ±0.50 ±0.28 ±0.55 ±0.56 ±0.15 ±0.09 ±0.19 ±0.13 P N.S. 5.001 5.001 N.S. 28 (6,6) 16.82 30.57 20.75 32.27 (6,7) 9.10 12.79 8.89 12.38 ±0.50 ±0.21 ±0.55 ±0.45 ±0.15 ±0.21 ±0.19 ±0.19 P s.001 5.001 5-001 5.001 84 (6,6) 17-77 3^.82 23.19 ' 42.20 (6,4) 9.02 13.00 9.76 19.67 ±0.21 ±0.27 ±0.48 ±0.88 ±0.17 ±0.56 ±0.17 ±0.83 P 5.001 5.001 5-001 5.001 210 (6,3) 19.09 38.52 28.15 42.30 (4,3) 8.60 14.74 7.52 36.19 ±0.29 ±0.73 ±0.52 ±0.50 ±0.19 ±0.23 ±0.18 ±1.29 P 5.001 5.001 5.001 5.001 a Values are means ± S.E. b Time i n days post-denervation c Number of normal and denervated animals tested r e s p e c t i v e l y - 97 -A P P E N D I X 4 : E x p e r i m e n t a l V a r i a b l e s I n t r o d u c t i o n T h e r e was a m a r k e d d i f f e r e n c e b e t w e e n t h e d e n e r v a t e d SOL a n d t h e EDL a t 1 d a y p o s t - d e n e r v a t i o n . T h e r e f o r e t h e p o s s i b i l i t y t h a t e x p e r i m e n t a l e r r o r was r e s p o n s i b l e f o r t h i s e f f e c t was i n v e s t i g a t e d . T h e t i m e t a k e n t o d i s s e c t t h e m u s c l e a n d t h e pH o f t h e K r e b s b a t h i n g t h e m u s c l e d u r i n g s t i m u l a t i o n w e r e t h e two v a r i a b l e s e x a m i n e d . R e s u l t s T h e r e was c o n s i d e r a b l e v a r i a t i o n i n t h e t i m e t a k e n t o d i s s e c t t h e m u s c l e s w h i c h r a n g e d f r o m 6 t o 55 m i n u t e s . A l t h o u g h t h e r e was a m a r k e d d i f f e r e n c e b e t w e e n t h e d e n e r v a t e d a n d n o r m a l g r o u p s i n t h e EDL 210 d a y s p o s t - d e n e r v a t i o n , t h e s a m p l e was t o o s m a l l t o c o n s i d e r t h i s s t a t i s t i c a l l y s i g n i f i c a n t ( T a b l e X I ) . T h e r e was a d i f f e r e n c e b e t w e e n t h e d e n e r v a t e d a n d n o r m a l S O L w i t h r e s p e c t t o t h e pH o f t h e b a t h i n g s o l u t i o n , w h i c h was s t a t i s t i c a l l y s i g n i f i c a n t a t 1 d a y p o s t - d e n e r v a t i o n ( T a b l e X I I ) . D i s c u s s i o n T h e f i n d i n g t h a t t h e pH o f t h e K r e b s s o l u t i o n was i n c r e a s e d i n t h e 1 d a y d e n e r v a t e d S O L e x p e r i m e n t s a l t h o u g h d i s t u r b i n g , f a i l s t o p r o v i d e a n e x p l a n a t i o n f o r t h e m a r k e d d e c r e a s e i n t e n s i o n i n t h e SOL s e e n a t t h a t t i m e , s i n c e a n i n c r e a s e i n pH h a s b e e n shown t o r e s u l t i n b o t h a n i n c r e a s e a n d a d e c r e a s e i n t e n s i o n ( P a n n i e r e t al.,1970; R o o s a n d B o r a n , 1981). TABLE XI Time taken to Dissect Soleus and EDL Muscles from Normal and Denervated Mice. a SOL EDL Time b ( , ) c Norm. Den. ( , ) Norm. Den. 1 P 28 P 84 P 210 P (6,6) 22.83 19.67 ±4.28 ±1.68 N.S. (6,6) 22.83 31.83 ±4.28 ±6.14 N.S. (6,6) 19.20 16.33 ±3.65 ±2.90 N.S. (6,3) 19.00 15.00 ±0.47 ±4.24 (5,6) (5,7) (6,4) (4,2) 20.80 14.83 ±2.50 ±2.65 N.S. 20.80 21.86 ±2.50 ±2.27 N.S. 12.33 17.00 ±1.71 ±3.86 N.S. 14.25 ±0.42 26.00 ±0.71 a Values are means ± S.E., i n minutes from s a c r i f i c e to i n s t a l l a t i o n i n muscle bath b Time i n days post-denervation c Number of normal and denervated animals tested r e s p e c t i v e l y f o r which data i s available * I n s u f f i c i e n t data f o r s t a t i s t i c a l analysis TABLE XII pH of Krebs Solution f o r Experiments on Soleus and EDL Muscles from Normal and Denervated Mice. a SOL EDL Time 0 ( , ) c Norm. Den. ( , ) Norm. Den. 1 P 28 P 84 P 210 P (5.5) 7.64 7.86 ±0.03 ±0.02 5-001 (5,4) 7.64 7.67 ±0.03 ±0.03 N.S. (6.6) 7.65 7-70 ±0.04 ±0.05 N.S. (6,2) 7.74 7.72 ±0.04 ±0.07 * (4,6) 7.73 ±0.03 N.S. (4,3) 7.73 ±0.03 N.S. (6,4) 7.75 ±0.04 N.S. (4,2) 7.63 ±0.03 7.79 ±0.02 7.64 ±0.02 7.69 ±0.05 7-75 ±0.05 a Values are means ± S.E. b Time i n days post-denervation c Number of normal and denervated animals tested respectively f o r which data i s available * Insufficent data for s t a t i s t i c a l analysis 

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