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Changes in motor end-plates resulting from muscle fibre necrosis and regeneration
Journal of the neurological Sciences, 1974,21 : 391--417
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© ElsevierScientificPublishing Company,Amsterdam - Printed in The Netherlands
Changes in Motor End-Plates Resulting from Muscle Fibre Necrosis and Regeneration A Light and Electron Microscopic Study of the Effects of the Depolarizing Fraction (Cardiotoxin) of Dendroaspbjamesoni Venom L. W. D U C H E N , B A R B A R A J. EXCELL, R. PATEL* AND BETTY S M I T H Departments of Neuropathology, Institute of Psychiatry and the Maudsley Hospital, De Crespiony Park, London SE5 8AF and of Physiology, Queen Elizabeth College, Campden Hill Road, London W8 7AH (Great Britain)
(Received 5 September, 1973)
INTRODUCTION The venoms of elapid snakes are mixtures of basic non-enzymic polypeptides which have varying modes of action and varying degrees of toxicity (see Lee 1972). Excell and Patel (1972) and Patel (1972) showed that the venom of Dendroaspisjamesoni could be separated by column chromatography into 14 fractions. One fraction (F8) caused a marked depolarization of frog muscle fibres, soon followed by degenerative changes, when tested in vitro. This fraction had an action similar to the depolarizing fraction (cardiotoxin) of various cobra venoms (Chang and Lee 1966; Earl and Excell 1972). It was of interest to study the in vivo effects of F8 to determine whether this cardiotoxin has a selective action on muscle fibres and whether changes in the morphology of the motor end-plate might occur as a result of a lesion primarily affecting the muscle cell. In particular it was of interest to know whether axonal sprouting could be induced by a necrotizing lesion of the muscle fibre. This paper reports the results of experiments in which a sublethal dose of cardiotoxin (F8) was injected directly into the leg muscles of the mouse and the subsequent degenerative and regenerative changes studied. It was found that the muscle fibres were selectively affected by this toxin leaving nerve terminals intact but that the morphology of the motor end-plates became altered in a characteristic way. A brief account of these findings has been given in a preliminary communication (Duchen, Excell, Patel and Smith 1973). This research was supported by grants from the National Fund for Research into Crippling Diseases, the Muscular Dystrophy Group of Great Britain and the Muscular Dystrophy Associationsof America, Inc. *Wellcome Trust Research Training Scholar. Present address: School of Medicine, Suva, Fiji.
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MATERIALS AND Mt!I'HODS
The venom of D. jamesoni was fractionated by column chromatography on CMSephadex C25 using two-stage gradient elution with phosphate buffer followed by desalting and lyophilization. Fourteen fractions were obtained. F8, the depolarizing (cardiotoxic) fraction, showed two bands on acrylamide gel electrophoresis. The methods of preparation and study of the activity of these fractions have been described in detail by Patel (1972). Toxicity tests of the various fractions showed that the greatest toxicity was present in F9, 10 and 12 and was associated with the postsynaptic (curare-like) block in neuromuscular transmission. The toxicity of F8 was only about one-twentieth that of the whole venom (standardized by the protein content of the fractions) a lethal dose (LDso) for a mouse being approximately 100 #g. It was determined that the venom contained no phospholipase A and that the depolarizing activity of F8 was heat-stable. A single injection of 20 #g of F8 dissolved in physiological saline was given, under ether anaesthesia, into the muscles of the right hindleg of albino mice, A 30 gauge hypodermic needle was introduced through the skin over gastrocnemius and pushed into the anterior compartment of the leg where half the dose of venom was injected. The needle was then slightly withdrawn and the remainder of the dose of venom was injected into the posterior muscles of the leg. More than 60 mice, both male and female, were used and were allowed to survive for varying periods of time ranging from 30 min to 6 months after the single injection of F8. For light microscopy tissues were examined in frozen and paraffin sections. Under ether anesthesia formol-calcium (10'~o formaldehyde in 1'),~i calcium acetate) was perfused through the heart. Serial frozen sections of the leg muscles were cut at 20 #m. These were stained by the method of Koelle and Friedenwald (1949) to demonstrate cholinesterase activity followed by the method of Namba, Nakamura and Grob (1967) using silver impregnation to demonstrate axons. For examination of paraffin-embedded tissues formol-calcium and formol-acetic-methanol (1 part formaldehyde, 1 part glacial acetic acid, 8 parts methanol) were used for fixation by perfusion. The whole limbs were decalcified in formic acid-sodium citrate solution and sectioned in longitudinal or transverse planes. Use of the "serial block" method of embedding tissues (Beesley and Daniel 1956) allowed examination of all muscles of the limb at different levels. Sections were stained with routine histological methods. Palmgren's (1948) method of silver impregnation was used to demonstrate axons in paraffin sections. For electron microscopy mice were anaesthetized with ether and perfused through the left ventricle with cold 3%o glutaraldehyde in phosphate buffer. Blocks were taken from the zones of innervation of soleus, tibialis anterior, extensor digitorium longus (EDL) and the periphery of gastrocnemius. After post-fixation in osmium tetroxide and staining with uranyl acetate, blocks were embedded in epoxy resin. Sections were cut in longitudinal and transverse planes and stained with lead citrate. Many mice given intramuscular injections of saline or phosphate buffer have been studied with both light and electron microscopy and no significant abnormalities found. Since the depolarizing fraction is heat stable and no antiserum was available more specific controls could not be used.
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RESULTS
After recovery from the ether anaesthesia, the animals showed temporary impairment of the use of the right hindleg for several hours and for the next 2 or 3 days the foot could not be used easily during climbing. However, the general condition of the animals was never seriously disturbed, no animals died as a result of the injection of 20/~g of the depolarizing fraction, and within a few days there was no apparent abnormality of movement of the limb.
Light Microscopy Muscle fibres Within 30 min of the injection, which was the shortest survival time studied, there were obvious abnormalities in the leg muscles. These abnormalities did not affect whole muscles and were seen only in irregular patches which affected parts of muscles. All the leg muscles were affected to a greater or lesser extent, and slow and fast muscle fibres seemed to be equally severely affected. The affected regions became very pale and were slightly swollen and very friable. Light microscopy showed that in places the internal structure of the muscle fibres was disorganised (Fig 1). Cross-striations were lost and the myofibrillar material was replaced by densely eosinophilic masses which were either homogeneous or in which a faint fibrillary structure could be seen. These dense masses were often sharply bordered and contiguous with patches of paler-stained segments of muscle fibre. The muscle fibres were not necrotic in their entirety, and areas which looked relatively normal lay
Fig. 1. Longitudinal section of tibialis anterior 1 hr after the local injection of cardiotoxin. Sarcolemmal tubes are intact but the myofibrillar structure is lost and replaced by eosinophilic clots and debris. One muscle fibre looks normal. HE, x 400.
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adjacent to necrotic zones. The sarcolemmal membrane seemed to be always intact so that where myofibrils had been lost the appearance was like that of an empty or fluid-filled tube. Within about 3 hr there was the beginning of an accumulation of polymorphonuclear leucocytes between the necrotic fibres. The inflammatory reaction became steadily more marked and by 24 hr there were masses of leucocytes almost suggesting abscess formation in some areas, particularly in tibialis anterior. By the second day the necrotic muscle fibres were partly filled with rounded mononuclear cells with eosinophilic granular or foamy cytoplasm while between the muscle fibres polymorphonuclear leucocytes were still present (Fig. 2). Patches of amorphous necrotic material lay within the muscle cells. By the third day, little necrotic debris remained within the muscle fibres which were now filled with cells.
Fig. 2. At 48 h soleus muscle fibres contain much necrotic debris and numerous rounded m o n o n u c l c m cells, probably mostly phagocytic in type. HE, x 400.
Many thin fibres with plump central nuclei were present. By the seventh day the continuity of the myofibrillar material was well established in most fibres though many showed areas of pale staining without cross-striations and many fibres containing abundant plump spindle-shaped cells were still present. Basophilia was not a feature of muscle fibres. A striking abnormality from this time onwards was the presence of long rows of nuclei within muscle fibres (Fig. 3). In some of the larger fibres, more than one row of nuclei were present. Transverse sections (Fig. 4) showed a single central nucleus in smaller fibres while in the larger ones several deeplysituated nuclei were seen as well as those normally peripherally placed beneath the sarcolemma. Abnormally situated nuclei were found in the longest surviving animals and seemed to be a permanent abnormality. They were useful as indicators of where necrosis or damage had taken place previously.
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In most muscles regeneration seemed practically complete within 3 weeks. Some focal lesions persisted for longer. These were small rounded granulomatous masses filling and expanding muscle fibres, with a central amorphous haematoxyphilic core surrounded by flattened epithelioid-type cells. Since the tissues had been de-
Fig. 3. Soleus at 7 days shows m a n y muscle fibres with rows of centrally placed nuclei. These are probably fibres which were necrotic and have regenerated. HE, x 400.
Fig. 4. Transverse section of tibialis anterior at 21 days. M a n y of the muscle fibres, particularly the larger ones, contain several internally placed nuclei. A single fibre has not completely regenerated and contains n u m e r o u s small cells. HE, x 400.
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calcified it was n o t certain w h e t h e r calcification h a d o c c u r r e d in some areas o f muscle necrosis but the a p p e a r a n c e did suggest that this had h a p p e n e d . The d i s t r i b u t i o n o f these lesions which persisted for m a n y weeks was consistently limited to regions p r e d o m i n a n t l y c o m p o s e d o f large fast-type muscle fibres, p a r t i c u l a r l y in the periphery o f g a s t r o c n e m i u s a n d in tibialis a n t e r i o r where they were c o m m o n e s t . T h e y were not seen in soleus or in regions o f o t h e r muscles c o m p o s e d o f slow fibres. Motor innervation
In all the a n i m a l s studied with either light or electron m i c r o s c o p y it c o u l d be o b s e r v e d that muscles h a d been affected in a p a t c h y irregular m a n n e r . This m e a n t that the necrosis involved some muscles in their zones o f i n n e r v a t i o n a n d o t h e r muscles in the end-plate-free areas, a n d the d i s t r i b u t i o n a n d the extent o f necrosis was never exactly the same. In order, therefore, to assess the l o n g - t e r m effects o f the d e p o l a r i z i n g fraction on i n n e r v a t i o n it was necessary to k n o w where the muscle
Fig. 5. Longitudinal frozen sections of gastrocnemius (,4) and tibialis anterior (B) of a normal rnoll~,, stained by the method of Koelle and Friedenwald to show cholinesterase activity, and by the silver imprcs,nation method of Namba et al., to demonstrate axons. Each preterminal axon (arrows) innervates :~ end-plate in which the cholinesterase reaction product is localized in the thick continuous branchhl~, gutters of the subneural apparatus, x 640.
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fibres had been necrotic at an early stage. This could be done without difficulty since the central nuclei, which indicated previous necrosis or damage to muscle fibres, seemed to remain permanently. The assessment of the morphology of endplates was therefore always done in areas where was either acute necrosis or, in the longer-surviving animals, signs of earlier damage. Areas with central nuclei could be identified in frozen sections taken at representative levels counter-stained with haematoxylin after the cholinesterase reaction and silver impregnation were completed. The preterminal myelinated motor axons and the axon terminals were studied in frozen and paraffin silver-impregnated sections. The normal appearance is shown in Fig. 5. At no stage in the experiment was there evidence of axonal damage or fragmentation. During the acute phase of muscle necrosis, intact axons lay among the necrotic muscle fibres and axonal terminals could be seen innervating these fibres. Although there was a disturbance of the normal orderly architecture of the tissues the terminals looked normal. Cholinesterase activity was demonstrable, even when the underlying muscle fibre was necrotic (Fig. 6). The intensity of cholinesterase activity was reduced for several days and the localization of the reaction product was not in the usual well-defined gutters, but in rather poorly-defined thin lines or smudges (Fig. 7). During the first 3 or 4 days many end-plates were irregularly shaped and oblong rather than rounded or oval in outline as in the normal. Occasionally, particularly in tibialis anterior, where granulomatous lesions often persisted in the necrotic muscle fibres, there were ultraterminal axonal sprouts. These sprouts were not found consistently and were quite unlike the sprouting previously found to be associated with a presynaptic block of transmission. (Duchen 1970; Duchen and Tonge 1973). Rather were they like the occasional ultraterminal axonal projection seen during the re-innervation of muscle after a peripheral nerve lesion.
Fig. 6. Tibialis anterior 24 hr after the injection of cardiotoxin. Axons are intact and the cholinesterase reaction product is visibleat the end-plates although the muscle fibres are necrotic. Method as in Fig. 5. x 400.
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Fig. 7. Gastrocnemius at 3 days shows normal-looking preterminal axons but weaker than normal cholinesterase activity. The reaction product is localized in thin lines or poorly defined smudges. Muscle fibres are severely disorganized. Method as in Fig. 5. x 640.
Fig. 8. In tibialis anterior at 8 weeks the cholinesterase reaction product at this end-plate is localized in numerous sub-units instead of in continuous gutters. The sites of cholinesterase activity seem to lie at many points along each axon terminal. Method as in Fig. 5. x 640.
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After the 10th day, when regeneration of muscle fibres was well advanced, the cholinesterase reaction at the end-plates became stronger and better-defined. However, from this time onwards the end-plates were seen as collections of "sub-units" showing strong cholinesterase activity arranged along the ramifications of the axon terminals (Fig. 8). In some end-plates, these "sub-units" looked like beads strung along the axon terminal with non-reactive areas between them. In other end-pJates the areas showing cholinesterase activity were located at the tips of the branches of axonal terminals giving the end-plates an "exploded" appearance (Fig. 9). In
Fig. 9. End-plates in tibialis anterior at 10 weeks remain abnormal in appearance. They appear expanded and each consists of an aggregation of spots or rings (subunits) of cholinesterase reaction product. M a n y of these subunits appear to be located at the tips of branches of axon terminals. Method as in Fig. 5. x 400.
soleus (Fig. 10) where end-plates are normally small, the aggregations of sub-units were usually compact while in tibialis anterior and gastrocnemius (Fig. 10) where end-plates are normally larger the clusters of sub-units were quite widely spread. Axon terminals seemed longer and more branched than usual. In many end-plates there was no cholinesterase activity for a short distance along the axon terminals from the point of branching of the preterminal axon (Figs. 9 and 10). Each aggregation of sub-units was therefore larger in area than a normal end-plate. It was clear, however, that each cluster of sub-units belonged to a single preterminal axon and was localized to one muscle fibre. There were no long axonal sprouts leading from one end-plate to new terminals on other muscle fibres. Further, although no quantitative estimation was made of the "terminal innervation ratio" (CoOrs and Woolf
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I-:ig. 10. End-plates at 4 months in the superficial region of gastrocnemius (A) where muscle fibres arc mainly fast in type and soleus (B) which consists of slow fibres, show similar morphological abnormalities although they differ in size. Method as in Fig. 5. x 400.
1959) it seemed that the basic pattern of motor innervation of muscles remained normal with a single preterminal myelinated axon innervating a single muscle fibre and scarcely any preterminal axonal branching being present. The abnormal morphology of the motor end-plates was found in all animals studied including those allowed to survive for 6 months or more. Abnormal endplates were found only in areas where there was evidence of previous damage and regeneration of muscle fibres. Where end-plates were normal in appearance the muscle fibres invariably showed no local evidence of previous damage, a finding which also applies to the electron microscopic studies.
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Electron Microscopy Muscle fibres The shortest time interval between the injection of cardiotoxin and fixation of tissues for electron microscopy was 30 min. Already by this time severe lesions were present within fibres of all the muscles examined (soleus, tibialis anterior, E D L and gastrocnemius). Slow and fast types of muscle fibre were equally severely affected. It is difficult to be certain of the exact sequence of events leading to the total disintegration of the internal structure of muscle fibres which was seen. One early change was the loss of Z-line structure while the orientation of thick and thin filaments was maintained (Fig. 11). In other muscle fibres the only change noted was the dilatation of the tubules of the sarcoplasmic reticulum. More severe lesions consisted in the disorganization of the myofilaments which were still recognizable and showed the remnants of a fibrillar arrangement. Many muscle fibres showed a total disruption of their internal structure. There were masses of a m o r p h o u s material in irregular clots, patches of clear, probably fluid-filled, spaces and dissolution of myofilaments. Mitochondria were swollen and their cristae irregularly condensed. In fast muscle fibres (such as gastrocnemius) masses of fragments of tubules were found. Muscle fibres were not affected in their entirety. In many instances there was a sharp demarcation between the part of the cell which was necrotic and the part remaining structurally intact. In other muscle fibres there was a transitional
Fig. 11. This muscle fibre from the superfical region of gastrocnemius at 5 hr after the injection of the cardiotoxin shows loss of Z-lines, the sites of which are shown by arrows. Myofilamentsand their fibrillar arrangement and M-lines are preserved. × 30,000.
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zone in which the arrangement of filaments was distorted, and the orderly transverse alignment of Z-lines lost. In all the necrotic muscle fibres the limiting basement membrane was intact. In many places the sarcolemmal membrane was absent so that the contents of the fibre (fluid, vesicles, clots of amorphous debris and remnants of tubules and mitochondria) were bounded and held in place by the basement membrane only (Fig. 12).
Fig. 12. In this transverse section of soleus at 30 min after the injection of cardiotoxin parts of two necrotic muscle fibres are shown. They contain debris, abnormal mitochondria and fragments of tubules. At some places (arrow) the sarcolemmal membrane is lost and only the basement membrane remains intact. × 15,000.
Within a few hours leucocytes were found adhering to the necrotic muscle fibres. By 2-3 days the sarcolemmal tubes were packed with cells which had the characteristics of macrophages - - dense nuclei, abundant rough endoplasmic reticulum, numerous "ruffled" cytoplasmic processes interdigitating with processes from adjacent cells, and phagosomes and abundant dense granular material, lipid droplets and vacuoles. In addition to these phagocytic cells there were others inside the sarcolemmal tubes with less dense nuclei and cytoplasm which contained myofilaments and which were obviously muscle-forming cells. Regeneration proceeded rapidly and by the 7th day most muscle fibres were composed of cells in which myofilaments were abundant though in places they were not organized in sarcomeres. During the regenerative phase, the sarcolemmal tubes could be identified, particularly in transverse sections, by the basement membrane which encircled and enclosed the clusters of myocytes and phagocytic cells. In many places the myocytes themselves were
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enclosed in a layer of basement membrane which was a branch of the outer membrane. In other places the myocytes were very closely apposed without any intervening basement membrane and in some places these cells seemed to be fusing together. A common finding at 7 days was the presence o f dense rod-like bodies of varying length and width composed of Z-line material. These bodies were usually seen where myofilaments were not arranged in parallel and where fibrils were not properly formed. Thin filaments were often seen attached to these bodies. In addition, dense bodies of Z-line material were occasionally found in places where fibrils and sarcomeres were well formed. During the stage of muscle regeneration another common finding was the presence of numerous dense rounded bodies beneath the sarcolemmal basement membrane (Fig. 13). These seemed most likely to be autophagic
Fig. 13. Regenerating muscle fibres at 3 days are very irregular in outline as shown in Xhis longitudinal section of a soleus fibre in which the surface is cut tangentially at m a n y places. Dense rounded bodies lie beneath the basement m e m b r a n e and m a y be lysosomes derived from within the muscle fibre, x 25,000.
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Fig. 14. A longitudinal section of extensor digitorum longus at 30 min after the injection of cardiotoxin shows that the internal structure of this muscle fibre is severely disrupted. Myofilaments are replaced by dense a m o r p h o u s clots and degenerating mitochondria, fragments of tubules, debris and nuclei remain within the muscle fibre. Axon terminals (arrows) are however normal in appearance and post-synaptic folds of the sarcolemmal m e m b r a n e are seen. x 10,000.
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vacuoles or lysosomes discharged from the interior of the muscle fibre. In the animals surviving for much longer periods there were abnormalities of muscle fibres which seemed to be permanent, as already noted with light microscopy. Centrally-placed nuclei and disarray of myofibrils with the formation of striated annulets ("Ringbinden"), were common findings. These abnormally-orientated myofibrils were found not only at the periphery of the muscle fibres where they formed a complete or partial ring, but also extended into the interior of the fibre so that longitudinally-orientated fibrils lay external to them.
Motor end-plates During the acute phases (30 min to 2 hr after the injection of cardiotoxinj when the muscle fibres became necrotic, axon terminals were normal in appearance (Fig. 14). They contained abundant mitochondria and vesicles and lay in close contact with the synaptic surface of the sarcolemmal membrane. At no time was there any evidence of axonal disintegration. The post-synaptic folds of the muscle fibre were normal in their appearance even though the internal structure of the fibre was totally disrupted. In these places the post-synaptic folds appeared to be suspended in fluidfilled pools (Fig. 15). The basement membrane material which normally fills the synaptic space and folds was identifiable and normal in appearance. At later stages the post-synaptic sarcolemmal membrane was often absent (Fig. 16), just as parts of the extra-junctional sarcolemma were lost wherever there was muscle necrosis. Post-synaptic folds were then lost and their position was marked only by the persistence of the basement membrane material which at first could be seen projecting into the muscle cell, but later, still forming an intact layer, became folded or rolled up on itself at the sites of the subneural folds. During the next few days when the muscle fibres were filled with phagocytic cells and regenerating myocytes the outline of the fibres became very irregular and the electron microscopic appearances very complex indeed. Motor nerve terminals were always of normal appearance and contained normal-looking vesicles and other organelles. The axonal terminals were now wrapped in Schwann cell processes and the axolemmal membrane made only irregular contacts with the basement membrane of the muscle fibre. The subneural basement membrane often showed patchy thickening or duplication, probably marking the sites of the original post-synaptic folds (Fig. 17). Within the confines of the muscle fibre i.e. within the basement membrane, there was often no muscle structure to be seen in the subneural position and the nerve terminals lay over the intracellular phagocytes with only the basement membrane of the muscle fibre between these cells and the axons (Fig. 18). At 7-14 days when the regenerative changes were well advanced axon terminals were enclosed within Schwann cell cytoplasm in some places while in other places on the same muscle fibre they lay in contact with the sarcolemmal basement membrane, an abnormal nerve-muscle relationship which persisted in the motor endplates of both slow and fast types of muscle fibre. Close contacts between different parts of the axon terminal and its muscle fibre were separated from each other by regions where the axon was wrapped in Schwann cell (Fig. 19). Another frequent finding was that of axon terminals enclosed within "tunnels" formed by sleeves of
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Fig. 15. At 30 min this muscle fibre (M) is severely disorganized but the axon terminal (A) is normal in appearance. It contains abundant synaptic vesicles and mitochondria which are structurally normal in contrast with the very abnormal mitochondria within the muscle cell. The post-synaptic folds (arrows) are present and the sarcolemmal membrane which forms them is intact, x 35,000
s a r c o l e m m a p r o j e c t i n g into the muscle fibre (Fig. 20). In these places the terminal, cut transversely or obliquely, was s u r r o u n d e d by the c y t o p l a s m o f the muscle cell. A x o n t e r m i n a l s lying in " t u n n e l s " are only o c c a s i o n a l l y seen in the n o r m a l a n d then usually only in o b v i o u s l y tangential sections o f muscle fibres. M a n y o f the nerve-muscle j u n c t i o n s showed a p a u c i t y o r absence o f s u b n e u r a l folds o f the post-
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Fig. 16. At this end-plate in tibialis anterior 2 hr after the injection of cardiotoxin the axon terminal (A) contains abundant vesicles and mitochondria while the underlying muscle fibre (M) contains only debris in this region. The post-synaptic sarcolemmal membrane seems to be deficient at some places (arrows) so that the structure of the subneural folds is not as well maintained as at earlier stages (cf. Fig. 15). This is similar to the loss o f sarcolemma in extra-junctional regions (cf. Fig. 12). x 30,000.
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Fig. 17. At 3 days an axonal terminal (A) is partially enclosed within Schwann cell processes (S). These interpose between axon and basement membrane of the synaptic cleft. The basement membrane projects into the "muscle fibre" in a series of thickenings (arrows) which mark the sites of post-synaptic folds from which the sarcolemmal m e m b r a n e has been lost. The cell in the subneural position is probably phagocytic in type. x 30,000.
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Fig. 18A. In this transverse section of a soleus fibre 3 days after the injection of cardiotoxin axon terminals (A) lie in contact with the basement m e m b r a n e of the muscle fibre but in the subneural position the cells have the characteristics of phagocytes. One cell (M) contains scattered myofilaments, x 15,000.
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Fig. 18B. Detail of fig. 18A showing an axon terminal (A) of normal appearance and containing a b u n d a n t vesicles and mitochondria. The axon lies in contact with the basement membrane of the muscle fibre. No sarcolemmal m e m b r a n e is seen and the cell beneath the axon is phagocytic in type. × 45,000
synaptic sarcolemmal membrane, both during early stages of muscle regeneration as well as long after regeneration was complete (Fig. 21). Irregular gaps and processes of Schwann cell lying between axolemma and sarcolemma were also frequently seen. Since these abnormalities were found in the animals allowed to survive for 6 months or more it seems that these end-plates remain permanently abnormal particularly in the deficiency of subneural folds. DISCUSSION
The cardiotoxic fraction of the venom ofD. jamesoni is free of post-synaptic blocking action and is free of phospholipase A activity since the whole venom is known to be free of this enzyme (Excell and Patel 1972; Patel 1972). The effects of F8 seem to be due to its highly selective action and, since the muscle necrosis was confined to the region of the injection with individual muscles involved to a varying extent,
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Fig. 19. At 7 days in soleus close contacts between axon (A) and regenerating muscle fibre (M) are shorter than normal. Part of the axon is wrapped in Schwann cell processes, x 30,000.
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Fig. 20. At 17 days in soleus an axon terminal lies in a "tunnel" of sarcolemma. Subneural folds are not seen. Expansion of the regenerating muscle fibre around the intact axon terminals may be the way in which this type of abnormal neuromuscular junction develops. × 30,000.
it is probable that the venom was rapidly bound to receptor sites and did not diffuse widely throughout the leg muscles. Muscle necrosis has been caused in experimental animals by numerous methods including thermal injury (Price, Howes and Blumberg 1964; Reznik and Engel 1970), crush injury (Allbrook 1962), the injection of alcohol and oil (Walton and Adams 1956), the injection of Clostridium welchii toxins (Aikat and Dible' 1956) as well as by the injection of the venoms of cobra (Stringer, Kainer and Tu 1971) and rattlesnake (Stringer, Kainer and Tu 1972). Muscle necrosis in man has also been observed after bites by the sea snake Enhydrina schistosa (Marsden and Reid 1961). The acute necrosis of muscle fibres seems to be similar in appearance whatever its method of production although the lesions caused by rattlesnake venom
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Fig. 21. This motor end-plate in soleus at 4 months remains abnormal in that the lengths of close synaptic contact are short and that post-synaptic folds of sarcolemmal membrane are few and poorly formed. x 30,000. in wh i ch the c r u d e w h o l e v e n o m was used {Stringer et al. 1972), were described as h a e m o r r h a g i c a n d there was also d e s t r u c t i o n o f the b a s e m e n t m e m b r a n e o f m u s c l e fibres, a b n o r m a l i t i e s w h ic h were n o t f o u n d in the p r e s e n t studies. T h e a p p e a r a n c e
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of the muscle fibres during the regenerative stages do not seem different from those described by many other authors (see Mauro, Shafiq and Milhorat 1970) and familiar problems arise in the identification and nomenclature of individual cells within regenerating damaged muscle fibres. There is no doubt however that at some stages during regeneration the cells lying immediately beneath the axon terminals, separated from them by basement membrane, are phagocytic in type as shown by their abundant rough endoplasmic reticulum, numerous Golgi apparatuses, phagosomes and complex "ruffled" cytoplasmic margins. It would be of interest to know whether during this stage of regeneration, the axon terminals, which are morphologically normal, contain and release transmitter and whether the muscle cell membrane is capable of response. Although some attention has been given to the influence of the motor innervation on the capacity for injured fibres to regenerate (Walton and Adams 1956) the effects of muscle injury on its innervation has been neglected. This neglect may have been due to the fact that trauma (crushing) or thermal injury may damage axons as well as muscle fibres. It was therefore of particular interest to determine whether cardiotoxin had any effect on axons and their terminals. Physiological studies on the depolarizing (cardiotoxic) fractions obtained from the venoms of both D. jamesoni (Patel 1972) and Naja nivea (Earl and Excell 1972) gave no evidence for any depolarization of the nerve terminals which would have been shown by a rise in the frequency of miniature end-plate potentials due to an outpouring of transmitter, at least prior to muscle depolarization in the frog. However, in this species, Chang and Lee (1966) showed that the cardiotoxic fraction from Naja naja atra venom caused a loss of conduction in the axons up to the terminals concomitant with severe muscle depolarization, indicating that some degree of axonal depolarization may be present. They suggest that this effect of cardiotoxin may be responsible for the reduction in acetylcholine output from rat phrenic nerve terminals in the presence of the crude venom (Su, Chang and Lee 1967). Mammalian nerve trunks are relatively resistant to block and in/n vitro experiments Chang, Chuang, Lee and Wei (1972) found that a minimum concentration of 100 /~g/ml cardiotoxin was required to block axonal conduction in the rat phrenic nerve trunk. In the present in vivo experiments a dose of 20 #g of cardiotoxin was injected into the mice, a dose selected to cause only local changes and to permit the study of the long term effects. Although an acute disturbance of axonal function near the terminals cannot be ruled out in these experiments the structure of pre-terminal myelinated axons and motor nerve terminals was normal in appearance. However no electrophysiological studies have been done in animals which have survived for any length of time and the characteristics of neuromuscular transmission are not known after regeneration is complete but end-plates are still abnormal in appearance. The changes in the end-plates occur in the following sequence. First there is the disorganization of the contents of the muscle cell and its resultant collapse. It seems that Schwann cells then extend down and enclose the axon terminals, due perhaps to loosening of nerve-muscle junctions since the surface of the muscle fibre becomes very irregular at his stage. The axon terminals probably increase in length and while regeneration of the muscle fibre is occurring, so that it re-expands as its content of
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myofilaments increases again, the contact between axon and sarcolemmal membrane becomes limited to short intermittent lengths. These small areas of close (synaptic) contact are separated from each other by parts of the axon terminalwhich are wrapped in Schwann cell processes and do not make synaptic contact. The normally smooth continuous contact between axon terminal and muscle fibre thus becomes broken up into a series of small synaptic sub-units. Each aggregation of sub-units is, however, confined to a single muscle fibre and is innervated by a single pre-terminal myelinated axon which is not branched. From the findings in the present studies it is possible to understand how the postsynaptic folds of the sarcolemma are lost during the acute stage of muscle necrosis. The reason why folds are not formed again after muscle regeneration is not clear. Previous studies of the formation of new end-plates in regenerating limb muscles of the newt (Lentz 1969) and after recovery from the effects of botulinum and tetanus toxins (Duchen 1971, 1973) showed that post-synaptic folds developed as the new nerve-muscle contacts matured over several weeks. In the present experiments it seems that there is merely a reorganization of the structure of the end-plates and that no new end-plates are formed in response to the disruption of the internal structure of the muscle fibres. The finding that end-plate abnormalities occur as a consequence of a lesion primarily affecting the muscle cell may be relevant to the study of the innervation of muscle in human diseases. CoErs (1955) and Co&s and Woolf (1959) showed that the end-plates were abnormal in patients with various types of myopathy (i.e. diseases considered to affect the muscle fibre primarily). In particular, complex spread-out terminal axonal arborizations were described. CoErs (1955) pointed out that the neuronal changes remained limited to the most distal part of the axon and that there was no excessive pre-terminal axonal branching. Morphologically abnormal end-plates have been described in cases of myasthenia gravis (see Woolf 1969). In this disease, known to be associated with a disorder of neuromuscular transmission, the end-plates were observed to be elongated but there was also much pre-terminal axonal branching. Abnormalities of the end-plates in the mouse were also found after recovery from the effects ofbotulinum toxin (Duchen and Strich 1968a; Duchen 1970, 1971) and tetanus toxin (Duchen and Tonge 1973) which are both known to cause a pre-synaptic block of transmission. In these experimental conditions new nerve-muscle junctions are formed as a result of sprouting from axonal terminals and are seen eventually as sub-units of varying shape and size scattered along the muscle fibres or spread over several adjacent fibres with abundant pre-terminal axonal branching. Studies of the innervation of muscle in motor neurone disease, poliomyelitis and peripheral neuropathies in man (Co6rs 1955; Co6rs and Woolf 1959) and in motor neurone disease in the mouse (Duchen and Strich 1968b) have shown that in all these conditions, in which muscle is partially denervated, pre-terminal branching of surviving axons is marked. It thus seems that pre-terminal branching, whatever the mechanism is by which it develops, is a constant histological feature of disease of the lower motor neurone whether primarily affecting the perikaryon of the anterior horn cell or the terminals of its axon. The findings in the present experiments show how abnormalities in the
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morphology of end-plates develop in response to a lesion primarily affecting the muscle cell and also show that axonal sprouting is not induced by this type of lesion. SUMMARY
The venom of Dendroaspisjamesoni (Jameson's mamba) was fractionated by column chromatography. One of the fractions (F8) caused rapid depolarization of muscle fibres when tested in vitro, an action similar to that of "cardiotoxins" of other snake venoms. A single injection of 20 #g of the depolarizing fraction was made directly into the muscles of one hindleg in mice. Light and electron microscopic studies were made of muscle fibres and motor end-plates of animals allowed to survive from 30 min to 6 months after the injection. The depolarizing fraction caused rapid and severe disorganization of the internal structure of muscle fibres affecting slow and fast types of fibre equally severely. Muscle fibres were affected in segments and not along their entire length. Early changes were the disappearance of Z-lines, the dissolution of myofilaments and degenerative changes in mitochondria, but the basement membranes of the muscle fibres remained intact. Regeneration of muscle took place rapidly and was usually complete within 2 to 3 weeks but centrally-placed nuclei, rows of nuclei and malorientation of myofibrils including the formation of striated annulets remained as permanent abnormalities and were used as indicators of where necrosis had occurred. Myelinated axons and motor nerve terminals were normal in appearance and contained abundant vesicles and mitochondria even when the muscle fibres they innervated were necrotic. The axonal terminals became enclosed within Schwann cell processes. After muscle regeneration was complete the end-plate morphology remained abnormal. With light microscopy the end-plates were seen as aggregations of sub-units, each aggregation being limited to a single muscle fibre and innervated by a single pre-terminal myelinated axon. The electron-microscopic appearance of the motor end-plates also remained abnormal and there was a striking absence of post-synaptic folds of the sarcolemmal membrane. The observations made in these experiments indicate that the cardiotoxin selectively damages muscle fibres leaving the motor innervation structurally intact and also show how morphological abnormalities may develop in motor end-plates as a result of a primary lesion of the muscle fibre. The appearances are similar to those occurring in the innervation of muscle in human myopathies. The absence or branching of preterminal axons and of sprouting from axon terminals is in contrast to the changes found in human and experimental conditions in which there is known to be a disorder of the lower motor neurone. REFERENCES AIKAT, B. K. AND J. H. DIBLE (1956) The pathology of Clostridium welchii infection, J. Path. Bact., 71 : 461~76. ALLBROOK, D. (1962) An electron microscopic study of regenerating skeletal muscle, J. Anat. ( L o n d . 96:137-152. BEESLEY,R. A. AND P. M. DANIEL (1956) A simple method for preparing serial blocks of tissue, J c/ill Path., 9: 267-268.
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CHANG, C. C. AND C. Y. LEE (1966) Electrophysiological study of neuromuscular blocking action of cobra neurotoxin, Brit. J. Pharmac. Chemother., 28 : 172-181. CHANG, C. C., S-T. CHUANG, C. Y. LEE AND J. W. WEI (1972) Role of cardiotoxin and phospholipase A in the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom, Brit. J. Pharmac. Chemother., 44: 752-764. COORS, C. (1955) Les variations structurelles normales et pathologiques de la jonction neuromusculaire, Acta neurol.psychiat, belg., 55: 741-866. COERS, C. AND A. L. WOOLF (1959) The Innervation of Muscle, Blackwell, Oxford. DUCHEN, L. W. (1970) Changes in motor innervation and cholinesterase localization induced by botulinum toxin in skeletal muscle of the mouse: differences between fast and slow muscles, J. Neurol. Neurosur#. Psychiat., 33: 40-54. DUCHEN, L. W. (1971) An electron microscopic study of the changes induced by botulinum toxin in the motor end-plates of slow and fast skeletal muscle fibres of the mouse, J. neurol. Sci., 14: 47-60. DUCHEN, L. W. (1973) The effects of tetanus toxin on the motor end-plates of the mouse: an electron microscopic study, J. neurol. Sci., 19: 153-167. DUCHEN,L. W. AND SABINAJ. STRICH(1968a) The effects of botulinum toxin on the pattern of innervation of skeletal muscle in the mouse, Quart. J. exp. Physiol., 53: 84-89. DUCHEN, L. W. AND SABINAJ. STRICH (1968b) An hereditary motor neurone disease with progressive denervation of muscle in the mouse: the mutant "wobbler", J. Neurol. Neurosurg. Psychiat., 31 : 535-542. DUCHEN, L. W. AND D. A. TONGE (1973) The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor end-plates in slow and fast skeletal muscle of the mouse, J. Physiol. (Lond.), 228: 157-172. DUCHEN, L. W., BARBARAJ. EXCELL, R. PATELAND BETTY SMITH (1973) Light and electron microscopic changes in mouse muscle fibres and motor end-plates caused by the depolarizing fraction (cardiotoxint of the venom of Dendroaspisjamesoni, J. Physiol. (Lond.), 234: 1-2P. EARL, JANET E. AND BARBARAJ. EXCELL(1972) The effect of toxic components ofNaja nivea (Cape cobra~ venom on neuromuscular transmission and muscle membrane permeability, Comp. Biochem. Physiol.. 41A: 597-615. EXCELL,BARBARAJ. AND R. PATEL (1972) Characterization of toxic fractions from Dendroaspis jamesoni venom, J. Physiol. (Lond.), 225: 29-30P. KOELLE, G. B. AND J. S. FRIEDENWALD (1949) A histochemical method for localizing cholinesterase activity, Proc. Soc. exp. Biol. (N.Y.), 70: 617-622. LEE, C. Y. (1972) Chemistry and pharmacology of polypeptide toxins in snake venoms, Ann. Rev. Pharmacol., 12: 265-286. LENTZ, T. L. 0969) Development of the neuromuscular junction, J. Cell Biol., 42: 431~43. MARSDEN,A. T. H. AND H. A. REID (1961) Pathology of sea-snake poisoning, Brit. reed. J., i: 1290-1293. MAURO, A., S. A. SHAFIQAND A. T. MILHORAT(Eds.) (1970) Regeneration of Striated Muscle and Myogenesis (Proceedings of an International Conference held under auspices of the Muscular Dystrophy Associations of America, New York, 1969) (International Congress Series, No. 218), Excerpta Medica, Amsterdam. NAMBA, T., T. NAKAMURAAND D. GROB (1967) Staining for nerve fiber and cholinesterase activity in fresh frozen sections, Amer. J. clin. Path., 47: 74-77. PALMGREN, A. (1948) A rapid method for selective silver staining of nerve fibres and nerve endings in mounted paraffin sections, Acta. zool. (Stockh.), 29: 377-392. PATEL, R. (1972) An Investigation of the Neurotoxic Components of the Venom of Dendroaspis jamesoni (Jameson's mamba), Ph. D. thesis, London University. PRICE, H. M., E. L. HOWES AND J. M. BLUMBERG(1964) Ultrastructural alterations in skeletal muscle fibres injured by cold, Lab. Invest., 13: 1264-1278. REZNIK, M. AND W. K. ENGEL (1970) Ultrastructural and histochemical correlations of experimental muscle regeneration, J. neurol. Sci., 11: 167-185. STRINGER, J. M., R. A. KAINERAND A. T. TU (1971) Ultrastructural studies of myonecrosis induced by cobra venom in mice, Toxicol. appl. Pharmacol., 18: 442-450. STRINGER, J. M., R. A. KAINERAND A. T. TU (1972) Myonecrosis induced by rattlesnake venom, Amer. J. Path., 67: 127-136. Su, C., C. C. C8ANG AND C. Y. LEE (1967) Pharmacological properties of the neurotoxin of cobra venom. In: F. E. RUSSELLAND P. R. SAUNDERS(Eds.) Animal Toxins, Pergamon, Oxford, pp. 259-267. WALTON, J. N. AND R. D. ADAMS(1956) The response of the normal, the denervated and the dystrophic muscle-cell to injury, J. Path. Bact., 72: 273-298. WOOLF, A. L. (1969) Pathological anatomy of the intramuscular nerve endings. In: J. N. WALTON (Ed.) Disorders of Voluntary Muscle, 2nd edition, Churchill, London, pp. 203-237.
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Changes in Motor End-Plates Resulting from Muscle Fibre Necrosis and Regeneration A Light and Electron Microscopic Study of the Effects of the Depolarizing Fraction (Cardiotoxin) of Dendroaspbjamesoni Venom L. W. D U C H E N , B A R B A R A J. EXCELL, R. PATEL* AND BETTY S M I T H Departments of Neuropathology, Institute of Psychiatry and the Maudsley Hospital, De Crespiony Park, London SE5 8AF and of Physiology, Queen Elizabeth College, Campden Hill Road, London W8 7AH (Great Britain)
(Received 5 September, 1973)
INTRODUCTION The venoms of elapid snakes are mixtures of basic non-enzymic polypeptides which have varying modes of action and varying degrees of toxicity (see Lee 1972). Excell and Patel (1972) and Patel (1972) showed that the venom of Dendroaspisjamesoni could be separated by column chromatography into 14 fractions. One fraction (F8) caused a marked depolarization of frog muscle fibres, soon followed by degenerative changes, when tested in vitro. This fraction had an action similar to the depolarizing fraction (cardiotoxin) of various cobra venoms (Chang and Lee 1966; Earl and Excell 1972). It was of interest to study the in vivo effects of F8 to determine whether this cardiotoxin has a selective action on muscle fibres and whether changes in the morphology of the motor end-plate might occur as a result of a lesion primarily affecting the muscle cell. In particular it was of interest to know whether axonal sprouting could be induced by a necrotizing lesion of the muscle fibre. This paper reports the results of experiments in which a sublethal dose of cardiotoxin (F8) was injected directly into the leg muscles of the mouse and the subsequent degenerative and regenerative changes studied. It was found that the muscle fibres were selectively affected by this toxin leaving nerve terminals intact but that the morphology of the motor end-plates became altered in a characteristic way. A brief account of these findings has been given in a preliminary communication (Duchen, Excell, Patel and Smith 1973). This research was supported by grants from the National Fund for Research into Crippling Diseases, the Muscular Dystrophy Group of Great Britain and the Muscular Dystrophy Associationsof America, Inc. *Wellcome Trust Research Training Scholar. Present address: School of Medicine, Suva, Fiji.
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MATERIALS AND Mt!I'HODS
The venom of D. jamesoni was fractionated by column chromatography on CMSephadex C25 using two-stage gradient elution with phosphate buffer followed by desalting and lyophilization. Fourteen fractions were obtained. F8, the depolarizing (cardiotoxic) fraction, showed two bands on acrylamide gel electrophoresis. The methods of preparation and study of the activity of these fractions have been described in detail by Patel (1972). Toxicity tests of the various fractions showed that the greatest toxicity was present in F9, 10 and 12 and was associated with the postsynaptic (curare-like) block in neuromuscular transmission. The toxicity of F8 was only about one-twentieth that of the whole venom (standardized by the protein content of the fractions) a lethal dose (LDso) for a mouse being approximately 100 #g. It was determined that the venom contained no phospholipase A and that the depolarizing activity of F8 was heat-stable. A single injection of 20 #g of F8 dissolved in physiological saline was given, under ether anaesthesia, into the muscles of the right hindleg of albino mice, A 30 gauge hypodermic needle was introduced through the skin over gastrocnemius and pushed into the anterior compartment of the leg where half the dose of venom was injected. The needle was then slightly withdrawn and the remainder of the dose of venom was injected into the posterior muscles of the leg. More than 60 mice, both male and female, were used and were allowed to survive for varying periods of time ranging from 30 min to 6 months after the single injection of F8. For light microscopy tissues were examined in frozen and paraffin sections. Under ether anesthesia formol-calcium (10'~o formaldehyde in 1'),~i calcium acetate) was perfused through the heart. Serial frozen sections of the leg muscles were cut at 20 #m. These were stained by the method of Koelle and Friedenwald (1949) to demonstrate cholinesterase activity followed by the method of Namba, Nakamura and Grob (1967) using silver impregnation to demonstrate axons. For examination of paraffin-embedded tissues formol-calcium and formol-acetic-methanol (1 part formaldehyde, 1 part glacial acetic acid, 8 parts methanol) were used for fixation by perfusion. The whole limbs were decalcified in formic acid-sodium citrate solution and sectioned in longitudinal or transverse planes. Use of the "serial block" method of embedding tissues (Beesley and Daniel 1956) allowed examination of all muscles of the limb at different levels. Sections were stained with routine histological methods. Palmgren's (1948) method of silver impregnation was used to demonstrate axons in paraffin sections. For electron microscopy mice were anaesthetized with ether and perfused through the left ventricle with cold 3%o glutaraldehyde in phosphate buffer. Blocks were taken from the zones of innervation of soleus, tibialis anterior, extensor digitorium longus (EDL) and the periphery of gastrocnemius. After post-fixation in osmium tetroxide and staining with uranyl acetate, blocks were embedded in epoxy resin. Sections were cut in longitudinal and transverse planes and stained with lead citrate. Many mice given intramuscular injections of saline or phosphate buffer have been studied with both light and electron microscopy and no significant abnormalities found. Since the depolarizing fraction is heat stable and no antiserum was available more specific controls could not be used.
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RESULTS
After recovery from the ether anaesthesia, the animals showed temporary impairment of the use of the right hindleg for several hours and for the next 2 or 3 days the foot could not be used easily during climbing. However, the general condition of the animals was never seriously disturbed, no animals died as a result of the injection of 20/~g of the depolarizing fraction, and within a few days there was no apparent abnormality of movement of the limb.
Light Microscopy Muscle fibres Within 30 min of the injection, which was the shortest survival time studied, there were obvious abnormalities in the leg muscles. These abnormalities did not affect whole muscles and were seen only in irregular patches which affected parts of muscles. All the leg muscles were affected to a greater or lesser extent, and slow and fast muscle fibres seemed to be equally severely affected. The affected regions became very pale and were slightly swollen and very friable. Light microscopy showed that in places the internal structure of the muscle fibres was disorganised (Fig 1). Cross-striations were lost and the myofibrillar material was replaced by densely eosinophilic masses which were either homogeneous or in which a faint fibrillary structure could be seen. These dense masses were often sharply bordered and contiguous with patches of paler-stained segments of muscle fibre. The muscle fibres were not necrotic in their entirety, and areas which looked relatively normal lay
Fig. 1. Longitudinal section of tibialis anterior 1 hr after the local injection of cardiotoxin. Sarcolemmal tubes are intact but the myofibrillar structure is lost and replaced by eosinophilic clots and debris. One muscle fibre looks normal. HE, x 400.
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adjacent to necrotic zones. The sarcolemmal membrane seemed to be always intact so that where myofibrils had been lost the appearance was like that of an empty or fluid-filled tube. Within about 3 hr there was the beginning of an accumulation of polymorphonuclear leucocytes between the necrotic fibres. The inflammatory reaction became steadily more marked and by 24 hr there were masses of leucocytes almost suggesting abscess formation in some areas, particularly in tibialis anterior. By the second day the necrotic muscle fibres were partly filled with rounded mononuclear cells with eosinophilic granular or foamy cytoplasm while between the muscle fibres polymorphonuclear leucocytes were still present (Fig. 2). Patches of amorphous necrotic material lay within the muscle cells. By the third day, little necrotic debris remained within the muscle fibres which were now filled with cells.
Fig. 2. At 48 h soleus muscle fibres contain much necrotic debris and numerous rounded m o n o n u c l c m cells, probably mostly phagocytic in type. HE, x 400.
Many thin fibres with plump central nuclei were present. By the seventh day the continuity of the myofibrillar material was well established in most fibres though many showed areas of pale staining without cross-striations and many fibres containing abundant plump spindle-shaped cells were still present. Basophilia was not a feature of muscle fibres. A striking abnormality from this time onwards was the presence of long rows of nuclei within muscle fibres (Fig. 3). In some of the larger fibres, more than one row of nuclei were present. Transverse sections (Fig. 4) showed a single central nucleus in smaller fibres while in the larger ones several deeplysituated nuclei were seen as well as those normally peripherally placed beneath the sarcolemma. Abnormally situated nuclei were found in the longest surviving animals and seemed to be a permanent abnormality. They were useful as indicators of where necrosis or damage had taken place previously.
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In most muscles regeneration seemed practically complete within 3 weeks. Some focal lesions persisted for longer. These were small rounded granulomatous masses filling and expanding muscle fibres, with a central amorphous haematoxyphilic core surrounded by flattened epithelioid-type cells. Since the tissues had been de-
Fig. 3. Soleus at 7 days shows m a n y muscle fibres with rows of centrally placed nuclei. These are probably fibres which were necrotic and have regenerated. HE, x 400.
Fig. 4. Transverse section of tibialis anterior at 21 days. M a n y of the muscle fibres, particularly the larger ones, contain several internally placed nuclei. A single fibre has not completely regenerated and contains n u m e r o u s small cells. HE, x 400.
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calcified it was n o t certain w h e t h e r calcification h a d o c c u r r e d in some areas o f muscle necrosis but the a p p e a r a n c e did suggest that this had h a p p e n e d . The d i s t r i b u t i o n o f these lesions which persisted for m a n y weeks was consistently limited to regions p r e d o m i n a n t l y c o m p o s e d o f large fast-type muscle fibres, p a r t i c u l a r l y in the periphery o f g a s t r o c n e m i u s a n d in tibialis a n t e r i o r where they were c o m m o n e s t . T h e y were not seen in soleus or in regions o f o t h e r muscles c o m p o s e d o f slow fibres. Motor innervation
In all the a n i m a l s studied with either light or electron m i c r o s c o p y it c o u l d be o b s e r v e d that muscles h a d been affected in a p a t c h y irregular m a n n e r . This m e a n t that the necrosis involved some muscles in their zones o f i n n e r v a t i o n a n d o t h e r muscles in the end-plate-free areas, a n d the d i s t r i b u t i o n a n d the extent o f necrosis was never exactly the same. In order, therefore, to assess the l o n g - t e r m effects o f the d e p o l a r i z i n g fraction on i n n e r v a t i o n it was necessary to k n o w where the muscle
Fig. 5. Longitudinal frozen sections of gastrocnemius (,4) and tibialis anterior (B) of a normal rnoll~,, stained by the method of Koelle and Friedenwald to show cholinesterase activity, and by the silver imprcs,nation method of Namba et al., to demonstrate axons. Each preterminal axon (arrows) innervates :~ end-plate in which the cholinesterase reaction product is localized in the thick continuous branchhl~, gutters of the subneural apparatus, x 640.
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fibres had been necrotic at an early stage. This could be done without difficulty since the central nuclei, which indicated previous necrosis or damage to muscle fibres, seemed to remain permanently. The assessment of the morphology of endplates was therefore always done in areas where was either acute necrosis or, in the longer-surviving animals, signs of earlier damage. Areas with central nuclei could be identified in frozen sections taken at representative levels counter-stained with haematoxylin after the cholinesterase reaction and silver impregnation were completed. The preterminal myelinated motor axons and the axon terminals were studied in frozen and paraffin silver-impregnated sections. The normal appearance is shown in Fig. 5. At no stage in the experiment was there evidence of axonal damage or fragmentation. During the acute phase of muscle necrosis, intact axons lay among the necrotic muscle fibres and axonal terminals could be seen innervating these fibres. Although there was a disturbance of the normal orderly architecture of the tissues the terminals looked normal. Cholinesterase activity was demonstrable, even when the underlying muscle fibre was necrotic (Fig. 6). The intensity of cholinesterase activity was reduced for several days and the localization of the reaction product was not in the usual well-defined gutters, but in rather poorly-defined thin lines or smudges (Fig. 7). During the first 3 or 4 days many end-plates were irregularly shaped and oblong rather than rounded or oval in outline as in the normal. Occasionally, particularly in tibialis anterior, where granulomatous lesions often persisted in the necrotic muscle fibres, there were ultraterminal axonal sprouts. These sprouts were not found consistently and were quite unlike the sprouting previously found to be associated with a presynaptic block of transmission. (Duchen 1970; Duchen and Tonge 1973). Rather were they like the occasional ultraterminal axonal projection seen during the re-innervation of muscle after a peripheral nerve lesion.
Fig. 6. Tibialis anterior 24 hr after the injection of cardiotoxin. Axons are intact and the cholinesterase reaction product is visibleat the end-plates although the muscle fibres are necrotic. Method as in Fig. 5. x 400.
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Fig. 7. Gastrocnemius at 3 days shows normal-looking preterminal axons but weaker than normal cholinesterase activity. The reaction product is localized in thin lines or poorly defined smudges. Muscle fibres are severely disorganized. Method as in Fig. 5. x 640.
Fig. 8. In tibialis anterior at 8 weeks the cholinesterase reaction product at this end-plate is localized in numerous sub-units instead of in continuous gutters. The sites of cholinesterase activity seem to lie at many points along each axon terminal. Method as in Fig. 5. x 640.
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After the 10th day, when regeneration of muscle fibres was well advanced, the cholinesterase reaction at the end-plates became stronger and better-defined. However, from this time onwards the end-plates were seen as collections of "sub-units" showing strong cholinesterase activity arranged along the ramifications of the axon terminals (Fig. 8). In some end-plates, these "sub-units" looked like beads strung along the axon terminal with non-reactive areas between them. In other end-pJates the areas showing cholinesterase activity were located at the tips of the branches of axonal terminals giving the end-plates an "exploded" appearance (Fig. 9). In
Fig. 9. End-plates in tibialis anterior at 10 weeks remain abnormal in appearance. They appear expanded and each consists of an aggregation of spots or rings (subunits) of cholinesterase reaction product. M a n y of these subunits appear to be located at the tips of branches of axon terminals. Method as in Fig. 5. x 400.
soleus (Fig. 10) where end-plates are normally small, the aggregations of sub-units were usually compact while in tibialis anterior and gastrocnemius (Fig. 10) where end-plates are normally larger the clusters of sub-units were quite widely spread. Axon terminals seemed longer and more branched than usual. In many end-plates there was no cholinesterase activity for a short distance along the axon terminals from the point of branching of the preterminal axon (Figs. 9 and 10). Each aggregation of sub-units was therefore larger in area than a normal end-plate. It was clear, however, that each cluster of sub-units belonged to a single preterminal axon and was localized to one muscle fibre. There were no long axonal sprouts leading from one end-plate to new terminals on other muscle fibres. Further, although no quantitative estimation was made of the "terminal innervation ratio" (CoOrs and Woolf
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I-:ig. 10. End-plates at 4 months in the superficial region of gastrocnemius (A) where muscle fibres arc mainly fast in type and soleus (B) which consists of slow fibres, show similar morphological abnormalities although they differ in size. Method as in Fig. 5. x 400.
1959) it seemed that the basic pattern of motor innervation of muscles remained normal with a single preterminal myelinated axon innervating a single muscle fibre and scarcely any preterminal axonal branching being present. The abnormal morphology of the motor end-plates was found in all animals studied including those allowed to survive for 6 months or more. Abnormal endplates were found only in areas where there was evidence of previous damage and regeneration of muscle fibres. Where end-plates were normal in appearance the muscle fibres invariably showed no local evidence of previous damage, a finding which also applies to the electron microscopic studies.
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Electron Microscopy Muscle fibres The shortest time interval between the injection of cardiotoxin and fixation of tissues for electron microscopy was 30 min. Already by this time severe lesions were present within fibres of all the muscles examined (soleus, tibialis anterior, E D L and gastrocnemius). Slow and fast types of muscle fibre were equally severely affected. It is difficult to be certain of the exact sequence of events leading to the total disintegration of the internal structure of muscle fibres which was seen. One early change was the loss of Z-line structure while the orientation of thick and thin filaments was maintained (Fig. 11). In other muscle fibres the only change noted was the dilatation of the tubules of the sarcoplasmic reticulum. More severe lesions consisted in the disorganization of the myofilaments which were still recognizable and showed the remnants of a fibrillar arrangement. Many muscle fibres showed a total disruption of their internal structure. There were masses of a m o r p h o u s material in irregular clots, patches of clear, probably fluid-filled, spaces and dissolution of myofilaments. Mitochondria were swollen and their cristae irregularly condensed. In fast muscle fibres (such as gastrocnemius) masses of fragments of tubules were found. Muscle fibres were not affected in their entirety. In many instances there was a sharp demarcation between the part of the cell which was necrotic and the part remaining structurally intact. In other muscle fibres there was a transitional
Fig. 11. This muscle fibre from the superfical region of gastrocnemius at 5 hr after the injection of the cardiotoxin shows loss of Z-lines, the sites of which are shown by arrows. Myofilamentsand their fibrillar arrangement and M-lines are preserved. × 30,000.
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zone in which the arrangement of filaments was distorted, and the orderly transverse alignment of Z-lines lost. In all the necrotic muscle fibres the limiting basement membrane was intact. In many places the sarcolemmal membrane was absent so that the contents of the fibre (fluid, vesicles, clots of amorphous debris and remnants of tubules and mitochondria) were bounded and held in place by the basement membrane only (Fig. 12).
Fig. 12. In this transverse section of soleus at 30 min after the injection of cardiotoxin parts of two necrotic muscle fibres are shown. They contain debris, abnormal mitochondria and fragments of tubules. At some places (arrow) the sarcolemmal membrane is lost and only the basement membrane remains intact. × 15,000.
Within a few hours leucocytes were found adhering to the necrotic muscle fibres. By 2-3 days the sarcolemmal tubes were packed with cells which had the characteristics of macrophages - - dense nuclei, abundant rough endoplasmic reticulum, numerous "ruffled" cytoplasmic processes interdigitating with processes from adjacent cells, and phagosomes and abundant dense granular material, lipid droplets and vacuoles. In addition to these phagocytic cells there were others inside the sarcolemmal tubes with less dense nuclei and cytoplasm which contained myofilaments and which were obviously muscle-forming cells. Regeneration proceeded rapidly and by the 7th day most muscle fibres were composed of cells in which myofilaments were abundant though in places they were not organized in sarcomeres. During the regenerative phase, the sarcolemmal tubes could be identified, particularly in transverse sections, by the basement membrane which encircled and enclosed the clusters of myocytes and phagocytic cells. In many places the myocytes themselves were
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enclosed in a layer of basement membrane which was a branch of the outer membrane. In other places the myocytes were very closely apposed without any intervening basement membrane and in some places these cells seemed to be fusing together. A common finding at 7 days was the presence o f dense rod-like bodies of varying length and width composed of Z-line material. These bodies were usually seen where myofilaments were not arranged in parallel and where fibrils were not properly formed. Thin filaments were often seen attached to these bodies. In addition, dense bodies of Z-line material were occasionally found in places where fibrils and sarcomeres were well formed. During the stage of muscle regeneration another common finding was the presence of numerous dense rounded bodies beneath the sarcolemmal basement membrane (Fig. 13). These seemed most likely to be autophagic
Fig. 13. Regenerating muscle fibres at 3 days are very irregular in outline as shown in Xhis longitudinal section of a soleus fibre in which the surface is cut tangentially at m a n y places. Dense rounded bodies lie beneath the basement m e m b r a n e and m a y be lysosomes derived from within the muscle fibre, x 25,000.
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Fig. 14. A longitudinal section of extensor digitorum longus at 30 min after the injection of cardiotoxin shows that the internal structure of this muscle fibre is severely disrupted. Myofilaments are replaced by dense a m o r p h o u s clots and degenerating mitochondria, fragments of tubules, debris and nuclei remain within the muscle fibre. Axon terminals (arrows) are however normal in appearance and post-synaptic folds of the sarcolemmal m e m b r a n e are seen. x 10,000.
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vacuoles or lysosomes discharged from the interior of the muscle fibre. In the animals surviving for much longer periods there were abnormalities of muscle fibres which seemed to be permanent, as already noted with light microscopy. Centrally-placed nuclei and disarray of myofibrils with the formation of striated annulets ("Ringbinden"), were common findings. These abnormally-orientated myofibrils were found not only at the periphery of the muscle fibres where they formed a complete or partial ring, but also extended into the interior of the fibre so that longitudinally-orientated fibrils lay external to them.
Motor end-plates During the acute phases (30 min to 2 hr after the injection of cardiotoxinj when the muscle fibres became necrotic, axon terminals were normal in appearance (Fig. 14). They contained abundant mitochondria and vesicles and lay in close contact with the synaptic surface of the sarcolemmal membrane. At no time was there any evidence of axonal disintegration. The post-synaptic folds of the muscle fibre were normal in their appearance even though the internal structure of the fibre was totally disrupted. In these places the post-synaptic folds appeared to be suspended in fluidfilled pools (Fig. 15). The basement membrane material which normally fills the synaptic space and folds was identifiable and normal in appearance. At later stages the post-synaptic sarcolemmal membrane was often absent (Fig. 16), just as parts of the extra-junctional sarcolemma were lost wherever there was muscle necrosis. Post-synaptic folds were then lost and their position was marked only by the persistence of the basement membrane material which at first could be seen projecting into the muscle cell, but later, still forming an intact layer, became folded or rolled up on itself at the sites of the subneural folds. During the next few days when the muscle fibres were filled with phagocytic cells and regenerating myocytes the outline of the fibres became very irregular and the electron microscopic appearances very complex indeed. Motor nerve terminals were always of normal appearance and contained normal-looking vesicles and other organelles. The axonal terminals were now wrapped in Schwann cell processes and the axolemmal membrane made only irregular contacts with the basement membrane of the muscle fibre. The subneural basement membrane often showed patchy thickening or duplication, probably marking the sites of the original post-synaptic folds (Fig. 17). Within the confines of the muscle fibre i.e. within the basement membrane, there was often no muscle structure to be seen in the subneural position and the nerve terminals lay over the intracellular phagocytes with only the basement membrane of the muscle fibre between these cells and the axons (Fig. 18). At 7-14 days when the regenerative changes were well advanced axon terminals were enclosed within Schwann cell cytoplasm in some places while in other places on the same muscle fibre they lay in contact with the sarcolemmal basement membrane, an abnormal nerve-muscle relationship which persisted in the motor endplates of both slow and fast types of muscle fibre. Close contacts between different parts of the axon terminal and its muscle fibre were separated from each other by regions where the axon was wrapped in Schwann cell (Fig. 19). Another frequent finding was that of axon terminals enclosed within "tunnels" formed by sleeves of
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Fig. 15. At 30 min this muscle fibre (M) is severely disorganized but the axon terminal (A) is normal in appearance. It contains abundant synaptic vesicles and mitochondria which are structurally normal in contrast with the very abnormal mitochondria within the muscle cell. The post-synaptic folds (arrows) are present and the sarcolemmal membrane which forms them is intact, x 35,000
s a r c o l e m m a p r o j e c t i n g into the muscle fibre (Fig. 20). In these places the terminal, cut transversely or obliquely, was s u r r o u n d e d by the c y t o p l a s m o f the muscle cell. A x o n t e r m i n a l s lying in " t u n n e l s " are only o c c a s i o n a l l y seen in the n o r m a l a n d then usually only in o b v i o u s l y tangential sections o f muscle fibres. M a n y o f the nerve-muscle j u n c t i o n s showed a p a u c i t y o r absence o f s u b n e u r a l folds o f the post-
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Fig. 16. At this end-plate in tibialis anterior 2 hr after the injection of cardiotoxin the axon terminal (A) contains abundant vesicles and mitochondria while the underlying muscle fibre (M) contains only debris in this region. The post-synaptic sarcolemmal membrane seems to be deficient at some places (arrows) so that the structure of the subneural folds is not as well maintained as at earlier stages (cf. Fig. 15). This is similar to the loss o f sarcolemma in extra-junctional regions (cf. Fig. 12). x 30,000.
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Fig. 17. At 3 days an axonal terminal (A) is partially enclosed within Schwann cell processes (S). These interpose between axon and basement membrane of the synaptic cleft. The basement membrane projects into the "muscle fibre" in a series of thickenings (arrows) which mark the sites of post-synaptic folds from which the sarcolemmal m e m b r a n e has been lost. The cell in the subneural position is probably phagocytic in type. x 30,000.
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Fig. 18A. In this transverse section of a soleus fibre 3 days after the injection of cardiotoxin axon terminals (A) lie in contact with the basement m e m b r a n e of the muscle fibre but in the subneural position the cells have the characteristics of phagocytes. One cell (M) contains scattered myofilaments, x 15,000.
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Fig. 18B. Detail of fig. 18A showing an axon terminal (A) of normal appearance and containing a b u n d a n t vesicles and mitochondria. The axon lies in contact with the basement membrane of the muscle fibre. No sarcolemmal m e m b r a n e is seen and the cell beneath the axon is phagocytic in type. × 45,000
synaptic sarcolemmal membrane, both during early stages of muscle regeneration as well as long after regeneration was complete (Fig. 21). Irregular gaps and processes of Schwann cell lying between axolemma and sarcolemma were also frequently seen. Since these abnormalities were found in the animals allowed to survive for 6 months or more it seems that these end-plates remain permanently abnormal particularly in the deficiency of subneural folds. DISCUSSION
The cardiotoxic fraction of the venom ofD. jamesoni is free of post-synaptic blocking action and is free of phospholipase A activity since the whole venom is known to be free of this enzyme (Excell and Patel 1972; Patel 1972). The effects of F8 seem to be due to its highly selective action and, since the muscle necrosis was confined to the region of the injection with individual muscles involved to a varying extent,
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Fig. 19. At 7 days in soleus close contacts between axon (A) and regenerating muscle fibre (M) are shorter than normal. Part of the axon is wrapped in Schwann cell processes, x 30,000.
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Fig. 20. At 17 days in soleus an axon terminal lies in a "tunnel" of sarcolemma. Subneural folds are not seen. Expansion of the regenerating muscle fibre around the intact axon terminals may be the way in which this type of abnormal neuromuscular junction develops. × 30,000.
it is probable that the venom was rapidly bound to receptor sites and did not diffuse widely throughout the leg muscles. Muscle necrosis has been caused in experimental animals by numerous methods including thermal injury (Price, Howes and Blumberg 1964; Reznik and Engel 1970), crush injury (Allbrook 1962), the injection of alcohol and oil (Walton and Adams 1956), the injection of Clostridium welchii toxins (Aikat and Dible' 1956) as well as by the injection of the venoms of cobra (Stringer, Kainer and Tu 1971) and rattlesnake (Stringer, Kainer and Tu 1972). Muscle necrosis in man has also been observed after bites by the sea snake Enhydrina schistosa (Marsden and Reid 1961). The acute necrosis of muscle fibres seems to be similar in appearance whatever its method of production although the lesions caused by rattlesnake venom
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Fig. 21. This motor end-plate in soleus at 4 months remains abnormal in that the lengths of close synaptic contact are short and that post-synaptic folds of sarcolemmal membrane are few and poorly formed. x 30,000. in wh i ch the c r u d e w h o l e v e n o m was used {Stringer et al. 1972), were described as h a e m o r r h a g i c a n d there was also d e s t r u c t i o n o f the b a s e m e n t m e m b r a n e o f m u s c l e fibres, a b n o r m a l i t i e s w h ic h were n o t f o u n d in the p r e s e n t studies. T h e a p p e a r a n c e
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of the muscle fibres during the regenerative stages do not seem different from those described by many other authors (see Mauro, Shafiq and Milhorat 1970) and familiar problems arise in the identification and nomenclature of individual cells within regenerating damaged muscle fibres. There is no doubt however that at some stages during regeneration the cells lying immediately beneath the axon terminals, separated from them by basement membrane, are phagocytic in type as shown by their abundant rough endoplasmic reticulum, numerous Golgi apparatuses, phagosomes and complex "ruffled" cytoplasmic margins. It would be of interest to know whether during this stage of regeneration, the axon terminals, which are morphologically normal, contain and release transmitter and whether the muscle cell membrane is capable of response. Although some attention has been given to the influence of the motor innervation on the capacity for injured fibres to regenerate (Walton and Adams 1956) the effects of muscle injury on its innervation has been neglected. This neglect may have been due to the fact that trauma (crushing) or thermal injury may damage axons as well as muscle fibres. It was therefore of particular interest to determine whether cardiotoxin had any effect on axons and their terminals. Physiological studies on the depolarizing (cardiotoxic) fractions obtained from the venoms of both D. jamesoni (Patel 1972) and Naja nivea (Earl and Excell 1972) gave no evidence for any depolarization of the nerve terminals which would have been shown by a rise in the frequency of miniature end-plate potentials due to an outpouring of transmitter, at least prior to muscle depolarization in the frog. However, in this species, Chang and Lee (1966) showed that the cardiotoxic fraction from Naja naja atra venom caused a loss of conduction in the axons up to the terminals concomitant with severe muscle depolarization, indicating that some degree of axonal depolarization may be present. They suggest that this effect of cardiotoxin may be responsible for the reduction in acetylcholine output from rat phrenic nerve terminals in the presence of the crude venom (Su, Chang and Lee 1967). Mammalian nerve trunks are relatively resistant to block and in/n vitro experiments Chang, Chuang, Lee and Wei (1972) found that a minimum concentration of 100 /~g/ml cardiotoxin was required to block axonal conduction in the rat phrenic nerve trunk. In the present in vivo experiments a dose of 20 #g of cardiotoxin was injected into the mice, a dose selected to cause only local changes and to permit the study of the long term effects. Although an acute disturbance of axonal function near the terminals cannot be ruled out in these experiments the structure of pre-terminal myelinated axons and motor nerve terminals was normal in appearance. However no electrophysiological studies have been done in animals which have survived for any length of time and the characteristics of neuromuscular transmission are not known after regeneration is complete but end-plates are still abnormal in appearance. The changes in the end-plates occur in the following sequence. First there is the disorganization of the contents of the muscle cell and its resultant collapse. It seems that Schwann cells then extend down and enclose the axon terminals, due perhaps to loosening of nerve-muscle junctions since the surface of the muscle fibre becomes very irregular at his stage. The axon terminals probably increase in length and while regeneration of the muscle fibre is occurring, so that it re-expands as its content of
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myofilaments increases again, the contact between axon and sarcolemmal membrane becomes limited to short intermittent lengths. These small areas of close (synaptic) contact are separated from each other by parts of the axon terminalwhich are wrapped in Schwann cell processes and do not make synaptic contact. The normally smooth continuous contact between axon terminal and muscle fibre thus becomes broken up into a series of small synaptic sub-units. Each aggregation of sub-units is, however, confined to a single muscle fibre and is innervated by a single pre-terminal myelinated axon which is not branched. From the findings in the present studies it is possible to understand how the postsynaptic folds of the sarcolemma are lost during the acute stage of muscle necrosis. The reason why folds are not formed again after muscle regeneration is not clear. Previous studies of the formation of new end-plates in regenerating limb muscles of the newt (Lentz 1969) and after recovery from the effects of botulinum and tetanus toxins (Duchen 1971, 1973) showed that post-synaptic folds developed as the new nerve-muscle contacts matured over several weeks. In the present experiments it seems that there is merely a reorganization of the structure of the end-plates and that no new end-plates are formed in response to the disruption of the internal structure of the muscle fibres. The finding that end-plate abnormalities occur as a consequence of a lesion primarily affecting the muscle cell may be relevant to the study of the innervation of muscle in human diseases. CoErs (1955) and Co&s and Woolf (1959) showed that the end-plates were abnormal in patients with various types of myopathy (i.e. diseases considered to affect the muscle fibre primarily). In particular, complex spread-out terminal axonal arborizations were described. CoErs (1955) pointed out that the neuronal changes remained limited to the most distal part of the axon and that there was no excessive pre-terminal axonal branching. Morphologically abnormal end-plates have been described in cases of myasthenia gravis (see Woolf 1969). In this disease, known to be associated with a disorder of neuromuscular transmission, the end-plates were observed to be elongated but there was also much pre-terminal axonal branching. Abnormalities of the end-plates in the mouse were also found after recovery from the effects ofbotulinum toxin (Duchen and Strich 1968a; Duchen 1970, 1971) and tetanus toxin (Duchen and Tonge 1973) which are both known to cause a pre-synaptic block of transmission. In these experimental conditions new nerve-muscle junctions are formed as a result of sprouting from axonal terminals and are seen eventually as sub-units of varying shape and size scattered along the muscle fibres or spread over several adjacent fibres with abundant pre-terminal axonal branching. Studies of the innervation of muscle in motor neurone disease, poliomyelitis and peripheral neuropathies in man (Co6rs 1955; Co6rs and Woolf 1959) and in motor neurone disease in the mouse (Duchen and Strich 1968b) have shown that in all these conditions, in which muscle is partially denervated, pre-terminal branching of surviving axons is marked. It thus seems that pre-terminal branching, whatever the mechanism is by which it develops, is a constant histological feature of disease of the lower motor neurone whether primarily affecting the perikaryon of the anterior horn cell or the terminals of its axon. The findings in the present experiments show how abnormalities in the
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morphology of end-plates develop in response to a lesion primarily affecting the muscle cell and also show that axonal sprouting is not induced by this type of lesion. SUMMARY
The venom of Dendroaspisjamesoni (Jameson's mamba) was fractionated by column chromatography. One of the fractions (F8) caused rapid depolarization of muscle fibres when tested in vitro, an action similar to that of "cardiotoxins" of other snake venoms. A single injection of 20 #g of the depolarizing fraction was made directly into the muscles of one hindleg in mice. Light and electron microscopic studies were made of muscle fibres and motor end-plates of animals allowed to survive from 30 min to 6 months after the injection. The depolarizing fraction caused rapid and severe disorganization of the internal structure of muscle fibres affecting slow and fast types of fibre equally severely. Muscle fibres were affected in segments and not along their entire length. Early changes were the disappearance of Z-lines, the dissolution of myofilaments and degenerative changes in mitochondria, but the basement membranes of the muscle fibres remained intact. Regeneration of muscle took place rapidly and was usually complete within 2 to 3 weeks but centrally-placed nuclei, rows of nuclei and malorientation of myofibrils including the formation of striated annulets remained as permanent abnormalities and were used as indicators of where necrosis had occurred. Myelinated axons and motor nerve terminals were normal in appearance and contained abundant vesicles and mitochondria even when the muscle fibres they innervated were necrotic. The axonal terminals became enclosed within Schwann cell processes. After muscle regeneration was complete the end-plate morphology remained abnormal. With light microscopy the end-plates were seen as aggregations of sub-units, each aggregation being limited to a single muscle fibre and innervated by a single pre-terminal myelinated axon. The electron-microscopic appearance of the motor end-plates also remained abnormal and there was a striking absence of post-synaptic folds of the sarcolemmal membrane. The observations made in these experiments indicate that the cardiotoxin selectively damages muscle fibres leaving the motor innervation structurally intact and also show how morphological abnormalities may develop in motor end-plates as a result of a primary lesion of the muscle fibre. The appearances are similar to those occurring in the innervation of muscle in human myopathies. The absence or branching of preterminal axons and of sprouting from axon terminals is in contrast to the changes found in human and experimental conditions in which there is known to be a disorder of the lower motor neurone. REFERENCES AIKAT, B. K. AND J. H. DIBLE (1956) The pathology of Clostridium welchii infection, J. Path. Bact., 71 : 461~76. ALLBROOK, D. (1962) An electron microscopic study of regenerating skeletal muscle, J. Anat. ( L o n d . 96:137-152. BEESLEY,R. A. AND P. M. DANIEL (1956) A simple method for preparing serial blocks of tissue, J c/ill Path., 9: 267-268.
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CHANG, C. C. AND C. Y. LEE (1966) Electrophysiological study of neuromuscular blocking action of cobra neurotoxin, Brit. J. Pharmac. Chemother., 28 : 172-181. CHANG, C. C., S-T. CHUANG, C. Y. LEE AND J. W. WEI (1972) Role of cardiotoxin and phospholipase A in the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom, Brit. J. Pharmac. Chemother., 44: 752-764. COORS, C. (1955) Les variations structurelles normales et pathologiques de la jonction neuromusculaire, Acta neurol.psychiat, belg., 55: 741-866. COERS, C. AND A. L. WOOLF (1959) The Innervation of Muscle, Blackwell, Oxford. DUCHEN, L. W. (1970) Changes in motor innervation and cholinesterase localization induced by botulinum toxin in skeletal muscle of the mouse: differences between fast and slow muscles, J. Neurol. Neurosur#. Psychiat., 33: 40-54. DUCHEN, L. W. (1971) An electron microscopic study of the changes induced by botulinum toxin in the motor end-plates of slow and fast skeletal muscle fibres of the mouse, J. neurol. Sci., 14: 47-60. DUCHEN, L. W. (1973) The effects of tetanus toxin on the motor end-plates of the mouse: an electron microscopic study, J. neurol. Sci., 19: 153-167. DUCHEN,L. W. AND SABINAJ. STRICH(1968a) The effects of botulinum toxin on the pattern of innervation of skeletal muscle in the mouse, Quart. J. exp. Physiol., 53: 84-89. DUCHEN, L. W. AND SABINAJ. STRICH (1968b) An hereditary motor neurone disease with progressive denervation of muscle in the mouse: the mutant "wobbler", J. Neurol. Neurosurg. Psychiat., 31 : 535-542. DUCHEN, L. W. AND D. A. TONGE (1973) The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor end-plates in slow and fast skeletal muscle of the mouse, J. Physiol. (Lond.), 228: 157-172. DUCHEN, L. W., BARBARAJ. EXCELL, R. PATELAND BETTY SMITH (1973) Light and electron microscopic changes in mouse muscle fibres and motor end-plates caused by the depolarizing fraction (cardiotoxint of the venom of Dendroaspisjamesoni, J. Physiol. (Lond.), 234: 1-2P. EARL, JANET E. AND BARBARAJ. EXCELL(1972) The effect of toxic components ofNaja nivea (Cape cobra~ venom on neuromuscular transmission and muscle membrane permeability, Comp. Biochem. Physiol.. 41A: 597-615. EXCELL,BARBARAJ. AND R. PATEL (1972) Characterization of toxic fractions from Dendroaspis jamesoni venom, J. Physiol. (Lond.), 225: 29-30P. KOELLE, G. B. AND J. S. FRIEDENWALD (1949) A histochemical method for localizing cholinesterase activity, Proc. Soc. exp. Biol. (N.Y.), 70: 617-622. LEE, C. Y. (1972) Chemistry and pharmacology of polypeptide toxins in snake venoms, Ann. Rev. Pharmacol., 12: 265-286. LENTZ, T. L. 0969) Development of the neuromuscular junction, J. Cell Biol., 42: 431~43. MARSDEN,A. T. H. AND H. A. REID (1961) Pathology of sea-snake poisoning, Brit. reed. J., i: 1290-1293. MAURO, A., S. A. SHAFIQAND A. T. MILHORAT(Eds.) (1970) Regeneration of Striated Muscle and Myogenesis (Proceedings of an International Conference held under auspices of the Muscular Dystrophy Associations of America, New York, 1969) (International Congress Series, No. 218), Excerpta Medica, Amsterdam. NAMBA, T., T. NAKAMURAAND D. GROB (1967) Staining for nerve fiber and cholinesterase activity in fresh frozen sections, Amer. J. clin. Path., 47: 74-77. PALMGREN, A. (1948) A rapid method for selective silver staining of nerve fibres and nerve endings in mounted paraffin sections, Acta. zool. (Stockh.), 29: 377-392. PATEL, R. (1972) An Investigation of the Neurotoxic Components of the Venom of Dendroaspis jamesoni (Jameson's mamba), Ph. D. thesis, London University. PRICE, H. M., E. L. HOWES AND J. M. BLUMBERG(1964) Ultrastructural alterations in skeletal muscle fibres injured by cold, Lab. Invest., 13: 1264-1278. REZNIK, M. AND W. K. ENGEL (1970) Ultrastructural and histochemical correlations of experimental muscle regeneration, J. neurol. Sci., 11: 167-185. STRINGER, J. M., R. A. KAINERAND A. T. TU (1971) Ultrastructural studies of myonecrosis induced by cobra venom in mice, Toxicol. appl. Pharmacol., 18: 442-450. STRINGER, J. M., R. A. KAINERAND A. T. TU (1972) Myonecrosis induced by rattlesnake venom, Amer. J. Path., 67: 127-136. Su, C., C. C. C8ANG AND C. Y. LEE (1967) Pharmacological properties of the neurotoxin of cobra venom. In: F. E. RUSSELLAND P. R. SAUNDERS(Eds.) Animal Toxins, Pergamon, Oxford, pp. 259-267. WALTON, J. N. AND R. D. ADAMS(1956) The response of the normal, the denervated and the dystrophic muscle-cell to injury, J. Path. Bact., 72: 273-298. WOOLF, A. L. (1969) Pathological anatomy of the intramuscular nerve endings. In: J. N. WALTON (Ed.) Disorders of Voluntary Muscle, 2nd edition, Churchill, London, pp. 203-237.