Regeneration of the septohippocampal pathways in adult rats is promoted by utilizing embryonic hippocampal implants as bridges

The ability of embryonic hippocampal tissue to promote regeneration of cholinergic axons in the septohippocampal system has been studied in adult rats. Strips of embryonic hippocampus, taken from 7–40 mm rat fetuses, were implanted into a 2–3 mm wide cavity which completely transected the septal cholinergic axons innervating the intrinsic hippocampus. The ingrowth of cholinergic fibres into the denervated host hippocampal formation was monitored by measuring the activity of the enzyme, choline acetyltransferase (ChAT), and by acetylcholine esterase (AChE) histochemistry. The results demonstrated a gradual, partial return of both ChAT enzyme activity and AChE-positive fibres in the initially denervated hippocampal formation of the adult recipient. Time-course studies indicated that this ingrowth progressed from the implant into the rostral tip of the host hippocampus, and continued caudally to cover the entire dorsal hippocampus by 3–6 months post-operative Although the regenerating AChE-positive fibres reached the hippocampal target in the recipient along abnormal routes, they reinnervated selectively the appropriate terminal areas within the host hippocampus and dentate gyrus, suggesting the presence of quite specific mechanisms to guide the regenerating axons back to their original targets. Lesions of the medial septum-diagonal band area of the host and horseradish peroxidase (HRP) injections into the host hippocampus, caudal to the implant, indicated that the origin of the regenerating axons was predominately from the ipsilateral ventral medial septum and diagonal band area of the host. The results provide evidence that axonal regeneration and reinnervation of a denervated target zone can be promoted by utilizing implants of embryonic CNS tissue to bridge a tissue defect between the target and the lesioned axonal stumps.     

Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents

Following a peripheral nerve injury, brain-derived neurotrophic factor (BDNF) and the p75 neurotrophin receptor are upregulated in Schwann cells of the Wallerian degenerating nerves. However, it is not known whether the endogenous BDNF is critical for the functions of Schwann cells and regeneration of injured nerve. Treatment with BDNF antibody was shown to retard the length of the regenerated nerve from injury site by 24%. Histological and ultrastructural examination showed that the number and density of myelinated axons in the distal side of the lesion in the antibody-treated mice was reduced by 83%. In the BDNF antibody-treated animals, there were only distorted and disorganized myelinated fibres in the injured nerve where abnormal Schwann cells and phagocytes were present. As a result of nerve degeneration in BDNF antibody-treated animals, subcellular organelles, such as mitochondria, disappeared or were disorganized and the laminal layers of the myelin sheath were loosened, separated or collapsed. Our in situ hybridization revealed that BDNF mRNA was expressed in Schwann cells in the distal segment of lesioned nerve and in the denervated muscle fibres. These results indicate that Schwann cells and muscle fibres may contribute to the sources of BDNF during regeneration and that the deprivation of endogenous BDNF results in an impairment in regeneration and myelination of regenerating axons. It is concluded that endogenous BDNF is required for peripheral nerve regeneration and remyelination after injury.     

Changes in the forms of the components of the troponin complex during regeneration of injured skeletal muscle

Using the immunoperoxidase technique, antibodies to the fast components of the troponin complex stained all regenerating cells after localized alcohol injury to rat skeletal muscle. Antibodies to slow troponin components stained only some of these cells. About 6 weeks after injury with the nerve intact, the fast and slow forms of the troponin components were located in different cells. During the later stages of regeneration, staining for myosin ATPase correlated with the staining with antibodies to fast and slow troponin components. A similar staining pattern was also observed in the early stages of regeneration of muscle denervated at the time of injury. In this case, antibodies to fast skeletal muscle troponin components continued to stain all the cells 10 weeks after injury. Injured denervated muscle cells stained equally dark by myosin ATPase after preincubation at pH 9.4 over this period. None of the regenerating myotubes in denervated muscle stained for myosin ATPase after preincubation at pH 4.3.     

MUSCLE FIBRE SPLITTING AND REGENERATION IN DISEASED HUMAN MUSCLE

In soleus muscle of rat, necrosis of muscle fibres within a grossly intact endomysium was produced by 60–70°C Ringer's solution injected into the space around the muscle. After 2 weeks regenerated fibres appeared to be fragments of 'split' fibres. In split fibres of patients with myopathy all stages of myogenesis were seen in electron micrographs. Hence splitting of muscle fibres is thought to indicate regeneration rather than degeneration. Based on the morphology of the clefts through split fibres and of normal fetal myogenesis a hypothetical mechanism for splitting of muscle fibres is proposed. Myoblasts, myotubes and myofibres occur within a common basement membrane separated only by plasma membranes and a 20 nm wide gap. In light microscopy they appear as a single fibre. Many meshes of the endomysial framework remain empty and condense around regenerating fibres. Myotubes and young myofibres fuse laterally or become detached and appear by light microscopy to be fragments of a longitudinally split fibre. Though encircled by thick endomysial sheaths, connective tissue between them is sparse. The site of fusion may be marked by a strand of mitochondria or by internal nuclei. Incomplete fusion results in branched fibres or short longitudinal clefts or cavities. The prerequisite of this hypothesis is that regeneration is focal and takes place in a grossly intact endomysium. Repeated death of muscle fibres and focal regeneration in human myopathic muscle lead to dissociation of parenchyma and connective tissue. The mechanism is similar to the formation of pseudolobuli in cirrhosis of the liver.     

MUSCLE TRANSPLANTATION BETWEEN NORMAL AND DYSTROPHIC MICE. 2. HISTOCHEMICAL STUDIES

Innervated and denervated auto- and crosstransplants, ranging in age from 1 hour to over 300 days, of normal and dystrophic tibialis anterior muscles in 129 ReJ mice have been examined histochemically for oxidative and glycolytic enzymes, ATPase, acetylcholinesterase, glycogen and nucleic acids. All transplants were devoid of glycogen and phosphorylase activity within 12 hours of transplantation. Fibre types were distinguished in the central areas of 2 day old transplants with NADH-TR and ATPase reactions, but at the periphery, where fibres were at a rnore advanced stage of degeneration, fibre typing was not evident and NADH-TR and ATPase reaction products were clumped. By 3 to 4 days most implanted fibres had broken down, but NADH-TR and ATPase activity were still evident. “yotubes were observed in areas of 3 day old transplants where muscle fibres had completely degenerated and these stained uniformly for RNA, NADH-TR and ATPase. Phosphorylase activity and glycogen were first observed in myotubes of 8 day old transplants. When examined before 12 days, myotubes of all transplants showed uniform NADH-TR, ATPase, phosphorylase and RNA. Subsequently NADH-TR, ATPase and PAS reactions showed progressive differentiation of type 1 and 2 fibres in innervated normal and dystrophic transplants in normal mice. All transplants in normal and in dystrophic mice, denervated at the time of transplantation, failed to show fibre type differentiation. Despite substantial regeneration before 25 days, all transplants in dystrophic mice subsequently underwent progressive degeneration. A similar pattern was observed in denervated transplants in normal mice. The few fibres that remained in these transplants showed uniform NADH-TR and ATPase activity, whilst phosphorylase and glycogen were absent. Innervated normal and dystrophic transplants in normal mice showed maintenance of differentiated type 1 and 2 fibres even after 300 days, thus resembling intact normal rather than intact dystrophic muscles. This study supports the view that murine muscular dystrophy may have a neural basis for its pathogenesis.     

The fate of dystrophin during the degeneration and regeneration of the soleus muscle of the rat

Summary Immunocytochemistry and Western blotting were used to monitor the fate of dystrophin in the soleus muscle of the rat during a cycle of degeneration and regeneration induced by inoculation of the muscle with the venom of Notechis scutatus scutatus (the Australian tiger snake). In control muscle dystrophin was localised close to the plasma membrane. Dystrophin began to break down 3–6 h after venom inoculation, giving a characteristic discontinuous labelling pattern. At 12 h dystrophin was absent from the plasma membrane, and by 1 day the architecture of the muscle fibres had completely broken down. By 2 days post inoculation regeneration had commenced. The regenerating myofibres possessed well-organised myofibrils and the plasma membrane was intact. Dystrophin was detected by Western blot at 3 days, but was not seen in sections until regeneration of the muscle was well advanced, at 4 days post inoculation. The results suggested that although dystrophin was present in the myofibres at 3 days, it was not incorporated into the plasma membrane until 4 days post inoculation. This may be due to the influence of the functional reinnervation of the regenerating fibres, which occurs at 4–5 days, or to the growing fibres reaching a critical diameter.Supported by the Muscular Dystrophy Group of Great Britain, the Wellcome Trust, and the MRC     

Myelinated nerve fibre regeneration in diabetic sensory polyneuropathy: correlation with type of diabetes

Observations were made on myelinated fibre regeneration in diabetic sensory polyneuropathy assessed in sural nerve biopsy specimens. These confirmed that regenerative clusters initially develop within abnormally persistent Schwann cell basal laminal tubes. The number of regenerating fibres, identified by light microscopy, was found to decline in proportion to the reduction in total myelinated fibre density. The relative number of regenerating fibres was significantly greater in patients with insulin-dependent as compared with those with non-insulin-dependent diabetes after correction for age. There was a slight negative correlation between the relative proportion of regenerating fibres and age, but this was not statistically significant. The progressive reduction in the number of regenerating fibres with declining total fibre density indicates that axonal regeneration fails with advancing neuropathy. The production of nerve growth factor (NGF) and NGF receptors by denervated Schwann cells is likely to be important for axonal regeneration. To investigate whether the failure of axonal regeneration could be related to a lack of NGF receptor production by Schwann cells, we examined the expression of p75 NGF receptors by Büngner bands immunocytochemically. In comparison with other types of peripheral neuropathy, p75 NGF receptor expression appeared to take place normally. It is concluded that failure of axonal regeneration constitutes an important component in diabetic neuropathy. Its explanation requires further investigation.These results were presented in part at a meeting of the European Association for the Study of Diabetes held in Düsseldorf in September 1994     

Regenerating nerve fibres do not displace the collateral reinnervation of cat teeth

Experiments in adults cats²² have shown that when the inferior alveolar nerve is sectioned and regeneration blocked, there is extensive collateral reinnervation from neighbouring nerves which reinnervate the denervated teeth. In the present experiments the fate of collateral nerve fibres supplying the teeth has been investigated following regeneration of the inferior alveolar nerve. In some experiments the inferior alveolar nerve was sectioned and regeneration temporarily blocked whilst in others the nerve was frozen but not sectioned, to allow a more complete restoration of normal properties after regeneration. The jaw opening reflex evoked by electrical stimulation of the left canine and incisor teeth was abolished by left inferior alveolar nerve section or freeze but returned within 3–9 weeks due to reinnervation by collateral nerve fibres. Regenerating inferior alveolar nerve fibres were allowed to reinnervate the teeth 12–15 weeks after the initial nerve injury. Twenty seven weeks after the initial nerve injury, pulpal nerve fibres supplying the teeth which had been denervated were present in the regenerated inferior alveolar nerve as well as the ipsilateral lingual and mylohyoid nerves and the contralateral inferior alveolar, lingual and mylohyoid nerves. Except for the ipsilateral inferior alveolar and lingual nerves, these nerves do not normally include pulpal fibres from these teeth. In these experiments, therefore, after inferior alveolar nerve section or freeze, the collateral reinnervation of tooth pulps was not withdrawn following the return of regenerating nerve fibres.     

Rat adrenal transplants are reinnervated: an invalid model of denervated adrenal cortical tissue

Adrenal autotransplantation is a widely used approach to investigate the potential for neural modulation of adrenal cortical function. It is believed that regenerating adrenal transplants are not reinnervated, thereby providing a model to investigate adrenal function in the absence of neural modulation. However, the hypothesis that adrenal transplants become reinnervated has not been directly tested. The purpose of the present study was to characterize the time course, extent, and nature of the reinnervation of the regenerating adrenal transplant and to assess whether the recovery of steroidogenic function and enzyme expression correlates temporally with the presence of innervation. Using immunohistofluorescent detection of tyrosine hydroxylase (TH), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP), the innervation of regenerating adrenals was assessed 14-30 days after transplantation of adrenal capsules beneath the kidney capsule in rats. Extensive reinnervation by TH-, NPY-, and VIP-positive fibres was present by 14 days after transplantation including regions of the adrenal capsule and cortex, with only minimal reinnervation by CGRP-positive fibres up to 30 days. TH- and NPY-positive chromaffin cells were also observed in the regenerating transplants. In addition, there was marked recovery of steroidogenic function and steroidogenic enzyme expression up to 30 days. The finding that nerve fibres are present in the transplants during the re-establishment of steroidogenic function and enzyme expression suggests that innervation may modulate the regeneration and functional recovery of adrenal transplants. In an attempt to prevent reinnervation of transplants, adrenal capsules were autotransplanted to denervated kidneys. Immunohistochemical analysis showed that, despite extensive denervation of the kidney tissue, the reinnervation and regeneration of the adrenal transplants still occurred. These data demonstrate the marked capacity of the regenerating adrenal to become reinnervated and reinforces the conclusion that adrenal transplants are an invalid model of denervated adrenal cortical tissue.     

Expression of utrophin (dystrophin–related protein) during regeneration and maturation of skeletal muscle in canine X–linked muscular dystrophy

The regulation of utrophin, the autosomal homologue of dystrophin, has been studied in the canine X–linked model of Duchenne muscular dystrophy. Dystrophic muscle has been shown to exhibit abnormal sarcolemmal expression of utrophin, in addition to the normal expression at the neuromuscular junction, in peripheral nerves, vascular tissues and regenerating fibres. To establish whether this abnormal presence of utrophin in dystrophic muscle is a consequence of continued expression following regeneration, or is attributable to a disease related up–regulation, the expression of utrophin was compared immunocytochemically with that of dystrophin, β–spectrin and neonatal myosin in regenerating normal and dystrophic canine muscle, following necrosis induced by the injection of venom from the snake Notechis scutatis. In normal regenerating muscle, sarcolemmal utrophin and dystrophin were detected concomitantly from 2–3 d post–injection, prior to the expression of β–spectrin. Down–regulation of utrophin was apparent in some fibres from 7 d, and it was no longer present on the extra–junctional sarcolemma by 14 d. Neonatal myosin was still present in all fibres at this stage, but dystrophin and β–spectrin had been fully restored. In dystrophic regenerating muscle, downregulation of utrophin occurred from 7 d, although it persisted on some fibres until 28 d, longer than in normal muscle. At 42 d, however, utrophin in dystrophic muscle was only detected in a population of small fibres thought to represent a second cycle of regeneration, with no immunolabelling of mature fibres. The results show that most utrophin is down–regulated in regenerating dystrophic fibres, prior to neonatal myosin, thus abnormal sarcolemmal expression of utrophin in dystrophic muscle is unlikely to be a continuation of the maturational process. Persistence of both utrophin and neonatal myosin, however, suggest a delay in the maturation of dystrophic muscle. In addition, a second cycle of degeneration and regeneration in dystrophic muscle does not occur whilst utrophin is still present, suggesting it may have a protective role against fibre damage and necrosis.     

Role of the basement membrane in the regeneration of skeletal muscle

In many experimental models of skeletal muscle damage and in human muscle disease, empty basement membrane tubes remain following the destruction of muscle fibres. In the present study we test the hypothesis that the empty basement membrane tubes play an essential role in the orientation of regenerating muscle fibres. Two groups of 15 Wistar rats were used. In one group, aqueous barium chloride (BaCl2) solution was injected into the right quadriceps muscle; in the other group, freshly prepared 2% trypsin solution was similarly injected. The different stages of muscle cell necrosis and regeneration were observed by histology, by immunofluorescence using an anti-basement membrane antibody, and by transmission (TEM) and scanning electron microscopy (SEM) in animals killed 1-77 days following injection. Although there was muscle fibre necrosis at sites of BaCl2 injection, empty basement membrane tubes were well preserved. Myoblasts grew along the empty basement membrane tubes and by 77 days, the regenerated muscle fibres at the site of the injection were well oriented. Trypsin not only destroyed muscle fibres but also destroyed the basement membrane tubes; in the early stages of regeneration the myoblasts were disorientated but by 77 days, regeneration was comparable to that seen in the barium chloride injected muscle. The results of this study suggest that preservation of empty basement membrane tubes is not essential for the orientation of regenerating myoblasts in skeletal muscle.     

Regeneration of myenteric plexus in the mouse colon after experimental denervation with benzalkonium chloride

Recent reports suggest a far greater plasticity in nerve tissue than previously believed. As the digestive tract is exposed to a variety of insults, this question is relevant to enteric nerves, but little is known about their ability to recover from damage. To address this problem, we ablated the myenteric plexus of the mouse colon with the detergent benzalkonium chloride (BAC) and followed the ensuing morphologic changes for up to 60 days by using light- and electron microscopy. We found that, 2 days after BAC application, the treated area was essentially devoid of intact nerve elements. From day 7, new nerve fibers were observed within the denervated region. This growth progressed until, at days 30-60, newly grown nerve fibers were present in most of this region, and the pattern of muscle innervation was similar to the normal one. At least part of these fibers originated at neurons within intact ganglia surrounding the denervated region. The cross-sectional area of neurons near the denervated region at day 14 was 52% greater than controls. Glial cells were closely associated with the regenerating nerve fibers. From day 14 onward, we observed undifferentiated cells and differentiating neurons in ganglia surrounding the denervated region, and by day 30, new neurons were present in the myenteric region, along with regenerating nerve fibers. We conclude that the myenteric plexus is endowed with a considerable ability of regeneration and plasticity. The results provide evidence for the presence of stem cells and for an adult neurogenesis in this plexus.     

NCAM, vimentin and neonatal myosin heavy chain expression in human muscle diseases

The intermediate filament protein vimentin, the neonatal isoform of the myosin heavy chain gene (MHCn), and the neural cell adhesion molecule (NCAM) are developmentally and/or neurally regulated molecules that reappear transiently after the induction of necrosis, or denervation. Immunostaining using antibodies against these molecules helps to identify regenerating and/or denervated muscle fibres even if they are not recognized by conventional staining procedures. This study examined the expression of vimentin, MHCn, and NCAM using immunohistochemistry in 82 biopsy specimens from muscular dystrophies, inflammatory myopathies, and neurogenic atrophies. Anti-vimentin labelled significantly more fibres than anti-MHCn staining in the inflammatory myopathies (P<0.03) but not in the muscular dystrophies (P=0.58) and neurogenic atrophies (P=0. 58). The fraction of NCAM+ fibres was always more elevated than vimentin+ or MHCn+ fibres. In the necrotizing myopathies, most NCAM+ fibres were regenerating ones (co-expressing vimentin). In neurogenic atrophies, half the NCAM+ fibres were regenerating and half of them were NCAM+/vimentin- and thus were considered to be denervated. Taken together, anti-vimentin staining detects a broader spectrum of regenerating fibres than anti-MHCn, at least in the inflammatory myopathies. The number of anti-NCAM labelled fibres in the necrotizing myopathies is similar, but not identical, to the number of regenerating fibres. Co-staining with anti-vimentin (or anti-MHCn) and anti-NCAM identifies a subset of fibres that is considered to be denervated.     

Muscle pathology in dysferlin deficiency

Dysferlin deficiency is being increasingly recognized in limb-girdle dystrophy and distal myopathy but its role in the development of muscle pathology is still poorly understood. For this purpose, 26 muscle biopsies from 25 dysferlinopathy patients were analysed by routine histochemistry and by immunohistochemistry with eight different antibodies, and scored for inflammatory response and type of cell infiltrate, fibre degeneration and regeneration, fibre type composition and severity of histopathological changes. In cases with an advanced-stage dystrophic pattern we observed type 1 fibre predominance exceeding 80%, suggesting a selective loss of type 2 fibres or a conversion process. The extent of muscle fibre regeneration and degeneration in dysferlinopathy was intermediate between sarcoglycanopathy and Duchenne dystrophy or myositis, suggesting a rather aggressive course of the disease. An increased inflammatory response was observed in the majority of our patients (16/26), who also showed an active dystrophic pattern. Type and localization of cellular infiltrates suggest that inflammatory reaction is secondary to necrosis. Major histocompatibility complex (MHC) class I molecules were overexpressed in dysferlinopathy, mainly in association with fibre phagocytosis and regeneration; their occasional expression in non-necrotic fibres might represent a marker of ongoing necrosis. Muscle inflammation might be triggered by the structurally altered membrane consequent to dysferlin defect.     

Regeneration distal to a prolonged conduction block.

In 6 baboons a tourniquet round the knee was used to produce a prolonged local conduction block. This was followed, within a few days, by a surgical crush of the tibial or deep peroneal nerve at the ankle, in order to produce Wallerian degeneration distally. Electrophysiological recordings from small foot muscles were then used to study the time-course of regeneration in motor fibres. When the results were compared with those from crushed but unblocked nerves of the opposite leg, there was no evidence that either reinnervation of muscles or the subsequent maturation of the regenerating motor nerve fibres was delayed by the prolonged proximal conduction block.     

EOSINOPHILIC FASCIITIS: AN ULTRASTRUCTURAL AND IMMUNOHISTOCHEMICAL STUDY OF THE INTERMEDIATE FILAMENT PROTEIN SKELETIN IN REGENERATING MUSCLE FIBRES

Thornell L.-E. & Bjelle A. (1981) Neuropathology and Applied Neurobiology 7, 435–449
Eosinophilic fasciitis: an ultrastructural and immunohistochemical study of the intermediate filament protein skeletin in regenerating muscle fibres
In a case of eosinophilic fasciitis with pronounced muscle involvement non-specific ultrastructural signs of degeneration were observed in areas of perifascicular atrophy. By combined immunohistochemical and electron microscopical techniques intermediate skeletin filaments were demonstrated. These filaments, when found in combination with nuclei with large nucleoli and an abundance of polyribosomes are a sign of regeneration of muscle fibres. This finding is a useful indicator in assessing the prognosis and therapy of muscle involvement in eosinophilic fasciitis.     

Reinnervation of a single vibrissa after nerve excision in the adult rat

After a survival time of 180 days following the excision of a 2 mm segment of the vibrissal nerve to the gamma straddler vibrissa in the adult rat, a retrograde fluorescent single-labelling experiment revealed that 46% of the injured vibrissal sensory neurones had regenerated their peripheral processes. Peripheral collateral sprouting was not involved in the reinnervation of the denervated gamma vibrissa, as proved by a retrograde fluorescent double-labelling experiment. The regenerating nerve fibres did not invade the intact neighbouring vibrissae of the gamma vibrissa, and the sensory nerve fibres of the intact vibrissae were not translocated to the denervated gamma vibrissa. Thus, the sensory function of the denervated gamma vibrissa was restored exclusively by the regeneration of the damaged vibrissal nerve.     

Regeneration of muscle axons in the frog is directed by diffusible factors from denervated muscle and nerve tubes

In the frog, peripheral muscle axons regenerate after a lesion to reinnervate the original synaptic sites on muscle fibers. Previous experiments in the frog have shown that satellite cells of the nerve tube direct the outgrowth of regenerating muscle axons over distances of many millimeters. In the present experiments, denervated muscle was used as a target for regenerating muscle axons. Muscle and satellite cells of the nerve tube also were placed in filters to determine if their influence on axonal outgrowth was exerted by diffusible factors. Filters were used with a pore size of 0.22 micron. With this pore size, target cells were isolated from physical contact with the surrounding cells; yet an exchange of fluids--and therefore of molecules released by the target cells--could occur across the filter. In the presence of denervated muscle or satellite cells of the nerve tube in filters, regenerating axons turn and grow toward the target cells. This influence on the direction of axonal outgrowth was produced over distances of 6 mm by muscles and 4 mm by cells of the nerve tubes. This directed outgrowth is in marked contrast to the random pattern of outgrowth in the absence of the targets. The present findings set the stage for tissue culture experiments in which the phenomena observed in vivo can be analyzed in terms of mechanisms. The present finding that denervated muscle attracts regenerating axons means that sufficient material may be available for the characterization and isolation of the relevant molecules.     

An ultrastructural study of the segmental uptake of horseradish peroxidase in the endplate region of denervated skeletal muscle fibres

The segmental uptake of horseradish peroxidase (HRP) in the endplate region of denervated skeletal muscle fibres has been studied ultrastructurally using a method for selecting single muscle fibres with high segmental peroxidase staining from denervated mouse tibialis anterior muscle. Segments containing large peroxidase positive phagosomes could already be seen 10-15 min after i.v. injection of HRP. Such segments were still present 24 h after HRP injection. The localization of phagosomes, deep in the fibres rather than immediately under the sarcolemma, suggests that the uptake occurs from t-tubuli. Vivid proliferation of t-tubuli, consisting of vesiculation, enlargement and encircling of cytoplasmic components, was also observed. The HRP accumulates in phagosomes of varying size and shape. Similar membrane-limited bodies without or with very weak peroxidase staining were also observed. The peroxidase-positive phagosomes participate in autophagic processes as suggested by their content of undegraded cellular material. Golgi profiles, which occurred deep in the muscle fibres, and enlarged components of the sarcoplasmic reticulum were frequently encountered in the segments. Myofibrillar degeneration occurs in the segments and progresses with time after denervation. The described segments may be related to the increased membrane turnover in denervated muscle fibres and/or they may be related to processes aimed at establishing new synaptic contacts.