Reinnervation of a striated muscle by vagal sensory axons

Fibres of the sterno-cleido-mastoid (s.c.m.) muscle normally innervated by effects of the accessory nerve have been reinnervated by afferent fibres of the vagus nerve after supranodose vagal-accessory nerve anastomoses or direct implantation of the vagus nerve into the s.c.m. in 58% of the rabbits, 60% of the cats and 75% of sheep in which experiments were performed. Afferents of the vagus growing from cell bodies of the nodose ganglion after severance of central connections can replace the efferent of motor supply to the muscle. Evidence that there was reinnervation of the s.c.m. muscle by vagal afferent fibres was provided from the observations that: (i) electrical stimulations of the anastomosed cervical vagus nerve elicited potentials in the s.c.m. muscle which were abolished by local anaesthesia or final section of the nerve proximal to the site of stimulation; (ii) discharges recorded as bursts of electromyographic potentials occurred during spontaneous movements of larynx, respiratory tract, oesophagus and stomach and on their mechanical or evoked stimulation; and (iii) horseradish peroxidase injected into the reinnervated s.c.m. muscle was detected in somata of ipsilateral nodose ganglia cells. The afferent fibres contributing to the reinnervation were confirmed to be cholinergic as transmission was blocked by gallamine and histochemical evidence obtained of cholinergic motor end-plates. Factors which may have limited the small extent of reinnervation--only one vagal sensory axon out of 600 is able to form functional connections--are discussed.     

Reinnervation of denervated skeletal muscle by central neurons regenerating via ventral roots implanted into the spinal cord

The reinnervation of denervated skeletal muscle by central axons regenerating via a ventral root implanted into the spinal cord was examined in rats. The 8th thoracic ventral root was severed and its distal end implanted into the ventro-lateral column of the spinal cord via a stab incision. In control animals the root was severed, but was not implanted into the stab incision. After 12-14 months the animals were examined electrophysiologically to determine the presence or absence of motor units in the 8th intercostal muscle which were reinnervated by centrally derived axons regenerating via the implant. Such units were found in implanted animals, but in none of the controls. Evidence that the motor units were reinnervated by central axons included the facts that the units could be activated either, (1) reflexly (i.e. trans-synaptically) by electrical stimulation of the dorsal roots or spinal cord, or (2) pharmacologically by either the intraspinal injection of glutamate or acetycholine, or by the systemic administration of strychnine. Great care was taken to ensure that the only feasible connection between the spinal cord and the 8th intercostal muscle was via the site of implantation. The EMG signals from the motor units were of large amplitude, typical of reinnervated muscle, and their individual activation resulted in discernible contractions of regions of the T8 intercostal muscle. We conclude that regenerating CNS neurons can be guided to innervate denervated skeletal muscle by the implantation of severed ventral roots into the spinal cord. The neuromuscular synapses formed are functional and persistent. The findings may be relevant to the restoration of function after nervous injuries, such as the avulsion of ventral roots.     

Abnormalities in the vagus nerve in canine acrylamide neuropathy

Dogs exposed to acrylamide develop a sensorimotor peripheral neuropathy and megaoesophagus. The presence of neuropathy was confirmed electrophysiologically and histologically. Hindlimb motor conduction velocity was reduced and there was a loss of large diameter myelinated fibres in the dorsal common digital nerve and the tibial nerve. The conduction velocity of vagal motor fibres innervating the thoracic oesophagus was not decreased; there was a reduction in the conduction velocity of the mixed nerve action potential of the vagus. Degenerating nerve fibres were observed in the vagus in the midthoracic region. The damage to vagal nerve fibres may be an important factor in the causation of megaoesophagus.     

Sensory denervation of the plantar lumbrical muscle spindles in pyridoxine neuropathy

Summary Male albino rats treated with excessive amounts of pyridoxine developed an impairment of neuromuscular function. The equatorial region of the plantar lumbrical intrafusal muscle fibres was studied in the electron microscope and the calibre of the nerve fibres was determined in semi-thin sections of the posterior tibial nerves. Degeneration of the primary sensory endings coincided with the onset of ataxia, and in more advanced stages of the neuropathy as well as after a 2-month treatment-free period the equatorial region was denervated. There was a corresponding decrease in the number of large nerve fibres. It is considered essential that primary sensory endings of lumbrical muscle spindles should be included in studies of distally accentuated sensory neuropathies.     

Reinnervation of denervated lumbar ventral roots and their target muscle by thoracic spinal motoneurons via an implanted nerve autograft in adult rats after spinal cord injury.

Intraspinally implanting a nerve autograft (NAG) to promote axonal regeneration toward periphery was investigated as a surgical treatment for spinal cord injury in adult rats. Fifteen animals underwent a left hemisection of the spinal cord at T12 level and an intradural section of all ipsilateral lumbar ventral roots. In repaired animals (n = 9), the electrophysiologically selected left L3 and L4 lumbar ventral roots supplying the quadriceps muscle were anastomosed to a NAG. The NAG was taken from the right peroneal nerve and then ventrolaterally implanted into the cord at a level 7 mm rostral to the hemisection. In the control group (n = 6), sectioned lumbar ventral roots were left unrepaired. Nine months later, the animals were assessed with clinical, electrophysiological, and histological examinations. Muscle action potential and motor evoked potential were obtained from the denervated/reinnervated quadriceps in all repaired animals, with a mean amplitude of 918.3+/-328.9 microV and 215.8+/-39.7 microV, respectively. Horseradish peroxidase retrograde labeling from the denervated/repaired lumbar ventral roots, performed in five repaired animals, showed that the mean of labeled neurons, ipsilaterally located in the thoracic ventral horn near the implantation site, was 145.8+/-111.7. Histological analysis showed numerous myelinated axons in the NAG and denervated/repaired lumbar ventral roots of all repaired animals. The study of neuromuscular junctions furthermore confirmed numerous newly formed endplates appearing in the denervated/reinnervated quadriceps. These changes were absent in the control animals. These data indicate that the rostral thoracic spinal motoneurons can innervate the caudal denervated/repaired lumbar ventral roots and the target quadriceps via an implanted NAG, thereby inducing some functional recovery in adult rats after lower thoracic spinal cord injury.     

DNA‐fragmentation and expression of apoptosis‐related proteins in experimentally denervated and reinnervated rat facial muscle

Muscle fibres may undergo apoptotic cell death in several neuromuscular disorders such as denervated muscle fibres in spinal muscular atrophies. We investigated DNA‐fragmentation ( in situ by the TUNEL‐method) and expression of apoptosis‐associated proteins in experimentally denervated and reinnervated rat facial muscle up to 24 weeks after surgery to evaluate the rate and time lapse of apoptotic muscle fibre loss. While denervated muscle displayed constantly high rates of DNA‐fragmentation, denervated and immediately reinnervated muscle showed a distinct decrease of primarily elevated DNA‐cleavage, finally resembling rates of normal controls. Denervated muscle fibres revealed strong immunoreactivity of the anti‐apoptotic proteins bcl‐2 and bcl‐xL, and the pro‐apoptotic factor bax. In reinnervated muscle fibres, only bcl‐2 was constantly up‐regulated while bcl‐xL and bax diminished after the 7th week. The present findings indicate that denervation may prompt muscle fibres to activate an intrinsic 'suicide' programme to undergo apoptosis. High levels of bcl‐2 after denervation may sustain cell survival until reinnervation, e.g. after accidental nerve damage or in neurodegenerative disorders. Furthermore, increasing levels of bcl‐2 are able to neutralize high apoptosis‐promoting bax levels. Interventions modifying DNA‐fragmentation and the expression of apoptosis‐related proteins may lead to new therapeutic concepts in denervating disorders of muscle in the absence of other primary therapies.     

Motor innervation of the striated oesophagus muscle: Part 1. Intramural distribution of the right and left vagus nerve in the rat oesophagus as revealed by the glycogen depletion technique

The muscularis propria of the rat oesophagus is entirely made up of striated muscle fibres. All fibres are of the same histochemical type, which is characterized by high activity of actomyosin ATPase, medium activity of oxidative enzymes and relatively strong reaction for phosphorylase. Prolonged stimulation (10 Hz, 30 min) of the vagus nerves causes depletion of the glycogen content of the oesophageal muscle fibres. This stimulation effect can be visualized by means of the PAS technique as well as by the histochemical reaction for phosphorylase.In 8 animals the right and in 8 animals the left vagus nerve were stimulated repetitively and the stimulated muscle fibres were identified in transverse sections of the oesophagi, stained for phosphorylase. The muscle fibres supplied by one vagus nerve are distributed all over the circumference of the oesophagus. In the upper third of the oesophagus stimulation of either vagus nerve depletes slightly less than 50% of the muscle fibres, whereas in the lower two-thirds the right vagus nerve seems to predominate to a certain degree.In 3 animals both vagus nerves were stimulated simultaneously. Bilateral stimulation produced a very extensive depletion. Only a few muscle fibres remained unaffected. Functional implications of the results, the question of polyneuronal innervation and the role of the myenteric plexus are discussed.     

Parvalbumin immunohistochemistry in denervated skeletal muscle

Parvalbumin is a calcium–binding protein which, in muscle, is mainly found in type 2B fibres, whereas type 1 fibres lack parvalbumin immunoreactivity. Previous studies have shown that this pattern is highly dependent upon motor neuron innervation and is modified in denervated, cross–reinnervated or chronic low–frequency stimulated muscles. In the present study, we have examined the modifications of parvalbumin immunocyto–chemistry in the anterior tibialis muscle of the rat at different intervals following section of the sciatic nerve. During the first 2 weeks after denervation, no changes in parvalbumin immunoreactivity were seen, although a global reduction of fibre diameter was observed. Three weeks after denervation, small angulated. strongly parvalbumin–immunoreactive fibres appeared. From the second month onwards, the pattern of parvalbumin immunohistochemistry was characterized by areas composed of small, strongly immunoreactive fibres separated by less atrophic areas displaying a normal chequerboard distribution of parvalbumin immunoreactivity. The increase of parvalbumin–immunoreactivity in denervated and reinnervated muscle, as seen in our study, indicates that important changes in parvalbumin distribution occurs in muscle fibres after denervation. These changes are probably produced in an attempt to bind the free cytosolic calcium which accumulates in denervated fibres, and further reinforces the role of parvalbumin in calcium homeostasis during denervation and reinnervation.     

Growth and denervation response of skeletal muscle fibers of newborn rats

The cross-sectional area of the fibers of hindlimb muscles of rats increased 10-40 times during the first 6 weeks after birth. Denervation at birth stopped the growth of the muscle fibers. The number of satellite cells decreased, and eventually all fibers vanished. Reinnervation, if any, was poor. Partial denervation did not induce collateral reinnervation. Some denervated gastrocnemius muscles were reinnervated and after 8-12 months contained hypertrophic fibers and signs of necrosis and regeneration. When soleus muscles were completely denervated and cross-reinnervated after 4 weeks by the peroneal nerve, only half as many fibers became reinnervated after neonatal denervation as compared to muscles denervated at the age of 4 weeks. The experiments suggest that immature muscle fibers are less apt to become reinnervated than mature fibers. The few reinnervated fibers may be overloaded and therefore hypertrophy and eventually necrotize. Regeneration is abortive because satellite cells are scarce. These results may be relevant for the understanding of neuromuscular disorders with early (fetal) onset.     

Enteric co-innervation of striated muscle fibres in human oesophagus

Abstract  Oesophageal striated muscle of several mammalian species receives dual innervation from both vagal motor fibres originating in the brain stem and enteric nerve fibres originating in myenteric ganglia. The aim of this study was to investigate this so-called enteric co-innervation in the human oesophagus. Histochemical and immunohistochemical methods combined with confocal laser scanning microscopy were utilized to study innervation of 14 oesophagi obtained from body donors (age range 47–95 years). In addition, the distribution of striated and smooth muscle in longitudinal and circular layers of the tunica muscularis was studied semiquantitatively. The upper half of the oesophagus was built up of both muscle types with a predominance (>50–60%) of striated muscle, whereas the lower half consisted of smooth muscle only. The majority of motor endplates was compact and ovoid. Enteric nerve fibres on ∼17% of motor endplates stained for neuronal nitric oxide synthase, vasoactive intestinal polypeptide, galanin and neuropeptide Y and were completely separated from vagal cholinergic nerve terminals. There was remarkable variability of co-innervation rates between striated muscle bundles with some reaching almost 50%. Myenteric neurons representing the putative source of enteric co-innervating nerve fibres, stained for all these markers, which were almost completely colocalized with NADPH-diaphorase. Our study provides evidence for enteric co-innervation of striated muscle in human oesophagus. From these and recent functional results in various rodent species, we suggest that this innervation component represents an integral part of an intramural reflex mechanism for local most likely inhibitory modulation of oesophageal motility.     

The location of neuromuscular junctions on regenerating adult mouse muscle in culture

The location of neuromuscular junctions which form in vitro between regenerating adult mouse muscle fibres and sections of embryonic mouse spinal cord was examined. The position of the original motor end-plates on the explanted muscle fibres was determined by using either rhodamine-labelled α-bungarotoxin (RαBT) binding to the acetylcholine receptors, or by stains to demonstrate acetylcholinesterase (AChE) also located at the end-plate. In this culture system, the explanted muscle fibres degenerate and regenerate to form new myotubes which develop cross-striations and contractions. The location of the newly-formed neuromuscular junctions in these mature cultures was then demonstrated using RαBT-binding to acetylcholine receptors, silver impregnation and cholinesterase techniques. Less than half the new neuromuscular junctions were at the original end-plate areas indicating that, at least in this system, junctions can form at sites other than those of the original end-plate.     

Recovery of rat skeletal muscles after partial denervation is enhanced by treatment with nifedipine

Following partial denervation of adult rat skeletal muscle intact axons sprout to reinnervate denervated muscle fibres and increase their territory. The extent of this increase is limited and may depend on the ability of axon terminals to form and maintain synaptic contacts with the denervated muscle fibres. Here we tested the possibility whether reducing Ca²⁺ entry into presynaptic nerve terminals through dihydropyridine sensitive channels may allow more nerve–muscle contacts to be formed and maintained. Hindlimb muscles of adult Wistar rats were partially denervated by removing a small segment of the L4 or L5 spinal nerve on one side. A nifedipine-containing silastic rubber strip was subsequently implanted close to the partially denervated soleus or extensor digitorum longus (EDL) muscles in some animals. In control experiments silastic strips which did not contain nifedipine were used. Several weeks later isometric contractions were recorded, to determine the effect of (a) partial denervation and (b) nifedipine treatment on force output and motor unit numbers. The tension produced by nifedipine treated partially denervated muscles was 82% and 79% of the unoperated contralateral value for soleus and EDL, respectively. This was significantly greater than in untreated muscles, which only produced 61% and 48%, respectively. Mean motor unit force was also significantly larger with nifedipine treatment. Histological analysis revealed that a significantly larger proportion of the total number of muscle fibres remained in nifedipine-treated partially denervated muscles (soleus, 90% and EDL, 101%) compared with untreated muscles (soleus, 51% and EDL, 66%). Thus the number of neuromuscular contacts was increased with nifedipine treatment.     

The pathology of the muscle spindle in myasthenia gravis.

Abnormalities in the morphology and motor innervation of the muscle spindles are described in 4 autopsied cases of myasthenia gravis. There were changes consistent with motor denervation in 5 of 118 spindles examined in sectioned material. In 65 teased, silver-impregnated spindles there was proliferation of the fusimotor innervation due to axonal sprouting occurring in the endings themselves. Motor end-plates on the intrafusal muscle fibres showed "dystrophic" changes similar to those previously described in extrafusal muscle in this disease. The sensory innervation was normal. The possible signifance of these findings is discussed.     

The origin and development of the vagal and spinal innervation of the external muscle of the mouse esophagus

Retrograde and anterograde tracing and immunohistochemical techniques were used to examine the origin of the extrinsic innervation, and the development of the vagal innervation to the mouse esophagus. Cholinergic nerve terminals were localised using an antiserum to the vesicular acetylcholine transporter and cholinergic cell bodies were localised using an antiserum to choline acetyltransferase. Cholinergic nerve terminals, which also contained calcitonin gene-related peptide, were present at the motor end plates in the external (striated) muscle of the esophagus. Following injection of Fast Blue into subdiaphragmatic or cervical levels of the esophagus, the only retrogradely-labelled cholinergic nerve cell bodies that also contained calcitonin gene-related peptide were found in the nucleus ambiguus. Neurons in the dorsal motor nucleus of the vagus, the nodose ganglia and dorsal root ganglia gave rise to a number of different types of nerve terminals within the myenteric plexus. Retrogradely-labelled neurons in the dorsal motor nucleus of vagus contained cholinergic markers only, nitric oxide synthase only or cholinergic markers plus nitric oxide synthase, retrogradely-labelled neurons in the dorsal root ganglia contained calcitonin gene-related peptide only, and a small number of retrogradely-labelled neurons in the nodose ganglia contained tyrosine hydroxylase. The development of the vagal innervation to the esophagus was examined following application of DiI to the vagus nerve of fixed mouse embryos. Anterogradely-labelled nerve fibres, which arose from both nodose ganglia and the medulla, were already present in the esophagus of embryonic day 12 (E12) mice. Some of the DiI-labelled vagal nerve fibres were present in among the smooth muscle cells of the external muscle layer prior to their transdifferentiation to striated muscle. We conclude that the neurons in the nucleus ambiguus that project to the esophagus differ from other extrinsic neurons in their chemistry as well as their targets within the esophagus. The development of the extrinsic innervation precedes the transdifferentiation of the external muscle to striated muscle, raising the possibility that, during development, smooth muscle of the esophagus is innervated transiently by vagal neurons.     

Regeneration of taste buds after reinnervation of a denervated tongue papilla by a normally nongustatory nerve

Taste buds degenerate and disappear after transection of their sensory nerve supply, and they differentiate anew from epithelial cells (e.g., lingual) following regeneration of sensory but not motor or autonomic axons. A controversy exists as to whether only gustatory sensory nerves can cause buds to reform or whether any sensory nerve can perform this function. This issue arose because the results of cross-innervation studies revealed a specificity whereas grafting data demonstrated a nonspecificity. A retest of specificity in the cross-reinnervation situation was performed by reinnervating the denervated vallate papilla of adult rat tongue with a sensory branch of the vagus nerve that is not normally gustatory. It was found that taste buds disappeared and remained lost from acutely and chronically denervated papilla. However, some buds were found 90–100 days after reinnervation by the normally nongustatory vagus nerve branch. Transection of the regenerated vagus nerve resulted in the loss of innervation and the degeneration of taste buds from reinnervated papilla indicating that this nerve had supported buds. These results show that a normally nongustatory nerve can induce the formation of taste buds after its axons grow into appropriate tissue. It appears that the ability to support taste buds is a nonspecific, rather than a specific, property of sensory nerve.     

Reinnervation of transplanted vas deferens by cholinergic nerves normally supplying skeletal muscle

The rat vas deferens was removed and either transplanted alongside the soleus muscle or into the bed of the soleus muscle that had previously been removed, and in this case the soleus nerve was connected to the transplant. The vas deferens reinnervated by the somatomotor nerve recovered the best. Contractions to transmural electrical stimulation could not be elicited from the denervated vas deferens, although noradrenaline and acetylcholine elicited contractions. The reinnervated vas deferens produced good contractile responses to transmural stimulation, and these were substantially reduced by a cholinergic muscarinic blocking agent, hyoscine, as compared to only a small reduction in the control vas deferens. Neostigmine potentiated the contraction of the transplanted vas deferens to a greater extent than that of the control. This indicated that a substantial component of the contractile response was produced by cholinergic fibres. Consistent with this was the finding that, while guanethidine blocked a greater proportion of the contraction in the control vas deferens, the contraction of the reinnervated transplant was less affected. Acetylcholine elicited a strong contraction in control vas deferens, but only a small response was obtained in the reinnervated transplant. However, the response to noradrenaline was greater in the transplant than in the control vas deferens. These results indicate that cholinergic nerves normally supplying skeletal muscle can reinnervate smooth muscle and that the alien somatomotor innervation altered the responsiveness of the smooth muscle of the vas deferens. Morphological studies confirm the shift from adrenergic to cholinergic fibres in the reinnervated vas deferens.     

Peripheral synaptogenesis in nerve parabiosis

One hundred and ninety-two rats were parabiotically coupled to test regeneration of the sciatic nerve of the “donor” rat in the denervated gastrocnemius of the “host.”. Proliferation of the implanted nerve was clearly demonstrated after 30 days. Histological and histochemical study revealed the formation of motor end-plates of increasing complexity and after survival of 300 days mature motor end-plates were observed. The electrophysiological study demonstrated that the muscle responded to stimulation of the nerve with normal stimulus. The immunological interaction did not interfere with the formation of “functional” motor end-plates and central nerve centers of the “donor” rat are capable of eliciting motor activity of the limb of the “host.”.     

Neural nature of the time-dependent factor(s) involved in motor reinnervation

The influence of a time interval between nerve transection and reimplantation into a foreign muscle on the effectiveness of reinnervation was studied in the rat. We evaluated the weight loss and the maximal twitch and tetanic tensions developed by the reinnervated muscle upon nerve stimulation 60 days after reimplantation (i) of an acutely severed nerve onto an acutely denervated muscle; (ii) of a chronically (20 days) severed nerve onto an acutely denervated muscle, and (iii) of an acutely severed nerve onto a chronically denervated muscle. The best recovery was obtained when a chronically severed nerve was implanted into an acutely denervated muscle. We conclude that a neural time-dependent factor(s) is involved in motor reinnervation.     

Inhibition of Experimental Seizures in Canines by Repetitive Vagal Stimulation

Repetitive electrical stimulation of the canine cervical vagus nerve interrupts or abolishes motor seizures induced by strychnine and tremors induced by pentylenetetrazol (PTZ). Tremors were defined as rhythmic alternating contractions of opposing muscle groups, exerting much less force than seizure contractions. Seizures were induced by injection boluses of strychnine or PTZ at 1- to 4-min intervals until sustained muscle activity was observed electromyographically (EMG). Vagal stimulation terminated seizures in 0.5-5 s. There were prolonged periods with no spontaneous EMG activity after stimulation. The period of protection was approximately four times the stimulation period. The antiseizure actions of vagal stimulation were not altered by transection of the vagus distal to the stimulating electrode. Optimal stimulus parameters were estimated: strength, approximately 20 V (electrode resistance 1-5 omega); frequency 20-30 Hz; duration, approximately 0.2 ms. These data suggest that the antiseizure effects derive from stimulation of small-diameter afferent unmyelinated fibers in the vagus nerve. These results may form the basis of a new therapeutic approach to epilepsy.     

Continuous low amplitude direct current stimulation of the crushed peripheral nerve accelerates the early recovery of choline acetyltransferase but not of acetylcholinesterase activity in fast and slow muscles

We investigated if continuous 1 μA direct current stimulation of the injured nerve, with the cathode electrode at the distal end of the nerve crush injury (cathode stimulation), accelerated the recovery of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity in transiently denervated extensor digitorum longus (EDL) and soleus (SOL) rat muscles. ChAT is a specific marker of cholinergic nerve terminals and may reflect axon ingrowth, and AChE reflects the re-establishment of neuromuscular junctions and recovery of muscle activity. Compared to sham operated animals, the cathode (CA) stimulated rats had a statistically significant larger ChAT activity in the EDL and SOL muscles on days 12 and 14 after nerve crush (P < 0.01, n = 6). The difference in ChAT activity between the groups decreased thereafter. Regarding recovery of muscle AChE, CA stimulation of the crushed sciatic nerve did not detectably accelerate the normalization of activity and pattern of AChE molecular forms in the EDL and SOL muscles. This means that the early rise in ChAT muscle activity in CA stimulated rats was not followed by an accelerated normalization of the neuromuscular transmission in the same group. It is more likely that the higher ChAT activity observed after cathode stimulation indicates a higher ChAT content in regenerating motor nerve endings, rather than a greater number of motor axons entering the muscles. It seems possible that cathode stimulation increased ChAT axonal transport, causing the early increase of ChAT content in the nerve endings. This raises the possibility that the axon transport and subsequent secretion of a trophic factor(s) from the nerve to the reinnervated muscle are enhanced as well, thus shortening the overall time of muscle force recovery in the absence of an appreciable acceleration of recovery of the neuromuscular transmission.