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.     

Reorganization of cranial sympathetic pathways following neonatal ganglionectomy in the rat

Postganglionic sympathetic innervation normally is distributed ipsilaterally to lateral cranial targets. However, contralateral outgrowth occurs following unilateral ganglionectomy in neonatal rats. This study was conducted to determine the prevalence, morphological features, ganglionic derivations, and temporal sequence of sympathetic reinnervation of denervated cranial targets. Unilateral superior cervical ganglionectomy of mature rats revealed exclusively ipsilateral distributions of catecholaminergic histofluorescent fibers to orbital targets (Meibomian gland, tarsal muscle, orbital muscle, iris, ciliary body, vasculature) and the circle of Willis, with the exception of the anterior cerebral artery. In mature rats following neonatal unilateral ganglionectomy, all targets were reinnervated by fibers displaying morphologies and target relationships similar to normal innervation, but with lower densities. Acute excision of the remaining superior cervical ganglion eliminated all fibers in 7 of 8 preparations. In adult rats receiving neonatal bilateral superior cervical ganglionectomies, cerebral vasculature was reinnervated consistently, and orbital targets contained fluorescent fibers in 6 of 16 cases, indicating that reinnervation can derive from other sources when superior ganglion outgrowth is prevented. Observations in developing rats revealed fibers along the cranial portion of the contralateral optic nerve sheath at 2-3 days postganglionectomy, and within the orbit at later ages, reaching the most distal targets by 14 days. It is concluded that widespread sympathetic reinnervation of orbital and cerebrovascular targets derives primarily from the contralateral superior ganglion. Orbital ingrowth apparently originates intracranially and enters the orbit by an atypical pathway within the optic foramen.     

Long-term delivery of FGF-6 changes the fiber type and fatigability of muscle reinnervated from embryonic neurons transplanted into adult rat peripheral nerve

Motoneuron death leads to muscle denervation and atrophy. Transplantation of embryonic neurons into peripheral nerves results in reinnervation and provides a strategy to rescue muscles from atrophy independent of neuron replacement in a damaged or diseased spinal cord. But the count of regenerating axons always exceeds the number of motor units in this model, so target-derived trophic factor levels may limit reinnervation. Our aim was to examine whether long-term infusion of fibroblast growth factor-6 (FGF-6) into denervated medial gastrocnemius muscles improved the function of muscles reinnervated from neurons transplanted into nerve of adult Fischer rats. Factor delivery (10 microg, 4 weeks) began after sciatic nerve transection. After a week of nerve degeneration, 1 million embryonic day 14-15 ventral spinal cord cells were transplanted into the distal tibial stump as a neuron source. Ten weeks later, neurons that expressed motoneuron markers survived in the nerves. More myelinated axons were in nerves to saline-treated muscles than in FGF-6-treated muscles. However, each group showed comparable reductions in muscle fiber atrophy because of reinnervation. Mean reinnervated fiber area was 43%-51% of non-denervated fibers. Denervated fiber area averaged 11%. FGF-6-treated muscles were more fatigable than other reinnervated muscles but had stronger motor units and fewer type I fibers than did saline-treated muscles. FGF-6 thus influenced function by changing the type of fiber reinnervated by transplanted neurons. Deficits in FGF-6 may also contribute to the increase in type I fibers in muscles reinnervated from peripheral axons, suggesting that the effects of FGF-6 on fiber type are independent of the neuron source used for reinnervation.     

Embryonic neurons transplanted into the tibial nerve reinnervate muscle and reduce atrophy but NCAM expression persists

OBJECTIVE: The aim of this study was to use the glycogen depletion technique to determine whether reinnervated muscle fibers could be distinguished from denervated muscle fibers by their size or by neural cell adhesion molecule (NCAM) expression. METHODS: Medial gastrocnemius muscles of five adult Fischer rats were reinnervated from embryonic neurons transplanted into the distal stump of the tibial nerve. Ten weeks later, the transplants were stimulated repeatedly to deplete reinnervated muscle fibers of glycogen. Areas of reinnervated (glycogen-depleted) muscle fibers were measured and assessed for NCAM expression. The areas of muscle fibers from reinnervated, denervated (n=5) and unoperated control muscles (n=5) were compared. RESULTS: Mean reinnervated muscle fiber area was significantly larger than the mean for denervated fibers (mean +/- SE: 40 +/- 6 and 10 +/- 1% of unoperated control fibers, respectively). NCAM was expressed in 55 +/- 7% of reinnervated fibers (mean +/- SE; range: 42-77%). The mean areas of reinnervated fibers that did or did not express NCAM were similar. NCAM was only expressed in some fibers in completely denervated muscles. DISCUSSION: Our data show that NCAM expression does not differentiate muscle denervation or reinnervation. Quantifying the area of large fibers did distinguish reinnervated muscle fibers from denervated fibers and showed that reinnervation of muscle from neurons placed in peripheral nerve is a strategy to rescue muscle from atrophy.     

Properties of medial gastrocnemius motor units and muscle fibers reinnervated by embryonic ventral spinal cord cells

Severe muscle atrophy occurs after complete denervation. Here, Embryonic Day 14-15 ventral spinal cord cells were transplanted into the distal tibial nerve stump of adult female Fischer rats to provide a source of neurons for muscle reinnervation. Our aim was to characterize the properties of the reinnervated motor units and muscle fibers. Some reinnervated motor units contracted spontaneously. Electrical stimulation of the transplants at increasing intensity produced an average (+/- SE) of 7 +/- 1 electromyographic and force steps. Each signal increment represented the excitation of another motor unit. These reinnervated units exerted an average force of 12.0 +/- 1.5 mN, strength similar to that of control fatigue-resistant units. Repeated transplant stimulation depleted 17% of the muscle fibers of glycogen, an indication of some functional reinnervation. Reinnervated (glycogen-depleted), denervated (no cells transplanted), and control fibers were of histochemical type I, IIA, or IIB. Fibers of the same type were grouped after reinnervation. The proportion of fiber types also changed. Reinnervated fibers were primarily type IIA, whereas most fibers in denervated and control muscles were type IIB. Reinnervated fibers of each type had significantly larger cross-sectional areas than the corresponding fiber types in denervated muscles. These data suggest that neurons with different properties can reside in the unusual environment of the adult rat peripheral nerve, make functional connections with muscle, specify muscle fiber type, and reduce the amount that each type atrophies.     

Functional plasticity in the sympathetic nervous system of the neonatal rat

The age-dependent effects of sympathetic superior cervical ganglionectomy on smooth muscle alpha-adrenoceptor contractile responses were investigated in Müller's muscle in the superior eyelid of the Sprague-Dawley rat. Ganglionectomies in juvenile (32 days) and adult (70 days) rats produced sustained sympathetic denervation, determined by absence of contractile responses to electrical stimulation of ipsilateral and contralateral sympathetic chains and reduced responses to indirect release of nerve terminal norepinephrine by tyramine. Denervation was associated with diminished contractile responses and postjunctional supersensitivity to alpha-adrenoceptor stimulation by methoxamine. In contrast, contractile responses in rats ganglionectomized as neonates (day 3) were greater than those of other groups and sensitivity to methoxamine was comparable to control values. Responses to tyramine suggested that noradrenergic reinnervation had occurred. Electrical stimulation of the ipsilateral chain did not elicit contractions; however, stimulation of the contralateral chain evoked contractions in all neonatally ganglionectomized preparations and responses were present by 20 days of age. Contralateral reinnervation was derived from postganglionic sympathetic neurons with axons in the internal carotid nerve. This pathway is probably not formed by collateral sprouting of neurons with bilateral projections at the time of surgery for contralateral stimulation did not evoke contractions in the neonate. It is concluded that Müller's muscle is reinnervated by sympathetic neurons in the contralateral chain after denervation in neonates but not in older animals, and it is suggested that the higher potential for plasticity is attributable to the immaturity of the neonatal sympathetic nervous system.     

Reinnervation of long-term denervated rat muscle freely grafted into an innervated limb

This experiment was designed to test the hypothesis that in the presence of regenerating nerve fibers long-term denervated skeletal muscle does not become reinnervated. This hypothesis was tested in rats by the transplantation of 22-month denervated extensor digitorum longus (EDL) muscles into the sites of EDL muscles in the contralateral, normally innervated legs. Two months after transplantation, the muscles contracted when stimulated via the motor nerve, and based on silver-acetylcholinesterase staining, all grafts possessed innervated motor end plates. Compared to values for control EDL muscles in old rats, the maximum force developed by standard free grafts in old rats was 19% and that of long-term denervated grafts was 7%. For standard free grafts, nerve stimulation produced a maximum force that was 81% of that produced by direct stimulation, and for control EDL muscles in young and old rats, the values were 96 and 90%, respectively. These results show that after long-term denervation rat muscles are capable of becoming functionally reinnervated, even though by the time of reinnervation the animals have attained an advanced age of 26 months.     

Transient and chronic neonatal denervation of murine muscle: a procedure to modify the phenotypic expression of muscular dystrophy

The extensor digitorum longus muscles of 14-d-old normal (129 ReJ ++) and dystrophic (129 ReJ dy/dy) mice were denervated by cutting the sciatic nerve. One denervation protocol was designed to inhibit reinnervation of the shank muscles, the other to promote reinnervation. Chronically denervated muscles (muscles that remained denervated for 100 d after nerve section) exhibited marked atrophy, but the number of myofibers in these muscles (1066 +/- 46 and 931 +/- 62 for the denervated normal and dystrophic muscles, respectively) was similar to the number of myofibers found in age-matched, unoperated normal muscles [922 +/- 28 (Ontell et al., 1984)] and was significantly greater than the number of myofibers found in age-matched dystrophic muscles [547 +/- 45 (Ontell et al., 1984)]. Similar effects on myofiber number were obtained when denervated muscles were allowed to reinnervate. Reinnervation of both normal and dystrophic muscles mitigated the marked atrophy that characterized chronically denervated muscles. The dystrophic reinnervated muscles appeared "healthier" than age-matched, unoperated dystrophic muscles, having 70% more myofibers, less myofiber diameter variability, substantially less connective tissue infiltration, and a greater amount of contractile tissue at their widest girths. The present study demonstrated that it is possible to alter the phenotypic expression of the histopathological changes associated with murine dystrophy, in dystrophic myofibers that are formed during fetal development, by subjecting the muscle to neonatal denervation.     

Nimodipine Suppresses Preferential Reinnervation of Mouse Soleus Muscles by Slow α-Motoneurons

Denervation of mouse soleus muscle followed by self-reinnervation causes a significant increase in slow twitch (type I) muscle fiber content, suggesting preferential reinnervation by slow α-motoneurons. Since intracellular Ca²⁺influences both axonal elongation rate and branching, we examined the process of self-reinnervation in mouse soleus muscles in the presence of the L-type Ca²⁺channel blocker nimodipine. Soleus muscles in both control and experimental animals were denervated by crushing the soleus nerve where it enters the muscle. Experimental animals received a daily i.p. injection of a 0.1% nimodipine solution beginning 4 days prior to denervation and ending 2 weeks postdenervation. At 2 months postdenervation reinnervated and contralateral muscles from both control and experimental animals were sectioned and histochemically stained for myosin ATPase to determine the percentage of slow twitch fibers in the muscles. It was found that, in agreement with previous experiments, untreated reinnervated muscles had a significantly higher percentage of slow twitch fibers than did their contralateral controls (91.3 versus 74.6%). However, in nimodipine-treated animals only a small, but not statistically significant, difference between reinnervated and contralateral control muscles was observed (76.5 versus 72.8%). These results suggest that Ca²⁺influx through L-type calcium channels in growing neurites may play a role in the outcome of the reinnervation process.     

Recovery of muscle after different periods of denervation and treatments

Three aspects of reinnervation and recovery of skeletal muscle following various periods of denervation were investigated: (1) the effect of duration of denervation; (2) the effect of hyperthyroidism on recovery; and (3) whether the muscle or the nerve limits recovery. The rat medial gastrocnemius (MG) nerve was cut and then resutured after 0, 3, 7, 21, or 56 days. In a second group of animals, the MG muscle was denervated and, in addition, the animal received triiodothyronine (T₃) supplementation during reinnervation. The third group of animals had the denervated MG muscle reinnervated by a larger number of newly transected foreign axons. The force produced by the reinnervated muscle depends on the period that the muscle was denervated. Recovery was impaired when the period of denervation exceeded 7 days. T₃ treatment did not benefit the return of force production, nor did providing the muscle with a larger number of newly transected axons.     

Transplantation of primary satellite cells improves properties of reinnervated skeletal muscles

Skeletal muscle demonstrates a force deficit after repair of injured peripheral nerves. We tested the hypothesis that transplantation of satellite cells into reinnervated rabbit tibialis anterior (TA) muscles improves their properties. Adult rabbits underwent transection and immediate suture of the common peroneal nerve. In order to provide an environment favorable for cell transplantation, TA were then made to degenerate by cardiotoxin injection, either immediately or after a 2-month delay, which is sufficient for muscle reinnervation. In both cases, the injured TA were transplanted with cultured satellite cells 5 days after induction of muscle degeneration. When cells were transferred immediately after nerve repair, drastic morphological and functional muscle alterations were observed. However, when the muscles were allowed to become reinnervated before cell transplantation, muscles were heavier and developed a significantly higher maximal force compared to denervated-reinnervated muscles. Thus, application of the cell therapy protocol improved properties of denervated muscles only when they were allowed to become innervated. These results, which represent the application of cell therapy to improve force recovery of reinnervated muscles, will be of significant interest in certain clinical contexts, particularly after immediate or delayed muscle reinnervation.     

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.     

Effect of electrotherapy on denervated muscles in rabbits: An electrophysiological and morphological study

The effects of electrotherapy on muscles denervated by crushing the sciatic nerve were studied in rabbits using electrophysiologic and morphologic techniques. The sciatic nerve was crushed in the thigh, just above its division into the lateral and medial popliteal nerves and the completeness of denervation was ascertained by EMG tests. Electrotherapy was carried out in six animals and six others were denervated controls; the contralateral leg of each animal was used as the normal control. The first appearance of signs of reinnervation was detected by means of EMG tests and the “reinnervation time” established. The animals were killed at the 50th day after nerve crush; the length of nerves below the crushed point was measured; soleus and extensor digitorum longus (EDL) muscles were removed and weighed. The percentages and the mean diameter of different muscle-fiber types in normal, control-denervated, and treated soleus and EDL muscles were calculated. No statistically significant difference was observed between electrophysiologic findings in control-denervated and treated rabbits. Electrotherapy was found to antagonize the weight loss of EDL muscles and the reduction of type 2b fiber diameters induced by denervation, but significantly accentuated the reduction of the mean diameter of type 1 fibers in EDL and soleus and increased the weight loss of denervated soleus muscles. The results obtained raise further doubts about the clinical utility of electrotherapy.     

Restoration of denervated skeletal muscle transplants after reinnervation in rats

The present study describes reinnervation and restoration of rat skeletal muscle denervated for the duration of 3, 6 or 12 months. Denervation of extensor digitorum longus (EDL) muscle was achieved by cutting and ligating the donor rat sciatic nerve in situ. At 3, 6 and 12 months, the denervated EDL muscles were removed and transplanted into an innervated normal leg of another rat. In addition, normal (i.e., no prior denervation) muscles were transplanted as controls for comparison. The muscles were analyzed at 4 and 12 weeks after transplantation. The EDL muscle weight and myofiber size decreased with extended denervation times. After transplantation, the muscles underwent regeneration and reinnervation, and recovered as determined by an increase in muscle mass and myofiber size. The 3-month denervated muscle regenerates recovered completely, and were similar to the non-denervated normal muscle regenerates. Reinnervation, and partial recovery of muscle weight and myofiber size was observed in 6- and 12-month denervated muscle transplants. These results document that while regeneration and reinnervation does occur in denervated muscles after transplantation, the extent of recovery is related to the duration of denervation.     

Glycosaminoglycans treatment increases IGF-I muscle levels and counteracts motor neuron death: A novel nonanticoagulant action

The present study shows that sciatic nerve crush in 2-day-old rats causes extensor digitorum longus (EDL) muscle atrophy and motor neuron loss and that treatment with glycosaminoglycans (GAGs) promotes muscle reinnervation, motor neuron survival, and markedly increases insulin-like growth factor-I (IGF-I) content in the denervated muscles. EDL muscle denervation-induced atrophy in saline-treated rats is progressive and reaches the greatest extent at 42 days after birth, which correlates with reduced EDL weight growth. There is also a partial reinnervation as shown by the number of reinnervated EDL muscle fibers (65.4% of control) and by the poor restoration of the indirect isometric twitch tension (62% of control) that is further reduced under tetanic stimulation (34% of control). The number of surviving motor neurons that innervate EDL muscle drops from 55 +/- 3 to 29 +/- 8. In GAGs-treated 42-day-old rats, the effects of neonatal nerve lesioning on EDL muscle atrophy and denervation are successfully reversed, and the isometric twitch tension and the capacity to hold tetanic stimulation are restored to almost control levels. The number of surviving EDL motor neurons is also increased to 43 +/- 4. Treatment with GAGs selectively affects IGF-I content in denervated hindlimb muscles, which is augmented from 7.02 +/- 0.71 ng/mg tissue to 25.72 +/- 0.7 in the EDL and from 3.2 +/- 0.18 to a robust 211 +/- 9.6 in the soleus.     

Modulation of parasympathetic neuron phenotype and function by sympathetic innervation

Selective sympathetic nerve dysfunction occurs during aging and in certain disease states. Here, we review findings concerning the effects of chronic sympathetic denervation on parasympathetic innervation to orbital target tissues in the adult rat. Long-term sympathetic denervation was induced by excising the ipsilateral superior cervical ganglion for 5-6 weeks prior to analyses. Following sympathectomy, pterygopalatine ganglion parasympathetic neurons show reduced nitric oxide synthase protein in their somata and projections to vascular targets. Laser Doppler measurements of ocular blood flow indicate that sympathectomy is also accompanied by reduced nitrergic vasodilatation. In the superior tarsal muscle of the eyelid, parasympathetic varicosities, normally, are distant to smooth muscle cells but make axo-axonal contacts with sympathetic nerves, consistent with physiological evidence showing only prejunctional inhibitory effects on sympathetically mediated smooth muscle contraction. Following sympathectomy, parasympathetic varicosities proliferate and closely appose smooth muscle cells, and this is accompanied by establishment of parasympathetic-smooth muscle excitatory neurotransmission. Many pterygopalatine parasympathetic neurons normally contain nerve growth factor (NGF) protein and express NGF mRNA. However, following chronic sympathectomy or elimination of sympathetic impulse activity, NGF mRNA and protein are markedly reduced, indicating that sympathetic neurotransmission enhances NGF expression in parasympathetic neurons. Together, these findings portray a striking dependency of parasympathetic neurons on sympathetic nerves to maintain normal phenotype and function. Sympathetic influences on parasympathetic neurons may be mediated, in part, through axo-axonal synapses. NGF synthesis and release by parasympathetic neurons may represent a molecular basis underlying the formation of these synapses, and up-regulation of NGF synthesis by sympathetic nerve activity may act to reinforce these associations.     

Myotrophic effects on denervation atrophy of hindlimb muscles of mice with systemic administration of nerve extract

Atrophy was assessed in denervated hindlimb muscles of adult mice which either were not otherwise treated or received daily intraperitoneal injections of extract of rats' sciatic nerves. After 7 days, the denervated muscles of injected animals exhibited significantly smaller decreases in wet weight, total protein and cross-sectional areas of muscle fibers relative to innervated contralateral control muscles. The effects of denervation and nerve extract on different muscles varied.     

Synaptic localization of a 66-kDa soluble protein from skeletal muscle: evidence for its developmental and neural regulation

Soluble proteins from normal, adult denervated, and developing rat muscles were studied in order to identify common molecular species undergoing developmental regulation and nerve dependence. Significant increases in 66- and 30-kDa proteins were found as a consequence of 14 days of denervation. Subsequent reinnervation restores normal adult levels. During development, high levels of the 66-kDa protein were found in neonatal muscles but slowly decreased concomitant with the following postnatal maturation period; the adult levels were reached at Postnatal Day (P) 21. From the immunocytochemical studies it is deduced that both proteins were concentrated mainly at the end-plate region in adult normal muscle. Following denervation, the proteins were found distributed over the entire cell. For the 66-kDa protein, a similar pattern of extensive distribution was seen in immature muscle. Although no data for functional implications for these proteins are available at present, the properties described here make them of interest in understanding nerve-muscle interactions.     

Influence of atrophy on the efficiency of muscle reinnervation

The effectiveness of motor reinnervation carried out after an interval of 20 days between nerve transection and reimplantation onto a foreign muscle was studied in the rat. Sixty days after reimplantation the compound action potential, the maximal indirect twitch and tetanic tensions, and weight loss were evaluated. The functional data demonstrated an incomplete recovery which differed from the complete functional restoration observed in an acutely denervated muscle reinnervated with a chronically severed nerve. Time-dependent changes induced in the muscle by denervation seem therefore to influence the efficacy of motor reinnervation.