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.     

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.     

Number of contractions to maintain mass and force of a denervated rat muscle

Within 5 weeks, denervated extensor digitorum longus (EDL) muscles of rats lose 66% of mass, 91% of force, and 76% of fiber cross-sectional area (CSA). We previously determined the parameters of electrical stimulation for denervated rat EDL muscles to generate tetanic contractions sufficient to maintain mass and force close to control values. Using these parameters, we tested the hypothesis that a range exists for number of contractions per day, below and above which values for mass, maximum force, and fiber CSA are lower than values for innervated control muscles. For 5 weeks, denervated EDL muscles were stimulated to generate between 25 and 5000 contractions daily with contractions separated by constant intervals of rest, repeated 24 h per day. Force was not maintained with fewer than 200 or more than 800 contractions daily, whereas mass and fiber CSA were not maintained with fewer than 50 contractions daily. Protocols of stimulation that maintain muscle mass and force during prolonged periods of denervation may minimize problems clinically associated with denervation atrophy.     

Muscle-nerve-muscle neurotization for the reinnervation of denervated somatic muscle

Muscle-Nerve-Muscle (MNM) is the reinnervation of a denervated (recipient) muscle via a nerve graft inserted into the belly of an innervated (donor) muscle. MNM is studied for the reinnervation of intrinsic denervated somatic skeletal muscle by evaluating both restored muscle contractile ability and innervation state. In a rat model, muscle function is tested following MNM neurotization from an innervated (donor), extensor digitorum longus muscle to a denervated (recipient), peroneus digit quinti (PDQ) muscle. PDQ muscle cross-sections labeled for neural cell adhesion molecule protein (NCAM), a marker for fiber denervation. MNM neurotization results in the recovery of PDQ muscle force generating capacity (58% of Normal-control) and a significantly lower percentage of residual muscle fiber denervation (38% denervated) compared with the Denervated-control (79% denervated) group. MNM neurotization reinnervates 62% of the previously denervated muscle fibers in the PDQ muscle. No decrement in force capacity is observed in the donor EDL muscle. Nerve grafting for MNM neurotization may restore modest contractile function to denervated muscle and reinnervate relatively more denervated muscle fibers than the Denervated-control.     

A specific force deficit exists in skeletal muscle after partial denervation.

Skeletal muscle demonstrates a specific force deficit after repair of injured peripheral nerves, microneurovascular muscle transfer, and normal aging. Because atrophy cannot account for deficits in specific force, other, unknown, mechanisms are responsible for the resulting muscle contractile dysfunction under these circumstances. We tested the hypothesis that a subpopulation of denervated fibers is partially or completely responsible for the specific force deficit after partial denervation of the rat extensor digitorum longus muscle (EDL). Adult Fisher rats underwent either sham exposure or partial transection of 80% of the cross-sectional area of the left deep peroneal nerve. After a 2-week recovery period, maximum isometric force (F(0)) was measured in situ and maximum specific force (sF(0)) was calculated for EDL from both control (n = 8) and partial denervation (n = 7) groups. Innervated fiber cross-sectional area (CSA(inn)) was measured directly from whole EDL cross sections after immunohistochemical labeling for neural cell adhesion molecule (NCAM), a marker of muscle fiber denervation. A corrected specific force value (sF(0-inn)) was calculated by normalizing F(0) to CSA(inn). Partial skeletal muscle denervation resulted in significant reductions in muscle mass, F(0), and sF(0). The percentage of muscle fibers expressing NCAM in the extrajunctional sarcolemma increased from 1.0 +/- 0.8% in control to 49 +/- 15% in partially denervated EDL muscles. A 62.7% deficit in EDL specific force was observed after partial denervation. Denervated muscle fibers accounted for 59.3% of this deficit, but sF(0-inn) still differed significantly between control and partially denervated muscles, with a 25.5% difference between groups. In partially denervated muscles, the specific force deficit is partially but not fully explained by a subpopulation of noncontractile, denervated fibers.     

Factors affecting denervation-like changes at the neuromuscular junction during aging

The ability to sustain synaptic transmission diminishes with aging. To determine whether this is accompanied by alterations in the structure of the synapse, end-plate architecture was examined in EDL muscles from 10- to 25-month rats. There was a significant age-related increase in end-plate area and a decrease in the number of nerve terminals per end plate. Furthermore, the percentage of end plates with ultraterminal sprouts increased dramatically with age. To compare the morphological changes associated with aging to the changes in response to denervation, EDL muscles from 10- and 25-month animals were partially denervated. Both end-plate area and ultraterminal sprouting increased, while terminal number decreased following denervation in the 10-month muscles. To determine whether the age- and denervation-associated changes were accompanied by alterations in muscle-derived nerve-outgrowth factors, in vitro assays were performed. Neurite outgrowth was quantified from embryonic motoneurons incubated with muscle extract, or grown on cryostat-cut muscle cross sections from 10- and 25-month innervated and denervated EDL muscles. Both aged and denervated muscles induced greater degrees of neurite outgrowth compared with younger innervated muscles. Innervation to the EDL was then examined, and signs of axonal degeneration were observed. It is suggested that aging is associated with alterations in the motor axon to the EDL muscle. These changes are manifest at the neuromuscular junction. In turn, the muscle responds as if it were in a state of functional denervation.     

Contractile and histochemical properties of sliced muscle grafts regenerating in normal and denervated rat limbs.

Rat extensor digitorum longus muscles were transversely sliced into 7–8 segments. The muscle slices were autografted back into their original beds. In one series the recipient limbs were normal and in the other, limbs were denervated. At postoperative intervals of 7, 14, 30, and 60 days, the contractile properties (Latency period, contraction and half relaxation times, time parameters of contraction of twitch and tetanus, and twitch and tetanic tension) and histochemical properties (succinic dehydrogenase and myofibrillar ATPase) were analyzed. Sliced grafts regenerating in normally innervated legs followed a typical conversion from slow to fast contraction times, whereas regenerates in denervated limbs remained slow. Histochemically, innervated regenerates developed a heterogeneous pattern of muscle fiber type staining during the second month, whereas different histochemical types of muscle fibers did not appear in noninnervated regenerates. As in ontogeny, denervation retards or prevents the full structural and functional differentiation of regenerating muscle fibers.     

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.     

Characteristics of cat skeletal muscles grafted with intact nerves or with anastomosed nerves

Grafting of 3-g extensor digitorum longus (EDL) muscles of cats may be made with (i) severence of the nerve with spontaneous reinnervation, termed standard grafts (ii) severence of the nerve with reinnervation facilitated by anastomosis of the nerve, termed nerve-anastomosed grafts; and (iii) preservation of the nerve, termed nerve-intact grafts. In previous studies, standard grafts developed a maximum isometric tetanic tension (P0) that was 22% of the value for control EDL muscles. We hypothesized that the low values of P0 resulted from incomplete reinnervation of muscle fibers. To test this hypothesis, EDL muscles were grafted in cats with nerves intact and with nerves anastomosed. In standard grafts differences were observed in both structure and function at 120 compared with 240 days after grafting. Characteristics of the nerve-intact and nerve-anastomosed grafts did not change significantly between 120 and 240 days and the data were pooled for comparisons with control EDL muscles. Nerve-anastomosed and nerve-intact grafts developed P0 values that were 34 and 64% of the control values, respectively. Nerve-intact grafts had a mass and fiber cross-sectional area not different from control EDL muscles. Compared with control values, all grafts had fewer fibers, more connective tissue, lower absolute and normalized P0, reduced capillary density, and increased fatigability. The greater P0 of nerve-intact compared with standard and nerve-anastomosed grafts supported our hypothesis that the degree of reinnervation is a factor that limits graft development. The presence of a necrotic core and the low tension development of even the nerve-intact grafts suggested that revascularization is a significant limitation as well.     

Free grafting of the extensor digitorum longus muscle in the rat after Marcaine pretreatment

Normal or predenervated extensor digitorum longus muscles in the rat were given one or two preinjections of a solution of Marcaine and hyaluronidase and then freely autografted into normally innervated or denervated limbs. This procedure caused the sarcolysis of virtually all the original muscle fibers within the graft, followed by a massive regenerative response that restored the normal number of muscle fibers to the grafted muscle. Contraction times of grafts were very slow when contractile responses were first elicited by direct stimulation of the muscles. In normally innervated limbs, contraction speeded up progressively until early in the second month when the speeds approached those of the normal muscle. Grafts placed into denervated legs followed a similar pattern of development of contraotile properties during the first 3 weeks, but after that time the nerveless grafts contracted progressively more slowly than their reinnervated counterparts. Mature grafts developed a histochemically heterogeneous pattern of muscle fiber types (Types I and II) after staining for adenosine triphosphatase and succinic dehydrogenase activity.     

Contractile properties of standard and nerve-intact muscle grafts in the rat

The contractile properties of standard and nerve-intact grafts of extensor digitorum longus muscles of rats were compared in vitro. Fourteen days after grafting, the time to peak tension and the half-relaxation times of nerve-intact grafts were shorter than those of standard grafts, but both were longer than control values. By 60 days, these variables attained normal values. At every sample period, the tetanic tensions of nerve-intact grafts were higher than those of standard grafts. Even at the early sampling periods, the twitch-tetanic tension ratios of nerve-intact grafts were close to normal values, whereas those of standard grafts were higher than normal. Stabilized nerve-intact grafts had a larger mass and greater maximum tetanic tension development than standard grafts, but were more fatigable. Compared with control EDL muscles, stable nerve-intact grafts show no differences except for lessened fatigability, whereas standard grafts demonstrate significant functional deficits.     

Effect of nerve extract on atrophy of denervated or immobilized muscles

Changes in denervated muscles are due to disuse caused by paralysis of the muscle and the loss of special neurotrophic substances. We determined the relative roles of these two factors in the production of atrophy in denervated rats' extensor digitorum longus (EDL) muscles. Muscles were denervated and/or immobilized (by fixation of the ankle) for 7 days. Some rats also received daily intramuscular injections of a saline extract of rats' sciatic nerves (2.0 mg protein/ml). Atrophy was assessed by measurement of wet weight, total protein, and cross-sectional areas of types IIA and IIB fibers (in sections stained for ATPase). Both denervation and immobilization produced significant decreases in weight, protein, and areas of fiber. The group of rats with denervated EDL muscles had significantly greater atrophy than the group with immobilized muscles. In another group, denervated EDL muscles had significantly greater atrophy than contralateral muscles which were immobilized. However, when denervated muscles were injected with nerve extract, they did not differ significantly from contralateral, noninjected, immobilized muscles. Comparisons of the group of rats in which one EDL was denervated with groups in which one muscle was immobilized or was denervated and injected with nerve extract, indicated that loss of trophic influence was responsible for about 40% of the decreases in wet weight, total protein, and cross-sectional area of type IIB fibers, and the remaining 60% was due to disuse. Loss of trophic influence was responsible for only about 5% of the atrophy of denervated type IIA fibers. Therefore, inactivity and loss of neurotrophic influence were responsible for the atrophy which occurred in denervated skeletal muscles, and these two factors influenced the two types of fiber differently. The component of denervation atrophy due to loss of trophic influence could be completely prevented by injection of substances extracted from peripheral nerves.     

Phosphorylase kinase activities in damaged mouse skeletal muscles

In the course of work in which the phosphorylase kinase (PhK)-deficient mouse was used as a model of a defined inherited myopathy, we measured the PhK activity in regenerated autografts of normal whole extensor digitorum longus (EDL) muscles. Initially, no PhK activity was found for up to 71 days after grafting. A more sensitive assay technique revealed PhK activity in regenerated normal grafts from 43 days after grafting, but the levels never reached those found in ungrafted normal muscle. PhK activity was also reduced in normal EDL muscles following either: denervation, or tenotomy, or denervation and devascularisation, or denervation, devascularisation and tenotomy, but the reduction was never as great as that observed in grafted muscle of equivalent age. PhK activity was also reduced in the tibialis anterior (TA) muscles of the myopathic C57B1/10 mdx strain of mouse, in which the skeletal muscles undergo persistent bouts of degeneration and regeneration, whilst retaining their vascular and nervous connections. It was concluded that the loss of PhK activity in grafted muscle is due to a combination of the effects of denervation, tenotomy and regeneration which occur on grafting.     

Myotrophic effects on denervated fast-twitch muscles of mice: correlation of physiologic, biochemical, and morphologic findings

Certain morphological, biochemical, and physiological parameters were assessed in fast-twitch muscles of 6-week-old mice with unilateral hindlimb denervation for 4 weeks. Some of the mice received daily injections (i.p.) of nerve extract throughout the period of denervation. Values from treated and untreated denervated muscles were compared with each other and with those from contralateral, innervated controls. The cross-sectional areas of denervated types IIA and IIATy muscle fibers were 45% and 28% greater, respectively, in muscles of treated than of untreated mice, which resulted in greater maximal tetanic tension. Injection with nerve extract did not influence the postdenervation reduction of phosphorylation of myosin light chain 2-fast nor the loss of posttetanic twitch potentiation, two parameters thought to be related. Denervation produced a significant decrease in relative content of cytosolic parvalbumin; however, this change was completely prevented by administration of nerve extract. This latter finding correlated with the amelioration of greater than 50% of the postdenervation prolongation of half-relaxation time of the twitch in treated than in untreated muscles. More than half of the prolongation of time-to-peak of the twitch was also prevented in denervated muscles of treated than of untreated mice.     

Energy reserves and chemical changes in denervated anterior and posterior latissimus dorsi muscles of the chicken

Postdenervation changes in most muscles, whether experimentally produced in animals or resulting from disease or injury in man, are characterized by a largely irreversible atrophy. One notable exception is the prolonged hypertrophy exhibited by the denervated anterior latissimus dorsi (ALD) muscle of the chicken. We have determined the concentrations of several chemical compounds and nitrogen fractions in the innervated and denervated latissimus dorsi muscles of the young chicken at various intervals after denervation. The posterior latissimus dorsi (PLD), a fast muscle, has significantly decreased ATP, phosphocreatine (PC), and total creatine (TC) concentrations 1 week after denervation. It also has less noncollagen nitrogen (NCN), as well as sarcoplasmic and fibrillar nitrogen; however, collagen nitrogen is increased and the muscle weighs 15% less than its contralateral control. The slow ALD, on the other hand, has not yet decreased its level of energy stores (ATP or PC) or TC at 3 weeks after denervation; nor are there any changes in NCN or any individual nitrogen fraction at 1 week. The hypertrophy of the denervated ALD reaches its maximum by the end of the first week, when it is 43% heavier than the control muscle. The weight gain in both innervated and denervated ALD are similar after that with the denervated muscle still 41% heavier at 3 weeks.     

Effect of denervation on overdevelopment of chloroquine-induced autophagic vacuoles in skeletal muscles

We studied how denervation affects the overdevelopment of autophagic vacuoles in muscles of chloroquine-treated rats. The number of autophagic vacuoles increased significantly in the chloroquine-treated soleus muscles after denervation as compared to similarly treated contralateral, innervated muscles. No vacuoles were present in the denervated and innervated muscles of saline-treated rats. After denervation, the autophagic vacuoles in chloroquine-treated muscle contained numerous glycogen particles and various heterogeneous materials. A biochemical study showed no significant difference in the activities of lysosomal proteases and hydrolases in the chloroquine- and salinetreated muscles after denervation, although these activities were markedly increased in comparison to the same activities in the contralateral, innervated muscles. Chloroquine treatment by itself did not, but denervation with or without chloroquine treatment did enhance the biochemical activities of lysosomal enzymes in the animals. We speculate that denervation induces the marked accumulation of autophagic vacuoles in chloroquine-incuced myopathy.     

Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles

The mitotic activity in muscles of growing rats and the effect of denervation were studied by means of continuous infusion of 5-bromo-2-deoxyuridine (BRDU). Denervated muscles after 10 weeks contained 20 to 60% fewer muscle nuclei than normal; BRDU labeled about 25% of the nuclei of normal soleus and extensor digitorum longus (EDL) and of denervated EDL muscles but only 5% in the denervated soleus muscle. Labeled nuclei persisted in denervated but not in normal muscles. After the main growth period, the turnover of myonuclei was at most 1 to 2% per week. The behavior of connective tissue nuclei was similar to that in muscle fibers. Infusion of BRDU had no effect on contractile properties. It is suggested that the exceptionally rapid atrophy of the denervated rat soleus associated with loss of satellite cells was due to loss of myonuclei and differentiation and fusion of satellite cells. The cause may possibly be that the phase of postdenervation fibrillation is shorter than in other muscles.     

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.     

Morphology of stable muscle grafts of rats: effects of gender and muscle type

Our purpose was to quantify morphological characteristics of extensor digitorum longus (EDL) and soleus (SOL) muscle grafts in female (N = 8) and male (N = 8) rats. Muscles were grafted orthotopically, with the nerve remaining intact, and were studied 56 days later. The mass of EDL and SOL grafts and control muscles of females was 60% to 65% of male values; this difference was directly related to gender differences in body mass. The fiber composition of EDL and SOL grafts did not differ from control, and no gender effects were noted. The mean fiber area (MFA) of control EDL and SOL muscles of females averaged 65% of male values. The MFA of grafts did not differ due to gender, and averaged 60% of control value for SOL and 70% for EDL grafts. We conclude there are no substantial differences in the regenerative capacity of EDL and SOL muscles grafted with the nerve intact.     

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.