Brain-Derived Neurotrophic Factor mRNA Levels Are Up-Regulated in Hindlimb Skeletal Muscle of Diabetic Rats: Effect of Denervation

In a previous study the levels of brain-derived neurotrophic factor (BDNF) mRNA were shown to be elevated in skeletal muscle of the diabetic rat compared with age-matched control animals. It was proposed that diabetes-induced changes in nerve function may initiate changes in nerve/muscle contact akin to those following denervation of target skeletal muscle. In this study hindlimb skeletal muscles were denervated by sciatic nerve crush or transection and the effect on BDNF mRNA levels in control and diabetic rats was observed using Northern blotting. Contralateral to the side of nerve injury, the levels of BDNF mRNA in soleus muscle of diabetic rats were higher than in controls (three- to sevenfold), as has been seen previously in diabetic rats without any axotomy. Sciatic nerve crush or transection, of 1 week or of 3 weeks duration, lowered the levels of BDNF mRNA by 50% in ipsilateral soleus muscle of diabetic rats. BDNF mRNA levels in contralateral gastrocnemius muscle were not similarly raised in diabetic rats compared with controls and nerve injury had no effect. In control animals, ipsilaterally, the BDNF mRNA levels of soleus muscle were raised approximately twofold at 1 week and were lowered by approximately 50% at 3 weeks following nerve injury. Neurotrophin-3 mRNA levels were reduced approximately 50% in soleus muscle of diabetic rats compared with control rats, and nerve injury had no significant effect. The specific up-regulation of BDNF mRNA in soleus muscle of diabetic rats is discussed in terms of a proposed diabetes-induced ischemia within hindlimb skeletal muscle, with a protective role for BDNF in muscle and/or nerve being introduced.     

Influence of locomotor training on the structure and myosin heavy chains of the denervated rat soleus muscle

OBJECTIVE: Mechanism of denervation atrophy remains poorly understood. In particular, the question about irreversibility of the late atrophy is still open. Therefore, in the present study, we investigated whether and how a passive movement can affect a progress of atrophy in rat soleus muscle. To address this issue, a locomotor training on a treadmill was applied to rats with their right hindlimb muscles denervated. METHODS: The hindlimb muscles were denervated by cutting the sciatic nerve. Starting either 7 days or 1 month after the surgery, the animals were trained on a treadmill. Two months after denervation, the soleus muscle was investigated using light and electron microscopy and biochemical methods. Control soleus muscles were obtained from non-trained animals: the untreated and the 2-month denervated. RESULTS: Locomotor training caused slight increase in denervated rat soleus muscle weight and significant increase in its fiber diameter. The training positively affected some of the factors that were believed to be the reasons of atrophy irreversibility, because of significant increase in the number of capillary blood vessels and muscle fiber nuclei with the concomitant decrease in the number of severely damaged muscle fibers and amount of collagen. Morphology of the contractile apparatus was also improved as more regular organization of sarcomeres and the hexagonal arrangement of myosin filaments was evident. Moreover, the amount of myosin heavy chains (MHC) significantly increased after training. The effects were more evident in the animals with longer training. CONCLUSION: Passive movement seems to attenuate some of the pathologic processes within the denervated muscle.     

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.     

Cordotomy-denervation interactions on contractile and myofibrillar properties of fast and slow muscles in the rat

Cordotomy-denervation interactions were studied on contractile and myofibrillar properties of slow (soleus) and fast (extensor digitorum longus) muscles of the rat. The spinal cord was transected midthoracically in neonatal (2-day-old) animals. Two months after birth, a unilateral transection of the sciatic nerve was carried out in both cordotomized and control animals. Five weeks after denervation, contractile properties were tested isometrically in vitro; myofibrillar properties were assessed by histochemical staining of the muscle fibers and by electrophoretic analysis of the myosin heavy chain composition. The following results were obtained: (i) In cordotomized animals the contraction time of the soleus was significantly shorter (-23.3% on average) than that in the control animals and this shortening was accompanied by a proportional slow-to-fast shift in myofibrillar properties. (ii) The extensor digitorum longus properties were not significantly different in the control and cordotomized animals. (iii) Denervation in control animals was followed by a marked increase of contraction and half-relaxation times in the extensor digitorum longus, whereas in the soleus only the half-relaxation time was significantly increased; myofibrillar properties in the soleus showed an appreciable slow-to-fast shift, whereas in the fast muscle the main change was an increase in type 2A fibers to the detriment of type 2B. (iv) In cordotomized animals, denervation caused the soleus contraction time to increase to control values, whereas myofibrillar properties shifted to an even faster pattern; in the extensor digitorum longus denervation caused the same changes seen in the control animals. The results showed that cordotomy at birth caused the soleus to develop as a faster muscle than in the control animals. The concurrent effects of cordotomy and denervation on the myofibrillar properties of the soleus suggest that the slow-to-fast change in these properties is a common consequence of the reduction in the level of motor activity. The opposite effects of the two experimental conditions in the soleus contraction time support the view that the contractile alterations that follow denervation mainly reflect alterations in the muscle activation process.     

Recovery of reinnervating rat muscle after cast immobilization

We evaluated the recovery of the reinnervating rat plantar flexor muscles after different periods of casting and then decasting the lower extremities. Four groups of 4-month-old, female Wistar rats underwent bilateral crush-denervation of the sciatic nerve at the sciatic notch. Two weeks after nerve crush, the hind legs of three groups of rats were immobilized with bilateral casts at the knee and ankle joints and the fourth group was a control group. Of the three casted groups, one was mobilized after 1 week and another group after 3 weeks of casting. The third experimental group remained casted until the end of 6 weeks. Six weeks after the nerve crush, all groups were evaluated for muscle weights of the soleus, plantaris, and gastrocnemius; absolute amounts of the myofibrillar, sarcoplasmic, and stromal proteins in the gastrocnemius; the fiber diameters and percent composition of type I and type II fibers in the soleus and plantaris; and the isometric contractile properties of the soleus muscle. Compared with the denervated control group, the experimental groups revealed the following: (i) Four weeks of casting caused a reduction in wet weight (range 30.6 to 40.4%, P less than 0.01) in the soleus, plantaris, and gastrocnemius muscles. Decasting led to an earlier recovery of the soleus than of the plantaris and gastrocnemius muscles. (ii) The myofibrillar protein returned to control values with 3 weeks of decasting but the stromal protein remained significantly elevated and the sarcoplasmic protein significantly depressed regardless of the period of mobilization. (iii) Except for the type I fibers in the plantaris, the remainder of the muscle fibers in the soleus and plantaris decreased in size due to casting. Only the type I muscle fibers of the soleus increased in size with longer periods of mobilization. (iv) Four weeks of casting significantly altered the maximum isometric twitch tension (-42.3%), contraction time (+17.1%), maximum tetanic tension (-38.1%), and half-fatigue time (+40.5%) in the soleus. The reinnervating soleus muscle appears to recover from the effects of casting sooner than the plantaris or gastrocnemius muscles.     

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.     

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.     

Increased phosphorylation of nuclear protein in myonuclei isolated from denervated skeletal muscle

The slow-twitch soleus muscle of the rat hindlimb was denervated by cutting the sciatic nerve in the midthigh on one side. A sham operation was performed on the contralateral side to provide a control soleus muscle. At 24, 48, 72, and 120 hours after these surgical procedures, the animals were sacrificed and the nuclei which were isolated from these muscles incubated in a phosphorylating medium containing [gamma-32P]ATP. At the 48 hour denervation period only, the in vitro phosphorylation of TCA-precipitable nuclear proteins was significantly increased compared to control values. Resolution of the SDS-solubilized nuclear proteins by polyacrylamide gel electrophoresis revealed that the increased phosphorylation was more marked in some phosphoproteins than others. The significance of these early denervation changes are discussed with respect to increases in the activities of the nuclear RNA polymerases and changes in the phosphorylation of cytosolic protein.     

Use of dexamethasone with TTX block of nerve conduction shows that muscle membrane properties are fully controlled by evoked activity

This paper provides further evidence that motorneurons control extrajunctional properties of skeletal muscles through the activity evoked in the muscle fibres. The experiments compare the amount of action potential resistance to tetrodotoxin (TTX resistance) in denervated soleus muscle with that in soleus whose nerve was crushed and then allowed to regenerate in the presence of a block of the sciatic impulse conduction. Measurements were taken after about 2–3 weeks to allow full reinnervation and recovery of trophic regulation by the nerve. Blocking sciatic impulse conduction with TTX solutions containing low doses of the anti-inflammatory drug dexamethasone induced values of extrajunctional TTX resistance identical to those caused by denervation. In contrast lower levels of TTX resistance were obtained with dexamethasone-free solutions or when the drug was administred through the systemic path rather than topically applied to the nerve. These results indicate that physiological neural regulatory signals other than activity do not participate to the regulation of extrajunctional properties of skeletal muscles. Furthermore the low levels of TTX resistance measured with dexamethasone-free blocks confirm our previous experiments indicating that reported differences between denervation and pure inactivity are attributable to incomplete suppression of nerve impulse conduction.     

Trophic effects on the contractile and histochemical properties of rat soleus muscle

Contractile and histochemical properties of rat soleus muscle were studied bilaterally after 2 or 4 weeks of denervation (DEN), which eliminates activity and non-activity-related influences, and chronic application of tetrodotoxin (TTX) to the motor nerve, which produces a completely inactive innervated muscle. After 2 or 4 weeks of disuse, the percentages of slow twitch oxidative fibers in both DEN- and TTX-treated soleus were reduced significantly and to similar extents. The dynamic contractile properties of TTX-treated and denervated muscles were similar to those of control muscle after 2 weeks, but by 4 weeks, parallel increases were seen in normalized rate of tension development and maximal shortening speed of these muscles. After either period of disuse, the atrophy of TTX-treated soleus was significantly less than that of denervated muscle. Atrophy was associated with correspondingly diminished capacity of denervated or TTX-disused muscles to generate tension after 2 weeks. By 4 weeks of treatment, the reduction in tension of denervated soleus was greater than its diminished size. These results imply the existence of a non-activity-related stimulus in TTX-inactivated muscles which slows the reductions in muscle mass and specific tension observed in denervated soleus muscle. In contrast, the similarities in dynamic speed-related properties and fiber type profiles between DEN- and TTX-treated rat soleus may be explained simply by the absence of neuromuscular activity.     

Recovery of the soleus muscle after short- and long-term disuse induced by hindlimb unloading: effects on the electrical properties and myosin heavy chain profile

The hindlimb unloading (HU) rat is a model of muscle disuse characterized by atrophy and slow-to-fast phenotype transition of the postural muscles, such as the soleus. We previously found that the resting sarcolemmal chloride conductance (gCl) that is typically lower in slow-twitch myofibers than in fast ones increased in soleus fibers following 1 to 3 weeks of HU in accord with the slow-to-fast transition of myosin heavy chain (MHC) isoforms. Nevertheless, the gCl already raised after a 3-day HU, whereas no change in MHC expression was detected. The present work evaluates the ability of soleus muscle to recover on return to normal load after a short (3 days) or long (2 weeks) disuse period. The changes observed after a 2-week HU were slowly reversible, since 3-4 weeks of reloading were needed to completely recover gCl, fiber diameter, MHC expression pattern, as well as the mechanical threshold Rheobase, an index of calcium homeostasis. After 3-day HU, the gCl increased homogeneously in most of the soleus muscle fibers and gCl recovery was rapidly completed after 4-day reloading. These results suggest different induction mechanisms for gCl augmentation after the short and long HU periods, as well as a possible role for gCl in the slow muscle adaptation to disuse.     

Effects of denervation on calpain and calpastatin in hamster skeletal muscles

Three leg muscles (soleus, extensor digitorum longus, and plantaris) of adult male golden Syrian hamsters were denervated by bilateral transecting of the sciatic nerve. Eighteen days after denervation, wet weights, amounts of soluble protein, and activities of wide-specificity calpain II, intermediate filament protein-specific calpain I, and calpastatin were measured by protein determination and enzyme and immunological assays. In comparison with control (nondenervated) muscles and depending on the specific muscle and protein, the activities of the calpains increased 1.3 to 1.9 times the control values, whereas the calpastatin decreased to one-half and one-third of control values. The muscle which showed the greatest increase in both calpain activities and the largest decrease in calpastatin activity was the plantaris (a fast-twitch, oxidative glycolytic muscle). The extensor digitorum longus (fast-twitch glycolytic) showed increases in calpain II activity similar to those in the plantaris, but smaller changes in calpain I and calpastatin. The soleus (slow-twitch, oxidative) showed the smallest changes in calpain II and calpastatin activities, although an increase in the calpain I activity was seen after denervation. These results suggest a possible relationship between the presence of fast-twitch, oxidative glycolytic fibers in a muscle and increased potential for intracellular proteolysis following denervation.     

Apigenin inhibits sciatic nerve denervation–induced muscle atrophy

Introduction: Apigenin (AP) has been reported to elicit anti‐inflammatory effects. In this study, we investigated the effect of AP on sciatic nerve denervation–induced muscle atrophy. Methods: Sciatic nerve–denervated mice were fed a 0.1% AP‐containing diet for 2 weeks. Muscle weight and cross‐sectional area (CSA), and the expression of atrophic genes and inflammatory cytokines in the gastrocnemius were analyzed. Results: Denervation significantly induced muscle atrophy. However, values for muscle weight and CSA were greater in the denervated muscle of the AP mice than the controls. AP suppressed the expression of MuRF1, but upregulated both myosin heavy chain (MHC) and MHC type IIb. AP also significantly suppressed expression of tumor necrosis‐alpha in the gastrocnemius and soleus muscles, and interleukin‐6 expression in the soleus muscle. Discussion: AP appears to inhibit denervation‐induced muscle atrophy, which may be due in part to its inhibitory effect on inflammatory processes within muscle. Muscle Nerve 58 : 314–318, 2018     

Myoglobin in rat hind limb muscles after denervation and during reinnervation

Radioimmunoassay of myoglobin (Mb) was performed in rat hind limb muscles after surgical denervation and during reinnervation following cryolesion of the sciatic nerve. Muscles of the contralateral leg served as controls. After resection of the sciatic nerve, decreased Mb concentrations were noted on the fourth day in the tibialis anterior, peroneus longus, and extensor digitorum longus (EDL) muscles. Thereafter, the levels increased up to the last observation on day 32. The increases in Mb levels in the tibialis anterior and EDL muscles were considerably more pronounced (305% and 324%, respectively) than in the peroneus longus and soleus muscles (148% and 137%, respectively). After cryolesion of the sciatic nerve, the Mb concentrations in the tibialis anterior, peroneus longus, and EDL muscles increased, reaching maximal values on days 16-21. The levels then decreased and normal values were observed 2 months postoperatively. The normalization of the Mb levels during reinnervation corresponded fairly well in time with the clinical recovery and neurophysiological findings observed in a previous study.     

Ca2+-activated protease in denervated rat skeletal muscle measured by an immunoassay

Right hind leg muscles of adult male rats were denervated by transecting the sciatic nerve, while muscles of the left leg served as a control. On days 4, 9, and 18 after denervation, the rats were killed and the extensor digitorum longus and soleus muscles removed. Extracts were prepared which contained the Ca2+-activated protease. The molecular weight of the protease in the extracts, measured immunologically on Western blots of SDS-polyacrylamide gels, was identical to that of purified standard protease and was unaffected by denervation. The amount of protease in each extract was measured by a solid phase immunoassay using monospecific IgG labeled with 125I, and the results were expressed as units of protease activity per milligram noncollagen protein. There were increases of 40 to 80% in the mean values obtained for both denervated muscles compared with controls. Because the protease is probably localized on the cell membrane, these modest increases are likely to be a consequence of a generalized over-development of muscle membranes which follows denervation.     

Effects of denervation on the distribution of myosin isozymes in skeletal muscle fibers

Polyamines were monitored in tenotomized and denervated rat soleus (slow-twitch) and gastrocnemius (fast-twitch) muscles as markers of the degeneration-regeneration process. Experimental tenotomy produced rapid muscle fiber degeneration followed by recovery and return to normal muscle histology. The tenotomized soleus muscle showed striking central lesions in many fibers which were maximal at 1 to 2 weeks, whereas only a few fibers with central lesions were detectable in gastrocnemius. Alterations in the concentration of the polyamines, putrescine, spermidine, and spermine, reflected the extent of pathologic lesion formation in response to tenotomy in the two muscles. After tenotomy, putrescine was elevated significantly for 2 weeks in the soleus muscle and spermidine and spermine increased to twofold that in the contralateral control within 2 weeks, returning toward normal thereafter with no significant differences at 5 weeks after tenotomy. In the tenotomized gastrocnemius muscle, which showed relatively few fibers with central lesions, there was no detectable putrescine between 1 and 6 weeks, and the increases in spermidine and spermine concentrations were less than twofold at all times measured. Alterations in polyamine concentrations in response to denervation were strikingly different. Putrescine was markedly elevated 1 to 2 weeks after denervation and thereafter returned to normal. In both muscles, spermidine and spermine concentrations remained elevated throughout the 8 weeks after denervation. Persistent elevations of polyamines in denervated muscle may reflect connective tissue hyperplasia, in contrast to the tenotomized muscle where the increases are probably due to the degeneration-regeneration process of the muscle fiber itself.     

Scanning electron microscopic study of denervated and reinnervated intrafusal muscle fibers in rats.

Isolated muscle spindles from lower lumbrical muscles of rats were used to study the 3-dimensional organization of intrafusal structures by scanning electron microscopy following (a) complete denervation, (b) reinnervation after a single crush lesion of the sciatic nerve, or (c) reinnervation after transection and immediate suture of this nerve. One week after complete denervation, previous sites of intrafusal motor endplates were transformed into sarcolemmal ovoid bulges. These bulges persisted in denervated muscle spindles up to 12 weeks. Regenerated motor nerve endings were detected on intrafusal muscle fibers 1 month, and thereafter following sciatic nerve crush injuries, and 3 months and later following transection and suture of the nerve. Furthermore, 3 different types of subsynaptic areas of motor nerve terminals were observed. The scanning electron microscopic technique also allowed visualization of splitting and fusion of intrafusal muscle fibers. The findings are discussed in view of their possible functional implications.     

Transient Reinnervation of Antagonistic Muscles by the Same Motoneuron

When reinnervation is allowed after a sciatic nerve cut in the adult rat, motoneuron axons may branch to innervate antagonistic muscles. This multiple innervation is widespread, but transient. Fourteen weeks after denervation isometric muscle contraction experiments and studies with anterograde transport of the fluorescent tracer Fast Blue showed that branches from the same motoneurons reached the distal part of the tibial nerve and either the soleus or the extensor digitorum longus muscles or both muscles. Retrogradely double-labeled motoneurons were found after injections of different fluorescent tracers into these muscles. Sixty-four to 88 weeks after the nerve cut, similar experiments showed that selective innervation was reestablished. The findings suggest a selective mechanism for axon withdrawal in an adult mammal.