失神经骨骼肌细胞凋亡：运动与感觉神经的比较

Skeletal muscle atrophy inevitably occurs in denervated skeletal muscle,and cell apoptosis plays an important role in skeletal muscle atrophy and degeneration.The present study established rat models of simple nerve injury by transecting the ventral or dorsal spinal nerve root and observed rat skeletal muscle cell apoptosis following simple motor nerve injury versus simple sensory nerve injury.Following skeletal muscle denervation for 10 weeks,cell apoptosis was detected in skeletal muscle,which was accompanied by obvious changes in rat behavior and electrophysiological responses.In addition,changes in cross-sectional area and average gray-scale of motor endplates of the gastrocnemius muscle were analyzed following sciatic nerve injury and motor nerve injury.Cell nuclei in denervated skeletal muscle tissue were more densely arranged than in normal skeletal muscle tissue.Cell nuclei were most dense in the sciatic nerve injury group,followed by the motor nerve injury group and the sensory nerve injury group.Fas/FasL expression and the number of apoptotic cells increased in denervated skeletal muscle,and apoptosis-related changes were observed.These findings suggested that motor and sensory nerves provided trophic actions following skeletal muscle and motor nerve injury,resulting in a greater influence on skeletal muscle atrophy than sensory nerve injury.Therefore,reconstruction of motor nerves should be preferentially considered for treating denervation-induced skeletal muscle atrophy.     

Morphological differences in skeletal muscle atrophy of rats with motor nerve and/or sensory nerve injury

Skeletal muscle atrophy occurs after denervation. The present study dissected the rat left ventral root and dorsal root at L4-6 or the sciatic nerve to establish a model of simple motor nerve injury, sensory nerve injury or mixed nerve injury. Results showed that with prolonged denervation time, rats with simple motor nerve injury, sensory nerve injury or mixed nerve injury exhibited abnormal behavior, reduced wet weight of the left gastrocnemius muscle, decreased diameter and cross-sectional area and altered ultrastructure of muscle cells, as well as decreased cross-sectional area and increased gray scale of the gastrocnemius muscle motor end plate. Moreover, at the same time point, the pathological changes were most severe in mixed nerve injury, followed by simple motor nerve injury, and the changes in simple sensory nerve injury were the mildest. These findings indicate that normal skeletal muscle morphology is maintained by intact innervation. Motor nerve injury resulted in larger damage to skeletal muscle and more severe atrophy than sensory nerve injury. Thus, reconstruction of motor nerves should be considered first in the clinical treatment of skeletal muscle atrophy caused by denervation.     

Reinnervation of the tibialis anterior following sciatic nerve crush injury: a confocal microscopic study in transgenic mice

Transgenic mice whose axons and Schwann cells express fluorescent chromophores enable new imaging techniques and augment concepts in developmental neurobiology. The utility of these tools in the study of traumatic nerve injury depends on employing nerve models that are amenable to microsurgical manipulation and gauging functional recovery. Motor recovery from sciatic nerve crush injury is studied here by evaluating motor endplates of the tibialis anterior muscle, which is innervated by the deep peroneal branch of the sciatic nerve. Following sciatic nerve crush, the deep surface of the tibialis anterior muscle is examined using whole mount confocal microscopy, and reinnervation is characterized by imaging fluorescent axons or Schwann cells (SCs). One week following sciatic crush injury, 100% of motor endplates are denervated with partial reinnervation at 2 weeks, hyperinnervation at 3 and 4 weeks, and restoration of a 1:1 axon to motor endplate relationship 6 weeks after injury. Walking track analysis reveals progressive recovery of sciatic nerve function by 6 weeks. SCs reveal reduced S100 expression within 2 weeks of denervation, correlating with regression to a more immature phenotype. Reinnervation of SCs restores S100 expression and a fully differentiated phenotype. Following denervation, there is altered morphology of circumscribed terminal Schwann cells demonstrating extensive process formation between adjacent motor endplates. The thin, uniformly innervated tibialis anterior muscle is well suited for studying motor reinnervation following sciatic nerve injury. Confocal microscopy may be performed coincident with other techniques of assessing nerve regeneration and functional recovery.     

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.     

Magnetic resonance imaging of mouse skeletal muscle to measure denervation atrophy

We assessed the potential of different MRI measures to detect and quantify skeletal muscle changes with denervation in two mouse models of denervation/neurogenic atrophy. Acute complete denervation and chronic partial denervation were examined in calf muscles after sciatic nerve axotomy and in transgenic SOD1^G93A mice, respectively. Serial T₂, diffusion tensor, and high resolution anatomical images were acquired, and compared to behavioral, histological, and electrophysiological data. Increase in muscle T₂ signal was first detected after sciatic nerve axotomy. Progressive muscle atrophy could be monitored with MRI-based volume measurements, which correlated strongly with postmortem muscle mass measurements. Significant increase in muscle fractional anisotropy and decreases in secondary and tertiary eigenvalues obtained from diffusion tensor imaging (DTI) were observed after denervation. In SOD1^G93A animals, muscle denervation was detected by elevated muscle T₂ and atrophy in the medial gastrocnemius at 10 weeks. Changes in T₂ and muscle volume were first observed in medial gastrocnemius and later in other calf muscles. Alterations in secondary and tertiary eigenvalues obtained from DTI were first observed in tibialis anterior and medial gastrocnemius muscles at age 12 weeks. We propose that MRI of skeletal muscle is a sensitive surrogate outcome measure of denervation atrophy in animal models of neuromuscular disorders, with potential applicability in preclinical therapeutic screening studies in rodents.     

Successful intramuscular neurotization is dependent on the denervation period. A histomorphological study of the gracilis muscle in rats

To characterize the extent to which reinnervation potential depends on the duration of denervation, intramuscular neurotization of the gracilis muscle was performed either immediately or 2, 4, 6, and 8 weeks after transection of the obturator nerve. For neurotization, the sciatic nerve was split into three fascicle groups and fixed intramuscularly. Muscle morphology after 6 weeks of regeneration was identified with anti-myosin immunohistochemistry and NADH staining. Newly formed motor endplates were characterized using acetylcholinesterase staining and electron microscopy. Wet muscle weight ratio indicated the functional state of synapses. Depending on the denervation period, three levels of regenerative outcome were evident. Best results were seen after immediate neurotization or after 2 weeks of denervation. Regeneration, although at a significantly lower level, also occurred after denervation periods of 4 and 6 weeks. Regeneration following neurotization after 8 weeks of denervation was negligible. Quantity and quality of motor endplate formation depended on the denervation period. Thus, in special clinical situations intramuscular neurotization within a distinct time window provides a good reconstructive option.     

Neurotrophic control of skeletal muscle phospholipids

The phospholipid composition of rat gastrocnemius muscles was assessed one to nine days after sciatic nerve transection was performed either close to the muscle (5-8 mm from the point of entrance of the nerve into the muscle for short-stump nerve sections) or far from it (30-35 mm central to the nerve's point of entrance for long-stump nerve sections). In both instances, denervation did not cause striking changes in total phospholipid content but resulted in a selective loss of 1,2-diacyl-sn-glycero-3-phosphorylethanolamine (phosphatidyl ethanolamine, GPE). The quantity of GPE in denervated muscles was found to be significantly less than that it controls (p less than 0.01) within one day for short-stump preparations, as compared to three days for long-stump preparations. Also, direct comparison of short- and long-stump GPE values showed significant differences (p less than 0.01) at all times from the first to the fifth day following denervation, with no difference detected thereafter. These results imply that maintenance of skeletal muscle GPE involves a neurogenic influence which is independent of nerve-evoked muscle activity. This conclusion may help us understand neuromuscular diseases in which perturbation of phospholipid components has been implicated.     

Characterization of matrix metalloproteinases in denervated muscle

In a nerve crush model of denervation, we examined muscle matrix metalloproteinase (MMP) expression, localization and activity. In normal muscle, MMP mRNA levels were low, and immunohistochemically MMPs were distributed around the muscle fibre with MMPs-3, -7 and -9 also staining at the neuromuscular junction. Seven days after nerve crush, muscle MMP immunoreactivity, especially MMP-12 and MMP-14, became irregularly distributed. At 20 days reinnervation of the muscle was observed, and some restitution of the normal pattern of immunoreactivity was noted concomitant with a higher level of MMP mRNA expression. In situ zymography showed that MMP activity was very weak in normal muscle whereas it was increased up to 40 days following denervation. Our results suggest that MMPs in muscle are involved in the tissue changes following denervation. Further experiments are required to test the hypothesis that MMP inhibition may be beneficial in protecting muscle from excessive remodelling following denervation and therefore improve reinnervation.     

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.     

Time course of muscle atrophy and recovery following a phenol-induced nerve block

Clinically, phenol is used often as a neurolytic agent to treat pain and spasticity. The purpose of this study was to examine the time course of denervation and recovery in several hindlimb muscles following application of a 5% aqueous solution of phenol to the sciatic nerve. Phenol was applied to the sciatic nerve of adult female rats either by intraneural or perineural injection. Axonal degeneration was evident within the sciatic nerve 2 days following phenol application, although variable amounts of damage were observed. By 2 weeks, the soleus and tibialis anterior had atrophied to 63% and 51% of control. Reinnervation of hindlimb muscles occurred between 2 and 4 weeks following the nerve block. Following denervation, the soleus became slower in that all of the fibers expressed the slow myosin heavy chain (MHC). At 5 months, maximum tension of the soleus was 74% of control and the muscle consisted of more fast fibers on average, some of which expressed IIx MHC. These data suggest that 5% phenol causes an injury to the nerve that is more severe than a crush injury, and that reinnervation of denervated muscles may be by motoneurons other than those that originally innervated the muscles. © 1996 John Wiley & Sons, Inc.     

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.     

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.     

成肌调节因子Myf-5在失神经骨骼肌萎缩不同时段的表达

背景：成肌调节因子Myf-5是参与肌肉发生过程分子调控、启动和维持骨骼肌细胞生长发育的重要基因,可能与失神经骨骼肌萎缩的发生有关。 目的：观察不同部位、不同时段骨骼肌失神经支配后成肌调节因子Myf-5基因的表达情况。 设计、时间及地点：随机对照动物实验,于2008-03/04在山西医科大学完成。 材料：选择健康8周龄雄性SD大鼠24只,随机分成4组,即假手术组(有神经支配)、去神经2 d组、去神经7 d组、去神经28 d组,每组6只。 方法：假手术组不切断坐骨神经,仅做假手术。去神经组右下肢后部中段切断坐骨神经1 cm以上,分别于去神经第2,7,28天用脊椎脱臼法处死大鼠,分离出右小腿的胫骨前肌、比目鱼肌、腓肠肌、跖肌标本。 主要观察指标：用反转录-聚合酶链反应技术检测各组肌肉Myf5 mRNA表达情况,抗Myf-5 多克隆抗体免疫组织化学染色(ABC 法),测量灰度值。 结果：去神经骨骼肌早期,Myf-5的mRNA在去神经支配后第2,7,28天均表达上调(P < 0.05)。Myf-5 抗体阳性染色细胞核数在SD大鼠骨骼肌失神经28 d时的肌卫星细胞中最多。 结论：Myf-5在大鼠失神经骨骼肌萎缩早期不同肌肉表达均为上调。大鼠骨骼肌失神经支配后早期肌卫星细胞中Myf-5表达上调。     

Muscle-specific beta-enolase concentrations after cross- and random innervation of soleus and extensor digitorum longus in rats

The concentration of beta-enolase, a highly specific marker of the skeletal muscle of rats, was determined in a slow-twitch muscle, the soleus (SOL) and a fast-twitch muscle, the extensor digitorum longus (EDL) after cross-innervation, random reinnervation, or denervation. The beta-enolase concentration is normally high in EDL and low in SOL. When the nerves entering into these muscles were cross-sutured, the beta-enolase concentration in EDL decreased and that in SOL increased to reach an almost equal value in 20 weeks and by the 35th week the SOL ultimately had a higher beta-enolase concentration than the EDL. When the sciatic nerve trunk was completely transected and sutured immediately, the beta-enolase concentration in EDL decreased and that of SOL increased; in 20 weeks SOL had a beta-enolase concentration similar to that of the EDL. When these muscles were denervated by cutting the sciatic nerve trunk, their beta-enolase concentrations were markedly lowered, but EDL still retained on the 12th week a beta-enolase value comparable to the normal SOL. Possible mechanisms behind the observed changes in beta-enolase concentration are discussed.     

经皮电刺激大鼠骨骼肌失神经肌萎缩时成肌调节因子基因的表达

背景：在去神经早期大鼠骨骼肌成肌调节因子(MyoD)表达明显上调,有明显延缓骨骼肌肌萎缩的作用。临床实验证实电刺激是治疗失神经肌萎缩的有效方法。尚未有实验证实电刺激对失神经肌萎缩MyoD表达的影响。 目的：验证电刺激对大鼠骨骼肌MyoD基因表达的影响。 设计、时间及地点：随机对照动物实验,于2008-07/11在山西医科大学动物实验中心完成。 材料：健康的SD大鼠36只,雌雄不限。随机分成3组,即空白对照组、去神经组、电刺激组,每组12只。 方法：空白对照组不做任何处理;去神经组和电刺激组大鼠制作右侧坐骨神经离断,腓肠肌失神经支配模型。用电刺激对电刺激组进行刺激,1次/d,30 min/次。分别于去神经第2,7,14,28天,处死大鼠,取小腿的腓肠肌肉标本。 主要观察指标：用反转录聚合酶链式反应技术检测MyoD mRNA的表达变化,免疫组织化学检测MyoD蛋白表达的变化。 结果：在去神经支配后第2,7,14,28天,去神经组和电刺激组标本中MyoDmRNA和蛋白含量表达上调,与空白对照组比较差异有显著性意义(P < 0.05),电刺激组表达高于去神经组(P < 0.05)。 结论：通过电刺激可以上调大鼠腓肠肌失神经模型MyoD的表达,说明电刺激是延缓骨骼失神经肌萎缩的有效方法。     

The influence of skeletal muscle reinnervation on experimentally induced myotonia

Earlier studies have shown that prior denervation of muscle prevents myotonia induced by 2,4-dichlorophenoxy acetic acid (2,4-D) both in vivo and in vitro. This work studied the effect of reinnervation on 2,4-D myotonia. Twenty Sprague-Dawley rats were injected with 2,4-D at specific intervals following unilateral sciatic nerve crushing; the gastrocnemius muscle on both sides was studied electromyographically to assess myotonia and to document denervation and reinnervation. All the rats gradually became amyotonic following denervation; myotonia reappeared during reinnervation. Myotonic discharges were no longer detectable 1 week after nerve crushing, but returned completely within 3 weeks. Blocking axoplasmic transport with colchicine had essentially the same effect on myotonia. A reciprocal temporal relationship was noted between the occurrence of fibrillations and myotonic discharges. These findings substantiate the view that innervation is essential to maintain the muscle membrane in a state that will support myotonic discharges.     

Persistence in degenerating sciatic nerve of substances having a trophic influence upon cultured muscle

Citrate-soluble proteins were extracted from normal and degenerating chicken sciatic nerves and added to chick embryonic skeletal muscle cultures to determine the trophic influences of the soluble proteins upon muscle cells. The soluble protein from normal or degenerating nerves was equally effective in promoting the protein synthesis, maturation, and long-term maintenance of muscle cells. Normal or degenerating nerve protein stimulated [¹⁴C]leucine incorporation into muscle cell protein and enhanced the morphologic maturation of muscle cells in culture. Furthermore, matured, cross-striated myotubes survived longer in the presence of normal or degenerating nerve protein than in control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on thin-layer gels revealed similar electrophoretic patterns for normal and degenerating nerve proteins. Electrophoretic gels also revealed the presence in these extracts of a protein with MW_R 84,000 which migrated in a manner identical to a neurotrophic protein which was recently purified from chicken sciatic nerves. The present results indicate that neural proteins having trophic influences upon muscle in vitro persist in degenerating sciatic nerve. We therefore conclude that the in vivo changes in muscle after denervation are not due to the depletion of trophic substances following nerve transection.     

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.