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.     

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.     

Age differences in morphology of reinnervation of partially denervated mouse muscle

The effect of age on the ability of motor neurons to develop and maintain an enlarged total axonal and synaptic volume was compared in soleus muscles of 5-8-month and 25-30-month mice, 30-120 d after partial denervation. Before and after partial denervation (transection of the L5 root), the total number of muscle fibers was the same in all muscles. However, in young animals, there was only some transient atrophy and hypertrophy mostly receded by 120 d, whereas in old muscle, a more prominent early atrophy was followed by persistent hypertrophy. Ectopic endplates were not found. In zinc-iodide-osmium (ZIO) stained preparations, muscle fibers with small nerve terminals were present at 60 d and were still present in old muscle at 120 d. Fluorescent staining of nerve terminals and acetylcholine receptors revealed that in young muscle, postsynaptic sites were nearly or completely reoccupied by 60 d. In old muscle, about 22% of former junctions were denervated, with the remainder minimally to fully reinnervated. At 60 d and thereafter, collateral sprouts originated from nodes of Ranvier in both young and old muscle and were remyelinated in young but mainly unmyelinated and remarkably tortuous in old animals. These results, confirmed with immunofluorescent strains for myelin basic protein and neurofilaments, account for many of the physiological findings (Jacob and Robbins, 1990). Motor unit size expanded 2.5 times in young and 2 times in old muscle at 60 d after partial denervation. However, the increment in total quantal output and nerve terminal volume per motor neuron was 60-100% greater than control in young but only 20-25% greater in old muscle, with little further recovery. This inability of the motor neuron in old mice to expand the field of innervation may reflect a limitation imposed by reduced axonal transport. The present findings may elucidate the muscle weakness in postpolio syndrome and amyotrophic lateral sclerosis.     

Changes in denervated skeletal muscle of amiodarone-fed mice

Prolonged dosing of mice with amiodarone produced a myopathy characterized by autophagic vacuolation and phospholipid inclusions. A previous morphological study had shown that amiodarone did not affect the rate of nerve regeneration after sciatic nerve crush. In the present study, reinnervation was assessed by the reappearance of miniature endplate potentials that confirmed that axonal regeneration and motor reinnervation was not affected by amiodarone. However, there was a marked delay in the recovery of motor function in the amiodarone-treated mice. Denervation was found to induce an extensive necrosis of muscle fibers in the deeper parts of fast-twitch muscles. Histochemical studies showed that type 1 fibers were spared, necrosis affecting mainly type 2 fibers with relatively high oxidative enzyme activity (fast-twitch oxidative fibers). Biochemical studies showed a significant increase in the amount of amiodarone and its metabolite in denervated muscle of amiodarone-treated mice when compared with contralateral, normally innervated muscles.     

Inhibition of caspases promotes long-term survival and reinnervation by axotomized spinal motoneurons of denervated muscle in newborn rats

We examined whether (1) a pan-caspase inhibitor, Boc-D-FMK, exerts long-term neuroprotective effects on spinal motoneurons (MNs) after root avulsion in neonatal rats and (2) whether the rescued spinal MNs regenerate their axons into a peripheral nerve (PN) graft and reinnervate a previously denervated target muscle. Eight weeks after root avulsion, 67% of spinal MNs remained in the Boc-D-FMK-treated group, whereas all MNs died in the sham control group. By 12 weeks postinjury, however, all Boc-D-FMK treated MNs died. In the regeneration experiment, a PN graft was implanted at different times after injury. The animals were allowed to survive for 4 weeks following the operation. Without caspase inhibition, MNs did not regenerate at any time point. In animals treated with Ac-DEVD-CHO, a caspase-3-specific inhibitor, and Boc-D-FMK, 44 and 62% of MNs, respectively, were found to regenerate their axons into a PN graft implanted immediately after root avulsion. When the PN graft was implanted 2 weeks after injury, however, MNs failed to regenerate following Ac-DEVD-CHO treatment, whereas 53% of MNs regenerated their axons into the graft after treatment with Boc-D-FMK. No regeneration was observed when a PN graft was implanted later than 2 weeks after injury. In the reinnervation study, injured MNs and the target biceps muscle were reconnected by a PN bridge implanted 2 weeks after root avulsion with administration of Boc-D-FMK. Eight weeks following the operation, 39% of MNs reinnervated the biceps muscle. Morphologically normal synapses and motor endplates were reformed in the muscle fibers. Collectively, these data provide evidence that injured neonatal motoneurons can survive and reinnervate peripheral muscle targets following inhibition of caspases.     

Perlecan and synaptophysin changes in denervated skeletal muscle

The present study observed sciatic nerve and gastrocnemius muscle changes in denervated rats using morphology methods,and assessed expression of perlecan,an extracellular matrix com-ponent,which is located at the skeletal muscle cell surface as acetylcholine esterase,as well as synaptophysin,a synaptic marker.Results showed degeneration and inflammation following transection of the sciatic nerve.In addition,the sciatic nerve-dominated skeletal muscle degen-erated with mild inflammation,indicating that skeletal muscle atrophy primarily contributed to denervation-induced nutritional disturbances.With prolonged injury time(1-4 weeks post-injury),perlecan expression gradually decreased and reached the lowest level at 4 weeks,but synap-tophysin expression remained unchanged after denervation.Results suggested that perlecan expression was more sensitive to denervation and reflected regional extracellular matrix changes following denervation.     

去神经支配骨骼肌萎缩的多种治疗

背景：骨骼肌去神经支配后出现肌萎缩和收缩功能的丧失,肌纤维发生与萎缩相关的一系列的形态学和组织学的变化。近年来随着技术水平及理论研究的进展,治疗效果有了较大的提高,但仍未达到满意的效果。 目的：总结并讨论近年来有关去神经支配骨骼肌萎缩的治疗新进展,为肌肉功能的恢复提供理论基础。 方法：由第一作者用计算机检索中国期刊全文数据库(CNKI：2000/2010)和Medline数据库(2000/2010),检索词分别为“去神经支配,骨骼肌,肌萎缩,治疗”和“denervated,skeletal muscles,atrophy,treatment”。共检索到135篇文章,按纳入和排除标准对文献进行筛选,共纳入30篇文章,从物理治疗、神经修复和植入、细胞移植、药物、基因和中药治疗等方面进行总结和分析。 结果与结论：近年来去神经支配骨骼肌的治疗取得了很大进展,但这些方法只能在一定程度上缓解骨骼肌萎缩,在一段时间内维持肌肉的生理功能。目前研究随着去神经支配骨骼肌萎缩机制的阐明,针对性的治疗措施将会取得更好的效果。     

Does electrical stimulation of denervated muscle,continued after reinnervation,influence recovery of contractile function?

The study was conducted to determine if daily electrical stimulation of denervated muscle, initiated the day following crush denervation and continued for 8 weeks (i.e., 5 weeks after presumptive reinnervation), would influence denervation-associated alterations in muscle size and in situ contractile properties of rat gastrocnemius. A stimulation protocol of brief, strong, isometric contractions was designed to maximize the beneficial effects as described by previous authors. By 8 weeks after crush, unstimulated muscles were still significantly lighter in wet weight, were tetanically weaker, and showed slower isometric contractile responses in situ than controls. Denervated muscles which had been stimulated daily were heavier and tetanically stronger (the latter not different from controls) than those in the nonstimulated group. Muscle weights from groups of animals killed at 2 or 4 weeks after nerve crush indicated the major benefit of stimulation occurred during this initial 4-week period. In situ fatigue properties were unaffected by denervation or stimulation. A protocol of electrical stimulation-evoked strong contractions, initiated soon after denervation and continued after reinnervation, was effective in attenuating the strength-related, but not speed-related, changes in neuromuscular function resulting from denervation. These latter changes are presumably the result of loss of “neurotrophic influence” and/or continuous low-tension muscle activity lost as a result of denervation.     

Remodeling of the cytoskeletal lattice in denervated skeletal muscle

The effect of denervation-induced atrophy on the cytoskeletal lattice in rat fast- and slow-twitch skeletal muscle has been investigated. Immunochemical analyses and immunofluorescence microscopy experiments employing monospecific antibodies to dystrophin, desmin, and α-tubulin were carried out on intact and denervated muscles. The relative cellular content of dystrophin and desmin were reduced in the soleus muscle (slow-twitch), while significant increases were shown in the gastrocnemius muscle (fast-twitch). In both muscles, α-tubulin levels increased up to 12-fold as a function of time compared to control values. Immunofluorescence microscopy revealed a distinct rearrangement of the microtubule network toward a predominantly longitudinal alignment, which was accompanied by an increase in the density of the fluorescence. It is concluded that the relative increase of the three structural proteins in the fast-twitch gastrocnemius muscle may be related to the apparent resistance of this muscle type to denervation-induced atrophy. The increased α-tubulin content in denervated slow- and fast-twitch muscles could be indicative of an adaptive mechanism designed to maintain the integrity of the muscle fiber in view of eventual regenerative activities. © 1996 John Wiley & Sons, Inc.     

Molecular species of acetylcholinesterase in denervated rat skeletal muscle

Acetylcholinesterase (AChE, E.C. 3.1.1.7) activity was investigated in homogenates of control and denervated rat extensor digitorum longus muscle. Denervation produced a large, rapid decrease in AChE activity. Calculated on a whole-muscle basis, this decrease was 18% at 2 days after denervation and 75% at 7 days. NaCl (1 m), 0.5% Triton X-100 extracts of extensor digitorum longus muscle were subjected to velocity sedimentation in sucrose gradients. Three peaks of AChE activity were found with sedimentation coefficients of 3.9 S, 9.9 S, and 16.0 S. In control muscles, the distribution of AChE activity among the three peaks was 39, 39, and 14%, respectively. Two days after denervation, this distribution was greatly altered, but the sedimentation coefficients remained the same. Whereas the AChE activity associated with the 3.9 S and 16.0 S peaks was reduced, that of the 9.9 S peak was increased. The greatest quantitative loss in AChE activity was associated with the 3.9 S peak. Seven days after denervation, the AChE activity of each of the three forms was greatly reduced.     

失神经支配的骨骼肌萎缩与防治

背景：随着当今医学的发展,失神经支配骨骼肌萎缩的防治已取得了显著的进步,但临床疗效仍不十分满意。 目的：对失神经支配骨骼肌萎缩防治方法的研究现状作一总结,试图寻找更为有效的失神经支配骨骼肌萎缩防治方法。 方法：以denervation,muscle atrophy,treatment为检索词,检索Medline数据库(1998-01/2008-01)。以失神经,肌萎缩,治疗为检索词,检索中国期刊全文数据库(1998-01/2008-01)、万方数据库(1998-01/2008-01)和《中国临床康复》杂志(1998-01/2008-01)。文献检索语种限制为英文和中文。以肌肉的耐力及收缩力、失神经的肌湿重和骨骼肌的修复情况为评价指标。纳入研究失神经支配骨骼肌萎缩的显微外科手术方法、物理疗法、生物和化学疗法、基因疗法。排除上述方法之外失神经支配骨骼肌萎缩的其他疗法。 结果与结论：周围神经损伤后,骨骼肌失神经支配将不可避免的发生萎缩。因此,探索失神经支配骨骼肌萎缩的防治方法,吸引了国内外许多学者的兴趣,必将成为21世纪周围神经领域内的重要任务和研究热点。显微外科手术、物理疗法、生物和化学疗法、基因疗法等都是失神经支配骨骼肌萎缩有效的防治手段。目前,该领域的研究已经呈现多角度、多方面的趋势。失神经肌萎缩的防治方面已经有了针对性的措施,但在改善微循环、防止细胞凋亡、抑制胶原过度生长以及如何应用基因治疗的方法在基因水平改变生肌调节因子的表达等方面,还有大量工作需要进行。随着组织工程学、细胞培养学、分子生物学、基因工程等方面的不断发展,防治失神经肌萎缩必定会有新的突破。     

Effect of swimming on reinnervation of rat skeletal muscle

There are no studies that define the optimum intensity or time to begin exercising reinnervating muscle. Through overwork of reinnervating muscle, accomplished by synergistic tenotomy, we developed a working hypothesis. This hypothesis was tested on a physiological model. In the present study, the sciatic nerve of five groups of rats was crushed. One group served as the control and the remaining animals were exercised by swimming with weights attached to their tails for one or two hours each day (early reinnervation group) or four weeks (late reinnervating group) after crush denervation. The evaluation of muscle weights, fibre types, and protein concentration indicate that intense swimming (two hours every day) does not enhance the repair of reinnervation muscle. An evaluation of total proteins suggests that a high workload may be hazardous in the early phase of reinnervation. This study tends to confirm the hypothesis that when there are too few contractile units, as presumably is the case in early reinnervation, exercise inhibits the reinnervation process.     

Apoptosis in young and old denervated rat skeletal muscle

Introduction: We investigated the apoptotic response to different degrees of denervation in young and older rats randomized into control (C), partial (PD), and complete denervation (CD) of muscles innervated by the sciatic nerve. Methods: Muscle wet weight to body weight (MWW/BW), myosin heavy chain (MHC) isoforms, and fiber cross‐sectional area were determined in gastrocnemius and soleus muscles. Apoptotic responses were determined by changes in myonuclei and expression of Bcl‐2 and BAX. Results: PD and CD resulted in significant reductions in MWW/BW and FCSA in both young and older rats. Older controls had greater apoptotic responses than young controls. Apoptotic responses were greater in PD and CD than in C in both age groups. No statistical interaction between denervation and age group was seen. Conclusions: Older age was associated with increased level of apoptosis, but older muscle was not more vulnerable to the effect of denervation. Muscle Nerve 55 : 262–269, 2017     

Regulation of increased acid proteinase in denervated skeletal muscle.

Acid proteolytic enzyme activity was determined in homogenates of control and denervated rat extensor digitorum longus muscle. With either undenatured or [³H]acetylated hemoglobin (pH3.5) as substrate, total enzyme activity in muscles denervated near the point of nerve entry was consistently increased 48 and 72 hr after surgery. A series of ‘near-far’ experiments was performed to determine if the length of nerve stump left attached to the denervated muscle would affect the time course of the elevation in proteolytic activity, but these experiments did not result in differences that could be attributed to the level of nerve section. Acid proteinase, presumably important in degradative processes, is clearly under some form of neural control. Whether the motor nerve exerts this influence by controlling the level of muscular activity or by additional means remains to be established.     

Effect of reinnervation on the degradation rate of junctional acetylcholine receptors synthesized in denervated skeletal muscles

Two populations of ACh receptors (AChRs) with different degradation rates have been shown to coexist in the postsynaptic membrane after denervation of the neuromuscular junction (NMJ). One population, consisting of the slowly degrading original AChRs inserted into the plasma membrane prior to denervation, has a degradation half-life (t1/2) of approximately 8 d. This degradation rate accelerates after denervation (to a t1/2 approximately 3 d), but can be decelerated back to the predenervation rate by reinnervation. The second population, the rapidly degrading new AChRs, which replace the degrading original AChRs at the NMJ after denervation, resembles embryonic AChRs, with a t1/2 of approximately 1 d. In the present study, we report that the degradation rate of these new junctional AChRs is unaltered for 3-6 half-lives after reinnervation. We further report that a small amount (less than 10%) of slowly degrading AChRs (t1/2 approximately 3 d) may also be synthesized in denervated muscle. We suggest that, unlike its effect on the original, slowly degrading AChRs, reinnervation does not modulate the degradation rate of the rapidly degrading new junctional AChRs. It merely regulates the ratio of rapidly to slowly degrading AChRs being synthesized and inserted at the NMJ.     

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.