Repetitive stretch suppresses denervation‐induced atrophy of soleus muscle in rats

This study was conducted to examine whether stretch‐related mechanical loading on skeletal muscle can suppress denervation‐induced muscle atrophy, and if so, to depict the underlying molecular mechanism. Denervated rat soleus muscle was repetitively stretched (every 5 s for 15 min/day) for 2 weeks. Histochemical analysis showed that the cross‐sectional area of denervated soleus muscle fibers with repetitive stretching was significantly larger than that of control denervated muscle (P < 0.05). We then examined the involvement of the Akt/mammalian target of the rapamycin (mTOR) cascade in the suppressive effects of repetitive stretching on muscle atrophy. Repetitive stretching significantly increased the Akt, p70S6K, and 4E‐BP1 phosphorylation in denervated soleus muscle compared to controls (P < 0.05). Furthermore, repetitive stretching‐induced suppression of muscle atrophy was fully inhibited by rapamycin, a potent inhibitor of mTOR. These results indicate that denervation‐induced muscle atrophy is significantly suppressed by stretch‐related mechanical loading of the muscle through upregulation of the Akt/mTOR signal pathway. Muscle Nerve, 2009     

Regenerative cell injection in denervated muscle reduces atrophy and enhances recovery following nerve repair

Introduction: Functional muscle recovery after peripheral nerve injury is far from optimal, partly due to atrophy of the muscle arising from prolonged denervation. We hypothesized that injecting regenerative cells into denervated muscle would reduce this atrophy. Methods: A rat sciatic nerve lesion was performed, and Schwann cells or adipose‐derived stem cells, untreated or induced to a “Schwann‐cell–like” phenotype (dASC), were injected into the gastrocnemius muscle. Nerves were either repaired immediately or capped to prevent muscle reinnervation. One month later, functionality was measured using a walking track test, and muscle atrophy was assessed by examining muscle weight and histology. Results: Schwann cells and dASC groups showed significantly better scores on functional tests when compared with injections of growth medium alone. Muscle weight and histology were also significantly improved in these groups. Conclusion: Cell injections may reduce muscle atrophy and could benefit nerve injury patients. Muscle Nerve 47: 691–701, 2013     

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     

Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1487–1496, 1997     

Lack of type 1 and type 2A myosin heavy chain isoforms in rat slow muscle regenerating during chronic nerve block

The degeneration regeneration process was induced by bupivacaine injection in innervated, denervated, and nerve-blocked rat soleus muscles. Nerve block was obtained by superfusion of the sciatic nerve with tetrodotoxin (TTX). Two weeks after bupivacaine injection, immunohistochemical and electrophoretical analyses showed the presence of type 1 myosin heavy chain (MHC) only in innervated regenerated muscles, type 2A in innervated and denervated, but not in TTX-paralyzed muscles, and type 2X under all experimental conditions. The presence of type 1 MHC in the innervated, and its absence in both denervated and TTX-paralyzed muscles were also verified immunohistochemically 1 week after bupivacaine injection. It is concluded that the nerve impulses play a determinant role in the expression of 1 and 2A MHC isoforms in the innervated regenerating muscle. The possible causes of the absence of the type 2A MHC isoform in the TTX-paralyzed muscles are discussed. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 226–232, 1998     

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。     

Effect of locomotor training on muscle performance in the context of nerve-muscle communication dysfunction

Introduction: The effects of locomotor training (LT) on skeletal muscle after peripheral nerve injury and acetylcholinesterase deficiency are not well documented. Methods: We determined the effects of LT on mouse soleus muscle performance after sciatic nerve transection with excision (full and permanent denervation), nerve transection (partial functional reinnervation), nerve crush (full denervation with full functional reinnervation), and acetylcholinesterase deficiency (alteration in neuromuscular junction functioning). Results: We found no significant effect of LT on the recovery of soleus muscle weight, maximal force in response to muscle stimulation, and fatigue resistance after nerve transection with or without excision. However, LT significantly increased soleus muscle fatigue resistance after nerve crush and acetylcholinesterase deficiency. Moreover, hindlimb immobilization significantly aggravated the deficit in soleus muscle maximal force production and atrophy after nerve crush. Conclusions: LT is beneficial, and reduced muscle use is detrimental for intrinsic muscle performance in the context of disturbed nerve–muscle communication. Muscle Nerve, 2012     

Butyrate prevents muscle atrophy after sciatic nerve crush

Introduction: Histone deacetylases (HDACs) have been implicated in neurogenic muscle atrophy, but the mechanisms by which HDAC inhibitors might have beneficial effects are not defined. Methods: We used sciatic nerve crush to determine the effect of butyrate on denervation‐induced gene expression and oxidative stress. Results: Butyrate treatment initiated 3 weeks before injury and continued 1 week after injury increases histone acetylation and reduces muscle atrophy after nerve crush. Butyrate delivered only after nerve crush similarly prevented muscle atrophy. Butyrate had no effect on the increase in histone deacetylase 4 (HDAC4) protein levels following nerve crush but prevented the increase in expression of myogenin, MuRF1, and atrogin‐1. Butyrate did not affect mitochondrial reactive oxygen species production, but it increased antioxidant enzyme activity, reduced proteasome activity, and reduced oxidative damage following nerve injury. Conclusions: These data suggest that HDAC inhibitors are promising pharmacological agents for treating neurogenic muscle atrophy. Muscle Nerve 52: 859–868, 2015     

Intermittent stretching induces fibrosis in denervated rat muscle

Introduction: Stretching (St) has been used for treating denervated muscles. However, its effectiveness and safety claims require further study. Methods: Rats were divided into: (1) those with denervated (D) muscles, evaluated 7 or 15 days after sciatic nerve crush injury; (2) those with D muscles submitted to St during 7 or 15 days; and (3) those with normal muscles. Muscle fiber cross‐sectional area, serial sarcomere number, sarcomere length, and connective tissue density were measured. MMP‐2, MMP‐9, TIMP‐1, TGF‐β1, and myostatin mRNAs were determined by real‐time polymerase chain reaction. MMP‐2 and MMP‐9 activity was evaluated by zymography. Collagen I was localized using immunofluorescence. Results: St did not prevent muscle atrophy due to denervation, but it increased fibrosis and collagen I deposition at day 15. St also upregulated MMP‐9 and TGF‐β1 gene expressions at day 7, and myostatin at day 15. Conclusions: Stretching denervated muscle does not prevent atrophy, but it increases fibrosis via temporal modulation of TGF‐β1/myostatin and MMP‐9 cascades. Muscle Nerve 53 : 118–126, 2016     

Proteomic analysis of mouse soleus muscles affected by hindlimb unloading and reloading

Introduction: Disuse muscle atrophy, induced by prolonged space flight, bed rest, or denervation, is a common process with obvious changes in slow‐twitch soleus muscles. Methods: Proteomic analysis was performed on mouse soleus subjected to hindlimb unloading (HU) and hindlimb reloading (HR) to identify new dysregulated proteins. Results: Following HU, the mass and cross‐sectional area of muscle fibers decreased, but they recovered after HR. Proteomic analyses revealed 9 down‐regulated and 7 up‐regulated proteins in HU, and 2 down‐regulated and 5 up‐regulated proteins in HR. The dysregulated proteins were mainly involved in energy metabolism, protein degradation, and cytoskeleton stability. Among the dysregulated proteins were fatty acid binding protein 3, α‐B crystalline, and transthyretin. Conclusions: These results indicate that muscle atrophy induced by unloading is related to activation of proteolysis, metabolic alterations toward glycolysis, destruction of myofibrillar integrity, and dysregulation of heat shock proteins (HSPs). The dysregulated proteins may play a role in muscle atrophy and the recovery process. Muscle Nerve 52 : 803–811, 2015     

Lack of caspase‐3 attenuates immobilization‐induced muscle atrophy and loss of tension generation along with mitigation of apoptosis and inflammation

Introduction: Immobilization by casting induces disuse muscle atrophy (DMA). Methods: Using wild type (WT) and caspase‐3 knockout (KO) mice, we evaluated the effect of caspase‐3 on muscle mass, apoptosis, and inflammation during DMA. Results: Caspase‐3 deficiency significantly attenuated muscle mass decrease [gastrocnemius: 28 ± 1% in KO vs. 41 ± 3% in WT; soleus: 47 ± 2% in KO vs. 56 ± 2% in WT; (P < 0.05)] and gastrocnemius twitch tension decrease (23 ± 4% in KO vs. 36 ± 3% in WT, P < 0.05) at day 14 in immobilized vs. contralateral hindlimb. Lack of caspase‐3 decreased immobilization‐induced increased apoptotic myonuclei (3.2‐fold) and macrophage infiltration (2.2‐fold) in soleus muscle and attenuated increased monocyte chemoattractant protein‐1 mRNA expression (2‐fold in KO vs. 18‐fold in WT) in gastrocnemius. Conclusions: Caspase‐3 plays a key role in DMA and associated decreased tension, presumably by acting on the apoptosis and inflammation pathways. Muscle Nerve 47: 711–721, 2013     

Adipose-derived stem cells delay muscle atrophy after peripheral nerve injury in the rodent model

Introduction: Injuries to peripheral nerves cause distal muscle atrophy. The effects of adipose-derived stem cell (ASC) injections into a muscle after injury were examined. Methods: A 1.5 cm defect in the rat sciatic nerve was created, resulting in gastrocnemius muscle atrophy. The nerve defect was repaired with autograft; DiR-labeled ASCs were injected into the gastrocnemius immediately postoperatively. Quantitation of gross musculature and muscle fiber area, cell survival, fibrosis, lipid deposition, inflammation, and reconstructive responses were investigated. Results: ASCs were identified in the muscle at 6 weeks, where injections showed increased muscle mass percentage retained, larger average fiber area, and less overall lipid content accumulated throughout the musculature. Muscles having received ASCs showed increased presence of interlukin-10 and Ki67, and decreased inducible nitric oxide synthase (iNOS). Discussion: This investigation is suggestive that an ASC injection into denervated muscle post-operatively is able to delay the onset of atrophy. Muscle Nerve 59:603–603, 2019     

FYN‐dependent muscle–immune interaction after sciatic nerve injury

Denervation of skeletal muscles results in loss of muscle mass and contractile force. Recent evidence suggests that local immune system activation plays a key role in these processes, but the mechanisms underlying muscle–immune system cross‐talk are not understood. The purpose of this study was to address the mechanisms by which muscle responds to denervation and to elucidate the specific role played by FYN in local immune system activation. We studied initial events taking place in the gastrocnemius of wild‐type and Fyn^?/? mice following sciatic nerve transection. Discontinuous sucrose gradient centrifugation was used to prepare lipid rafts at different time‐points (1, 7, and 14 days) after surgery. Activation of FYN, cytokine expression (IL‐1β and TNF‐α), and T‐cell activation (CD3 and IL‐15) were followed by in vitro kinase assays, enzyme‐linked immunoassay (ELISA), Western blotting, and immunoprecipitation. Sciatic nerve injury resulted in increased SRC kinase activity in gastrocnemius lipid rafts. Production of both IL‐1β and TNF‐α was increased, peaking after 1 day, followed after 7 and 14 days by upregulation of IL‐15 and CD3 expression and the development of caveolin‐3 and CD3 complexes. The integrity of lipid rafts and the upregulation of SRC kinase activity, cytokine expression, and T‐cell activation and cross‐talk with muscle cells following denervation were abolished in Fyn^?/? mice. The integrity of FYN‐dependent lipid rafts is required for local immune system activation within denervated muscle, and lipid rafts are implicated in orchestrating muscle–immune‐cell cross‐talk. These results are likely to provide new insights into the therapy of neuromuscular injury. Muscle Nerve, 2010     

The rate of nutrient supply to normal and denervated slow and fast muscle, and its relation to muscle blood flow

Muscle blood flow, glucose uptake, and the ability of muscle to concentrate an inert amino acid have been studied in vivo using a variety of radiosotopes. The rate of blood flow is four times greater in the slow soleus muscle than in the fast gastrocnemius muscle of the rat, but the rate of deoxyglucose uptake is 14 times higher in the slow muscle relative to the fast muscle. Slow muscle is also able to accumulate the inert amino acid, α-aminoiso-butyric acid, more effectively than does fast muscle. A week after denervation, the rate of blood flow through denervated muscles was increased 30- to 40-fold over control values. However, deoxyglucose uptake was not significantly elevated in the gastrocnemius and was reduced to 20% of control levels in the soleus. The ability of the denervated muscles to concentrate α-aminoisobutyric acid was also impaired. These changes could not be accounted for in terms of an altered inulin space. There is no correlation between the rate of muscle blood flow and the ability of muscle to extract blood-borne nutrients. Impaired transport mechanisms precede and may initiate the major morphological changes that follow muscle denervation.     

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。     

Biometrical and histochemical comparison between extra- and intra-fusal muscle fibres in denervated and re-innervated rat muscle

Summary Biometrical and histochemical changes in extra- and intrafusal muscle fibres were determined in twenty adult rats during denervation and re-innervation. The right sciatic nerve was severed and at regular intervals the denervated gastrocnemius, soleus and peroneal muscles were examined and compared with the corresponding muscles of the non denervated limb.Although the extra- and intra-fusal muscle fibres show the same histochemical changes, the atrophy of the intra-fusal muscle fibres is insignificant compared with the atrophy of the extra-fusal muscle fibres in the denervation phase. This could be explained by the small size of the intra-fusal fibres and the increased fluid resorption of the spindle.In the re-innervation phase the same histochemical changes occur in both types of muscle fibres.     

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.     

Effects of denervation on glycoproteins of rabbit gastrocnemius and soleus muscles

Rabbit gastrocnemius and soleus differ in speed of contraction, in acetylcholine sensitivity, and in calcium uptake into fragmented sarcoplasmic reticulum. Following denervation, the gastrocnemius changes with respect to speed of contraction, acetylcholine sensitivity, and calcium uptake, whereas the soleus changes much less. Glycoproteins are important constituents of cell membranes, and it was felt that these constituents may reflect differences between innervated and denervated muscles. Concentration of protein-bound sialic acid was 0.75 nmoles/mg protein in fat free residue of innervated gastrocnemius and 1.4 nmoles/mg protein in fat free residue of innervated soleus. The concentration of protein-bound hexosamines was 4.0 nmoles/mg protein in gastrocnemius and 5.8 nmoles/mg protein in soleus. Following denervation for fifteen days, the protein-bound NANA (N-acetylneuraminic acid) of gastrocnemius increased from 638 nmoles/muscle to 920 nmoles/muscle; whereas soleus protein-bound NANA did not change (168 nmoles/muscle versus 180 nmoles/muscle). The total protein bound hexosamines did not change significantly in either denervated gastrocnemius or soleus. The data suggest the synthesis de novo of sialic acid-containing glycoproteins or the enhanced transfer of sialic acid to preformed proteins in denervated gastrocnemius but not in soleus.