Midkine-deficient mice delayed degeneration and regeneration after skeletal muscle injury

Midkine (MK), a heparin-binding growth factor, was previously found to be expressed in the rat myotube-forming stage. We investigated MK gene-deficient (Mdk^−/−) mice in terms of skeletal muscle degeneration and regeneration after injury by bupivacaine injection into the tibialis anterior muscle. Injured muscles showed intense inflammatory cell infiltration. Myotubes, myofibers with centrally located nuclei in their cytoplasm, were significantly smaller in Mdk^−/− mice than in wild type (Mdk^+/+) mice 7 days after injury (p = 0.02). The distribution of myotube sizes showed quantitative differences between the two groups at 5 and 7 days, but not at 14 days. Many small myotubes were found in the regenerative area of Mdk^−/− mice compared with that of Mdk^+/+mice 5 and 7 days after injury. The expression of Iba1, a macrophage marker, was significantly lower in Mdk^−/− mice 3 days after injury (p = 0.01). The number of desmin-positive cells like myoblasts in Mdk^−/− mice was significantly fewer than that in Mdk^+/+ mice 3 days after injury. Our results suggested that deletion of MK results in a delay in regeneration, preceded by decelerated migration of macrophages to the damaged area, and that MK has a role in cell differentiation and maturation after skeletal muscle injury.     

NS-398, a cyclooxygenase-2-specific inhibitor, delays skeletal muscle healing by decreasing regeneration and promoting fibrosis

Nonsteroidal anti-inflammatory drugs are often prescribed after muscle injury. However, the effect of nonsteroidal anti-inflammatory drugs on muscle healing remains primarily controversial. To further examine the validity of using these drugs after muscle injury, we investigated the working mechanism of NS-398, a cyclooxygenase-2-specific inhibitor. In vitro experiments showed that NS-398 inhibited the proliferation and maturation of differentiated myogenic precursor cells, suggesting a detrimental effect on skeletal muscle healing. Using a mouse laceration model, we analyzed the in vivo effect of NS-398 on skeletal muscle healing at time points up to 4 weeks after injury. The in vivo results revealed delayed muscle regeneration at early time points after injury in the NS-398-treated mice. Compared to controls, lacerated muscles treated with NS-398 expressed higher levels of transforming growth factor-beta1, which corresponded with increased fibrosis. In addition, transforming growth factor-beta1 co-localized with myostatin, a negative regulator of skeletal muscle growth. We also found reduced neutrophil and macrophage infiltration in treated muscles, indicating that the delayed skeletal muscle healing observed after NS-398 treatment could be influenced by the anti-inflammatory effect of NS-398. Our findings suggest that the use of cyclooxygenase-2-specific inhibitors to treat skeletal muscle injuries warrants caution because they may interfere with muscle healing.     

Heat stress promotes skeletal muscle regeneration after crush injury in rats

Influences of heat stress on skeletal muscle regeneration were examined in experimental rats. After crush injury to the Extensor digitorum longus muscle (EDL) of the left hindlimb, animals were randomly divided into non-heat and heat groups. In the latter, packs filled with hot water (42 °C) were percutaneously applied to the injured EDL muscle for 20 min to the front of the lower leg, soon after the injury. During the early stages of muscle regeneration, due to the heat stress, secondary degeneration at the injured site progressed faster, and migration of macrophages, proliferation and differentiation of satellite cells were facilitated. At 14 and 28 days after the injury, the ratio of regenerating muscle fibers exhibiting central nuclei in the heat treated group was significantly lower than that in the non-heat group, and cross sectional area in the heat group was evidently larger than that in the non-heat group. Moreover, in the heat group, the ratio of collagen fiber area at 14 and 28 days after the injury was smaller than in the non-heat group. Together, these findings suggest that acceleration of degeneration processes by heat stress soon after injury is likely to promote skeletal muscle regeneration and inhibit collagen deposition.     

Electron-microscopic study of capillary regeneration in skeletal muscle after mechanical injury

Bulletin of Experimental Biology and Medicine -     

Nrf2 augments skeletal muscle regeneration after ischaemia–reperfusion injury

Skeletal muscles harbour a resident population of stem cells, termed satellite cells (SCs). After trauma, SCs leave their quiescent state to enter the cell cycle and undergo multiple rounds of proliferation, a process regulated by MyoD. To initiate differentiation, fusion and maturation to new skeletal muscle fibres, SCs up‐regulate myogenin. However, the regulation of these myogenic factors is not fully understood. In this study we demonstrate that Nrf2, a major regulator of oxidative stress defence, plays a role in the expression of these myogenic factors. In both promoter studies with myoblasts and a mouse model of muscle injury in Nrf2‐deficient mice, we show that Nrf2 prolongs SC proliferation by up‐regulating MyoD and suppresses SC differentiation by down‐regulating myogenin. Moreover, we show that IL‐6 and HGF, both factors that facilitate SC activation, induce Nrf2 activity in myoblasts. Thus, Nrf2 activity promotes muscle regeneration by modulating SC proliferation and differentiation and thereby provides implications for tissue regeneration.Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Macrophage activation and skeletal muscle healing following traumatic injury

Following injury to different tissues, macrophages can contribute to both regenerative and fibrotic healing. These seemingly contradictory roles of macrophages may be related to the markedly different phenotypes that macrophages can assume upon exposure to different stimuli. We hypothesized that fibrotic healing after traumatic muscle injury would be dominated by a pro‐fibrotic M2a macrophage phenotype, with M1 activation limited to the very early stages of repair. We found that macrophages accumulated in lacerated mouse muscle for at least 21 days, accompanied by limited myofibre regeneration and persistent collagen deposition. However, muscle macrophages did not exhibit either of the canonical M1 or M2a phenotypes, but instead up‐regulated both M1‐ and M2a‐associated genes early after injury, followed by down‐regulation of most markers examined. Particularly, IL‐10 mRNA and protein were markedly elevated in macrophages from 3‐day injured muscle. Additionally, though flow cytometry identified distinct subpopulations of macrophages based on high or low expression of TNFα, these subpopulations did not clearly correspond to M1 or M2a phenotypes. Importantly, cell therapy with exogenous M1 macrophages but not non‐activated macrophages reduced fibrosis and enhanced muscle fibre regeneration in lacerated muscles. These data indicate that manipulation of macrophage function has potential to improve healing following traumatic injury. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

VEGF enhances intraneural angiogenesis and improves nerve regeneration after axotomy

Whilst there is an increased understanding of the cell biology of nerve regeneration, it remains unclear whether there is a direct interrelationship between vascularisation and efficacy of nerve regeneration within a nerve conduit. To establish this is important as in clinical surgery peripheral nerve conduit grafting has been widely investigated as a possible alternative to the use of nerve autografts. The aim of this study was to assess whether vascular endothelial growth factor (VEGF), a highly specific endothelial cell mitogen, can enhance vascularisation and, indirectly, axonal regeneration within a silicone nerve regeneration chamber. Chambers containing VEGF (500–700 ng/ml) in a laminin‐based gel (Matrigel) were inserted into 1 cm rat sciatic nerve defects and nerve regeneration examined in relation to angiogenesis between 5 and 180 d. Longitudinal sections were stained with antibodies against endothelial cells (RECA‐1), axons (neurofilament) and Schwann cells (S‐100) to follow the progression of vascular and neural elements. Computerised image analysis demonstrated that the addition of VEGF significantly increased blood vessel penetration within the chamber from d 5, and by d 10 this correlated with an increase of axonal regeneration and Schwann cell migration. The pattern of increased nerve regeneration due to VEGF administration was maintained up to 180 d, when myelinated axon counts were increased by 78% compared with plain Matrigel control. Furthermore the dose‐response of blood vessel regeneration to VEGF was clearly reflected in the increase of axonal regrowth and Schwann cell proliferation, indicating the close relationship between regenerating nerves and blood vessels within the chamber. Target organ reinnervation was enhanced by VEGF at 180 d as measured through the recovery of gastrocnemius muscle weights and footpad axonal terminal density, the latter showing a significant increase over controls (P < 0.05). The results demonstrate an overall relationship between increased vascularisation and enhanced nerve regeneration within an acellular conduit, and highlight the interdependence of the 2 processes.     

Ischaemia–reperfusion modulates inflammation and fibrosis of skeletal muscle after contusion injury

Regeneration of skeletal muscle following injury is dependent on numerous factors including age, the inflammatory response, revascularization, gene expression of myogenic and growth factors and the activation and proliferation of endogenous progenitor cells. It is our hypothesis that oxidative stress preceding a contusion injury to muscle modulates the inflammatory response to inhibit muscle regeneration and enhance fibrotic scar formation. Male F344/BN rats were assigned to one of four groups. Group 1: uinjured control; Group 2: ischaemic occlusion of femoral vessels for 2 h followed by reperfusion (I‐R); Group 3: contusion injury of the tibialis anterior (TA); Group 4: I‐R, then contusion injury. The acute inflammatory response (8 h, 3 days) was determined by expression of the chemokine CINC‐1, TGF‐β1, IFN‐γ and markers of neutrophil (myeloperoxidase) and macrophage (CD68) activity and recruitment. Acute oxidative stress caused by I‐R and/or contusion, was determined by measuring GP91^phox and lipid peroxidation. Muscle recovery (21 days) was assessed by examining the fibrosis after I‐R and contusion injuries to the TA with Sirius Red staining and quantification of collagen I expression. Consistent with our hypothesis, I‐R preceding contusion increased all markers of the acute inflammatory response and oxidative stress after injury and elevated the expression of collagen. We conclude that ischaemia‐induced oxidative stress exacerbated the inflammatory response and enhanced fibrotic scar tissue formation after injury. This response may be attributable to increased levels of TGF‐β1 and diminished expression of IFN‐γ in the ischaemic contused muscle.     

Dose-response relationship of mesenchymal stem cell transplantation and functional regeneration after severe skeletal muscle injury in rats

Various therapeutic strategies that aim to influence clinical outcome after severe skeletal muscle trauma have been considered. One such method, the local transplantation of stem cells, has been shown to improve tissue regeneration. The number of cells required for successful regeneration, however, remains unclear. The aim of this study was therefore to examine the correlation between the number of transplanted bone marrow-derived mesenchymal stem cells (MSCs) and the resulting muscle function. One week after inducing an open crush trauma in 34 female Sprague Dawley rats, increasing quantities of autologous MSCs (0.1 x 10(6), 1 x 10(6), 2.5 x 10(6), and 10 x 10(6) cells) or saline solution (control group) were transplanted into the left soleus muscle of the rat hind limb. At 4 weeks posttrauma, the outcome was assessed by measuring muscle contraction forces following an indirect fast twitch and tetanic stimulation. A logarithmic dose-response relationship was observed for both maximum twitch and tetanic contraction forces (R(2) = 0.9 for fast twitch [p = 0.004]; R(2) = 0.87 [p = 0.002] for tetanic contraction). The transplantation of 10 x 10(6) cells resulted in the most pronounced improvement of muscle force. MSC therapy represents a promising new tool for the treatment of skeletal muscle trauma that shows potential for aiding in the prevention of severe functional deficiencies. The logarithmic dose-response relationship demonstrates the association between the number of transplanted cells and the resulting muscle forces, as well as the amount of MSCs required for promoting muscular regeneration.     

The mitotic clock in skeletal muscle regeneration, disease and cell mediated gene therapy

The regenerative capacity of skeletal muscle will depend on the number of available satellite cells and their proliferative capacity. We have measured both parameters in ageing, and have shown that although the proliferative capacity of satellite cells is decreasing during muscle growth, it then stabilizes in the adult, whereas the number of satellite cells decreases during ageing. We have also developed a model to evaluate the regenerative capacity of human satellite cells by implantation into regenerating muscles of immunodeficient mice. Using telomere measurements, we have shown that the proliferative capacity of satellite cells is dramatically decreased in muscle dystrophies, thus hampering the possibilities of autologous cell therapy. Immortalization by telomerase was unsuccessful, and we currently investigate the factors involved in cell cycle exits in human myoblasts. We have also observed that insulin-like growth factor-1 (IGF-1), a factor known to provoke hypertrophy, does not increase the proliferative potential of satellite cells, which suggests that hypertrophy is provoked by increasing the number of satellite cells engaged in differentiation, thus possibly decreasing the compartment of reserve cells. We conclude that autologous cell therapy can be applied to specific targets when there is a source of satellite cells which is not yet exhausted. This is the case of Oculo-Pharyngeal Muscular Dystrophy (OPMD), a late onset muscular dystrophy, and we participate to a clinical trial using autologous satellite cells isolated from muscles spared by the disease.     

miRNAs在骨骼肌损伤修复过程中的调控作用

不合理的运动方式经常会引起骨骼肌损伤,骨骼肌损伤后,成体生肌性干细胞(主要是卫星细胞)被激活,进而增殖分化,与原来肌纤维融合完成修复过程。miRNAs是一种在后转录水平调节基因表达的非编码RNAs,在骨骼肌损伤与修复过程中主要通过靶向抑制一些转录因子、转录激活途径关键酶、细胞通讯连接重要蛋白以及各种信号通路中关键蛋白的翻译而发挥调控作用。为了更好地了解miRNAs在骨骼肌损伤修复过程中的作用,本文通过对miRNAs在骨骼肌损伤与修复过程中卫星细胞不同生物学状态以及信号通路等方面的作用和研究现状做一综述。     

脊髓损伤后骨骼肌变化及预后的研究进展

脊髓损伤是脊柱骨折严重的并发症。由于椎体的移位或碎片骨突出于椎管内,使脊髓产生不同程度的损伤。在脊髓损伤之后,其效应器之一,骨骼肌将不可避免的发生一些相应的变化:凋亡、萎缩、运动单位的改变;肌肉兴奋性、收缩能力的改变;肌球蛋白重链的表达以及3-磷酸甘油脱氢酶活性的改变等,这些变化影响脊髓损伤的预后及患者的生活质量。目前针对脊髓损伤后骨骼肌变化的研究方法主要是从运动训练及电刺激这两方面。     

miRNA-206及miRNA-26a在周围神经卡压后骨骼肌纤维化过程中的表达

目的 探讨大鼠坐骨神经慢性卡压损伤后其支配的腓肠肌组织内miRNA-206、miRNA-26a的表达变化及其意义。方法 2020年5月至2020年7月,选取50只成年雄性SD大鼠;大鼠右侧后腿采用Mackinnon建立的坐骨神经卡压模型方法对坐骨神经行硅胶管卡压术,作为手术组（实验组）;对大鼠左侧后腿仅分离暴露坐骨神经,作为假手术组（对照组）。于卡压术后2、4、6、8、10周时间点随机取大鼠10只,取其双后腿腓肠肌,观察腓肠肌组织萎缩及纤维化程度,并行组织形态学观察,RT-PCR定量测定miRNA-206、miRNA-26a、转化生长因子-β（TGF-β）、Ⅰ型胶原蛋白（COL-Ⅰ）的表达。结果 坐骨神经慢性卡压损伤后,手术组与假手术组相比,腓肠肌组织湿重明显减轻,肌肉横截面积变小,肌肉组织胶原纤维增生,miRNA-206及miRNA-26a的表达先降低后升高,4周时表达最低,后表达逐渐升高;TGF-β及COL-Ⅰ含量升高,两组比较差异统计学意义（P＜0.05）。结论 周围神经慢性卡压损伤可致支配骨骼肌发生纤维化病变,TGF-β及COL-Ⅰ表达升高,miRNA-206及miRNA-26a在此过程中表达先降低后升高,提示miRNA-206及miRNA-26a在周围神经卡压致骨骼肌萎缩及纤维化过程中起到重要作用。     

The role of oxidative stress in skeletal muscle injury and regeneration: focus on antioxidant enzymes

Reactive oxygen species (ROS) are generated in skeletal muscle both during the rest and contractile activity. Myogenic cells are equipped with antioxidant enzymes, like superoxide dismutase, catalase, glutathione peroxidase, γ-glutamylcysteine synthetase and heme oxygenase-1. These enzymes not only neutralise excessive ROS, but also affect myogenic regeneration at several stages: influence post-injury inflammatory reaction, enhance viability and proliferation of muscle satellite cells and myoblasts and affect their differentiation. Finally, antioxidant enzymes regulate also processes accompanying muscle regeneration—induce angiogenesis and reduce fibrosis. Elevated ROS production was also observed in Duchenne muscular dystrophy (DMD), a disease characterised by degeneration of muscle tissue and therefore—increased rate of myogenic regeneration. Antioxidant enzymes are consequently considered as target for therapies counteracting dystrophic symptoms. In this review we present current knowledge regarding the role of oxidative stress and systems of enzymatic antioxidant defence in muscular regeneration after both acute injury and persistent muscular degeneration.     

被动训练促进失神经肌萎缩模型大鼠骨骼肌结构和功能的恢复

文题释义：肌肉萎缩：是指横纹肌营养障碍,肌肉纤维变细甚至消失等导致的肌肉体积缩小。多由肌肉本身疾患或神经系统功能障碍所致,病因主要有：神经源性肌萎缩、肌源性肌萎缩、失用性肌萎缩和其他原因性肌萎缩。肌肉营养状况除肌肉组织本身的病理变化外,更与神经系统有密切关系。脊髓疾病常导致肌肉营养不良而发生肌肉萎缩。 肌卫星细胞：是一类存在于肌细胞基底膜与肌膜之间的成体干细胞,作为肌源性干细胞在肌肉组织损伤后,能够在激活后发挥良好的增殖、分化能力,在骨骼肌损伤的修复和再生过程中发挥重要作用。 背景：炎症细胞或炎性因子参与失神经损伤后骨骼肌肌卫星细胞的增殖和分化,在失神经骨骼肌肌组织病理过程中起着重要的作用。 目的：研究被动康复训练对失神经萎缩大鼠骨骼肌结构、功能以及肌动蛋白和炎症因子表达的影响。 方法：将30只SD大鼠平均分为假手术组、模型组和训练组,模型组及训练组大鼠暴露坐骨神经并剪断,假手术组只暴露而不剪断坐骨神经。造模后2个月始用自制滚筒对训练组大鼠进行被动康复训练2个月,用肌肉湿质量比和BBB评分评估肌肉萎缩的程度及运动功能,苏木精-伊红染色观察肌纤维微细结构及横截面积,免疫组化染色检测各组腓肠肌肌动蛋白及肿瘤坏死因子α、白细胞介素6及白细胞介素1β表达。实验经沈阳医学院实验动物福利伦理委员会的审批,批准文号为SYYXY2015010601。结果与结论：①训练组BBB评分高于模型组;②训练组腓肠肌湿质量高于模型组但肌纤维的横截面积却低于模型组(P < 0.001,P < 0.05),训练组腓肠肌肌动蛋白表达高于模型组(P < 0.001);③训练组炎症因子肿瘤坏死因子α、白细胞介素6及白细胞介素1β的表达水平低于模型组(P < 0.001或P < 0.05);④结果说明,被动训练有助于失神经萎缩肌肉结构和功能的恢复,降低炎症因子的水平防止肌肉的进一步萎缩,提高骨骼肌的肌力。 ORCID: 0000-0002-9303-8651(王世杨) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Respiratory muscle injury, fatigue and serum skeletal troponin I in rat

To evaluate injury to respiratory muscles of rats breathing against an inspiratory resistive load, we measured the release into blood of a myofilament protein, skeletal troponin I (sTnI), and related this release to the time course of changes in arterial blood gases, respiratory drive (phrenic activity), and pressure generation. After ∼1.5 h of loading, hypercapnic ventilatory failure occurred, coincident with a decrease in the ratio of transdiaphragmatic pressure to integrated phrenic activity ( P _di /∫Phr) during sighs. This was followed at ∼1.9 h by a decrease in the P _di /∫Phr ratio during normal loaded breaths (diaphragmatic fatigue). Loading was terminated at pump failure (a decline of P _di to half of steady-state loaded values), ∼2.4 h after load onset. During 30 s occlusions post loading, rats generated pressure profiles similar to those during occlusions before loading, with comparable blood gases, but at a higher neural drive. In a second series of rats, we tested for sTnI release using Western blot–direct serum analysis of blood samples taken before and during loading to pump failure. We detected only the fast isoform of sTnI, release beginning midway through loading. Differential detection with various monoclonal antibodies indicated the presence of modified forms of fast sTnI. The release of fast sTnI is consistent with load-induced injury of fast glycolytic fibres of inspiratory muscles, probably the diaphragm. Characterization of released fast sTnI may provide insights into the molecular basis of respiratory muscle dysfunction; fast sTnI may also prove useful as a marker of impending respiratory muscle fatigue.     

Delayed post‐injury administration of C5a improves regeneration and functional recovery after spinal cord injury in mice

The activation of a complement system can aggravate the secondary injury after spinal cord injury (SCI). However, it was reported recently that the activation of a complement could have both a secondary injury and a neuroprotective effect, in which C5a is the most important factor, but there is no direct evidence for this dual effect of C5a after SCI. In order to investigate the potential neuroprotective effect of C5a after SCI, in this study ectogenic C5a was injected intraperitoneally before/after SCI in vivo, or administrated to mechanically injured neurones in vitro; following this, neurone apoptosis, neurite outgrowth, axonal regeneration and functional recovery were investigated. The in‐vivo experiments indicated that, following treatment with C5a 24 h before or immediately after injury, locomotor function was impaired significantly. However, when treatment with C5a took place 24 h after injury, locomotor function improved significantly. In‐vitro experiments indicated that a certain concentration of C5a (50–100 nM) could inhibit caspase‐3‐mediated neurone apoptosis by binding to its receptor CD88, and that it could even promote the neurite outgrowth of uninjured neurones. In conclusion, delayed post‐injury administration of C5a within a certain concentration could exert its neuroprotective effect through inhibiting caspase‐3‐mediated neurone apoptosis and promoting neurite outgrowth of uninjured neurones as well. These data suggest that C5a may have opposite functions in a time‐ and concentration‐dependent manner after SCI. The dual roles of C5a have to be taken into account when measures are taken to inhibit complement activation in order to promote regeneration after SCI.