We thank the Animal House and the Integrated Microscopy Facilities of IGB-CNR. This work has benefited from research funding from the European Community's Seventh Framework Programme in the project ENDOSTEM (Activation of vasculature associated stem cells and muscle stem cells for the repair and maintenance of muscle tissue, grant agreement number 241440), the Italian Ministry of Education-University-Research (MIUR-PRIN2 010-2011) to G.M. and S.B. and PON Cluster IRMI to G.M., and the CARIPLO foundation to G.M. and S.B.

所有实验均严格按照动物研究机构的指导方针进行，并按照有关动物实验的法律公共卫生，动物健康，营养和健康的意大利卫生部食品安全主管部门批准。颈椎脱位，程序可以根据IACUC或等值要求各不相同，从机构的机构。 1.心脏毒素注射在胫骨前肌<ol><li>通过在启动过程之前，稀释在无菌磷酸盐缓冲盐水（PBS）中或在水中的心脏毒素原液制备10μM的工作心脏毒素（CTX）的溶液中。 注意:CTX溶于水以1毫克/毫升。制备70微米原液。分装和储存在-20°C。用于该协议CTX是具有大约7 000道尔顿的分子量cardiotoxins的混合物。 注意:建议穿双层手套，口罩的卫生和安全眼镜（或者安全眼镜，建议罩下工作），并要非常小心，以避免与解决任何联系，即使稀释。 </li><li>麻醉氯胺酮和赛拉嗪（80毫克/公斤和10毫克/体重kg）或其它经批准的可用麻醉剂的腹膜内注射小鼠。 </li><li>用鼠标面朝上，喷后肢用70％的乙醇。可视化的胫骨前（TA）肌肉的准确位置，用手术刀的帮助下，切割毛发走在小腿的前部，膝盖下。 注意:TA肌肉源于胫骨的近侧主体，下降横向于骨，并在脚踝结束。其长肌腱伸入穿过脚踝脚（ 见图1A）。 </li><li>通过拉动柱塞背面慢慢绘制〜100μL的该溶液放入注射器。 </li><li>去除气泡，直立持注射器（与针向上）。轻拉活塞，然后敲击注射器一边帮附着在管的内部的气泡来向上。慢慢地按下柱塞释放出大量气泡。液体滴几滴将走出针。在这一点上，请务必重新挑液体成管，而不是分散的，给予心脏毒素的毒性。 </li><li>撤回溶液进入注射器，直至达到所需的剂量。注射的每个TA肌肉CTX溶液20微升。 </li><li>由以下的胫骨为指导的位置，从朝向膝盖腱插入在TA的中心针。由于TA是一个薄组织，进行注射足不出户过深（插入针2/3毫米深，在大约10°到20°角），避免超越肌肉本身（ 图1A）。 </li><li>不要拔出针头在注射后立即，但等待2 - 3秒，以防止泄漏CTX的年龄从肌肉下降。 </li><li>在手术后，放置在加热板（在37℃）的笼子，直到小鼠是清醒的，因为麻醉剂和所用的乙醇降低体温。 </li></ol> 2.胫前隔离注意:肌肉可以在不同的时间点心脏毒素注射后根据实验要求中分离出来。 <ol><li>颈椎脱位牺牲鼠标。 </li><li>喷用70％乙醇的后肢。在实验过程中，动物可固定到支撑保持下来。 </li><li>切在近端肌腱（脚以上）的电平的皮肤，插入皮肤下剪刀的刀片之一，并切开皮肤向上，在膝盖的方向。随着产钳的帮助下，拉起皮肤完全暴露的肌肉。小心去除所有的皮肤和从感兴趣的区域中的头发。 </li><li>消除慢慢捏或切割筋膜 （与日在尖镊子或细微的胫骨（ 图1B）的相对侧解剖剪刀）肌肉的极端周边。一旦打破，温和地去除薄尖镊子的帮助下筋膜 ;避免损坏下面的肌肉。 注意:与其他肌肉和器官中，TA由筋膜 （腿的深筋膜），结缔组织的薄片是位于深皮肤下，并需要被除去以容易分离下面的肌肉覆盖。 </li><li>可视化远端TA肌腱和伸指肌腱的脚上面，这是并列的。趾长伸肌（EDL）肌的腱位于略低于TA肌腱。成年小鼠的筋是肉眼可见的。 </li><li>除去以下以下两种技术中的一种对TA肌肉: <ol><li>切细微解剖剪刀既筋，和他们拉向近侧，牵着摹用钳子筋。分开的两个筋，在相反的方向轻轻拉动它们的肌肉分开。 </li><li>薄尖镊子的帮助下，通过将腱本身（ 图1C，上图）所示的尖端分离从伸指肌腱远端TA肌腱。轻轻滑动的TA低于镊子将其从下面的肌肉（ 图1C，底部图片）分开。 </li><li>保持肌肉下用镊子把它微微抬起，切TA肌腱，轻轻地向上拉，直到只有膝盖以下附后（ 图1D，左图）的区域。切TA膝盖以下，之后用剪刀（ 图1D，右图）肌肉的边缘。 注意:如果肌肉保持小幅连接到两侧，用细微解剖剪刀，帮助卸下。 注意:如果镊子不要轻易适合TA下方，它是可能的筋膜尚未完全除去，它仍然是对肌肉的顶部可见。在这种情况下，继续彻底删除前筋膜 。 </li></ol></li></ol> 3.新鲜冰冻肌肉技术<ol><li>放置黄蓍胶或软木的切片的类似粘合剂冷冻化合物（ 例如，最佳切削温度（OCT）化合物）的量小。冷冻前标签在反面的软木塞的切片，如果需要的话（ 图2A）。 </li><li>为了获得肌肉的横截面，水槽的TA进入黄蓍胶通过插入远端肌腱和外而使肌肉的约3/4，并确保有一个垂直位置的肌肉相对于所述软木塞（ 图2A）。 </li><li>填充的铝罐（可选地，一个金属杯或玻璃烧杯），用异戊烷。暂停异戊烷的可以含有杜瓦LIQUIð氮，不允许液氮进入罐。 注意:异戊烷需要达到适当的温度（-150〜-160℃）的组织的最佳的冻结。当一些白色固体颗粒形成在罐的底部和异戊烷变得略微粘稠达到正确的温度。 注:固体颗粒形成之前不要冷冻样品，因为它可以导致冻结文物。另一方面，如果异戊烷变得完全固体，把它在室温下，直到它被解冻，确保一些白色颗粒仍然存在。 </li><li>使用Mixter镊子，迅速浸软木与肌肉入异戊烷，保持肌肉定位向下，并确保释放软木塞当它完全浸入在液体中。以这种方式，而检体浮起，只有软木的反面应在液体中可见。 </li><li>离开SAmple在异戊烷1分钟。 </li><li>使用Mixter镊子（或类似镊子），取肌肉和浸入其在液氮中至少2分钟。 </li><li>单独包装的标本在标有铝箔，并立即将它们放置在干冰（-70℃），或直接将其存储在-80℃冰箱中。 注意:在整个过程中，从步骤3.4至3.7，这是非常重要的是要保持正确的温度，并防止解冻样本（ 如 ，转移到-80℃的冰箱中）。在不受控制的解冻和水微晶是在组织内本，并最终导致工件的形成或肌纤维的击穿的再冻结的温度升高的结果（可视化的组织切片中的纤维的中心大白色空穴）。 </li></ol> 4.冷冻肌肉的低温恒温器切片<ol><li>设置低温恒温室温度tØ-20〜-22℃。 </li><li>把试样存根，刀片，并在低温恒温室中的样品，使它们平衡至少30分钟。 </li><li>放置在试样存根冷冻化合物（ 例如，OCT化合物中）少量的，并与该冷冻化合物固化前的试样浸入软木。在此期间，将在刀片保持器的刀片。 </li><li>使用所提供的螺钉来定向与该叶片夹持器（ 图2B）的试样保持器对准试样和刀片。 </li><li>切片厚度设为10微米。进行到切割组织。为驱动轮的每一匝，试样保持器前进朝向叶片的受控距离。 </li><li>放置在偏振幻灯片（每张幻灯片8至10节）（ 图2C）的部分。 </li><li>切片后，存放在幻灯片-80℃。 注:样品可以储存在-80℃冰箱和用于furthe重用- [R切片，如果妥善处理和储存。 </li></ol> 5，常规病理染色（苏木曙红渍） 注意:几个组织学染色可以在肌肉部分根据分析来进行。对于形态和形态分析的常规病理染色是苏木曙红（H＆E）染色重铬酸。苏木染色核深紫色。核染色复染与曙红（粉红/红色），其染色嗜酸性结构，例如在细胞质中的肌纤维。 <ol><li>完全风干冷冻切片为10个部分 - 在室温下15分钟，误入染色托盘中的幻灯片，并把托盘染色低谷。 </li><li>染色1分钟用苏木。 </li><li>打下相当薄弱自来水喷下的染色槽10分钟洗出苏木。 </li><li>浸入载玻片5分钟，在95％的乙醇中，如果曙红的醇溶液将使用（跳到吨他在水性曙红溶液的情况下，工序）。 </li><li>染液1分钟用曙红溶液。 </li><li>用清水冲洗，以消除过度伊红的解决方案。 注意:与任一苏木或曙红染色的最佳时机应为每个新的批次来限定。 </li><li>脱水的样品在梯度乙醇系列:50％和70％（每几秒钟）。迅速浸入在95％乙醇，随后2的变化在100％乙醇，每次5分钟。 </li><li>明确在2变化二甲苯中，每5分钟或更长的时间。这个步骤必须在化学通风橱中进行。 </li><li>装入滑动与基于二甲苯的安装平台，施加在滑动安装介质，并用盖玻片覆盖几滴。安装介质也可以应用到盖玻片和盖玻片放在滑动。 <ol><li>避免形成该载玻片和盖玻片之间的气泡。为了消除气泡，保持滑板垂直（安装后），挤出多余的MO通过在钝钳（或类似的钝工具）盖玻片轻轻按压unting介质和气泡。 </li></ol></li><li>保持滑动罩下几个小时或过夜以让二甲苯在进行分析之前完全蒸发。 </li><li>通过使用每鼠的至少一个全肌肉部的非重叠序列图像执行的H＆E染色的再生肌切片的形态和形态测定分析。捕捉图像与明场显微镜，用放大20倍。 注:建议5只小鼠的最小数量，以获得测量值的统计学意义。 注:图像分析软件可用于这一目的，比如免费下载软件ImageJ的（http://imagej.nih.gov/ij/）。 </li></ol>

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The authors have nothing to disclose.

这里，我们描述了协议来诱导骨骼肌（ 即，CTX的肌内注射）急性损伤。它被广泛地用作一种强大的工具来研究骨骼肌再生的动态体内 。 CTX注射诱导的肌纤维，它是由肌膜的去极化和纤维12的收缩而引起的变性，并触发，导致肌肉再生事件的级联。骨骼肌是在注射和损伤后所要求的时点解剖，根据实验的要求，并用于后续的组织学分析。解剖的肌肉可以被冻结，夹杂在冷冻保...

哺乳动物成人骨骼肌是由多核肌细胞（肌纤维）是专门为收缩fascicules群体形成。每个肌纤维是细长的合胞体，由肌膜（质膜）和含有肌原纤维，这是由定期多次组织收缩蛋白（肌动蛋白和肌球蛋白丝）所包围。在成年生活中，在休息的条件下，骨骼肌有其肌细胞核1非常低的营业额;的确，肌细胞核，这是位于肌纤维的周围，肌膜下，在细胞周期的G0期被捕并且无法增殖1,2。 骨骼肌必须重新生成以下的损伤，组织重塑的几个事件紧密相关的互相后达到稳态的特有能力。急性损伤或外伤之后，变性诱导，随后通过再生过程涉及不同的细胞群，包括肌肉细胞的常住人口中，卫星细胞（SCS）。事实上，在没有任何环境刺激的，卫星细胞是处于静止状态，位于肌膜和基底层3,4之间的专门利基。以下内容的损伤或疾病，旺成为激活，增殖，迁移到受损区域，并最终分化，从而引发新形成肌纤维5。活化的SC建立与不同细胞群，主要炎症细胞，这是在创伤6-8的部位招募串扰。这种串扰允许细胞遵循稳压范例通过该分子信号驱动结构上的修改，最终导致动态平衡9。此外旺，炎症和间质细胞，血管生成过程，并重新支配的事件也都参与其中，以协调的方式采取行动，以修复此高度组织化和Specialized结构。 有在研究骨骼肌再生的各个方面，不仅要了解肌肉的生理机能，同时也提高需要全过程的深入了解治疗策略的极大兴趣。几个实验的方法是目前可用来研究不同细胞群体，信号通路，以及所涉及的分子机制的标识和功能。急性损伤的小鼠模型代表一个强有力的工具来研究这个过程的许多方面。不同常用的技术来诱导急性肌肉损伤允许研究遵循再生过程在体内 ，从非常早期阶段的过程中结束。这个协议描述从肌肉注射蛇毒来源的心脏毒素的（CTX），其诱导myolysis并触发再生过程中，最多的组织样本的分析的步骤。继CTX注射，MICE可以在不同的时间点，这取决于实验要求被牺牲，并且骨骼肌可以解剖并进行进一步的分析处理。最后，我们描述了组织切片的染色方案来执行形态学观察和基本定量分析。该协议允许对急性骨骼肌再生的体内以高度重现的方式10的研究。

<table><tbody><tr><td>Cardiotoxin from&amp;nbsp;&lt;em&gt;Naja mossambica mossambica&lt;/em&gt;</td><td>SIGMA ALDRICH</td><td>C9759</td><td></td></tr><tr><td>Syringe For Insulin BD Micro-Fine+ Needle 30 G x 8 mm - Da 0.3 mL</td><td>BD</td><td>324826</td><td></td></tr><tr><td>Tragacanth Gum</td><td>MP BIOMEDICALS,LLC</td><td>104792</td><td></td></tr><tr><td>2-Methylbutane (Isopentane)</td><td>SIGMA ALDRICH</td><td>78-78-4.</td><td></td></tr><tr><td>OCT Killik Solution For Inclusion Cryostat</td><td>Bio-optica</td><td>&amp;nbsp;05-9801</td><td></td></tr><tr><td>Feather Microtome Blade S35</td><td>Bio-optica</td><td>&amp;nbsp;01-S35</td><td></td></tr><tr><td>Glass Slide Superfrost Plus</td><td>Menzel-Gl&amp;auml;ser</td><td>09-OPLUS</td><td></td></tr><tr><td>Dumon #5 Mirror Finish Forceps&amp;nbsp;</td><td>2BIOLOGICAL INSTRUMENTS</td><td>11251-23</td><td></td></tr><tr><td>Scissors Straight Sharp/Sharp</td><td>2BIOLOGICAL INSTRUMENTS</td><td>15024-10</td><td></td></tr><tr><td>Scissors Noyes Straight</td><td>2BIOLOGICAL INSTRUMENTS</td><td>15012-12</td><td></td></tr><tr><td>Fine Iris Scissors Straight Sharp/Sharp 10.5 cm</td><td>2BIOLOGICAL INSTRUMENTS</td><td>14094-11</td><td></td></tr><tr><td>Eukitt</td><td>Bio-optica</td><td>09-00100</td><td></td></tr><tr><td>Slide Coverslip</td><td>BIOSIGMA</td><td>VBS651</td><td></td></tr><tr><td>Xylene</td><td>SIGMA ALDRICH</td><td>214736</td><td></td></tr><tr><td>Ethanol 100%</td><td>sigma-Aldrich</td><td>02860-2.5L</td><td></td></tr><tr><td>Hematoxyline</td><td>J.T. BAKER</td><td>3873</td><td></td></tr><tr><td>Eosin</td><td>SIGMA ALDRICH</td><td>HT110116</td><td></td></tr><tr><td>Cryostat</td><td>LEICA</td><td>CM3050 S</td><td></td></tr></tbody></table>

induction of acute skeletal muscle regeneration by cardiotoxin injection

骨骼肌再生是发生在成人骨骼肌中响应损伤或疾病的生理过程。急性损伤诱导的骨骼肌再生是研究参与肌肉再生以及机制和不同玩家的事件一种广泛使用的，强大的模型系统。实际上，这个过程的详细知识是为了更好地理解的病理状况导致骨骼肌变性必需的，而且它在确定新的靶向治疗策略有助于。目前的工作描述了一个详细的和可重复的协议，并通过单次肌注的心脏毒素（CTX）诱导小鼠急性骨骼肌再生。 CTX属于家庭的蛇毒毒素和导致肌纤维的myolysis，最终触发再生事件。骨骼肌再生的动力学是由肌肉切片的组织学分析评价。该协议还示出了用于解剖，冷冻，并切割该胫骨前肌，以及，被广泛用于后续形态学和形态分析例程苏木曙红染色的实验程序。

H＆E染色允许在特定时间点的再生处理的形态的骨骼肌再生过程中进行评价。 图3显示了对野生型小鼠的受伤的TA肌肉进行时间过程分析。肌肉已经分离在CTX注射后3,7，15，和30天，如在图3A中图式。 H＆E染色横截面有代表性的图片显示肌肉修复的动力随着时间的推移（ 图3B-E）。在伤后第3天，肌肉的结构体系结构被完全破坏，并且两个...

Watch this Scientific Journal Video about Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection at JoVE.com

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

该原稿描述了详细的协议来诱导成年小鼠和肌肉的随后的操作，如解剖，冷冻，切割，常规染色，和肌纤维横截面积分析急性骨骼肌再生。

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

induction-acute-skeletal-muscle-regeneration-cardiotoxin

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

21.9K 次观看.  Institute of Genetics and Biophysics "A. Buzzati Traverso", CNR. 该原稿描述了详细的协议来诱导成年小鼠和肌肉的随后的操作，如解剖，冷冻，切割，常规染色，和肌纤维横截面积分析急性骨骼肌再生。 

视频: Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

一个小鼠模型，通过神经肌肉电刺激肌肉训练

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

科研

医学

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

生物工程

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

通过心肌毒素损伤和在 Vivo 中注射自传递 siRNA 在骨骼肌再生过程中的卫星细胞功能分析

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

遗传学

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

发育生物学

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

人骨骼肌活检手术使用修改伯格斯坦技术

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

微创肌肉植入 (MIME)--一种促进供体细胞介导的肌的新实验技术

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

生物学

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

基于 Ding's Roll 操作治疗大鼠模型肌肉损伤的按摩器

Mimicking Ding's Roll Method on Notexin-Induced Muscle Injury in Rats

Ding's roll method is one of the most commonly used manipulations in traditional Chinese massage (Tuina) clinics and one of the most influential contemporary Tuina manipulations in China. It is based on the traditional rolling method commonly used in the one finger Zen genre and named Ding's roll method. Due to its anti-inflammatory and blood circulation-promoting effects, Ding's rolling method has sound therapeutic effects on myopathy. Because of the large area of force applied to human skin, Ding's roll method is challenging to perform on experimental animals with small skin areas, such as rats and rabbits. Additionally, the strength of Tuina applied to the human body differs from that applied to experimental animals, so it may happen that the strength is too high or too low to achieve the therapeutic effect of Tuina during the experiment. This experiment aims to create a simple massager suitable for rats based on Ding's rolling manipulation parameters (strength, frequency, Tuina duration). The device can standardize manipulation in animal experiments and reduce the variation in Tuina force applied to different animals due to subjective factors. A rat model of notexin-induced skeletal muscle injury was established, and plasma injury markers creatine kinase (CK) and fatty acid binding protein 3 (FABP3) were used to assess the therapeutic effect of Tuina on skeletal muscle injury. The results showed that this Tuina massager could reduce the levels of CK and FABP3 expression and slow down the degree of skeletal muscle injury. Therefore, the Tuina massager described here, mimicking Ding's roll method, contributes to standardizing Tuina manipulation in experimental research and is of great help for subsequent research on the molecular mechanism of Tuina for myopathy.

作者聚焦：使用自制按摩器对大鼠进行推拿按摩 

This protocol describes a simple device that mimics Ding's roll method, establishes a rat model of skeletal muscle injury, and uses hematoxylin-eosin staining to observe the pathology of damaged tissue and enzyme-linked immunosorbent assay to detect changes in serum damage markers.

通过注射心脏毒素急性骨骼肌再生的诱导

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