自然科学与工程研究理事会，加拿大研究支持。

1。介绍<ol><li>在Macintosh实验室已使用腓肠肌内侧肌肉准备好几年了，而在这之前，整个腓肠肌准备，菲尔·加德纳博士开发的。 </li></ol> 2。麻醉<ol><li>成年Sprague-Dawley大鼠（200-300克）通常用在我们的实验室的研究在原位的收缩性能。将大鼠可被约束在一个有机玻璃装置，市售的，或者通过用毛巾覆盖，和保持。 </li><li>我们使用氯胺酮/赛拉嗪，（100毫克·ml -1的 ，每个在85:15）的混合，和管理0.1毫升每100g大鼠体重肌内2,3。戊巴比妥钠（50至60毫克·kg -1时 ，intraperiteneal）或异氟烷（2-3.5％，吸入）也可以使用4。 </li><li>虽然麻醉效果，一个精确的重量可以以下方式获得的左后肢剃光。这也是一个不错的添e是确保所有的电子设备的开启和准备。这包括计算机，应变放大器，油加热器和其他。 </li><li>入住的退货反射反应定期和根据需要补充麻醉（0.05至0.1毫升每100克）。 </li></ol> 3。启动手术<ol><li>我们用的是有机玻璃固定手术大鼠的平台上。简单的胶带做这项工作。确保动物左后肢伸展，右前肢。加入一滴润滑剂的眼睛。我们使用石蜡油。否则，眼睛会干出氯胺酮麻醉。 </li><li>一个小的手术灯是有用的。这一次发射足够的热量，以帮助保持动物的体温。或者，可用于水加热毯。检查，以确保角膜或脚趾，捏反射消失，然后再继续。无菌技术是没有必要的，因为该过程是急性。动物不会从麻醉中恢复过来。我们安乐死的大鼠麻醉过量（0.2毫升，心内）时，所有的程序完成。 </li><li>第一切口是通过皮肤从脚跟到脊柱。我们使用剪刀，因为切口的深度可以控制，以及剪刀被用于分离从下层组织的皮肤。大鼠具有良好的止血作用，所以，只要你避免大血管，出血将是有限的。让暴露的表面覆盖，尽可能用等渗生理盐水浸泡的纱布。 </li><li>后分离从底层的结缔组织，皮肤表面的肌肉层被切断。请务必不要去太深刻，你不希望损害坐骨神经或血管，或肌肉的利益。开始在腓肠肌和削减大约是在同一直线上的皮肤切口。皮克下定位，避免坐骨神经。一旦你的神经，你可以遵循的路径与您的切口，但仍保持远高于神经。有一个明确的缝之间亩粒子。定位，并切断沿，对膝盖缝。观察和避开血管。 </li></ol> 4。准备先导孔股骨骨针<ol><li>切在尾部方面股骨一层薄薄的肌肉，露出光秃秃的骨骼。使用手持式旋转刀具，钻头，或销副，（0.9毫米的碳素钢BUR），通过一个小规模的试点孔皮质，刚刚进入髓质。不要钻得太深，或将导致失血过多。这个洞以后将用于放置骨针固定股骨上的肌动描记基地。 </li></ol> 5。隔离腓肠肌内侧肌肉的神经支配<ol><li>接下来的步骤需要在解剖显微镜。找到腘神经消失的地方，后面的腓肠肌内侧肌肉。轻轻摊开的各个分支机构，并切断所有不支配腓肠肌内侧肌肉。可以通过微刺激神经支配。于氏的舞台，也确保肤浅的坐骨神经分支被切断。 </li><li>现在距离坐骨神经清除结缔组织，所以它可以溜进的神经套（后下）。请务必轻轻治疗神经。任何延伸的神经可导致inexcitability，过早地结束你的实验。 </li></ol> 6。隔离跟腱和腓肠肌<ol><li>偶尔帮助剪刀，钝性分离，可用于从其他组织分离腓肠肌。从跟腱，跖肌腱被拉出捆绑和削减。领带只是用来帮助保持肌腱和右后可以切去肌腱被切断，从一个相当大的长度的腓肠肌跖分离。将＃1丝线结扎跟腱周围，在一个平结领带。不要拉得太紧，否则的肌腱会损坏。我们使用骨咬骨钳，切割calcan<html> EUS，留下一小块骨头仍连接到跟腱。保持切削表面是水平的，以确保骨被切断，不只是肌腱。这将确保，可以贴在跟腱的肌动描记。 </li><li>沿着底面的腓肠肌，比目鱼肌可以看出。同样，钝性分离，可用于分离比目鱼肌，腓肠肌。我们要隔离的腓肠肌内侧，所以它是唯一仍然连接到跟腱的肌肉。切比目鱼肌肌腱的末端。然后你就可以分离的内侧和外侧的腓肠肌，外侧的肌腱切断，只留下跟腱的腓肠肌内侧肌附着。拉出腓肠肌外侧远离内侧，沿其长度的50％左右。超出此长度时，纤维交叉和损坏将导致进一步拉。检查，以确保的肌肉是免费的结缔组织。 </li></ol>E"&gt; 7。切胫骨<ol><li>将柄连字左右，只是上面的中点。这需要很紧，不削减到组织。第二个循环之前搭售有助于防止打滑。再次使用一个平结保证的。 </li><li>用一个小锯，切小腿的路程。这种切割应该是大约中间沿胫骨。 </li><li>尖锐的探针插入到髓质的胫骨。该探针将被用来固定的胫骨上的肌动描记基地。 </li><li> 2弹性件连接到皮肤上，使用米歇尔剪辑。将被使用的这些和附加的弹性件，保持皮肤周围的肌肉以形成一个容器，将充满温热的石蜡油。 </li></ol> [可选] 8。插入sonometric晶体（可选步骤）5 <ol><li>将动物加热垫低设置。直肠探头，在这个时候，如果尚未插入，可以插入。使用一小滴石蜡油润滑。 </li><li>使用微刺激，确定肌肉内的端部的分册。 21号针捅到的部位的肌肉的起源和插入。 </li><li>针，和密封件与兽医 - 键手术胶水由入孔滑动sonometric晶体。使用一个很小的一滴胶水，它被放置在水晶。应用胶水剪纸一块成锐角，可以精确地在施加胶水。我们正在把一个额外的晶体在插入的分册，确定由微刺激。 </li></ol> [继续] 9。在设备安装（见图1） <ol><li>放置鼠的肌动描记的基础上，面向杆与胫骨探头。将探头放置在支架，固定腿。 </li><li>拉扯皮肤在两侧，以形成一个容器，并与弹性件固定。 </li><li>设置钻头（1/16英寸）到股骨，利用导频孔手术准备期间创建的。确保您支持的断裂的股骨股骨的前部，当你把钻头（针副），可能会导致过早终止实验。贴上销副到横栏固定股骨。 </li><li>将远端残端的坐骨神经通过的神经刺激袖口（阴极向肌肉）和掖背部紧贴的肌肉的起源。将刺激这些电线。 </li><li>将跟腱的杆，其具有其上的应变仪传感器。配合暖和，但后期调整留出一些空间。可以肯定的肌肉的取向是垂直于与杆。 </li><li>由温石蜡油的皮肤填充的容器。 </li><li>打开灯具的散热和位置锡箔制成的防护罩在头上的动物，和力传感器。额外的屏蔽箔可能是必要的，以确保动物的核心温度不EXCEED 38°C。直到从腓肠肌内侧部和力传感器之间的连接被删除的字符串的松弛肌肉的长度可以调整。 </li></ol> 10。设定最大电压和基准长度<ol><li>而肌肉热身，成立了刺激和测试刺激电压。这是重要的，以确保在非常简短的脉冲持续时间的刺激设置。我们使用50微秒。最大电压应该是小于1 V 0.5 V开始，增加电压，直到抽搐幅度不增加。最大电压是激活所有电机单元的最低电压。我们通常会刺激在最高电压的两倍，或3 V，以较高者为准。 </li><li>通常情况下，实验将开始与肌肉的长度，让最大的抽搐收缩。抽搐是一个单一的刺激脉冲。肌肉长度增加约1毫米另一抽搐。这作为只要抽搐振幅的重复正在增加。一旦抽搐幅度的降低，其长度将返回到一个幅度最大抽搐。 </li><li>在此初始设置的长度，我们测试的系统与我们所说的一个"精调强直收缩"。刺激器提供脉冲在200 Hz时为500毫秒。这种刺激的交付将导致在一个完全融合的强直收缩。由肌肉产生的力拧紧所有的连接，包括对肌肉的结。耗散的增效4经过适当的休息，肌肉的基准长度是复位（参见9.2部分）。一般，空调强直收缩将允许一些肌束长度缩短，使肌肉将有一点点地取回的长度，使振幅最大抽搐收缩被拉伸。 </li></ol> 11。启动实验<ol><li>一个典型的实验包括长度adjustm耳鼻喉科和/或与各种图案的刺激的刺激。数据收集可能涉及正义的力量或力量雄厚，拥有长度，肌束长度和肌电图。 </li></ol> [可选] 12。力频率的关系<ol><li>设置的的列车持续时间足够长的时间达到高原的力量，在任何频率下的利益。在内侧腓肠肌达到200 Hz时的最大等长收缩力。火车时间必须在至少200毫秒，不再是更好的低频率，但会导致一些疲劳，如果使用一些收缩力频率之间的关系，以确定全系列。我们通常使用0，20，40，60，80，100和200赫兹的收缩。的频率范围内，将允许全方位的力频率之间的关系被描述。之间的收缩必须允许一个适当的休息，避免过度疲劳（1-10分钟）。 </li></ol> 13。部队长的关IP 6 <ol><li>与估计的最优长度先前建立的力长度之间的关系，可以通过系统调整的肌肉的长度从-4毫米到4毫米使用一个伺服电机装置的确定。这应该是最大的刺激（200 Hz）的使用，但我们发现，很短暂的收缩会产生相同的真正的最佳长度为7。次极大收缩，如抽搐，可以使用，但是，这将产生一个不同的长度依赖的力8。 </li><li>测定的力 - 长度关系的一个重要方面是需要估计的被动的力在哪些测量的力发生在肌束长度。主动力的计算公式为总力之间的差异和相应的被动式力。由于并联的弹性结构承受被动的力量，这些都是在系列与系列弹性结构，被动力必须减少总力的贡献系列埃拉STIC结构被拉长在一个收缩3。一旦被动的力为所有相关的肌肉的长度是已知的，合适的被动式力可估计分册长度使用sonomicrometry的连续测量，或者通过估计的测量系统，并在串联结构的肌肉 - 肌腱单位遵守。如果不这样做，编制和峰值力的最佳长度，被低估了。 </li></ol> 14。力-速度关系9 <ol><li>如果你想确定的力 - 速度关系，则需要附加力传感器系统，它允许任一负载或长度变化率的控制。最简单和最具成本效益的方法是使用空气压力限制缩短至一个受控负载。肌肉在我们的系统中，安装在杠杆的支点的一侧上的空气压力阻碍的另一侧的长度变化。的长度传感器NEEDS被雇用，这样您就能够检测到肌肉长度变化在等渗收缩。这的安排允许afterloaded收缩与气动阻力。双舱可以允许释放的等渗负载的等长收缩。总共应获得15-20收缩，近卸载到最大等长收缩力的负荷范围。一个方程拟合数据时，不应该使用负载在90％以上的等轴测9。 </li></ol> 15。代表性的成果: 在图2中给出的样品收缩。这些收缩力频率的关系来说明。这些收缩活动的峰力计算，并绘制对频率的主动力，得到图3;力频率的关系。图2中的数据可以拟合方程:AF = C /（1 + E（（AF）/ B）+ D），AF主动力，F是频率和，<html> b，c和d为常数。 对于大鼠腓肠肌内侧肌肉，半最大频率的刺激通常是在非疲劳肌肉2 50-60赫兹。结果将上述公式所描述的线紧密贴合。 <img alt="图1" src="/files/ftp_upload/3167/3167fig1.jpg" /> 图1的装置和肌肉设置:气动设置被显示在左边，在右边等距。当动态收缩所需的（适于从13）时，在左侧所示的杠杆系统附加的转译表。步进电机控制的一台计算机（适于从14）。 <img alt="图2" src="/files/ftp_upload/3167/3167fig2.jpg" /> 图2叠加等长收缩:20，40，60，80，100和200赫兹。 200 Hz的收缩的振幅是8.13 N。 les/ftp_upload/3167/3167fig3.jpg"/&gt; 图3。力频率关系:主动收缩力，在图2中绘制的线表示的最佳拟合线。

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental Contractil Properties of Skeletal Muscle Following a stroke in a Rat Model. Master's Thesis. , (2008).</li><li>MacNaughton, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Length dependence of Fatigue and of Repetitive Contractions. Master's Thesis. , (2005).</li></ol>

没有利益冲突的声明。

质量好收缩的结果，可以得到照顾，在手术准备，​​牢固地安装在设备和良好的优质电子产品。当学生学习此手术，一些常见的单包括:伸展坐骨神经，破坏血流量，和大量出血。神经必须小心处理，以防止损坏。你会知道你已损坏的神经，如果最大强直力的最佳长度基本上是小于在图3中，或如果刺激需要最大限度地激活所有电动机单元的电压是大于5伏，所示。它是相对容易的，以避免血管这肌肉在手术期间提供服务。影响这些船只发生时，钻头被放置在尾部的股骨表面的。如果先导孔是不股骨的平坦表面上，钻头可能打滑。当发生这种情况时，有一种可能性腘血管浏览打乱。如果设置后血池周围的肌肉，它是一个迹象，表明你已经破坏了这些船只。如果大静脉被切断，不绑，失血过多也可能发生。需要注意的是大静脉的踝关节周围。 原位肌肉准备是一个有价值的方法，肌肉收缩性能的研究。单电机单位可以激活10，但通常所有电机设备被激活同步。这是一个相对于正常的异步激活，发生自愿电机单位招聘的缺点，所以代表的限制。然而，积极的一面是，同步激活可以量化的所有运动单位的平均响应。 有两种方法已被使用，以避免同步激活。一种是使用的电极压脉袋与几个对刺激电线。这允许与每对的运动单元的一部分的激活，刺激，可以通过对旋转以实现异步启动。激活此方法可以结合与anodal块11尝试激活运动单位根据大小原则12在适当的序列。在这种方法中，所有的电动机单元与近侧的电极对被激活，并且一个块被施加直接电流刺激。块刺激的幅度可以是调制，以抑制不希望激活电动机单元。显然块影响大轴突在最低电压，并逐步影响更小的单位。 在原地大鼠腓肠肌准备的是一个有价值的生理的方法来研究骨骼肌肉的收缩与健康和疾病的生化特性。

<table border="1"><tbody><tr><th>的设备名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>剪</td><td></td><td></td><td>好品质的宠物电推剪</td></tr><tr><td>手术灯</td><td>动力卢梅</td><td></td><td>会做的几种</td></tr><tr><td>肌动描记器</td><td></td><td></td><td>自定义建</td></tr><tr><td>刺激</td><td>草</td><td> S-88 </td><td>会做的几种</td></tr><tr><td>应变计放大器</td><td> CWE </td><td> PM-1000 </td><td></td></tr><tr><td>遥测温度计</td><td> YSI </td><td> YSI-400 </td><td></td></tr><tr><td>机器人平台</td><td> Arrick机器人</td><td> MD-2 </td><td></td></tr><tr><td> Sonometric放大器</td><td> Sonometrics </td><td> Sonolab </td><td></td></tr><tr><td>计算机和数据采集</td><td> PC与NI蟒蛇的路</td><td></td><td>定制软件（LabVIEW中） </td></tr><tr><td>缸体加热器</td><td>实验室</td><td>多块</td><td></td></tr><tr><td>神经袖口</td><td></td><td></td><td>订制</td></tr><tr><td>式微</td><td></td><td></td><td>订制</td></tr></tbody></table>

procedures for rat in situ skeletal muscle contractile properties

有很多情况下，理想的是获得生理情况下下的骨骼肌的收缩反应:正常流通，完整的整个肌肉，在体温下。这包括研究的收缩反应，如强直后电位，楼梯和疲劳。此外，疾病，淘汰，受伤，训练和药物治疗的后果，感兴趣的可以。本视频演示了适当的程序，设置和使用这宝贵的肌肉准备。 要建立这种准备，必须在动物麻醉，外科手术分离腓肠肌内侧肌肉，完整无缺的起源。必须小心，以保持的血液和神经耗材。长款的坐骨神经被清除的结缔组织，并切断近端。所有分支的远端残端不支配腓肠肌内侧肌肉被切断。远端神经残端插入背板特德到袖口内衬不锈钢刺激线。被切断，留下一小块骨头仍连接到跟腱跟骨。可以插入sonometric晶体和/或电极的肌电图。由金属探针在股骨和胫骨的固定，防止肌肉原点的运动。跟腱附着到的力传感器，和在两侧的松弛的皮肤被拉起以形成一个容器，与温热的石蜡油填充。油分布均匀热和蒸发热损失降到最低。一种热电灯上被引导的肌肉，肌肉和大鼠被允许温热至37℃。虽然它正在预热，最高电压和最佳的长度可以被确定。这些都是重要的初始条件为完整的整肌任何实验。实验包括标准的收缩特性，如力频率的关系，力长度的关系，以及力 - 速度关系的决心。 </P&gt; 手术隔离照顾，固定的起源的肌肉和校准力传感器的肌肉 - 肌腱单位，和正确的数据分析，高品质的测量可以得到这个肌肉准备。

Watch this Scientific Journal Video about Procedures for Rat in situ Skeletal Muscle Contractile Properties at JoVE.com

Procedures for Rat in situ Skeletal Muscle Contractile Properties

该视频演示了手术的准备工作需要研究的大鼠腓肠肌内侧肌肉准备的收缩反应<em在原位</em&gt;。这种准备允许在生理条件下对骨骼肌收缩性能的测量。该动物麻醉，并在其远端从周围组织分离肌肉。跟腱被连接到一个力传感器，使肌肉的收缩反应的测量在37℃用一个完整的循环。

对大鼠的过程<em在原位</em骨骼肌收缩性能

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

procedures-for-rat-in-situ-skeletal-muscle-contractile-properties

Research

JoVE Journal

Biology

Procedures for Rat in situ Skeletal Muscle Contractile Properties

28.5K 次观看.  University of Calgary. 该视频演示了手术的准备工作需要研究的大鼠腓肠肌内侧肌肉准备的收缩反应

视频: Procedures for Rat in situ Skeletal Muscle Contractile Properties

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

在体内微循环在骨骼肌内重要的显微镜测量

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

科研

生物学

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

组织分流和冻结的骨骼肌疾病模型

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

检测和DNA损伤分析小鼠骨骼肌<em&gt;原位</em&gt;使用TUNEL法

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

大鼠骨骼肌中纤维-脂肪源性祖细胞（FAPs）和肌源性祖细胞（MPs）的鉴定、分离和表征

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...