我们感谢玉田县甘图3使用。这项工作得到了国家科学基金会0420971;史密斯学院布莱克斯利，Wilens和霍姆斯的资金和霍华德休斯医学研究所。

1。样品制备<ol><li>从小鼠为起始原料，使用新鲜，闪光灯，冻结或RNAlater处理的组织。离开前出发Kimwipe吸干多余的防腐剂。 </li><li>删除所有周围肌肉的肌腱和筋膜，并直言不讳地在寒冷的房间（4℃）2分钟冷却玻璃板与单刃剃须刀刀片的组织，直到该组织是切碎或粉状。在预先称重的离心管收集样品，并确定组织的重量。使用为您的起始原料组织不超过10毫克。 </li><li>添加45μL，每1毫克剁碎组织提取缓冲液（8 M尿素，50毫米数码地面电视，4％CHAPS，0.2％的载体两性电解质5 / 8，0.0002％溴酚蓝），在常温下。如果体积大于350μL，加350μL的最初和同质化（步骤1.4），再加入其余缓冲区，以尽量减少飞溅。 </li><li>均质的组织为15秒，7次机动杵（Kontes公司）4℃，彼此之间的同质化冰的冷却两分钟。 </li><li>离心30分钟，在15600 XG样品在4 ° C。保存上清液并测量其体积。细胞碎片颗粒被丢弃。 </li><li>上清液中的蛋白质沉淀用冰冷的试剂级丙酮（3倍V：V比）。涡管15秒。离心15600为10秒XG形成的颗粒，并在提取缓冲液重悬与Kontes电机和聚丙烯搅拌棒（BioSpec产品） </li><li>重复步骤1.6。 </li><li>无论是进行蛋白质样品的浓度估计或存放于-20 ° C </li></ol> 2。蛋白质浓度估计使用洛瑞检测7或BCA法测定8（皮尔斯）;约4.5毫克毫升-1的目的。 3。等电聚焦<ol><li>删除一个ReadyStrip IPG胶条（11厘米，pH值5-8，Bio - Rad公司）从-20 ° C，并允许它在室温下平衡10-15分钟。 </li><li>涡样品和吸管600-800微克的蛋白质样品（总体积200μL），一个在一个样品中的补液托盘通道的后缘的直线，留在每年年底的约1厘米。 </li><li>使用钳，剥离IPG胶条的塑料盖 - 确保只能处理地带的两端，避免任何与凝胶接触。注意的地带基本结束，并在左侧的托盘位置。将样品的顶部下来的IPG胶条胶边，避免诱捕气泡下方。允许它为一小时，以补充水分。 </li><li>覆盖与2.5毫升矿物油（BioRad公司）。盖盖子的托盘，并留下在室温下过夜补充水分。 </li><li>重点托盘通道两端放置在一纸灯芯，和每一个10μL的微孔水浸湿。 </li><li>取出IPG胶条，并保持垂直约10秒钟，沥干油。印迹胶Backing与一个Kimwipe。 </li><li>将下来，基本结束左聚焦托盘的IPG胶条胶边。盖2.5毫升矿物油的地带。 </li><li>放入托盘PROTEAN IEF细胞（BIO - RAD）和计划3个步骤协议（见表1）在默认情况下电池温度20℃，最大电流为50μA/条，并没有补液时间。 <table border="1"><tbody><tr><td></td><td>电压</td><td>时间</td><td>伏特小时</td><td>舷梯</td></tr><tr><td>第1步</td><td> 250 </td><td> 20分钟</td><td></td><td>线性</td></tr><tr><td>第2步</td><td> 8000 </td><td> 2.5小时</td><td></td><td>线性</td></tr><tr><td>第3步</td><td> 8000 </td><td></td><td> 40000 </td><td>快速</td></tr><tr><td>共有</td><td></td><td> 6.5小时</td><td> 40000 </td><td></td></tr></tbody></table>表1。三步协议PROTEAN IEF细胞IPG胶条为11厘米。 </li><li>以基金的使用钳细胞IPG胶条。吸干与Kimwipe的塑料支持，并放置在一个干净的补液托盘IPG胶条胶侧。可以覆盖补液托盘和包装用保鲜膜，并存储在 - 80 ° C或进行。 </li></ol> 4。 SDS聚丙烯酰胺凝胶电泳<ol><li>转移到一个11厘米的平衡托盘的条状（凝胶的一面朝上）。 </li><li>添加4毫升减少缓冲区（6 M尿素，2％SDS，0.05 M的Tris / HCl进行pH值8.8，20％甘油，2％的数码地面电视）的渠道和平衡轻微摇晃10分钟的地带。 </li><li>卸下减少缓冲区，烷基化缓冲（6 M尿素，2％SDS，0.05 M的Tris / HCl进行pH值8.8，20％甘油，2.5％碘乙酰胺）添加4毫升和平衡轻微摇晃10分钟的地带。 </li><li>冲洗浸泡STRI IPG胶条p简要1 × SDS电泳缓冲液（25毫米的Tris，甘氨酸192毫米，0.1％SDS，pH值8.2）。 </li><li>条形凝胶的一面向上奠定到11厘米的背面板的标准预制10.5％-14％的Tris - HCl SDS聚丙烯酰胺凝胶（Bio - Rad公司），所以它轻轻推，它使得与SDS凝胶充分接触;确保被困两者之间的凝胶表面无气泡。 </li><li>熔化的琼脂糖溶液1毫升（0.5％琼脂糖，25毫米三，192毫米甘氨酸，0.1％SDS，溴酚蓝）的IPG胶条覆盖，让琼脂糖固化5分钟。 </li><li>负载5μL蛋白标记的10-225 kDa的（USB接口，P / N：76740）成分子量标准单井。 </li><li>在室温下，在200 V或在寒冷的房间（4℃）使用溴酚蓝染料前端迁移到监控电泳凝胶在SDS缓冲液（25毫米的Tris，甘氨酸192毫米，0.1％SDS，pH值8.2）运行。 </li><li>从塑料演员取出凝胶染色考马斯亮蓝轻轻摇晃过夜R - 250染色（甲醇45.5％，45.5％dH2O，9.0％醋酸，0.25％考马斯亮蓝R - 250）。 </li><li>两次动摇Destain 1的溶液（9.0％乙酸甲醇45.5％，45.5％DH 2 O）的凝胶为3小时，每Destain 2（5％甲醇，90％DH 2 O 5％乙酸）过夜。存放在7％醋酸凝胶进行分析。 </li></ol> 5。凝胶成像和分析<ol><li>捕捉图像的使用数量的一个软件程序，经营VersaDoc成像系统（BioRad公司）凝胶。作物在实验中所有的凝胶均匀的图像区域。 </li><li> PDQuest 8.0软件程序装入裁剪图像来创建一个实验集。按照实验设置向导来定义整个凝胶的现场检测和现场匹配的参数。检查和完善现货，如果需要手动匹配的结果。 </li><li>凝胶进行定量和统计分析，检测与TW匹配的蛋白质点O -倍或更高的统计学意义的差异表达，两者之间的凝胶组。创建与这些利益点分析，并削减他们使用EXQuest当场切割机（BioRad公司），胰蛋白酶消化的LC - MS为基础的蛋白质鉴定。 </li></ol> 6。凝胶中的胰蛋白酶消化对于这一步修改后的皮尔斯 - 凝胶胰蛋白酶解包（＃89871X，皮尔斯）协议使用。 <ol><li>加入200μL的脱色液（25毫米碳铵，50％乙腈）凝胶插头。涡和样品在37 ° C时30分钟，用颤抖的。小心取出并丢弃的解决方案。 </li><li>重复步骤6.1。 </li><li>加入30μL新鲜配制的减少缓冲区（50毫米三[2 - 羧乙基]膦，碳铵22.5毫米）管含有凝胶的插头和孵育10分钟，在60 ° C。 </li><li>允许样品冷却，然后取出并丢弃减少缓冲区。</li><li>加入30μL烷基化缓冲区（100毫米碘乙酰胺，20毫米的碳酸氢铵，立即箔纸包裹的管使用前的准备。样品在室温下孵育1小时在黑暗中。 </li><li>取下并丢弃的烷基化缓冲区。洗净，加入脱色的解决方案，以每管200μL凝胶插头。样品在37 ° C下15分钟。 </li><li>取下并丢弃的脱色液，重复清洗。 </li><li>加入50μL乙腈凝胶插头，并培育他们在室温下10分钟，让他们干收缩，然后小心地除去乙腈。 </li><li>再次重复步骤6.8。凝胶块应该看看白电和小。 </li><li>允许在Centrivap浓缩为10分钟（Labconco，型号EX1245）干凝胶件。 </li><li>膨胀的凝胶块，加入10μL激活胰蛋白酶溶液（10毫微克/μL胰蛋白酶，碳铵25毫米）。在室温下孵育15米inutes。 </li><li>添加25μL消化缓冲液（25毫米碳酸氢铵）管。在室温下用颤抖的隔夜孵育样品。 </li><li>超声10分钟的样品（布兰森，浴sonicator模型2510）和自旋去除上清到一个新的管前。保存上清。 </li><li>为了进一步提取的多肽，添加在室温下5分钟，用颤抖的10μL0.1％甲酸和孵化。超声10分钟的样品，收集上清，并结合步骤6.13上清保存在。 </li></ol> 7。微尺度脱盐肽提取<ol><li>删除PepClean的C - 18离心柱（ThermoFisher）根据制造商的说明的帮助肽提取碳酸氢铵等无机盐。 </li><li>两次与20μL，70％的乙腈洗脱肽，蒸发Centrivap浓缩离心干燥1小时和重悬样品在10μL的0.1％甲酸MS分析肽。 </li></ol> 8。 HPLC耦合质谱的蛋白质鉴定<ol><li>单独的5μL肽混合物的高效液相色谱（HPLC）超过40分钟，用0.​​2％甲酸一个Pepswift单片的PS - DVB列（戴安）线性2-50％乙腈梯度耦合到纳流我源到一个离子阱质谱仪（LCQ DECA XP中最大，赛默飞世尔科技）在流量400 NL / min的提示。 </li><li>检测400之间的肽MS1的信号 - 1400 m / z和允许3 MS2的测序CID动态排斥孤立的最激烈的离子峰。 </li><li>对于蛋白质鉴定，分析通过烷基化半胱氨酸修改了静态和动态修改（丝氨酸，苏氨酸，酪氨酸）磷酸化，甲基化的一个SEQUEST鼠标参考数据库检索（BioWorks软件，热尔科技）的肽结果TION（组氨酸），氧化（蛋氨酸）的ADP -核糖基化（精氨酸）和N -末端乙酰。应用保守的匹配标准（例如，设置为2阿拉伯马格里布联盟和1.00片段宽容，蛋白质中含有至少2肽传递Xcorr VS充电状态过滤器的肽宽容[Z = 1 / X = 1.5，Z = 2 / X = 2.00，Z = 3 / X = 2.50，Z = 4 / X = 3.00]和p的概率&lt;0.05），5，手动检查信心蛋白质鉴定的质谱图。 </li></ol> 9。代表性的成果： 男性和女性未行使的，小鼠骨骼肌（肱二头肌）提取和分离到一个二维地图5，第一层次的肌肉比较蛋白质组（图2）。一旦使用高分辨率数字成像可视化的，在每个检测到的约800个蛋白点。使用男性的蛋白质组图谱作为基准，很显然，有众多的景点，纷纷改变在女性的蛋白质组。当场强度<html>措施中蛋白质金额的变化（图3）。变化超过2倍（向上或向下）的蛋白质点的身份统计学意义（P &lt;0.05），与一个n = 5只，是由氨基酸序列，使用液相色谱耦合质谱分析。这些结果表明，女性表现出糖的能量代谢酶和肌酸激酶（CK）的异构体，肌肉在不同的能源供应系统的丰度减少。人类和动物显示较高的男性在休息和下面的练习9,10血清CK水平，但血清CK水平并不一定相关的肌原纤维破坏11,12量。这在CK的细胞丰度在小鼠肱二头肌的两性异形是一种新型的finding5可能回答的生理不同的血清CK水平。 数字： g1.jpg“/&gt; 图1比较蛋白质组学的协议的一个整体示意图。该协议分为三组：样品制备，样品分析和蛋白质鉴定。这三组协议的两端也是合理的暂停的地方，尽管最好的结果是得到整体的协议是，如果不停止。 <img alt="图2" src="/files/ftp_upload/3536/3536fig2.jpg" /> 图2比较雌性小鼠肱二头肌蛋白点相对男性在肱二头肌的相同点（O绿色圆圈）。斑点，增加大于或等于女性的2倍盘旋红色（o）和下降小于或等于2倍盘旋蓝（O）。 <img alt="图3" src="/files/ftp_upload/3536/3536fig3.jpg" /> 图3凝胶点强度分析：X轴，女运动前（对照组，N = 5）; Y轴，女单一回合0小时时间点组（N = 5）。回归线：相关系数= 0.919;斜率= 0.976;拦截= -0.0293。以上的红线和蓝线以下的斑点变化超过+ / - 2倍。

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没有利益冲突的声明。

的LC / MS蛋白质组学方法，这里介绍的骨骼肌蛋白质组第一级的快速分析是最可靠和可重复性的协议。它允许一个比较合理的权宜之计性别特异性。低丰度蛋白会要求肌肉样本分馏，以消除许多尽可能的收缩蛋白，从而提高了低丰度蛋白。调整蛋白质组的配置文件，可以完成不同的pH值范围IPG胶条和备用的比例梯度凝胶，如果需要的话。更敏感的荧光染料存在，但我们建议每个实验室确定这些污渍在你的系统的线性度。对于蛋白表达的更严格的分析DIGE系统（二维凝胶电泳系统，GE医疗集团生命科学版）可用于，然而，这确实增加了一个复杂程度的方法。此外，本文所描述的协议，可以使用最ANI正常的基因组已经测序，以及来自任何来源的蛋白质的从头测序的组织，只要它可以解决一个双向凝胶上，因为除了使用高纯度的酶可以产生重叠的酶片段以胰蛋白酶。其他组织来源的样本，可能需要提取方法一些改动，特别是如果膜和疏水蛋白，然而，我们不得不使用本议定书的心脏，肝，肾和脑组织良好的效果。其他翻译后修饰可能通过修改质谱数据库检索标准进行检测。总之，我们提出了一个合理详细的蛋白质组谱分析的初步协议。

<table border="1"><tbody><tr><th>试剂名称</th><th>类型</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> PepClean的C - 18旋转 列</td><td>耗材</td><td> ThermoFisher 科学</td><td> PI - 89870 </td><td></td></tr><tr><td> Pepswift单片 列（100um的× 5厘米） </td><td>耗材</td><td>戴安</td><td> 162348 </td><td></td></tr><tr><td>标准预铸 10.5％-14％的Tris - HCl SDS 聚丙烯酰胺凝胶</td><td>耗材</td><td> BioRad公司</td><td> 345-0106 </td><td></td></tr><tr><td> Readystrip IPG胶条</td><td>耗材</td><td> BioRad公司</td><td>不同pH值 范围</td><td></td></tr><tr><td>乙酸</td><td>试剂</td><td> Fisher Scientific则</td><td> A465 - 1 </td><td></td></tr><tr><td>丙酮</td><td>试剂</td><td> Pharmco </td><td> 329000 </td><td></td></tr><tr><td>乙腈</td><td>试剂</td><td> Fisher Scientific则</td><td> A955 </td><td></td></tr><tr><td>一garose </td><td>试剂</td><td> BioRad公司</td><td> 163-2111 </td><td>覆盖 解决方案</td></tr><tr><td>碳酸氢铵</td><td>试剂</td><td> Fluka公司</td><td> 40867 </td><td></td></tr><tr><td>溴酚蓝</td><td>试剂</td><td> Fisher Scientific则</td><td> BP114 </td><td></td></tr><tr><td>生物LYTE两性电解质</td><td>试剂</td><td> BioRad公司</td><td></td><td>不同pH值 范围</td></tr><tr><td> CHAPS </td><td>试剂</td><td> USB </td><td> 13361 </td><td></td></tr><tr><td> Commassie蓝R - 250 </td><td>试剂</td><td> ThermoFisher 科学</td><td> 20278 </td><td></td></tr><tr><td>二硫苏糖醇（DTT） </td><td>试剂</td><td> USB </td><td> 15395 </td><td></td></tr><tr><td>甲酸</td><td>试剂</td><td> ThermoFisher 科学</td><td> 28905 </td><td></td></tr><tr><td>甘油</td><td>试剂</td><td> Sigma - Aldrich公司</td><td> G6279 </td><td></td></tr><tr><td>甘氨酸</td><td>试剂</td><td> USB </td><td> 16407 </td><td></td&gt; </tr><tr><td>碘乙酰胺</td><td>试剂</td><td> Sigma - Aldrich公司</td><td> I6125 </td><td></td></tr><tr><td>甲醇</td><td>试剂</td><td> Fisher Scientific则</td><td> A452 </td><td></td></tr><tr><td>矿物油</td><td>试剂</td><td> BioRad公司</td><td> 163-2129 </td><td></td></tr><tr><td>十二烷基硫酸钠</td><td>试剂</td><td> Sigma - Aldrich公司</td><td> L6026 </td><td></td></tr><tr><td> Tris碱</td><td>试剂</td><td> Sigma - Aldrich公司</td><td> 93349 </td><td></td></tr><tr><td>三[2 carboexyethyl] 膦</td><td>试剂</td><td> ThermoFisher 科学</td><td> 77720 </td><td></td></tr><tr><td>胰蛋白酶Endoproteinase TPCK处理，ms级</td><td>试剂</td><td>刺穿</td><td> 90055 </td><td>修改</td></tr><tr><td>尿素</td><td>试剂</td><td> USB </td><td> 75826 </td><td></td></tr><tr><td>水</td><td>试剂</td><td>伯迪克＆ 杰克逊</td><td> 365 </td><td></td></tr><tr><td> Centrivap选矿厂</td><td> TOOL </td><td> Labconco </td><td></td><td></td></tr><tr><td> Exquest当场刀</td><td>工具</td><td> BioRad公司</td><td></td><td></td></tr><tr><td> LCQ德卡XP最大离子 阱质谱仪</td><td>工具</td><td> ThermoFisher 科学</td><td></td><td></td></tr><tr><td>电动杵</td><td>工具</td><td> Kontes公司</td><td></td><td></td></tr><tr><td>聚丙烯搅拌</td><td>工具</td><td> BioSpec产品</td><td></td><td></td></tr><tr><td>棒</td><td></td><td></td><td></td><td></td></tr><tr><td> PROTEAN IEF细胞</td><td>工具</td><td> BioRad公司</td><td></td><td></td></tr><tr><td> Sonicator </td><td>工具</td><td>布兰森</td><td></td><td></td></tr><tr><td>测量师加高效液相色谱法 系统</td><td>工具</td><td> ThermoFisher 科学</td><td></td><td></td></tr><tr><td> VersaDoc成像 系统</td><td>工具</td><td> BioRad公司</td><td></td><td></td></tr></tbody></table>

skeletal muscle gender dimorphism from proteomics

总值在骨骼肌的收缩主要是由确定的收缩蛋白的数量相对较少，但该组织也显着适应环境因素，如抵抗运动和废用性萎缩肥大。因此，它具有重塑和适应压力（热，缺血，重金属等 ）2,3。肌肉而延长，所谓的离心收缩4，可能会发生损坏由肌肉发力。收缩蛋白可能会损坏等exertions需要修复，退化和/或人工合成的，这些功能是不是收缩蛋白的一部分，但其他的要少得多丰富的蛋白质在细胞内。要确定什么样的蛋白质子集是改善这类损害，必须建立一个全球性的蛋白质组运动前5，然后依次行使后确定的差蛋白的表达，从而突出适应损伤和修复的候选蛋白。此外，大多数骨骼肌的研究已进行物种的雄性，因此可能无法代表女性的肌肉。 在这篇文章中，我们提出了一个重复性从男性和女性的肌肉中提取的蛋白质，以及高分辨率数字成像6双向凝胶电泳分离方法。这提供了一个点（和随后确定的蛋白质），显示协议统计学意义（P &lt;0.05），2倍的增加或减少，从控制状态出现或消失。然后切除，胰蛋白酶消化和高压液相色谱耦合质谱仪（LC / MS）蛋白质鉴定（LC / MS / MS），5分离。这种方法（图1），可用于几乎任何修改（肝，脑，听到许多组织吨等 ）。

Watch this Scientific Journal Video about Skeletal Muscle Gender Dimorphism from Proteomics at JoVE.com

Skeletal Muscle Gender Dimorphism from Proteomics

一个直接的方法设置隔离并确定在骨骼肌中表达最丰富的蛋白质的身份。从10毫克肌肉的二维凝胶约800点，发现了这样的性别特定蛋白表达的决心。这些方法将给予在相同的结果，大多数组织。

骨骼肌蛋白质组学性别异形

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

skeletal-muscle-gender-dimorphism-from-proteomics

Research

JoVE Journal

Medicine

12.4K 次观看.  Smith College. 一个直接的方法设置隔离并确定在骨骼肌中表达最丰富的蛋白质的身份。从10毫克肌肉的二维凝胶约800点，发现了这样的性别特定蛋白表达的决心。这些方法将给予在相同的结果，大多数组织。

视频: Skeletal Muscle Gender Dimorphism from Proteomics

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

离体骨骼肌肌的力学分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

科研

医学

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

胫骨神经切断术 - 一个标准化的模型失神经诱导小鼠骨骼肌

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

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.

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

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 ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

骨骼肌疾病的定量磁共振成像

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人的<em&gt;股外侧肌</em&gt;骨骼肌活检使用韦尔 - Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

诱导和人类劳力骨骼肌损伤评估

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析：一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

小鼠骨骼肌形态及纤维型成分的半自动化分析

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with &#039;One Stop Shop&#039; Near-infrared Spectroscopy

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

"一站式" 近红外光谱对骨骼肌神经血管的耦合、氧化能力及微血管功能的作用

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...