这项工作是支持国家机构的健康（AR-49419，DD），肌肉萎缩症协会（DD），美国国立卫生研究院，培训津贴T90DK70105（CH）的补助。

1。评价的收缩和被动的EDL肌体外的属性 EDL肌肉的收缩和被动性质的测量前体内体外肌肉测试系统中使用的极光科学。请参考表1的材料和设备。 1.1设备准备<ol><li>组装组织器官浴通过的oxytube固定到水套的组织浴中。将组装的浴到肌肉的安装装置。连接气体管线到oxytube。水循环线固定到水套的组织浴中，并放置在针入熔池排水阀。 </li><li>开启循环水浴中，并调整温度至30℃7。允许5 PSI（磅每平方英寸）的95％O 2 -5％CO 2通过oxytube流动。填写浴林格氏缓冲。至少平衡缓冲10分钟以稳定的气体流量的通过调整oxytube阀。 </li><li>开启工具（刺激，双模式控制杆系统，和信号接口）。根据制造商的指示，加载动态的肌肉控制（DMC）软件。 </li></ol> 1.2 EDL肌肉解剖所有的动物研究机构动物管理和使用委员会的批准。 <ol><li>麻醉的小鼠腹腔注射2.5微升/ g体重的麻醉剂的鸡尾酒（参考材料部分）。在整个手术过程中，被检查的深度镇静进行掐脚趾。 10％的初始麻醉剂量给药时，需要保持动物在麻醉状态下的补充。刮后肢。身体核心温度保持在37°C之前，解剖过程通过将鼠标上的一个加热垫。通过不断测量直肠TEMPERAT的监测体温尤尔使用热探测器。 </li><li>将鼠标置于仰卧在夹层板（ 图1）。剥离腿部皮肤暴露的后肢肌肉。 Sylgard的块中使用了两个裁缝销，股薄肌的脚和其他固定的腿。鼠标本体上方放置一个热灯保持核心体温在37℃。不断superfuse所有外露的肌肉林格氏缓冲。通过真空线，沥干多余的缓冲。 </li><li>暴露在立体显微镜下解剖脚的皮肤对远端的TA肌腱和韧带伸。轻轻地取出筋膜覆盖的TA肌肉。切伸韧带，到释放远端TA的肌腱。 </li><li>把远端的TA的肌腱和使用它的TA肌肉剥离。小心地取出TA在其近侧附着的肌肉。将一块薄薄的林格氏缓冲液浸湿的棉EDL肌肉吸收的TA肌肉血管破裂出血引起的。使用真空线，以除去多余的缓冲液和血液。 </li><li>配合双方结然后使用面包丝线缝合在肌腱结（MTJ）（ 图2）的前端的EDL肌由一个环结。在远侧部的近端EDL肌暴露股二头肌做一个切口。重复结（ 图2）相同的一组在近端的EDL肌腱的MTJ。将杠杆臂钩的近侧或远侧结使用相同的缝合线与双平结。切断剩余的缝合线。 </li><li>切优越的的近端缝合打结的近端EDL肌腱。抬起EDL肌肉的钩子和切下的血管的肌肉。把EDL肌肉的后肢远端缝合打结删除远端EDL肌腱逊色。与林格氏缓冲液浸湿的棉片覆盖外露的后肢。 </li><li>将钩杆臂。垂直对齐的肌肉在两个电极之间。固定的的远端缝合线固定后。抬起组织浴淹没的肌肉，林格氏缓冲。使用双粗/细翻译阶段调整至1.0g的静息张力，并允许肌肉平衡至少10分钟。 </li></ol> 1.3测量的EDL肌肉的收缩和被动性使用表2中的参数设置的DMC的每个下面的测量软件。使用肌肉的动态分析仪（DMA）软件分析数据。 1.3.1测量的EDL肌肉的收缩性能<ol><li>刺激的EDL肌肉三次在150Hz与相距60秒，以稳定的肌肉8。 </li><li>在不同的休息紧张刺激的EDL肌肉，以确定最佳的长度（LO）。的最佳长度是在该肌肉的最大收缩张力的长度。允许的肌肉，2分钟，放松身心。 </li><li>调整至芦静息张力。在单一的刺激，测量肌肉力量。确定的绝对抽搐力（PT），峰值张力（TPT），半舒张时间（½RT）的Pt。 2分钟使肌肉放松。 </li><li>调整至芦静息张力。测量强直肌肉力量，产生不同的刺激频率（50，80，100，120，150和200赫兹）。确定的绝对最大强直肌肉力量（PO）肌肉力量达到最大。测量TPT和半RT宝9。 </li><li>让肌肉放松5分钟。调整至芦静息张力。应用10个周期的离​​心收缩周期之间休息2分钟。每个周期的离​​心收缩后，计算出相对的宝力损失。 </li><li>分离设备和EDL肌肉，减少肌腱的缝合部位。确定肌湿重，计算肌肉剖面积（CSA）6,10。 </li></ol> 1.3.2测量的无源特性的EDL肌<ol><li>对侧EDL肌肉解剖，并将其附加的装置，如第1.2节中描述的步骤2至7。 </li><li>主题EDL肌肉一个六步的拉伸协议，其中的肌肉紧张到160％罗与罗10％的增量。分析的应力-应变曲线6。 </li><li>通过测量在以下时间帧的应力松弛率（SRR）的拉伸后，保持在10％罗肌肉评估粘性特性的EDL肌：从峰值到0.1秒后的峰（峰峰值），从0.1到0.2秒从0.2到0.5秒的页，页，从0.5至1秒pp和从1到1.5秒的页。 </li><li>在研究结束时，安乐死颈椎脱位和/或斩首的鼠标时，鼠标仍然在麻醉状态下。分离设备和EDL肌肉，减少肌腱的缝合部位。确定肌湿重，计算交叉本身的肌肉ctional面积（CSA）6，10。 </li></ol> 2。 原位的TA肌肉的收缩性能的评价肌肉原位测试系统采用极光科学的TA肌肉的收缩性能。请参考表1的材料和设备。 2.1设备准备<ol><li>升温至37°C的热控制的动物阶段使用的循环水浴中。 </li><li>开启工具（刺激，双模式控制杆系统，和信号接口）。根据制造商的指示，装入DMC软件。 </li></ol> 2.2的TA肌肉原位测力<ol><li>麻醉鼠标，刮胡子的后肢和第1.2节中所描述的步骤1至3的TA肌肉暴露。 </li><li>打一个双平结髌骨周围的韧带USI毫微克的面包丝线缝合。配合的前端TA肌肉的MTJ（ 图2）由一个环结双平结，然后，配合使用相同的缝合线从远侧TA肌腱结留下〜10 mm的环路的另一双平结。将所述第二侧循环的双平结。 </li><li>删除的管脚的后肢和定位的动物容易。公开的股​​二头肌。做一个切口，在中线透露坐骨神经。打一个双平结的坐骨神经周围的近端。修剪缝合线的一侧，并切断神经优于结。轻轻地，拉坐骨神经向膝盖缝合线，并清除周围的结缔组织免费〜5毫米的长度。不要在此过程中，舒展神经，Ringer缓冲，不断superfuse神经。 </li><li>准备对侧TA的肌肉中所描述的步骤1至3。量的一个暴露的后肢的Ri用一块NGER的缓冲液中浸泡的棉花。预温（37°C）林格氏缓冲液，同时不断superfuse后肢。在真空中去除多余的缓冲线。 </li><li>定位的动物平台上的动物容易。将膝盖夹紧支架的动物平台，并确保两个膝盖髌骨韧带缝合线与双平方海里的金属针。 Sylgard的块中使用的裁缝针引脚双脚。固定在的动物平台上的热控制的阶段。热灯定位保持动物芯体温度在37℃下</li><li>固定电极支架的动物平台，奠定了坐骨神经上的电极使用的缝合线。保持电极远离后肢肌肉。把裸露的后肢远端的TA肌腱的在MTJ缝合网站。附加的前端TA肌腱的缝合线套环，以该杆臂钩。一个温暖的林格氏缓冲液浸湿的棉，覆盖外露的后肢肌肉。 </li></ol><ol><li>使用表2中的参数设置DMC软件。按照第1.3.1节中描述的相同的协议来确定的TA肌肉的收缩性能。使用的DMA软件分析数据。 </li><li>收缩特性测量后，分离的远端TA，肌腱缝合环矫平机臂钩。删除的TA肌肉。确定肌湿重，计算CSA 10。 </li><li>测量对侧TA肌肉的收缩性能，根据上述的步骤1至3。在研究结束时根据机构的指导方针，实施安乐死的鼠标。 </li></ol>

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没有利益冲突的声明。

在这个协议中，我们已经说明了生理实验测量EDL肌肉的收缩性能的TA肌肉的收缩和被动属性。肌肉生理学研究的一个主要问题是目标肌肉的氧合。对于大的肌肉（如的TA肌肉）， 在原位方法是优选的，因为氧的扩散从林格氏缓冲液可能不会达到在一个中心的肌肉在体外测定。 在原位的方法并没有扰乱正常血液供给和缺氧-相关的人工的效果是可以避免的。的EDL肌肉的生理学研究中最常用的肌肉之一。可以实现在体外系统足够的氧的整个肌肉，因为小规模的肌肉。另外， 在体外系统中，提供了一个封闭的环境，来操纵的离子的浓度（的Ca 2 +，Na +和K +）和化学CALS（ATP和葡萄糖）是必要的以获得最佳的肌肉力产生。这提供了一个很好的机会来研究这些变量的影响力生产。 四肢肌肉的收缩和被动性质的准确测量是研究骨骼肌功能的关键。这些属性的变化特点通常被认为是各种肌肉疾病的标志。这些参数的变化也很重要的指标，以确定​​是否一个实验性的治疗是有效的或不。

<table border="1"><tbody><tr><td> 材料 </td><td> 生产厂家 </td><td></td><td> 规格和意见 </td></tr><tr><td>组织器官浴</td><td> radnoti LLC，加利福尼亚州，美国</td><td></td><td>的组织浴水套（猫＃158351-LL），氧气的分散筒（猫＃160192），鲁尔阀（CAT＃120722） </td></tr><tr><td>循环水浴</td><td>默飞世尔科技，美国马萨诸塞州沃尔瑟姆， </td><td></td><td></td></tr><tr><td>混合气体</td><td> Airgas的国家，美国北卡罗来纳州夏洛特， </td><td></td><td> 95％O 2和5％CO 2的 </td></tr><tr><td> 在体外肌肉功能检测设备</td><td>科学，极光，极光，加拿大</td><td></td><td>该系统由刺激器（型号＃701A），一个双模式的杠杆系统（型号＃300C或305C），一个信号接口（型号＃604B）和一个测试APparatus（＃800A型），垂直安装组织器官浴</td></tr><tr><td> 在体外肌肉功能分析软件</td><td></td><td></td><td>动态肌肉控制（DMC）软件和动态肌肉的控制数据分析软件（DMA） </td></tr><tr><td>鼠标麻醉鸡尾酒混合在0.9％NaCl </td><td>请参考机构的指引</td><td></td><td>氯胺酮（25毫克/毫升），甲苯噻嗪（2.5毫克/毫升）和乙酰丙嗪（0.5毫克/毫升）。在整个手术过程中，可能需要的初始剂量为10％的补充以保持动物麻醉下。 </td></tr><tr><td> SYLGARD </td><td>世界精密仪器</td><td>猫＃SYLG184 </td><td></td></tr><tr><td>一个特制的有机玻璃夹层板</td><td>在内部设计</td><td></td><td>请参考图1 </td></tr><tr><td>暖气灯</td><td>张照明公司，波士顿，MA，USA </td><td></td><td> 15瓦的灯泡，以保持鼠标的温暖，在解剖</td></tr><tr><td>林格氏缓冲</td><td>化学品购自默飞世尔科技，美国马萨诸塞州沃尔瑟姆， </td><td></td><td> MM：1.2成分的NaH 2 PO 4（猫S369），1硫酸镁4（猫M63），4.83氯化钾（CAT＃P217），137氯化钠（CAT＃217），24 碳酸氢钠 （猫S233），2 氯化钙 （猫＃C79）和10血糖（猫＃D16）。分别溶解化学品混合在上面列出的顺序。存放于4℃ </td></tr><tr><td>立体解剖显微镜</td><td>尼康，梅尔维尔，NY，USA </td><td></td><td></td></tr><tr><td>解剖工具</td><td>精细的科学工具，福斯特市，CA，USA </td><td></td><td>粗钳，粗剪，细镊子（直和45°角） </td></tr><tr><td>编织丝线缝合＃4-0 </td><td>苏SofSilk USSCTures的，诺瓦克，CT，USA </td><td>猫＃SP116 </td><td></td></tr><tr><td>一个特制的不锈钢钩</td><td>小零件， </td><td></td><td> 2英寸长的S / S 304V（0.18“直径）力传感器305C或2.5”的S / S 304V（0.012“直径）传感器300C（猫＃ASTM A313） </td></tr><tr><td> 原位肌肉功能分析系统</td><td>科学，极光，极光，加拿大</td><td></td><td>刺激器（型号＃701B），一个双模式的杠杆系统（型号＃305C），信号接口（型号＃604A）和热控制脚踏板装置（型号：809A的系统（809B， 在原位的鼠标装置）包括） </td></tr><tr><td> 在体外肌肉功能分析软件</td><td>科学，极光，极光，加拿大</td><td></td><td>动态肌肉控制（DMC）软件和动态肌肉的控制数据分析软件（DMA） </td></tr><tr><td>一个特制的TA试验动物平台</td><td>在内部设计</td><td></td><td>请参照图2 </td></tr><tr><td>一个特制的不锈钢钩</td><td>小零件， </td><td>猫＃ASTM A313 </td><td> 0.5英寸长的S / S 304V（0.18“直径） </td></tr><tr><td>客户定制25G铂电极</td><td> Chalgren企业，旅游Gilroy，CA </td><td></td><td>焊接2 0.016“厚的铂丝的两个24G电线</td></tr></tbody></table> 表1。材料和设备。

evaluation of muscle function of the extensor digitorum longus muscle ex vivo and tibialis anterior muscle in situ in mice

身体运动主要是由骨骼肌的机械功能。骨骼肌是由众多的护套肌肉结缔组织的肌纤维束。每个肌纤维包含许多的肌原纤维，沿其长度的肌纤维纵向运行。肌原纤维，是肌肉的收缩装置的，它们是由被称为肌节的反复收缩单位。一个肌节单元包含的Z盘和肌联蛋白的蛋白质的肌动蛋白和肌球蛋白丝间隔。骨骼肌的机械功能是由肌肉的收缩和被动特性定义。收缩性能进行了表征肌肉收缩，肌肉松弛产生的力量和时间的过程中产生的力的大小。影响肌肉收缩（如肌动蛋白和肌球蛋白丝，钙稳态的，ATP / ADP比值等之间的相互作用）的任何因素，影响收缩properties。被动的属性是指弹性和粘性性能（刚度和粘度）的肌肉在收缩的情况下。确定这些属性是由细胞外和细胞内的结构部件（如titin的）和结缔组织（主要由胶原）1-2。收缩和被动性的肌肉功能是不可分割的两个方面。例如，是通过屈肘的肌肉收缩的前室的上臂和在后室的上臂的肌肉被动伸展。为了真正地了解肌肉功能，收缩和被动性质进行研究。 肌肉的收缩和/或被动的力学性能，经常会损害在肌肉疾病。一个很好的例子是杜氏肌营养不良症（DMD），严重的肌肉萎缩疾病引起的抗肌萎缩蛋白的不足3。抗肌萎缩蛋白是一种细胞骨架蛋白A在稳定肌细胞膜（肌膜），肌肉收缩时的4。在的情况下，肌营养不良蛋白，肌膜损坏力的传递过程中产生的剪切力。此的膜撕裂启动连锁反应，导致肌肉细胞的死亡和损失的收缩机械。作为结果，减少肌肉力量和死肌纤维所取代的纤维化组织5。这以后的变化会增加肌肉僵硬6。这些变化的精确测量提供了重要的指导作用，以评估疾病进展，并确定新基因/细胞/药物干预的治疗效果。在这里，我们提出了两种方法来评估趾长伸肌（EDL）的肌肉和胫骨前肌（TA）肌肉的收缩性能的收缩和被动的机械性能。

下面的结果是从我们以前的报告6,9表示。数据表示为平均值±标准误差的平均值， 表3示出了在正常的BL10和肌营养不良蛋白缺失的小鼠（MDX）在4至6个月的年龄的EDL肌肉的形态性质的图4示出了代表性的收缩和被动特性的EDL BL10 和 mdx小鼠的肌肉。以下条款，包括具体的（绝对力量除以通过的CSA）的抽动力（ 图4A），具体的最大强直收缩力（ 图4B），TPT和半RT的绝对最大强直收缩力的EDL肌的收缩性能（ 图4C和 D）。 TPT和½，RT也可以被计算出绝对的抽搐力。的应力-应变曲线（ 图4E）和SRR（ 图4F）重新用于描述的EDL肌肉的被动特性。 缺乏抗肌萎缩蛋白的收缩和被动的EDL肌6,9的性能有显着的影响。具体的抽搐和MDX EDL肌强直收缩力显着减少。 TPT是显着更快，而½RT是显着慢于对 mdx EDL肌肉。的应力-应变曲线表明，刚度显着增加的 mdx EDL肌。 MDX EDL肌肉也产生了显著高得多的抵抗力量（被动压力）达到峰值应力前，后的应力峰值下降得更快。另外，在SRR显着较高的 mdx EDL肌相比的BL10 EDL肌肉。 统计分析由学生t检验分析两组之间的统计意义。对于s在多个组中，单向或双向ANOVA分析，Bonferroni法事后分析tatistical意义，建议使用SAS软件（SAS公司，卡里，NC）。差异被认为是显着的， 当 p &lt;0.05。 表1。材料和设备。 <table border="1"><tbody><tr><td class="jove_content_small"> 实验 </td><td class="jove_content_small"> 静息张力（克） </td><td class="jove_content_small"> 脉冲频率（Hz） </td><td class="jove_content_small"> 脉冲宽度（ms） </td><td class="jove_content_small"> 刺激持续时间（ms） </td><td class="jove_content_small"> 拉伸长度 </td><td class="jove_content_small"> 拉伸持续时间（ms） </td><td class="jove_content_small"> 伸长率 </td><td class="jove_content_small"> 评论 </td></tr><tr><td colspan="9"> 1。收缩和被动的EDL肌体外的性能评价 </td></tr><tr><td colspan="9"> 1.3.1测量的EDL肌肉的收缩性能 </td></tr><tr><td> 1。热身</td><td> 1.0 </td><td> 150 </td><td> 0.2 </td><td> 300 </td><td></td><td></td><td></td><td>休息的肌肉，刺激的60秒之间。这些初步的强直收缩的肌肉后面进行的测量稳定。 </td></tr><tr><td> 2。最佳肌肉长度（LO） </td><td> 0.5，1.0，1.5和2.0 </td><td> 1 </td><td> 0.2 </td><td> 300 </td><td></td><td></td><td></td><td>让肌肉放松每一个刺激的30秒之间。使用数字卡尺测量肌肉的最佳长度。 </td></tr><tr><td> 3。单TWITCH力（PT） </td><td>罗静息张力调整</td><td> 1 </td><td> 0.2 </td><td> 300 </td><td></td><td></td><td></td><td></td></tr><tr><td> 4。强直肌肉力量</td><td>罗静息张力调整</td><td> 50，80，100，120，150和200的</td><td> 0.2 </td><td> 300 </td><td></td><td></td><td></td><td> 1分钟之间刺激使肌肉放松。确定的频率产生的最大绝对强直收缩力（PO）。 </td></tr><tr><td> 5。离心收缩</td><td>罗静息张力调整</td><td>使用的频率产生的最大强直收缩力（宝） </td><td> 0.2 </td><td> 700 </td><td> 10％劳</td><td>最后的刺激持续时间为200 ms </td><td> 0.5桢/秒</td><td> 10个周期与周期之间休息2分钟，重复离心收缩。 </td></tr><tr><td> 6。 CSA的EDL肌</TD&gt; <td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> CSA =（肌肉质量（g）/ [1.06克/厘米3×（罗×0.44）]，1.06克/厘米3，是肌肉的密度和0.44是EDL肌纤维长度至Lo比。 </td></tr><tr><td colspan="9"> 1.3.2测量的无源特性的EDL肌 </td></tr><tr><td> 1。六步拉伸协议</td><td>罗静息张力调整</td><td></td><td></td><td></td><td> 10％劳</td><td></td><td> 2厘米/秒</td><td>重复拉伸协议，增量罗的10％至160％，罗达到。帆都鼓满1.5秒之间舒展周期。 </td></tr><tr><td> 2。 SRR </td><td>罗静息张力调整</td><td></td><td></td><td></td><td> 10％劳</td><td></td><td> 2厘米/秒</td><td> SSR的计算方法是将不同的应力与TIME经过两个时间点之间的时间框架。 </td></tr><tr><td colspan="9"> 2.3测量的TA肌肉的收缩性能 </td></tr><tr><td> 1。热身</td><td> 4.0 </td><td> 150 </td><td> 0.2 </td><td> 300 </td><td></td><td></td><td></td><td>休息的肌肉，刺激的60秒之间。 </td></tr><tr><td> 2。最佳肌肉长度（LO） </td><td> 3.0，4.0，5.0，6.0和7.0 </td><td> 1 </td><td> 0.2 </td><td> 300 </td><td></td><td></td><td></td><td>让肌肉放松每一个刺激的30秒之间。使用数字卡尺测量肌肉的最佳长度。 </td></tr><tr><td> 3。 CSA的TA肌肉</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> CSA =（肌肉质量（g）/ [1.06克/厘米3×（LO×0.6）]。0.6的TA肌肉纤维长度罗岭蒂奥。 </td></tr></tbody></table> 表2中。参数的EDL和TA的肌肉的机械性能的评价。 <table border="1"><tbody><tr><td> 应变 </td><td> 年龄（月） </td><td> 车身重量（g） </td><td> EDL的重量（mg） </td><td> EDL罗（毫米） </td><td> ：EDL CSA（毫米2） </td></tr><tr><td> BL10 </td><td> 6 </td><td> 32.03±0.57 </td><td> 13.90±0.77 </td><td> 14.09±0.04 </td><td> 2.12±0.12 </td></tr><tr><td> MDX </td><td> 6 </td><td> 35.44±0.42 * </td><td> 16.73±0.42 * </td><td> 13.93±0.05 * </td><td> 2.57±0.07 * </td></tr></tbody></table> 表3中。的EDL肌肉的形态性质的*，该值在的mdx小鼠年龄匹配的BL10小鼠显着不同。 <img src="/files/ftp_upload/50183/50183fig1.jpg" alt="图1" fo:src="/files/ftp_upload/50183/50183fig1highres.jpg" fo:content-width="5.5in"/> 图1。示意图的定制鼠标夹层板的夹层板是由一个半英寸厚的有机玻璃制作的机构店。 <a href="https://www.jove.com/files/ftp_upload/50183/50183fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/50183/50183fig2.jpg" alt="图2" fo:src="/files/ftp_upload/50183/50183fig2highres.jpg" fo:content-width="4in"/> /&gt; 图2。一系列的数字图像显示的步骤追平了双平结，然后在MTJ由一个环结。星号，EDL肌肉，箭，远端肌腱EDL肌肉。 <img src="/files/ftp_upload/50183/50183fig3.jpg" alt="图3" fo:src="/files/ftp_upload/50183/50183fig3highres.jpg" fo:content-width="6in"/> /&gt; 图3。定制的平台， 在原位 TA肌肉功能分析示意图的。有机玻璃动物，平台和的不锈钢膝盖持有人被设计为安装在原位鼠标装置的809B，不锈钢棒（MPR-2.0猫锡斯基尤，Grants Pass，俄勒冈），通用电极支架（猫＃MXB，锡斯基尤，Grants Pass，俄勒冈）;§，电极附件棒（猫MPR-3.0，锡斯基尤，Grants Pass，俄勒冈）; Sylgard的块。 <a href="https://www.jove.com/files/ftp_upload/50183/50183fig3large.jpg" target="_blank">点击这里查看大图</a> 。 /&gt; 图4。代表性结果的EDL肌的收缩特性的EDL肌的收缩和被动特性。 其特征在于具体抽搐力（A），具体强直动力（B），峰值张力（C）和半弛豫时间（D）的时间。由应力 - 应变档案中（E）和SSR的被评估的EDL肌肉的被动特性。 *，，mdx小鼠有显着不同的年龄相匹配的BL10小鼠。

Watch this Scientific Journal Video about Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice at JoVE.com

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

四肢肌肉收缩和被动的机械性能的变化是重要的肌肉疾病的生物标志物。这手稿描述生理检测，以测量这些特性在小鼠趾长伸肌和胫骨前的肌肉。

伸趾长肌的肌肉功能评价<em&gt;体外</em和胫骨前肌<em在原位</em在小鼠

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

evaluation-muscle-function-extensor-digitorum-longus-muscle-ex-vivo

Research

JoVE Journal

Biology

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

41.8K 次观看.  University of Missouri. 四肢肌肉收缩和被动的机械性能的变化是重要的肌肉疾病的生物标志物。这手稿描述生理检测，以测量这些特性在小鼠趾长伸肌和胫骨前的肌肉。

视频: Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

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

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

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 ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

体外评估对离体骨骼肌的收缩，疲劳性和交替“

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

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 ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

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 ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

平滑肌功能中分离出来的组织浴，应用药理学研究测量

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

小鼠主动脉夹层损伤：高效<em&gt;体内</em&gt;平滑肌细胞增殖和内皮功能模型

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

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 ...