The authors would like to thank Dr. Hannah Baker for her extensive work in optimizing this procedure.

所有动物人道待遇，所有的协议是由弗吉尼亚IACUC的大学认可。 1.设备的准备<ol><li>确保所有机器都连接正确。 </li><li>打开电脑，其次是高功率双相刺激和双模杠杆系统。 </li><li>此时，将动物与2％异氟醚供给的麻醉室中，并把该加热元件上，以使平台被加热到37℃。 </li><li>放置电极在70％乙醇，使涂布在聚四氟乙烯（PTFE）的提示被淹没并同时设置设备和软件将被消毒。 </li><li>找到并在桌面上打开杠杆系统控制软件。 注意:这将是执行功能测试所需的软件。 </li></ol> 2.软件安装<ol><li>一旦程序被打开（ 图1A），更改参数在设置菜单中为所需的值下S代表即时兴奋剂。 注意:在这个协议中，所有参数都保持在与"运行时间（s）"，它被改变为180秒（ 图1B）异常的预设水平。 </li><li>创建设置菜单下自动保存的文件夹。 </li><li>找到标有"自动保存基地"一个类型能够窗口。输入样品的名称，例如"RAT1 - 日期 - 时间点"。直接将"自动保存基地"类型能够窗口的左侧，单击框"启用自动保存"。 </li><li>在控制屏幕的顶部，选择"序"。一个新的窗口将打开。在新窗口的底部，选择"打开序列"。一个新的窗口将打开。选择预制序列，然后单击确定。与序列参数，包括频率，刺激的持续时间和休息时间的协议列表将在指定的窗口发展:序列编辑器（ 图1C）。点击"加载序列" - &gt;＆＃34，关闭窗口"。 </li><li>要查看实时电流和刺激，选择"文件" - &gt;"实时数据监控"。一个新的窗口将打开。 </li><li>在新的实时数据窗口，格式屏幕用于通过使用自动缩放功能，或手动输入屏幕上显示的最大和最小y值的测试。 </li></ol> 3.动物建立注:所有测力是那些一个11周大的Lewis大鼠的。有肌肉质量和力量产量（牛顿）之间的线性关系。因此，作为鼠的增加年龄，由腿产生的力的值应增加。 <ol><li>确保动物在麻醉的正常飞机从麻醉室后，再取出。彻底去除脚踝上，并使用电推剪实验腿骨盆之间的外侧所有的头发。 注:麻醉平面正确实现当动物我S非响应于一个脚趾捏。有必要按照各机构的动物护理和使用委员会提出的准则。 </li><li>放置在仰卧位的动物，以确保动物的鼻子安全地处于麻醉鼻锥体，以便它保持在麻醉的足够的深度。 </li><li>调节踏板装置由三个独立的旋钮（ 图2）的位置。使用旋钮（A和B）来调整脚踏板，放置在其最左和最低的位置分别在踏板装置。同时留下余地购买操作这将使动物的脚的正确定位。在这个位置上，可以使用在轨道的左侧的旋钮移动装置朝向或从实验者更远，使得动物腿位于一个直平面。 </li><li>清洁用碘酒和酒精的三个转变的腿。碘应保持腿部30秒。 </li><li>调整动物或平台（ 图2A中的D），以便扩展腿确保脚的鞋底和脚踏板之间的完全接触。 </li><li>用医用胶布，对固定支板（ 图2D）动物的脚。至关重要的是，脚后跟是对踏板和整个脚的底部齐平是平坦的，在测试过程中不会从板去除。 </li><li>定位夹紧机构以稳定的腿。通过转动扳手推到足够程度，减少腿部运动并锁住它的稳定销。 </li><li>在该位置，使用旋钮C到移动装置或朝向或远离实验者使得踝关节，胫骨和股骨谎言在一条直线上（ 图2C）。确保该腿与脚踏板平行。使在球场上和在装置的背面的细旋钮调整，缓慢移动的踝使脚和胫骨是在90°的位置。 </li><li> Continue移至腿部所以股骨和胫骨是在90度垂直的角度（ 图2B）。在这一点上，该动物是准备的电极。 </li></ol> 4.电极的放置<ol><li>通过点击标有"即时兴奋剂"橙色按钮，激活"即时兴奋剂"。 </li><li>放置两个电极表面上的胫骨前的近端和直到尖峰看出实况显示器上移动电极尖端。理想地，尖峰应该在0.4 N. 注意:电极应放在相邻并正交腓神经的平面，这反过来，横向从膝盖并垂直于胫骨上运行。 </li><li>插入一针远远不够，皮尔斯真皮，勉强进入肌层。四处移动另一个电极，直到尖峰看到约0.6 N.插入针现场监视器上，并用夹子爱好或医用胶布它们夹紧到位。 </li><li>一个djust粗，微调找到最大力量输出。 </li><li>对高功率双相刺激，就会有在中心两个旋钮。一个标为"RANGE"和其他"调整"。打开"RANGE"旋钮，所需的最大电流。 注:峰将在幅度缓慢增加，最大电流被确定为在其中三个连续刺激导致相同收缩反应的水平。抵制转高于所需的电流;最大电流会刺激整个肌肉收缩，但任何更高的电流会导致肌肉邻国的招聘和潜在的拮抗剂为好。 </li><li>旋转"调整"钮，将用于刺激肌肉的"RANGE"的百分比。在这一点上，力应改为1.0左右N.这可能需要在电流的增加或减少。 </li><li>重新检查电极，以确保他们的安全。停止即时兴奋剂。 </li><li>在"实时数据"窗口中，单击"启动序列。" </li><li>继续回到控制屏幕，并单击"分析"按钮位于橙色的"即时兴奋剂"按钮上方监测曲线。强直曲线应开始采取各地的60赫兹刺激的形状。 </li></ol> 5.整理刺激和清理<ol><li>序列完成后，取出电极并擦拭干净，用70％的酒精。放置电极的封面。 </li><li>解开膝盖夹紧并关闭麻醉。除去从麻醉气体的动物，并放置在俯卧位的动物，仍然在加热垫。维持100％O 2的老鼠为后异氟醚气体已被关闭，以保持含氧大鼠几分钟。动物最初可能会移动，但直到动物恢复了意识不返回动物回到笼子里。如果肌肉酸痛被发现在恢复，由动物保健委员会规定的应给予NSAID的剂量。 </li><li>关闭所有在步骤1.2中列出的设备，关闭软件，并继续数据分析。 </li><li>擦拭平台和脚踏板。 </li></ol> 6.数据分析进行数据分析，以适应本实验室设计，并根据实验室协议的顺序:注意。分析值，重要性的数据点，并且该过程的其他方面将变化取决于用户的意图。 <ol><li>打开数据分析软件。 </li><li>单击高通量菜单上在一个时间，以使多个数据文件（样品）的分析。选择"强制频率"分析。 </li><li>点击"选择文件"按钮，然后根据需要打开多个保存的数据文件。 </li><li>选择光标放置方法中的"说明书"。 注意:这将允许用户分析所有的DAT的一个所希望的时间戳记内，而不是在程序自动选择分析位置。 </li><li>结束光标时间戳值更改为2.单击"分析"按钮（ 图1D）。 </li><li>保存表和分析使用电子表格中的数据，点击"保存表到ACSII按钮，将保存文件，并可以在以后的电子表格打开。 </li><li>打开电子表格保存的数据文件。 </li><li>创建标记的"绝对最大"一个附加列，并确定基线和每个样品的最大值之间的差。这将提供在每个频率所产生的总的最大的力。 </li><li>为了确定扭矩，由杠杆臂的长度乘以每个力值。 注意:在这种情况下，这将被动物的脚长度来表示。该协议使用30毫米的平均实验确定值。用户现在已经确定了马云的价值观在每个频率产生ximum转矩。 </li><li>绘制这些值作为转矩频率曲线，或者，通过在所有刺激频率的动物产生的最大转矩。 注意:此可以识别并用作样品之间的比较的单点。 </li></ol>

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The authors have nothing to disclose.

此协议演示了用于对大鼠后肢的前脚部隔室进行体内肌肉功能测试相对简单的方法。肌肉功能测试的其他形式，包括体外 和原位协议，也可以提供有关肌肉生理重要信息。然而， 在体内的功能测试的意义在于它非侵入性的性质，而且它最准确地概括肌肉刺激内源性机制的事实。对于这两种体外 和原位检测，肌腱和/或肌肉被暴露，因此，必须保持湿润或浸没41,42， 在体内试验中删除可以由所需的外科手术引起的创伤和炎症的混杂变量用于原位肌肉功能测试;如果实验的目的是研究抗炎和细胞过程，这是特别重要<s达&gt; 43。此外， 在体内测试 需要很少的外科技术人员作为肌肉不从其周围隔离的，并且不要求精确结降低肌肉/腱打滑（如为的情况下在原位或离体试验）41。此外，具有足够的实际应用中，正确放置电极的速度，并能够迅速做出调整，以达到最大的力生产的肌肉将确保协议完成是迅速并且两个内的动物和不同用户的相同设备39的reproducible-能力。是有益与整个前小腿部件的评估开始如图所示，在不易进入的协同肌（EDL和HL），用于对TA肌肉的更直接调查的切除之前。使用这种方法，我们可以相当迅速地实现技术的掌握。尽管这里描述的过程说明和突出的力fr的效用equency协议以诱导破伤风和确定由肌肉产生的最大的力，用户应确定能最佳通知其具体实验（S）和研究目标功能测试的类型（多个）。 有迹象表明，应仔细以确保最佳的和可重复的实验结果由肌肉的各种刺激参数进行的，即，一致最大力量生产几个关键步骤。几个关键特征在图2中概述的，然而，适当放置和刺激电极的稳定是腓总神经再现的最大刺激的绝对先决条件。在这方面，在电极应表面放置。即，如果电极的位置是太深，一是风险拮抗肌的直接电刺激，从而减少前脚部室的观察收缩反应的幅度。此外，该两个电极应放在尽可能靠近彼此接近可行减少周围的皮肤和结缔组织的电阻。在一般情况下，靠近膝盖和内侧到腿部直接追踪胫前到它满足腓肠肌的边缘的电极定位常常产生足够的力的生产。这也确保了电极相邻和正交放置在腓神经，这反过来又，垂直于胫骨和横向向下从膝盖腿运行的平面。然而，在动物之间的解剖学自然变化，需要时刻保持警惕，以确保电极位置是在逐案基础上进行了优化。正因为如此，有与由用户的体验显著减弱电极放置有关的试验和错误的一定水平。电极刺入皮肤应尽量少，以减少肿胀和炎症的次数，从而减少了我asured力的生产。这是取决于其中针最初放置，但建议移动针头的两倍或以下特别是在周围的膝盖骨的区域。最后，一旦电极被放置在动物的腿部，小的调整，可以在腿部的定位，并通过所述电极提供的电流进行。这应该同时监测从单一抽搐产生的力来完成。除了电极放置，调整，也可以以跨电极输送的电压制成。然而，在这里所描述的设置，它作为一种方法来提高输出力增加电压时要谨慎使用，因为增加的电压会刺激神经支配的拮抗肌是很重要的。 有迹象表明，必须进行监测，以确保电极放置仍然保持最佳状态的三个关键技术问题。首先，将麻醉动物的脚必须牢固地锚定到脚踏板装置，其测量肌肉力产生（ 图2）。如果脚不牢固地固定，由肌肉产生的真实力可以不完全转换为力传感器。不稳定脚固定也引入失去电极的最佳放置为超出正常肌肉收缩运动的风险（ 即脚移动从脚板的距离）可以从它们的浅位置引起电极的位移或完全去除它们。这两种情况下会降低测量的力。第二，在动物的身体应完全仰卧并在直平面（ 图2）对准。动物身体的正确位置防止因呼吸腿部轻微抖动，同时还最大限度地减少了腿和骨盆的扭动，实现更好的位置和刺激电极的连续接触。膝关节三，正确定位和固定是criti校准以确保该腿保持稳定，因此，有助于稳定刺激电极的最佳放置，以允许腓总神经一致活化。 有迹象表明，应该强调的几个附加分。首先，将商业肌肉杠杆系统被设计为在左腿执行测试，但是设置可被修改为在右腿执行测试为好。第二，肌肉杠杆系统可基于动物的大小进行选择，所以用户应该确保所用的平台是足够来测量，并支持由所选择的动物模型产生的力。为设备平台可检验的肌肉都仅限于那些诱发足部的跖分机或背屈。第三，它应该再次强调的是，电极位置可以是具有挑战性的，需要耐心和实践掌握的技术。电极也很快变得迟钝经常使用，所以它是有帮助的几个备用小号ETS一次变得难以表面刺破皮肤。第三，本报告中所描述的协议利用特异性刺激序列和数据分析程序。肌肉杠杆系统控制软件和数据分析软件，它提供可以回答许多其他实验问题，并因此数据，其效用延伸超出了本文中概述。因此，我们鼓励用户探索超越本文介绍的软件协议（S）的限制。尽管存在这些轻微的局限性， 在体内肌肉功能测试是一个功能强大的方法来确定骨骼肌的健康和收缩能力，因为它是微创并且可以在多种场合下进行，在延长的时间范围内，在相同的动物。总之，这种类型的维修工具的使系统特别善于测试对于在大鼠后肢骨骼肌损伤或疾病的新疗法的效果。

骨骼肌在响应损伤或疾病1,2修理一个显着的内在能力。在实验上，此再生反应的鲁棒性得到了很好的在动物模型中通过研究，例如记载，骨骼肌损伤，修复和再生myotoxins（ 例如 ，心脏毒素）3-7的涂布后的时间过程。更具体地，经过广泛的心脏毒素诱导的肌肉损伤，再生是通过卫星细胞介导的（肌纤维8的38-67％），则驻留干细胞的成熟，最终成为功能的肌纤维4,9-13。最终的结果是增加了后期的损害健康，力产生肌肉组织14-16功能再生。虽然细节超出了报告的范围，对于肌肉再生的机理基础上体现多种细胞类型，从利用canoni多谱系精心打造的事件校准信号通路到两个组织发育和形态发生5,17-21至关重要。重要的是，myotoxin诱导再生是通过这一事实使该细胞外基质，神经元神经支配和血管灌注保持结构完整以下心脏毒素诱导的肌肉损伤3,8,22。与之形成鲜明对比，这些关键组织的结构和组件，根据定义，在VML损伤的情况下完全不存在;其中，组织坦诚的损失，由于多种原因，导致永久性的功能和外观缺陷23-25。 不管用以下VML损伤与myotoxin诱发肌肉损伤比较肌肉修复和再生，改善了骨骼肌再生和修复的机械基础的理解相关联，在多种情况下的额外的挑战，将由的生物利用率得到更好的服务在与纵向的组合相关动物模型中肯的功能性措施ssessments。如本文中所讨论的，大鼠后肢的研究提供了一个极好的模型系统为此。更具体地，前脚部室的肌肉（胫前，趾长伸肌（EDL）和hallicus长肌（HL）），这是负责足部背屈，很容易识别和操纵。此外，它们是由大血管（髂和分支机构）提供服务，并通过神经（坐骨神经和分支机构，包括腓）运行26-28腿的长度支配。因此，人们可以使用大鼠后肢模型直接评估体内骨骼肌功能/病理学，或评价对相应骨骼肌功能在血管或神经病理相关的改变的更间接影响。在这两种情况下，疾病的严重程度，以及治疗的功效可被确定为肌肉力量的生产（扭矩）和相应的脚微米的功能ovement 29-34。 理想的情况下，力的测量是伴随着组织学研究和基因表达分析，以更严格的评估骨骼肌的结构和分子的状态。基本组织学和免疫组织化学，例如，能够回答关于肌肉大小，肌肉纤维取向，细胞外基质组合物，核的位置，细胞数，和蛋白质定位的问题。基因表达分析，反过来，是必要的用于识别可能影响/调节肌肉纤维，疾病状态，和代谢活性的成熟的分子机制。虽然这些方法提供了重要的信息，它们通常表示终端端点，以及最重要的是，它们不能直接解决骨骼肌的功能的能力，并且因此，是相关的，而不是致病。然而，当组织学研究和基因表达分析与功能结合测量电评估ES，那么，力生产和功能再生机制可以最准确地识别。 在这点上，产生一个肌肉的能力上的力可在体外测定，原位或体内 。所有这三种方法都有优点和局限性。 在体外试验中，例如，肌肉完全隔离，并从动物的身体中移除。通过去除血管和神经该供给肌肉的影响，可以在严格控制的外部环境35来确定所述组织的收缩能力。 原位肌测试允许肌肉被分离，如在体外制剂，但是，神经支配和血供保持不变。 原位实验模型的好处是，它允许在神经支配和血液供应最小扰动36被研究个体肌肉。同时在体外 和原位实验，药物治疗可以更直接地，而不必考虑任何周围组织或循环系统的所测量的收缩反应37冲击的影响施加。然而， 在体内的功能测试中，如本文中所描述的，是为在其天然环境38评价肌肉功能的至少侵入性技术，并且可以随着时间的推移重复进行（ 即 ，纵向）。因此，这将是下文讨论的焦点。 在这方面，插入感兴趣的肌肉，或供应其运动神经附近经皮电极，提供的电信号的肌肉。换能器然后测量指示由预定，定制软件协议在激活的肌肉产生的长度或力的变化。从这些数据中，肌肉的物理性质可以被确定。这些包括用于CE-频率，最大破伤风，力 - 速度，硬度，长度张力，和疲劳。肌肉长度或力也可保持恒定，以使肌肉收缩等长或等渗。重要的是，这些实验性协议可以迅速地进行，容易反复和customized-所有而将动物麻醉并用的小时恢复期到几天。一个单一的动物可在体内进行力量测试多次，从而使疾病模型或治疗平台/技术评估的纵向研究。 如本文所述，在具有高功率组合，商业肌肉杠杆系统，双相刺激器被用于在体内肌肉功能测试，以执行经由刺激来评价大鼠后肢的胫骨前肌的与脚的背屈的贡献腓总神经。我们已经开发出专门设计用于评价再生医学/ Ti的一个协议继鼠的TA肌肉创伤性损伤VML工程ssue技术肌肉的修复。应当指出; EDL的和HL需要以具体评价的TA肌肉（它们占以下腓神经刺激（电晕测得的总胫骨前转矩的约15％-20％被解剖出的前脚部隔间。等 ，2013年） ）。因为这种方法提供了肌肉生理/功能的全面的纵向分析，它可以在许多其它类型的生理调查以及各种疾病或治疗领域39棚重要机械洞察力。例如， 在体内肌肉功能测试是适用于运动生理学，缺血/再灌注研究，肌病，神经损伤/神经病和血管，肌肉减少症，和肌营养不良40研究。

<table><tbody><tr><td>Isothesia</td><td>Henry Schein Animal Health</td><td>05260-04-04</td><td></td></tr><tr><td>Isoflurane Vaporizer-Funnel Fill</td><td>Vet Equip</td><td>911103</td><td></td></tr><tr><td>Inlet Adaptor for Vaporizer</td><td>Vet Equip</td><td>911124</td><td></td></tr><tr><td>Outlet Adaptor for Vaporizer</td><td>Vet Equip</td><td>911125</td><td></td></tr><tr><td>Tabletop Anaesthesia Machine</td><td>Vet Equip</td><td>901801</td><td></td></tr><tr><td>Compressed oxygen gas</td><td>Praxair</td><td>N/A</td><td></td></tr><tr><td>VaporGuard Activated Charcoal Filter</td><td>Vet Equip</td><td>931401</td><td></td></tr><tr><td>T/Pump Professional water heater</td><td>Stryker</td><td>N/A</td><td>set on Continuous Therapy Time at 38/100 for temperature</td></tr><tr><td>Transpore Surgical Tape</td><td>3M</td><td>1527S-1</td><td>rip in half to make thinner strips</td></tr><tr><td>A5 Golden animal clippers</td><td>Oster</td><td>078005-050-002</td><td></td></tr><tr><td>Povidone-Iodine Solution</td><td>Aplicare</td><td>82-227K</td><td></td></tr><tr><td>Alcohol Swabs</td><td></td><td></td><td></td></tr><tr><td>200 proof Ethanol</td><td>Decon labs</td><td></td><td>diluted to 70% with deionized water</td></tr><tr><td>cotton tipped applicators</td><td>Puritan</td><td>836-WC</td><td></td></tr><tr><td>Teflon coated electrodes-Monopolar needle electrode</td><td>Chalgren Enterprises</td><td>111-725-24TP</td><td></td></tr><tr><td>servomotor</td><td>Cambridge Technology</td><td>Model 6650LR</td><td></td></tr><tr><td>Dual Mode Lever System</td><td>Aurora Scientific Inc</td><td>Model 305C-LR-FP</td><td>contact manufacturer to order</td></tr><tr><td>Signal Interface</td><td>Aurora Scientific Inc</td><td>Model 604A</td><td></td></tr><tr><td>High-Power, Bi-Phase Stimulator</td><td>Aurora Scientific Inc</td><td>Model 701C</td><td></td></tr><tr><td>Data analysis software</td><td>Aurora Scientific Inc</td><td>DMAv5.110 software</td><td></td></tr><tr><td>Muscle lever system control software</td><td>Aurora Scientific Inc</td><td>DMCv5.400 software</td><td></td></tr></tbody></table>

applications of in vivo functional testing of the rat tibialis anterior for evaluating tissue engineered skeletal muscle repair

尽管骨骼肌，永久的功能和/或化妆品缺陷（ 例如 ，容积肌肉损失（VML）从外伤，疾病和各种先天性的，遗传性和获得的条件得到的是相当普遍的。组织工程和再生医学的技术有巨大的再生能力势，以提供一个治疗方案，但是，在与相关的功能性的措施纵向评估组合生物学相关动物模型的利用是改善的再生治疗学的发展用于治疗VML状损伤是至关重要的。在这方面，一个商业肌肉杠杆系统可用于测量长度，张力，力和骨骼肌速度参数。我们使用这个系统，结合一个高功率，双相刺激器，来测量响应于前小腿隔室的体内活化力产大鼠后肢。我们PREVIously使用该设备来评估VML损伤的胫骨前肌（TA）肌肉功能的影响，以及功能恢复的下列治疗受伤的TA肌肉与我们的组织工程肌肉修复（TEMR）技术的程度。对于这样的研究中，麻醉大鼠的左脚被牢固地锚定到连接到一个伺服马达脚板，和腓总神经是由两个经皮针形电极刺激引发肌肉收缩和脚的背屈。腓神经刺激诱发的肌肉收缩在一定范围的刺激频率（1-200赫兹）的测定，以确保有效的生产的最终高原，其允许准确测定的峰强直力。除了VML损伤的程度以及功能恢复的治疗之后的程度进行评估，这种方法可以方便地应用于研究肌肉生理和病理生理的不同方面。这种做法笑ULD帮助提高治疗的更合理的发展对肌肉的修复和再生。

强直曲线可以用于区分亚最佳结果最佳结果。该曲线通常开始形成，在60赫兹的频率。要取得良好效果的关键因素是刺激肌肉，使其产生其最大力量和破伤风期间保持这种力量的能力。理想曲线应该有一个不间断，锋利，垂直于刺激的时间上去，接着用最少的振荡平面平台期，和不间断的，尖锐的垂直下降在刺激终止期间（ 图4）。从理想曲线偏差是肌肉疲劳时指示（ 图5D）或肌肉没有被正确刺激产生最大力量（ 图5B - C）。后者一般不正确的电极放置结果导致肌肉纤维最大的招聘失败stimula期间化。一个显着特点，可以让研究人员确定一个非理想曲线是不正确的电极放置或病理变化的结果对肌肉强直曲线是否完整（融合）或不完整的（非融合）。一个未融合的，不完全强直曲线表示该电极被放错位置，导致肌肉没有遇​​到一个最大收缩。可以观察到在肌肉的病理学变化的例子作为最大收缩减少与对照相比，或者更快速地迷彩一个收缩反应为。 三种不同类型的在这个过程期间获得的峰代表不同的电极和腿的位置，并能够在图3中可以看到，第一峰将围绕0.4N并且当正确的电极放置在表面上测定的皮肤（ 图发生图3A）。第二组峰具有ħigher振幅，通常约0.5-0.6N（ 图3B），并且当在电极刺穿穿过真皮发生。这些获得后，腿和脚被调节以最大化动力生产，这是实现当峰值幅度增大到约1N或更大（ 图3C）。在这一点上，即时兴奋剂可以关闭和顺序可以开始。这些准则确保准确和可重复的结果，并在整个协议的关键检查点。 最后的结果可在根据用户从力试验和实验设计中提取的信息不同的方式来表示。在这个协议中，最大的力在所有刺激的频率的测量，然而其它的数据点可以是用于特定研究者或应用很重要的。一个例子是刺激在该强直曲线初具规模的频率。 ŧ他的数据可以比从以前或以后实验中得到的相同的动物，或用于不同处理组之间的比较的其他结果。力生产可以通过身体质量进行标准化来计算等角力和提供的年龄对所观察到的最大收缩的影响的更公正的评估。虽然不同的体重和年龄的动物将产生各种最大的力，当被正确地执行的程序的强直曲线的形状应是所有组之间是一致的。 <img alt="图1" src="/files/ftp_upload/54487/54487fig1.jpg" /> 图1:杠杆系统控制和数据分析软件进行分析概述打开程序时，控制软件（ 一 ）概述。 （B）参数"即时兴奋剂"。 （C）实例序列力频率的刺激。 （D </st荣&gt;）从分析软件的高通量力频率分析有代表性的数据。应该指出的是，例如序列和数据分析的过程是特定于该协议，并不代表全系列通过本软件提供的序列和输出。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54487/54487fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54487/54487fig2.jpg" /> 图 2: 在大鼠的定位和安置在设备脚 （A） 关键方面的老鼠是在牢固地固定在踏板的左脚仰卧位。由脚，腿和大腿做出了正确的角度盘旋。 （B）中由脚踝创建的直角被突出显示。 （C）的腿部应<html>在直平面对齐从脚到身体。 （D）的电极位置是平行和垂直于腓总神经的平面。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54487/54487fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54487/54487fig3.jpg" /> 图3: 代表峰演示正确的电极放置的重要性，以最大力量生产 （A）基线峰值强直性放置太肤浅电极观察反应。 （B）与插在正确的位置电极较大的峰值。从更大的波峰信号正确的电极放置到最佳的序列前峰值幅度为腿和脚的位置（C）过渡的最佳调整。/ftp_upload/54487/54487fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54487/54487fig4.jpg" /> 图 4: 在100赫兹最优强直曲线 ，该曲线上升和急剧下降并具有平坦平台期。这个例子说明正确电极放置和最大力量的刺激。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54487/54487fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54487/54487fig5.jpg" /> 图 5: 在100赫兹获得次优强直曲线 （A）按照松弛代表性实施例中 ，该曲线骤降基线以下。这表明刺激的拮抗剂。 （B - D）这些图都是不正确的电极放置和不平等的招聘肌纤维的结果。高原阶段表现出较大的波动（B），上升斜率（C），或向下倾斜（D）。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54487/54487fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair at JoVE.com

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

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

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

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

applications-vivo-functional-testing-rat-tibialis-anterior-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

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

13.7K 次观看.  University of Virginia. We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration. 

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

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

科研

医学

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

无胸​​腺大鼠模型工程前交叉韧带移植评估

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

生物工程

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

大鼠双侧髌腱损伤模型中干细胞治疗的评价

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

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

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

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

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

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 ...

生物学

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

小鼠肌功能的无创评价

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

循环机械负荷诱导大鼠跟腱肌腱病

A Passive Ankle Dorsiflexion Testing System for an In Vivo Model of Overuse-induced Tendinopathy

Tendinopathy is a chronic tendon condition that results in pain and loss of function and is caused by repeated overload of the tendon and limited recovery time. This protocol describes a testing system that cyclically applies mechanical loads via passive dorsiflexion to the rat Achilles tendon. The custom-written code consists of pre- and post-cyclic loading measurements to assess the effects of the loading protocol along with the feedback control-based cyclic fatigue loading regimen. 
We used 25 Sprague-Dawley rats for this study, with 5 rats per group receiving either 500, 1,000, 2,000, 3,600, or 7,200 cycles of fatigue loads. The percentage differences between the pre- and post-cyclic loading measurements of the hysteresis, peak stress, and loading and unloading moduli were calculated. The results demonstrate that the system can induce varying degrees of damage to the Achilles tendon based on the number of loads applied. This system offers an innovative approach to apply quantified and physiological varying degrees of cyclic loads to the Achilles tendon for an in vivo model of fatigue-induced overuse tendon injury.

作者聚焦：在肌腱病研究中整合机械和生物分析 

This protocol presents a testing system used to induce quantifiable and controlled fatigue injuries in a rat Achilles tendon for an in-vivo model of overuse-induced tendinopathy. The procedure consists of securing the rat's ankle to a joint actuator that performs passive ankle dorsiflexion with a custom-written MATLAB script.

用于过度使用诱导的肌腱病 体内 模型的被动踝关节背屈测试系统

Tendinopathy is a chronic tendon condition that results in pain and loss of function and is caused by repeated overload of the tendon and limited recovery ...

制造小鼠肌腱组合体以研究疾病中的细胞相互作用

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

作者聚焦：通过开发小鼠组合来了解细胞机制来推进肌腱研究

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

中的应用

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一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

无胸腺大鼠模型工程前交叉韧带移植评估

大鼠双侧髌腱损伤模型中干细胞治疗的评价

伸趾长肌的肌肉功能评价

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用于过度使用诱导的肌腱病体内模型的被动踝关节背屈测试系统

用于过度使用诱导的肌腱病体内模型的被动踝关节背屈测试系统

用于过度使用诱导的肌腱病体内模型的被动踝关节背屈测试系统

工程肌腱组件以探测疾病和修复中的细胞串扰

工程肌腱组件以探测疾病和修复中的细胞串扰

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一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

无胸​​腺大鼠模型工程前交叉韧带移植评估

大鼠双侧髌腱损伤模型中干细胞治疗的评价

伸趾长肌的肌肉功能评价

小鼠肌功能的无创评价

用于过度使用诱导的肌腱病 体内 模型的被动踝关节背屈测试系统

用于过度使用诱导的肌腱病 体内 模型的被动踝关节背屈测试系统

用于过度使用诱导的肌腱病 体内 模型的被动踝关节背屈测试系统

工程肌腱组件以探测疾病和修复中的细胞串扰

工程肌腱组件以探测疾病和修复中的细胞串扰

无胸腺大鼠模型工程前交叉韧带移植评估

用于过度使用诱导的肌腱病体内模型的被动踝关节背屈测试系统

用于过度使用诱导的肌腱病体内模型的被动踝关节背屈测试系统

用于过度使用诱导的肌腱病体内模型的被动踝关节背屈测试系统