这项工作是由国家卫生研究院资助K08NS065157（TAF），此外支持，宾夕法尼亚肌肉劳损，关节炎研究所奖号码P30AR050950，肌肉骨骼及皮肤疾病（TAF和史蒂文与舍雷尔）支持这项工作。最后，施赖纳斯儿科研究中心种子资金（TAF），支持这项工作。我们要感谢年轻的博士，金的儿子，初步证明她的援助，在整个安装过程和Amy A.金生产的草图图1。

1。动物主题 1.1。治疗<ol><li>所有动物的程序应与本地机构的动物护理和伦理委员会的批准，并按照使用和卫生准则委员会和国家机构，采取措施以减少疼痛和不适。 </li><li>我们的小鼠饲养温度控制在12小时的反向光明与黑暗周期条件下，而美联储鼠标议员和水随意。 </li><li>对于成人再生的研究，老鼠应该是年龄至少6周时，坐骨神经进行。这个年龄段是超出修剪polyneural神经肌肉接头时发生的时间。 </li><li>在这些实验中，我们用6-8周龄的C57BL / 6小鼠，从查尔斯河收购。当比较后坐骨神经再生（即：轴突生长率）应该是相同的小鼠品系，为轴突再生的差异已经不不同的近交系15,16之间。如果要使用小鼠基因操纵，窝控制是最合适的。 </li></ol> 1.2。术前准备<ol><li>动物深感麻醉手术通过腹腔注射氯胺酮（100毫克/千克）和甲苯噻嗪（10毫克/公斤）的鸡尾酒。每个动物也收到了美洛昔康皮下注射（10毫克/公斤），以减少手术后的疼痛。 </li><li>都躯仔细剃光使用外科快船（Roboz，RC-5903）和脱毛奈尔脱毛膏（在当地的药店）完成的。 </li><li>使用无菌棉头涂药器和手术优碘擦洗尔科技（19066452），皮肤洁净。 </li><li>眼药膏（费希尔科学，19082795）被应用到眼睛，用无菌棉头涂药器。 </li><li>鼠标被放置在一个干净的不锈钢板，根据已置于预先加热homeothermic毯系统（哈佛仪器，507222F）。动物的温度保持在37°C。 </li><li>所有的四肢被录制下来，采取对称，使膝关节使身体（图1，A组）与直角定位后肢的照顾。 </li><li>外科领域覆盖无菌的悬垂性。所有仪器消毒的高压灭菌器或热珠灭菌（精细的科学工具，18000-45）和医生戴着口罩，隔离衣，无菌手套。 </li></ol> 2。重现坐骨神经神经压榨<ol><li>准备后，横跨中线（图1）的半圆形切口皮肤。皮肤轻轻从底层肌肉解剖，折叠的出路在手术过程中保持。这是保持湿润，在手术过程中使用0.1毫升无菌生理盐水（Hospira公司，0409-4888-20）的应用。 </li><li>开放筋膜平面之间的臀大肌和二头肌femori的前头部小号揭示了坐骨神经（图1，A组）。为控制外科手术，对侧坐骨神经应揭露和动员，但保持不变。臀部肌肉，然后再反对和缝合使用6-0编织丝，非吸收性缝合线（Roboz位SUT-1073-11）。 </li><li>实验坐骨神经，然后以同样的方式，在地方可视化（图1，B组），以纾缓拉钩暴露。拉钩是消毒后才能使用。 注：虽然市售牵引系统，他们往往相当昂贵。我们能够做出一个满意的牵引系统，使用廉价的硬件供应和昆虫针（见材料部分）。 </li><li>坐骨神经，然后轻轻地从周围的结缔组织组织使用虹膜切除术剪刀释放。 </li><li>使用罚款5/45（精细的科学工具，11251-35）镊子，神经被放置在底部的超精细的止血钳颚（太精细科学LS 13020-12）。三个分册都是按顺序排列，而不是彼此的顶部（图1，插图乙）。止血钳已刻有商标，从他们的尖端在1.5毫米。坐骨神经的最外层部分被放置在此之前暗恋的标志线。这确保了统一宽度的挤压，神经不超过延长夷为平地，由于破碎力时的止血钳的钳口。如果超越钳尖端的神经延伸的神经将只能部分粉碎。 </li><li>粉碎的神经垂直45毫米，第三脚趾，作为衡量一个线程，接近坐骨神经的路径。神经被压死15秒一次止血钳在3人次。护理是采取不舒展神经。当止血重开，整个神经应该是半透明的在暗恋网站。 </li><li>已经预先浸的止血钳的第二对（第一）粉状碳（Fisher Scientific则，C272-500）是用来庆祝粉碎网站。神经被压死，15秒3次点击在同一暗恋网站。无碳标识应超出最初的暗恋的边界。如果需要精确的美眉网站的标志，这一点尤为重要。在使用之前，碳粉消毒紫外线照射两小时，并在其后使用无菌技术处理。 <ol><li>预先浸碳钳，但排除在手术部位广泛碳，钳开奶粉中碳，然后轻轻关闭（但不点击）关闭，并用无菌纱布擦去碳以外的止血。镊子进行检查核实，破碎表面均匀地涂上碳粉下至少3倍的放大倍率。如果有必要，他们正在重新浸泡和消灭。 </li></ol></li><li>臀部肌肉重新反对和缝合在对侧同样的方式。 </li><li>最后，皮肤切口封闭使用9毫米反射剪辑（世界精密仪器，500346; APPLIER：500345）。如果发现9毫米的反射剪辑限制运动，规模较小的反射剪辑或6-0缝线（Roboz，被测-1073-11），可以用来代替。 </li></ol> 3。手术后护理<ol><li>程序之后，动物被放置在37°C，直到他们表现出的迹象，在加热垫。 </li><li>然后，他们搬回到他们的家庭笼，易于凝胶和湿食品的形式在地板上的水和食物。 </li></ol> 4。半薄膜的制备<ol><li>过量的巴比妥（300毫克/千克）的腿部肌肉被删除暴露坐骨神经*。神经留在原地，后身是沉浸在2％多聚甲醛，2％和0.1米冰磷酸盐缓冲30分钟gluteraldehyde。 *如果完成整个安装肌肉准备，肌肉有收获前暴露坐骨神经。 </li><li>小心取下的神经，只处理近端的照顾。然后神经是固定后，在另外三个小时固定液。 </li><li>神经后固定在0.1 M磷酸缓冲冲洗三次。 </li><li>正沉浸在2％四氧化锇在0.1 M磷酸缓冲一小时的神经。 </li><li>神经，然后脱水顺序沉浸在越来越浓乙醇（50％，70％，80％，95％，100％，100％，100％）。每次浸泡15分钟。 </li><li>脱水神经培养环氧丙烷两次，每次三分钟。 </li><li>然后神经沉浸在1:1的环氧丙烷的混合物和嵌入至少6小时，812（通常是隔夜）。 </li><li>然后神经沉浸在2:1的环氧丙烷的混合物和嵌入812过夜。 </li><li>最后，神经沉浸在812纯嵌入了六个小时，然后在适当的模具和BA嵌入式在60°C KED为48小时。 </li><li>从远端神经残端从粉碎用玻璃刀，并用甲苯胺蓝染色Ultracut开普敦超薄切片（徕卡）的网站设置距离1.0微米的部分被切断。薄片也可以制作电子显微镜超微结构检查。 </li></ol> 5。整个山肌制备<ol><li>第一小鼠牺牲与过量的巴比妥（300毫克/千克）和后肢的膝盖删除。 </li><li>四个后肢肌肉被删除分析：胫骨前肌（TA），趾长伸肌（EDL）的比目鱼肌，腓骨长。 </li><li>删除所有的肌肉都通过仔细解剖，并通过他们的结缔组织固定黑色sylgard（费希尔科学，NC9492579）涂层菜。他们在PBS冲洗，然后固定在4％多聚甲醛为30分钟。 </li><li>电讯局长在PBS冲洗，嵌入在华侨城（Fisher Scientific则14-373-65），丙酮和干冰浴中迅速冻结。 <ol><li>它储存于-80°C薄切片的情况下，整个安装准备失败。在一般情况下，电讯局长是太充足的整个坐骑厚。 </li></ol></li><li>的EDL，比目鱼肌，腓骨长在PBS 3×10分钟放置在0.1 M甘氨酸*（尔科技，AC12007-0010;在PBS稀释），30分钟冲洗，并再次在PBS清洗3×10分钟。然后，他们在冰冷的100％的甲醇整整 5分钟，在-20°C间淬火，在PBS冲洗3×10分钟，沐浴在荧光标记的α-银环蛇（PBS稀释1:200），30分钟。肌肉冲洗3×10分钟，PBS在2％BSA的1小时块（嘉里建设，50-61-00）和0.2％的Triton X-100（点科学公司，9002-93-1），在PBS，在4°C过夜，而在相同的2％BSA/0.2％的Triton块稀释的初级抗体鸡尾酒摇摆。 * GLycine和阻塞的解决方案是肌肉收获的当天和前在室温下搅拌30-60分钟使用。 <ol><li>为了纪念轴突和神经肌肉突触，我们使用相结合的鼠标单克隆神经丝标记（Covance公司，SMI-312R;稀释1:1000），鼠标单克隆突触囊泡标记（SV2 DSHB，稀释1:1000），和若丹明共轭阿尔法 - 银环蛇（Sigma-Aldrich公司，1:200稀释）。为了纪念反应的雪旺氏细胞，我们使用兔反GAP-43（Novus公司生物制品，NB300-143，稀释1/500）。 IgG1亚型特定的荧光标记的羊抗鼠二抗和DyLight 649标记的驴抗兔（的杰克逊ImmunoResearch，稀释1:200），可视化的主要抗体。 </li></ol></li><li>翌日，肌肉在PBS冲洗3×10分钟，孵育二抗稀释1:200（2％BSA/0.2％Triton的阻塞解决方案）。然后，他们在PBS冲洗，FO 2×10分钟llowed由5分钟的浸泡中的DAPI（Invitrogen公司，D3571;稀释在去离子水至300纳米），另外10分钟的PBS冲洗。 </li><li>每一块肌肉，然后解剖上，黑色sylgard（尔科技，NC9492579）涂层培养皿。最初的肌腱被删除插入点到肌肉，然后肌肉变薄剥去室内肌纤维（图3，小组交流）。已采取审慎措施，以保持外表面的肌肉，包括终板带（图3，面板的DF）。所产生的肌肉安装在加幻灯片与vectashield（媒介实验室的H-1000）和22（Fisher Scientific则12-550-15）x 40毫米透明指甲油密封盖玻片（Fisher Scientific则12-548-5C）双方。当幻灯片上终板带触点的盖玻片上。 </li></ol> 6。测量再生<ol><li>可再生估计在所有三个肌肉（腓骨，EDL的，比目鱼）。我们经常前胺再生神经损伤后14天，在这一个相当大的比例NMJs重新支配的时间。较早和稍后的时间点也合适，取决于被要求的科学问题。在每一块肌肉，重新支配整个终板带进行检测。只有EN脸表面NMJs得分。在这种方式中，至少有200个肌肉NMJs可快速检测。 </li><li>得分肌肉再次神经支配，我们确定了“再支配的比例”。分母是α-银环蛇毒素的结合和雪旺氏细胞GAP-43免疫标记的失神经支配神经肌肉接头的数量。分子标记neurofilament/SV2免疫重新支配NMJs的数量。如果需要，可能会被列为部分（SV2）α-银环蛇毒素的完整覆盖或完全（SV2α-银环蛇的全覆盖）重新支配NMJs。为重新支配与失神经NMJs的例子见图3，G和H板。 </li></ol> 7。代表结果<img alt="图1" src="/files/ftp_upload/3606/3606fig1.jpg" /> 图1。 重要的神经损伤后肢的解剖示意图。A.一个半圆形的皮肤切口，已经露出底层肌肉，臀部肌肉已被分离，坐骨神经透露（步骤2.4以上）。箭头表示近似暗恋网站。拉钩安置作为一般指引，并在每个手术，以减轻对坐骨神经的可视化方法的止血钳挤压调整乙（插图）：坐骨神经的位置上下颚之前暗恋的止血钳（步骤2.5以上）。单行本标记，以证明他们是相邻的水平，但不垂直，在粉碎。虽然3 FASCicles在此图中的标记，也可能会看到第四个分册，腓关节分支。为坐骨神经的分支模式的更详细的解剖图分册远端粉碎点，请参阅Greene的大鼠的解剖图188 17。 <img alt="图2" src="/files/ftp_upload/3606/3606fig2.jpg" /> 图2。一个在就地和甲苯胺蓝染色，半薄大鼠坐骨神经部分碳显着暗恋网站。在原位粉碎网站（左后肢）的一个例子。黑碳表示暗恋网站。星号标记的胫神经支配大腿肌肉和服务作为一个有用的标志性建筑在暗恋手术的一个分支。坐骨神经的胫骨分工中概述了腓蓝色，绿色，和黄色。B.半薄切片腓展示一个完整的挤压止血力量进行PS。C.半薄片，展示一个不完整的挤压与直角钳进行。在这两种图像退化髓鞘型材标记的箭头。在图 C箭头标记保存髓鞘型材的例子幸免轴突集群。比例尺为10μm。 <img alt="图3" src="/files/ftp_upload/3606/3606fig3.jpg" /> 图3全安装肌肉示意图交流：和代表NMJs的。的EDL（ 一 ），腓骨长肌（B）和比目鱼（c）在去除后肢肌肉渲染图像。 右后肢肌肉所示。膝盖和踝关节的解剖方向还包括以供参考。肌腱的各色白光和肌肉上的位置时，概述了坚实的黑线。他们概述了一个黑色的虚线下方的肌肉时，他们延长。裁员是在红色虚线所示。切割网站，如蓝色箭头所示的肌肉剥离，随后变薄。间伐后，肌肉分离从引脚切割固定肌腱周围什么DF：从整个安装肌肉渲染图像后，去除结缔组织和随后的变薄。每一块肌肉终板带概述GH：比目鱼整装肌肉坐骨神经展示（高）重新支配（G）和失神经肌肉路口粉碎后14天。在这些面板，NMJs，轴突，雪旺氏细胞可视化如上所述，在第5.5.1节。乙酰胆碱受体是红色的，绿色轴突，雪旺氏细胞处理蓝色。在面板Ğ，箭头指示的NMJ已经由一个轴突重新支配领域。在面板 H箭头表明，GAP-43阳性的雪旺氏细胞的过程。请注意没有轴突。比例尺为10μm。

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R., Anderson, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+axonal+regeneration+in+the+central+and+peripheral+nervous+systems+of+the+NG2-deficient+mouse.">Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse.</a> BMC Neurosci. 8, 80-80 (2007).</li><li>Thornton, M. R., Mantovani, C., Birchall, M. A., Terenghi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+N-CAM+and+N-cadherin+expression+in+axotomized+and+crushed+rat+sciatic+nerve.">Quantification of N-CAM and N-cadherin expression in axotomized and crushed rat sciatic nerve.</a> J. Anat. 206, 69-78 (2005).</li><li>Beer, G. M., Steurer, J., Meyer, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardizing+nerve+crushes+with+a+non-serrated+clamp.">Standardizing nerve crushes with a non-serrated clamp.</a> J. Reconstr. Microsurg. 17, 531-534 (2001).</li></ol>

我们什么都没有透露。

我们已经提出了一个方法来获取可靠完整的坐骨神经与精确标记的美眉现场。如前所述，坐骨神经是一种常见的周围神经损伤的小鼠和大鼠模型。虽然每个美眉的方法都有其优点和缺点，我们发现，这种方法产生一个完整的挤压，很容易被一个特殊的设备（如特殊夹具等）的最低标记。 粉碎方法仪器用于小鼠坐骨神经的最常见的是5号直角钳。虽然它有可能获得一个完整的压榨，用这种方法，我们发现，这种仪器产生，尤其是在一个新的用户手中变量美眉（太少的力量离开幸免轴突或神经太大撕裂），。 其他不常见的方法，用于生产挤压包括小动脉瘤夹，在液氮冷却的镊子，并speciallŸ制造，校准，无锯齿夹9,12,5,7。这些文书的每个人都可以可靠地产生一个完整的迷恋。我们选择了止血钳，因为仪器是很容易获得，产生一个可靠的挤压，和允许的美眉网站的明确标志。止血钳的一个缺点是，我们不能轻易衡量粉碎过程中产生的力。然而，一次测试，止血钳将可靠地产生一个完整的美眉，可以很容易地替换相同的镊子如果镊子被损坏。 无论选择的方法进行粉碎，粉碎过程中最关键的部分，也许是验证神经损伤的完整性。在一个新的用户手中，这一点尤为重要。半薄神经远端的损伤部位（图2）分析提供了一个简单的方法，这样做，可以发现一个完整的美眉（图2，B组）或不完整的美眉（图2，C组）。 18。然而，不再迷恋倍或压碎，显着降低的雪旺氏细胞内膜会改变的速度和/或再生的成功。 标记方法两个可行的美眉网站标志着碳粉以外的方法是通过10-0 13,19坐骨神经外膜缝合，分子探针，尤金，或1 FluoSpheres </SUP&gt;。 10-0缝合严重标志着美眉网站，但需要更高层次的显微外科技能，比碳标记的变异缝合结果不太精确边界的美眉网站标识的应用。研究再生率时，必须准确标明边界的美眉网站下面的损伤。我们没有调查FluoSpheres;然而，他们遇到的爱恋现场标记的精确度和持久性的标准。他们是特别有用的时候成像在体内再生YFP的鼠标线1。对于我们而言，我们发现碳充足和廉价的标记，容易在随后的半薄或电子显微镜评价额外的好处。此外，我们已经发现，碳标记持续至少六个星期，使其适合较长时间的实验。 粉碎后的再生分析一个完整的investigaTION结合后粉碎再生功能，电生理和形态学评估5,16。再生后的形态学评估也许是最常见的。这些评估可以分为那些评估延迟生长，轴突生长率和特异性神经支配16。在这份报告中，我们提出了一个整体的安装程序，可用于快速评估形态的再生（图3）肌肉。在这种分析中，α-银环蛇和雪旺氏细胞GAP-43标记的失神经支配NMJs重新支配神经丝，SV2，α-银环蛇毒素标记的路口进行量化和比较。 GAP-43显着上调后神经的雪旺氏细胞和很好的识别失神经末端雪旺氏细胞在神经肌肉交界处20。虽然GAP-43是由轴突表示，神经丝同时使用容易区分雪旺氏细胞GAP-43阳性的轴突。此外，因为肌肉深处NMJs可能不容易标记抗体（α-银环蛇毒素的小分子相比）雪旺氏细胞，GAP-43免疫重要的是证实了的神经肌肉交界处的失神经支配的地位和提高精度时计失神经NMJs。 这种方法提供了一种简单而有效的方法，以评估一旦整个肌肉终板带的神经支配的地位。他人已经使用THY-1-CFP（23）/ 9000-GFP的小鼠表达青色荧光蛋白在轴突和雪旺氏细胞的GFP跟进后坐骨神经2胫前重新支配。一个警告，必须指出，虽然。这种方法依赖于神经丝和SV2免疫量化重新支配。这是可能的，轴突生长，可畅通，但随后表达SV2可能会推迟21。因此，在一些实验，这将是重要的确认延迟再生，不是Delayed突触SV2（即突触前分化）帐户的表达观察到的表型。

<table border="1"><tbody><tr><td> 名称（描述/数量） </td><td> 供应商 </td><td> 目录编号 </td></tr><tr><td>迷你快船号0000刀片</td><td> roboz </td><td> RC-5903 </td></tr><tr><td>奈尔头发卸妆（9盎司） </td><td>教会和德怀特有限公司，公司</td><td> N / A </td></tr><tr><td>优碘外科磨砂（1加仑） </td><td> Fisher Scientific则</td><td> 19066452 </td></tr><tr><td>眼药膏（1盎司） </td><td> Fisher Scientific则</td><td> 19082795 </td></tr></tbody></table> 手术工具： <table border="1"><tbody><tr><td> 名称（描述/数量） </td><td> 供应商 </td><td> 目录编号 </td></tr><tr><td>福斯特250热珠消毒</td><td>罚款科学工具</td><td> 18000-45 </td> &lt;/ TR&gt; <tr><td>虹膜剪刀（11厘米长） </td><td>世界精密仪器</td><td> 500216 </td></tr><tr><td>帕兹 - 史密斯钳（直;18厘米的; 1X2牙） </td><td>罚款科学工具</td><td> 11024-18 </td></tr><tr><td>麦克弗森Vannas剪刀（5毫米刀片） </td><td>世界精密仪器</td><td> 14124 - G </td></tr><tr><td>杜蒙＃5镊子 - Dumoxel标准提示</td><td>罚款科学工具</td><td> 11252-30 </td></tr><tr><td>杜蒙＃5/45钳 - Dumoxel小费标准</td><td>罚款科学工具</td><td> 11251-35 </td></tr><tr><td>精细止血超（直光滑的内表面） </td><td>罚款科学工具</td><td> 13020-12 </td></tr><tr><td>粉末活性炭（500克） </td><td> Fisher Scientific则</td><td> C272-500 </td></tr><tr><td>大小6-0缝线的C-1针（不育，丝绸，黑色，编织，非吸收性; 18“的长度;盒36） </td><td> roboz </td><td>位SUT-1073-11 </td></tr><tr><td>反射的剪辑APPLIER（9毫米剪辑） </td><td>世界精密仪器</td><td> 500345 </td></tr><tr><td> 9mm不锈钢的反射剪辑（100/box） </td><td>世界精密仪器</td><td> 500346 </td></tr></tbody></table> 动物护理： <table border="1"><tbody><tr><td> 名称（描述/数量） </td><td> 供应商 </td><td> 目录编号 </td></tr><tr><td> 0.9％氯化钠注射液（不含防腐剂; 20毫升） </td><td> Hospira公司</td><td> 0409-4888-20 </td></tr><tr><td>完整的柔性探头Homeothermic毛毯系统（中等，115伏，60赫兹） </td><td>哈佛仪器</td><td> 507222F </td></tr></tbody></table> 手术Platform和拉钩： （在当地的五金商店购买） <table border="1"><tbody><tr><td> 名称（描述/） </td><td> 数量 </td></tr><tr><td>不锈钢平台（铣削〜12“X12”X1 / 8“） </td><td> 1 </td></tr><tr><td>按钮磁铁（例如：Eclipse的E825） </td><td> 3 </td></tr><tr><td>不锈钢螺栓（长3“按钮磁铁决心直径） </td><td> 3 </td></tr><tr><td>不锈钢螺母（大小以适合螺栓） </td><td> 9 </td></tr><tr><td>橡皮筋（轻张力） </td><td> 3 </td></tr><tr><td>昆虫销（两端弯曲，形成挂钩） </td><td> 3 </td></tr></tbody></table> 半变薄： <table border="1"><tbody><tr><td> 名称（描述/数量） </td><td> 供应商&lt;/ STRONG&gt; </td><td> 目录编号 </td></tr><tr><td>多聚甲醛（1公斤） </td><td> Sigma-Aldrich公司</td><td> P6148-1KG </td></tr><tr><td>无水磷酸氢钠（500克;用于制备磷酸盐缓冲液） </td><td> Fisher Scientific则</td><td> S375-500 </td></tr><tr><td>无水磷酸二氢钠（1公斤;用于制备磷酸盐缓冲液） </td><td> Fisher Scientific则</td><td> AC38987-0010 </td></tr><tr><td>戊二醛（50％; 10×10毫升） </td><td>特德·佩拉，公司</td><td> 18431 </td></tr><tr><td>四氧化锇（4％水溶液，10×10毫升） </td><td>特德·佩拉，公司</td><td> 18465 </td></tr><tr><td>环氧丙烷（450毫升） </td><td>特德·佩拉，公司</td><td> 18601 </td></tr><tr><td>嵌入812（包，硬质块/图像对比度高） </td><td>电子显微镜科学</td><td> 14120 </td></tr><tr><td> toluidiNE蓝（25克） </td><td>特德·佩拉，公司</td><td> 19451 </td></tr></tbody></table>整个山肌肉的制备及免疫组化： <table border="1"><tbody><tr><td> 名称（描述/数量） </td><td> 供应商 </td><td> 目录编号 </td></tr><tr><td>多聚甲醛（1公斤） </td><td> Sigma-Aldrich公司</td><td> P6148-1KG </td></tr><tr><td>无水磷酸氢钠（500克;用于制备磷酸盐缓冲液） </td><td> Fisher Scientific则</td><td> S375-500 </td></tr><tr><td>无水磷酸二氢钠（1公斤;用于制备磷酸盐缓冲液） </td><td> Fisher Scientific则</td><td> AC38987-0010 </td></tr><tr><td> 10％BSA稀释液/封闭液（200毫升） </td><td> kirkegaard佩里实验室，公司</td><td> 50-61-00 </td></tr><tr><td>的Triton X-100（100毫升） </td&gt; <td>点科技有限公司的</td><td> 9002-93-1 </td></tr><tr><td>甘氨酸，98％（1公斤） </td><td> Fisher Scientific则</td><td> AC12007-0010 </td></tr><tr><td>组织TEK的Cryo-OCT复合（12个案例; 4盎司瓶） </td><td> Fisher Scientific则</td><td> 14-373-65 </td></tr><tr><td> sylgard陶氏170（2磅包） </td><td> Fisher Scientific则</td><td> NC9492579 </td></tr><tr><td>不锈钢昆虫销，大小为1（100/pkg） </td><td>罚款科学工具</td><td> 26001-40 </td></tr><tr><td>四甲-A银环蛇毒素（0.5毫克） </td><td> Sigma-Aldrich公司</td><td> T0195-.5MG </td></tr><tr><td>单克隆抗体对SMI-312（0.1毫升） </td><td> Covance公司</td><td> SMI-312R </td></tr><tr><td>单克隆抗体反对SV2（0.1毫升） </td><td>发展研究杂交瘤细胞银行（DSHB） </td><td> SV2 </td></tr><tr><td>兔聚GAP-43单克隆抗体对</td><td> Novus公司生物制品</td><td> NB300-143 </td></tr><tr><td> flourescein标记的羊抗鼠IgG，Fcγ小类1比</td><td>杰克逊ImmunoResearch </td><td> <a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=115-095-205&amp;Attributes=Yes&amp;Quantity=1">11 5-095-205</a> </td></tr><tr><td> DyLight 649共轭的驴抗兔IgG </td><td>杰克逊ImmunoResearch </td><td> <a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=711-495-152&amp;Attributes=Yes&amp;Quantity=1">711-495-152</a> </td></tr><tr><td> 4&#39;,6-diamidino-2-苯基吲哚（DAPI，dilactate; 10毫克） </td><td> Invitrogen公司</td><td> D3571 </TD&gt; </tr><tr><td> vectashield安装介质（10毫升） </td><td>媒介实验室</td><td>的H-1000 </td></tr><tr><td> superfrost加上载玻片（白尺寸：75×25毫米; 144包） </td><td> Fisher Scientific则</td><td> 12-550-15 </td></tr><tr><td>高级盖眼镜fisherfinest（尺寸：40×22毫米; 1盎司包。） </td><td> Fisher Scientific则</td><td> 12-548-5C </td></tr></tbody></table>

reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis

再生周围神经系统（PNS）被广泛研究人类疾病的相关了解PNS的神经元，从而有可能阐明中枢神经系统再生1失败安装强大的再生反应。坐骨神经（axonotmesis）的是在啮齿类动物2周围神经损伤的最常见的车型之一。粉碎中断所有轴突，但雪旺氏细胞基部叶片，使再生的最佳3,4保留。这使得研究者，研究正是越来越轴突的互动能力与雪旺氏细胞和基底椎板4。大鼠一般都首选的动物模型实验神经压榨。它们被广泛使用，其损毁坐骨神经提供了一个合理的近似人类神经病变5,4。虽然在规模小于大鼠神经，鼠神经有许多相似的特质。但最重要的，谅解备忘录E型是由于转基因株系的广泛的可用性日益成为宝贵的，现在允许一个关键的单个分子神经再生6，7的详细解剖。此前研究者已经使用了多种方法来产生神经挤压或损伤，包括简单的直角钳，冷藏镊子，止血钳，血管钳，研究者设计的夹8,9,10,11,12。研究者还利用各种方法标记的损伤部位，包括缝合，碳粒子和荧光珠13,14,1。我们描述的方法来获取准确和持久性标志的使用罚款的止血钳和随后的碳爱恋标志着网站的爱恋现场1再现完整的坐骨神经。作为我们描述的坐骨神经过程的一部分，我们还包括肌肉整体的一个比较简单的方法安装，我们使用到随后量化再生。

Watch this Scientific Journal Video about Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis at JoVE.com

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

在这份报告中，我们描述了一种方法来粉碎小鼠坐骨神经。这种方法使用现成的止血钳和轻松，重现产生完整的坐骨神经。此外，我们描述了一种方法来准备适合分析坐骨神经后神经再生肌肉整个坐骑。

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

reproducible-mouse-sciatic-nerve-crush-subsequent-assessment

Nanyang Technological University

Research

JoVE Journal

Neuroscience

33.7K 次观看.  Temple University. 在这份报告中，我们描述了一种方法来粉碎小鼠坐骨神经。这种方法使用现成的止血钳和轻松，重现产生完整的坐骨神经。此外，我们描述了一种方法来准备适合分析坐骨神经后神经再生肌肉整个坐骑。

视频: Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

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. ...

科研

医学

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

神经科学

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative to understand the cellular and molecular changes involved in maintaining and repairing NMJs. For this purpose, we have developed a method to reliably and consistently examine regenerating NMJs in mice. This nerve injury method involves crushing the common fibular nerve as it passes over the lateral head of the gastrocnemius muscle tendon near the knee. Using 70 day old female mice, we demonstrate that motor axons begin to reinnervate previous postsynaptic targets within 7 days post-crush. They completely reoccupy their previous synaptic areas by 12 days. To determine the reliability of this injury method, we compared reinnervation rates between individual 70 day old female mice. We found that the number of reinnervated postsynaptic sites was similar between mice at 7, 9, and 12 days post-crush. To determine if this injury assay can also be used to compare molecular changes in muscles, we examined levels of the gamma-subunit of the muscle nicotinic receptor (gamma-AChR) and the muscle-specific kinase (MuSK). The gamma-AChR subunit and MuSK to are highly upregulated following denervation and return to normal levels following reinnervation of NMJs. We found a close relationship between transcript levels for these genes and innervation status of muscles. We believe that this method will accelerate our understanding of the cellular and molecular changes involved in repairing the NMJ and other synapses.

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

腓神经损伤方法:可靠检测，以确定和测试因素修复神经肌肉接头

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different methods of simulating postoperative physiotherapy are presented. Multiple standardized strategies are used to assess motor and sensory recovery using an MN model of peripheral nerve lesion and repair, thus permitting easy comparison of the results. Several options are included for providing a postoperative physiotherapy-like environment to rats that have undergone MN injuries. Finally, the paper provides a method to evaluate the recovery of the MN using several noninvasive tests (i.e., grasping test, pin prick test, ladder rung walking test, rope climbing test, and walking track analysis), and physiological measurements (infrared thermography, electroneuromyography, flexion strength evaluation, and flexor carpi radialis muscle weight determination). Hence, this model seems particularly appropriate to replicate a clinical scenario, facilitating extrapolation of results to the human species.
Although the sciatic nerve is the most studied nerve in peripheral nerve research, analysis of the rat MN presents various advantages. For example, there is a reduced incidence of joint contractures and automutilation of the affected limb in MN lesion studies. Furthermore, the MN is not covered by muscle masses, making its dissection easier than that of the sciatic nerve. In addition, MN recovery is observed sooner, because the MN is shorter than the sciatic nerve. Also, the MN has a parallel path to the ulnar nerve in the arm. Hence, the ulnar nerve can be easily used as the nerve graft for repairing MN injuries. Finally, the MN in rats is located in the forelimb, akin to the human upper limb; in humans, the upper limb is the site of most peripheral nerve lesions.

Presented is a protocol to produce different types of median nerve (MN) lesions and repair in the rat. Additionally, the protocol shows how to evaluate the functional recovery of the nerve using several noninvasive behavioral tests and physiological measurements.

大鼠中位神经再生评价的功能与生理方法

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

自动步态分析，评估带外周神经或脊髓挫伤的啮齿动物的功能恢复

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

最大等轴泰塔尼奇力测量大鼠的蒂比亚利斯前肌肉

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

单边坐骨神经粉碎损伤后大鼠运动行为的多因素评估

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Traumatic Peripheral Nerve Injury in Mice

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve and denervating muscle have long been a goal in musculoskeletal research. Many traumatically injured limbs have nerve trauma that defines the long-term patient outcome. Over several years, precise methods of producing microsurgical nerve injuries have been developed, including crush, lacerations, and nerve-gap grafting, allowing for reproducible outcome assessments. Moreover, newer methods are created for calibrated crush injuries that offer clinically relevant correlations with outcomes used to assess human patients. The principles of minimal manipulation to ensure low variability in nerve injury allow for adding still more associated tissue injuries into these models. This includes direct muscle crush and other components of limb injury. Finally, atrophy assessment and precise analysis of behavioral outcomes make these methods a complete package for studying musculoskeletal trauma that realistically incorporates all the elements of human traumatic limb injury.

<meta charset="utf8"></head><body>小鼠外伤性周围神经损伤

The present protocol describes traumatic peripheral nerve injuries (TPNIs), including precisely calibrated crush, strictly aligned and misaligned laceration, as well as grafted and non-grafted gaps of the sciatic nerve in mice. Custom-designed sensors are developed to gauge nerve trauma, induced with commonly available tools to ensure reproducible post-TPNI outcomes.

小鼠外伤性周围神经损伤

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve ...