这项工作是由美国国立卫生研究院资助/ NINDS NS26380和NS55976，克里斯托弗和Dana里夫基金会和丹尼尔Heumann为脊髓研究基金的支持。德雷克塞尔大学医学院脊髓研究中心提供支持来完成这项工作的核心设施。

1）显微手术的准备工作<ol><li>手术站需要用稀释的漂白解决方案之前，设置仪器和辅助材料，清洁和消毒。蒸压工具将1天手术前，在无菌容器中。打开热珠的灭菌器，用于去除从程序在不同的动物之间的文书的病原体和微生物污染物（精细科学的工具）。 </li><li>插入热障使用在手术过程中保持体温。设置用于控制出血的棉签和纱布。准备凝胶泡沫的小（小于2mm 3）微止血和无菌生理盐水溶液浸泡使用的片断。 </li><li>放置在感应室和异氟醚麻醉大鼠（5％氧气气化，流量2.0升/分钟）。删除动物室编制表，剃一个大面积的皮草从预定的手术领域。请用70％异丙醇开始在该领域的中心向外辐射，以同样的方式擦洗Polyhydroxidine解决方案（Xenodine）。这种洗3次重复序列。 </li><li>广场上的热屏障，覆盖鼻子和嘴吸入锥的动物，异氟醚减少3％和0.2 L / min的流速。子皮肌肉注射（注射剂和镇痛丁丙诺啡0.05毫克/公斤），提供适当的抗生素剂量（氨苄青霉素）。 </li><li>医生会Xenodine解决方案擦洗他们的手，用清水冲洗干净。无菌手术衣，将安装到外科医生将干手用无菌巾，无菌手套。这个过程是遵循在这个文本中描述的每个外科手术。 </li><li>由一名助理，并覆盖无菌文书的盖子将被删除，仪器将被放在一个无菌的，涵盖了外科医生。 </li></ol> 2）横断的PN促进变性<ol><li>与动物趴在其一侧的直线切口，从对膝关节的股骨的头部保持平行，约3mm的股骨。通过肤浅的股二头肌肌肉沿皮肤切口线切割，揭露底层股外侧肌。 </li><li>用剪刀在股外侧膝盖附近的一个小切口，并扩大对股骨的头部。再次用剪刀轻轻分开股外侧直到连体坐骨神经的胫骨和腓骨神经分支的可视化（平行运行的股骨）。 </li><li>跟踪这些分支对股骨的头部，直到确定坐骨神经主干。正下方的坐骨神经分叉，用细镊子仔细分离结缔组织（外膜）周围的胫骨神经分支（这是较大的分支）。 </li><li>一旦隔离滑胫神经周围6-0丝线结扎，并拧紧。使用微型剪刀样的神经喙结扎。穿过股外侧肌的缝合线，并配合一个结。这将提供一个容易的胫神经切断结束位置时的神经是要7-10天后收获。 </li><li>嫁接前的胫神经变性提供了一个更适合轴突生长比一个新鲜的神经准备的环境。 </li><li>股外侧肌用6-0丝线缝合关闭。股二头肌肌用6-0丝线缝合关闭。关闭与米歇尔的伤口剪辑皮肤。关闭异氟醚的流量，去除热障笼吸入鼻锥和地方的动物，并返回到首页笼，一旦警觉和活跃。 </li></ol> 3）拆除通知书段<ol><li>麻醉动物，剃须和清洁预定的手术视野如上所述（1.3）。广场上的热障的动物。 </li><li>除去伤口剪辑和独立以前的皮肤切口。穿过浅肌层缝合揭露股外侧肌，并通过缝合切断这种肌肉。 </li><li>跟踪缝合线，切断胫神经和神经缝合删除。用细镊子，解剖一个15毫米长的胫骨神经的外膜，并穿过这个末端。取出的神经节段，沉浸在无菌汉克斯平衡盐溶液。 </li><li>如果这是一个异种移植实验Euthasol腹腔注射的动物安乐死。如果这是一个自体移植的实验中，腿部肌肉和皮肤被关闭如2.6中所述）并准备为颈脊髓损伤的动物，如下所述。 </li></ol> 4）宫颈半横断损伤和移植第1天<ol><li>麻醉动物，剃须和清洁INTENDED外科领域如上所述（1.3）。广场上的热障的动物。管理抗生素和止痛药，如上述（1.4）。 </li><li>找到枕骨缺口和突出T2背椎过程，使切口之间的这些外部的地标全长。穿过中线肤浅斜方肌和独立的基础的颈椎菱形肌肉。除了通过插入一个吊具保持肌肉。 </li><li>深部supraspinal肌肉，应切背C4，C5和C6椎体过程。使用rongeurs执行C5的部分椎板切除术，揭露整个右侧和脊髓左侧内侧部分。作为一个具有里程碑意义的脊髓中线的使用突出的背静脉。 </li><li>使用一个微型手术刀刀片或30号针头刺破硬脑膜，并扩展到C5脊髓切口。请在底层蛛网膜下腔和软脑膜小切口。 </li><li>随着拉玻璃微导管开始吸脊髓背侧，右侧，使用温和的压力和微钳分开的组织和促进其清除。通过脊髓中间和腹侧部分继续创建一个2毫米长腔，完成从背到腹，从中线向外侧脑膜。 </li><li>放入腔达到止血盐水浸泡过的凝胶泡沫。 </li><li>删除段周围神经的Hanks液。轻轻放在附近剥离喙墙半切病变腔从近端神经节段的3mm和外膜。安全段10-0通过硬脑膜外膜缝合，然后缝合硬脑膜移植物植入结束封闭。 </li><li>夹心2件硅胶保护膜，并在稍后的时间点容易隔离（Biobrane）之间的移植。广场旁边的C6，C7和T1椎体过程。不要放在附近的远端神经结束脊髓或肌肉组织。 </li><li>在4-0缝合伤口剪辑接近皮肤切口层密切肌肉。 </li><li>将动物热屏障，并返回家笼笼，一旦警觉和活跃。 </li></ol>第21天<ol start="11"><li>麻醉动物，剃须和清洁预定的手术视野如上所述（1.3）。将动物热障和管理抗生素和止痛药，如上述（1.4）。通过削减肤浅缝线周围神经移植的硅橡胶膜暴露。仔细游离的长度从硅橡胶膜使用精细镊子和弹簧剪刀和反映从外科领域的神经。 </li><li>执行部分椎板切除右侧C7椎体过程。皮尔斯（4.4）以上脑膜和愿望，准备在1 毫米 3背象限病变腔C7脊髓。用盐水浸泡过的凝胶泡沫，以达到止血，然后与硫酸软骨素酶ABC浸泡凝胶泡沫取代。治疗病变部位15分钟，每5分钟提供新鲜的硫酸软骨素酶浸泡凝胶泡沫。 </li><li>封面与硅橡胶膜的神经移植的外部长度。关闭用4-0缝线和切开皮肤伤口剪辑的表浅肌肉。将动物热屏障，并返回家笼笼，一旦警觉和活跃。 </li></ol>日23 <ol start="14"><li>麻醉动物，剃须和清洁预定的手术视野如上所述（1.3）。将动物热障和管理抗生素和止痛药，如上述（1.4）。通过削减肤浅缝线周围神经移植的硅橡胶膜暴露。请勿打扰的嫁接，而是揭露C7嫁接部位，使用拉玻璃套管汉密尔顿注射器microinject脊髓约1毫米，并置移植到脊髓远端右侧的硫酸软骨素酶ABC加入1μl。 </li><li>轻轻地揭下从远端神经节段2毫米的神经外膜和C7病灶腔放在附近本月底。安全段10-0通过硬脑膜外膜缝合。 </li><li>关闭用4-0缝线和切开皮肤伤口剪辑的表浅肌肉。将动物热屏障，并返回家笼笼，一旦警觉和活跃。 </li></ol> 5）电生理记录的动作电位在脊髓移植和远端<ol><li>要确定如果是通过传输到PNG再生轴突的电活动，我们必须首先揭露和孤立的嫁接。在此过程中，必须特别小心，以避免物理损坏移植，这是移植物的位置后，硅橡胶膜的使用是很重要的。麻醉动物，剃须和清洁预定的手术视野如上所述（1.3）。广场日Ë动物热障。 </li><li>开场后的皮肤和浅表肌肉分开，硅橡胶膜条确定和仔细解剖免费的移植。 </li><li>使用rongeurs删除立即延髓和尾鳍的PNG脊髓并置站点背椎过程。请注意，不要去打扰太多在这种方法的移植。在这两个级别的硬脑膜缝。 </li><li>约1厘米喙C5的移植脊髓并置站点插入单丝（铂铱混合）进入脊髓病变同侧刺激电极，深度约1mm。小心地抬到一个双极钩状电极移植中心部分。提供10μA单一的刺激脉冲100μsec期限，并确定是否有通过行驶到移植再生轴突的动作电位。 </li><li>开关连接线的位置，使钩电极成为刺激电极插入到同位的PNG脊髓和试图记录动作电位在脊髓脊髓远端和单线。 </li><li>在实验结束后提供1mA的刺激100μsec期限的火车，在50Hz时为1小时总。安乐死的动物1小时后，灌注4％多聚甲醛和脊髓组织的CFO在被激活的神经元再生轴突在PNG的免疫细胞化学检测过程。 </li></ol> 6）逆行标记神经元的再生轴突成PNG和轴突顺行标签，进入脊髓再生<ol><li>电措施，没有实验动物，它可以识别轴突的生长，在脊髓移植和终止过程中的源。麻醉动物，剃须和清洁预定的手术视野如上所述（1.3）。将动物和热障的PNG暴露在6.1以上。 </li><li>用微型剪刀削减通过嫁接的中点，剥去外膜移植物的切完。体现削减两端远离对方打下每个单独的表上的封口膜。 </li><li>将一个真正的蓝光近端切结束标签神经元轴突的PNG浸泡凝胶泡沫的脱脂棉。将凝胶泡沫纱布浸泡在生物素化葡聚糖胺（BDA）的远端切断结束标签轴突延长移植到脊髓背。 </li><li> 3 - 4D后安乐死的动物，灌注4％多聚甲醛和过程的大脑和脊髓的真正的蓝色标记的神经元或北京经济技术开发区组织化学标记轴突荧光显微镜组织切片。 </li></ol> 7）胸完整的横断损伤和移植这提供了一个以上的颈椎半横断损伤模型的替代方法。这里产生更严重，双侧损伤和周围神经段进入病灶腔跨度和延髓和脊髓损伤尾椎树桩上。 第1天<ol><li>麻醉动物，剃须和清洁预定的手术视野如上所述（1.3）。广场上的热障的动物。管理抗生素和止痛药，如上述（1.4）。 </li><li>确定最后一根肋骨插入点的T12椎体水平。 T11，T10和T9椎体过程，从这个角度喙皮肤切口。通过浅筋膜穿过连接这三个过程的supraspinal肌肉。 </li><li> T10椎体的过程中执行完整的椎板切除术（覆T12脊髓节段）。使用一个微型手术刀刀片或30号针头刺破硬脑膜，在T12脊髓延长切口。请在底层蛛网膜下腔和软脑膜小切口。 </li><li>随着拉玻璃微导管开始双边吸T12脊髓，用温柔的压力和微型镊子分开的组织和促进其清除。通过脊髓中间和腹侧部分继续创建一个3毫米长腔，是完全背腹之间的横向脑膜。 </li><li>用盐水浸泡过的凝胶泡沫，以达到止血，然后与硫酸软骨素酶ABC浸泡凝胶泡沫取代。治疗病变部位15分钟，每5分钟提供新鲜的硫酸软骨素酶浸泡凝胶泡沫。 </li><li>汉克的解决方案中删除段周围神经。轻轻地反映近端神经节段4毫米的外膜，切割长度紧贴适合脊髓一侧延髓和尾鳍树桩之间。广场旁边的第一嫁接段的第二部分，apposed到另一侧的脊髓。缝合硬脑膜封闭在植入移植，并用小硬脑膜一块硅橡胶膜。 </li><li>在4-0缝合伤口剪辑接近皮肤切口层密切肌肉。 </li><li>将动物热屏障，并返回家笼笼，一旦警觉和活跃。 </li></ol>第3天<ol start="9"><li>麻醉动物，剃须和清洁预定的手术视野如上所述（1.3）。将动物热障和管理抗生素和止痛药，如上述（1.4）。通过肤浅的缝合线切割硅橡胶膜覆盖嫁接部位暴露。请勿打扰的嫁接，但而暴露脊髓骶管的移植和使用拉玻璃套管汉密尔顿注射器microinject脊髓约1毫米，并置移植到脊髓远端两侧的硫酸软骨素酶ABC加入1μl。 </li><li>关闭用4-0缝线和切开皮肤伤口剪辑的表浅肌肉。将动物热屏障，并返回家笼笼，一旦警觉和活跃。进行电刺激，记录和示踪标签5）和6）以上。 </li></ol> 8）代表性的成果当此过程优化周围神经段将紧密结合起来，与主机的脊髓。升序和降序脊髓轴突将进入移植，移植物的长度在一个相对直线平行增长，并延伸到每天约1毫米的速度移植末端。到达末端后，轴突会穿透相邻的脊髓，如果软骨素已被用于去除抑制蛋白多糖从周围的疤痕组织。将由一个由脊髓神经元的反应，刺激轴突内移植的电生理及免疫细胞化学检测功能连接。功能恢复的解剖学的相关性进行评估跟踪超出神经移植到脊髓神经轴突的数量和长度。

<ol>
	<li>Houle, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+the+potential+for+chronically+injured+neurons+to+regenerate+axons+into+intraspinal+peripheral+nerve+grafts.">Demonstration of the potential for chronically injured neurons to regenerate axons into intraspinal peripheral nerve grafts.</a> Exp. Neurol. 113, 1-9 (1991).</li><li>Ye, J. H., Houle, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+the+chronically+injured+spinal+cord+with+neurotrophic+factors+can+promote+axonal+regeneration+from+supraspinal+neurons.">Treatment of the chronically injured spinal cord with neurotrophic factors can promote axonal regeneration from supraspinal neurons.</a> Exp. Neurol. 143, 70-81 (1997).</li><li>Houle, J. D., Tom, V., Mayes, D., Wagoner, G., Phillips, N., Silver, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+an+autologous+peripheral+nerve+bridge+and+matrix+modification+by+chondroitinase+allows+robust+functional+regeneration+across+a+hemisection+lesion+of+the+adult+rat+spinal+cord.">Combining an autologous peripheral nerve bridge and matrix modification by chondroitinase allows robust functional regeneration across a hemisection lesion of the adult rat spinal cord.</a> J. Neurosci. 26, 7405-7415 (2006).</li><li>Tom, V., Houle, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraspinal+microinjection+of+chondroitinase+ABC+following+injury+promotes+axonal+regeneration+out+of+a+peripheral+nerve+graft.">Intraspinal microinjection of chondroitinase ABC following injury promotes axonal regeneration out of a peripheral nerve graft.</a> Exp. Neurol. 211, 315-319 (2008).</li></ol>

<ol><li>实验的可重复性，它是重要的，在动物脊髓损伤的水平是一致的。因此，至关重要的是，确定相应的椎椎板切除过程。因为我们使用的是这项研究的一个半切或切断病变模型，有没有病变的大小或含糊之处，具体脊髓束是否受伤或幸免。 </li><li>周围神经移植前预先堕落，因为这启动消化外围的轴突再生轴突生长提供空间，因为它会激活雪旺氏细胞产生多种神经营养因子支持轴突长入，这是必要的。 </li><li>初...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>10-0 silk suture</td>
<td>&nbsp;</td>
<td>ArosSurgical</td>
<td>T5A10N10</td>
<td>&nbsp;</td>
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<td>6-0 silk suture</td>
<td>&nbsp;</td>
<td>McKesson</td>
<td>2693</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>&nbsp;</td>
<td>McKesson</td>
<td>483549</td>
<td>&nbsp;</td>
<tr>
<td>Antibody to cFos</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F7799</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated dextran Amine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D7135</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphin (.3mg/ml)</td>
<td>&nbsp;</td>
<td>McKesson</td>
<td>12496075701</td>
<td>&nbsp;</td>
<tr>
<td>Chondroitinase ABC</td>
<td>&nbsp;</td>
<td>Associates of Cape Cod</td>
<td>100332-1A</td>
<td>&nbsp;</td>
<tr>
<td>Euthasol</td>
<td>&nbsp;</td>
<td>Webster Veterinary</td>
<td>07-805-9296</td>
<td>&nbsp;</td>
<tr>
<td>Hanks Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>21-021-CV</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>209-1966</td>
<td>&nbsp;</td>
<tr>
<td>Michel Wound Clips</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>12040-02</td>
<td>&nbsp;</td>
<tr>
<td>Neurotrace Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>N7167</td>
<td>&nbsp;</td>
<tr>
<td>True Blue</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T5891</td>
<td>&nbsp;</td>
<tr>
<td>Xenodine</td>
<td>&nbsp;</td>
<td>Webster Veterinary</td>
<td>92201</td>
<td>&nbsp;</td>
<tr>
<td>Hot Bead Sterilizer</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Forced Exercise Wheel</td>
<td>&nbsp;</td>
<td>Lafayette Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TreadScan System</td>
<td>&nbsp;</td>
<td>Clever System</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Infinite Horizon Impact Device</td>
<td>&nbsp;</td>
<td>Precision Systems and Instrumentation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Stimulation Device</td>
<td>&nbsp;</td>
<td>MagStim Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

combining peripheral nerve grafting and matrix modulation to repair the injured rat spinal cord

外伤性脊髓损伤（SCI）的会导致死亡的神经元，运动和感觉神经纤维（轴突）与大脑的沟通途径和中断的中断。我们研究的目标之一是促进轴突再生，恢复整个病变部位的连接。而要做到这一点，我们制定了周围神经（PN）的坐骨神经段是直接放在之间的脊髓受损的结束或用于整个病变的桥梁形式嫁接技术。这种方法有几个优点比其他神经组织移植，再生轴突可以对一个具体的目标区域的指示，再生轴突的数量和源代码很容易被追踪技术确定，移植可用于电生理实验来衡量功能的恢复与轴突在移植，并有可能使用的自体神经，以减少移植排斥反应的可能性。在我们的实验室中，我们已经完成的自体（捐赠者和接受者是同一种动物）和异源（捐赠者和接受者是不同的动物）移植与比较的结果。这种方法已成功地用于急性和慢性损伤的情况下。再生轴突到达末端的PN移植往往不能延伸到脊髓背，所以我们使用的硫酸软骨素microinjections，降低与SCI的地区周围的疤痕组织相关抑制分子。与此同时，我们发现，提供外源性生长和营养分子，鼓励到距离较远的脊髓神经轴突再生。移植后的几个月，我们执行各种解剖，行为和电生理测试，以评估在脊髓受伤的动物功能的恢复。在不同程度的损伤，并在不同的物种，这种实验方法已成功地用于在几个脊髓损伤模型（小鼠，大鼠和猫）。重要的是，周围神经移植的方法是有效地促进急性和长期受伤的神经元的再生。

Watch this Scientific Journal Video about Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord at JoVE.com

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

脊髓外伤，会扰乱大脑的沟通。要恢复失去的连接，我们利用了周围神经移植提供一个底层与神经营养因子和基质调节酶，再生纤维结合，以消除抑制分子，促进长途增长。

结合周边神经移植和矩阵调制修复受伤的大鼠脊髓

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of ...

combining-peripheral-nerve-grafting-matrix-modulation-to-repair

Research

JoVE Journal

Biology

16.3K 次观看.  Drexel University College of Medicine. 脊髓外伤，会扰乱大脑的沟通。要恢复失去的连接，我们利用了周围神经移植提供一个底层与神经营养因子和基质调节酶，再生纤维结合，以消除抑制分子，促进长途增长。

视频: Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

科研

生物学

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

扫描浸软组织的电子显微镜可视化的细胞外基质

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

压和外尿道括约肌测量在清醒大鼠植入导管和电极允许重复测量

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

以血管内炎组织调节为重点, 利用肠系膜孤立动脉评价血管张力

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and subsequent bladder wall remodeling. To facilitate assessment of the cellular composition of the bladder wall in non-injured control and spinal cord injured mice, we developed an optimized dissociation protocol that supports high cell viability and enables the detection of discrete subpopulations by flow cytometry.
Spinal cord injury is created by complete transection of the thoracic spinal cord. At the time of tissue harvest, the animal is perfused with phosphate-buffered saline under deep anesthesia and bladders are harvested into Tyrode&#8217;s buffer. Tissues are minced prior to incubation in digestion buffer that has been optimized based on the collagen content of mouse bladder as determined by interrogation of publicly available gene expression databases. Following generation of a single cell suspension, material is analyzed by flow cytometry for assessment of cell viability, cell number and specific subpopulations. We demonstrate that the method yields cell populations with greater than 90% viability, and robust representation of cells of mesenchymal and epithelial origin. This method will enable accurate downstream analysis of discrete cell types in mouse bladder and potentially other organs.

The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry.

脊髓损伤后小鼠泌尿膀胱分子分析的单细胞分离方法

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and ...