DPS acknowledges past and present support in part from grant #2665 and #2934, respectively, from the PVA Research Foundation. PKS is an Alberta Innovates &#8211; Health Solutions Scientist, operating funds were provided by the Leblanc Chair for Spinal Cord Research, University of Calgary.

注：所有动物的程序都在通过在路易斯维尔大学的机构动物护理和使用委员会设定的指导方针进行，秉承联邦法规。 1.准备低钙和2毫米的Ca 2+人工脑脊液（学联）的灌流液<ol><li>制备2倍低的Ca 2+（0.1毫摩尔）库存C缓冲区，2倍正常的Ca 2+（2mM的）股票缓冲剂，和2x库存B缓冲液，如表1所记载的单个储液允许的离子含量修饰（ 例如，低或零的Ca 2+），并且可以储存在4℃下进行长达一个月。 </li><li>解剖时心脏内灌注，加入100 ml 2倍低钙股票C和100毫升2X B股成塑料瓶，使1X低钙学联。混合存储一夜之间在4°C或作出新的。 </li><li>对于先体外后体内脊髓IMAG期间灌注荷兰国际集团，加入500毫升2倍正常的Ca 2+股票A和500毫升的2倍B股为1升的玻璃瓶，产生1个学联。混合存储一夜之间在室温或作出新的。 </li><li>调节pH脑脊液缓冲器至7.4。同时保持脑脊液在室温下，将低的Ca 2+脑脊液在冰上，然后气泡两个缓冲器与卡波金气（95％O 2; 5％CO 2）30分钟前，在灌注期间使用和连续进行。 </li></ol> 2.准备离体成像商会<ol><li>换一个加热毯（无自动关闭）各地学联瓶，调整热量低/中设置。 </li><li>插入一个专用的脑脊液灌注线成连接到灌注泵和直列加热器是由一个双极温度控制器和反馈温度探头控制， 如图1A中的瓶子。 </li><li>安装一台显微镜阶段插入（152×102毫米），双光子激发/ conf目录OCAL显微镜阶段配有溢出容器。 </li><li>放置一个大型的开放式浴室灌注腔（宽×长×高12×24毫米x 8毫米）到溢出容器来容纳和提供新鲜的含氧学联连续流向体外脊髓（ 见图1B一个合适的前体内成像室设计）。 </li><li>直列加热器的金属输出端口连接到使用合适的管道的离体成像室。放置一个薄吸收垫的离体成像室下方，以帮助在成像期间保持缓冲器/组织的温度。 </li><li>坚持体外成像室使用可拆卸黏粘性的溢出容器/台插入底部。粘性粘性的延展性将允许根据需要，以确保目标的光路垂直于组织表面的腔室，以进行调整。如有必要，可使用一个靶心级别调整室。 </li><li>安全的邻近该腔室的流体入口直列加热器反馈温度探测器和一个不锈钢吸管连接到在腔室出口处的真空线。磁带和适当的微量持有者在这方面是有用的。打开真空源上。 </li><li>打开灌注泵开始填充成像室与含氧脑脊液。将灌注泵每分钟1.5-2毫升。 </li><li>通过调整真空控制线学联在成像室的容积。有足够的脑脊液中的成像室，以覆盖脊髓节段，并允许水浸渍物镜和组织表面之间有足够的工作距离是很重要的。髓鞘和轴突，可以产生不利影响，如果该组织的部分没有完全新鲜的含氧学联淹没，导致实验者诱发文物。 </li><li>调整直列加热器温度控制器，使灌流液温度在成像室是接近室温。 </li></ol> &lt;P类=“j​​ove_title”&gt; 3。成人小鼠颈脊髓解剖<ol><li>通过注入经腹腔注射麻醉戊巴比妥钠（200毫克/千克）的过量安乐死的成年6-10周龄THY1 -YFP转基因小鼠（用适当的菌株，在背根神经节细胞荧光蛋白的表达）。 </li><li>一旦在麻醉状态下的正常水平（ 例如，没有反应，捏脚趾和损失瞬目反射），仔细刮皮肤上的脊髓和脑外科快船。使用优碘和70％的乙醇垫清洁皮肤。 </li><li>喷用70％乙醇胸部/腹部区域，然后使用冷冻，卡波金鼓泡低钙灌注受试者transcardially 2+脑脊液。 （参见图1C的建议剥离台式成立）。 </li><li>由于肝脏相形见绌，用小夹子固定灌注针插入心脏。把题目交给访问的剃背侧主体和擦拭用70％乙醇的剃的区域。 </li><li>使背部皮肤切口沿着用解剖剪，以暴露脑和脊柱（ 图2A）从鼻子开始髋中线。通过将销的鼻子和臀部保护的准备。 </li><li>从这点上使用解剖显微镜，牢牢贯穿头骨的背表面处嗅球的水平。插入的剪刀尖端插入切口的侧向边缘之一，并沿着黄芩双边直到小脑的边缘切割。 注意：不要完全切除黄芩，因为它是以后需要从整个解剖脊髓解除覆组织（ 见图 2b）。 </li><li>提起黄芩，露出大脑。轻轻切interparietal骨（位于小脑）的左和右边缘，并将其拉回尾端以暴露脑干/脊髓（ 图2C </stroNG&gt;）。 </li><li>使用以45°角的表面保持细尖剪刀切椎骨双边只是不如后神经根。 注：避免与明亮的白色背柱接触。这些纤维是那些将被成像（ 图2D），并且可以很容易损坏。 </li><li>解除脊柱的黄芩和背表面用细尖镊子，轻轻拉动上覆组织在一块作为切口以暴露脊髓。继续椎骨切到上胸椎水平隔离颈髓用于后续的成像（ 图2E）的1-2厘米的段。 </li><li>用剪刀剪下平行组织/骨脊柱和清晰的组织柱下方。做出这些削减尽可能水平。这是很重要的，该组织平躺在腔室，使得所述帘线是用于成像的水平。 </li><li>使用＃11手术刀横切脑干（过去终端）和的竹叶纤维束的纤维在胸上段的水平隔离颈脊髓节段（ 见图2F）。用剪刀剪下组织/骨这些相同的两个点。孤立的组织应包含脊柱涵盖尾脑干，颈膨大，和上胸段脊髓（ 图2G）的2/3。 </li><li>把孤立的脊柱成卡波-冒泡，冷冻低钙培养皿2+脑脊液。如果需要的话，修剪脊柱下方的组织，直到它位于平盘。 </li><li>分离的脊柱转移到离体成像室，并逐渐增加灌注液温度用1小时〜36 - 37℃。保持成像在这个温度。 </li></ol> 4.放置离体脊髓到成像室和髓鞘标签与尼罗红<ol><li>小心确保脊柱在i-maging室用合适的接头片按住改性与垂直网格平台由细莱卡线程（删除网格平台的中间螺纹以允许脊髓空间;参见图3A）。 </li><li>解冻的尼罗红的等分试样（5mM储备在DMSO和0.22微米的过滤;可在4℃下，或6个月，在-20℃下在黑暗中储存1个月）。刚刚加入尼罗红到成像室之前，降低灌注泵的流量，并调整真空管线，以确保在该室中的流体总是覆盖脊髓。 </li><li>添加5-10微升尼罗红原液到成像室附近的线（ 图3B）的尾端。用1毫升吸管轻轻拌匀尼罗红在整个室。允许尼罗红留在腔室1-2分钟，然后将真空线返回并调节灌注泵以每​​分钟1.5-2毫升连续灌注脊髓。 </li><li>小心抬起显微镜阶段并对准目标的脊髓节段的中心和内侧上背列，直到水浸泡的目的是从腔室中的脑脊液的表面约10毫米。使用显微镜物镜转换器调焦轮慢慢把目标，直到它轻轻触及学联的表面。使用落射荧光光源向背侧列聚焦成的视图（ 图3C）的字段。 </li></ol>鼠标脊髓TPLSM和激光诱导脊髓损伤5. 离体成像（Lisci酒店） 注：后静脉提供了有用的标记物，以居中的组织作为竹叶纤维束纤维（从背根神经节的细胞体发起和从T6升到并沿着脊髓的中线下方）平行于该血管中。较厚的YFP +颈脊神经后根的预测也提供了一个有用的指标作为纤维升降的横向到Y指纹+竹叶纤维束纤维的直径小。 <ol><li>一旦脊髓组织适当排列，切换到激光扫描模式进行升序竹叶纤维束髓纤维基线成像。激发二者的YFP和尼罗红，用调谐到950纳米的波长（〜17毫瓦在25X，1.1 NA物镜的出口测量）和适当的分光镜（DM）和带通滤波器的双光子激光源，以隔离荧光发射荧光团的（我们使用五十零分之五百二十五DM560，60分之600DM640和七十〇分之六百八十五，对YFP和尼罗红分离成黄 - 橙，和扩展红色通道，分别地）。 <ol><li>如果轴突的3％以上的显示期间基线成像（30-60分钟）的轴突球状体，丢弃该制剂由于实验者诱发工件。 </li></ol></li><li>诱发Lisci酒店通过放大来看，以30.3X场（创建〜20微米直径的消融）。调谐激光器至800nm​​，并且增加激光的功率，以〜110毫瓦样品。允许看激光扫描完成5场，以确保纤维完全切断。 </li><li>确认Lisci酒店通过改变激光设置恢复到成像设置（950纳米，17毫瓦的样品，2.08X）和扫描组织以可视化的消融。验证在消融的直径和该初级烧蚀轴突完全横断（ 见图3D）。使用显微镜及Z的捕获时间推移设置记录轴突和髓磷脂损伤达8-10小时+损伤后的动态响应。 注意：如果消融术是不完整的（ 即纤维横断不全），弃去准备，作为测定原发性和继发损伤的机制将被混淆。此外，分离的脊髓节段可以被成像到24小时后Lisci酒店只有轻度至中度Lisci酒店无关的组织损伤。 </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White+matter+NMDA+receptors:+an+unexpected+new+therapeutic+target.">White matter NMDA receptors: an unexpected new therapeutic target.</a> Trends Pharmacol Sci. 28, 561-566 (2007).</li><li>Baltan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+anoxia:+a+tale+of+two+white+matter+tracts.">Surviving anoxia: a tale of two white matter tracts.</a> Crit Rev Neurobiol. 18, 95-103 (2006).</li><li>Stirling, D. P., Stys, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+axonal+injury:+internodal+nanocomplexes+and+calcium+deregulation.">Mechanisms of axonal injury: internodal nanocomplexes and calcium deregulation.</a> Trends Mol Med. 16, 160-170 (2010).</li><li>Wang, H., Fu, Y., Zickmund, P., Shi, R., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+anti-stokes+Raman+scattering+imaging+of+axonal+myelin+in+live+spinal+tissues.">Coherent anti-stokes Raman scattering imaging of axonal myelin in live spinal tissues.</a> Biophys J. 89, 581-591 (2005).</li><li>Beirowski, B., Nogradi, A., Babetto, E., Garcia-Alias, G., Coleman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+axonal+spheroid+formation+in+central+nervous+system+Wallerian+degeneration.">Mechanisms of axonal spheroid formation in central nervous system Wallerian degeneration.</a> J Neuropathol Exp Neurol. 69, 455-472 (2010).</li><li>Shi, R., Asano, T., Vining, N. C., Blight, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+membrane+sealing+in+injured+mammalian+spinal+cord+axons.">Control of membrane sealing in injured mammalian spinal cord axons.</a> J Neurophysiol. 84, 1763-1769 (2000).</li><li>Stirling, D. P., Cummins, K., Wayne Chen, ., R, S., Stys, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axoplasmic+reticulum+Ca(2+)+release+causes+secondary+degeneration+of+spinal+axons.">Axoplasmic reticulum Ca(2+) release causes secondary degeneration of spinal axons.</a> Ann Neurol. 75, 220-229 (2014).</li></ol>

The authors have nothing to disclose.

我们描述的成像体外脊髓髓鞘轴突（ 即，薄束纤维束）结合激光诱导脊髓损伤，研究主要和次要髓轴突变性的动态进展随时间。 离体的表面的成像的方法脊髓克服了许多与体内成像相关的，如运动伪影和实验者诱发缺氧在长时间成像会议的潜在的并发症。此协议隔离使用含有低的Ca 2+和避免了覆轴突纤维的损伤，在背柱或腹侧白质带制剂一个经常遇到的挫折含?...

轴突变性是发病的一个显着原因跨越多个神经系统疾病，包括神经外伤，中风，自身免疫和神经退行性疾病。不像外周神经系统（PNS），中枢神经系统（CNS）的轴突具有有限的能力来再生一旦由于内在的和外在的障碍（ 即，抑制分子，以在髓鞘退化期间瘢痕形成产生和释放的轴突生长）1受伤-7。尽管一些这些障碍已被广泛研究，治疗干预的目的是防止中枢神经系统轴突变性，促进强劲的轴突再生，并恢复功能可连接地，仍然有限。 一旦从它们的体细胞中分离的轴突发生退化称为沃勒变性，其特征是轴索肿胀，球体的形成和最终破碎（8综述）的刻板过程。在相反，所述近端残端保留在与横断外围轴突胞体的连续性，形成一个膨胀的端部，模具回郎飞的最近的节点，然后可以开始生长锥的形成，一个重要的先决条件必需的后续轴突再生9-11。与此相反，许多中央轴突的近端轴突末梢形成特性“endbulbs”或回缩灯泡，不能形成生长锥，而是缩回远离损伤位点的地方，他们伤害12-15后保持数月。除了主轴突损伤，附加轴索损害/损失也可能发生于在很大程度上从初始损伤幸免轴突。最初幸免轴突此延迟轴突损失被称为次级轴突变性。 CNS轴突损伤这个固有响应呈现功能轴突再生更加难以实现的目标，在脑和脊髓。 虽然公顷轴突损伤（ 例如，球体的形成，回缩灯泡）的llmarks已良好表征从验尸组织和轴突变性的实验模型中，这些动态过程的分子机制的阐明已受到限制。这些研究大多依赖于静态终结点意见，即天生没有捕捉到随着时间的推移个人轴突响应。虽然外源施加轴索示踪剂是有益的，从静态部分，并在实时成像澄清轴索反应，遗传编码的轴突标记的可用性极大地提高了可视化实时轴突使用荧光显微镜的能力。的确，从Kerschensteiner和同事开创性报告中使用大鼠Thy1-GFP-S小鼠在送他们的预测在脊髓的背神经元列的子集，编码绿色荧光蛋白最初提供的轴索变性和再生的直接证据体内线16。实时成像方法，使用双光子激光扫描显微镜（TPLSM）和兴趣细胞的基因荧光蛋白标记继续提供直接证据和机械洞察到许多不同的动态过程，如轴突变性， 钙离子信号，轴突再生，星形胶质细胞生理学，小胶质细胞生理学和损伤反应17-25。 在对比轴突，很少是已知的髓鞘反应到损伤的实时性。髓鞘是由中枢神经系统和雪旺氏细胞在PNS少突胶质细胞产生和维持白质的重要​​组成部分。髓鞘绝缘99％的轴突的表面，并通过这样做提供了支持高效快速的跳跃式脉冲传播，最近由Buttermore 等人 26审查的高电阻，低电容保护覆盖物。捕捉髓鞘的动态响应损伤我们使用溶剂化，亲脂性荧光染料尼罗红27。此活力染色的溶剂化性质允许其发射光谱是依赖于物理化学环境28,29的光谱的移位。这些性质是有用的洞察axomyelinic损伤机制，并且可以使用适当选择分光镜和发射滤光片被可视化，或者使用光谱显微镜27解决。例如，尼罗红的发光光谱是蓝移的极性较小的，富含脂质的环境中，例如在脂肪细胞和正常CNS髓磷脂（峰值发射〜580-590纳米）27中。与此相反，在〜625纳米的形成为轴突endbulbs这种活体染料的发光光谱的峰进行轴突顶梢枯死27。虽然特别是在endbulbs与正常髓鞘这些光谱变化背后的确切机制尚不清楚，这种光谱变化可能揭示潜在的改变蛋白质的积累，或混乱导致暴露的疏水性的结合位点27。 而在体内成像是观察脊髓性轴索损伤动力在他们自然环境的最终指标，它在技术上是具有挑战性的，需要大量的专业知识手术，并经常重复手术暴露背列可能会引入实验工件（ 如炎症和疤痕形成）。此外，昂贵的设备，通常需要以允许悬浮液的显微镜物镜下一个完整的动物和定位。动物需要进行仔细的监测，以及确保它们保持温暖，以确保流体被补充，并保证有缺氧由于长时间麻醉成像会议迹象。后者是非常重要的，因为轴突和髓鞘绝对需要不断的灌注和充足的氧气水平来保持活力。然而，这往往是没有报道或在大多数体内研究监测，以日期。此外，运动伪影由于心脏和呼吸（异氟烷麻醉成年鼠：〜300-450次每分钟（BPM）是最佳的维持97-98％的氧饱和度（正常率〜632 BPM）和〜55-65呼吸每分钟（正常率为〜每分钟163次呼吸），分别）） 在体内脊髓成像化妆过程中遇到的30区分使用高分辨率荧光显微镜挑战，因为即使是最快的激光扫描原发性和继发性轴索损伤反应不可避免地受到这些运动文物。进展超快共振扫描仪结合的可植入椎骨刚性框架窗口可以允许在清醒的动物的鼠脊髓的成像，但更快的扫描时间不可避免地降低了信号噪声比恶化图像质量。作为目前用于脑成像在脊髓的成像技术进一步改进可以克服许多这样的障碍，并限制由INA引入潜在的困惑dequate组织灌注， 如，31-33。 很多东西被知道关于脑白质生理和白质损伤的机制已利用体外或视神经，周围神经和脊髓白质34-41条体外制剂白质决定的。这些准备工作继续推进我们的脑白质损伤机制的知识，因为它们允许控制在变化的环境因素，在活组织控制应用药物和试剂，使用电功能的评估，和轴突的直接荧光显微镜观察和髓鞘。然而，一些以前的方法，观察脊髓背柱条或腹白质条轴突在拆除阶段，可能会影响密切相轴突响应不可避免地伤害面神经轴突。为了充分利用该实验操作上面，避免损坏已经在调查RY纤维，我们使用的体外脊髓型颈椎病的模型，因为它可以防止电源线背方面的直接接触。因此，隔离在软脑膜和邻近浅表背柱轴突的架构仍然是可行的，泰然自若。 在这里，我们描述了一个相对简单的方法，使中央有​​髓轴突的直接可视化，因为它们对一个病灶性损伤的实时动态响应多达8 - 损伤后10+小时。激光诱导脊髓损伤（Lisci酒店）模型允许保持空间受限随着时间的推移原发性和继发性轴索损伤的机制为主要病变（消融部位）之间的差异。开浴成像室可以访问治疗干预，试剂递送和环境操作。推定axomyelinic保护剂可以通过直接观察与冗长和昂贵的实验涉及的组织的过程可以快速评估实时荷兰国际集团，切片，染色，图像采集和分析，从而提供了一个有用的代理模型测试实验药剂中活的动物时，必须评估急性反应和保护性操作。

<table border="0" cellpadding="0" cellspacing="0" width="968">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">Material/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:113px;">Catalog Number</td>
			<td style="width:341px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large bath chamber with slice supports</td>
			<td>Warner Instruments</td>
			<td>RC-27L</td>
			<td>For ex vivo imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Slice Supports</td>
			<td>Warner Instruments</td>
			<td>SS-3</td>
			<td>For ex vivo imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Slice hold-down for RC-27L and RC-29 chambers</td>
			<td>Warner Instruments</td>
			<td>SHD-27LP/10</td>
			<td>For ex vivo imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">Suction Tube, Series 20 Classic Design, left handed</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:113px;">ST-1L</td>
			<td>For ex vivo imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solution In-line heater/cooler</td>
			<td>Warner Instruments</td>
			<td>SC-20</td>
			<td>To regulate perfusate temperature during imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">Bipolar temperature controller</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:113px;">CL-100</td>
			<td>To regulate perfusate temperature during imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">Liquid Cooling System</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:113px;">LCS-1</td>
			<td>To regulate perfusate temperature during imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">Cable assembly for heater controllers</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:113px;">CC-28</td>
			<td>To regulate perfusate temperature during imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">Replacement bead thermisitor for CC-28 cable</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:113px;">TS-70B</td>
			<td>To regulate perfusate temperature during imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">Magnetic holder with suction tubing</td>
			<td style="width:139px;">Bioscience Tools</td>
			<td style="width:113px;">MTH-S</td>
			<td>To hold the stainless steel vacuum suction tubing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">Adjustable holder</td>
			<td style="width:139px;">Bioscience Tools</td>
			<td style="width:113px;">MTH</td>
			<td>To hold the temperature probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">clear silicone sealant</td>
			<td></td>
			<td></td>
			<td>For ex vivo imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">superglue</td>
			<td></td>
			<td></td>
			<td>For ex vivo imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">thin plexiglass strips</td>
			<td></td>
			<td></td>
			<td>For ex vivo imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">nile red</td>
			<td style="width:139px;">Life Technologies</td>
			<td>N-1142</td>
			<td>For labeling myelin</td>
		</tr>
	</tbody>
</table>

an ex vivo laser-induced spinal cord injury model to assess mechanisms of axonal degeneration in real-time

受伤的中枢神经系统轴突不能再生，经常收回远离损伤部位。轴突从最初幸免受伤以后可能发生二次轴索变性。缺乏生长锥形成，再生，和附加有髓轴突突起损耗脊髓内极大地限制了神经恢复以下损伤。评估脊髓髓鞘轴突如何中央到损伤响应，我们开发了一种体外活脊髓模型利用表达黄色荧光蛋白在轴突和一个联络和高度可再现激光诱导脊髓损伤的转基因小鼠，记录的命运轴突和髓磷脂（亲脂性的荧光染料尼罗红）随时间使​​用双光子激发时间推移显微镜。如急性弥漫性轴索损伤，轴索回缩，和髓鞘变性动态过程研究的最佳实时。然而，挫伤为主受伤和运动伪影的非焦点自然界中在体内脊髓成像化妆区分使用高分辨率的显微镜具有挑战性的原发性和继发性轴索损伤反应。此处所描述的体外脊髓模型模拟的临床相关挫伤/压缩诱导轴突病理学的几个方面，包括轴索肿胀，球体形成，轴突横断，和周围的轴突溶胀提供了有用的模型来研究在实时这些动态过程。这种模式的主要优点是极好的时空分辨率，可以让初次损伤的直接损伤轴突和继发性损伤机制之间的差异;试剂直接向灌注液沐浴帘线控输注;精确的改变的环境氛围（ 例如，钙，钠离子，已知的贡献者轴突损伤，但几乎不可能在体内操纵）;和小鼠模型也提供了一个优点，因为它们提供了一个机会，以可视化和操纵基因鉴定细胞群和亚细胞结构。在这里，我们将介绍如何隔离和图像从小鼠活脊髓捕捉急性弥漫性轴索损伤的动态。

的一个适当的实验室设置隔离，保持生存能力需要，和图像的信息的体外脊髓示于图1的显微镜需要配备一个可调谐脉冲飞秒激光器，适当dicroics和发射滤波器，和一个水浸渍物镜具有高数值孔径（≥1.0）。以确保在解剖脊髓存活，程序应该在冷冻含氧低的Ca 2+脑脊液的存在下，使足够的Ca 2+为隔离轴突膜以密封42来执行。如果不这样做可能会导致axomyelinic...

Watch this Scientific Journal Video about An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time at JoVE.com

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

an-ex-vivo-laser-induced-spinal-cord-injury-model-to-assess

Research

JoVE Journal

Neuroscience

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

10.8K 次观看.  University of Louisville. We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability. 

视频: An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

侧向液压冲击：创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

科研

神经科学

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

初级量子神经元轴突运输的实时成像点标记的BDNF

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

M&amp;#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

在体内挫伤脊髓损伤后影像血管动力学的双色法

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

脊髓损伤 wistar 大鼠跑步机的作业训练

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

小鼠完全转型脊髓损伤的诱导

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...