Name: an ex vivo laser-induced spinal cord injury model to assess mechanisms of axonal degeneration in real-time
Uploaded: 2014-11-25T14:00:00.000+00:00
Duration: 11 min 18 s
Description: 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

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>

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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><tbody><tr><td>Large bath chamber with slice supports</td><td>Warner Instruments</td><td>RC-27L</td><td>For ex vivo imaging chamber</td></tr><tr><td>Standard Slice Supports</td><td>Warner Instruments</td><td>SS-3</td><td>For ex vivo imaging chamber</td></tr><tr><td>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><td>Suction Tube, Series 20 Classic Design, left handed</td><td>Warner Instruments</td><td>ST-1L</td><td>For ex vivo imaging chamber</td></tr><tr><td>Solution In-line heater/cooler</td><td>Warner Instruments</td><td>SC-20</td><td>To regulate perfusate temperature during imaging</td></tr><tr><td>Bipolar temperature controller</td><td>Warner Instruments</td><td>CL-100</td><td>To regulate perfusate temperature during imaging</td></tr><tr><td>Liquid Cooling System</td><td>Warner Instruments</td><td>LCS-1</td><td>To regulate perfusate temperature during imaging</td></tr><tr><td>Cable assembly for heater controllers</td><td>Warner Instruments</td><td>CC-28</td><td>To regulate perfusate temperature during imaging</td></tr><tr><td>Replacement bead thermisitor for CC-28 cable</td><td>Warner Instruments</td><td>TS-70B</td><td>To regulate perfusate temperature during imaging</td></tr><tr><td>Magnetic holder with suction tubing</td><td>Bioscience Tools</td><td>MTH-S</td><td>To hold the stainless steel vacuum suction tubing&amp;nbsp;</td></tr><tr><td>Adjustable holder</td><td>Bioscience Tools</td><td>MTH</td><td>To hold the temperature probe</td></tr><tr><td>clear silicone sealant</td><td></td><td></td><td>For ex vivo imaging chamber</td></tr><tr><td>superglue</td><td></td><td></td><td>For ex vivo imaging chamber</td></tr><tr><td>thin plexiglass strips</td><td></td><td></td><td>For ex vivo imaging chamber</td></tr><tr><td>nile red</td><td>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

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

轴突的相互作用与小胶质细胞和巨噬细胞在小鼠背柱破碎损伤活体成像

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Photothrombosis引起的局部脑缺血脊髓损伤的小鼠模型

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

医学

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 Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

猪缺血/再灌注模型中脊髓微灌注的实时评估

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

受控半自动激光诱导损伤研究斑马鱼幼虫脊髓再生

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...

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