This project was funded by a grant from the Multiple Sclerosis Society of Canada and the Alberta Innovates - Health Solutions CRIO Team program. MBK is a recipient of studentships from Alberta Innovates - Health Solutions and the Multiple Sclerosis Society of Canada. SKJ is funded by a graduate student support grant from the Alberta endMS Regional Research and Training Center of the Multiple Sclerosis Society of Canada. The authors wish to acknowledge Dr. Jan van Minnen and the Regeneration Unit in Neurobiology core facility for training and use of equipment.

注：在此过程中使用的动物按照加拿大议会动物护理（CCAC）的指导方针照顾。伦理学批准了卡尔加里大学的动物护理委员会。 1.准备注射器注射<ol><li>溶解溶血卵磷脂至在磷酸盐缓冲盐水的1％溶液（PBS; pH 7.4）中并储存在-20℃以小等分试样（75微升）。解冻一小瓶至RT。 注意：如果溶血卵磷脂是未溶解，超声处理所述管中的超声波清洗器（40千赫）约30分钟，形成均匀的溶液。 </li><li>处理好与格外小心预拉玻璃毛细管，以避免损坏细腻技巧。旋10微升注入注射器的螺母和螺纹它在毛细管后跟2套圈的平坦端，确保套圈的配合端部对齐和毛细是紧贴在锥形管头（ 图1）。冲洗isoprop注射器基酒精和取出活塞。一旦干燥，紧绷螺丝针组件手到注射针筒。 </li><li>首先将金属毂针起动注射器。皮尔斯的橡胶盘与针和向下滑动到基座上。填写吸注射器溶血卵磷脂。轻踏吸注射器直至液体是在针的尖端可见。这确保气泡不会被引入到注入注射器。 </li><li>插入起动注射器的金属毂针头插入注入注射器。做一个坚定的密封，橡胶盘，慢慢地踩下吸式注射器，直到毛细管填充与解决方案的提示。小心地取出吸注射器同时按下填充注射针筒的筒体而不引入气泡。将活塞插入所述注入注射器，并确保从毛细管的前端溶液流动，柱塞被轻轻按下。 注：如果有任何气泡在帽可见illary或注入注射器，该注射器制剂必须重复从开头。在注射仪器连续流体对于确保精确注射量。 </li><li>安装完成注入注射器立体定向显微的手臂。这样就完成设备将能够无需重新填充之前注入15-20只动物。 </li><li>丢弃的注射器吸剩下的溶血卵磷脂。撤回并踩下异丙醇几次，拆下金属毂针。等待几个小时为剩余的异丙醇，在吸注射器填充之前再次蒸发。 </li></ol> 2.准备动物的手术过程注：此过程被描述为女性C57BL / 6小鼠，8-10周龄。 <ol><li>麻醉动物用腹膜内注射氯胺酮（200毫克/千克）和赛拉嗪（10毫克/千克）或每机构动物护理法规。该计划动物如果使用注射麻醉是在麻醉下进行约1小时。 </li><li>测试动物深受牢牢捏脚麻醉。经过适当麻醉动物不会回应的压力。 </li><li>使用快船，刮胡子的动物，靠近耳朵的背侧一个2-3厘米的2区。要小心，不要损坏耳朵。 </li><li>擦拭区域的清洁与加到纱布垫70％的乙醇。确保所有修剪头发已经从该地区被删除。消毒用碘的区域。 </li><li>适用凡士林眼睛，以防止整个过程中干燥。 </li><li>保持动物在加热的回收室，直到准备开始的过程。 </li></ol> 3.执行手术过程注：确保所有步骤的程序足够的无菌技术。这包括正确使用手套，头饰，面具，和窗帘的。所有工具应该进来之前联系消毒吨与动物。 <ol><li>移动的动物到立体定位框架，背侧，升高在中间由折叠的纸巾部分夸大脊柱的曲率。固定手臂和尾部与手术胶带和固定头牙钳。稳定耳棒是没有必要的此过程。 </li><li>用手术刀做一个3厘米中线切口，开始仅低于耳朵和切割在尾方向。 </li><li>找到2个大的脂肪结构之间的鸿沟，并用细镊子双手各拉这些分开。传播拉钩打开手术区域。 </li><li>在手术显微镜，找到T2椎体的突出生长过程（注：此功能是C57BL / 6小鼠品系的特征）。通过叠加肌肉组织进行钝性分离封闭弹簧剪刀，以更好地可视化T2。使用镊子，感觉T3和T4的硬表面，以确认正确的解剖位置。 </li><li>使用弹簧剪刀，使T3和T4之间的结缔组织浅侧向切口（2-3毫米深）。由于在小鼠脊柱的上部胸椎部的椎骨之间的自然间距中，椎板切除术是没有必要的，揭示脊髓。要留意的太深削减刺破和损坏电源线。 注：在这一步小程度的出血是常见的。如果发生这种情况，保持海绵矛入区域，直到出血消退（30-60秒）。 </li><li>可视化的脊髓。它将覆盖着厚厚的一层硬膜可见，如果还没有削减这脑膜层而露出电源线。一个突出的血管运行尾/喙通过脊髓的大致中线。 注：此血管不应该被用来作为一个具有里程碑意义的中线。相反，适当的照明应显示灰度白质边界侧翼的背柱，这些应该被用来估计中线。 </li><li>如果硬脑膜保持不变，让温柔的侧面擦伤了32克的金属针，直到它被清除。目标是去除硬脑膜而不是切割剩余底层脑膜，这是不一样厚，更难看到。 注：脑脊液推出表示违反蛛网膜的，虽然这可能会发生没有机械损坏的组织，累积脑脊液应用海绵矛被移除以更好地可视化电源线的表面上。 </li><li>移动注入注射器到位并慢慢降低，直到毛细管的尖端刚好触及脊髓立即横向中线的任一侧。锁定到位的手臂。 </li><li>使用Z方向立体定向臂的分级测量作出基线位置测量。从这个读数，减去1.3毫米。使用快速而浅的向下运动刺穿组织，然后小心地将毛细管，直到新的测量为止。可选：如果需要的话，病变在背柱可通过在中线同一穿孔动作和0.3毫米（更多信息参见讨论）的深度来制造。 注意：这些值是特定的T3和T4之间的注射。如果选择加入到在脊髓的任何其它位置进行注射时，这些值必须来自任何可用小鼠脑图谱。 </li><li>使用显微压低溶血卵磷脂进入脊髓腹侧白质。使1旋转微操作的每5秒，2分钟，得到在0.5微升的最终体积。留在原地的毛细管2额外分钟，以防止液回流，然后小心地取出毛细管。 </li><li>配合单一缝合肌肉/脂肪组织覆盖脊柱。使用非间断缝合关闭皮肤。应用更多碘切口部位。 </li><li>将动物在加热的恢复室，直到它恢复，然后将其放回笼子。应用止痛药后操作上按机构照顾动物的规定。额外的术后护理通常不是必要的，因为动物是完全动态，并能自我喂食和饮水，尽快为他们从麻醉中恢复。 </li><li>重复该过程其余的动物。 注：使用熟练，操作上可以在每个动物的10-​​15分钟内完成，特别是第二个人收紧的缝合线的帮助。相同的玻璃毛细管，可用于约15-20手术前尖变钝，应予以替换。如所述的，用注射的PBS进入脊髓，代替溶血卵磷脂控制手术可以相同进行。我们建议不要在清洁毛细管供将来使用。 </li></ol> 4.组织处理和分析<ol><li>牺牲动物用氯胺酮（500毫克/千克）和赛拉嗪（25毫克/千克）在所希望的时间点腹膜内过量。病变通常发展在FOLL由于方式：1-3天，积极脱髓鞘; 3-7天，OPC招聘; 7-10天，少突胶质细胞分化; 10-21日，活动髓鞘再生2,10,11。 </li><li>为了制备组织用于组织学，首先执行一个穿心灌注12用20毫升室温的PBS随后在PBS20毫升冰冷的4％多聚甲醛。对于准备树脂包埋组织的半自动或超薄切片，请参阅步骤4.9。 </li><li>采用弧形骨剪刀通过每个椎骨起始于脊柱的颈椎端和工作向下到较低胸椎水平切断除去脊髓。修复脊髓O / N在PBS的4％多聚甲醛在4℃。切换帘线以在PBS中的30％的蔗糖在4℃下至少72小时。 </li><li>确定注射部位作为对脊髓的背侧表面的异常。切割3毫米片组织（与注射部位的中心）和对准件在含有最佳切割温度cryomolds（OCT）共mpound。暂停cryomolds的下侧中的2-甲基丁烷和干冰冷冻混合物，直到OCT是完全冻结。储存在-80°C，直到准备部分。 </li><li>部上低温恒温器的脊髓在20微米的宽度到显微镜载玻片，并允许在空气中干燥O / N保存的幻灯片在-20℃之前。 </li><li>检测用髓鞘13的铬花青组织学染色病灶的位置和大小。完成所有步骤，在RT。风干载玻片30分钟。地方的幻灯片中的每个1分钟清除剂为1分钟，随后再水化在梯度乙醇溶液（100％，95％，90％，70％，50％，水）。 <ol><li>地方的幻灯片在15分钟羊毛铬花青溶液中，然后用1分钟的洗涤用水。区分在0.5％氢氧化铵，持续10秒，随后是1分钟洗涤用水。在脱水梯度乙醇解决方案，持续1分钟每个，盖玻片与安装介质，并且图像明亮场英里（如上所述反序）croscope。 </li></ol></li><li>用免疫组化可视化轴突和髓鞘。风干载玻片30分钟。脱水用在RT梯度乙醇（水，50％，70％，90％，95％，100％）为每2分钟，再反向顺序返回到水中。这一步会导致个别髓鞘环14更大的分辨率。 <ol><li>方框，用10％山羊血清和0.25％的Triton X-100的PBS在RT下60分钟的非特异性抗体的相互作用。在PBS中孵育载玻片上O / N在4℃下稀释的初级抗体（1,000小鼠抗SMI312 1：2000，兔抗MBP 1）。 </li><li>为5分钟，用PBS于室温洗5次。在PBS中孵育载玻片上，在室温60分钟稀释的二抗（500的Alexa 488抗兔1：500，Alexa的546抗小鼠1）。为5分钟，用PBS于室温洗5次。摩滑动使用GELVATOL和图像上的荧光显微镜盖玻片。 </li></ol></li><li>用免疫组化可视化的少突胶质细胞谱系的细胞。遵循ST程序EP 4.7，省略脱水/再水化工序中，并用10％马血清代替山羊血清。使用下列一抗稀释液：山羊抗PDGFRα1:100，小鼠抗CC1 1：200，兔抗OLIG2 1：200。使用下面的二级抗体稀释度：Alexa的488抗山羊1：500，Alexa的594抗小鼠1：500，Alexa的647抗兔1：500。 </li><li>为了制备组织为半或超薄切片，首先执行一个穿心灌注12用20毫升室温的PBS，随后20毫升冰冷的4％多聚甲醛/ 1％在PBS中的戊二醛。按照步骤4.3中所述取出脊髓。修复O / N在4％多聚甲醛的PBS / 1％的戊二醛在4℃。 </li><li>确定注射部位如在步骤4.4中所述。切1mm的片含有注射部位的组织。对病变的任一侧附加1毫米块如果需要，可以制备为好。 </li><li>在通风橱中，加入10倍体积的1％的四氧化锇/ 1.5％亚铁氰化钾到在冰上的每个块60分钟。 注：四氧化锇是一种剧毒物质，应格外小心。在用四氧化锇接触的所有材料应置于玉米油（两倍体积为四氧化锇溶液）进行中和。 </li><li>用0.2M二甲胂酸盐洗帘线3次在室温10分钟，在玉米油丢弃。脱水用梯度乙醇溶液（水，50％，70％，90％，2倍，95％，2倍，100％，2倍与氧化丙烯），用于在RT下10分钟。 </li><li>在树脂嵌入线按照制造商的说明。 </li><li>削减块对超微和安装部分到水滴在显微镜载玻片。地方上滑动的热板上（约50℃）至干。 </li><li>通过在50℃下加入数滴1％甲苯胺蓝/ 2％硼砂溶液到载玻片10-15秒形象化髓鞘。用清水冲洗干净，干燥，盖玻片与安装介质。在明亮的视野显微镜的图像部分。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demyelination+and+remyelination+of+the+caudal+cerebellar+peduncle+of+adult+rats+following+stereotaxic+injections+of+lysolecithin,+ethidium+bromide,+and+complement/anti-galactocerebroside:+a+comparative+study.">Demyelination and remyelination of the caudal cerebellar peduncle of adult rats following stereotaxic injections of lysolecithin, ethidium bromide, and complement/anti-galactocerebroside: a comparative study.</a> Glia. 25 (3), 216-228 (1999).</li><li>Mei, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropillar+assays+as+a+high-throughput+screening+platform+for+therapeutics+in+multiple+sclerosis.">Micropillar assays as a high-throughput screening platform for therapeutics in multiple sclerosis.</a> Nat. Med. 20 (8), 954-960 (2014).</li><li>Powers, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remyelination+reporter+reveals+prolonged+refinement+of+spontaneously+regenerated+myelin.">Remyelination reporter reveals prolonged refinement of spontaneously regenerated myelin.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (10), 4075-4080 (2013).</li><li>Smith, K. J., Blakemore, W. F., McDonald, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+remyelination+restores+secure+conduction.">Central remyelination restores secure conduction.</a> Nature. 280 (5721), 395-396 (1979).</li><li>Jeffery, N. D., Blakemore, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+deficits+induced+by+experimental+spinal+cord+demyelination+are+abolished+by+spontaneous+remyelination.">Locomotor deficits induced by experimental spinal cord demyelination are abolished by spontaneous remyelination.</a> Brain. 120 (Pt 1), 27-37 (1997).</li><li>Duncan, I. D., Brower, A., Kondo, Y., Curlee, J. F., Schultz, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+remyelination+of+the+CNS+leads+to+functional+recovery.">Extensive remyelination of the CNS leads to functional recovery.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (16), 6832-6836 (2009).</li></ol>

The authors have nothing to disclose.

已经开发了许多动物模型来研究MS，最可辨别的实验性自身免疫性脑脊髓炎（EAE）模型。在EAE，啮齿动物的免疫接种髓鞘肽的片段，并进行炎症病变的发展体现在上行性麻痹。虽然这种模式已经为免疫调节药物MS临床前测试非常有用，它不是理想的研究髓鞘主要有三个原因：为半自动或超薄组织处理时首先炎性病灶的位置是随机和定位病变部分可以是具有挑战性的。第二个是髓鞘再生发生在一个特定的时间过程，以及一个单一的EAE病变的确切年龄不能在没有连续的非侵入性磁共振成像是已知的。第三是髓鞘再生是下列药物治疗中的EAE可能不是药物的主要结果自然发生的现象，在啮齿动物，和髓鞘再生的证据，但在代替减少炎症的继发现象。 制造脱髓鞘另一种常见的方法是通过引入铜螯合剂cuprizone在饮食实现。这将导致广泛的脱髓鞘，最主要的是在胼胝体。有在研究胼胝体作为站点髓鞘再生的原因如下限制：首先，轴突直径（因此髓鞘厚度）比其它CNS区域更小，并且因此薄remyelinated护套可以是从那些从未脱髓鞘难以区分。其次，因为鼠标胼胝体中含有&gt; 70％的无髓鞘的轴突16，它可以是不清楚的一个remyelinated段是否损坏髓鞘或从头合成髓鞘的成人，这通常发生在17的真正修复。 这是我们的信念，为研究髓鞘再生的最佳模式是直接注射毒素，无论是溶血卵磷脂，ethid鎓溴化物，或其他人进入尾脑梗18或脊髓白质。前者位置仅通过精确3维立体定向注射来实现，并且被限制为较大的啮齿动物（大鼠）由于小脑梗的小尺寸。这不包括转基因小鼠的研究脱离和髓鞘的广泛资源。脊髓，但是，包含了许多大型白质是方便手术。在延髓胸椎段椎骨之间的空间允许对脊髓的曝光，而不需要一个椎板切除，这是在尾胸外科手术的一个必要步骤。特异性靶向腹侧白质的优点在于，所述的轴突比背侧白质均匀地较大，使得髓鞘再生的量化较少模糊的任务类似于与胼胝体相关的挑战。另外，腹侧白质组成一个更大的吨ARGET区注入;几百微米横向中背区将使毛细管柱外，而同偏差腹侧仍然会产生显着的脱髓鞘病灶。一些协议注入溶血卵磷脂到两个背侧和相同的动物19的腹侧列。这样可以提高正确毛细管放置两者的可能性和量化的病灶中较少的动物的数量。而呈现的当前数据是从8-10周龄的动物在操作的时候，我们还采用对8-10个月大的小鼠，其中髓鞘再生被描述为显着更慢4同样地进行了成功。 髓鞘的定量是不是一个平凡的事业。中央教条断定，remyelinated段长度较短和更薄的平均比健康同行，从而得到比的剖半直径：计算（轴突直径轴突+髓鞘直径除以）ř超薄切片已经成为标准程序。然而，已知的是remyelinated段加厚随时间2，并使用髓鞘再生少突胶质细胞的转基因报告最近的研究表明，许多节间最终成为从控制器20区分。量化病灶内成熟少突胶质细胞的数量是一种间接的方法来测量修复，因为少突胶质细胞是能够使茎节的宽一些，而髓鞘再生-取决于模型的一个显著比例使用-可以发生从雪旺氏细胞3。当然，由于髓鞘已被链接到恢复跳跃式传导21，修复的最终指标是神经功能缺损的功能恢复。而髓鞘再生已在若干物种22,23被链接到的功能恢复，它并没有成为在小鼠溶血卵磷脂研究的标准程序。这很可能是由于缺乏明显的observ的无论从背腹或病变能赤字，相比于更强大的脱髓鞘模型如EAE，甚至cuprizone。我们认为，从溶血卵磷脂注射液，和随后的恢复与髓鞘产生的功能缺陷，会用细感觉运动功能的敏感测试只能观察到。 “髓鞘”要么一起上面列出，虽然是一个粗暴的方法途径的动物模型的考研搜索，显示溶血卵磷脂（109）相比，EAE（188）和cuprizone（197）更少的搜索命中。如果我们的论点，即溶血卵磷脂脱髓鞘是研究髓鞘再生上级的做法，为什么最少的讨论？或许是忐忑，使用这种方法源于技术难度在执行手术的信仰。实际上，此过程是快速，成本效益，并且是不超过常规组织剥离比较困难，需要的材料都得到商业上出售。我们希望，这个协议证明了那些希望这个强大的模型添加到他们的剧目为研究髓鞘修复的精彩和扩大的领域非常有用。

多发性硬化症（MS）是中枢神经系统（CNS）的特征在于免疫细胞浸润和含有失去髓鞘，少突胶质细胞，轴突和神经斑块的慢性脱髓鞘疾病。大多数患者具有疾病过程包括炎症复发伴有广泛的神经系统症状，随后缓解期的。超过一半的患者最终没有明显的反复，但持续的神经衰落过渡到继发进展阶段。据认为，这逐步恶化是由于轴索损害和损失，有助于在由慢性脱髓鞘一部分。战略，以恢复丢失的髓鞘因此被认为有前途的治疗方法，以延缓疾病进展，或许逆转神经系统症状。 髓鞘再生是在CNS的内源性修复反应，由此从招募少突胶质细胞前体细胞中产生新的髓鞘（OPCs的）分化成髓鞘形成少突胶质细胞。髓鞘再生已显示在动物模型中是相当健壮1-3，然而，它的效率下降与4岁。事实上，髓鞘发生在人类身上，虽然它是不完整的，在大多数MS患者5。为MS所有目前可用的药物主要针对该疾病的异常免疫组分和，而在减少复发有效，不显着地延缓疾病的进展。下一代的用于MS的管理的治疗策略将为了防止两个复发和进展6结合在免疫调节与内源性髓鞘再生的增强进展。 一种方法来研究DE-和髓鞘再生在CNS涉及直接喷射洗涤剂溶血磷脂酰胆碱（溶血卵磷脂）进入脊髓白质1,3,7的。这个过程产生了很好的特点demyelinat荷兰国际集团的伤害主要由巨噬细胞/小胶质细胞浸润和活化8,9，无功星形胶质细胞增生，轴突动态平衡/轴索损伤，和OPC增殖和迁移10的扰动。病变可预见的演变过了几个星期的期限，并最终能够完全髓鞘再生的。这种方法已在研究参与DE-和髓鞘再生事件的编排是特别有用的。此外，它已被采纳为候选疗法的临床前测试的工具以下脱髓鞘损伤加速修复。

<table border="0" cellpadding="0" cellspacing="0" width="815">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:199px;">Company</td>
			<td style="width:163px;">Catalog Number</td>
			<td style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">spring scissors</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:163px;">15004-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:163px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">retractor</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:163px;">17003-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">clippers</td>
			<td style="width:199px;">Philips</td>
			<td style="width:163px;">QG3330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">heating recovery chamber</td>
			<td style="width:199px;">Peco Services</td>
			<td style="width:163px;">V1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">surgical tape</td>
			<td style="width:199px;">3M</td>
			<td>1527-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scalpel handle</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scalpel blade</td>
			<td style="width:199px;">Feather</td>
			<td>No. 15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sponge spear</td>
			<td style="width:199px;">Beaver Visitec</td>
			<td>581089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5-0 Vicryl sutures</td>
			<td style="width:199px;">Ethicon</td>
			<td>J511G</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">curved ToughCut spring scissors</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td>15123-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32 gauge metal needle</td>
			<td style="width:199px;">BD</td>
			<td style="width:163px;">305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">needle holders</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:163px;">12002-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">angled forceps</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:163px;">11251-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cotton tipped applicator</td>
			<td style="width:199px;">Puritan</td>
			<td style="width:163px;">806-WC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gauze pads</td>
			<td style="width:199px;">Safe Cross First Aid</td>
			<td style="width:163px;">3763</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">10 &#956;L syringe</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:163px;">7635-01</td>
			<td style="width:239px;">make sure to purchase the microliter, not gastight syringe</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">compression fitting</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:163px;">55750-01</td>
			<td style="width:239px;">contains the 2 ferrules and removable nut, but the nut that comes with the 10 &#956;L syringe is a tighter fit</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">priming kit</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:163px;">PRMKIT</td>
			<td style="width:239px;">contains the priming syringe, removable hub needle and the rubber discs</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">pre-pulled glass capillaries</td>
			<td style="width:199px;">WPI</td>
			<td style="width:163px;">TIP10TW1 (pack of 10)</td>
			<td style="width:239px;">contains capillaries with 10 &#956;m inner diameter. 30 &#956;m inner diameter also work (TIP30TW1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stereotactic frame</td>
			<td style="width:199px;">David Kopf instruments</td>
			<td style="width:163px;">Model 900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasonic cleaner</td>
			<td>Fisher Scientific</td>
			<td>FS-20</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Tissue-Tek optimal cutting temperature (OCT) compound</td>
			<td style="width:199px;">VWR</td>
			<td style="width:163px;">25608-930</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Tissue-Tek intermediate cryomold</td>
			<td style="width:199px;">VWR</td>
			<td style="width:163px;">25608-924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cryostat</td>
			<td>Leica</td>
			<td>CM1900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bright field microscope</td>
			<td>Olympus&#160;</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ultramicrotome</td>
			<td>Leica EM UC7</td>
			<td>EM UC7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lysophosphatidylchoine</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:163px;">L1381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-methylbutane</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:163px;">M32631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton x-100</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:163px;">X-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">goat serum</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:163px;">G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse anti-SMI312 antibody</td>
			<td style="width:199px;">Covance</td>
			<td style="width:163px;">SMI-312R</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit anti-MBP antibody</td>
			<td style="width:199px;">Abcam</td>
			<td style="width:163px;">AB40390</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat anti-PDGFR&#945; antibody</td>
			<td style="width:199px;">R&#38;D Systems</td>
			<td style="width:163px;">AF1062</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit anti-Olig2 antibody</td>
			<td style="width:199px;">Millipore</td>
			<td style="width:163px;">AB9610</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse anti-CC1 antibody</td>
			<td style="width:199px;">Calbiochem</td>
			<td style="width:163px;">OP80</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Alexa Fluor 488 goat anti-rabbit IgG</td>
			<td style="width:199px;">Life technologies</td>
			<td style="width:163px;">A-11008</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Alexa Fluor 546 goat anti-mouse IgG</td>
			<td style="width:199px;">Life technologies</td>
			<td style="width:163px;">A-11003</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Alexa Fluor 488 donkey anti-goat IgG</td>
			<td style="width:199px;">Jackson Immuno</td>
			<td style="width:163px;">705-546-147</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Alexa Fluor 594 donkey anti-mouse IgG</td>
			<td style="width:199px;">Jackson Immuno</td>
			<td style="width:163px;">715-586-151</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Alexa Fluor 647 donkey anti-rabbit IgG</td>
			<td style="width:199px;">Jackson Immuno</td>
			<td style="width:163px;">711-606-152</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td>Oxoid</td>
			<td>BR0014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isopropyl alcohol</td>
			<td>Sigma</td>
			<td>109827</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ketamine</td>
			<td>CDMV</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">xylazine</td>
			<td>CDMV</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iodine</td>
			<td>West Penetone</td>
			<td>2021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vaseline petroleum jelly</td>
			<td>VWR</td>
			<td>CA05971</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sucrose</td>
			<td>Sigma</td>
			<td>S5016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eriochrome Cyanine R</td>
			<td>Sigma</td>
			<td>32752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sulfuric acid</td>
			<td>Sigma</td>
			<td>320501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iron(III) chloride</td>
			<td>Sigma</td>
			<td>157740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ammonium hydroxide</td>
			<td>Sigma</td>
			<td>320145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acrytol</td>
			<td>Leica Biosystems</td>
			<td>3801700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Citrisolv</td>
			<td>Fisher Scientific</td>
			<td>22-143-975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">horse serum</td>
			<td>Sigma</td>
			<td>H0146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glutaraldehyde</td>
			<td>Electron Micrscopy Sciences</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">osmum tetroxide</td>
			<td>Electron Micrscopy Sciences</td>
			<td>19150</td>
			<td>highly toxic</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">potassium hexacyanoferrate (II) trihydrate</td>
			<td>Sigma</td>
			<td>P3289</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">corn oil</td>
			<td>Sigma</td>
			<td>C8267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyvinyl alcohol</td>
			<td>Sigma</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glycerol</td>
			<td>Sigma</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cacodylic acid</td>
			<td>Electron Micrscopy Sciences</td>
			<td>12300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">propylene oxide</td>
			<td>Electron Micrscopy Sciences</td>
			<td>20401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EMBED kit</td>
			<td>Electron Micrscopy Sciences</td>
			<td>14120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Toluidine Blue O</td>
			<td>Sigma</td>
			<td>T3260</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium tetraborate decahydrate</td>
			<td>Sigma</td>
			<td>S9640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:199px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eriochrome cyanine solution</td>
			<td style="width:199px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ingredient</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td>Amount to add</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eriochrome Cyanine R</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td style="width:239px;">0.8 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sulfuric acid</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td style="width:239px;">400 mL 0.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iron(III)chloride</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td style="width:239px;">20 mL 10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td style="width:239px;">80 mL</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:216px;">*add eriochrome cyanine to sulfuric acid, followed by iron(III) chloride and water. Solution can be kept after use. Make fresh once per year.</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelvatol</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ingredient</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td>Amount to add</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td>140 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyvinyl alcohol</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td>20 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glycerol</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td>40 g</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">*mix well, place at&#160; 37 &#176;C overnight, centrifuge at 1960xg 30 min, aliquot into 40 tubes</td>
			<td style="width:199px;"></td>
			<td style="width:163px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental demyelination and remyelination of murine spinal cord by focal injection of lysolecithin

多发性硬化是特征在于含有丢失少突胶质细胞，髓鞘，轴突，神经元和斑块形成的中枢神经系统的炎性脱髓鞘疾病。髓鞘是一种内源性修复机制，由此新髓鞘产生扩散，招聘和少突胶质细胞前体细胞的分化成随后髓鞘形成少突胶质细胞，并且有必要保护轴突从进一步的损害。目前，所有的治疗多发性硬化症的治疗目标疾病的异常免疫组件，它降低炎症复发，但不阻止进展为不可逆的神经衰退。因此，当务之急是髓鞘再生，促进战略，将开发出可延缓疾病进展，或许逆转神经系统症状。脱髓鞘的几种动物模型存在，包括实验性自身免疫性脑脊髓炎和curprizone;但是，也有LIMItations在将其用于研究髓鞘再生。更可靠的方法是焦点注射毒素进入中枢神经系统，包括洗涤剂溶血卵磷脂进入啮齿动物的脊髓白质。在这个协议中，我们表明，涉及注入溶血卵磷脂到小鼠的腹侧白质的手术过程是快速，成本效益，并且需要比那些市售没有额外的材料。这个过程是很重要的，不仅用于研究参与的髓鞘再生过程中的正常事件，但也可作为临床前工具用于筛选候选髓鞘再生促进治疗剂。

焦注射溶血卵磷脂到腹侧白质的产生离散的脱髓鞘病灶可检测超过大约3毫米（ 图2）的距离。病变芯为髓鞘（MBP）和轴突（SMI312）的免疫组织化学染色表明，已经在第7天（ 图3）被剥离髓鞘的轴突。通过14天，许多轴突通过MBP阳性环，这表明髓鞘再生的发生包围。染色的少突胶质细胞谱系（PDGFRα，OLIG2，CC1）中的细胞，存在于细胞的第14天相比，至7天，以及相比的OPCs成熟少突胶质细胞的分布两者的总数显著增加（ 图4） 。与此相一致的发现，甲苯胺蓝染色半薄切片揭示的薄髓鞘在该很少在第7天（ 图5）检测到的14天的存在，这表明这些remyelinated我nternodes。 该过程是动物之间高度重复性。当沉重的呼吸改变的固定位置发生变化毛细血管这通常不是一个问题，有足够的镇静作用。损坏轴突似乎是最小的，除非是在病变部位，由于最早使用的模型1的已经描述的中心。我们认为，这是从玻璃毛细管机械性损伤，因为它也可观察到的在PBS中注射对照组。然而，变异性变小的倾向，并且我们和其他人使用的相似的方法检测到的实验条件下用少至4只动物每组15之间的差异。 <img alt="图1" src="/files/ftp_upload/52679/52679fig1.jpg" /> 图1.大会注入注射器。（A）的注射针筒的螺母拧到吨<html>他的玻璃毛细管的平端，接着是2套圈使得其配合端互锁。一旦毛细管牢固地贴身在锥形套箍，该组件被拧紧的手到注入注射器的端部。（B）的片A的橡胶盘的中心与连接到该起动注射器的金属针头毂，并将其向下滑动到基。（℃）抽出的溶血卵磷脂溶液进入吸注射器（D）的轻踏吸注射器直至溶血卵磷脂的第一滴是在针的尖端可见。（E）将吸注射器插入注入的筒注射器，使得一个牢固的密封用橡胶盘。轻踏溶液直至它运行在毛细管的末端。仔细撤回起动注射器，同时保持压力在柱塞除去金属毂针而不引入气泡进入注入注射器。上传/ 52679 / 52679fig1large.jpg“目标=”_空白“&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52679/52679fig2.jpg" /> 图2.代表性溶血卵磷脂病变沾上铬花青。连续切片（相隔400μm的间隔）的特性的溶血卵磷脂病变的第14天染色的羊毛铬花青形象化髓鞘（蓝色）。需要注意的是脱髓鞘被限制腹侧白质和该病变跨越约3毫米延髓/尾方向。比例尺= 1毫米。 <a href="https://www.jove.com/files/ftp_upload/52679/52679fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52679/52679fig3.jpg" /> 图3.轴突和髓鞘溶血卵磷脂模型。 （A）假（PBS）7天注入脊髓显示，被髓鞘环包围着健康的轴突。（B）溶血卵磷脂病灶7天显示裸露的轴突和髓鞘退化（C）在第14天，轴突（标注有白色箭头的例子）的比例与髓鞘环的再现有关。比例尺= 10微米。 <a href="https://www.jove.com/files/ftp_upload/52679/52679fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52679/52679fig4.jpg" /> 图4.少突胶质谱系细胞的溶血卵磷脂模型。 （A，C）在7天，有PDGFRα+的OPC的更大的代表性（白条），相对于CC1 +少突胶质细胞（红色条）;酒吧内的数字代表百分比。 OLIG2被探测到确认少突胶质谱系细胞的染色。（B，C）在第14天，有是在少突胶质细胞谱系细胞则为7天（P &lt;0.05，双尾t检验的总数一显著增加，n = 4的每组）。也有少突胶质细胞对OPCs的分布为14天，而到7天（P &lt;0.0001，Fisher精确检验）一显著增加。比例尺= 10微米。每个字段被捕获在60X的原始放大倍率。值是平均值±SD。 *表示P &lt;0.05。 <a href="https://www.jove.com/files/ftp_upload/52679/52679fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52679/52679fig5.jpg" /> 图5.髓鞘再生的溶血卵磷脂模型。 （A）截甲苯胺蓝染色的HEA半薄腹白质部分lthy小鼠显示轴突在宽范围内口径与各个髓鞘的厚度。（B）在第7天，缺乏髓鞘的是（右下）观察到相邻的未受影响的区域。（c）在14天，薄薄地髓鞘（标注有红色箭头的例子）出现在整个病变，指示remyelinated段。比例尺= 10微米。 <a href="https://www.jove.com/files/ftp_upload/52679/52679fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin at JoVE.com

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

实验脱髓鞘和小鼠脊髓髓鞘再生由溶血卵磷脂的焦点注射

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

experimental-demyelination-remyelination-murine-spinal-cord-focal

Research

JoVE Journal

Neuroscience

26.7K 次观看.  Hotchkiss Brain Institute at University of Calgary. Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

视频: Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury.
The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion.
In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.

Severe spinal cord injuries often result in tissue defects. Two possibilities are described to successfully bridge such gaps to promote tissue adaptation, regenerative responses and functional improvement in rats via implantation of a mechanical microconnector system after acute injury and five weeks after complete spinal cord transection.

实验策略急，慢性损伤后桥的损伤脊髓组织大差距

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the ...

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 ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

由亲炎细胞因子的内脑注射诱导的广度脑皮质脱叶的鼠模型

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

直接注射慢病毒载体突出大鼠脊髓中的多种运动途径

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

在Vivo直接重新编程居民胶质细胞到内神经通过病毒载体的脑内注射

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

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 ...