German Legal Casualty Insurance (DGUV), Research Commission of the Medical Faculty of the Heinrich-Heine-University

对动物的安全性和舒适性制度指导方针坚持和遵守者介绍了德国动物保护法（国办，环保和消费者保护北莱茵威斯特伐利亚，北威州LANUV所有外科手术前和手术后的动物护理）。 1.雌性Wistar大鼠的胸脊髓全横断（220 - 250克） <ol><li> 脊髓的制备 <ol><li>使用异氟烷吸入麻醉（2 -以1比在O 2 / NO 2的异氟醚3％:2）和卡洛芬（皮下[SC] 5毫克/千克）的推注。建议卡洛芬和阿片丁丙诺啡（0.02毫克/千克SC）的结合。手术开始时，眼睑反射性地用棉签和缩足反射性地用钳子捏刺激光触不再遵守。 </li><li>放置在加热毯动物在37℃保持身体temperatu重新手术并把眼膏眼睛上时避免在麻醉下干燥。 </li><li>刮动物的背部，并准备与皮肤消毒皮肤。 </li><li>切开皮肤在沿着胸椎中线4厘米的外科刀片并将其打开。从这一步，用消毒手术器械的所有程序（灭菌或浸泡消毒）。 </li><li>缩回使用小肌肉夹紧胸椎上方的肌肉。 </li><li>通过谨慎地剪裁的小骨片，直到椎骨是平的删除在胸8级（TH8）和TH9用咬骨钳棘突。 </li><li>使用镊子解剖解除在棘突Th7时脊柱并用咬骨钳，直到椎板切除术是在TH8和TH9执行从尾夹小块椎骨至喙。暴露硬脑膜而不通过一次仔细除去椎骨的只有少数和非常小的碎片损坏它。</li><li>钳由2稳定在夹子棘突Th7时和TH10骨干，提升动物从椎骨脱开呼吸运动。 </li></ol></li><li> 在胸科级8/9完全性脊髓横断 <ol><li>提起用细镊子硬脑膜，切在横向方向精眼科剪硬脑膜。 </li><li>握住细镊子硬脑膜的横向切口处插入一个脊柱钩子到硬膜和蛛网膜之间的subarachnoidic空间。避免刺不与任何钳或脊髓钩软或硬膜脑膜破损。 </li><li>缓慢旋转钩把它所有沿脊髓组织，小心不要刺入硬脑膜（PIA仍然完好）。 </li><li>抬起约1脊髓 - 向上2毫米，直到一个间隙在脊髓组织和硬膜之间的腹侧看到。 </li><li>将细眼剪刀插进空间betweeñ硬脑膜和软切脊髓，而脊柱钩子被留在原处。 </li><li>解除脊髓的两个断端用两个镊子和视觉确保完全横断。 </li><li>为了控制损毁动物和动物的慢性损伤，附近间断缝合硬脑膜与单丝非吸附9.0线程。 </li><li>对于慢性病灶遵循部分1.6。 </li></ol></li><li> 彩信植入 <ol><li>放置MMS损伤部位与两管躺在椎骨的每个横向侧的上方和它降低到病变腔。 </li><li>缝合管，以在与非吸收性4-0螺纹椎骨侧的肌肉，以确保MMS的稳定化。确保在此步骤中，使用镊子彩信的固定。 </li><li>用一对细剪刀切取下MMS连接器引脚。 </li><li>关闭硬脑膜彩信以上和9.0螺纹缝合它。 </li><li>附加一个管到真空泵，并且密封另一个由夹紧。 </li><li> 经由开口管用真空泵施加到MMS平缓负压，抽吸脊髓树桩到MMS内腔。申请几分钟的负压（最大为10分钟），并通过传感器监测（250 - 350毫巴）。 </li><li>切接近彩信管和取出管。与步骤1.6继续。 </li></ol></li><li> 脊髓组织切除包括初始伤后第5周的慢性损伤疤痕 <ol><li>按照步骤1.1.1 - 1.1.5。 </li><li>由棕黄色外观和硬组织对脊髓的顶端标识疤痕组织。轻轻地用细镊子持有和细剪刀去除瘢痕组织的表层。用这种方法，重新打开椎板切除的部位，露出含有脊髓损伤区域中的组织。停止准备当缝合硬脑膜肉眼识别。 </li><li>夹紧骨干2稳定夹在旋转OU的过程Th7时和TH10，然后提高动物从椎骨脱开呼吸运动。 </li><li>用小纸板尺子测量脊髓区域要被切除（长度:4毫米），标记与横向切口此相应组织区域的边缘，以允许随后的切除组织。 </li><li> 经切割和愿望的组合祛疤组织。取出已经从脊髓与横向切口分离组织。使用轻柔抽吸每当它足以允许去除组织。此外，每当瘢痕组织的质地僵硬，使组织的愿望太困难了，用细剪刀剪去，并删除该组织。 </li><li>将止血明胶海绵一块（大约5 x 5毫米×5毫米的立方体）进入组织间隙，直到出血补贴。因为它是浸有液体的明胶海绵的大小会立即收缩。 </li></ol></li><li> ImplaPEG 600 ntation <ol><li>制备1毫升纯的未稀释的PEG 600的注射用加热至37℃。 </li><li>除去明胶海绵。 </li><li>将足量（约5 - 7微升）的PEG为用10微升的注射器或10微升吸管的差距。 </li><li>小心覆盖密封剂/硬脑膜更换一块（约5mm×4mm）所的区域。 </li><li>固定在密封剂到肌肉组织周围与组织胶几滴。上放置的PEG-填充切除间隙顶部的密封剂。防止密封剂的滑移，可使用组织胶的小滴密封剂的角部固定到周围的肌肉组织。注意:避免到脊髓组织的组织胶泄漏！ </li></ol></li><li> 组织和术后护理闭幕 <ol><li>缝合肌肉和皮肤层进行间断缝合层编织吸附4-0线程</li><li>注入2×2.5毫升氯化钠（NaCl[0.9％]）在36℃的SC为手术后补液。 5毫升NaCl的单次注射将伸展动物的皮肤和可能会导致不必要的伤害的动物。直到完全意识恢复不要让动物无人值守。 </li><li>注入每日卡洛芬（SC 5毫克/公斤），用于在手术后至少2天。房子动物单笼中的第2天，随后在2组 - 3。 </li><li>申请的第一个手术后一周的抗生素治疗（恩诺沙星每日口服给药）。 </li><li>做人工膀胱轻轻喙尾鳍抚摸超过该动物的腹部以每天两到三倍排尿。要小心，不要动动物的脊柱和尾巴不举的动物。在整个生存时间每天手动作废完全spinalized动物的膀胱。必须小心放置食物颗粒和水瓶，其中可通过对动物没有达到的高度站在他们的后爪。虽然在后四分之一的功能被削弱，动物移动和手术后立即积极探索笼子。建议保持大鼠有点大男子主义团体。 </li><li>对于慢性病灶留疤切除前5周内动物的存活时间。按照步骤1.4。 </li></ol></li></ol>

<ol>
	<li>Ramer, L. M., Ramer, M. S., Bradbury, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoring+function+after+spinal+cord+injury:+towards+clinical+translation+of+experimental+strategies.">Restoring function after spinal cord injury: towards clinical translation of experimental strategies.</a> The Lancet. Neurology. 13 (12), 1241-1256 (2014).</li><li>McDonald, J. W., Howard, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repairing+the+damaged+spinal+cord:+a+summary+of+our+early+success+with+embryonic+stem+cell+transplantation+and+remyelination.">Repairing the damaged spinal cord: a summary of our early success with embryonic stem cell transplantation and remyelination.</a> Prog. Brain Res. 137, 299-309 (2002).</li><li>Yoon, S. H., 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=Complete+spinal+cord+injury+treatment+using+autologous+bone+marrow+cell+transplantation+and+bone+marrow+stimulation+with+granulocyte+macrophage-colony+stimulating+factor:+Phase+I/II+clinical+trial.">Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: Phase I/II clinical trial.</a> Stem Cells. 25 (8), 2066-2073 (2007).</li><li>Brotchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+spinal+cord+tumor+resection.">Intrinsic spinal cord tumor resection.</a> Neurosurgery. 50 (5), 1059-1063 (2002).</li><li>Estrada, V., Tekinay, A., Muller, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+ECM+mimetics.">Neural ECM mimetics.</a> Prog. Brain Res. 214, 391-413 (2014).</li><li>Tetzlaff, W., 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=A+Systematic+Review+of+Cellular+Transplantation+Therapies+for+Spinal+Cord+Injury.">A Systematic Review of Cellular Transplantation Therapies for Spinal Cord Injury.</a> J.Neurotrauma. 28 (8), 1611-1682 (2010).</li><li>Brazda, N., 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=A+mechanical+microconnector+system+for+restoration+of+tissue+continuity+and+long-term+drug+application+into+the+injured+spinal+cord.">A mechanical microconnector system for restoration of tissue continuity and long-term drug application into the injured spinal cord.</a> Biomaterials. 34 (38), 10056-10064 (2013).</li><li>Estrada, V., 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=Long-lasting+significant+functional+improvement+in+chronic+severe+spinal+cord+injury+following+scar+resection+and+polyethylene+glycol+implantation.">Long-lasting significant functional improvement in chronic severe spinal cord injury following scar resection and polyethylene glycol implantation.</a> Neurobiol. Dis. 67, 165-179 (2014).</li><li>Rasouli, A., 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=Resection+of+glial+scar+following+spinal+cord+injury.">Resection of glial scar following spinal cord injury.</a> J.Orthop.Res. 27 (7), 931-936 (2009).</li><li>Schira, J., 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=Significant+clinical,+neuropathological+and+behavioural+recovery+from+acute+spinal+cord+trauma+by+transplantation+of+a+well-defined+somatic+stem+cell+from+human+umbilical+cord+blood.">Significant clinical, neuropathological and behavioural recovery from acute spinal cord trauma by transplantation of a well-defined somatic stem cell from human umbilical cord blood.</a> Brain. 135, 431-446 (2011).</li></ol>

The authors have nothing to disclose.

这里有两个不同的手术方法呈现给弥补后（1）急性完全横断和MMS植入和（2）慢性脊髓损伤和纤维瘢痕切除加PEG基质植入脊髓组织的空白。这两种策略导致组织保存和轴索再生，以及对治疗的动物的显著运动功能改善。彩信手术后该公司缝合硬脑膜植入脊髓内的MMS充足的固定是关键技术的一步。 彩信持有进一步治疗潜力由于其实现的内部微通道系统，其允许治疗活性液体局部灌注进入病灶芯通过， 例如 ，一个附加的渗透性微型泵7。其预期未来的临床应用，彩信材料应是生物可吸收。目前，连接器系统的基于丙交酯材料组成的制造是被测试。此外，具有电子导体材料的MMS的涂层将为了治疗电场施加到损伤脊髓建立。 作为慢性脊髓病变，另外一种策略其次，由于手术切除疤痕后，分离脊髓树桩的身体紧张出现太高如果几个毫米的间隙必须交叉。出人意料的是，低分子量的PEG 600被证明是高度合适的生物聚合物来填充产生的间隙。它允许一个稳定的组织桥促进血管生成和有益的细胞类型的细胞侵入的形成。据推测，PEG600的物理性质，像它的粘度，扮演重要的角色所观察到的效果，因为其它的PEG-类型的较高或较低的分子量和/或粘度分别不是有益的。 桥接急性损伤后的间隙较小的新型连接器系统导致早在4周时损伤后的明确功能改善。相应的动物达到BBB得分大约7到那个时候和MMS的动物和对照组之间存在明显的差异是明显的。长期受伤而接受了手术切除疤痕和PEG-植入也恢复比未治疗的对照显著好转，但改善是显着的大多是在稍后的时间点（后约16周）的动物。这些观察可能的伤害的性质进行说明（ 即 ，急性与慢性和1mM对4毫米的组织缺损）。在较大的病变的情况下，轴突横跨病变部位的再生生长需要较长的时间段。此外，它很可能是不使用后肢的较长时间周期会导致更高程度的退化的事件，因此，在更短的突出功能改进。 慢性SPI后，PEG处理未来的优化纳尔，脊髓损伤，通过组合方法，用生长促进（干）细胞如脐带血细胞10 在体内 ，目前正在测试的PEG的例如 ，附加的播种。

创伤性损伤的脊髓不仅导致轴突，但它进一步的结果，其中阻碍任何再生响应组织缺损的损失（综述见1,2）。脊髓组织通常是通过继发变性导致囊肿形成或孔和周围的病变区域丢失。最具实验性的治疗措施集中在不完全性脊髓损伤局部一样横断，挤压或挫伤损伤健康组织的剩余边缘。对于像创伤性意外或外科手术，肿瘤一样切除导致总横断完全性损伤，只有极有限的治疗选择今天3,4可用。完成横断后，脊髓断端回缩组织结果技工紧张，而使脊髓小的差距。大多数战略集中在填充组织，细胞或矩阵5,6这一空白。 这里，不同的策略提出，使用一种新的microconnector装置7分离的树桩即重新适应。为了重新适应两个树桩，机械力已被应用作为轻微的负压来完成这个（ 图1）。机械microconnector系统（MMS）是聚甲基丙烯酸甲酯（PMMA）的与蜂窝状孔（ 图1A）和设置有一个出口管道系统中的多通道系统。它被注入到从大鼠（ 图1C）完整脊髓横断造成的组织间隙。一个管可连接到一个真空泵来施加负压到MMS（ 图1D）。压力拉动断开脊髓树桩到MMS的蜂窝状孔，其具有微结构化壁以保持组织就位时压力被释放（ 图1B）。管可以在手术后原封不动并连接到一个渗透性微型泵，以便注入的物质进入病灶核心（ 图1E-F）。 除了脊髓另一种类型的从手术移除脊髓肿瘤的或固体慢性损伤疤痕导致几毫米，这不能由MMS迄今克服大组织间隙完全损伤的结果的一种急性横断。大多数患者脊髓损伤的慢性损伤受损。在这些患者中，一个充分发展的瘢痕占据病变芯。手术切除病灶疤痕是其中SCI实验后8,9，目前调查处理的概念。而切除过程本身可以不产生相当大的额外伤害来执行，需要得到的组织间隙与合适的基质，它允许并促进组织的再生，并且在脊髓损伤的特定情况下，神经纤维再生桥接维护和促进运动功能。它是发现，低分子量聚乙二醇（PEG 600）是用于此目的的非常合适的材料。其缺乏免疫原性和非常低的粘度允许平滑集成到周围的组织。生物聚合物的插入单独促进有益细胞，包括内皮细胞，周雪旺氏细胞，和星形胶质细胞，以及入侵-非常重要的-降和通过紧凑髓鞘8升纤维束以及它们ensheathment的轴突的再生和伸长率。发现这些再生的反应由持久功能改进陪同。切除瘢痕组织和PEG 600的随后植入的组合呈现桥接实质性脊髓组织缺损的安全和简单的，但非常有效的手段。

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			<td height="21" style="height:21px;">Self-retaining retractor&#160;</td>
			<td>Fine Science Tools</td>
			<td>17008-07</td>
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			<td height="21" style="height:21px;">Aluspray&#160;</td>
			<td>Selectavet</td>
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experimental strategies to bridge large tissue gaps in the injured spinal cord after acute and chronic lesion

后一个脊髓损伤（SCI），其中阻碍轴突再生病变芯的疤痕形成。侮辱脊髓肿瘤切除，或创伤性意外事故在促进一般组织修复以及神经纤维再生生长进入及以后的受影响的区域援助造成组织缺损后弥合受伤的部位。两个实验性治疗策略提出:（1）一种新型microconnector装置植入到一个尖锐而完全横断胸大鼠脊髓重新适应切断脊髓组织树桩，并经过SCI网站（2）聚乙二醇灌装慢性损伤大鼠疤痕切除术。此模型中的慢性脊髓损伤是一个完整的脊髓横断其处理之前造成5周。这两种方法都最近取得非常有希望的成果，促进轴突再生，有利于细胞的浸润和功能改进脊髓损伤的啮齿动物模型。 机械microconnector系统（MMS）是带有出口管道系统聚丙烯酸甲酯（PMMA）构成的多通道系统施加负压到MMS管腔从而拉动脊髓树桩成蜂窝结构的孔。其植入到1毫米的组织间隙后的组织被吸入到设备。另外，MMS的内壁微结构更好组织粘连。 在慢性脊髓损伤的方法的情况下，脊髓组织 - 包括疤痕填充病变区域 - 被切除超过4毫米长的区域。显微瘢痕切除后所得腔充满聚乙二醇（PEG 600），其被发现是提供细胞侵入，血管再生，轴索再生，甚至在体内紧凑髓鞘极好底层。

组织保存，轴突再生和脊髓急性全横断后植入彩信的功能效益 据证实，彩信急性植入稳定完全脊髓横断树桩，降低了组织（ 图2A 与 B）的收缩。作为可视化通过在矢状切面三色染色，在病灶芯纤维化瘢痕的绿色结缔组织染色比MMS植入动物（ 图2C）致密得 ​​多和更突出在控制损毁动物（ 图2D）。有趣的是，有在植入与对照损伤动物MMS可视通过免疫组织学染色针对ED-1在长的存活时间没有观察到的差异在巨噬细胞聚集在病变部位（未示出）。 图3A）。此外，内腔被血管化的MMS（ 图3B）和轴突结构血管接近被发现（未示出）。旷场巴索-蒂-布雷斯纳汉运动得分（BBB）的评估，在2和4周揭示MMS注入动物相对于对照损毁动物显著功能改善（ 图3C黑线）（ 图3C中灰线）手术后。 细胞浸润，血管重建术，轴突再生和疤痕切除和PEG植入后慢性脊髓损伤大鼠的功能改善 在慢性损伤模式这两个部分的，完整的胸脊髓横断的LS五周手术切除病灶疤痕最初受伤后没有造成任何额外的检测伤害或不适的动物。瘢痕切除和PEG 600的后续插入导致组织的再生这是在基质内可检测的（ 图4）。此外，细胞外胶原鞘的沉积-这是典型的疤痕组织的基底膜小梁-相比仅病变的对照（ 图4B）的PEG-治疗（ 图4C）后不如突出。相反只病变控制的慢性瘢痕（未示出），填充的基质切除网站是由轴突生长促进细胞切除后入侵。已经早一个星期后切除和处理几个有利的细胞类型中的矩阵区域可以被识别，例如内皮细胞（其被发现存在于再生血液vessels [ 图4D]），星形胶质细胞（ 图4F）和外周雪旺氏细胞（ 图4G）。切除后五周的PEG处理过的区域充满了无数的轴突型材（ 图 4E）。 该PEG矩阵促进轴突无数（ 图4D，E，G）的 ​​大幅增长再生。随着跟踪研究和免疫组化染色，各种升序和降序轴突的人口可确定其再生只进不出，但也超越了PEG-填充区域8。传输的EM具有长生存期（8个月）分析动物的处理区域的不仅证实雪旺氏细胞的存在，而且还发现了聚乙二醇基质8内的再生轴突的紧凑髓鞘。再生轴突型材经常被与血管生成的相关领域（ 图4D）。免疫组化染色证实说，那些在治疗切除地区发现的轴突分布密切与雪旺细胞有关，似乎由这些细胞已经在切除和移植后8月初的时间点有髓。 一位长期（8个月）行为研究表明慢性疤痕切除和PEG-治疗（ 图5）后大幅持久运动功能改进。 <img alt="图1" src="/files/ftp_upload/53331/53331fig1.jpg" /> 图1.彩信设计及操作原理 （ 一 ）彩信摄影图片，（B）MMS侧壁的粘接面的微观结构，比例尺:50微米，（CF）示意图:（C）implantatio彩信进入脊髓损伤，（D）的真空施加到吸组织成蜂窝结构体（红色箭头:负压）的N，（E）的粘合力保持在靠近脊髓树桩以仅一个距离在管腔通过4个内部微通道的药理学物质几微米，（F）分布（黑色箭头指向在微通道1 - 4）。输注是通过在入口端口蓝色箭头所描绘。在D中-电子仅MMS的一半被示出。从Brazda 等人 ，2013 7授权转载爱思唯尔。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53331/53331fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53331/53331fig2.jpg" /> MMS小鬼后图2.脊髓组织保存lantation。在6脊髓组织，RESP。与（A）总横断后7个月，无彩信植入物（B）。在（B）中的箭头表示在（A）中对应于MMS（箭头）的植入部位的脊髓区域。注意在与此相反的保存完好的结构与未处理的动物（B）的脊髓组织的（A）中的收缩。 MMS注入（℃）对对照动物后矢状脊髓切片的三色染色，而不植入物（D）。绿色:结缔组织，红:细胞质，黑色:nucleus.The病灶中心填充有在控制损毁动物（D，黑色箭头示出了病变震中）疤痕组织（绿色），而在处理过的唯一的边际瘢痕形成围绕MMS明显后14天（C）的动物。注意，MMS由蜂窝结构体的这是如果削减20微米切片组织处理过程中冲了出去。比例尺为（A，B）在（A），用于（C，D）的（D）:1毫米。从Brazda 等人 2013年7许可改性爱思唯尔。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53331/53331fig2large.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/53331/53331fig3.jpg" /> 图3.轴突再生，再血管化和开放现场自发得分彩信植入后。（A）与泛轴突标记（PAM）在矢状脊髓节5周完成脊髓横断和MMS植入后磷酸化神经丝免疫组织化学染色。彩信管腔由星号表示。彩信的墙壁（W）是由虚线标记线。请注意，在以前组织缺乏彩信流明众多染色轴突。比例尺:100微米。 （B）中的血管在MMS管腔免疫组织化学染色（血管性血友病因子vWF的]染色鉴定，绿色）。 （ 三 ）BBB运动评分植入彩信评估（黑线，N = 9） 与对照受伤的动物（灰线，N = 6）。左和右后肢的平均BBB（每组平均值）描绘与标准偏差。统计学显著差异是由星号（曼 - 惠特尼秩和检验，P &lt;0.05）标记。从Brazda 等人2013年7许可改性爱思唯尔。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53331/53331fig3large.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/53331/53331fig4.jpg" /> 图4。PEG矩阵促进疤痕以下切除治疗组织再生和有益细胞浸润。（A）苏丹黑染色显示，切除的间隙（不含苏丹黑染色）大部件在第1周后切除充满了组织。 （B，C）纤维疤痕病变与IV型胶原1周后切除的染色。密集的疤痕存在于动物的控制，而PEG处理的动物显示弱得多，更明显的免疫染色。血管生成（D）面积（WWF）含有轴突再生1周后切除术（与神经丝[NF]标识）的配置文件。 （E）许多轴突已经在5周后切除成长为治疗部位。 （F，G）这两种胶质纤维酸性蛋白阳性（GFAP +）星形胶质细胞和S100 +雪旺氏细胞侵入PEG矩阵，后者在与regenerat密切联系发现后1周治疗后ING轴突了。比例尺:（A）1毫米; （CF）100微米，（G），50微米。 （BG）:改编自埃斯特拉达等人在2014年8爱思唯尔的权限<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53331/53331fig4large.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/53331/53331fig5.jpg" /> 慢性脊髓损伤，疤痕切除和PEG-治疗后运动功能图5.改进。修改后的BBB（mBBB）运动成绩PEG处理的评估（PEG，黑色菱形，N = 13％至14时间点） 与控制动物，它共收到脊髓横断无疤痕切除术（TX，白色三角形，N =每个时间点13-14）慢性脊髓损伤后。场均得分mBBB均值+标准误差片面曼 - 惠特尼U检验，* P≤0.05，** P≤0.01，*** P≤0.001;从埃斯特拉达等人修改2014年8爱思唯尔的权限，WPL =周后初期病斑，WPR =周后切除术。错误吧= SEM（标准平均误差）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53331/53331fig5large.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 Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion at JoVE.com

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

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 ...

experimental-strategies-to-bridge-large-tissue-gaps-injured-spinal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.9K 次观看.  Heinrich-Heine-University Medical Center. 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.

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

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二:外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

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

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 degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

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 ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

在大鼠颈脊髓弥散成像

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

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

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

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 ...

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 ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

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) ...