Name: experimental strategies to bridge large tissue gaps in the injured spinal cord after acute and chronic lesion
Uploaded: 2016-04-05T16:00:00.000+00:00
Duration: 9 min 14 s
Description: 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

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...

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。

<table><tbody><tr><td>PEG 600 Ph Eur&amp;nbsp;</td><td>Merck/VWR&amp;nbsp;</td><td>8,170,041,000</td><td></td></tr><tr><td>Gelastypt gelatine sponge&amp;nbsp;&amp;nbsp;</td><td>sanofi Aventis</td><td>PZN-8789582</td><td></td></tr><tr><td>Nescofilm Sealant&amp;nbsp;</td><td>Roth</td><td>2569.1</td><td></td></tr><tr><td>Baytril</td><td>Bayer</td><td></td><td></td></tr><tr><td>Rimadyl (Carpofen)</td><td>Pfizer</td><td></td><td></td></tr><tr><td>Forene (Isoflurane)</td><td>Abbvie</td><td></td><td></td></tr><tr><td>Kodan (skin disinfectant)</td><td></td><td></td><td></td></tr><tr><td>Histoacryl (tissue glue)</td><td></td><td></td><td></td></tr><tr><td>Friedman-Pearson Rongeur, 1 mm cup, straight&amp;nbsp;</td><td>Fine Science Tools</td><td>16020-14</td><td></td></tr><tr><td>Two-in-one Micro Spatula - 12 cm&amp;nbsp;</td><td>Fine Science Tools</td><td>10091-12</td><td></td></tr><tr><td>Dumont #7 Forceps - Inox Medical&amp;nbsp;</td><td>Fine Science Tools</td><td>11273-20</td><td></td></tr><tr><td>Dumont #5/45 Forceps - Inox Medical&amp;nbsp;</td><td>Fine Science Tools</td><td>11253-25</td><td></td></tr><tr><td>Spinal cord hook&amp;nbsp;</td><td>Fine Science Tools</td><td>10162-12</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>14078-10</td><td></td></tr><tr><td>Clamp&amp;nbsp;</td><td>Aesculap</td><td>EA016R</td><td></td></tr><tr><td>Ethicon Vicryl 4-0</td><td></td><td></td><td></td></tr><tr><td>Bepanthen Augen- und Nasensalbe</td><td>Bayer</td><td></td><td></td></tr><tr><td>Anatomical forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11000-13</td><td></td></tr><tr><td>Self-retaining retractor&amp;nbsp;</td><td>Fine Science Tools</td><td>17008-07</td><td></td></tr><tr><td>Skin clamp&amp;nbsp;</td><td>Fine Science Tools</td><td>13008-12</td><td></td></tr><tr><td>Aluspray&amp;nbsp;</td><td>Selectavet</td><td></td><td></td></tr></tbody></table>

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）。...

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 ...

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Universidad de Cartagena UDEC

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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

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

控制宫颈裂伤损伤小鼠

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

科研

医学

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

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.

Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance growth.

结合周边神经移植和矩阵调制修复受伤的大鼠脊髓

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

生物学

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

神经科学

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

在实验颈脊髓损伤协同使用神经前体细胞，并自组装肽的

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

调查器官脊髓联合培养物中生长多电极阵列功能再生

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

氟 TrFE 导管内许旺细胞移植桥断大鼠脊髓残肢促进轴突再生跨越间隙

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

成年大鼠脊髓侧性横断与不对称行为评估

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

Development of Combinatorial Therapeutics for Spinal Cord Injury using Stem Cell Delivery

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in the US, and it represents an immeasurable public health concern. Research has been conducted to provide effective therapy; however, SCI is still considered incurable due to the complex nature of the injury site. A variety of strategies, including drug delivery, cell transplantation, and injectable biomaterials, are investigated, but one strategy alone limits their efficacy for regeneration. As such, combinatorial therapies have recently gained attention that can target multifaceted features of the injury. It has been shown that extracellular matrices (ECM) may increase the efficacy of cell transplantation for SCI. To this end, 3D hydrogels consisting of decellularized spinal cords (dSCs) and sciatic nerves (dSNs) were developed at different ratios and characterized. Histological analysis of dSCs and dSNs confirmed the removal of cellular and nuclear components, and native tissue architectures were retained after decellularization. Afterward, composite hydrogels were created at different volumetric ratios and subjected to analyses of turbidity gelation kinetics, mechanical properties, and embedded human adipose-derived stem cell (hASC) viability. No significant differences in mechanical properties were found among the different ratios of hydrogels and decellularized spinal cord matrices. Human ASCs embedded in the gels remained viable throughout the 14-day culture. This study provides a means of generating tissue-engineered combinatorial hydrogels that present nerve-specific ECM and pro-regenerative mesenchymal stem cells. This platform can provide new insights into neuro-regenerative strategies after SCI with future investigations.

In this study, nerve-mimetic composite hydrogels were developed and characterized that can be utilized to investigate and capitalize on the pro-regenerative behavior of adipose-derived stem cells for spinal cord injury repair.

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in ...

生物工程

A Minimally Invasive, Fast Spinal Cord Lateral Hemisection Technique for Modeling Open Spinal Cord Injuries in Rats

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique eliminates laminectomy by removing two spinous processes and lifting, then tilting the caudal vertebral arch. The surgical area opens up without the need for laminectomy. Lateral hemisection is then performed with direct visible control under a microscope. The trauma is minimized, requiring only a small bone wound. 
This technique has several advantages: it is faster and, therefore, less of a burden for the animal, and the bone wound is smaller. Because the laminectomy is eliminated, there is less chance for unwanted injury to the spinal cord, and there are no bone splinters that can cause problems (bone splinters embedded in the spinal cord can cause swelling and secondary damage). The vertebral canal remains intact. The main limitation is that the hemisection can only be performed in the intervertebral spaces. 
The results show that this technique can be performed much faster than the traditional surgical approach, using laminectomy (11 min vs. 35 min). This technique can be useful for researchers working with animal models of open spinal cord injury as it is widely adaptable and does not require any additional specialized instrumentation.

Here, we describe a new, fast technique modeling open spinal cord injury in rats that eliminates laminectomy. Lateral hemisection is performed while viewing through a microscope. The technique is versatile and can also be used in the cervical, thoracic, and lumbar regions of the spinal cord of other animals.

一种微创、快速脊髓侧向半切技术，用于模拟大鼠开放性脊髓损伤

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique ...