The Authors acknowledge the economic support by FAIP (Federazione Associazioni Italiane Paraplegici), &#8220;Neurogel-en-Marche&#8221; Foundation (France), Fondazione &#8220;La Colonna&#8221;.

注:所有的程序批准了米兰大学的审查委员会，并会见了意大利指南实验动物符合欧共体指令日期为1986年11月（609分之86/ EEC）。 1.准备细胞移植注:在第5次和在培养中对这些实验的第九通道之间使用的神经干细胞;被贴上了移植前的测试对于文化增殖和分化能力。免疫细胞化学12确定差异化的程度。 <ol><li>重悬的细胞以1×10 6个细胞/ 150微升（移植每只小鼠1×10 6个细胞）的浓度。因为过量的细胞所需的注射器装载目的准备至少1.2×10 6细胞每只小鼠。 </li><li>在10毫升的锥形瓶中洗涤细胞用神经干细胞的培养基13的3倍。在每洗涤工序中，沉淀的细胞通过离心（500×g离心5分钟，RT）。 </li><li>在最后的旋转之前计数细胞。 </li><li>离心细胞（500×g离心5分钟），然后吸出上清液，注意不要以除去任何细胞，但留下不超过25微升上清液。 </li><li>通过加入1ml稀释剂的C向细胞沉淀重悬在温和移液制备2倍的细胞悬浮液。 </li><li>立即染色前，准备了2倍染料溶液（4×10 - 6 M）中稀释下通过加入4微升PKH26乙醇染料溶液到1ml稀释液C在一个管中，充分混合以分散。 </li><li>迅速加入1毫升2×细胞悬浮到1ml 2×染料溶液，并立即通过吹打混匀（最终细胞密度将是1.2×10 7个细胞/ ml和2×10 - 6M的 PKH26）。 </li><li>孵育细胞/染料悬液1-5分钟。 </li><li>通过加入等体积的停止染色（2-毫升）在HBSS中的1％BSA溶液孵育1分钟。 </li><li>离心机细胞（500 XG 10分钟），并小心地取出上清。 </li><li>重悬细胞沉淀在10ml的HBSS和离心机（500×g离心5分钟）的。 </li><li>用10ml完全培养基洗细胞沉淀2次以上，以确保去除未结合的染料。 </li><li>重悬细胞沉淀在10ml细胞回收，细胞生存力和荧光强度的评估完全培养基。如果需要的细胞用于移植，洗涤，并在3.3×10 5个细胞/ 50微升的浓度重悬他们在无菌生理溶液。 </li></ol> 2.准备手术<ol><li>清洁和消毒的手术设备。 </li><li>通过用无菌试剂擦拭准备手术区域。成立了IH撞击装置。 </li><li>准备麻醉设备:主动气体清除剂，含有VetScav过滤称重装置，连续流动感应商会，氧气的基因rator，低流量O 2米的小鼠。清洁感应腔和手术过程中使用的掩模。 </li><li>麻醉动物，用2.5％（体积/体积）的异氟烷在氧气中（1L /分），并等待5分钟的流动诱导的药物生效后。检查晶须抽搐或如果有缓慢后肢撤回响应于夹持足垫。在手术过程中，降低了异氟醚浓度为2.0％（体积/体积）的异氟烷在氧气中（1升/分钟）。 </li></ol> 3.准备小鼠外科手术和移植<ol><li>保持雄性成年CD1小鼠（25-30克）至少3天的标准条件下的实验（22±2°C，湿度65％，与上午08时00分之间的人造光至08:00时）之前。 注:从手术准备，​​缝合，整个过程需时约40分钟。 <ol><li>为了识别动物，标志着尾由防水彩色墨水的手段。 </li></ol></li><li>使用ELECTRIC推剪切小鼠的背部毛从颈部，大约在T2的水平，到腰区。 </li><li>用碘溶液和乙醇（在无菌水中70％）消毒准备好的区域。 </li><li>治疗与子皮下（SC）注射200μl的庆大霉素（1毫克/毫升在无菌盐水中的溶液）的动物。 </li><li>适用的眼用润滑剂包括动物的眼睛，以防止干燥，并注入丁丙诺啡（皮下，0.03毫克/公斤），以减轻疼痛。 </li></ol> 4.椎板切除术<ol><li>放置在幻灯片温暖鼠标，以避免在手术过程中低温的问题。定位与背侧的动物。 </li><li>使一个纵切口，用手术刀在感兴趣的区域中，从T7至T12。 </li><li>持皮肤和浅表脂肪垫通过使用标准钳子（通常在第 5 次和第 6 次胸椎背侧进程之间的空间中找到）。 </li><li>算该容器作为T6下的过程然后移动到T7。 </li><li>将鼠标的腹侧下一点轴承以提高脊柱的曲率。通过与格雷夫钳子阻止它固定的脊髓。 </li><li>通过使用手术刀，直到接触层手术刀头背表面切椎旁肌两侧从T7和T10椎体水平。 </li><li>用手术刀剔掉从T7交界处，直到T10。停止在T8和T9的刺状突起之间的空间。切T8-T9和T9-T10之间的组织与微剪刀。 <ol><li>用咬骨钳去除T9过程。通过仔细地刮走的肌肉层与微剪刀暴露的交界处。继续下去，直到骨头暴露。 </li></ol></li><li>使用镊子，从薄层除去肌肉和打开椎骨之间的小空间。轻轻插入微型剪刀骨下，削减双方的椎板和删除这部分用钳子。 </li><li>与F移除椎板orceps揭露线。确保不留下任何免费或锯齿状的碎骨落后;用咬骨钳去除它们。 </li><li>使用小尖镊子除去骨膜以及任何骨碎片或肌肉可以是附近的切口。 </li><li>取出T9背过程中的上半部分，并继续向IH冲击器设备协议。 </li></ol> 5. IH冲击器设备协议（挫伤） <ol><li>鼠标放置在的IH冲击器设备的稳定平台的中间。 </li><li>阻止与两个合资定位臂与稳定的平台连接两个牙钳动物（左臂为胸椎，右手臂颈椎）。 </li><li>使用喙臂把握喙椎体（T8）的横向边缘。 </li><li>操纵尾臂以相同的方式来把握的T10椎体。 </li><li>将稳定平台的设备，降低头（直径0.75毫米），为接近帘线尽可能不接触它。 </li><li>发起冲击前，抬起头三个完整圈。 </li><li>与撞击器设置为递送的60 kdyn的力，在100毫米/秒执行挫伤。 </li></ol> 6.缝线和后护理<ol><li>缝合切口用4/0可吸收缝合线。在覆盖层去除的部位暴露脊髓;通过小针的方式缝合的组织在切割的末端。把两针上面，立即和脊髓照射下的网站。 </li><li>收使用两个或三个反射剪辑，而不会夹断底层肌肉皮肤。 </li><li>水合小鼠手术后用2ml生理盐水溶液皮下注射在后腰。 </li><li>鼠标放置回一个预热笼，以避免在手术恢复体温过低。地方笼上加热垫。 </li><li>通过检查监督，在急性期后损伤的小鼠大小膀胱，缝线和动物体重，一天两次。轻轻挤压膀胱（每天两次，连续7天），以避免尿路的感染（尿液可能是阴天，血性或含有任何沉淀物）。 </li><li>每天用生理盐水溶液注射治疗的小鼠一次（2毫升两天手术后皮下注射）和抗生素（庆大霉素0.2毫升;皮下注射）五天后的损伤。 </li><li>治疗小鼠术后镇痛丁丙诺啡（0.1毫克/千克;每天两次）处理3天后的损伤。 </li></ol> 7.尾静脉注射细胞注意:在下面的步骤用于注入细胞到尾静脉的步骤是证明。细胞可以也给予了椎管内移植用立体框架15-16，或枕大池17。要考虑到其他类型的细胞可以用这种方法进行移植，如间充质基质细胞是很重要的（ 例如 ，骨髓间充质干细胞，脂肪来源的干细胞，羊水细胞）。此外，其他的治疗方案，如纳米粒子可经尾静脉对脊髓损伤后注入。 <ol><li>用70％的乙醇和PBS清洗后再使用针头。处理的针和注射器只与无菌手套。 </li><li>重悬的细胞在试管和负载75微升细胞向0.3 ml注射器（29克针和0.33毫升注射器）。 </li><li>确保没有气泡细胞悬液中存在。保持注射器在水平位置，以避免细胞沉淀。 </li><li>将鼠标灯具的散热下方扩张尾静脉。 </li><li>抢鼠标轻轻就拉进了鼠标阻挡器可视化的侧面尾静脉的狭窄的蓝线。 </li><li>清洁用酒精棉签尾巴。一旦静脉被可视化，抓住尾部静脉中指左手和拇指之间。 </li><li>把针表面从地平线15°角，并确保斜角到了。 </li><li>注射50微升的细胞以0.33微升/秒的速率。注射后，以10秒的延迟针的回缩。收回针头缓慢，注意细胞悬液的可能外流。 </li><li>清洁注射器在负载之间步7.1。 </li></ol> 8.行为测试和后肢功能<ol><li>鼠标放置在开放的领域。 </li><li>挫伤与根据巴索鼠标规模（BMS）18上的旷场试验后评价运动功能和后肢恢复。 注:神经功能必须定期伤后4周18评定，从第3天。 </li></ol> 9.灌注<ol><li>在实验期结束时，通过腹膜内注射戊巴比妥钠（65毫克/千克）麻醉动物。用脚趾捏到evaluatE中的麻醉水平，并继续后，才鼠标反应迟钝的有害刺激。 </li><li>抑制动物在手术平面上的仰卧位。 </li><li>通过体壁和腹壁只是肋骨下方切开。分离肝脏隔膜。 </li><li>使用剪刀剪开膜片以暴露胸膜腔。 </li><li>切肋骨两侧，直到锁骨。 </li><li>用止血钳剑突软骨，然后将止血钳在头上。 </li><li>保持心脏的倒数第三与钳横向平面内。针插入左心室。 </li><li>用止血钳的心脏。此固定针，并防止泄漏。 </li><li>用剪刀剪断右心房。 </li><li>使PBS以通过动物泵（18毫升/分钟）。保持整个缓冲器输注期间该压力（3-4分钟;对应于约70毫升PBS）中。继续下去，直到心脏是干净的。</li><li>切换活塞以允许固定液（4％多聚甲醛在蒸馏水中的4％PFA）通过泵。允许在4％PFA通过动物泵为大约8-10分钟（300毫升）中。逐渐增加，达到一最大30毫升/分钟的压力。一个硬化的肝脏是一个成功灌注的最佳适应证。 </li></ol> 10.组织收集和处理，组织学和Iimmunohistochemistry <ol><li>解剖脊髓从T5到L1。然后后修复组织的6ml在4％PFA（O / N，4℃）。 </li><li>把相同的组织中，在4℃下，用30％的蔗糖的溶液72小时，以冷冻保护它，并防止在冷冻过程中晶体的形成。 </li><li>用干冰迅速冷冻的线，并将其存储在-80℃。 </li><li>用15微米厚的低温恒温器的方式部分和收集部分到玻片上，继续进行免疫。 </li><li>冲洗切片用200μl的PBS各S立德（3次;每次5分钟，RT）。 </li><li>透每个幻灯片用200μl的10％NGS和0.2％的Triton X-100的PBS中进行1小时，在室温。 </li><li>冲洗用200微升PBS每张幻灯片（3次;每次5分钟，RT）的部分。 </li><li>阻断非特异性位点用200μl阻断对滑动溶液（5％NGS; 0.1％的Triton X-100的PBS）中30分钟（RT）。 </li><li>孵育各滑动用200μl初级抗体O / N的在4℃下（稀抗体在5％NGS;在PBS中的0.1％的Triton X-100）。 </li><li>用200μlPBS中的每个滑动洗切片（3次;每次5分钟; RT）下。 </li><li>孵育2小时在RT适当的二级抗体。 </li><li>用200μlPBS中的每个滑动洗切片（3次;每次5分钟; RT）下。 </li><li>染色细胞核用200μl的4&#39;，6- diamidin -2- phenilindole吲哚（DAPI）（1μg/ ml的终浓度，在室温下10分钟）。 </li><li>通过使用FluorSave试剂安装和共聚焦显微镜分析。 注:在控制测定，初级抗体必须被省略，且具有同等的浓度相同亚类的无关的IgG替换。微管相关蛋白2的第一抗体被使用。 </li></ol>

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M., 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=Basso+Mouse+Scale+for+locomotion+detects+differences+in+recovery+after+spinal+cord+injury+in+five+common+mouse+strains.">Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.</a> J Neurotrauma. 23 (5), 635-659 (2006).</li><li>Hofstetter, C. P., 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=Allodynia+limits+the+usefulness+of+intraspinal+neural+stem+cell+grafts;+directed+differentiation+improves+outcome.">Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome.</a> Nat Neurosci. 8 (3), 346-353 (2005).</li><li>Bottai, D., Madaschi, L., Di Giulio, A. M., Gorio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability-dependent+promoting+action+of+adult+neural+precursors+in+spinal+cord+injury.">Viability-dependent promoting action of adult neural precursors in spinal cord injury.</a> Mol. Med. 14 (9-10), 634-644 (2008).</li><li>Bottai, D., 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=Embryonic+stem+cells+promote+motor+recovery+and+affect+inflammatorycell+infiltration+in+spinal+cord+injured+mice.">Embryonic stem cells promote motor recovery and affect inflammatorycell infiltration in spinal cord injured mice.</a> Exp Neurol. 223 (2), 452-463 (2010).</li></ol>

作者宣称没有竞争经济利益。

在本文中，我们描述了在70克迪涅（重度）的力的方法来获得外伤性脊髓损伤的可重复使用的模型是无限的地平线撞击。使用较大的力的范例（80克迪涅），我们可能会导致不幸的是具有较高的小鼠的死亡率相关联的更严重的伤害。为了避免这个问题，我们一般选择温和的力量范式（70克迪涅），使用功能和较低的死亡率逐渐复苏关联到一个可重复的病变。为了产生这样的稳定​​损伤是非常重要的，要特别注意到撞击平台上的动物的正确固定;尤其是脊髓必须集中在冲击尖端，和冲击的两个臂必须彼此平行。此外，必须注意采取阻断与撞击镊子动物中，当椎骨可以粉碎并帘线可以由镊子的顶端受到损坏。阿尼玛的定位升的椎板切除术后是至关重要的，并且在此过程中的动物的处理也很重要。特别注意，也必须给予椎板程序，这必须始终在相同的位点和对于相同的扩展来执行。当椎板期间执行这些过程，另一个方法的问题并监控是当使用微型剪刀，咬骨钳，镊子或切割骨头，并通过去除薄层的自由帘线损坏电源线的风险的降低，消除了其余的横向骨突起和碎片，并除去骨膜。使用微型剪刀和咬骨钳与尖端朝上将减少遇到上述问题的风险。 这种方法的一个重要的限制是高概率的动物可能发展在损伤后时期的内部和外部严重泌尿系统感染。外部感染是由于损伤小鼠莫无力已经与后肢重量的支持。与此相反，内部尿路感染可能由受伤小鼠无法独立地排尿引起。为了避免这些问题，这是绝对必要的，以与所指示的抗生素注射动物，并在第一个星期的动物护理程序，每天检查膀胱的尺寸的两倍。在水合状态和重量必须仔细期间损毁后的前三个星期进行检查。 施加描述的方法，我们能够获得的那是通过一个特定的行为测试由巴索和他的同事（BMS）18开发评价后肢功能再现的赤字。紧接在损伤后是功能后肢损失完成，之后是一个逐渐恢复是在第一个2-3周的最重要的。行为恢复达到较高水平时损伤小鼠成年PM-的NPC（ 图1治疗</str翁&gt;）。我们还连续切片的细胞处理的小鼠的脊髓中，在伤后4周，并检测PKH26阳性的PM-NPC的通过共聚焦显微镜在病变（ 图3）的边缘的手段。我们估计，至关重要的接枝PM-NPC的总数大于所述接枝胚胎干细胞和成人神经干细胞如先前报道由我们的研究小组20-21。在损毁和移植后四周，大部分的PM-NPC的有较大的机构，并具有延长了由特异性抗体免疫染色完全向MAP-2（ 图4）的树突状突起。 外伤性脊髓损伤这种模式的主要优点是损伤的标准化。功能后肢复苏再现时间相关的曲线也取得。这样的再现性，能够以限定证明型的原理研究用于研究治疗，包括移植细胞的再生医学studies的情况下的数量减少。此外，脊髓损伤的病理生理学的几个方面可以进行更详细的分析。

脊髓损伤（SCI）是由高能量创伤样机动车事故中最常见的损伤，跌伤，运动和暴力1。在严重的SCI，伤害力摧毁或损害的神经组织，引起神经功能突然丧失。外伤性脊髓损伤经常发生在10和40岁之间的青壮年。这极大地影响了患者的精神和身体状况，并导致巨大的经济影响，社会2。在急性期的治疗方法往往仅限于高剂量糖皮质激素，手术稳定和减压，以减轻可能进一步损害3-4，但是SCI后，这些方法在运动恢复中的作用仍存在争议。除了 ​​急性组织丢失，跌打损伤和退行性变引起脱髓鞘多种细胞类型5-6和死亡继发性机制的激活。功能恢复程度可以了10被关联到幸免脑白质的损伤部位7的程度。 脊髓损伤的动物模型，可以使用这两种，调查组织损伤的生物反应，并测试潜在的治疗方法。此外，人体病理学的有用的动物模型不仅具有重现该条件的某些方面，但还必须提供超过直接的临床观察和实验的优点。脊髓损伤的最广泛使用的模型包括挫伤或压迫损伤传递到外科手术暴露脊髓8。受控体重下降挫伤的发展代表了SCI研究史上的一个重要里程碑。俄亥俄州立大学脊髓研究中心一直追求可用于诱导脊髓与冲击的由计算机9控制参数的特定压缩设备的技术挑战。这原本是专为使用无线网络日大鼠;后来它被修改申请对小鼠10。这种方法的优点是，损伤的生物力学能在深入研究以上和损伤的参数可以在一个更完整的方式，以获得一个重现的实验模型来限定，因此，允许的效果更精确的评估测试治疗对功能恢复的过程。 许多研究已评估了移植的效果在脊髓损伤模型11的各种干细胞。我们最近分离出的子脑室区（SVZ）几个小时成人神经干细胞的老鼠捐助12-13死后。此过程提供了神经干细胞，称为后验神经前体（PM-NPC的），这似乎是在再生医学的方法用于治疗脊髓损伤有利的群体。在本文中，我们将证明:i）在细胞标记协议的重要示踪剂PKH26，II）的外科学的iCal过程在外伤性脊髓损伤执行，以及iii）在静脉内（IV）标记的细胞的施用。另外，在本工作中，我们表明，移植的细胞移植到脊髓损伤部位并分化大多成微管相关蛋白（MAP）2阳性的细胞。此外，该分化伴有促进后肢功能的稳定的恢复。

<table><tbody><tr><td>PKH26GL-1KT&amp;nbsp;</td><td>Sigma</td><td>091M0973</td><td></td></tr><tr><td>Infinite horizon (IH) Impactor device&amp;nbsp;</td><td>Precision Systems and Instrumentation, LLC</td><td>Model 0400 Serial 0171</td><td></td></tr><tr><td>Gentamycin 10 mg/ml</td><td>Euroclone</td><td>ECM0011B</td><td>1 mg/ml in sterile saline solution</td></tr><tr><td>Isoflurane-Vet 250 ml</td><td>Merial</td><td>B142J12A</td><td></td></tr><tr><td>Blefarolin POM OFT 10 g</td><td></td><td></td><td></td></tr><tr><td>Slide Warmer</td><td>2Biological Instruments</td><td>HB101-sm-402</td><td></td></tr><tr><td>Scalpel, size 10</td><td>Lance Paragon</td><td>26920</td><td></td></tr><tr><td>Small Graefe Forceps</td><td>2Biological Instruments</td><td>11023-14</td><td></td></tr><tr><td>Rongeur</td><td>Medicon Instruments</td><td>07 60 07</td><td></td></tr><tr><td>Micro scissors</td><td>2Biological Instruments</td><td>15000-00</td><td></td></tr><tr><td>Absorbable sutures (4/0)</td><td>Safil Quick</td><td>C0046203</td><td></td></tr><tr><td>Hemostat</td><td>2Biological Instruments</td><td>13014-14</td><td></td></tr><tr><td>Reflex 7 wound clip applicator</td><td>2Biological Instruments</td><td>12031-07</td><td></td></tr><tr><td>7 mm Reflex wound clips</td><td>2Biological Instruments</td><td>12032-07</td><td></td></tr><tr><td>NGS</td><td>Euroclone</td><td>ECS0200D</td><td></td></tr><tr><td>Triton X 100</td><td>Merck Millipore</td><td>1086431000</td><td></td></tr><tr><td>Anti Microtubule Assocoated Protein&amp;nbsp; (MAP) 2</td><td>Millipore</td><td>AB5622</td><td></td></tr><tr><td>Alexa Fluor 488</td><td>Invitrogen</td><td>A11008</td><td></td></tr><tr><td>FluorSave Reagent&amp;nbsp;</td><td>Calbiochem</td><td>345789</td><td></td></tr><tr><td>Neural stem cells medium</td><td></td><td></td><td>DMEM-F12 medium (Euroclone) containing 2 mm l-glutamine (Euroclone), 0.6% glucose (Sigma-Aldrich), 9.6 gm/ml putrescine (Sigma-Aldrich), 6.3 ng/ml progesterone (Sigma-Aldrich), 5.2 ng/ml sodium selenite (Sigma-Aldrich), 0.025 mg/ml insulin (Sigma-Aldrich), 0.1 mg/ml transferrin (Sigma-Aldrich), and 2 &amp;mu;g/ml heparin (sodium salt, grade II; Sigma-Aldrich), bFGF (human recombinant, 10 ng/ml; Life Technologies) and EGF (human recombinant, 20 ng/ml; Life Technologies)&amp;nbsp;</td></tr><tr><td>DMEM-F12</td><td>Euroclone</td><td>ASM5002</td><td></td></tr><tr><td>l-glutamine</td><td>Euroclone</td><td>ECB3000D</td><td></td></tr><tr><td>glucose</td><td>Sigma-Aldrich</td><td>G8270-100G</td><td></td></tr><tr><td>putrescine</td><td>Sigma-Aldrich</td><td>P5780-25G</td><td></td></tr><tr><td>progesterone</td><td>Sigma-Aldrich</td><td>P6149-1MG</td><td></td></tr><tr><td>Sodium-selenite</td><td>Sigma-Aldrich</td><td>S9133-1MG</td><td></td></tr><tr><td>transferrin</td><td>Sigma-Aldrich</td><td>T 5391</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I1882</td><td></td></tr><tr><td>Heparin sodium-salt</td><td>Sigma-Aldrich</td><td>H0200000</td><td></td></tr><tr><td>bFGF</td><td>Life Technology</td><td>PHG0024</td><td></td></tr><tr><td>h-EGF</td><td>Life Technology</td><td>PHG6045</td><td></td></tr><tr><td>Syringe 0.33 cc 29 G</td><td>Terumo</td><td>MYJECTOR&amp;nbsp;</td><td></td></tr><tr><td>buprenorphine</td><td>Schering Plough SpA</td><td>TEMGESIC</td><td></td></tr><tr><td>eye gel</td><td>Bausch &amp;amp; Lomb</td><td>LIPOSIC</td><td></td></tr></tbody></table>

neural stem cell transplantation in experimental contusive model of spinal cord injury

脊髓损伤是一种破坏性的临床状况，其特征在于神经机能障碍的复合体。脊髓损伤的动物模型，可以同时使用，调查的生物反应损伤和测试潜在疗法。挫伤或压迫损伤传递到外科手术暴露脊髓是最广泛使用的模型的病理状况。在此报告的实验挫伤通过使用无穷水平（1H）撞击的设备，它允许创建一个可再现损伤动物模型中，通过具体的损伤参数定义进行。干细胞移植通常被认为用于固化本衰弱病症的潜在有用的策略。大量的研究已经评估移植了各种干细胞的影响。在这里，我们证明了脊髓损伤后尾静脉注射细胞在CD1小鼠的适应方法。总之，我们提供的程序:一）细胞标记无线网络第一个重要的示踪物，ⅱ）手术前护理小鼠，ⅲ）执行的挫伤性脊髓损伤，和验尸神经前体的四）静脉内给药。这个挫伤模型可以用于评估在再生医学的方法的有效性和干细胞移植的安全性。

移植的细胞的总数为1×10 6个细胞，并分成三个连续注射在尾静脉。我们在50μl磷酸盐缓冲液（PBS）施用3.3×10 5个细胞。在30分钟内，进行第一次注射损伤后，第二6小时后和病变后的最后18小时。的18小时SCI后一个时限给药PM-NPC的选择通过血脑屏障的渗透性最优，此时14进行测定。来评价干细胞注射的效果有阳性对照椎板切除术动物将是有用（N = 14）和PBS注射的动物作为阴性对照组（n = 14）。 PM-的NPC提高恢复后肢功能，迁移到病变部位和分化的MAP-2阳性细胞 在T9挫伤造成后肢功能瞬时丧失其次是渐进克radual恢复（ 图1）。在2-3周，PBS处理的小鼠受伤改进和后肢功能达到了3点的BMS（对应于足底放置有或没有配重支承或偶尔，频繁，或一致的背侧步进爪的，但不是足底步进18）的。取而代之的是，同样的观察期间内，以PM-NPC的治疗受伤的小鼠表现出较高的回收率，达到4.5分的BMS（相当于频繁或一致足底步进未经协调，或频繁的或一致的跖一些协调步进）。行为的改善尤其明显在脊髓损伤后第7天和第14天之间的期间。的异常性疼痛般的前肢超感性19没有任何迹象在整个30天的观察期内录得在任何时候以任何实验组。 最嫁接的PM-NPC的，标有PKH26（ 图2），在该边缘积累病变形成集群从他们管理的初期（ 图3）。然后移植细胞沿着病变边缘和在一个更为分散的方式在那里分化，逐渐假设神经元的非对称的细胞构象迁移。在损害和移植后30天，PM-NPCS的细胞体尺寸增大，并且在大多数细胞中的树突状突起是显而易见的，并通过特异性抗体免疫染色完全向MAP-2（ 图4）。实现形态复杂性和阳性对MAP2由移植的PM​​-NPC的很可能不是由于融合存活宿主脊髓神经细胞，这是显而易见的，它们明显不同的形态和没有两个核中的任何单个标记的细胞。 <img alt="图1" src="/files/ftp_upload/52141/52141fig1highres.jpg" /> 图1:PM-NPC的改善功能RECOV红霉素在受伤的动物的开放领域运动是测试用于运动功能恢复18的决心。动物挫伤前一天测试，在BMS规模拿下9分。在第一天的损伤后，在损伤的动物，在BMS评分下降到零。对损伤的小鼠后肢功能的恢复表现出显着和持久的改善，当动物与PM-NPC的处理。在双盲进行了分析，并且每个组由14只动物。值代表平均±SEM。我们确定通过ANOVA试验接着Tukey的检测后的装置的统计差异。 *** P &lt;0.001; ** P &lt;0.01，对PBS中。 <img alt="图2" src="/files/ftp_upload/52141/52141fig2highres.jpg" /> 图2 PM-NPCS的PKH26标记后用PKH26标记过程，PMNPCS是VISU<html> alized与实时图像显微镜EVOS佛罗里达州和显微照片被采取同样的手段（比例尺= 50微米）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52141/52141fig2large.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/52141/52141fig3highres.jpg" /> 图3.本地化的PM-的NPC在病灶部位。PKH26标记PM-NPC的（红色）在整个病变部位的第30天其静脉注射后的边缘存在。该图片代表1鼠标，但至少5动物（比例尺= 50微米），得到类似的图像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52141/52141fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 负载/ 52141 / 52141fig4highres.jpg"/&gt; 在移植的PM-NPC的大多数 PKH26标记PM-NPC的（红色） 图4. MAP2的表达获得一个神经元状与树突状突起和已分化的MAP-2阳性细胞（绿色）。细胞核被染成蓝色（DAPI）。该图片代表1鼠标，但至少5动物（比例尺= 25微米），得到类似的图像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52141/52141fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury at JoVE.com

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

脊髓损伤是一种创伤性条件，导致严重的发病率和死亡率高。在这项工作中，我们详细描述脊髓损伤的小鼠，随后通过神经干细胞的移植挫伤模型。

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 ...

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12.9K 次观看.  University of Milan. 脊髓损伤是一种创伤性条件，导致严重的发病率和死亡率高。在这项工作中，我们详细描述脊髓损伤的小鼠，随后通过神经干细胞的移植挫伤模型。 

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

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

椎管内针对肌萎缩性脊髓侧索硬化症和外伤性脊髓损伤颈腹角细胞移植

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

科研

医学

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

挫伤模型脊髓严重损伤的保护作用

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

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

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

发育生物学

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. ...

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 ...

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 ...

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

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

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

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

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

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

脊髓器官型培养物中 h-SC-NES 细胞的长期植入

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs.
Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (&#8805;30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

作者聚焦:用于推进脊髓再生疗法的长期脊髓切片培养

In this paper, we provide a reproducible method to generate and maintain long-term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies.

长期小鼠脊髓器官型切片培养作为验证脊髓损伤细胞移植的平台

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches ...