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>

<ol>
	<li>. Cord Injury Statistical Center: spinal cord injury facts and figures at glance Available from: <a target="_blank" href="https://www.nscisc.uab.edu/PublicDocuments/fact_figures_docs/Facts%202013.pdf">https://www.nscisc.uab.edu/PublicDocuments/fact_figures_docs/Facts%202013.pdf</a> (2013)</li><li>Yip, P. K., Malaspina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+trauma+and+the+molecular+point+of+no+return.">Spinal cord trauma and the molecular point of no return.</a> Molecular Neurodegeneration. 7, 6 (2012).</li><li>Fehlings, M. G., Cadotte, D. W., Fehlings, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+systematic+reviews+on+the+treatment+of+acute+spinal+cord+injury:+a+foundation+for+best+medical+practice.">A series of systematic reviews on the treatment of acute spinal cord injury: a foundation for best medical practice.</a> J Neurotrauma. 28 (8), 1329-1333 (2011).</li><li>Furlan, J. C., Noonan, V., Cadotte, D. W., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+decompressive+surgery+of+spinal+cord+after+traumatic+spinal+cord+injury:+an+evidence-based+examination+of+pre-clinical+and+clinical+studies.">Timing of decompressive surgery of spinal cord after traumatic spinal cord injury: an evidence-based examination of pre-clinical and clinical studies.</a> J Neurotrauma. 28 (8), 1371-1399 (2011).</li><li>Sekhon, L. H., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology,+demographics,+and+pathophysiology+of+acute+spinal+cord+injury.">Epidemiology, demographics, and pathophysiology of acute spinal cord injury.</a> Spine. 26 (24), 2-12 (2001).</li><li>Gorio, 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=Recombinant+human+erythropoietin+counteracts+secondary+injury+and+markedly+enhances+neurological+recovery+from+experimental+spinal+cord+trauma.">Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma.</a> Proc Natl Acad Sci U S A. 99 (14), 9450-9455 (2002).</li><li>Windle, W. F., Clemente, C. D., Chambers, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+formation+of+a+glial+barrier+as+a+means+of+permitting+a+peripheral+nerve+to+grow+into+the+brain.">Inhibition of formation of a glial barrier as a means of permitting a peripheral nerve to grow into the brain.</a> J Comp Neurol. 96 (2), 359-369 (1952).</li><li>Young, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+contusion+models.">Spinal cord contusion models.</a> Prog Brain Res. 137, 231-255 (2002).</li><li>Stokes, B. T., Noyes, D. H., Behrmann, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electromechanical+spinal+injury+device+with+dynamic+sensitivity.">An electromechanical spinal injury device with dynamic sensitivity.</a> J Neurotrauma. 9 (3), 187-195 (1992).</li><li>Jakeman, L. B., 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=Traumatic+spinal+cord+injury+produced+controlled+contusion+in+mouse.">Traumatic spinal cord injury produced controlled contusion in mouse.</a> J Neurotrauma. 17 (4), 299-319 (2000).</li><li>Sahni, V., Kessler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapies+for+spinal+cord+injury.">Stem cell therapies for spinal cord injury.</a> Nat Rev Neurol. 6, 363-372 (2010).</li><li>Marfia, G., 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=Adult+neural+precursors+isolated+from+post+mortem+brain+yield+mostly+neurons:+an+erythropoietin-dependent+process.">Adult neural precursors isolated from post mortem brain yield mostly neurons: an erythropoietin-dependent process.</a> Neurobiol Dis. 43 (1), 86-98 (2011).</li><li>Gritti, 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=Multipotent+neural+stem+cells+reside+into+the+rostral+extension+and+olfactory+bulb+of+adult+rodents.">Multipotent neural stem cells reside into the rostral extension and olfactory bulb of adult rodents.</a> J Neurosci. 22 (2), 437-445 (2002).</li><li>Whetstone, W. D., Hsu, J. Y., Eisenberg, M., Werb, Z., Noble-Haeusslein, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Neurosci Res. 74 (2), 227-239 (2003).</li><li>Gonzalez-Lara, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+cellular+magnetic+resonance+imaging+to+track+the+fate+of+iron-labeled+multipotent+stromal+cells+after+direct+transplantation+in+a+mouse+model+of+spinal+cord+injury.">The use of cellular magnetic resonance imaging to track the fate of iron-labeled multipotent stromal cells after direct transplantation in a mouse model of spinal cord injury.</a> Mol Imaging Biol. 13 (4), 702-711 (2010).</li><li>Ottobrini, L., 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=Magnetic+resonance+imaging+of+stem+cell+transplantation+in+injured+mouse+spinal+cord.">Magnetic resonance imaging of stem cell transplantation in injured mouse spinal cord.</a> Cell R4. 2 (3), e963 (2014).</li><li>Janowwski, 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=Neurotransplantation+in+mice:+The+concorde-like+position+ensures+minimal+cell+leakage+and+widespread+distribution+of+cells+transplanted+into+the+cistern+magna.">Neurotransplantation in mice: The concorde-like position ensures minimal cell leakage and widespread distribution of cells transplanted into the cistern magna.</a> Neuroscience Letter. 430 (2), 169-174 (2008).</li><li>Basso, D. 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?...

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。

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			<td style="width:119px;">15000-00</td>
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			<td style="width:119px;">12031-07</td>
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			<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 &#956;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)&#160;</td>
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			<td height="21" style="height:21px;width:287px;">Insulin</td>
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			<td height="21" style="height:21px;width:287px;">Heparin sodium-salt</td>
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			<td style="width:119px;">H0200000</td>
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			<td style="width:119px;">PHG0024</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">h-EGF</td>
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		</tr>
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			<td height="21" style="height:21px;width:287px;">Syringe 0.33cc 29G</td>
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			<td height="42" style="height:42px;width:287px;">buprenorphine</td>
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		</tr>
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			<td height="21" style="height:21px;width:287px;">eye gel</td>
			<td style="width:115px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">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?...

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 ...

neural-stem-cell-transplantation-experimental-contusive-model-spinal

Research

JoVE Journal

Medicine

12.8K 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

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

在大鼠脊髓损伤后的急性和慢性触觉测试

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

单侧颈脊髓损伤的挫伤模型，利用无限的地平线冲击

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

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 ...

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

一种新的椎稳定制备方法挫伤脊髓损伤

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

控制皮层影响模型创伤性脑损伤

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

无线电遥测系统，以监测大鼠脊髓横断和胚胎神经干细胞移植在心血管功能

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

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. ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

脊髓压迫损伤的校准钳型

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...