这项工作是由从克里斯托弗和戴纳·里夫基金会，医学研究理事会，欧洲研究理事会ECMneuro和NHMRC剑桥生物医学研究中心的资助。我们想表达我们最深切的感谢海琳·梅勒范"T Spijker和贾斯泰纳·巴拉特在拍摄过程中的技术援助。我们要感谢伊丽莎白·莫洛内博士和乔斯特·弗哈根教授（荷兰神经科学研究所）在AAV生产协助。

以下所有动物的程序是按照英国动物（科学程序）法案1986年如果不熟悉这些程序进行的，请向当地/国家法规检查并启动协议前寻求兽医的意见。 1.选择动物的合适的应变注:背根挤压伤引起的轰动和爪的传入神经阻滞的损失。前爪传入神经阻滞常见的不良影响包括过度修饰，自残，和爪自残。 <ol><li>获得动物的程序。 注:对于大鼠，更具活性的菌株，例如利斯特-带帽和Wistar，具有相比于更服帖菌株，如Lewis和的Sprague-Dawley传入神经阻滞后爪自残的发生率较高。如果可能的话，最温顺的应变，刘易斯，应始终优先考虑。应该特别活跃的菌株所必需的实验由于遗传米odification或特定行为的考核要求，兽医治疗应到位，以满足预期的不利影响。 </li></ol> 2.制备用于注射的病毒注意:处理所有病毒，根据生物学和实验室安全规定。 <ol><li>稀释或病毒浓缩至2×10 12基因组拷贝（GC）的滴度/毫升以下单个病毒包装协议9。 注意:在此，在病毒的Dulbecco氏磷酸盐缓冲盐水（D-PBS），用5％的蔗糖稀释。滴度可以具有被优化取决于病毒，启动子的类型，构建体大小，和靶向的细胞类型。 </li><li>可选:有色染料（ 例如，固绿）添加1％，为以后的注射方便的可视化病毒解决方案。轻轻搅拌溶解。 </li><li>保持立即使用在冰上将该溶液（在4小时内）或在-80℃冷冻机中长长期储存。 </li></ol> 3.执行动物的术前准备注:由于手术的极度侵袭，无菌技术应该在任何时候都可以使用。 <ol><li>准备和消毒之前，要启动手术的所有手术工具和脊柱立体框架。例如，消毒在134℃的高压釜中的工具。使用一套全新的消毒工具，对每个动物。 </li><li>麻醉动物。按捏爪子，以测试撤回反射或者通过触摸为眼睑反射角膜确认正确的麻醉。 <ol><li>对于使用吸入麻醉如异氟烷成年大鼠，使用4％异氟烷在2.0L /氧气感应分钟。在整个手术维护，使用2 - 3％异氟烷在1.5L /分钟的氧，改变异氟烷浓度根据动物的呼吸模式，以避免过度或不足的剂量。 </li></ol></li><li>上CE动物最初麻醉，记录动物的术前体重。 </li><li>对于宫颈DRG注射，从耳朵之间剃去毛发上颈部刚好远侧的肩胛骨。消毒用消毒产品剃光面积。 </li><li>注入2毫升盐水皮下对动物的侧腹，具有止痛的适当剂量（ 例如，5毫克/公斤卡洛芬）和抗生素（ 例如，5mg / kg的恩氟沙星）沿。 </li><li>应用眼膏两眼，以防止在手术过程中角膜干燥。 </li><li>进行到手术在37℃下放置在动物下方的加热垫脊柱立体定位框架上。 </li></ol> 4.注射DRG和表演背根挤压伤注:这是一个非常精细的外科手术。明智的做法是先练上几个动物尸体推进到活的动物在手术前与解剖熟悉。 <ol><li>大号ocate在皮肤上的突出C2和T2棘突。使用10号解剖刀刀片（一旦皮肤被打开，白色，纤维状组织中线应该是肌肉的第一层上可见的C2和T2棘突之间的皮肤切口;肌肉的第一层具有"果冻状"结构）。 </li><li>使肌肉的沿使用解剖刀刀片的白色中线所述第一层上的类似尺寸的切口。不要超越突出T2棘突，因为有来自附近还有降主动脉的主要动脉分支。 注:可以从这一步骤起任何地方发生大量出血。始终止血，并用消毒棉签或手术吸收海绵，允许在任何时候都清晰的可视清除血液。与该过程继续进行"盲目地"可以导致对脊髓，DRG，或背根不希望的损坏，从而导致意外的不利影响在动物以后。 </li><li>缩回使用两个牵引器，一个吻侧放置和一个尾部肌肉的第一层;肌肉的第二层，具有纹状外观，应该是可见的。 </li><li>定位肌肉（其中可以观察到两个纵向肌肉由薄的，膜状组织连接）的所述第二层的中线。解剖使用一对microscissors以分离两个纵向肌肉的膜状组织。避免使用手术刀刀片如果可能的话，因为这可能导致不必要的肌肉损伤和出血。 </li><li>相应地调整牵开器，以暴露薄肌覆盖脊柱的第三层。棘突可通过用一对镊子的过度的肌肉的第三层轻轻触摸感觉到。 </li><li>使肌肉的第三层上的一个小切口使用microscissors，轻轻刮去使用刮匙或手术刀以侧向的方式清楚地暴露椎骨从骨肌。如果需要的话，削减一些肌肉或肌腱的客场让拉米nectomy容易。 </li><li>以暴露左C5 - C8的DRG，执行对C4左半侧椎板 - 通过仔细移除该薄片的一部分，并使用一对细咬骨钳椎弓根T1椎骨。在DRG靠近椎骨横突孔。 注:C3 - C7椎体没有突出的棘突。该C5 DRG位于C4和C5的椎骨之间，所述C6 DRG是C5和C6椎骨之间，等等，而C8 DRG是C7和T1椎骨之间。没有C8椎骨，尽管C8 DRG的存在和C8脊髓节段。 </li><li>一旦足够的DRG的已经暴露于注射，通过将装配有定制的，33号钝针头到立体定位注射器保持器的病毒填充微升注射器准备注射器。注射前，用30号针头斜面，使每个目标DRG的小肤浅的开放，以帮助注射针头的插入。 </li><li>插入33克由AuGe针入DRG的轻轻转动旋钮调整立体坐标的中心。不要过度插入针，因为这可能导致流体从DRG的腹侧泄漏出来。如果发生泄漏，立即调整针的位置。 注:数字立体坐标不被使用;但是，它有利于利用框架保持针注射。 </li><li>注入病毒1μL成使用输注注射泵以0.2微升/分钟的每个DRG。等待抽出针头之前进一步进行3分钟。在注射过程中，如果病毒解决方案包含彩色染料的DRG会慢慢变色。 </li><li>要执行并发C5 - C8背根挤压伤，挤压各根为10秒三次，每次使用一对细尖镊子（波恩微），相对完全钳子的端部;在组织中的白线应该出现在粉碎网站。不要去更深比用FORC要求EPS，因为这可能导致向腹侧根损害。 </li><li>继注入和/或挤压，确保没有出血或小件收涨动物之前在切口部位留下骨头碎片。如果优选的话，放置一小块吸收手术海绵的在暴露的脊髓和背根神经节的顶部。 </li><li>允许肌肉的第三层，以自然缩回到脊柱不缝合。松散地缝合在第二层上的两个纵向肌肉用可吸收6-0缝合线材料（≈3间断缝合）。缝合肌肉的所述第一层（5≈间断缝合）用可吸收缝合线6-0的材料。 </li><li>缝合皮肤可吸收5-0缝合线材料（≈10间断缝合）。 注:确保缝合不太紧。皮肤的鼓胀是过度收紧的迹象，可能会导致不适的动物。 </li><li>如果手术过程中发生大量出血，皮下注入1 - 2盐水中的溶液，以从动物补充流体的损失下当地法规所允许的。 </li><li>提供食用保湿凝胶，并允许动物从麻醉中完全恢复，返回动物返回到保持区之前至少1个小时进行定期监测。 </li><li>保持动物为至少3周以获得最佳的转基因表达，以评估感觉轴突再生。 </li></ol> 5.表演动物的术后护理<ol><li>第一个星期后手术过程中向动物提供食物捣烂柔软的棉被褥。如果需要的话，管理附加剂量的止痛和抗生素的使用符合当地/国家法规和兽医建议恢复提供帮助。 </li><li> 10日 - 7后取下皮肤上的缝线。 注:该手术的常见不良反应包括血清肿或血肿的切口部位的形成，划伤皮肤痕迹，由于引起瘙痒内部吸收缝合线，微妙的感觉或运动障碍，和自残的背根挤压伤后deafferented爪子迹象。对于实质上比预期更严重的任何福利问题，应立即寻求兽医的意见。 </li></ol> 6.执行顺行CTB注射轴突跟踪注:建议进行霍乱毒素B亚基（CTB）轴突跟踪一周之前组织收集。 <ol><li>准备1％CTB的解决方案，根据制造商的说明书。 </li><li>可选:1％有色染料添加到了后来就注射方便的可视化解决方案。轻轻搅拌溶解。 </li><li>麻醉动物（见步骤3.2.1），并通过胶带肢体表稳定左前爪。 </li><li>手动和缓缓注入1％CTB的1μL皮下注射到无毛足垫的中心，并使用微升注射器装配无线四位数字TH定制的，33号短针。 注:注射前，最好先用30号斜口针，使皮肤上的小口浅，这与注射针插入助攻。 </li><li>让动物动物返回到保持区域之前从麻醉中完全恢复。 </li></ol> 7.收集组织<ol><li>为了收集用于免疫组织化学的病毒注射的DRG和脊髓，施用麻醉剂的过量和穿心灌注用磷酸缓冲盐水，随后冷的4％多聚甲醛的动物。仔细对脊柱执行完全椎板切除术，以收集在显微镜下被固定的组织。与组织制备，切片，并且处理继续进行根据需要进行分析4，5，6。 注:将回收的切口部位应该是显而易见的，由s的存在所观察到的汽车组织。 </li><li>可替代地，以收集在体外培养的病毒注射的DRG，与经批准的方法入道牺牲动物，例如二氧化碳的浓度上升。小心解剖DRG在显微镜下，确保无菌条件只要有可能。与组织培养根据需要4，5，6，10进行。 </li></ol>

<ol>
	<li>Chew, D. J., Fawcett, J. W., Andrews, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenges+of+long-distance+axon+regeneration+in+the+injured+CNS.">The challenges of long-distance axon regeneration in the injured CNS.</a> Prog Brain Res. 201, 253-294 (2012).</li><li>Wu, A., Lauschke, J. L., Morris, R., Waite, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+rat+forepaw+function+in+two+models+of+cervical+dorsal+root+injury.">Characterization of rat forepaw function in two models of cervical dorsal root injury.</a> J Neurotrauma. 26 (1), 17-29 (2009).</li><li>Hocquemiller, M., Giersch, L., Audrain, M., Parker, S., Cartier, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-Associated+Virus-Based+Gene+Therapy+for+CNS+Diseases.">Adeno-Associated Virus-Based Gene Therapy for CNS Diseases.</a> Hum Gene Ther. 27 (7), 478-496 (2016).</li><li>Cheah, 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=Expression+of+an+Activated+Integrin+Promotes+Long-Distance+Sensory+Axon+Regeneration+in+the+Spinal+Cord.">Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord.</a> J Neurosci. 36 (27), 7283-7297 (2016).</li><li>Andrews, M. R., 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=Alpha9+integrin+promotes+neurite+outgrowth+on+tenascin-C+and+enhances+sensory+axon+regeneration.">Alpha9 integrin promotes neurite outgrowth on tenascin-C and enhances sensory axon regeneration.</a> J Neurosci. 29 (17), 5546-5557 (2009).</li><li>Tan, C. 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=Kindlin-1+enhances+axon+growth+on+inhibitory+chondroitin+sulfate+proteoglycans+and+promotes+sensory+axon+regeneration.">Kindlin-1 enhances axon growth on inhibitory chondroitin sulfate proteoglycans and promotes sensory axon regeneration.</a> J Neurosci. 32 (21), 7325-7335 (2012).</li><li>McCarty, D. M., Young, S. M., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+adeno-associated+virus+(AAV)+and+recombinant+AAV+vectors.">Integration of adeno-associated virus (AAV) and recombinant AAV vectors.</a> Annu Rev Genet. 38, 819-845 (2004).</li><li>Mason, M. R., 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=Comparison+of+AAV+serotypes+for+gene+delivery+to+dorsal+root+ganglion+neurons.">Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons.</a> Mol Ther. 18 (4), 715-724 (2010).</li><li>Hermens, W. T., 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=Purification+of+recombinant+adeno-associated+virus+by+iodixanol+gradient+ultracentrifugation+allows+rapid+and+reproducible+preparation+of+vector+stocks+for+gene+transfer+in+the+nervous+system.">Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.</a> Hum Gene Ther. 10 (11), 1885-1891 (1999).</li><li>Kappagantula, S., 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=Neu3+sialidase-mediated+ganglioside+conversion+is+necessary+for+axon+regeneration+and+is+blocked+in+CNS+axons.">Neu3 sialidase-mediated ganglioside conversion is necessary for axon regeneration and is blocked in CNS axons.</a> J Neurosci. 34 (7), 2477-2492 (2014).</li><li>Alto, L. T., 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=Chemotropic+guidance+facilitates+axonal+regeneration+and+synapse+formation+after+spinal+cord+injury.">Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury.</a> Nat Neurosci. 12 (9), 1106-1113 (2009).</li><li>Bonner, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafted+neural+progenitors+integrate+and+restore+synaptic+connectivity+across+the+injured+spinal+cord.">Grafted neural progenitors integrate and restore synaptic connectivity across the injured spinal cord.</a> J Neurosci. 31 (12), 4675-4686 (2011).</li><li>Wang, R., 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=Persistent+restoration+of+sensory+function+by+immediate+or+delayed+systemic+artemin+after+dorsal+root+injury.">Persistent restoration of sensory function by immediate or delayed systemic artemin after dorsal root injury.</a> Nat Neurosci. 11 (4), 488-496 (2008).</li><li>Wong, L. E., Gibson, M. E., Arnold, H. M., Pepinsky, B., Frank, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemin+promotes+functional+long-distance+axonal+regeneration+to+the+brainstem+after+dorsal+root+crush.">Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush.</a> Proc Natl Acad Sci U S A. 112 (19), 6170-6175 (2015).</li><li>Tanguy, Y., 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=Systemic+AAVrh10+provides+higher+transgene+expression+than+AAV9+in+the+brain+and+the+spinal+cord+of+neonatal+mice.">Systemic AAVrh10 provides higher transgene expression than AAV9 in the brain and the spinal cord of neonatal mice.</a> Front Mol Neurosci. 8, 36 (2015).</li><li>Foust, K. D., Poirier, A., Pacak, C. A., Mandel, R. J., Flotte, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+intraperitoneal+or+intravenous+injections+of+recombinant+adeno-associated+virus+type+8+transduce+dorsal+root+ganglia+and+lower+motor+neurons.">Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons.</a> Hum Gene Ther. 19 (1), 61-70 (2008).</li><li>Vulchanova, 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=Differential+adeno-associated+virus+mediated+gene+transfer+to+sensory+neurons+following+intrathecal+delivery+by+direct+lumbar+puncture.">Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.</a> Mol Pain. 6, 31 (2010).</li><li>Gray, S. 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=Directed+evolution+of+a+novel+adeno-associated+virus+(AAV)+vector+that+crosses+the+seizure-compromised+blood-brain+barrier+(BBB).">Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB).</a> Mol Ther. 18 (3), 570-578 (2010).</li><li>Fagoe, N. D., Attwell, C. L., Kouwenhoven, D., Verhaagen, J., Mason, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+ATF3+or+the+combination+of+ATF3,+c-Jun,+STAT3+and+Smad1+promotes+regeneration+of+the+central+axon+branch+of+sensory+neurons+but+without+synergistic+effects.">Overexpression of ATF3 or the combination of ATF3, c-Jun, STAT3 and Smad1 promotes regeneration of the central axon branch of sensory neurons but without synergistic effects.</a> Hum Mol Genet. 24 (23), 6788-6800 (2015).</li></ol>

James Fawcett is a paid consultant for Acorda Therapeutics and Novartis.

在本文中，我们提出了一种一步一步的指导，在成年大鼠的下颈脊髓进行DRG注射和背根挤压伤。由于这是一个非常侵入性和精细的外科手术，我们强烈建议所有潜在用户推进到活的动物在手术前获得足够的培训和实践。用户应该熟悉不仅与脊髓的解剖结构，而且还与周围的肌肉组织，椎体骨质结构，和血管。理想情况下，主管用户应该能够执行以最小的损伤的过程向周围的组织，通过而不诱导脊髓任何损害去除椎骨的部分进行清洁的椎板切除术。由于脊髓损伤明显，脊髓小病变可以有整个神经系统普遍不利影响。此外，经历了一个"干净"手术的动物不太可能受到意外后遭受因此，手术并发症和福利问题，并不太可能有希望的实验时间点之前被牺牲。 施用病毒进入神经系统中，有施用的几个可能的路线:静脉15内，腹膜内16，鞘内17，或直接注射到靶4,8。尽管静脉内和腹膜内注射是相对非侵入性的，血-脑屏障的交叉可能是一个问题18，这些路由导致非特异性转导，这不会是在一个专门的轴突再生研究是有用的。类似地，用于鞘内注射至蛛网膜下隙，更侵入性的管理途径，CNS中的许多神经元和非神经元细胞类型可以被转导的，这可能产生非特异性或断开圆盾Ť效果。因此，直接注射病毒进入DRG是一个有利的选择，并可能导致更高的转染效率比其他方法。该选项的主要缺点，但是，是外科手术，这需要专门培训的侵袭。 一旦用户已经掌握所需的手术技巧，该协议提供了极大的灵活性量。在一个轴突再生的研究中，动物可以结合研究了其他的技术，例如在体内电生理和感觉运动行为测试，而收集的组织可以用于解剖分析或组织培养4。这些技术，具有变化的实验时间点的组合，例如，可以被用来研究变性或不同纤维亚型，如NF200，CGRP，和IB4的再生的挤压伤4之后的进展。根据实验requiremeNTS，一个或另一个的所呈现的程序可以单独执行。例如，单独注射DRG可以用于轴突跟踪实验，而单独的背根挤压伤可以在需要前爪传入神经阻滞任何研究中使用。此外，用户还可以改变病毒和用于注射的转基因产物的类型，确切DRG被注入，并且确切的背根受伤。如果适用，也可以使用该协议来进行细胞移植或药理学施用到DRG。上获取的手术技巧的基础上，有经验的用户可进行到其它的技术，如DRG注射在腰部区域（ 例如，进入L3 - L5评估后肢功能）19或背柱破碎损伤，进一步研究脊髓功能5。 总之，我们认为，DRG注射和背根挤压伤是一个有用的模型来研究感觉轴突REGeneration。尽管进行专门培训，以进行微创手术的要求，协议是灵活的，用户和潜在用户可以修改很多地方，以满足他们的实验要求。这些程序可以作为那些寻找合适的动物模型感觉轴突再生的研究奠定了基础。

神经系统损伤后实现轴突再生是一项艰巨的任务1。研究轴突再生的中枢神经系统（CNS）的失败，研究人员利用神经损伤模型过多。由于中枢神经系统的地区不同，它使用一个适当的解剖模型来研究轴突再生是非常重要的。通过使用合适的模型，研究人员可以制定基于损伤的严重程度，感兴趣的神经元细胞类型的特定处理，并评估再生所需的脊髓束，而不是"一个对所有人"的治疗策略。 在脊髓损伤，例如，最衰弱的症状，从感觉和运动的损失造成的。感觉丧失是由在上行感觉通路造成的损害，而运动的损失是由该下行运动路径造成的损害。由于这些TW之间的蜂窝和解剖学上的差异Ø途径，许多有针对性的轴突再生的研究只专注于一个或另一个途径，与逻辑，即任一成功恢复将是巨大的利益给患者。在本文中，我们提出一种使用直接背根神经节（DRG）注射用病毒载体和在成年大鼠作为模型来研究感觉轴突再生的下颈脊髓并发背根挤压伤的协议。 DRG感觉神经元负责中继感官信息，如触觉和疼痛，从外围到中枢神经系统。在脊髓中的感觉神经元的轴突长突起作为一个很好的模型来研究长途轴突再生。此外，由于啮齿动物可以生存感觉传导通路的病变，如背根挤压伤以最小的福利并发症，研究人员可以研究中枢神经系统轴突再生，而无需完全损伤脊髓。一个四C5 - C8（宫颈升伊维尔基尼5 - 8）背根挤压伤已被证明是用于前爪传入神经阻滞2的有用模型。此外，背根挤压伤提供了一个"更清洁"的模型来研究轴突再生比直接脊髓损伤，因为它是由其他因素简单如胶质瘢痕的形成。 重编程为神经元再生状态使用病毒基因治疗已越来越多地被视为对许多神经系统疾病3有前途的治疗策略。研究表明携带生长促进蛋白的转基因可以实现鲁棒轴突再生与行为恢复4，5，6的腺相关病毒（AAV）载体的应用。在引发免疫应答和转导非分裂细胞，如神经元的能力AAV的表观低致病性，使它用于基因治疗的最佳载体。另外，重组AAV形式用于治疗。在这种形式中，它不能其病毒基因组整合到宿主基因组7，减少相对于其他的病毒载体，如慢病毒插入诱变的风险。这使得AAV基因治疗应用的安全选择。 作为一个包含DRG感觉神经元的细胞体，它是病毒的基因治疗管理的最合适的解剖目标，研究和/或促进感觉轴突再生。在比较不同的AAV血清型和慢病毒的研究中，AAV血清型5（AAV5）被证明是最有效的在当直接注射到DRG 8超过至少12周的时间过程转导的DRG神经元。此外，AAV可以实现40％以上的转导效率，转导所有DRG神经元亚型，如大直径神经丝200kDa的（NF200）阳性神经元和小直径降钙素基因相关肽（CGRP） -或同工凝集素B4（IB4）阳性神经元4,8。 由于DRG注射和背根挤压伤的手术过程是非常侵入性的细腻，我们认为，这篇文章将帮助新用户学习在一个非常有效的方式方法。在这篇文章中，我们将展示来自成年大鼠注射对照病毒AAV5-GFP（绿色荧光蛋白）到C6四个星期后有代表性的结果 - C7病种付费用并发C5 - C8背根挤压伤。这种模式特别适用于谁的研究人员正在研究利用病毒基因疗法来促进感觉轴突再生。

<table><tbody><tr><td>Fast Green FCF dye</td><td>Sigma-Aldrich</td><td>F7258</td><td>For visualizing colorless solution. Recommended concentration: 1%</td></tr><tr><td>Cholera Toxin B subunit</td><td>List Biological Laboratories</td><td>104</td><td>For anterograde axonal tracing. Recommended concentration: 1%</td></tr><tr><td>IsoFlo</td><td>Zoetis</td><td>115095</td><td>Inhalation anesthetic (active ingredient: isoflurane)</td></tr><tr><td>Baytril 2.5% injectable</td><td>Bayer</td><td>05032756093017</td><td>Antibiotic (active ingredient: enrofloxacin). Manufacturer&#039;s recommended dosage: 10 mg/kg</td></tr><tr><td>Carprieve 5.0% w/v</td><td>Norbrook</td><td>02000/4229</td><td>Analgesic (active ingredient: carprofen). Manufacturer&#039;s recommended dosage: 4 mg/kg</td></tr><tr><td>Lacri-Lube</td><td>Allergan</td><td>PL 00426/0041</td><td>Eye ointment</td></tr><tr><td>Olsen-Hegar Needle Holder</td><td>Fine Science Tools</td><td>FST 12502-12</td><td></td></tr><tr><td>Friedman Pearson Rongeur Curved 0.7 mm Cup</td><td>Fine Science Tools</td><td>FST 16121-14</td><td></td></tr><tr><td>Bonn Micro Forceps</td><td>Fine Science Tools</td><td>FST 11083-07</td><td>For performing dorsal root crush injury</td></tr><tr><td>Tissue Separating Scissors</td><td>Fine Science Tools</td><td>FST 14072-10</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>FST 14058-11</td><td></td></tr><tr><td>Micro-Adson Forceps</td><td>Fine Science Tools</td><td>FST 11018-12</td><td></td></tr><tr><td>Goldstein Retractor</td><td>Fine Science Tools</td><td>FST 17003-03</td><td></td></tr><tr><td>Vannas Spring Scissors (straight)</td><td>Fine Science Tools</td><td>FST 15018-10</td><td></td></tr><tr><td>SURGIFOAM Absorbable Gelatin Sponge</td><td>Ethicon</td><td>1972</td><td>For bleeding control</td></tr><tr><td>Microliter Syringe RN701 (10 &amp;mu;L)</td><td>Hamilton</td><td>80330</td><td></td></tr><tr><td>Custom-made Removable Needle (for DRG injection)</td><td>Hamilton</td><td>7803-05</td><td>33 gauge, 38 mm, point style 3</td></tr><tr><td>Custom-made Removable Needle (for CTB injection)</td><td>Hamilton</td><td>7803-05</td><td>33 gauge, 10 mm, point style 3</td></tr><tr><td>UltraMicroPump with SYS-Micro4 Controller</td><td>World Precision Instruments</td><td>UMP3-1</td><td></td></tr></tbody></table>

dorsal root ganglion injection and dorsal root crush injury as a model for sensory axon regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

作为代表性，呈现与所连接的DRG的横向脊髓部分直接显示该协议的有效性在转导DRG神经元和注射对照病毒，AAV5-GFP后过四星期跟踪在脊髓感觉轴突，入C7 DRG而不背根挤压伤（ 图1A）。在这两个背柱和脊髓背角神经轴突表达GFP（ 图1B），以及注入的DRG（ 图1C）中的细胞体和轴突。所述楔形核的额外的解剖分析，在脑干中的感觉轴突终端，揭示了正顺行CTB跟踪（ 图1D）。 当DRG注射用一个完整的C5-C8背根挤压伤执行的并发，脊髓或CTB-正极端子没有GFP阳性神经轴突S IN的楔形核中观察到（ 图2A）。然而，值得指出的是金黄地鼠感觉轴突仍然可以再生到在一个完全粉碎背根，这是一个PNS环境背根进入区，但不超出到脊髓4,5（ 图2A）。在所注入的病毒包含潜在促生长蛋白的转基因的情况下，标记的轴突在脊髓的存在可表示再生或不完整的背根挤压伤（ 图2B）。这两个结果之间区分，在楔形核CTB轴突跟踪应分析。在楔形核CTB-正极端子的存在突出的不完全损伤（ 图2C）的可能性，而没有CTB-正极端子的提示部分再生进入脊髓，作为再生轴突很可能不能生长的整个距离到达楔形核（ 图2D）。迄今为止，成功的感觉轴突再生的楔形核问题大多出现在案件高宫颈损伤11，12或13神经营养因子，14的应用。显示不完全损伤的迹象，任何动物应该被排除在轴突再生的研究。 <img alt="图1" src="/files/ftp_upload/55535/55535fig1.jpg" /> 图1:DRG 注射没有背根挤压伤。 （AC）表示AAV5-GFP注射后四周脊髓GFP阳性轴突（A），包括背柱和背角（B）和细胞体在DRG（C）脊髓部。 &lt;强&gt;（D）CTB-阳性感觉轴突终端在楔形核一周后CTB注射。比例尺是650微米（AC）和250微米（D）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55535/55535fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55535/55535fig2.jpg" /> 图2: 评估背根挤压伤和轴突再生。 （A）一个完整的背根挤压伤导致在楔形核脊髓或CTB-阳性轴突终末没有标记的轴突。金黄地鼠的感觉轴突可以再生至背根进入区，但不超过到脊髓。 （BD）标记的轴突的脊髓中的存在表示要么不完全损伤或regenerati上（B）。用额外的分析，CTB-正极端子在楔形细胞核中的存在表明不完全损伤（C），而其不存在表明完全损伤和潜在的部分再生到脊髓（D）。比例尺为250μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55535/55535fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration at JoVE.com

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

dorsal-root-ganglion-injection-dorsal-root-crush-injury-as-model-for

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.7K 次观看.  University of Cambridge. This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

视频: Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

染色质免疫沉淀从以下轴索损伤的背根神经节组织

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

科研

神经科学

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

生物工程

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

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

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

背根神经节神经元和区分脂肪干细胞:一种<em&gt;在体外</em&gt;共培养模型来研究周围神经再生

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

医学

Sheath-Preserving Optic Nerve Transection in Rats to Assess Axon Regeneration and Interventions Targeting the Retinal Ganglion Cell Axon

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury.
The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments.

This protocol describes an optic nerve transection that preserves the optic nerve sheath in rats. Hydrostatic pressure from microinjections into the optic nerve generate a complete transection, allowing for sutureless reapposition of the transected optic nerve ends and direct targeting of the axonal compartment in a transection model.

背根神经节内注射和背根挤压伤为感官轴突再生模型

摘要

探索更多视频

此视频中的章节