笔者想感谢埃里克·瓦斯科对他的援助在PDMS基板的准备，墉王滨海马塔博士在密歇根大学实验室进行有益的建议和DRG隔离的培训博士和马克Tuszynski博士和博士为有益的建议和协议为SC隔离。W.玛丽·坎帕纳在加州大学圣地亚哥分校。这项研究是由美国国家科学基金会（CBET 0941055和CBET 1510895），美国国立卫生研究院（R21CA176854，R01GM089866和R01EB014986）的部分资助。

对于细胞的分离所有的程序是由机构动物护理和使用委员会在密歇根州立大学获得批准。 1.预拉伸各向异性表面的制备<ol><li>混合10:基和固化剂的1溶液，将混合物倒入一个组织培养皿（直径12cm）。用4900毫克碱和490毫克的固化剂的总交联混合物。 </li><li>保持在真空下的凝胶混合物20分钟以除去气泡。 </li><li>放置在烘箱中的凝胶混合物过夜固化在60℃下。 </li><li>的PDMS膜固化之后，使用等离子体清洁/刻蚀系统在165毫托3分钟和O 2的65sccm的流处理表面用氧等离子体。 </li><li>切下一块从盘矩形薄膜（5×3.5厘米），而被认识到是哪一方氧处理，确保氧治疗面朝上，并固定到拉伸框架。放置在一个框架<html>拉伸阶段并通过在阶段转动旋钮直到它达到10％伸长均匀伸展​​;在较长轴9（或某些其他预定拉伸伸长率可以从拉伸阶段被直接读出）。拧紧框架上的螺钉固定伸展。 </li><li>取下阶段框架。确保膜表面是干燥，无粉尘，然后将硅氧烷室在膜允许腔室的粘性侧紧紧附至膜。 注:硅胶室提供了一个良好的保持在PDMS膜细胞培养基。该装置的设计示于图1。 </li><li>消毒用UV表面10分钟。 </li><li>加入1 ml聚-L-赖氨酸（PLL）（在磷酸盐缓冲盐水0.01％），以在腔室中与PDMS表面，等离子处理的6小时内，在37℃下孵育2小时以播种DRG神经元之前。这增强细胞附着。 </li></ol>DRG乐"&gt; 2。隔离<ol><li>准备标准生长培养基的DRG神经元。 </li><li>之前的隔离，高压灭菌的隔离设备和过滤器的试剂。添加5毫升隔离缓冲成一个6孔板的一个孔中，并将板在冰上。通过加入1毫升的胶原酶A（500U /毫升）至9毫升0.05％胰蛋白酶-EDTA（1毫克/毫升）制备出10毫升离解培养基中，筛选用0.22微米的过滤器，并置于冰上的解决方案。 </li><li>使用十到十二5 - 天的Sprague-Dawley大鼠7为隔离。喷用75％异丙醇的幼仔，并且通过断头牺牲。 </li><li>切去皮肤覆盖从背面脊髓和移除脊柱周围的任何多余的组织。在手术放大镜手术射灯上，从颈部做切口第一，然后沿两边用剪刀脊柱一截。从分离幼仔的尸体脊柱和去除多余的肌肉。然后，用剪刀沿经度切脊椎和使用镊子克轴完全打开脊椎和吸管抽取脊髓cord.Remove尖端创建用于隔离缓冲病种付费转移到无菌的15毫升管一个更大的开口。 </li><li>用细镊子，取出骨口袋里的神经节，并收集大约10 - 双方16 DRG。修剪神经根和神经节转移到冰冷的分离缓冲液。 </li><li>从吸管取出提示创建用于隔离缓冲病种付费转移到无菌的15毫升管一个更大的开口。化学解离后，离心神经节在900 XG和4℃5分钟。 </li><li>让组织沉降到试管底部并轻轻取出顶部的隔离缓冲器和加入10 mL离解培养基进入管。 </li><li>孵育在解离介质的解剖组织在一个37℃的水浴中1小时，同时摇动管每5 - 10分钟。 </li><li>后化学离解，离心机在900 XG的神经节和4℃5分钟。 </li><li>除去上清液并重新悬浮在10ml的标准生长培养基和涡流沉淀。 </li><li>离心离解的细胞作为第2.9指示一次。 </li><li>除去上清液，再暂停于12ml标准培养基和旋涡的沉淀。 </li></ol> 3. DRG的文化对预拉伸面<ol><li>之前接种细胞，从室移除PLL方案，并用无菌水和空气干燥冲洗PDMS表面。 </li><li>重新悬浮细胞后，让1的悬浮液组 - 2分钟，以允许碎屑沉降到管底部。添加1.5毫升细胞悬浮液到各伸展腔，并在37℃下孵育5％的CO 2。 </li><li>为了消除神经胶质细胞，在第1天在体外 （DIV），加入10微升（或15微升）氟脱氧2尿苷和尿苷（FDU-U）股票soluti混合物上（见材料数据表 ）至各孔中。 7小时后，更换新鲜标准生长培养基这种介质和放置预拉伸培养装置在37℃培养箱，用5％ 的 CO 2。 </li><li>在细胞培养期间（2 - 3周），通过用新鲜培养基置换一半的用过的培养基改变媒体每两天。注意:培养2周拉伸未拉伸的表面上后，纯化的SC被添加到腔室和一个星期（步骤4.7）中培养。 </li></ol> 4.预拉伸表面雪旺细胞（SCS）与DRG神经元共培养<ol><li>由坎帕纳博士的研究小组先前描述的15种姓隔离。 <ol><li>简单地说，从1日龄的小狗隔离旺。收集和分离坐骨神经在化学（胰蛋白酶EDTA和胶原酶A）和机械（18号针头/ 10 ml注射器）15。 </li><li>种子离解的细胞分化成多聚-D-赖氨酸（PDL）共ated T25烧瓶中，并培养在37℃，5％ 的 CO 2。在第5天，用抗Thy 1.1抗体和兔补体纯化通过抗体选择的细胞，并传代培养纯化的细胞通路2 - 4含Dulbecco氏改良的Eagle培养基（DMEM），10％胎牛血清使用培养基（FBS）的，1％青霉素/链霉素（佩恩/链霉素），21微克/ ml牛垂体提取物（BPE），和4μM的毛喉素。 </li></ol></li><li>取下的SC培养基，加入5 ml 0.05％胰蛋白酶-EDTA到烧瓶中。孵育所述细胞在37℃在5％CO 2的2 - 3分钟。 </li><li>检查用10X物镜，看他们是否从烧瓶抬起，然后加入5毫升培养培养基中，并与在烧瓶中的细胞混合在光学显微镜下观察细胞。 </li><li>将细胞悬浮液添加到15ml离心管中并离心，在200 xg离心和20℃5分钟。 </li><li>去除上清，悬浮颗粒的7ml标准DRG增长MEDIA。 </li><li>取10微升细胞悬液到0.5 ml离心管，用10微升台盼蓝的混合，再算上用使用10X物镜光学显微镜下血球细胞数。通过加入DRG生长培养基稀释细胞悬浮液，以每毫升5,000个细胞。 </li><li>从DRG文化在拉伸室中取出0.5毫升培养基中，将0.5ml SC悬浮于DRG文化。 </li><li>培养在37℃和5％的CO 2。更改细胞1周媒体每两天通过用DRG新鲜标准生长培养基替换一半的用过的培养基。 1周共培养后，过程中的细胞通过免疫组织化学10。 </li></ol>

<ol>
	<li>Liu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Layer-by-Layer Films for Biomedical Applications. , 525-546 (2015).</li><li>Faweett, J. W., Keynes, 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=Peripheral+Nerve+Regeneration.">Peripheral Nerve Regeneration.</a> Annu Rev Neurosci. 13, 43-60 (1990).</li><li>Li, Y., Field, P. M., Raisman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+adult+rat+corticospinal+tract+by+transplants+of+olfactory+ensheathing+cells.">Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells.</a> Science. 277, 2000-2002 (1997).</li><li>Geller, H. M., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+bridge:+Engineering+spinal+cord+repair.">Building a bridge: Engineering spinal cord repair.</a> Exp Neurol. 174, 125-136 (2002).</li><li>Totoiu, M. O., Keirstead, H. S. <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+injury+is+accompanied+by+chronic+progressive+demyelination.">Spinal cord injury is accompanied by chronic progressive demyelination.</a> J Comp Neurol. 486, 373-383 (2005).</li><li>Chua, J. 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=Extending+neurites+sense+the+depth+of+the+underlying+topography+during+neuronal+differentiation+and+contact+guidance.">Extending neurites sense the depth of the underlying topography during neuronal differentiation and contact guidance.</a> Biomaterials. 35, 7750-7761 (2014).</li><li>Dowell-Mesfin, N. 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=Topographically+modified+surfaces+affect+orientation+and+growth+of+hippocampal+neurons.">Topographically modified surfaces affect orientation and growth of hippocampal neurons.</a> J Neural Eng. 1, 78-90 (2004).</li><li>Baek, S., Gleason, R. L., Rajagopal, K. R., Humphrey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+small+on+large:+Potential+utility+in+computations+of+fluid-solid+interactions+in+arteries.">Theory of small on large: Potential utility in computations of fluid-solid interactions in arteries.</a> Comput Method Appl M. 196, 3070-3078 (2007).</li><li>Liu, C., 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=Effect+of+Static+Pre-stretch+Induced+Surface+Anisotropy+on+Orientation+of+Mesenchymal+Stem+Cells.">Effect of Static Pre-stretch Induced Surface Anisotropy on Orientation of Mesenchymal Stem Cells.</a> Cell Mol Bioeng. 7, 106-121 (2014).</li><li>Liu, C., 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+impact+of+pre-stretch+induced+surface+anisotropy+on+axon+regeneration.">The impact of pre-stretch induced surface anisotropy on axon regeneration.</a> Tissue Eng Part C Methods. , (2015).</li><li>Berns, E. 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=Aligned+neurite+outgrowth+and+directed+cell+migration+in+self-assembled+monodomain+gels.">Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels.</a> Biomaterials. 35, 185-195 (2014).</li><li>Kidambi, S., Lee, I., Chan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+neuron/astrocyte+co-culture+on+polyelectrolyte+multilayer+films:+A+template+for+studying+astrocyte-mediated+oxidative+stress+in+neurons.">Primary neuron/astrocyte co-culture on polyelectrolyte multilayer films: A template for studying astrocyte-mediated oxidative stress in neurons.</a> Adv Funct Mater. 18, 294-301 (2008).</li><li>Xia, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neurite+growth+of+rat+dorsal+root+ganglion+neurons+and+increased+colocalization+with+Schwann+cells+on+aligned+poly(methyl+methacrylate)+electrospun+nanofibers.">Directed neurite growth of rat dorsal root ganglion neurons and increased colocalization with Schwann cells on aligned poly(methyl methacrylate) electrospun nanofibers.</a> Brain Research. 1565, 18-27 (2014).</li><li>Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+growth+of+integrated+axon+tracts:+Extremes+and+exploitations.">Stretch growth of integrated axon tracts: Extremes and exploitations.</a> Prog Neurobiol. 89, 231-239 (2009).</li><li>Mantuano, E., Jo, M., Gonias, S. L., Campana, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+Density+Lipoprotein+Receptor-related+Protein+(LRP1)+Regulates+Rac1+and+RhoA+Reciprocally+to+Control+Schwann+Cell+Adhesion+and+Migration.">Low Density Lipoprotein Receptor-related Protein (LRP1) Regulates Rac1 and RhoA Reciprocally to Control Schwann Cell Adhesion and Migration.</a> Journal of Biological Chemistry. 285, 14259-14266 (2010).</li><li>Kim, B., ET, K. P., Papautsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stability+of+plasma+oxidized+PDMS+surfaces.">Long-term stability of plasma oxidized PDMS surfaces.</a> Conf Proc IEEE Eng Med Biol Soc. 7, 5013-5016 (2004).</li><li>Lopera, S., Mansano, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-Based+Surface+Modification+of+Polydimethylsiloxane+for+PDMS-PDMS+Molding.">Plasma-Based Surface Modification of Polydimethylsiloxane for PDMS-PDMS Molding.</a> ISRN Polymer Science. 2012, (2012).</li><li>Chandra, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organosilicon Materials. , (2013).</li><li>Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+poly(dimethylsiloxane)+surface+modification+to+control+cell+adhesion.">Simple poly(dimethylsiloxane) surface modification to control cell adhesion.</a> Surf Interface Anal. 41, 11-16 (2009).</li><li>Moore, M. 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=Multiple-channel+scaffolds+to+promote+spinal+cord+axon+regeneration.">Multiple-channel scaffolds to promote spinal cord axon regeneration.</a> Biomaterials. 27, 419-429 (2006).</li><li>Clarke, J. C., 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=Micropatterned+methacrylate+polymers+direct+spiral+ganglion+neurite+and+Schwann+cell+growth.">Micropatterned methacrylate polymers direct spiral ganglion neurite and Schwann cell growth.</a> Hearing Res. 278, 96-105 (2011).</li><li>Pfister, B. 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=Development+of+transplantable+nervous+tissue+constructs+comprised+of+stretch-grown+axons.">Development of transplantable nervous tissue constructs comprised of stretch-grown axons.</a> J Neurosci Methods. 153, 95-103 (2006).</li></ol>

The authors do not have any conflict of interest to disclose.

为了诱导对预拉伸的表面轴突对准，存在两个关键步骤:1）的PDMS膜必须是平坦的和均匀的厚度;和2）的神经胶质细胞，必须从DRG中移除。混合的PDMS和交联剂并在烘箱中固化后，交联的PDMS凝胶应保持在平坦的工作台上，并仔细处理，以免任何倾斜。的PDMS膜的氧等离子处理之后，应当锁相环涂层6小时内，由于表面的等离子体处理后的亲水性（细胞附着必需）衰减随时间。因为在等离子体处理被施加到膜的一侧，必须小心剥离PDMS断菜后​​可采取以确保正确的侧被放置在框架上，也就是说，在等离子体处理后的表面应朝上为细胞重视。 所述的DRG位于骨口袋即沿着脊柱对齐内部。由于椎骨的颜色是白色年轻幼仔，难以从椎骨区分的DRG。在这种情况下，以放置接近斑点光脊柱是重要的。根据亮，按病种付费，而骨依然白色不透明变成透明。重要的是要保持在冰上，在生理pH解剖缓冲器中，由于pH值影响神经细胞的生存力是重要的。分离的组织是非常粘稠的，而且往往转移到一个离心管中，当粘到枪头。从而切割吸管的非常尖，以产生一个较大的开口便于传输。 在某些协议，有添加解离缓冲液之前与离体组织所需的一个更离心步骤。代替这一步，我们的协议要求的组织中的解剖缓冲器沉降几分钟，这减少了额外离心步骤，否则将影响细胞的生存力和密度。细胞密度也取决于上幼仔的数量处死，并且因此重要的是要在每次保持幼仔的一致数目。也有与该DRG神经元，从而有丝分裂抑制剂，FDU-U，加入到培养基中以1 DIV抑制神经胶质细胞的生长沿分离胶质细胞。这通常用于在神经元的生长前期以抑制神经胶质细胞，但也有一些协议喜欢arabinofuranosylcytidine（阿糖胞苷），并FDU-U的组合。加入FDU-U的摩尔浓度到培养中的不同的协议不同，取决于分离的细胞的密度。我们发现有丝分裂抑制剂可以添加多次（在步骤3.3中指定的浓度），如需要（如果胶质细胞存在），其全部DRG培养期间。它一般不建议在，因为它影响到DRG神经元的连接任何给定的时间添加大量（超过15微升）。 等离子体处理提高面传热Ë亲水性和PLL涂料提供了充电表面轴突启动附件。根据在参考文献中，等离子体处理诱导亲水性将衰减随着时间的推移，在干燥条件16-17下。然而PDMS将经受与水的液体的条件下缓慢反应，并会从是疏水亲水18逐渐变化。因此，尽管等离子处理衰减随着时间的推移，它不应该影响我们的结果。对于PLL涂层，根据从供应商的产品信息，如果放在有利的条件下它是多年来稳定。一旦细胞附着在锁相环涂层，它们一般不会分离。此外，在PLL涂层有助于与初始附着，并随着细胞生长它们分泌细胞外基质（ECM）蛋白质，其进一步帮助他们保持锚定于表面19。未来的设计改​​进可能包括嵌入肽进入PD交联时的MS的网络，这将消除在等离子体处理和PLL涂层的步骤。 该协议，在此，描述了产生表面各向异性成功诱导轴突排列，生长和髓鞘形成的工程方法。该方法可通过两种方式进行实验展开。首先，将各向异性表面可以应用在培养创建神经组织，并在体外神经束然后可以在体内被移植。第二，表面各向异性的概念可以纳入，可以在体内被用来增强从损伤部位和髓鞘形成从主机的SC的再生轴突的取向移植支架的设计。相比这需要多个和复杂的装置20-22，以及在加入的生长因子的其他方法，我们的平台是相对简单和容易应用，并且仅依赖于预拉伸，以提高这两种轴突阿里gnment和髓鞘形成，而不需要生长因子的加法。此外，我们已在两个MSC和DRG神经元这很容易被应用于其它类型的细胞的实验设计测试预拉伸的这一概念。采用目前的设计，以调查对2维表面轴突生长，但是在未来的设备可以被升级为使3- dimenstional细胞培养物的测试。在神经修复和移植未来的应用可以纳入预拉伸到从含有控释药物性质沿交联的肽可生物降解的天然或合成材料制成的可移植的支架。

在神经损伤，近端和远端神经断端经常由神经束的直接调整防止由于病灶部位1-2。通常情况下，轴突束是由轴突高度有序的排列和捆绑，形成连通的复杂网络。然而，神经再生是由于缺乏组织轴突3-4对准一个混乱的过程。因此，为了产生足够数量的再生该桥接病灶部位的轴突，有必要以诱导良好的组织轴突对齐。此外，伴随脱髓鞘由于在损伤部位的髓鞘细胞的死亡神经损伤。由于脱髓鞘强烈损害神经轴突尚存的导电能力，靶向治疗脱髓鞘或促进髓鞘是神经损伤后5功能恢复显著。因此，这个协议的目的是为了说明工程方法，解决这两个问题神经再生。 面各向异性，当沿着不同的轴测量，在材料的物理或机械性能被定义为一个差，已经应用影响细胞对准，生长和迁移6-7。除了地形，还有其他的方法来诱导各向异性。先前，我们研究了通过聚二甲基硅氧烷（PDMS）膜的机械静态预拉伸诱导的表面的各向异性。 "强加于大小变形超级"的理论预测，将细胞在拉伸方向感测有效刚度与垂直方向不同，并且在有效刚度这种差异是由于表面各向异性8。间充质干细胞（MSC）上的预拉伸的PDMS膜培养能够通过积极地拉动面，其结果，以感测的各向异性，在预拉伸方向9对齐。同样地，各向异性表面芳从一个机械预拉伸的表面伊辛影响对准，以及生长和髓鞘形成背根神经节的（DRG）轴突10。在这里，我们提供了一个静态预拉伸PDMS基板上诱导表面各向异性以增强轴突再生10的协议。 为了激发轴突对齐，用需要的模式拓扑特征，通过报道排列的纤维和渠道6,11-12提供联系指导，被证明有利于轴突排列11,13。但是，报道了通过拓扑特征，如纤维，频道和图案化诱导轴突对准技术，无法延长并增加轴突的厚度。与此相反，逐步机械拉伸导致更长和更厚的轴突与该拉伸14的幅度增大拉伸方向轴突对齐。然而， 在体内的结合有动力的电机装置不可行。与此相反，静态预拉伸引起的各向异性是不太复杂，并且可以更容易地并入未来支架设计用于体内应用。 在这个协议中，静态预拉伸细胞培养系统用于诱导表面各向异性无拓扑特征。预拉伸培养系统由PDMS膜，可拉伸帧和拉伸阶段，随后该膜被固定到框架和预定的拉伸幅度施加在拉伸阶段。预拉伸表面上培养了21天新鲜分离的DRG神经元中轴突对准和厚度进行监测。接着，雪旺细​​胞（SC）上的对准轴突为髓鞘监测共培养。通过采用预拉伸诱导表面各向异性，我们能够提高MSC和背根神经节9-10，分别对准细胞分化和轴突对齐增长。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane &#65288;PDMS)</td>
			<td>Dow Corning</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium 1X</td>
			<td>GibcoBRL</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement 50X</td>
			<td>GibcoBRL</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax-I 100X</td>
			<td>GibcoBRL</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumax-I</td>
			<td>GibcoBRL</td>
			<td>11020-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Nerve Growth Factor-7S</td>
			<td>Invitrogen</td>
			<td>13290-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>GibcoBRL</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA/1mM EDTA</td>
			<td>GibcoBRL</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Trevigen</td>
			<td><a href="http://www.trevigen.com/item/2/7/64/220/Cultrex_PolyLLysine/">3438-100-01</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>p-6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-2 deoxy-uridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
			<td>Isolation Buffer</td>
		</tr>
		<tr>
			<td>Type I Collagenase</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11885</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30080.03</td>
			<td></td>
		</tr>
		<tr>
			<td>BPE</td>
			<td>Clonetics</td>
			<td>CC-4009</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Calbiochem</td>
			<td>344270</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone chamber</td>
			<td>Greiner bio-one</td>
			<td>FlexiPERM ConA</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaning/etching system</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Thy 1.1 antibody</td>
			<td>Sigma- Aldrich</td>
			<td>M7898</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Complement</td>
			<td>Sigma- Aldrich</td>
			<td>S-7764</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard growth medium</td>
			<td></td>
			<td></td>
			<td>For 500 ml Neurobasal Medium 1X, add 10 ml of B-27 50X, 5 ml of Glutamax-I 100X,</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 &#956;l of NGF-- 7S (50 ug/ml).</td>
		</tr>
		<tr>
			<td>FDU and Uridine stock solution</td>
			<td></td>
			<td></td>
			<td>FDU 100mg in 10ml of ddw (10mg/ml), filter in the hood and divided in 500ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Uridine 5g in 166.7ml of ddw (33mg/ml), filter in hood, divide in 200ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Take 61.5ul of FDU (10mg/ml) and 20.5ul of Uridine(33mg/ml), and add 4918ul of ddw to a final stock concentration,</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>then divide in 1 ml aliquots and store at -20 &#186;C.</td>
		</tr>
	</tbody>
</table>

an approach to enhance alignment and myelination of dorsal root ganglion neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

预拉伸的细胞培养系统促进DRG轴突对准10。 DRG神经元中培养到预拉伸和未拉伸的表面12天。轴突染色为β-III微管蛋白，以证明其对准。 图2轴突取向后培养12天的预拉伸和未拉伸的PDMS基底进行比较。背根神经节轴突对齐平行于拉伸方向的，而他们表现出随机取向并形成未拉伸的PDMS衬底上的互连网络。 除了诱导轴突排列，预拉伸引起的各向异性增强轴突震颤。对拉伸的表面上形成轴突束对准的轴突​​，其从连接的轴突网络不同的未拉伸的表面上观察到。预拉伸表面上的这些束状束类似于神经组织TR在行为体内 。 图3显示了在培养21天前的拉伸，拉伸表面DRG神经元轴突。 如图3示出，聚集在一起的预拉伸衬底上的轴突以形成束，其中出现了类似束状大片。此建议预拉伸表面是能够促进广泛轴突生长在预拉伸的方向，这是在增强的神经组织的再生重要。 为了评估髓鞘，雪旺均与对准轴突共培养。培养2周拉伸未拉伸的表面上的DRG神经元之后，纯化旺加入到腔室和一个星期中培养。在图4中 ，共培养的轴突沾满β-III微管蛋白（绿色），以及雪旺与P0（红色）染色。 P0为成熟的SC的标志物，也是髓鞘的组分，以及考虑髓鞘的指标。有绿色和红色显著共定位的预拉伸表面上，暗示旺附着到轴突拉伸表面上的，但不能在未拉伸的表面，其中在绿色和红色的污渍是随机分布的。预拉伸的表面增强轴突对准，束状状道的形成，和SCS到再生轴突的附着，所有关键为髓鞘形成。 <img alt="图1" src="/files/ftp_upload/54085/54085fig1.jpg" /> 图1:原理图的预拉伸过程 （a）所有 PDMS膜被夹在那些滑动帧的两条。该装置的框架是约7"×5";括号约为5"×1;对括号夹具大约3"×25"，和杯是约1.5"直径的宽开口的顶部，1"开放的底部，以及约0.25＆＃34;高。 （ 二 ）将帧上的拉伸阶段中，将滚筒转动申请10％伸长，然后通过拧紧螺丝固定条带的位置。 （ 三 ）从伸展台上卸下框架，并附硅腔到膜。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54085/54085fig1large.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/54085/54085fig2.jpg" /> 图2: 轴突对齐上预拉伸和未拉伸的表面接种的（ 一 ）静态预拉伸表面上12天的DRG神经元，与接种（b）的未拉伸表面上12天的DRG细胞相比的荧光图像。轴突在预拉伸方向上排列，而轴突未拉伸控制表面上随机排列。轴突小号沾满抗鼠β-III微管蛋白主要抗体，并使用10X物镜共聚焦显微镜成像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54085/54085fig2large.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/54085/54085fig3.jpg" /> 图3: 轴突生长厚度在拉伸与无拉伸PDMS基板 （ 一 ）拉伸及（b）在未拉伸的表面21天DRG轴突的相位对比图像。拉伸表面上的轴突形成厚束，而相对于未拉伸表面上的轴突形成相互连接的轴突网络。图像用使用10X物镜反演相衬显微镜拍摄。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54085/54085fig3large.jpg" target="_blank">请点击此处查看人</a>arger版本这个数字。 <img alt="图4" src="/files/ftp_upload/54085/54085fig4.jpg" /> 图 4: 共培养与对齐的DRG轴突旺上拉伸与未拉伸的PDMS基质中培养2周的拉伸和未拉伸的表面上后，纯化旺加入到腔室和一个星期中培养。 （ 一 ）β-III微管蛋白（绿色）染色拉伸表面上的轴突; （ 二 ）β-III微管蛋白的重叠（绿色）拉伸表面上，P0（红色）染色; （ 三 ）β-III微管蛋白（绿色）染色未拉伸的表面上的轴突; β-III微管蛋白（绿色）（ 四 ）覆盖未拉伸的表面上，P0（红色）染色。上的预拉伸的表面，红色和绿色的污渍共定位，表明的SC连接到并可能髓鞘的轴突。在未拉伸表面上，红色和绿色的污渍是随机分布的，这表明在SCS不附着到轴突。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54085/54085fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons at JoVE.com

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

一种方法来提高背根神经节的对齐和髓鞘

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

an-approach-to-enhance-alignment-myelination-dorsal-root-ganglion

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.4K 次观看.  Michigan State University. This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

视频: An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

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. ...

科研

神经科学

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 ...

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 ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

在癌神经侵袭的体外模型:背根神经节型号

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

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.

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 ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomy 对猪腰背根神经节通路和注射的治疗

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

背根神经节分离与原代培养对神经递质释放的研究

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

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 ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...