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?...

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><tbody><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/1 mM EDTA</td><td>GibcoBRL</td><td>25300-054</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Trevigen</td><td>&lt;a href=&quot;http://www.trevigen.com/item/2/7/64/220/Cultrex_PolyLLysine/&quot;&gt;3438-100-01&lt;/a&gt;</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&amp;rsquo;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, 2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 &amp;mu;l of NGF-- 7S (50 &amp;mu;g/ml).</td></tr><tr><td>FDU Uridine stock solution</td><td></td><td></td><td>FDU 100 mg in 10 ml of ddH&lt;sub&gt;2&lt;/sub&gt;O (10 mg/ml), filter in the hood and divided in 500 &amp;mu;l aliquots and store at -20 &amp;ordm;C. Uridine 5 g in 166.7 ml of ddH&lt;sub&gt;2&lt;/sub&gt;O (33 mg/ml), filter in hood, divide in 200 &amp;mu;l aliquots and store at -20 &amp;ordm;C.
Take 61.5 &amp;mu;l of FDU (10 mg/ml) and 20.5 &amp;mu;l of Uridine(33 mg/ml), and add 4,918 &amp;mu;l of ddH&lt;sub&gt;2&lt;/sub&gt;O to a final stock concentration, then divide in 1 ml aliquots and store at -20 &amp;ordm;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?...

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

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

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

隔间文化背根神经节神经元的制备及维修

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

科研

生物学

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 ...

神经科学

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 ...

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 ...

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 ...

用于研究 DRG 培养中神经元超敏反应的高级体外模型

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

作者聚焦：提高慢性疼痛治疗效果的体外和体内模型

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

在多室装置中收获、包埋和培养背根神经节以研究外周神经元特征

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

生物工程

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

大鼠背根神经节外植体和雪旺细胞共培养中外周轴突的体外髓鞘形成

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

通过免疫淘选富集成年小鼠背根神经节神经元培养物

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as ...

一种方法来提高背根神经节的对齐和髓鞘

摘要

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此视频中的章节

隔间文化背根神经节神经元的制备及维修

培养成年的背根神经节神经元轴突再生的遗传研究

背根神经节神经元和区分脂肪干细胞:一种

背根神经节内注射和背根挤压伤为感官轴突再生模型

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

在多室装置中收获、包埋和培养背根神经节以研究外周神经元特征

在多室装置中收获、包埋和培养背根神经节以研究外周神经元特征

在多室装置中收获、包埋和培养背根神经节以研究外周神经元特征

大鼠背根神经节外植体和雪旺细胞共培养中外周轴突的体外髓鞘形成

通过免疫淘选富集成年小鼠背根神经节神经元培养物