Name: hydraulic extrusion of the spinal cord and isolation of dorsal root ganglia in rodents
Uploaded: 2017-01-22T15:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents at JoVE.com

We would like to acknowledge David Kiel, Aarhus University, for filming and editing. VirtualDub software was used for processing of microscope video sequences. We thank the Danish Research Institute of Translational Neuroscience - DANDRITE, Nordic EMBL Partnership, for access to equipment and the Aarhus University Research Foundation, EU 7FP project PAINCAGE, Det Frie Forskningsrad (DFF) and The Lundbeck Foundation for funding.

所有的动物都完全符合丹麦和欧洲法规进行处理。批准文号:2012-15-2934。 1.将注射器的脊髓液压挤压的制备<ol><li>对于成年大鼠，用10毫升的注射器。对于成年小鼠，用5毫升注射器。对于幼崽，用2.5毫升的注射器。 </li><li>调节非滤波器枪头普遍适用于2-200微升移液器通过修整枪头的大端，直到它牢固地装配到注射器。放置在注射器调整枪头。对于幼崽，用23 G或类似针头，而不是一个调整枪头。 </li><li>抽吸冰冷无菌PBS，并留下在冰上注射器直至进一步使用。 </li></ol> 2.安乐死<ol><li>不要执行颈椎脱位，因为这会扰乱椎骨渲染挤出是不可能的。 </li><li>对于成年大鼠/小鼠，安乐死使用气体如CO 2或isofluRANE。这些物质都是有害的。在通风橱中进行安乐死，并根据机构指南处理物质。 （注意: 二氧化碳 :H280，P410，P403;异氟烷:H361d，P260，P281，P280） <ol><li>为了确保啮齿动物是死的，按捏用镊子一爪子确保没有反射。杀头使用大剪刀的啮齿动物。对于幼崽，斩首安乐死。 </li></ol></li></ol> 3.脊柱隔离<ol><li>水洒到皮毛来控制头发。 </li><li>使用剪刀，剖开沿脊柱的皮毛在远侧方向和由两侧切割沿过去骨盆骨脊柱隔离脊柱。远端切断脊髓骨盆带一把剪刀。 注:延髓 - 尾轴被表示为在整个协议近端 - 远端轴。 <ol><li>使用剪刀，以避免proximal-修剪脊柱大多数和远端，大部分地区，因为这些可能会导致脊柱过S形成功脊髓挤压。确保脊髓是在两端是可见的。如果脊髓是不可见的，用剪刀修整脊柱直到脊髓变得可见。 </li></ol></li></ol> 4.脊髓液压挤压<ol><li>放置一个培养皿（100毫米直径）上的冰填充用无菌PBS。 </li><li>通过在脊柱的弯曲部（最近端）施加食指，从而矫直脊柱尽可能拉直脊柱。 <ol><li>对于幼崽，避免弯曲脊柱，椎骨就会被打乱渲染脊髓挤出是不可能的。 </li></ol></li><li>插入枪头在脊柱的最远端（23政针幼仔，或者18G的针对成年小鼠）。如果正确插入，尖端在脊髓腔内稳定。 </li><li>确保脊柱被拉直尽可能。应用稳定的压力，并挤压脊髓成冰的培养皿。确保脊髓通过保持它在PBS直到进一步的处理保持湿润冰上。 </li></ol><img alt="图1" src="/files/ftp_upload/55226/55226fig1.jpg" /> 图1: 代表脊髓。液压挤压脊髓。从左到右依次为:成年大鼠，成人鼠标，鼠标小狗。腰椎放大标志着框。 <a href="http://ecsource.jove.com/files/ftp_upload/55226/55226fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 5.确定背根神经节（病种付费） <ol><li>拆分在用剪​​刀两个相等纵向部分脊柱并将分割脊柱在显微镜下。 </li><li>识别T13椎体段由<html>定位最远端的肋脉附着部位的脊柱注意不要混淆与附近的肋间神经的肋脉。最远侧肋脉附着到T13椎骨段的近端部分和T13 DRG位于远侧到椎骨T13和近侧到椎骨L1上。 </li><li>识别远端方向伴随的DRG。对于成年大鼠，按病种付费相伴出现白色带清晰可见，背，腹根。对于成年小鼠，伴随病种出现白色带清晰可见，背，腹根。对于幼崽，伴随病种出现明显的领域。 </li></ol><img alt="图2" src="/files/ftp_upload/55226/55226fig2.jpg" /> 图2:在脊柱的DRG鉴定脊髓的挤出后。脊柱已被划分为允许的DRG的可视化由箭头所示。成年大鼠和小鼠的小狗为例。<html> REF ="http://ecsource.jove.com/files/ftp_upload/55226/55226fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 6.按病种付费隔离的<ol><li>根据具体的DRG数目的培养皿（直径30毫米），以进行分离， 例如 ，L3，L4，L5。用冰冷的无菌PBS填充培养皿放置冰上。 </li><li>采用微型剪刀，剪背，腹根部尽量靠近病种付费，尽量释放出来，同时避免损坏病种付费。 </li><li>随着封闭镊子尖，轻轻舀出按病种付费，并将它们传送到相应编号的培养皿。 </li></ol><img alt="图3" src="/files/ftp_upload/55226/55226fig3.jpg" /> 图3:代表隔离病种付费。从左到右依次为:成年大鼠，成人鼠标，鼠标小狗。"_blank"&gt;请点击此处查看该图的放大版本。 7.处理组织中进行进一步的分析<ol><li> Western印迹<ol><li>隔离感兴趣的组织区域， 例如脊髓腰膨大。如果需要的话，脊髓分离成同侧和对侧侧面和进一步进入背，腹角。 </li><li>放置在裂解缓冲液中的组织，如包含在冰上适当的酶抑制剂的TNE裂解缓冲液（10mM Tris碱，150mM的氯化钠，1mM的EDTA，1％NP40双蒸H 2 O）。 </li><li>使用均质机械研磨杵在冰上组织约30秒。 </li><li>离心16,000 xg离心在4℃下15分钟，并根据标准方案5,6使用上清液用于进一步的处理或保留上清液在-20℃直至进一步使用。 </li></ol></li><li> RNA分析<ol><li>隔离感兴趣的组织。 </li><li>立即放置在RNA的稳定溶液中的组织稳定用于存储的RNA。 </li><li>根据标准方法7分离RNA。 </li><li>转换的RNA到cDNA和根据标准方法7进行定量PCR。 </li></ol></li><li>免疫组化<ol><li>对于新鲜冷冻的组织（适用于脊髓）: <ol><li>粉碎干冰块对应粉两汤匙的量准备干冰粉末。覆盖在布干冰块，并用锤粉碎立方体。放置干冰块上一层银箔（大约5厘米×5厘米）和盖用干冰粉末银箔。 </li><li>放置用镊子选择用于进一步分析，在干冰粉末的脊髓区域，让它单元冻结和脊髓转移到冷冻管中。保持干冰在任何时候或在-80存储°C，直到我们进一步即</li><li>脊髓转移到低温恒温器（-20℃）。如果需要的话，修剪低温恒温器内的脊髓。放置脊髓中，以便将其附加到试样圆盘嵌入低温恒温器内的材料。 </li><li>切断脊髓中的期望的厚度， 例如 5微米，并收集在玻璃板上。加热用手指玻璃板之前立即向组织收集更好附着。请低温管任何剩余的组织于-80°C，直到进一步使用。 </li><li>留在里面低温恒温器玻璃板的部分，直到准备固定后。 </li><li>后固定通过浸入切片在4％PFA中于室温下10分钟。 </li><li>执行根据标准方案8染色。 </li></ol></li><li>固定组织的冷冻切片（适用于脊髓背根节和） <ol><li>修复脊髓和背根节在4％PFA（2小时脊髓，对小鼠的DRG 1.5小时，大鼠的DRG 4小时）在4℃（放置脊椎在水平位置线，以防止它从在弯曲位置固定）。 </li><li>在4℃转移组织至25％w / v的蔗糖过夜。所述组织可以存储在25％（重量）补充有10％的叠氮化钠的几个液滴以防止微生物污染在4℃直至进一步使用/ v的蔗糖。如果需要的话，修剪脊髓弥补腰膨大仅在硅板或相似的。 </li><li>使用纸巾的前端，吸从组织过量的蔗糖溶液。 </li><li>填补了低温模具时半包埋材料。按任何气泡与工具低温模具的两侧，如封闭镊子。 </li><li>使用镊子放置在包埋材料的组织，并确保期望的组织的方向。 </li><li>与包埋材料完全填充模具低温，推动任何气泡远离组织尽可能（ 例如，通过使用封闭的镊子）。 </li><li>填补了培养皿英寸（100毫米在直径）与2-甲基丁烷。这种物质是有害的。执行在通风橱这一步，并根据机构的指导方针处理物质（注意:H224，H304，H336，H411，P210，P280，P273，P301，P331，P304 / P340，P309 / P310）。 </li><li>将干冰一层培养皿上，并添加两个干冰块培养皿。放置在培养皿的低温模具和允许包埋材料完全冻结。 </li><li>保持干冰嵌入组织在任何时候，或在-80℃包裹在封口膜，直到进一步的处理。 </li><li>上切割一个低温恒温器（-20℃）中所希望的厚度的组织， 例如 5微米。 </li><li>执行根据标准方案8染色。 </li></ol></li><li>固定组织的石蜡包埋（适用于脊髓背根节和） <ol><li>在4℃（放置脊髓修复脊髓和背根节在4％PFA（2小时脊髓，对小鼠的DRG 1.5小时，大鼠的DRG 4小时）在水平位置，以防止其在弯曲位置固定）。 </li><li>在4℃转移组织至25％w / v的蔗糖过夜。所述组织可以存储在25％（重量）补充有10％的叠氮化钠的几个液滴以防止微生物污染在4℃直至进一步使用/ v的蔗糖。 </li><li>隔离感兴趣的组织区域， 例如脊髓腰膨大。 </li><li>填补嵌入模具中途用石蜡。 </li><li>通过将其上的嵌入机的冷却面积稍微硬化石蜡降温嵌入模具简要。这允许更好的组织定位。 </li><li>放置在组织中在嵌入模具中的所需取向。填写与石蜡嵌入模具，并立即冷却。 </li><li>放置含石蜡包埋模具在4℃过夜，以让它完全硬化和除去含有来自嵌入模具的组织的石蜡块。 </li><li>切段Ø呐切片机在所希望的厚度， 例如 5微米。 </li><li>执行根据标准方案8染色。 </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

如果脊柱被干扰， 例如 ，通过颈椎脱位，脊髓将挤出期间分割。如果脊髓无法挤出，脊柱可以稍微在两端修剪和挤压尝试可以重复。在情况下，需要进行进一步的分析整个脊髓， 即由所述颈膨大以及腰膨大，脊柱应仅略微修整。如果需要作进一步的分析只腰膨大，脊髓应根据本协议进行修整。脊柱应该用指尖来缓解挤压被拉直。 该协议适用于所有年龄段的啮齿类动物，并通过这里的成年小鼠（8周），鼠标小狗（5天）和成年大鼠（10周）的例子。根据动物种类和菌株，胸椎和腰椎部分的数目可以变化9 </sup&gt; 10，11。根据不同的蛋白质要分析，成年啮齿动物可与安乐死之前酶抑制溶液灌注。如果执行涉及片面神经损伤的实验，脊髓可以分成同侧和对侧侧面采用超细镊子挤压之后立即。此外，每个侧面可以分成背，腹侧面。组织然后可以用于进一步的分析， 例如 Western印迹处理。 穿心灌注固定带之前，脊髓挤出的PFA应该避免，因为这使脊髓非柔性和防止脊髓液压挤出。液压脊髓挤压将撕下背根。对于实验中附后根是必要的，建议椎板切除术。此外，脊膜由液压挤压，这可以通过标准椎板避免丢失<sup系列="外部参照"&gt; 3。 也可以使用代替冰冷的PBS含氧人工脑脊液（ACSF）执行的脊髓和背根神经节分离的水力挤出。使用ACSF的允许分离的组织保存在一个更好的生理环境，这是在随后的电生理记录12,13的情况下是特别重要的。到ACSF替代可以是含有1克/升葡萄糖用于产生主DRG培养物14的PBS中。 脊髓液压挤压是一个比通过椎板脊髓隔离的传统方式，减少组织处理时间，因此减少蛋白质损坏的风险显著更快的方法。灌注固定剂前分离由椎板脊髓可以减少清扫期间和最终去除的过程中的组织损伤的风险从脊柱脊髓。然而，组织固定排除其适用性分析，如蛋白印迹。液压挤压产量结构未受损组织3适用于更广泛的分析。 按病种付费的一致鉴定可能很困难。然而，这是用于组织分析必需的， 如以下坐骨神经损伤。由编号，根据相对于肋脉其本地化的DRG，病种可以一致认定。脊髓组织的染色以及的DRG可以通过执行协议步骤7中概述的图示组织治疗的变化进行优化。

该方法的总的目标是在脊髓隔离以及鉴定和分离的DRG 1,2。脊髓液压挤压是一个比通过椎板脊髓隔离，通过一次破椎骨一个体现的传统方式显著更快的方法，这种方法可以减少组织损伤造成的解剖过程3,4的风险。的DRG可能难以辨认。正确识别是组织分析非常重要， 如下面的坐骨神经损伤。由编号，根据相对于肋脉其本地化的DRG，病种可以一致认定。 组织由这里展示的技术分离可应用于范围广泛的分析，包括Western印迹5，6，7定量PCR和免疫组化染色8。 当识别的DRG，其采取到已知脊髓节段的数目随动物种类和菌株9，10，11，以改变帐户是重要的。在小鼠中进行该方法的优点是高数量的可用遗传修饰的变体和相对低的壳体费用。在用大鼠的优点是相对高的组织的产量和如果涉及神经损伤，该过程将与尺寸有所缓解。

<table><tbody><tr><td>Bonn scissors, extra fine, straight, 8.5 cm</td><td>F.S.C. (Fine Science Tools)</td><td># 14084-08</td><td>For cutting open the spinal cord prior to isolation of DRGs (adult mouse, adult rat); Other manufacturer may be used</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td># 5427 R</td><td>For centrifugation of homogenized tissue</td></tr><tr><td>Cryostat microtome</td><td>Leica Biosystems</td><td># CM 3050 S</td><td>For cutting the embedded tissue prior to staining; Other model and manufacturer may be used</td></tr><tr><td>Forceps, Dumont, # 3</td><td>F.S.C. (Fine Science Tools)</td><td># 11231-30</td><td>For handling of spinal cord after extrusion; Other manufacturer/type may be used</td></tr><tr><td>Forceps, Dumont, # 5</td><td>F.S.C. (Fine Science Tools)</td><td># 11252-20</td><td>For isolation of DRGs (adult mouse, adult rat, pup); Other manufacturer may be used</td></tr><tr><td>Isoflurane (furane) IsoFlo Vet 100%</td><td>Abbott</td><td># 002185</td><td>For euthanization; Other manufacturer may be used; CAUTION: toxic</td></tr><tr><td>Iso-pentane GPR rectapur</td><td>VWR chemicals</td><td># 24872.298</td><td>For snap-freezing of tissue; Other manufacturer may be used; CAUTION: toxic</td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td># RM 2155</td><td>For sectioning of paraffin embedded tissue; Other model and manufacturer may be used</td></tr><tr><td>Paraffin wax pellets</td><td>Sigma-Aldrich</td><td># 76243</td><td>For paraffin embedding; Other manufacturer may be used</td></tr><tr><td>Paraffin tissue embedding station</td><td>Leica Biosystems</td><td># EG1160</td><td>For paraffin embedding of tissue for later sectionning; Other model and manufacturer may be used</td></tr><tr><td>Pellet pestel, motor cordless</td><td>Sigma-Aldrich</td><td># Z359971-1EA</td><td>For mechanical homogenization of isolated tissue prior to Western blotting; Other manufacturer may be used</td></tr><tr><td>Petri dish, 35 mm</td><td>Thermo Fischer Scientific</td><td># 121V</td><td>For storage of isolated DRGs; Other manufacturer and size may be used</td></tr><tr><td>Petri dish, 100 mm</td><td>Sigma-Aldrich</td><td># P7741</td><td>For spinal cord extrusion; Other manufacturer and size may be used</td></tr><tr><td>Phosphatase inhibitor, Phosstop</td><td>Sigma-Aldrich</td><td># 04906845001</td><td>Phosphatase inhibitor cocktail for addition to TNE-lysis buffer if needed; Other manufacturer may be used</td></tr><tr><td>Pipette tip, 1-200 &amp;mu;L, no filter</td><td>Sarstedt</td><td># 70.1189.105</td><td>For hydraulic extrusion; Other manufacturer may be used</td></tr><tr><td>Protease inhibitor, Complete</td><td>Sigma-Aldrich</td><td># 05892791001</td><td>Protease inhibitor cocktail for addition to TNE-lysis buffer if needed; Other manufacturer may be used</td></tr><tr><td>RNAlater solution</td><td>Sigma-Aldrich</td><td># R0901</td><td>For RNA stabilization for storage; Other manufacturer may be used</td></tr><tr><td>Rneasy Protect Mini KiT</td><td>Qiagen</td><td># 74124</td><td>For RNA isolation; Other manufacturer may be used</td></tr><tr><td>Scalpel; Swann-Morton surgical blade no. 11</td><td>Swann-Morton</td><td># REF0203</td><td>For isolation of spinal cord lumbar area; Other manufacturer/type may be used</td></tr><tr><td>Scissors, straight, type 3, 25 mm cutting edge</td><td>Bochem</td><td># 4070</td><td>For isolation of spine; Other manufacturer/type may be used</td></tr><tr><td>Scissors, 130 mm cutting edge</td><td>Hounisen</td><td># 1902.0130</td><td>For isolation of spinal cord (adult rat)</td></tr><tr><td>Spring scissors, straight, 8 mm cutting edge</td><td>F.S.C. (Fine Science Tools)</td><td># 15009-08</td><td>For cutting open the spinal column prior to isolation of DRGs (pup) and for isolation of DRGs (adult mouse, adult rat, pup); Other manufacturer may be used</td></tr><tr><td>Standard syringes, 2.5 mL, 5 mL, 10 mL</td><td>Terumo</td><td># SS02SE1; # SS05SE1; # SS10SE1</td><td>For hydraulic extrusion; Other manufacturer may be used</td></tr><tr><td>Stereomicroscope MZ12.5 with objective 1X and eyepiece 10X</td><td>Leica</td><td># 10446370</td><td>Other manufacturer/type may be used</td></tr><tr><td>Sterile PBS</td><td>GIBCO</td><td># 10010-015</td><td>For hydraulic extrusion; Can also be made according to standard protocols</td></tr><tr><td>Syringe needle 23 G x 1&quot;, 0.6 x 25 mm</td><td>Terumo Neolus</td><td># NN-2325R</td><td>For hydraulic extrusion in pups; Other manufacturer/type may be used</td></tr><tr><td>Syringe needle 18 G x 1 1/2, 1.2 x 40 mm</td><td>Terumo Neolus</td><td># NN-1838S</td><td>Alternative to pipette tip for hydraulic extrusion in adult mice; Other manufacturer may be used</td></tr><tr><td>Tissue-Tek</td><td>Sakura</td><td># 4583</td><td>Tssue embedding material for later cryosectionning</td></tr><tr><td>TNE-lysis buffer</td><td>VWR chemicals</td><td># 10128-582</td><td>For tissue lysis prior to Western blotting; Other manufacturer may be used; Can also be made according to standard protocols</td></tr></tbody></table>

hydraulic extrusion of the spinal cord and isolation of dorsal root ganglia in rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

液压挤出脊髓显示图1中所示的平滑无损表面。腰膨大可以很容易地被识别为脊髓加厚区域，在图1装箱。脊髓的背侧和腹侧双方可以通过肉眼按照形态来识别。在图1中 ，腹侧朝向观察者，通过沿着整个脊髓一个明显的中线识别。沿着脊髓两更广线允许背侧的鉴定。在最远端，马尾有时保持在成年大鼠和成年小鼠附着。 个别的DRG只能被识别，而位于脊柱， 图2。以下隔离， 图3，各个病种不能由外观识别。如果需要的话，它因此是在分离重要的是让他们清楚地分开。分离后，脊髓和背根节可以被处理和染色如在步骤7.3中概述，并在图4表示。 图4a示出了背根神经节段，其中所述的神经元和其周围卫星细胞是清晰可见的免疫组织化学染色。通过正确识别的背根神经节，可以分析对受伤的神经元的胞体以及在其周围的卫星细胞坐骨神经损伤的效果。 图4b示出了液压挤出脊髓，其中所述组织是完整的部分的边缘光滑反映的Nissl染色。 <img alt="图4" src="/files/ftp_upload/55226/55226fig4.jpg" /> 图4:代表性的组织切片。一 。 DRG节的免疫组化染色。 B点 。尼氏染色sectio液压挤压脊髓的n个。 <a href="http://ecsource.jove.com/files/ftp_upload/55226/55226fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents at JoVE.com

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

背根神经节的啮齿动物的脊髓和隔离的液压挤压

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

hydraulic-extrusion-spinal-cord-isolation-dorsal-root-ganglia

Nanyang Technological University

Research

JoVE Journal

Neuroscience

47.8K 次观看.  Aarhus University. Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage. 

视频: Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

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 ...

科研

神经科学

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.

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 ...

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 ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

多尾根结鱼巨噬细胞的快速隔离

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

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

Harvesting DRG from Rat Spine: A Procedure to Extract Dorsal Root Ganglion from the Spine of a Rat Model

Source: Schmidt, L. B. et al. <a href="https://www.jove.com/video/59296" target="_blank">The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the extraction of dorsal root ganglion or DRG from a harvested rat spine for downstream applications.

从大鼠脊柱中采集 DRG:从大鼠模型脊柱中提取背根神经节的程序

Source: Schmidt, L. B. et al. The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer. J. Vis. Exp. (2019)
...

JoVE Encyclopedia of Experiments

癌症研究

Head and Neck Cancer

Assays & techniques

Spinal Cord Extraction: A Protocol to Isolate Murine Spinal Cord for In Vitro Studies

Source: Na&#39;ara, S. et al. <a href="https://www.jove.com/video/52990" target="_blank">In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model.</a> J. Vis. Exp. (2016)
In this video, we describe a step-by-step protocol to isolate spinal cord from a mouse model for in vitro studies.

脊髓提取:分离小鼠脊髓用于体外研究的方案

Source: Na&#39;ara, S. et al. In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model. J. Vis. Exp. (2016)
In this video, we ...

In vivo studies

Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells

Source: Tsui, Y, et al. <a href="https://app.jove.com/v/55794">Hypoxic Preconditioning of Marrow-derived Progenitor Cells as a Source for the Generation of Mature Schwann Cells.</a> J. Vis. Exp. (2017).
This video demonstrates a procedure for isolating dorsal root ganglion (DRG) neurons from rat embryos and co-culturing them with Schwann cell precursors to generate mature Schwann cells. The isolated DRGs are processed to isolate neurons and glial cells. The isolated cells are cultured to purify the neurons, which are then co-cultured with Schwann cell-like cells (SCLCs) in a co-culture medium. Interaction with the DRG neurons leads to the maturation of SCLCs into Schwann cells.

分离 DRG 神经元并与雪旺细胞前体共培养以生成雪旺细胞

Generation of Fate-committed Schwann Cells via Coculture with DRG Neurons
1. Preparation of purified rat DRG neurons

	Autoclave all dissection tools ...

背根神经节的啮齿动物的脊髓和隔离的液压挤压

摘要

探索更多视频

此视频中的章节

培养成年的背根神经节神经元轴突再生的遗传研究

一种方法来提高背根神经节的对齐和髓鞘

背根神经节内注射和背根挤压伤为感官轴突再生模型

多尾根结鱼巨噬细胞的快速隔离

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

从大鼠脊柱中采集 DRG:从大鼠模型脊柱中提取背根神经节的程序

脊髓提取:分离小鼠脊髓用于体外研究的方案

分离 DRG 神经元并与雪旺细胞前体共培养以生成雪旺细胞