FZ型从NIH（R01NS064288）和克雷格H.尼尔森基金会的赠款，以支持这项工作。

1。盖玻片，培养基，消化酶的制备<ol><li> 12毫米的圆形＃1盖玻片用于神经元的文化。清洁的盖玻片，10％HCL一夜之间随后用蒸馏水和去离子水超声波清洗3次（20分钟/次）。清洗盖玻片存储在70％乙醇以供将来使用。在每次实验中，盖玻片是空气干燥，并放置到培养皿。 </li><li>涂层干燥的盖玻片，100μL涂层溶液含有100微克/毫升聚-D-赖氨酸和10微克/毫升层粘连工作的解决方案添加到每个盖玻片转移到37℃培养箱培养板。 1-2小时后，被删除的涂层解决方案和盖玻片用无菌1×PBS洗涤3次。 </li><li>准备培养基，最低基本培养基（MEM）的5％胎牛血清（FBS）1X青霉素，链霉素溶液（500单位的青霉素辅以林链霉素和500微克），1X GlutaMAX我的补充，和抗有丝分裂剂含有20μM的5 - 氟-2 - 脱氧和20μM尿苷（复旦/ R）的。无血清培养基，胎牛血清被替换与补充B27的。 </li><li>胶原酶准备一个解决方案所具有的MEM粉末溶解胶原酶的工作浓度为1毫克/毫升。为胰蛋白酶，的1X TrypLE的快速使用。 </li></ol> 2。成年小鼠DRG神经元的解剖和收获<ol><li>安乐死的6 - 10周龄成年小鼠后，去除背部皮肤和切断整个脊柱。清洗删除脊柱1×PBS的2-3倍。 </li><li>引脚删除脊柱腹朝上，夹层板和感觉神经暴露在解剖显微镜下仔细去除肌肉。木材一级连接到背根节神经厚，很容易找到。因此，对于大多数实验只腰背根节收获。 </li><li>通过每个椎体沿中心线与小剪刀剪下，小心地取出椎间盘。使用镊子分裂每个椎体暴露脊髓。 </li><li>解剖木材背根节，用钳子举起每个腰神经向脊髓跟踪找到相关的腰病种付费（从L1到L6）。 </li><li>从每个连接的末梢神经，背，腹根弹簧剪刀切断背根神经节，并存储在MEM培养基离心管置于冰上。更多的病种付费在脊髓水平不同（ 如胸病种付费）可以以类似的方式，必要时解剖。 </li></ol> 3。成年小鼠DRG神经元的消化和离解<ol><li>收集所有的解剖病种付费后，将用1毫升胶原酶一个解决方案MEM培养基，在37°C孵育90分钟的离心管。 </li><li>取代胶原酶一个解决方案，以便用500μl1X TrypLE快递lution和孵化，在37°C的15-20分钟。 </li><li>删除的TrypLE的Express解决方案和用1毫升准备好的文化中期的3倍（含5％FBS）的病种付费。 </li><li>加入600μL培养基，轻轻吸取向上和向下磨碎使用1毫升蓝色为20-30倍组织，毕业于枪头。 </li><li>研磨后，允许非游离组织落户底部的离心和细胞悬液转移至10毫升无菌管。另外600μL培养基添加和重复的研磨步骤，直到大部分组织是分离的。获得的细胞悬液包含两个神经元和非神经细胞。在大多数情况下，从6背根节（〜5×10 4）细胞用于一个电反应。 </li></ol> 4。通过电穿孔神经元遗传操纵<ol><li>准备转染小鼠神经元混合DNA质粒（〜10微克Amaxa核转染的解决方案解决方案）或siRNA寡核苷酸（〜0.2 nmol之间）作出100转每微升的最终体积。 </li><li>在室温下为7分钟680转离心分离细胞，弃上清，尽可能。加入准备转染的解决方案，并轻轻吸取向上和向下与200μL枪头重新挂起细胞的3-4倍。 </li><li>转让与转染溶液电试管混合细胞悬液和electroporate使用的Amaxa Nucleofector系统方案的G-013的细胞。 </li><li>电击后，立即加入500μL预温（37℃）培养液中含有胎牛血清试管和涂在所需的细胞密度培养板转移到所有的解决方案（约600μL）。实验需要再悬浮和再镀，塑料培养皿上培养神经元直接在高密度（10000-20000细胞/孔）。为实验，直接检查轴突生长，神经元是镀金的ONTO在较低的密度（3000-5000细胞/孔）镀膜玻璃盖玻片。培养板放入培养箱（37℃，5％CO 2）。 </li><li>电镀时，神经元连接到基板后的四个小时，轻轻地用500μl新鲜和预热的培养基更换培养基（其中包含的Amaxa核转染的解决方案），并返回为更多的文化板的孵化器（37°C ，5％CO 2）。都培养基中含有胎牛血清或无血清培养基可用于在这一步。 </li></ol> 5。培养成人背根神经节神经元轴突生长分析<ol><li> DNA质粒转染的神经元，基因表达利益（ 如绿色荧光蛋白），可以观察到，早在几个小时后电。对于与siRNA的转染的神经元，我们通常等待3-4天，以允许足够的内源性蛋白的消耗。培养的神经细胞可以直接固定在VARIO轴突生长分析我们的时间点（电击后1-4天），或重新暂停和重新镀分析轴突再生（见下文）。 </li><li> RNAi介导的损失函数研究，培养的神经细胞可以重新暂停，重新镀允许轴突再生神经元，其中有针对性的蛋白已经耗尽。这样做，取代旧的培养基1 ML预热的新鲜培养基和吸管轻轻地，重新暂停从培养板连接的神经元的6-10倍的高密度培养的神经元。由于非神经细胞（ 例如雪旺氏细胞）附加更严格的比神经细胞的培养皿中，重新悬浮细胞主要是神经元。 </li><li>重新悬浮神经元转移到离心管中，轻轻磨碎的10-15倍，重新分解成单细胞悬液的细胞团块。 </li><li>重制版到新制备的盖玻片再分离的神经元密度低（3000-5000细胞/孔），培养过夜（16-24小时）。 </li></ol> 6。固定，免疫荧光染色，荧光成像<ol><li>抽吸的介质，并添加4％多聚甲醛（PFA）（200μL/孔），在室温15-20分钟，修复细胞。 1X 3次PBS固定细胞，然后洗净。 </li><li>吸出PBS及加入封闭液（1％牛血清白蛋白，0.1％的Triton X-100，2.0％正常山羊血清中的1X PBS）在室温下60分钟的固定神经元。 </li><li>标签轴突，神经细胞是免疫与抗神经丝或反βIII的微管蛋白抗体染色。这样做，把对每个盖玻片石腊膜初级抗体溶液30μL-βIII微管蛋白抗体（1:1200稀释）下降。反转盖玻片，放置到主抗体溶液在室温下60分钟。 </li><li>返回到原来的培养板盖玻片，用1X PBS洗3次。 </li><li>重复的相同的程序为二级安tibody。 </li><li>的盖玻片，用蒸馏水洗3次，然后装入载玻片，盖玻片与安装的解决方案（ 如延长Gold抗淬灭）。 </li><li>染色的神经元可以与任何荧光显微镜系统配备一台数码相机成像。轴突长度测量和图像分析软件进行分析。 </li></ol> 7。代表结果在没有任何增值外生长因子的情况下，成年DRG神经元通常开始增长轴突先镀后48小时。常常表现出的轴突分支的形态（ 图1）。相比之下，再镀神经元开始电镀后只有几个小时延长轴突，轴突伸长分支（ 图2），大大减少。这些结果表明，再镀神经元损毁神经元的空调有着相似的性质。通过使用这种方法，我们最近进行亏损的功能研究，探讨从成人的背根神经节神经元在体外轴突再生相关的转录因子c-Jun在轴突生长的作用。结果表明，电，一组4种不同的siRNAs，针对不同地区的c-Jun（ON-TARGETplus）显着减少成年DRG神经元中c-Jun的蛋白水平，转染后第3天（ 图3）9。当神经元重新镀和培养过夜，从c-Jun的击倒神经轴突生长，显着降低（控制：348.37±16.21毫米; SI-C君：262.32±15.69微米， 图3）9。这些结果表明，培养的成年DRG神经元提供了一个有用的模型系统研究从成人神经轴突生长。 <img alt="图1" src="/files/ftp_upload/4141/4141fig1.jpg" /> 图1。成年小鼠DRG神经元培养密度低的为3天。具有抗染色的神经元 - ＆BETA;三，微管蛋白的抗体。请注意，大多数轴突显示分支形貌。比例尺：125微米。 <img alt="图2" src="/files/ftp_upload/4141/4141fig2.jpg" /> 图2。再镀成年鼠背根神经节神经元后，3天中文化。 （一）培养成人DRG神经元的高密度为3天。 （二）再镀成年DRG神经元培养过夜低密度。请注意，大多数轴突显示拉长形态与小轴突分支。比例尺：250微米和125微米在B <img alt="图3" src="/files/ftp_upload/4141/4141fig3.jpg" /> 图3。从体外成人DRG神经元的轴突生长c-Jun的作用。 （一）西方blot分析c-Jun的成年鼠背根神经节神经元的c-Jun的siRNAs的电击后。结果表明：c-Jun的水平显着降低。 （二）长大后重新电镀，过夜培养与EGFP转染的控制神经元的长轴突。 （三）公司的反fection的c-Jun的siRNA和EGFP受损成年后重新电镀，过夜培养背根神经节神经元的轴突生长。红：Tuj-1染色;绿色：绿色荧光蛋白。比例尺：125微米。这些成果已发表 ​​在Saijilafu等9。

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没有利益冲突的声明。

成年DRG神经元在体内和体外的周围神经损伤后轴突再生强劲，从而提供了一个有用的系统研究在成年动物的神经轴突再生。成年DRG神经元在体外培养成为一种广泛使用的方法来研究的分子机制轴突再生的调节。 在体外培养成年小鼠DRG神经元的过程中所提出的在这里可以迅速和有效的再生轴突生长的遗传研究。再悬浮和再镀过程是亏损的功能研究特别有用，因为它允许轴...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>的MEM </td><td> Invitrogen公司</td><td> 11090-081 </td></tr><tr><td>聚-D-赖氨酸氢溴酸</td><td> Sigma-Aldrich公司</td><td> P6407 </td></tr><tr><td>层粘连蛋白</td><td> Invitrogen公司</td><td> 23017-015 </td></tr><tr><td> 5 - 氟-2-脱氧</td><td> Sigma-Aldrich公司</td><td> F0503 </td></tr><tr><td>尿苷</td><td> Sigma-Aldrich公司</td><td> U3003 </td></tr><tr><td>胶原酶一个</td><td>罗氏公司</td><td> 10103578001 </td></tr><tr><td> TrypLE快递</td><td> Invitrogen公司</td><td> 12604-013 </td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 10270-098 </td></tr><tr><td>青霉素，链霉素（100X） </td><td> Invitrogen公司</td><td> 15140-122</td></tr><tr><td> GlutaMAX我（100X） </td><td> Invitrogen公司</td><td> 35050-038 </td></tr><tr><td>盖玻片（＃1） </td><td>电子显微镜科学</td><td> 72196-12 </td></tr><tr><td> 24孔细胞培养板</td><td>碧迪</td><td> 35-3047 </td></tr><tr><td> 1×PBS </td><td>敏达</td><td> 21-040-CV </td></tr><tr><td>无菌蒸馏水和去离子水</td><td>敏达</td><td> 25-055-CV </td></tr><tr><td>小鼠神经元nucleofector和电套件</td><td>龙沙</td><td> VPG-1001 </td></tr><tr><td>对c-Jun的ON TARGETplus的siRNA </td><td> Dharmacon的</td><td> L - 043776 </td></tr><tr><td>反 - βIII微管蛋白抗体（Tuj的1） </td><td> Covance公司</td><td> MMS-435P </td></tr><tr><td>延长黄金抗淬灭安装解决方案</td><td> Invitrogen公司</td><td> P36930 </td></tr></TBODY&gt; </table>

genetic study of axon regeneration with cultured adult dorsal root ganglion neurons

这是众所周知的，成熟的神经元在中枢神经系统（CNS）不能再生它们的轴突受伤后，由于减少的内在能力，以支持在1,2成熟的中枢神经系统轴突生长和充满敌意的环境。相比之下，在成熟的神经元周围神经系统（PNS）的再生容易受伤后3。成年猫背根神经节（DRG）神经元是众所周知的有力周围神经损伤后再生。每背根神经节神经元增长1轴突从胞体，分支成两个轴突分支：外围分支支配外围目标和中央部门，延伸到脊髓。损伤的背根神经节的末梢神经轴突大量的轴突再生的结果，而中央在脊髓轴突再生的受伤后不佳。但是，如果发生周围性轴索损伤脊髓损伤（这个过程被称为空调病变），中央轴突再生前是greatlŸ提高4。此外，中央DRG神经元轴突共享降皮质脊髓轴突充满敌意的环境。在一起，这是假设，控制成年DRG神经元的轴突再生的分子机制可以利用，以提高中枢神经系统轴突再生。因此，现在广泛使用的成人DRG神经元作为一个模型系统研究再生轴突生长5-7。 在这里，我们描述了一个成人，DRG神经元培养轴突再生的基因研究在体外可以使用的方法。在这个模型成年DRG神经元的基因通过电穿孔介导的基因转染6,8操纵。转染神经元的DNA质粒或SI / shRNA的，这种方法能够使增益和亏损的功能试验，以调查任何在成年DRG神经元的轴突生长的基因利益的作用。当神经元与SI / shRNA的转，有针对性的内源性蛋白通常耗尽后3-4天，在文化，在这期间，强大的轴突生长已经发生，减少损失的功能研究有效。为了解决这个问题，这里介绍的方法，包括一个染后的再悬浮和再镀步，使轴突从神经元的目标蛋白质的情况下重新长出。最后，我们利用体外模型研究轴突再生相关基因c-Jun的作用，在调解成年DRG神经元的轴突生长提供了一个例子。

Watch this Scientific Journal Video about Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons at JoVE.com

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

一个<em&gt;在体外</em用培养的成年小鼠背根神经节神经元的轴突再生的遗传研究模型描述。方法包括re-suspension/re-plating的步骤，允许进行遗传操作的神经元的轴突重新生长。这种做法是轴突再生功能丧失使用RNAi为基础的蛋白质击倒的研究特别有用。

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 ...

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Research

JoVE Journal

Neuroscience

26.4K 次观看.  Johns Hopkins University School of Medicine. 一个在体外

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

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 ...

科研

神经科学

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 ...

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 ...

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 ...

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 ...

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 ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

使用初级培养的希波坎帕神经元来研究Axon初始片段的组装

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...