我们衷心感谢中西部大学的影像核心设施 (MWU) 和学生组 [Chanmoly、克里斯托弗 Dipollina、达里尔 Giambalvo 和凯西 Sigerson] 在细胞培养和成像工作中的贡献。这项研究工作得到了 MWU 的内部拨款资助, 用于中频和研究开办基金 V.T。

所有的程序, 包括使用这些动物都得到了机构审查委员会批准的议定书 (IACUC-中西部大学) 的批准。
1. 从小鼠胚胎中收获根背根
<ol><li>
用窒息法弄死成年小鼠 (CO2), 然后斩首。立即进行手术切除椎体柱。
	<ol>
<li>用细剪刀切开皮肤层背, 露出椎柱。通过切开柱子两侧的肋骨, 通过骶骨将椎柱与其他动物分开, 来隔离椎柱。</li>
		<li>使用针/针将椎体柱 (腹侧向上) 安装在手术垫上。</li>
	</ol>
</li>
	<li>用细剪刀在椎体两侧进行双切口, 以暴露椎管内侧。</li>
	<li>在手术显微镜下 (放大率设置为 4X), 用剪刀轻轻地移动一侧的脊髓以暴露对侧背脊髓根部, 并沿周围神经的背根定位节段。每个神经节部分隐藏在椎间孔内。</li>
	<li>要收割根背节, 用一把钳子捏住脊髓和神经节之间的背部根部, 然后轻轻地从椎间孔中拔下神经节。</li>
	<li>将第二个钳放在脊柱神经周围的背节上, 并将背节与脊髓神经和脊柱根一起拉。</li>
	<li>将收集的背节放置在35毫米培养皿中, 其中含有3毫升的冰血清游离介质 (可以持续森林)34。</li>
	<li>在干玻璃培养皿中转移每个单独的背根, 在手术显微镜下, 用刀片清洁和修剪剩余的纤维和连接在背节上的结缔组织。根背节很容易被识别为一个凸出透明的结构沿白色脊柱神经/根部。血管经常被发现围绕着背节。</li>
	<li>把清洗后的背根放在一个新的培养皿中, 其中包含冰冷的森林控制介质。</li>
	<li>稀释凝胶蛋白混合物 (参见材料表) 在冰冷的森林间, (1:1)。</li>
	<li>用12井板预涂10/20 µL 的凝胶蛋白混合物将背根背板前体, 并将其放在孵化器内37摄氏度和 5% CO2为30-60 分钟。</li>
	<li>轻轻地添加 1.5-2 毫升的可持续森林管理, 以覆盖整个外植体和维持在培养条件下的外植 (37 °c 和 5% CO2)。 注意: 这是一个关键的步骤, 因为根背节是锚定在玻璃盘子只由聚合凝胶蛋白混合物。聚合时间和吹打技能是避免漂浮的关键。</li>
	<li>每72小时改变生长的根节的培养基, 让根背根生长, 只要需要。</li>
</ol>2. 将单个细胞神经元与根神经节隔离
<ol><li>将所有在1.5 毫升无菌管中收集的根背节放置在1.2 毫升的 F12 培养基中, 含1.25 毫克胶原酶 IV, 并在37°c 和 5% CO2上孵化, 45 分钟. 在第一次孵化后重复此步骤, 再进行45分钟。</li>
	<li>在37摄氏度和 5% CO2后, 在胶原酶 IV 治疗后立即用2毫升含有胰蛋白酶 (0.025%) 的 F12 培养基对外植体进行30分钟治疗。</li>
	<li>孵育2毫升的 F12 培养基含有胎牛血清 (血清; 33%) 在37°c 和 5% CO2 15 分钟。</li>
	<li>用2毫升的 F12 介质清洗外植体三次, 然后用玻璃吸管机械地将其分离, 直到介质变阴。 注: 本程序涉及从动物身上收集干净/修剪过的背根, 如前节所述。在进行外植体机械分离时, 要温和, 不要使用过度的力, 因为它会导致工作材料的溢出和丢失。</li>
	<li>过滤分离细胞培养通过0.22 µm 过滤器去除任何杂质和多余的结缔组织。离心过滤细胞裂解在 (2500 x g) 为2分钟。</li>
	<li>移除上清和并用重悬细胞颗粒在500µL 的 neurobasal 培养基中含有补充的神经元培养 (1x), 抗生素混合物 (1x), l-谷氨酸 (0.5 毫米) 和神经生长因子 (5 µg/毫升) (见材料表)。</li>
	<li>将离解的细胞板在层粘连蛋白涂层的覆盖物上 (50 µg/毫升; 参见材料表) 在首选的细胞密度。使用单元格计数器确定单元格的密度。 注: 我们在2.5万细胞/封面幻灯片上镀细胞。这种技术允许神经元和胶质细胞的分离。与 pseudounipolar 神经元共培养的胶质成分对神经元的存活起着至关重要的作用。</li>
</ol>3. 根背节外植体和背节衍生细胞的 HSV-1 感染
注: 这项工作是严格遵循生物安全 level-2 (BSL-2) 的要求, 我们有一个设备齐全的实验室, 由中西部大学生物安全委员会批准。本研究使用了 HSV-1 的一株科斯菌株。请采取适当的测量和安全预防措施, 根据当地机构的指导方针, 如果使用病毒菌株。
<ol><li>确定并准备在可持续森林媒体中正确稀释的病毒, 以感染该模型。本研究使用的病毒是 HSV-1 的科斯菌株。当使用根背节衍生的分离细胞, 用1单位的感染 (语言) 的感染, 这意味着病毒数量等于细胞数量。</li>
	<li>如果感染根背节外植体, 请使用病毒 (例如, 1万病毒) 的数量, 因为不能确定外植体中的确切数量的细胞。</li>
	<li>将外植体/细胞置于不育细胞板或管中, 其中含有可持续可持续的培养基和病毒的混合物。 注: 我们使用2.5万病毒的封面幻灯片包含2.5万细胞 (1 语言)。为了感染外植体, 我们使用1万病毒。</li>
	<li>将细胞板或管放置在37摄氏度, 以便感染发生;病毒感染的时间可能因病毒传染性而异。 注: 使用邻硝基硝基苯基半乳糖苷 (ONPG) 和 5-溴-4-氯-3-哚-galactopyranoside (X-加仑) 测定法确认病毒进入,26。</li>
</ol>4. 免疫荧光
<ol><li>修复磷酸盐缓冲盐水 (PBS) 制备的4% 福尔马林中的外植体和单细胞样品。清洗样品3次, 每10分钟在 PBS。</li>
	<li>用所需的原抗体孵化样品 (抗β蛋白, 抗感光, 抗硫酸硫酸盐 (HS), 和/或抗糖蛋白 D (gD) 抗体; 请参见材料表(稀释) 在 PBS 缓冲器中稀释, 其 0.3%-X (PBST) 和10正常山羊血清%。将样品存放在4摄氏度。</li>
	<li>每3次用 PBS 清洗样品10分钟。在适当的二级抗体 (488 和/或 Cy3) 的室温下孵育1小时的样品; 请参阅 PBS 中稀释的稀释材料表。</li>
	<li>每3次用 PBS 清洗样品10分钟。在安装步骤之前, 在 PBS 中用赫斯特染料 (1.5 µM) 孵化样品20分钟。</li>
	<li>使用荧光安装介质和盖玻片在玻璃幻灯片上安装样品。</li>
</ol>

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G., Arvin, A., 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> Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. , (2007).</li><li>Fueshko, S., Wray, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LHRH+cells+migrate+on+peripherin+fibers+in+embryonic+olfactory+explant+cultures:+an+in+vitro+model+for+neurophilic+neuronal+migration.">LHRH cells migrate on peripherin fibers in embryonic olfactory explant cultures: an in vitro model for neurophilic neuronal migration.</a> Dev Biol. 166 (1), 331-348 (1994).</li><li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+heparan+sulphate+in+inflammation.">The role of heparan sulphate in inflammation.</a> Nat Rev Immunol. 6 (9), 633-643 (2006).</li><li>Zhang, X., Wang, B., Li, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+heparan+sulfate+and+heparanase+in+neuroinflammation.">Implications of heparan sulfate and heparanase in neuroinflammation.</a> Matrix Biol. 35, 174-181 (2014).</li><li>Du, X., 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=Local+GABAergic+signaling+within+sensory+ganglia+controls+peripheral+nociceptive+transmission.">Local GABAergic signaling within sensory ganglia controls peripheral nociceptive transmission.</a> J Clin Invest. 127 (5), 1741-1756 (2017).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li></ol>

作者没有什么可透露的。

前体背节模型对于研究各种事件 (如神经元-胶质细胞相互作用) 以及微环境对神经元和胶质代谢的影响 (37) 极为有用。此外, 根背节模型可作为一种成本效益高的工具, 通过开发前体内系统进行急性慢性和潜伏感染阶段或某种疾病, 以此解决有关致病机制和相关标记的相关问题。此外, 在根背节外植体中的小分子筛查库可以利用这种植块模型进行药物开发。体外...

在这篇手稿中, 我们报告了一个方法, 以获得一个脊髓的前体模型的小鼠神经根神经节模型系统作为一种保存的组织样的微环境, 以调查广泛的生物学反应, 从神经胶质的污辱范围相互作用, 可塑性, 炎症标志物, 病毒感染。此外, 我们进一步制定了一个协议, 以建立一个主要的共同文化的根神经节衍生的单一感官神经元和卫星细胞。
根背节是位于中枢神经系统外的卫星灰色物质单元, 沿脊髓神经的脊柱脊髓根部。神经节, 位于椎间孔附近, pseudounipolar 的感觉神经元和卫星胶质细胞。pseudounipolar 神经元的特点是一个单一的突起, 它分裂成一个外围过程, 将躯体和内脏的输入从外围目标带到细胞体, 以及将感官信息从细胞体提交中枢神经系统的中心过程。结缔组织胶囊定义并分离出中枢神经系统的神经元和胶质细胞的外围簇。无产后细胞迁移到或从根背根曾经被描述和一个地方干细胞利基负责的神经性事件发生在整个生命中1。因此, 这个模型特别适合研究成人神经发生, axonogenesis, 创伤性病变的反应, 细胞死亡2,3,4,5,6,7 ,8,9 。
在 neuroregeneration 领域中, 从体内和说明体外中收获的根神经节重现 axonotmesis, 这是一种损伤状态, 其中轴突完全切断, 神经元细胞体与支配目标10 分离。 ,11。众所周知, 周围神经损伤会导致节根节内的基因表达减少和增加, 许多这些变化是再生过程的结果, 但许多可能也是由于免疫应答或来自非神经细胞的另一种反应。通过使用一个孤立的根神经节的前体系统, 这些复杂性被删除, 机械通路可以更容易地调查。
除了它在向中枢神经系统传递感官输入的中心作用外, 许多神经递质的受体丰富, 包括 GABA12,13,14,15在神经元躯体的水平以及神经元间交叉激励的证据可能表明, 根背根是成熟的感官输入的初步集成商 16, 17.这些新发现给根背节外植体提供了类似于其他 "微型脑" 模型的微型神经元网络系统的特性, 这是神经组织特异的 organoids 用于更广泛的实验领域的调查和治疗神经疾病的方法18,19。这些证据连同根背根是一个离散和明确定义的神经组织簇包围的结缔组织胶囊, 使它成为一个合适的器官为前体移植。
培养小鼠背根是一种有吸引力的多细胞选择模型的人 pathophysiologies 由于结构和遗传相似的物种之间。此外, 一个大的转基因小鼠菌株储存库, 是非常有利于未来的机械研究。突起延长在开发期间和在损伤以后需要机械相互作用在成长锥体和基体之间20,21。纳米和微阵列基材已被用作工具, 以指导突起的产物, 并表明他们的能力, 以响应地形特征在他们的微环境。神经元已经被证明可以生存, 坚持, 迁移, 并定向他们的轴突, 以导航表面特征, 如凹槽在基板22,23。然而, 这些研究通常利用培养的细胞系, 很难预测主要神经细胞将如何响应明确定义的, 物理提示在体内或前体。
本建议使用的小鼠背根的前体外植体模型模拟了生长轴突周围的真实细胞细胞相互作用和生物化学线索。在许多不同的实验范式, 从轴突再生, 干细胞生产, 到 neuroinflammation, 根背根外植体模型继续作为一个宝贵的工具来调查病毒感染和潜伏期方面的感官神经节24,25,26,27。
神经系统 (NS) 一般是病毒感染的目标28,29,30。大多数病毒感染上皮和内皮细胞表面, 并通过周围神经感觉和运动纤维从表面组织到 NS 的途径。特别是, 在上皮细胞最初感染后, 单纯疱疹病毒1型 (HSV-1) 在感觉神经节中建立终生潜伏期, 最好是31,32的背根。HSV-1 neuroptropic 的感染的能力最终导致神经系统疾病的33。

<table><tbody><tr><td>Adult Mice NIH/Swiss</td><td>Harlan Laboratories</td><td></td><td></td></tr><tr><td>35mm petri dish</td><td>Cell Treat</td><td>229635</td><td></td></tr><tr><td>Matrigel ECM</td><td>Sigma-Aldrich</td><td>E1270</td><td>gelatinous protein mixture</td></tr><tr><td>F12 Media</td><td>Gibco</td><td>11765-054</td><td>*Part of SFM media</td></tr><tr><td>Collagenase IV</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>25200-056</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F6178</td><td></td></tr><tr><td>0.22um filter</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>Neurobasal media</td><td>Gibco</td><td>10888-022</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504-044</td><td>Supplement for neuronal culture</td></tr><tr><td>PSN antibiotics</td><td>Gibco</td><td>15640-055</td><td>*Part of SFM media&lt;br/&gt; Antibiotic mixture</td></tr><tr><td>L-glutamate</td><td>Sigma-Aldrich</td><td>G7513</td><td>*Part of SFM media</td></tr><tr><td>NGF</td><td>Alomone Labs</td><td>N-100</td><td>Nerve growth factor</td></tr><tr><td>Laminin coated coverslide</td><td>Neuvitro</td><td>GG-14-Laminin</td><td></td></tr><tr><td>ONPG subtrate</td><td>Pierce</td><td>34055</td><td></td></tr><tr><td>X-gal</td><td>Invitrogen</td><td>15520034</td><td></td></tr><tr><td>Antibody anti-B-tubulin</td><td>Sigma-Aldrich</td><td>T8328</td><td>1:2000 dilution</td></tr><tr><td>Antibody anti-peripherin</td><td>Millipore</td><td>AB1530</td><td>1:1000 dilution</td></tr><tr><td>Hoechst dye</td><td>Thermo Fisher</td><td>62249</td><td>1.5 &amp;micro;M final concentration</td></tr><tr><td>Anti-heparan sulfate</td><td>US Biological</td><td>H1890-10</td><td>0.180555556</td></tr><tr><td>Anti gD antibody</td><td>Virostat</td><td>196</td><td>1:10 dilution</td></tr><tr><td>BSA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A2153-100G</td><td>*Part of SFM media</td></tr><tr><td>BME</td><td>Gibco</td><td>21010-046</td><td>*Part of SFM media</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021-1KG</td><td>*Part of SFM media</td></tr><tr><td>KIT (Insulin-transferrin-Selenium-A)</td><td>Gibco</td><td>51300-044</td><td>*Part of SFM media</td></tr><tr><td>Vitamin-C</td><td>Sigma-Aldrich</td><td>A4403</td><td>*Part of SFM media</td></tr><tr><td>Putrescine</td><td>Sigma-Aldrich</td><td>P7505</td><td>*Part of SFM media</td></tr><tr><td>488 (goat anti-mouse)</td><td>Life Technologies</td><td>A11029</td><td></td></tr><tr><td>Cy3 (goat anti-rabbit)</td><td>Jackson Immunoresearch laboratories</td><td>111-165-003</td><td></td></tr><tr><td>Normal Goat serum&amp;nbsp;</td><td>Vector</td><td>S-1000</td><td></td></tr><tr><td>Formalin Solution</td><td>Sigma-Aldrich</td><td>HT5014-120ML</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-031</td><td></td></tr><tr><td>Triton-X</td><td>Sigma-Aldrich</td><td>T9284-500ML</td><td></td></tr><tr><td>VectaShield</td><td>Vector</td><td>H-1500</td><td>Flurescence mount</td></tr><tr><td>Diamond White Glass Coverslides</td><td>Globe Scientific</td><td>1380-20</td><td></td></tr></tbody></table>

adult mouse drg explant and dissociated cell models to investigate neuroplasticity and responses to environmental insults including viral infection

本协议描述了鼠源性背根神经节 外植体的原生模型, 并介绍了分离的感官神经元和胶质细胞的与后向的联合培养。这些都是有用的和多才多艺的模型, 以调查各种生物学反应的生理和病理条件的周围神经系统 (三七), 从神经胶质相互作用, 可塑性,neuroinflammation 和病毒感染。与单纯的单细胞模型相比, 根背节外植体的使用具有很科学的优越性。例如, 作为一种脊髓的文化, 根背节外植体允许在整个神经元网络中进行 "前体" 转移, 包括在所有神经元和胶质功能中发挥重要作用的胞内微环境。此外, 在几天内还可以保持根背根节外植体, 并且培养条件可能会因需要而受到扰动. 此外, 收获的根背根可以进一步分离成一个体外的原始感觉神经元和卫星胶质细胞的共培养, 以研究神经元-胶质相互作用, neuritogenesis, 轴突锥相互作用与胞外微环境, 更一般, 与神经元代谢相关的任何方面。因此, 背节-外植体系统提供了极大的灵活性, 以成本效益的方式研究与生物、生理和病理条件有关的广泛事件。

可塑性和神经元-环境相互作用的多个方面可以用根神经节和一个分离细胞培养模型来研究。我们开始的研究, 通过隔离根背节外植体和根背节衍生的离解细胞, 以示意性代表在图 1。组织和单细胞模型可以通过使用多种分子技术, 如免疫荧光, 西方印迹, 基因组分析, 和其他分析技术, 取决于实验设计和目的的性质。首先, 我们使用我们的根背根外植体模型来研究生物化学线索对...

Watch this Scientific Journal Video about Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection at JoVE.co

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

在本报告中, 强调了脊髓培养和分离的原代培养的小鼠背根神经节的优势, 以调查与神经元-胶质细胞相互作用的广泛机制, 可塑性,neuroinflammation 和对病毒感染的反应。

成年小鼠背根节外植体和分离细胞模型调查可塑性和对包括病毒感染在内的环境侮辱的反应

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

adult-mouse-drg-explant-dissociated-cell-models-to-investigate

Research

JoVE Journal

Neuroscience

21.4K 次观看.  Midwestern University. 在本报告中, 强调了脊髓培养和分离的原代培养的小鼠背根神经节的优势, 以调查与神经元-胶质细胞相互作用的广泛机制, 可塑性,neuroinflammation 和对病毒感染的反应。

视频: Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em&gt;在体外</em&gt;髓鞘测定

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

科研

发育生物学

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

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

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 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 ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

小鼠脚垫接种模型，研究病毒引起的神经炎反应

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

免疫与感染

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 ...

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 ...

成年小鼠背根节外植体和分离细胞模型调查可塑性和对包括病毒感染在内的环境侮辱的反应

摘要

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小鼠脚垫接种模型，研究病毒引起的神经炎反应

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

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