Name: coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration
Uploaded: 2020-11-02T15:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration at JoVE.com

这项工作得到了SAF2017-82736-C2-1-R项目的财政支持，该项目由西恩西亚·埃因诺瓦西翁部长到MTM-F，由弗朗西斯科·德维托里亚基金会向JS提供。

注:动物实验已获国家和机构生物伦理委员会批准。
1. ihOEG （Ts12 和 Ts14） 文化
注:此程序是在组织培养生物安全柜的无菌条件下完成的。
<ol><li>准备 表 1中提供的 50 mL ME10 OEG 文化介质。</li>
	<li>准备5毫升的DMEM/F12-FBS，如 表1所示，在15毫升圆锥管。</li>
	<li>在37°C的清洁水浴中加热两种介质15分钟。</li>
	<li>在干净的水浴中，在37°C下解冻 Ts12 和 Ts14 细胞小瓶。</li>
	<li>在第 2 步准备的 DMEM/F12-FBS 培养介质中重新悬浮并添加细胞。</li>
	<li>离心机5分钟，200 x g。</li>
	<li>渴望超人。</li>
	<li>添加 500 μL 的 ME10 介质并重新悬念颗粒。</li>
	<li>准备一个 p60 细胞培养盘与 3 mL 的 ME10 和添加蜂窝悬架， 向下。</li>
	<li>移动以将细胞均匀地分布在板上。</li>
	<li>培养细胞在37°C在5%二氧化碳。 注:到达汇合后，至少必须再做一段，以优化培养的细胞。90% 的汇合是需要之前播种他们的封面上为 co 文化。汇合p-60的平均细胞数为7×10 5，Ts14为2.5×10 6，Ts12细胞系为2.5×10 6。 Ts12 和 Ts14 细胞线应每 2-3 天通过一次。</li>
</ol>2. 为检测准备 ihoeg （Ts12 和 Ts14）
注:此步骤必须在 RGN 解剖和培养前 24 小时完成。
<ol><li>用 10 微克/mL 多 L-赖氨酸 （PLL） 治疗 12 mm é 盖片 1 小时。 注:封口可以在PLL解决方案中过夜。</li>
	<li>用 1x 磷酸盐缓冲盐水 （PBS） 清洗盖片，三次。</li>
	<li>将 Ts12 和 Ts14 ihoEG 细胞从 p60 细胞培养皿中分离。<ol>
<li>将 4 mL 的 DMEM/F12-FBS 培养介质 （表 1） 添加到 15 mL 圆锥管中。在37°C的清洁水浴中加热。</li>
		<li>从盘子中取出介质，用 1 mL 的 1x PBS-EDTA 清洗电池一次。</li>
		<li>在OEG细胞中加入1毫升的尝试-EDTA，在37°C、5%CO2下孵育3-5分钟。
</li>
		<li>用 p1000 移液器收集细胞，并将它们转移到第 3.1 步准备的介质中。</li>
		<li>离心机5分钟，200 x g。</li>
		<li>渴望超人。</li>
		<li>添加 1 mL 的 ME10 介质并重新悬念颗粒。</li>
		<li>在血细胞计中数细胞数。</li>
	</ol>
</li>
	<li>种子 80，000 Ts14 细胞或 100，000 Ts12 细胞/井到盖片在 24 井板在 500 μL 的 ME10 介质.</li>
	<li>培养细胞在37°C在5%二氧化碳，为24小时。</li>
</ol>3. 视网膜组织解剖
注:2个月大的雄性威斯塔鼠被用作RGN来源。两个视网膜（一只老鼠），用于24口井细胞盘的20口井。使用前高压外科材料。帕潘分离套件是商业购买的（材料表）。按照提供商的重组说明操作。在 5 mM 库存中重组 D、L-2-氨基-5-磷新星酸 （APV），并准备阿利库特。
<ol><li>在检测当天，准备以下媒体。<ol>
<li>准备一个 p60 细胞培养菜与 5 ml 的冷 Ebss （小瓶 1 的木瓜分离套件） 。</li>
		<li>准备一个 p60 细胞培养菜与重组小瓶 2 （帕帕因） 的木瓜分离套件加上 50μL 的 APV. 然后，添加 250 μL 的重建小瓶 3（DNase 加 5μL 的 APV）。</li>
		<li>在无菌管混合 2.7 mL 小瓶 1 与 300 μL 小瓶 4 （白蛋白-卵巢蛋白酶抑制剂）.添加 150μL 的瓶 3 （DNase） 和 30μL 的 APV。</li>
		<li>准备20 mL的神经底-B27介质（NB-B27），如 表1所示。</li>
	</ol>
</li>
	<li>用二氧化碳窒息来牺牲老鼠。</li>
	<li>用断头台斩首来取下头部;把它放在一个100毫米的培养皿中，在将乙醇喷入层压流罩之前，先用70%的乙醇喷头。</li>
	<li>用剪刀切老鼠的胡须，这样它们就不会干扰眼睛的操纵。</li>
	<li>用钳子抓住视神经，拔出眼球，足以用手术刀在眼睛上切口。</li>
	<li>取出镜片和活泼的幽默，拉出视网膜（橙色般的组织），而眼睛的其余层留在里面（包括色素上皮层）。</li>
	<li>将网膜放入第 3.1.1 步准备的 p60 细胞培养盘中。</li>
	<li>将网膜转移到第 3.1.2 步准备的 p60 细胞培养盘，然后用手术刀将其切成大约大小和 1 mm 的小块。</li>
	<li>转移到15 mL塑料管。</li>
	<li>将组织孵育30分钟，在37°C的加湿孵化器中，在5%的CO2下，每10分钟搅拌一次。</li>
	<li>用玻璃巴斯德移液器上下吹笛，分离细胞团块。</li>
	<li>在200 x g 下将电池悬架离心5分钟。</li>
	<li>丢弃超纳坦并灭活木瓜，在步骤 3.1.3 中准备的溶液中重新悬浮细胞颗粒。（2只眼睛1.5 mL）。</li>
	<li>小心地将此单元悬架吹入 5 mL 的重组小瓶 4 中。</li>
	<li>离心机在200 x g 5分钟。</li>
	<li>在离心时，从OEG 24油井电池板中完全去除ME-10介质（先前在第2步中准备），并替换为每口井500μL的NB-B27介质。</li>
	<li>丢弃超纳坦，在NB-B27介质的2mL中重新悬浮细胞。</li>
	<li>板 100 μL 的视网膜细胞悬架，每孔的 m24 板，到 PLL 处理或 OEG 单层盖片上。</li>
	<li>在 NB-B27 介质中将培养物保持在 37 °C，96 小时保持 5% CO 2。</li>
</ol>4. 免疫污染
<ol><li>96小时后，通过在培养介质（600 μL）（PFA最终浓度2%）中加入1倍PBS中4%的准甲醛（PFA）来修复细胞10分钟。</li>
	<li>从 24 多井板中取出介质和 PFA，并在 1x PBS 中再次添加 500 μL 的 4% 甲醛 （PFA）。孵化10分钟。</li>
	<li>丢弃固定器，用 1x PBS 清洗 3 次 5 分钟。</li>
	<li>在PBS（PBS-TS）中以0.1%的Triton X-100/1%FBS块30-40分钟。</li>
	<li>准备PBS-TS缓冲区的主要抗体如下:SMI31（针对MAP1B和NF-H蛋白）单克隆抗体（1:500）。514 （识别 MAP2A 和 B 蛋白） 兔子多克隆反塞 （1:400）。</li>
	<li>将原发抗体添加到培养物中，并在4°C下过夜孵育。</li>
	<li>第二天，丢弃抗体，用1x PBS清洗盖片，3次，5分钟。</li>
	<li>准备PBS-TS缓冲区中的次要抗体如下:对于SMI-31，抗鼠标亚历克萨氟488（1:500）。对于 514，反兔子亚历克萨-594 （1:500）。</li>
	<li>在RT，在黑暗中用相应的荧光二次抗体孵育细胞1小时。</li>
	<li>在黑暗中用 1x PBS 清洗盖片 3 次，5 分钟。</li>
	<li>最后，安装带安装介质（材料表）的盖片，并保持在4°C。 注:如有必要，可执行带有 DAPI 的荧光核染色（4，6-迪米迪诺-2-苯酚）。安装前，用 DAPI（1x PBS 中的 10 微克/mL）在黑暗中孵育细胞 10 分钟。用 1x PBS 清洗盖片 3 次，最后用安装介质安装盖片。</li>
</ol>5. 轴再生量化
注:样品在表观荧光显微镜的40倍目标下进行量化。每次治疗至少应在随机字段上拍摄 30 张照片，其中至少需要 200 个神经元。每个实验至少应重复三次。
<ol><li>量化轴突（SMI31阳性中性）神经元相对于RGN总种群的百分比（与MAP2A/B 514神经元身体和树突的阳性免疫保持一起确定）。</li>
	<li>量化轴再生指数或平均轴长（μm/神经元）。此参数定义为所有已识别轴突的长度（以 μm）之和，除以计数神经元的总数，无论它们是否呈现轴突。轴长度是使用图像软件 ImageJ （NIH-USA） 的插件神经元J 确定的。</li>
	<li>使用适当的软件计算平均值、标准偏差和统计意义。</li>
</ol>

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R., Wanner, I. B., Phelps, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cell-neurite+alignment+enhances+neurite+outgrowth+in+scar-like+cultures.">Olfactory ensheathing cell-neurite alignment enhances neurite outgrowth in scar-like cultures.</a> Experimental Neurology. 269, 93-101 (2015).</li><li>Wigley, C. B., Berry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+adult+rat+retinal+ganglion+cell+processes+in+monolayer+culture:+comparisons+between+cultures+of+adult+and+neonatal+neurons.">Regeneration of adult rat retinal ganglion cell processes in monolayer culture: comparisons between cultures of adult and neonatal neurons.</a> Brain Research. 470 (1), 85-98 (1988).</li><li>Sonigra, R. J., Brighton, P. C., Jacoby, J., Hall, S., Wigley, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+olfactory+nerve+ensheathing+cells+are+effective+promoters+of+adult+central+nervous+system+neurite+outgrowth+in+coculture.">Adult rat olfactory nerve ensheathing cells are effective promoters of adult central nervous system neurite outgrowth in coculture.</a> Glia. 25 (3), 256-269 (1999).</li><li>Hayat, S., Thomas, A., Afshar, F., Sonigra, R., Wigley, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+olfactory+ensheathing+cell+signaling+mechanisms:+effects+on+their+support+for+neurite+regrowth+from+adult+CNS+neurons+in+coculture.">Manipulation of olfactory ensheathing cell signaling mechanisms: effects on their support for neurite regrowth from adult CNS neurons in coculture.</a> Glia. 44 (3), 232-241 (2003).</li><li>Kumar, R., Hayat, S., Felts, P., Bunting, S., Wigley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differences+and+interactions+between+phenotypic+subpopulations+of+olfactory+ensheathing+cells+in+promoting+CNS+axonal+regeneration.">Functional differences and interactions between phenotypic subpopulations of olfactory ensheathing cells in promoting CNS axonal regeneration.</a> Glia. 50 (1), 12-20 (2005).</li><li>Moreno-Flores, M. 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A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proceedings of the National Academy of Sciences of the United States of America. 80 (19), 6126-6130 (1983).</li><li>S&#225;nchez Martin, C., D&#237;az-Nido, J., Avila, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+a+site-specific+phosphorylation+of+the+microtubule-associated+protein+2+during+the+development+of+cultured+neurons.">Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons.</a> Neuroscience. 87 (4), 861-870 (1998).</li><li>Reshamwala, R., Shah, M., Belt, L., Ekberg, J. 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作者没有什么可透露的。

OEG移植在CNS损伤部位被认为是一个有希望的治疗CNS损伤，因为它构成亲神经再生属性7，8，9。然而，根据组织来源-嗅觉粘膜（OM-OEG）与嗅球（OB-OEG）-或捐赠者的年龄，这种能力存在相当大的差异26，31，33，36。因此，在开始体内研究之前，必须有一个简单且可重复的体外模型来检测给定OEG样本的神经再生能力。在此协议中，成年大鼠的轴突射 RGN 被共同培养到 OEG 的单层上进行检测。随后通过免疫荧光对RGN轴突和体外分泌标记进行分析，以评估RGN轴位再生。
初步检测的困难是OEG的来源。在这项工作中，我们使用可逆不朽的人类OEG（ihOEG）克隆线，以前建立和特点，我们组15，16，17，18，提供无限供应的同质OEG。其中两个ihOEG细胞系是Ts14，它保持了原始培养物和Ts12的再生能力，Ts12是一种低再生系，在这些实验中用作低再生控制18然而，尽管存在技术限制，以扩大人类原发性OEG细胞，它们也可以从鼻内窥镜活检-OM-或，在OB-OEG的情况下，从尸体捐赠者获得。
单层OEG培养物的制备是一个关键的过程，因为太多的细胞可能导致培养物从盘子中分离。因此，在 OEG 准备进行检测之前，建议用户根据其大小和分裂速率确定要镀入的细胞的最佳数量。
另一个关键问题是视网膜解剖后视网膜组织分离。分离组合中孵化后，必须分解组织碎片。如果做得太激烈，细胞将被摧毁，但如果做得太弱，组织碎片将完好无损。为了获得均匀的细胞悬浮物，我们建议填充和清空巴斯德移液器10-15倍，中间直径尖，同时避免冒泡。带宽尖端的巴斯德移液器可使用邦森燃烧器缩小范围。
为了评估不同胶质种群培养成人神经元轴突再生的能力，我们确定96小时是最适合目标的时间间隔，因为:1） 在不干扰OEG单层的情况下保持文化存活时间最长:2）现在是神经元长出轴突足够长的时间，以揭示不同OEG种群或其他非再生细胞（即成纤维细胞12、13、14、15、16、17、18、32、33）的再生能力差异。确定再生过程的时间过程当然很有趣，因为它可以在共同培养的较短时间内提供关于不同胶质种群的差异再生特性的信息。在我们手中，对于再生胶质，72-96 h之间的时间过程对于所有细胞系来说都非常相似，尽管轴突在72小时（未发布的数据）时更短。此外，96小时的共同培养，允许研究OEG依赖机制的成人轴突再生12，14。
在轴再生量化过程中，在盖片的不同随机区域以 400 增强（40 倍目标）拍摄至少 30 张照片非常重要，但要遵循所拍摄神经元的完整轴突。因此，实验者必须在所选区域拍摄串行照片，以测量真正的轴突长度。
还开发了其他体外方法来评估OEG再生功能。在这些模型中，OEG应用于不同神经元起源的培养物，并针对胶质培养，对中性形成和拉长进行了19、20、21、22、23、24、25、26、27的检测。然而，再生检测依赖于胚胎或产后神经元，这些神经元具有受伤的成人神经元所缺乏的内在可塑性。这个模型包括成人轴突再生在OEG线的培养与成人视网膜结节神经元（RGN）克服这一缺点。此外，我们正在解剖成人视网膜，因为我们在解剖过程中切除了视神经和轴突，我们获得神经元身体清洁骨髓，以进行培养。这是与成人CNS的其他部分的区别，其中骨髓可以阻碍非常与解剖，以获得清洁的神经元为培养。
根据威格利等人最初开发的28、29、30、31，我们强调协议的以下改进。首先，使用补充B27的神经平衡介质作为OEG-RGN培养介质，允许神经元细胞的生长，并积极影响实验的可重复性。其次，我们使用轴突隔间的特定标记来描述和量化轴突再生:第三，我们使用额外的直接参数，平均轴突长度/神经元，评估OEG的轴突生长再生潜力。
总之，我们认为这是一种简单、可重复、省时和中等成本的检测，不仅有助于评估 ihOEG 的神经再生潜力，而且还因为它可以扩展到 OEG 或其他胶质细胞的不同来源。此外，在翻译到体内或临床研究之前，它可以用作OEG或胶质样本神经再生潜力概念的宝贵证明。

成人中枢神经系统（CNS）神经元在受伤或患病后再生能力有限。促进CNS再生的一个共同策略是移植，在损伤部位，诱导轴突生长的细胞类型，如干细胞，施万细胞，星形细胞或嗅觉包裹胶质（OEG）细胞1，2，3，4，5。 
OEG源自神经峰6，位于嗅觉粘膜和嗅球中。在成人中，嗅觉神经元由于环境暴露而定期死亡，并被新分化的神经元所取代。OEG包围和引导这些新的嗅觉轴突进入嗅球，并建立新的突触，他们的目标在CNS7。由于这些生理属性，OEG已被用于CNS损伤的模型，如脊髓或视神经损伤，其神经再生和神经保护特性被证实为8，9，10，11。几个因素已被确定为负责这些细胞的亲再生特征，包括细胞外基质蛋白酶生产或分泌神经营养和轴突生长因子12，13，14。
鉴于扩大原OEG细胞的技术局限性，我们以前建立并定性了可逆不朽的人类OEG（ihOEG）克隆系，它提供了无限的均匀OEG供应。这些 ihOEG 细胞源自原始培养物，这些培养物来自验尸中获得的嗅觉球茎。它们通过端粒酶催化亚单（TERT）和上基因Bmi-1的转导而永生，并与SV40病毒大T抗原15、16、17、18一起改良。其中两个ihOEG细胞系是Ts14，它保持了原始培养物和Ts12的再生能力，Ts12是一种低再生线，在这些实验中用作低再生控制。
为了评估OEG在神经损伤后促进轴突再生的能力，已经实施了几个体外模型。在这些模型中，OEG应用于不同神经元起源和中性形成和拉长的培养物——对胶质文化的反应——被检测。这些神经元来源的例子有新生儿大鼠皮质神经元19，从皮质组织对大鼠胚胎神经元进行划伤20，大鼠视网膜外泄21，大鼠下丘脑或海马产后神经元22，23 ，产后大鼠鼻脊髓根结节神经元24个，产后小鼠皮质脊髓神经元25个，人类NT2神经元26个，或产后脑皮质神经元对反应性星形细胞疤痕样培养27个。
然而，在这些模型中，再生检测依赖于胚胎或产后神经元，这些神经元具有受伤的成人神经元所缺乏的内在可塑性。为了克服这个缺点， 我们提出了一个成人轴突再生模型在OEG线的培养与成人视网膜结节神经元（RN），基于一个最初由威格利等人28，29，30，31和修改和使用我们组12，13，14，15，16，17，18，32，33。简言之，视网膜组织从成年大鼠中提取，用木瓜消化。视网膜细胞悬浮液随后被镀在多晶硅处理的盖片上或 Ts14 和 Ts12 单层上。培养物在修复前保持96小时，然后进行轴突（MAP1B和NF-H蛋白）34和体血体（MAP2A和B）35标记的免疫荧光。轴突再生被量化为轴突神经元的百分比，关于RGN的总数，轴再生指数被计算为每个神经元的平均轴突长度。此协议不仅可用于评估 ihOEG 的神经再生潜力，而且可以扩展到 OEG 或其他胶质细胞的不同来源。

<table><tbody><tr><td>antibody 514</td><td>Reference 34</td><td></td><td>Rabbit polyclonal antiserum, which recognizes MAP2A and B.</td></tr><tr><td>antibody SMI-31</td><td>BioLegend</td><td>801601</td><td>Monoclonal antibody against MAP1B and NF-H proteins</td></tr><tr><td>anti-mouse Alexa Fluor 488 antibody</td><td>ThermoFisher</td><td>A-21202</td><td></td></tr><tr><td>anti-rabbit Alexa Fluor 594 antibody</td><td>ThermoFisher</td><td>A-21207</td><td></td></tr><tr><td>B-27 Supplement</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>D,L-2-amino-5-phosphonovaleric acid</td><td>Sigma</td><td>283967</td><td>NMDA receptor inhibitor</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>Nuclei fluorescent stain</td></tr><tr><td>DMEM-F12</td><td>Gibco</td><td>11320033</td><td>Cell culture medium</td></tr><tr><td>FBS</td><td>Gibco</td><td>11573397</td><td>Fetal bovine serum</td></tr><tr><td>FBS-Hyclone</td><td>Fisher Scientific</td><td>16291082</td><td>Fetal bovine serum</td></tr><tr><td>Fluoromount</td><td>Southern Biotech</td><td>0100-01</td><td>Mounting medium</td></tr><tr><td>ImageJ</td><td>National Institutes of Health (NIH-USA)</td><td></td><td>Image software</td></tr><tr><td>L-Glutamine</td><td>Lonza</td><td>BE17-605F</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Gibco</td><td>21103049</td><td>Neuronal cells culture medium</td></tr><tr><td>Papain Dissociation System</td><td>Worthington Biochemical Corporation</td><td>LK003150</td><td>For use in neural cell isolation</td></tr><tr><td>PBS</td><td>Home made</td><td></td><td></td></tr><tr><td>PBS-EDTA</td><td>Lonza</td><td>H3BE02-017F</td><td></td></tr><tr><td>Penicillin/Streptomycin/Amphotericin B</td><td>Lonza</td><td>17-745E</td><td>Bacteriostatic and bactericidal</td></tr><tr><td>Pituitary extract</td><td>Gibco</td><td>13028014</td><td>Bovine pituitary extract</td></tr><tr><td>Poly -L- lysine (PLL)</td><td>Sigma</td><td>A-003-M</td><td></td></tr></tbody></table>

coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration

嗅觉包裹胶质 （OEG） 细胞从嗅觉粘膜一直定位到嗅球的嗅觉神经层 （ONL）。在整个成年生活中，它们是新产生的嗅觉神经元的轴突生长的关键，从拉米纳预言家到ONL。由于其亲再生特性，这些细胞已被用于促进脊髓或视神经损伤模型的轴突再生。
我们提出了一个体外模型，以检测和测量神经损伤后OEG神经再生能力。在这个模型中，可逆不朽的人类OEG（ihOEG）被培养为单层，视网膜从成年大鼠中提取，视网膜结石神经元（RGN）被共同培养到OEG单层。96小时后，通过免疫荧光分析RGN中的轴突和索马托内皮标记，并量化带轴突的RN数量和平均轴突长度/神经元。
此协议比依赖胚胎或产后神经元的其他体外检测具有优势，它评估成人组织中的OEG神经再生特性。此外，它不仅可用于评估 ihOEG 的神经再生潜力，而且可以扩展到 OEG 或其他胶质细胞的不同来源。

在此协议中，我们提出了一个体外模型，以检测神经元损伤后的OEG神经再生能力。如图1所示，OEG源是一个可逆的不朽的人类OEG克隆细胞系-Ts14和Ts12-，它源自原始文化，从验尸15，17，18获得的嗅球。视网膜组织从成年大鼠身上提取，消化后，视网膜结节神经元（RGN）悬浮物镀在PLL处理的盖片上或iHOEG单层、Ts14或 Ts12上。培养物在修复前保持96小时。通过免疫荧光分析轴突和索马托德内皮标记，并量化轴再生。
Ts14 OEG 标识通过免疫识别进行评估，其中带有描述在"护套胶质"（图 2）中表示的标记，如 S100 β（图 2A）和维门汀（图 2B）:还分析了GFAP表达式，以丢弃星形细胞污染（图2C）。如图所示，Ts14 表示 S100 β和维门汀，但不是 GFAP。
在轴再生测定中，Ts14再生能力与 RGN-OEG 培养中的 Ts12 进行比较，使用 PLL 基板作为负控制（图 3）。与 Ts12 细胞或 PLL（图 3D，E）上镀入的神经元相比，在 Ts14 单层上共同培养的神经元中，轴突细胞的百分比和再生轴突的平均长度都显著提高。代表性图像显示，RGN缺乏在PLL或Ts12细胞（图3A，B）上再生轴突的能力，而Ts14刺激RGN（3C）轴突的生长。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61863/61863fig01v2.jpg"> 图1:大鼠视网膜结节神经元图与嗅觉包裹胶质细胞培养，作为成人轴突再生的模型。不朽的人类OEG（ihOEG）克隆细胞系-Ts12和Ts14-源自嗅觉球茎的原始文化。来自成年大鼠的视网膜结节神经元被镀在PLL治疗的盖片（负控制）或Ts14或Ts12单层上。培养物在修复前保持96小时，并通过免疫荧光分析轴突和体血淋淋的标记。具有轴突和平均轴突长度/神经元的神经元的百分比被量化为检测RGN轴位再生。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61863/61863fig01v2large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61863/61863fig02v2.jpg"> 图2:ihOEG细胞系Ts14的身份。文化中 Ts14 的免疫荧光图像，标有抗 S100 β（面板 A、绿色）和维门汀（面板 B、红色）。还分析了GFAP表达式（C面板，红色）以丢弃星形细胞污染。核沾染了 DAPI （蓝色）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61863/61863fig02v2large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61863/61863fig03.jpg"> 图3:用成人视网膜结节神经元（RGN）在OEG系的共生培养物中进行轴突再生的检测。（A-C）免疫荧光图像显示带有 514 抗体的 somatodendritic 标签，该抗体识别微管相关蛋白 MAP2A 和 B，红色，并带有与轴突特异性 SMI31 绿色抗体，对抗 MAP1B 和 NF-H 蛋白质。绿色箭头表示 RGN 轴突（SMI31 阳性:绿色），黄色箭头表示神经元身体和树突（514 正:红色和黄色）。（D， E）图形显示显示轴突和轴突再生指数的神经元百分比的平均值和标准偏差，该参数反映了每个神经元轴突的平均轴突长度 （μm）。在随机字段上拍摄至少 30 张图片（40 倍），并针对每个细胞样本进行量化。实验在三倍，从三个不同的大鼠（N =3），视网膜组织从两只眼睛汇集，与每个实验条件的重复（每个胶质种群测试）。星号表示统计学意义: *p < 0.05， **p < 0.01， ***p < 0.001， NS: 非意义 （ANOVA 和临时 Tukey 测试比较为 Ts14 vs Ts12、Ts14 vs PLL 和 Ts12 vs PLL 量化的参数）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61863/61863fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

Watch this Scientific Journal Video about Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration at JoVE.com

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

我们提出了一个体外模型，以评估神经损伤后嗅觉包裹胶质（OEG）神经再生能力。它基于OEG单层的轴突成人视网膜结节神经元（RGN）的共生和随后的轴再生研究，通过分析RGN轴突和声乐内皮标记。

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 ...

coculture-axotomized-rat-retinal-ganglion-neurons-with-olfactory

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.8K 次观看.  Universidad Francisco de Vitoria. 我们提出了一个体外模型，以评估神经损伤后嗅觉包裹胶质（OEG）神经再生能力。它基于OEG单层的轴突成人视网膜结节神经元（RGN）的共生和随后的轴再生研究，通过分析RGN轴突和声乐内皮标记。

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

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

研究胶质细胞轴突的生长异质性的影响，使用一个新的共培养法

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

科研

神经科学

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

在哺乳动物的中枢神经系统视神经横断后的实验操作方法

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

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 ...

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 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 ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

医学

Sheath-Preserving Optic Nerve Transection in Rats to Assess Axon Regeneration and Interventions Targeting the Retinal Ganglion Cell Axon

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury.
The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments.

This protocol describes an optic nerve transection that preserves the optic nerve sheath in rats. Hydrostatic pressure from microinjections into the optic nerve generate a complete transection, allowing for sutureless reapposition of the transected optic nerve ends and direct targeting of the axonal compartment in a transection model.

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as ...

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, ...

Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

Source: Portela-Lomba, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61863">Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to isolate retinal ganglion neurons (RGNs) from a rat&#39;s retina and coculture them with olfactory ensheathing glia (OEG) cells. This method allows for studying the neuro-regenerative potential of OEGs after neural injury.

视网膜神经节神经元与嗅鞘神经胶质细胞的共培养

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Co-culturing of a Dorsal Root Ganglion Explant with Schwann Cells for Neuron Myelination

Source: Blusch, A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64768">In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells.</a> J. Vis. Exp. (2023)
This video demonstrates the coculturing model of a dorsal root ganglion explant and Schwann cells. In coculture conditions, ascorbic acid promotes the differentiation of Schwann cells into myelinating forms. Myelinating Schwann cells wrap the axons multiple times to create a myelin sheath, which is essential for nerve function.

背根神经节外植体与雪旺细胞共培养用于神经元髓鞘形成

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Schwann cell culture

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