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. T., Lim, F., Mart&#237;n-Bermejo, M. J., D&#237;az-Nido, J., Avila, J., Wandosell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+olfactory+ensheathing+glia+promote+axonal+regeneration+of+rat+retinal+ganglion+neurons.">Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons.</a> Journal of Neurochemistry. 85 (4), 861-871 (2003).</li><li>Garc&#237;a-Escudero, V., 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=Patient-derived+olfactory+mucosa+cells+but+not+lung+or+skin+fibroblasts+mediate+axonal+regeneration+of+retinal+ganglion+neurons.">Patient-derived olfactory mucosa cells but not lung or skin fibroblasts mediate axonal regeneration of retinal ganglion neurons.</a> Neuroscience Letters. 509 (1), 27-32 (2012).</li><li>Sternberger, L. 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. A. K., St. John, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+cell+purification+and+determination+of+cell+purity:+crucial+aspects+of+olfactory+ensheathing+cell+transplantation+for+spinal+cord+repair.">Reliable cell purification and determination of cell purity: crucial aspects of olfactory ensheathing cell transplantation for spinal cord repair.</a> Neural Regeneration Research. 15 (11), 2016-2026 (2020).</li></ol>

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?...

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?...

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

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

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 ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

间接神经元的星形胶质细胞共培养试验:一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

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 ...