Adult spinal cord mixed cultures were first developed using rats with grant support from an NIH/NIDA R01 award (PI De Leo) and an NIH T32 training grant (PI Green). These methods were further adapted for mouse spinal cords with support from the following grants: NIH/NIDA 5K01DA023503 (PI Cao), NIH/NINDS 5R21NS066130 (PI Cao), and NIH/NIGMS P20GM103643 (PI Meng). The authors would also like to thank Dr. Alejandro M. S. Mayer, Department of Pharmacology, Chicago College of Osteopathic Medicine, for his technical help in obtaining microglia-rich mixed glial cells.

下面的协议被批准的机构动物护理和使用委员会（IACUC）在新英格兰的大学。以下协议是从4成年小鼠脊髓制备胶质细胞培养。 1.在无菌条件下文化胡德溶液的制备<ol><li>制备培养基:Eagle氏培养基（cDMEM）含DMEM的完全Dulbecco氏修改（用4.5克/升葡萄糖），10％胎牛血清（FBS），2mM的L-谷氨酰胺，100IU / mL青霉素，100毫克/毫升链霉素，250纳克/毫升两性霉素B，和50μM2-巯基乙醇（2-ME;见1.1.1）。混合和过滤消毒所有其他组件，然后添加FBS。存储培养基在4℃ <ol><li>制备50mM的2-ME /磷酸盐缓冲盐水（PBS）原液:添加100μL的浓缩2-ME（14.3 M）到28.6毫升1×PBS中，并过滤消毒（该溶液可以储存在4℃下数月）。 üSE 1毫升的2-ME原液每毫升1000培养基。 </li></ol></li><li> 密度梯度介质的制备 <ol><li>准备一个股票等渗密度梯度媒体解决方案（ 例如，100％珀）从1份10X常规无菌PBS和9份无菌股票密度梯度介质（ 如，珀）。 </li><li>稀释定期1×无菌PBS的100％的密度梯度介质（在1.2.1。制造），使20％的密度梯度介质（ 例如，10毫升100％的密度梯度介质的用40mL 1×PBS中），并储存在4 ℃。制备细胞培养物之前，使20％的密度梯度介质至室温（RT）。 </li></ol></li><li> 解决方案从木瓜蛋白酶解离系统准备 注: 木瓜蛋白酶解离系统所用的材料表来识别。其它相似的解离试剂盒（包括在内部装配）也可以使用。所有COMPONENTS必须是无菌的。 <ol><li>添加32毫升的Earle氏平衡盐溶液（EBSS）中的卵类粘蛋白的抑制剂混合物（粉末）。 </li><li>加入5毫升的EBSS的一个木瓜蛋白酶小瓶（粉末）。将在37℃水浴中的木瓜蛋白酶小瓶10分钟或直至木瓜蛋白酶溶解。 </li><li>添加EBSS 500微升一个DNA酶小瓶（粉）。轻轻混匀。 </li><li>添加脱氧核糖核酸酶溶液（以上）的木瓜蛋白酶小瓶的250微升。 </li><li>计算所需的上述木瓜蛋白酶/ DNA酶的混合物（每鼠脊髓木瓜蛋白酶/ DNA酶混合物的约800微升）的合计量和所需要的混合物转移到50 mL管中用于组织消化（步骤3.2）。存储在原始小瓶剩在4℃至少两周。 </li><li>保持冰从包的所有组件，直到需要。 </li></ol></li><li>制备脊髓收集管:一种无菌的15毫升管用Hank氏平衡盐溶液（HBSS 5mL的;从所述木瓜蛋白酶解离系统，0;收集脊髓）和一个无菌的15毫升管1X PBS（对于填补了5毫升注射器，见步骤2.4）。此外，5毫升的HBSS准备一份无菌培养皿英寸（35 mm或60毫米），保持它在培养罩。 </li></ol> 2.脊髓收藏注:所有设备必须是无菌的。由IACUC批准的指定区域执行收集。 <ol><li>获得收获小鼠脊髓以下工具:直锐/钝器18厘米的手术剪，一个12厘米的标准手术刀（＃3固体），碳钢无菌手术刀片＃10，小动物斩首，12厘米的标准曲线钳，和无菌20G针连接到一个5毫升的注射器。 </li><li>安乐死用CO 2对动物和消毒鼠标头并用70％乙醇（EtOH中）的后区。 </li><li>动物斩首的刽子手。就后腰中间纵切口，显露肌肉层。使通过在臀部的水平脊椎动物列干净的横断与直无菌手术剪（18厘米）（通过覆盖脊椎动物列肌肉层也切）。 </li><li>将动物对新鲜一次性擦拭片。小心地将20G的针（连接到一个5毫升的注射器填充有无菌1×PBS中（步骤1.4））到脊柱朝喙侧。按住向下动物紧密，迅速推向注射器。整个脊髓应该从子宫颈年底问世。收集脊髓到含有HBSS 15毫升管。 </li><li>重复步骤2.4小鼠的其余部分。每只动物之间，消毒用70％乙醇所有使用的仪器。 注意:通常情况下，一个单一的12孔板可以为每4小鼠脊髓（见4.3）成立。 </li></ol> 3.单细胞悬液的制备注:执行步骤3和4的培养罩把一切都无菌。 <ol><li>传输所有脊髓入含有HBSS的皮氏培养皿（步骤1.4）。削减每个脊髓成许多精美的，小件用无菌剪刀和镊子。转移脊髓组织用无菌镊子或10毫升吸管含有制备的木瓜蛋白酶/ DNA酶的酶混合物（步骤1.3.5）的50-mL锥形管（避免加入HBSS的酶混合物，因为这将进一步稀释制备酶溶液，并可能导致酶的性能降低）。 </li><li>然后振荡试管混合。在37℃下1小时管在培养箱/摇床圆周振荡在150转。 </li><li>涡管再次大力磨碎用5毫升吸管进一步推动分离组织中的酶溶液。 </li><li>在RT细胞悬液转移到15毫升管中并离心，在300×g离心5分钟。 </li><li>在离心过程中，混合2.7毫升EBSS与重组白蛋白卵类粘蛋白INH的300微升在无菌管ibitor溶液（1.3.1）。加入DNA酶溶液（步1.3.3）的150微升。 </li><li>离心后，除去上清，悬浮与上述制备（步骤3.5）的溶液中的细胞沉淀。涡流以及打破细胞沉淀。 </li><li>添加3mL的重组白蛋白的卵类粘蛋白抑制剂溶液（步骤1.4.1），以细胞悬浮液。离心细胞，在70×g离心在RT 6分钟。除去上清液（包含膜碎片）。 </li></ol>从单细胞悬液髓鞘4.进一步去除<ol><li>在800 XG在RT轻轻加入8毫升20％的密度梯度介质（在步骤1.2.2制备）到含有细胞沉淀管中，涡流破坏沉淀，离心所述细胞30分钟无制动 。小心除去碎片（主要是髓鞘）和上清液的顶层，但保留沉淀。 </li><li>要除去密度梯度的残余，由resuspendi洗细胞纳克用8毫升稀释cDMEM的（1份cDMEM和2份HBSS）中的细胞沉淀。离心在400×g离心10分钟，将细胞在4℃除去上清液并以相同的方式与稀释cDMEM（上文）再次洗涤细胞。保持冰的细胞，直到他们播种。 </li><li>除去上清，重悬在培养基中的细胞沉淀（cDMEM供给与2-ME（在步骤1.1中制备））。对于一个12孔板中，使用3毫升×4（使用小鼠数）+ 2毫升= 14毫升介质。这将确保有对整个板足够的细胞悬浮液（12个孔），并提供了可用于确定每孔的平均细胞数和培养物的小胶质含量额外井。如果将被用于其它类型的培养容器，按比例计算所需的培养基的总体积。 </li><li>添加1毫升细胞悬液到12孔平板的每个孔中。 </li><li>孵育细胞在含有5％CO 2 35.9 摄氏度 。 </li><l我&gt;第D 1更改媒体（通过抽吸去除旧的媒体），然后每3 - 4天之后（通常是改变D中的媒体:1，4，8，11，然后用细胞第12天）。 注:在第1天，将培养可以含有由于从脊髓组织中的残余物髓鞘显著的碎片;因此，建议改变第1天传媒（请参阅有关更多信息的讨论）。培养物准备好D之治疗12 - 14细胞通常是在D 12 80％汇合，并可以用D 14通常为接近100％汇合时，对D 12，有每孔约100,000细胞在12孔平板。 </li></ol><img alt="图4" src="/files/ftp_upload/54801/54801fig4.jpg" /> 图2:在不同时间建立的混合胶质细胞培养物混合后的神经胶质细胞的代表性图像从成年C57BL / 6小鼠中制备初级脊髓混合胶质。代表图像显示神经胶质培养的电镀后的进展。在第1天，一些细胞附着到培养板上，但它们仍然是多为圆形。也有许多漂浮细胞和显著碎片。在第4天，大部分细胞附着到培养板上。细胞似乎网状可见的过程。细胞零星分布和文化在这个时候约20-30％汇合。在第8天，文化是50-60％之间的融合。文化的部分地区有细胞的大片。文化的其他领域有细胞的生长稀疏。补丁中的某些细胞采取了"广场样"外观。第12天，培养物是在等于或高于80％汇合（此处示出的图像中的约90％汇合）。细胞在此时生长的对数期，天之间12-14是使用胶质细胞进行实验的最佳时间。 <a href="http://ecsource.jove.com/files/ftp_upload/54801/54801fig4large.jpg" target="_blank">请点击这里查看</a>更大的版本这个数字。 <ol start="7"><li>检查使用通过标准荧光激活细胞分选（FACS）染色方案11从额外孔细胞（步骤4.3），使用抗小鼠CD45抗体和抗小鼠的CD11b单克隆抗体的组合中的小胶质细胞的内容。 CD45 +细胞CD11b +细胞被识别为小胶质细胞。 </li></ol>

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The authors have nothing to disclose.

它是执行所有步骤，包括媒体，以获得实验之间重复性的结果改变时间表-以一致的方式非常关键。下面的步骤是获得优良的品质成人混合胶质尤为关键。 酶消化是该协议的一个重要方面。至关重要的是，该组织片是为了获得单细胞悬浮液充分消化。然而，过度消化将导致显著较少的细胞。每个实验需要执行导频测试，以确定基于所述类型用于孵化小瓶的确切消化时间，每次?...

慢性疼痛是一种严重的健康问题，影响到在美国的约100万成年人，具有高达635 $ 1十亿预计每年成本。越来越多的证据表明脊髓神经胶质细胞的神经性疼痛，最具破坏性的种慢性疼痛2中的一个发展的显著贡献。一个适当的体外培养体系将在细胞和分子水平深入研究神经胶质细胞的作用显著帮助。 目前，研究人员使用的体外神经胶质培养系统包括从新生小鼠或大鼠大脑皮质组织来源主要是神经胶质细胞和永生化从新生小鼠衍生的神经胶质细胞系或大鼠原胶质细胞。新生细胞含有未分化细胞显著数字，可以进一步分化成神经胶质细胞（主要为星形胶质细胞和小胶质细胞），从而提供一bundant细胞实验使用3。然而，我们以前的研究已经表明，新生儿脑胶质细胞从成人脊髓神经胶质细胞在脂多糖（LPS）刺激的反应显著不同。例如，白细胞介素（IL）-4显示的增强的LPS诱导的一氧化氮的抑制作用（NO）产生的成年大鼠脊髓神经胶质相比新生儿大脑胶质4。此外，趋化因子产生刺激后的由神经肽的型材，降钙素基因相关肽（CGRP），是新生小鼠脑部的神经胶质细胞之间无可争议不同（在参考文献中描述的方法。5）和成年小鼠脊髓神经胶质细胞（ 图1）。建立的细胞系是易于使用和维护，可以在很短的时间内提供大量的细胞。在一般情况下，永生化细胞系中产生或者使用病毒介导的永生化系统（如广泛使用的小胶质细胞系BV2）6,7或以下第自发变换电子识别（如星形胶质细胞系的C8-D1A和小胶质细胞系的C8-B4）8,9的细胞系具有优异的学习星形细胞和小胶质细胞单独的分子特征。然而，从细胞系所获得的结果总是需要在原代细胞或在体内条件进一步验证。还应当指出的是，没有发生过一个从啮齿动物的脊髓神经胶质衍生的神经胶质细胞系的报告。 以帮助调查脊髓神经胶质细胞中的神经性疼痛，是从成年小鼠脊髓衍生已通过适应用来产生成年大鼠混合胶质细胞培养4先前报道的方法开发了一种培养系统，5的发展中的作用该小鼠脊髓神经胶质文化进一步细化最近10和更详细的这篇文章中介绍。成人脊髓混合胶质细胞培养Ç一个与来自适合的特定的研究的需要选出的菌株的小鼠建立的，并且细胞能够在培养物中维持长达21天后的培养开始。这种方法可以在神经性疼痛的研究中使用，以及在涉及脊髓内的病理变化，如肌萎缩性侧索硬化症（ALS），人免疫缺陷病毒（HIV）相关的感觉神经病，和多个各种神经疾病的调查硬化症（MS）。

<table border="0" cellpadding="0" cellspacing="0" width="2053">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Dulbecco&#8217;s modification of Eagle&#8217;s media (DMEM) with 4.5g/L glucose</td>
			<td style="width:277px;">Lonza</td>
			<td style="width:119px;">12-709F</td>
			<td style="width:1168px;">The cDMEM media is a standard, widely used culture media. Individual researcher can decide where to purchase the DMEM and all other component used to make cDMEM media.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">L-Glutamine (100x)</td>
			<td style="width:277px;">Lonza</td>
			<td style="width:119px;">17-605E</td>
			<td>The L-glutamine is a standard, widely used component for various culture media. Individual researcher can decide where to purchase L-glutamine.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Antibiotic-Antimycotic Solution (100x)</td>
			<td style="width:277px;">Corning-Mediatech</td>
			<td style="width:119px;">30-004-CI</td>
			<td>This is a combination of penicillin, streptomycin and Amphotericin formulated to contain 10,000 units/ml penicillin G, 10 mg/ml streptomycin sulfate and 25 &#181;g/ml amphotericin B. Individual researcher can decide where to purchase individual component.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">2-mercaptoethanol (2-ME)</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:119px;">M3148</td>
			<td>A BioReagent, suitable for cell culture, molecular biology and electrophoresis.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Papain dissociation system</td>
			<td style="width:277px;">Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>Individual components of this kit can be purchased separately.&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Percoll</td>
			<td>GE Health Care</td>
			<td style="width:119px;">17-0891-01</td>
			<td>Percoll is sold as sterile solution. Undiluted Percoll can be re-autoclaved if needed.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Lab-line incubator/shaker&#160;</td>
			<td style="width:277px;">Barstead/Lab-line</td>
			<td style="width:119px;">MaxQ4000&#160;</td>
			<td>This is the incubator/shaker we have currently. Other types of shakers can be used instead.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Lipopolysaccharides, Salmonella Minnesota Re595&#160;</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:119px;">L-9764</td>
			<td>Other strains of LPS can also induce glial responses. The magnitude of the responses may vary.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse IL-6 DuoSet ELISA&#160;</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY406</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse TNF-alpha DuoSet ELISA</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY410</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse IP-10 (CXCL10) DuoSet ELISA</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY466</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse MCP-1 (CCL2) ELISA Opteia set</td>
			<td style="width:277px;">BD Biosciences</td>
			<td style="width:119px;">555260</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse chemokine Q-Plex</td>
			<td style="width:277px;">Quansys Biosciences</td>
			<td style="width:119px;">120251MS</td>
			<td>This product was available for in-house assay at the time. Instead, we sent out samples to the manufacturer for analysis.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">APC-anti-mouse-CD45 (clone 30-F11)</td>
			<td style="width:277px;">eBioscience</td>
			<td style="width:119px;">17-0451</td>
			<td>Antibody of the same clone but from other vendors can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">FITC-anti-mouse-CD11b (clone M1/70)</td>
			<td style="width:277px;">eBioscience</td>
			<td style="width:119px;">11-0112</td>
			<td>Antibody of the same clone but from other vendors can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Accuri C6 flow cytometer</td>
			<td style="width:277px;">BD Biosciences</td>
			<td style="width:119px;">BD Accuri C6&#160;</td>
			<td>Each individual Researcher can use any flow cytometer he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">FlowJo software</td>
			<td style="width:277px;">Tree Star, Inc.</td>
			<td style="width:119px;">FlowJo7.6.5</td>
			<td>Each individual Researcher can use any analysis software he or she prefers.</td>
		</tr>
	</tbody>
</table>

preparation of primary mixed glial cultures from adult mouse spinal cord tissue

它已被广为接受的脊髓神经胶质反应显著到神经性疼痛的发展作出贡献。已通过体外细胞培养系统中获得关于在细胞和分子水平胶质活动巨大的信息。所用的体外系统，主要包括从新生儿脑皮质组织来源的原神经胶质细胞和永生化细胞系。然而，这些系统可能无法反映体内脊髓神经胶质细胞的特性。为了使用培养体系，更好地反映体内状况进一步调查脊髓神经胶质细胞在周围神经损伤引起的神经性疼痛发展中的作用，我们的实验室已经发展到建立原发性脊髓混合胶质细胞培养从方法成年小鼠。简单地说，脊髓从成年小鼠收集，并通过木瓜蛋白酶消化后髓鞘去除与加工窝点性梯度网上平台。单细胞悬浮液是在完整的Dulbecco补充有2-巯基乙醇（2-ME）改良的Eagle培养基（cDMEM）培养在35.9℃。这些培养条件为混合胶质细胞的生长进行了优化。在这些条件下，细胞准备用于12之间实验 - 14天（细胞通常是在这段时间对数期）建立培养物（D 0）之后，并且可以被保持在培养条件达到D 21。该培养系统可以用于研究脊髓神经胶质细胞的刺激时与各种物质和试剂的反应。除了神经性疼痛，这种系统可用于研究在涉及脊髓神经胶质细胞的病理改变其他疾病胶质反应。

该方法可用于从小鼠和大鼠制备混合胶质细胞。每孔中的培养的D 12启动后一个12孔板的平均总细胞数应该是相对稳定的，与每孔周围100,000个细胞时细胞从小鼠脊髓的。从该方法获得的神经胶质细胞可以在被设计为检查时的物质和感兴趣剂施用成人脊髓神经胶质反应实验中使用。 图3提供了从一个典型的实验中的细胞因子和趋化因子反应的例子，其...

Watch this Scientific Journal Video about Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue at JoVE.com

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

神经性疼痛的发展涉及脊髓神经胶质细胞的病理变化。从成年脊髓组织的，设计用于在体外研究这些细胞中的胶质可靠的培养体系缺乏。因此，我们在这里展示了如何建立从成年小鼠的脊髓组织的主要胶质细胞混合培养。

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

preparation-primary-mixed-glial-cultures-from-adult-mouse-spinal-cord

Research

JoVE Journal

Neuroscience

10.5K 次观看.  University of New England. 神经性疼痛的发展涉及脊髓神经胶质细胞的病理变化。从成年脊髓组织的，设计用于在体外研究这些细胞中的胶质可靠的培养体系缺乏。因此，我们在这里展示了如何建立从成年小鼠的脊髓组织的主要胶质细胞混合培养。 

视频: Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

斜脊髓切片用于神经前根刺激的制备

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递:对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

从新生小鼠中磁分离小胶质细胞用于原代细胞培养

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

小鼠轻度创伤性脑损伤的电磁控制闭头模型

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

摘要

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从胚胎脊髓连合神经的解剖和文化

斜脊髓切片用于神经前根刺激的制备

斑马鱼

脊髓神经元从新生小鼠中分离和培养

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

拉米内切手术和脊髓窗口植入小鼠

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

神经干细胞向大鼠和小鼠大脑的动脉内传递:对脑缺血的应用

从新生小鼠中磁分离小胶质细胞用于原代细胞培养

小鼠轻度创伤性脑损伤的电磁控制闭头模型

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

摘要

探索更多视频

此视频中的章节

从胚胎脊髓​​连合神经的解剖和文化

斜脊髓切片用于神经前根刺激的制备

斑马鱼

脊髓神经元从新生小鼠中分离和培养

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

拉米内切手术和脊髓窗口植入小鼠

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

神经干细胞向大鼠和小鼠大脑的动脉内传递:对脑缺血的应用

从新生小鼠中磁分离小胶质细胞用于原代细胞培养

小鼠轻度创伤性脑损伤的电磁控制闭头模型

从胚胎脊髓连合神经的解剖和文化