作者没有确认。

根据科罗拉多大学机构动物护理和使用委员会的指导方针，对该手术中的动物进行护理和治疗。 准备解决方案<ol><li>在适当的温度下准备并储存所有溶液，如表1所示。 </li></ol> 2.涂层孔和滑块注意:神经元不能很好地粘附在塑料或玻璃表面上。 <ol><li>在分离神经元前一天，用无菌的24孔培养板的孔用0.5mL聚-D-赖氨酸（PDL; 表2 ）包被，并将其留在层流罩O / N中。 注意:优选的方法是使用24孔板并仅涂覆中心孔，因为来自外围孔的蒸发趋于更加加速并导致不一致的结果。此外，在t之前，玻璃载玻片可以放置在孔内o涂层。神经元将附着到涂覆的玻璃片上，并可以稍后去除以进行进一步的显微成像。 </li><li>在隔离当天，从井中取出PDL。用无菌水清洗几分钟，然后取出任何剩余的水，并使其干燥1小时（见下文）。 </li><li>干燥后，用层粘连蛋白涂覆载玻片（ 表2 ）。 注意:约10μL层粘连蛋白（10μg/μL）足以涂覆无菌24孔板的每个孔。将其与340μL培养基（总体积:350μL）混合以覆盖整个孔。让它在室温下在培养罩中静置2小时，然后在包被细胞之前吸出。 </li></ol>收获脊髓注意:所有仪器均应进行高压灭菌（135°C和30 psi，4 x 7 min循环），以进行无菌处理。 <ol><li>将1-3天龄的C57BL / 6小鼠小鼠安乐死在带有异氟烷的室内。等待30秒后停止运动，捏腿，确认缺乏反应。 </li><li>使用剪刀将头部与身体分开，小狗处于俯卧位。 </li><li>稳定后腿或手臂在手术台上，背面朝用户。 </li><li>使用弯曲的虹膜剪刀剪掉皮肤。 </li><li>从腰部上方的腰部区切开脊髓，然后切开胸部两侧，将其与身体分开。 注意:这需要从内脏器官仔细解剖脊髓，以避免对其他器官的无意损伤（ 图1a ）。 </li><li>在含有5mL0.2μm过滤器灭菌的磷酸盐缓冲盐水（PBS）的3×10cm培养皿中依次洗涤10s以除去多余的组织。 </li><li>将22 G针头和注射器装满5 mL过滤灭菌的PBS到脊柱的尾端，颅内冲洗，使脐带to退出第四培养皿（ 图1b ）。 </li><li>在15 mL管中收集脊髓，并加入5 mL HABG（ 表1 ）。小心避免压碎脊髓。 </li><li>对垃圾中的每只小狗重复步骤3.1-3.8。 注意:理想情况下，这个过程每个脊髓需要不到30分钟，以确保健康的神经元分离。 </li></ol>隔离神经元注意:以下步骤应在层流罩中进行。预计熟悉基本的无菌技术。 <ol><li> 组织分娩 <ol><li>取包含脊髓的管，轻轻晃动，悬挂组织。 </li><li>将组织从管中倒入60毫米玻璃培养皿和骰子用剃刀刀片，以产生大小为0.5毫米的细碎片。 </li><li>将大孔移液器的组织转移到含有5mL HABG的15mL管中。 </li><li>放在一个30℃水浴30分钟，以使细胞在该温度下平衡。将细胞保持在振动器上，足够使其悬浮在流体中。 注意:此步骤是为了避免在从冰到消化介质转移时对细胞的冲击。保持细胞在30°C有助于减少细胞死亡与另外增加的新陈代谢在37°C。 </li></ol></li><li>准备消化培养基（ 表1 ）。 </li><li> 准备密度梯度（表1）。 <ol><li>在4个不同的15 mL管中制备4层中的每一层，如表1所示 。 </li><li>从每层加入1 mL到一个新的15 mL管中。从底部的第1层开始，并依次添加，直到到达顶层的第4层。加入时避免干扰层。 </li></ol></li><li>从步骤2.2洗涤PDL涂覆的板。用无菌水洗涤数分钟，然后取出任何剩余的水使其干燥1小时。 </li><li> 将组织转移至消化培养基。 <ol><li>从30℃的摇床水浴中取出含有组织的管，并使其沉降数分钟。 </li><li>从37°C水浴中取出消化介质管，然后将其吸入皮肤锁定注射器。 </li><li>从含有组织的管吸出多余的HABG。 </li><li>在注射器上使用一个leur-lock 0.2μm过滤器，将消化液添加到含有组织的管中。 </li><li>将管置于30°C水浴中30分钟。保持细胞晃动刚好足以让它们悬浮在液体中。 注意:重要的是不要将细胞保持在消化介质中太长时间，或者使温度过高，这可能导致过度消化，导致组织悬浮在凝胶状混合物中。 </li></ol></li><li>在此期间，如步骤2.3所示涂覆层粘连蛋白。 </li> &lt;执行研磨（ 即从组织中分离细胞）。 <ol><li>从摇动的30°C水浴中取出管子，使其沉降数分钟。 </li><li>吸出过量消化介质。 </li><li>将组织悬浮于2 mL HABG中。 </li><li>使用狭窄的移液管，捣碎10次，持续45秒。 注意:这可能是最关键的一个步骤，如果不能正确完成，可以显着降低产量。 <ol><li>将组织吸入移液管，并立即将内容物倒空。 </li><li>避免引入空气，因为它会显着降低可行的产量。 注意:理想的移液管是9"玻璃移液管，移液管的顶端应进行火花抛光，使粗糙表面光滑，然后将其放置在二氯二甲基硅烷（DMDCS）在氯仿中的1:20溶液中，然后留下O / N，然后取出移液管，风干用高压釜灭菌。 注意:DMDCS和氯仿是高度易燃的，硅化应在通风橱中进行。 </li></ol></li><li>吸出顶部2 mL上清液，并将其置于标有"收集"的新型15 mL管中。 </li><li>重复步骤4.7.3-4.7.5两次（细胞收集管应至少有6 mL）。 </li><li>将收集管内容物慢慢转移到步骤4.2中制备的梯度管中，避免了梯度的破坏。 </li></ol></li><li>此时，从冰箱中取出以前制备的神经基质培养基（ 表1 ），并使其在37°C浴中温热。 </li><li> 净化神经元。 <ol><li>在800 xg和22℃下离心梯度管15分钟。 </li><li>用移液管收集所需的层（ 图2 ），并置于新的15 mL管中。对于最高纯度的神经n分离（ 即 &gt; 90％），收集层3.为了获得较少的纯度（ 即 &gt; 70-80％），收集层2和3。 </li><li>通过向新收集的层中加入5mL HABG来稀释密度梯度。 </li><li>在22℃下以200xg离心2分钟。 </li><li>弃去上清液，重新悬浮在5 mL HABG中，轻轻摇匀，使细胞悬浮。 </li><li>在22℃离心2分钟200×g。 </li><li>弃去上清液，重悬于3 mL神经巴氏培养基中，轻轻摇匀，重悬细胞。 </li></ol></li><li> 计数细胞 <ol><li>取10μL溶液，现在用神经巴氏培养基中的细胞，并与10μL台盼蓝混合。 </li><li>将10μL该混合物置于玻璃计数室中。 </li><li>使用标准的玻璃计数室，计算四个4×4象限中每一个中的细胞数。添加所有细胞计数（n），多层数2（稀释因子），除以4（计数的象限数），乘以3（神经基底培养基的体积），并乘以10 4以获得细胞/ mL的细胞浓度。 <img alt="方程" src="/files/ftp_upload/55856/55856eq0.jpg" /></li></ol></li><li> 将培养板上的细胞种植 <ol><li>将细胞悬浮液稀释至1毫升神经巴氏培养基中的30万个细胞。 注意:根据上述步骤获得的细胞浓度，加入另外的神经基底培养基以获得3×10 5个细胞/ mL的终浓度。方程式为C 1 V 1 = C 2 V 2 ，其中C 1是从收获获得的细胞的初始浓度; V 1为3mL;如上所述，C 2为3×10 5个细胞/ mL。 V 2求解方程。加入使溶液中细胞总体积相等所需的神经巴培养液体积V 2 。 </li><li>轻轻晃动将细胞分布在溶液中，并向涂覆的24孔板中的每个孔加1 mL。 </li></ol></li></ol>

<ol>
	<li>Qayumi, A. K., 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=Animal+model+for+investigation+of+spinal+cord+injury+caused+by+aortic+cross-clamping.">Animal model for investigation of spinal cord injury caused by aortic cross-clamping.</a> J Invest Surg. 10 (1-2), 47-52 (1997).</li><li>Fang, B., 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=Ischemic+preconditioning+protects+against+spinal+cord+ischemia-reperfusion+injury+in+rabbits+by+attenuating+blood+spinal+cord+barrier+disruption.">Ischemic preconditioning protects against spinal cord ischemia-reperfusion injury in rabbits by attenuating blood spinal cord barrier disruption.</a> Int J Mol Sci. 14 (5), 10343-10354 (2013).</li><li>Taira, Y., Marsala, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+proximal+arterial+perfusion+pressure+on+function,+spinal+cord+blood+flow,+and+histopathologic+changes+after+increasing+intervals+of+aortic+occlusion+in+the+rat.">Effect of proximal arterial perfusion pressure on function, spinal cord blood flow, and histopathologic changes after increasing intervals of aortic occlusion in the rat.</a> Stroke. 27 (10), 1850-1858 (1996).</li><li>Stoppini, L., Buchs, P. -. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-182 (1991).</li><li>Ahlemeyer, B., Baumgart-Vogt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocols+for+the+simultaneous+preparation+of+primary+neuronal+cultures+of+the+neocortex,+hippocampus+and+cerebellum+from+individual+newborn+(P0.+5)+C57Bl/6J+mice.">Optimized protocols for the simultaneous preparation of primary neuronal cultures of the neocortex, hippocampus and cerebellum from individual newborn (P0. 5) C57Bl/6J mice.</a> J Neurosci Methods. 149 (2), 110-120 (2005).</li><li>Brewer, G. J., Torricelli, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+neurons+and+neurospheres.">Isolation and culture of adult neurons and neurospheres.</a> Nat Protoc. 2 (6), 1490-1498 (2007).</li><li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">Rat hippocampal neurons in dispersed cell culture.</a> Brain Res. 126 (3), 397-425 (1977).</li><li>Graber, D. J., Harris, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+culture+of+spinal+motor+neurons+from+rat+embryos.">Purification and culture of spinal motor neurons from rat embryos.</a> Cold Spring Harb Protoc. 2013 (4), 319-326 (2013).</li><li>Anderson, K. N., Potter, A. C., Piccenna, L. G., Quah, A. K., Davies, K. E., Cheema, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+motor+neurons+from+the+newborn+mouse+spinal+cord.">Isolation and culture of motor neurons from the newborn mouse spinal cord.</a> Brain Res Prot. 12 (3), 132-136 (2004).</li><li>Gingras, M., Gagnon, V., Minotti, S., Durham, H. D., Berthod, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocols+for+isolation+of+primary+motor+neurons,+astrocytes+and+microglia+from+embryonic+mouse+spinal+cord.">Optimized protocols for isolation of primary motor neurons, astrocytes and microglia from embryonic mouse spinal cord.</a> J Neurosci Methods. 163 (1), 111-118 (2007).</li><li>Brewer, G. J., 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=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35 (5), 567-576 (1993).</li><li>Fang, B., 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=Ischemic+preconditioning+protects+against+spinal+cord+ischemia-reperfusion+injury+in+rabbits+by+attenuating+blood+spinal+cord+barrier+disruption.">Ischemic preconditioning protects against spinal cord ischemia-reperfusion injury in rabbits by attenuating blood spinal cord barrier disruption.</a> Int J Mol Sci. 14 (5), 10343-10354 (2013).</li><li>Su, M., Zhong, W., Ren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dependent+protection+of+reseveratrol+against+spinal+cord+ischemic-reperfusion+injury+in+rats.">Dose-dependent protection of reseveratrol against spinal cord ischemic-reperfusion injury in rats.</a> Trop J Pharm Res. 15 (6), 1225-1233 (2016).</li><li>Haapanen, H., 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=Remote+ischemic+preconditioning+protects+the+spinal+cord+against+ischemic+insult:+An+experimental+study+in+a+porcine+model.">Remote ischemic preconditioning protects the spinal cord against ischemic insult: An experimental study in a porcine model.</a> J Thorac Cardiovasc Surg. 151 (3), 777-785 (2016).</li><li>Conrad, M. F., Ye, J. Y., Chung, T. K., Davison, J. K., Cambria, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+complications+after+thoracic+aortic+surgery:+long-term+survival+and+functional+status+varies+with+deficit+severity.">Spinal cord complications after thoracic aortic surgery: long-term survival and functional status varies with deficit severity.</a> J Vasc Surg. 48 (1), 47-53 (2008).</li><li>Wong, D. R., 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=Delayed+spinal+cord+deficits+after+thoracoabdominal+aortic+aneurysm+repair.">Delayed spinal cord deficits after thoracoabdominal aortic aneurysm repair.</a> Ann Thorac Surg. 83 (4), 1345-1355 (2007).</li><li>Freeman, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-2+agonist+attenuates+ischemic+injury+in+spinal+cord+neurons.">Alpha-2 agonist attenuates ischemic injury in spinal cord neurons.</a> J Surg Res. 195 (1), 21-28 (2015).</li><li>Freeman, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+protection+via+alpha-2+agonist-mediated+increase+in+glial+cell-line-derived+neurotrophic+factor.">Spinal cord protection via alpha-2 agonist-mediated increase in glial cell-line-derived neurotrophic factor.</a> J Thorac Cardiovasc Surg. 149 (2), 578-586 (2015).</li></ol>

作者没有披露。

这种技术允许脊髓神经元的可靠培养。一旦熟练掌握了这项技术，大概需要3.5h才能完成。我们已经能够在大约4小时内从2个独立的小窝（总共16只小鼠）中进行神经元的分离。可行性的关键步骤是能够精通从小鼠中提取脊髓。产量允许电镀几个孔，并能够在各种条件下测试神经元。我们已经能够在成熟后治疗神经元，并使用蛋白质印迹分析可靠地评估蛋白质表达。此外，在孔内使用玻璃载玻片进?...

<html>了解脊髓病理学需要在宏观和微观层面上使用各种模型。大型和小型动物模型1,2,3用于脊髓疾病和损伤的体内研究。 在体内研究这些问题的优点是脊髓分析局限于全脊髓匀浆或组织切片4 。当在其驻留神经元和周围神经胶质中分离脊髓中的特定反应和目标时，这产生了一些歧义。遗传操纵的小鼠越来越多的可用性允许对细胞和分子水平的生物学进行更详细的研究。因此，这里使用新生儿小鼠模型，允许在体外研究脊髓神经元的独特性质和生物学。 在体外分离和维持神经元并不是特别简单，成年啮齿动物的皮层组织的神经元分离技术相对丰富，似乎导致大量的孤立神经元（ 即数百万） 5 ，相反，由于组织质量较小，脊髓组织中神经元的产量较低8,9,10 ，另外在小鼠中，新生儿隔离技术相对较少脊髓神经元和现有的方法受到较低的神经元产量（ 即数百个）的限制9或需要分离胚胎小鼠的费力和资源性重的技术10 。 在这个协议中，我们使用一种技术，允许从新生小鼠脊髓的成本和资源有效的隔离大量的神经元。如以前发表的技术中常见的，我们使用木瓜蛋白酶作为酶蛋白酶，允许从脊髓组织释放神经元5,6 。此外，我们使用密度梯度进行精细细胞分离，其先前已被证明是有效的6,10 。虽然培养细胞的培养基可以根据我们的经验和以前发表的11来改变 ，但补充新鲜的B27培养基补充剂已被证明对神经元寿命至关重要。神经元通常可行达10天，允许进行治疗。

<table border="0" cellpadding="0" cellspacing="0" width="1357">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Hibernate A Medium - 500 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td style="width:107px;">A1247501</td>
			<td style="width:805px;">https://www.thermofisher.com/order/catalog/product/A1247501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hibernate A Minus Calcium - 500 mL</td>
			<td style="width:116px;">Brainbits</td>
			<td>HA-Ca</td>
			<td>http://www.brainbitsllc.com/hibernate-a-minus-calcium/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax 100X - 100 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>35050061</td>
			<td>https://www.thermofisher.com/order/catalog/product/35050079</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement 50X - 10 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>17504044</td>
			<td>https://www.thermofisher.com/order/catalog/product/17504044</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Papain, Lyophilized - 100 mg</td>
			<td style="width:116px;">Worthington</td>
			<td>LS003119</td>
			<td>http://www.worthington-biochem.com/pap/cat.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal A Medium - 500 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>10888022</td>
			<td>https://www.thermofisher.com/order/catalog/product/10888022</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>15140122</td>
			<td>https://www.thermofisher.com/order/catalog/product/15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine hydrobromide - 5 mg</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/p6407?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Laminin - 1 mg</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>23017015</td>
			<td>https://www.thermofisher.com/order/catalog/product/23017015</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue - 20 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>T8154-20ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/t8154?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OptiPrep Density Gradient Medium - 250 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>D1556-250ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/d1556?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichlorodimethylsilane (DMDCS, Sigma Silicoat)</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>440272-100ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/440272?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>288306-1L</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Glass Pippette - 9&#34;</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:107px;">13-678-20C</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/cls7095d9?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette bulb - 5 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>Z186678-3EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/z186678?lang=en&#38;region=US&#38;cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BRAND&#174;&#160;Petri dish, glass - 60x15 mm</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>BR455717-10EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/br455717?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile 24 Well Cell Culture Plate</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>M8812-100EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/m8812?lang=en&#38;region=US</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hausser Hemacytometer (glass counting chamber)</td>
			<td style="width:116px;">Fischer Scientific</td>
			<td>02-671-6</td>
			<td>https://www.fishersci.com/shop/products/hausser-bright-line-phase-hemacytometer-hemacytometer/026716</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Slides - 12 mm sterile cover glass - uncoated</td>
			<td>Neuvitro</td>
			<td>GG-12-1.5-Pre</td>
			<td>http://www.neuvitro.com/german-coverslip-12mm-diameter.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NeuN Rabbit Monoclonal Antibody - 100 &#181;L</td>
			<td style="width:116px;">Abcam</td>
			<td>ab177487</td>
			<td>After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluded to 1:200 for 18 hours in 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAP-2 Mouse Monoclonal Antibody - 50 &#181;L</td>
			<td style="width:116px;">Abcam</td>
			<td>ab11267</td>
			<td>After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluded to 1:500 for 18 hours in 4 &#176;C</td>
		</tr>
	</tbody>
</table>

spinal cord neurons isolation and culture from neonatal mice

我们提出了脊髓神经元分离和培养的方案。神经元从新生儿C57BL / 6小鼠获得，并在出生后第1-3天分离。一只小鼠垫，通常从一个繁殖对出生的4-10只小狗被聚集一个实验，并且在用异氟烷治疗安乐死后，从每只小鼠单独收集脊髓。将脊柱解剖出来，然后将脊髓从柱上释放出来。然后将脊髓切碎以增加允许神经元和其他细胞从组织中释放的酶蛋白酶的递送的表面积。然后使用研磨将细胞释放到溶液中。随后将该溶液以密度梯度分级，以分离溶液中的各种细胞，允许分离神经元。从一个垃圾组可以分离出约1-2.5×10 6个神经元。然后将神经元接种到涂有粘合剂的孔上rs允许适当的成长和成熟。神经元在生长和培养基中需要约7天达到成熟度，此后可用于治疗和分析。

使用这种技术，单个垃圾（4-10只幼仔）允许分离适合种植在培养板上的1-2.5个10 6个神经元。通常，以上述浓度（ 即 30万个细胞/ mL）接种4-8个孔。 图3显示了在低（ a ）和高（ b ）放大光学显微镜下培养一周后在该浓度下神经元的出现。然而，我们也能够以高达500,000个细胞/孔和低至100,000个细胞/孔的浓度培养?...

Watch this Scientific Journal Video about Spinal Cord Neurons Isolation and Culture from Neonatal Mice at JoVE.com

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

这项研究提出了一种从WT新生小鼠中分离神经元的技术。它需要从新生小鼠中仔细地解剖脊髓，然后通过机械和酶切割将脊髓组织中的神经元分离出来。

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 ...

spinal-cord-neurons-isolation-and-culture-from-neonatal-mice

Research

JoVE Journal

Neuroscience

19.0K 次观看.  University of Colorado Anschutz Medical Campus. 这项研究提出了一种从WT新生小鼠中分离神经元的技术。它需要从新生小鼠中仔细地解剖脊髓，然后通过机械和酶切割将脊髓组织中的神经元分离出来。 

视频: Spinal Cord Neurons Isolation and Culture from Neonatal Mice

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

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

侧向液压冲击:创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

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. ...

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 ...

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 ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

从产前Islmn:GFP转基因小鼠的奥库罗运动、特罗赫利尔和脊柱运动神经元的分离与培养

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

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 ...

脊髓神经元从新生小鼠中分离和培养

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

侧向液压冲击:创伤性脑损伤小鼠模型

从产前小鼠海马神经元的分离和培养

鸡胚胎脊髓片培养协议

分离和游离感觉神经元的文化活动从鸡胚

斑马鱼

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

头脊髓和薄片体外制备心律失常活性

从产前Isl^mn:GFP转基因小鼠的奥库罗运动、特罗赫利尔和脊柱运动神经元的分离与培养

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

脊髓神经元从新生小鼠中分离和培养

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

侧向液压冲击:创伤性脑损伤小鼠模型

从产前小鼠海马神经元的分离和培养

鸡胚胎脊髓片培养协议

分离和游离感觉神经元的文化活动从鸡胚

斑马鱼

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

头脊髓和薄片体外制备心律失常活性

从产前Islmn:GFP转基因小鼠的奥库罗运动、特罗赫利尔和脊柱运动神经元的分离与培养

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

从胚胎脊髓连合神经的解剖和文化

从产前Isl^mn:GFP转基因小鼠的奥库罗运动、特罗赫利尔和脊柱运动神经元的分离与培养