Supported by NIH Grants NS14837, R01 NS38537, R24 HD050838 to Dr. Michael E. Selzer; Shriners Research Grant SHC-85220 to Dr. Michael E Selzer; and Shriners Research Grant SHC-85310 to Dr. Michael I. Shifman. Dr. Ant&#243;n Barreiro-Iglesias was supported by the Fundaci&#243;n Barri&#233; (Spain) and the Xunta de Galicia (Galicia, Spain).

表1为在本协议中使用的所有材料。 实验获得批准的机构动物护理和使用委员会在坦普尔大学。 1，动物<ol><li>获得野生型幼虫海七鳃鳗（Petromyzon马里努斯 L.），10 - 23厘米的长度-从流喂养密歇根湖，从支流到特拉华河（宾夕法尼亚州），或者流于缅因州（4 7岁）。 </li><li>在实验室中，幼虫保持在50组 - 100动物50加仑淡水坦克在16℃。符合碎石英寸坦克的幼虫在挖洞，水是木炭过滤，空调与空气的石头至少48小时将七鳃鳗在他们之前，去除余氯等杂质。没有必要对喂动物，其中回收自己的碎屑，最多使用的当天。 </li></ol> 2，完全性横断<ol><li>脊髓手术前准备有以下组成的七鳃鳗林格液:110 mM氯化钠，2.1 mM的氯化钾，2.6毫米氯化钙2，1.8毫米氯化镁2和10毫米的Tris缓冲液; pH值7.4。 </li><li>要执行脊髓横断，将幼虫在一个小容器进行麻醉，在冰冷的林格液1％三卡因甲磺酸盐。 </li><li>一旦麻醉的地方在陪替氏培养皿的一半填充的Sylgard（聚硅氧烷）的动物，并使用4个昆虫针（0.15毫米直径），以保持在位置上的动物的手术（一个被放置在动物的口鼻部，一个在尾和两个在第 5 次鳃的水平）。填补了培养皿中几乎达到了林格液的顶部。放菜的幼虫上的冰而进行体视显微镜下手术。 </li><li>要执行脊髓横断背，中线，作一纵切口，用手术刀（＃11）在第 5 次的水平鳃形象化脊髓。最初的皮肤是通过将手术刀刀片的尖端朝上，然后将肌肉组织被切得很平缓，直至脊髓从上面可以看到的开放。时尚的昆虫针两个挂钩，并用它们来保持身体的墙壁打开，而执行脊髓横断。 </li><li>用一把剪刀（Castroviejo＃8）完全横切在第 5 次鳃水平脊髓，垂直插入剪刀脊髓。脊髓已被完全地与一个干净的切割横断。可视化的体视显微镜下脊髓切割端，以确认该脊髓已被完全切断。然后，通过使用一对镊子的（＃5）重新对准脊髓的切断端部的横断后。 </li><li>在进行脊髓横断关闭背侧体壁的伤口有一对细钳子并放置冰上幼虫1小时，以允许伤口吨后Ø干。放一张滤纸冰和幼虫间和七鳃鳗林格液浸泡。低温保存的动物活的同时，它使伤口空气干燥，因此，有助于防止感染。 </li><li>然后转移幼虫小坦克（每罐1幼虫）用冰冷的水24小时。 </li><li>脊髓横断后检查损毁幼虫1天到行为上确认，没有任何动静尾椎受伤的部位。脊髓横断被认为是完整的，如果幼虫能只移动其头部吻端到损伤部位（在第 5 次鳃的水平）。如果是这种情况的幼虫现在可传送到罐（每罐1幼虫）与淡水，在室温（23 ℃），使它们恢复为所选定的时间点。 </li><li>在恢复期间换水，每2至3天。 </li></ol> 3，脑解剖和制备脑原位杂交<ol><li>在受伤后的所需时间点重新麻醉幼虫海七鳃鳗和地点和引脚他们在陪替氏培养皿用林格氏溶液来进行脑解剖如上。 </li><li>解剖大脑了，先作一横切口的鼻孔和松果体器官之间的手术刀。从这里开始利用一对Castroviejo剪刀，同时保持其与钳子切割周围的脑组织。 </li><li>一旦脑露出，通过使用一对镊子取出脉络丛和使用钩子来传播该动物的头部和暴露的脑神经。然后，切断脊髓横向与Castroviejo剪刀。而使用其他钳子切颅神经解剖大脑出了钳子抓住脊髓。 </li><li>解剖后，把大脑的一小块硅胶并保持在适当的位置插入2项等销嗅球和1脊髓。然后，切断后并沿背中线与剪刀的大脑cerebrotectal合缝和偏转翼片横向和管脚他们平与4昆虫针（ 图2）的聚硅氧烷。 </li><li>用脑转移片硅氧烷与含在磷酸缓冲盐溶液4％多聚甲醛（pH 7.4）中而固定，在室温北部沿海地区为3小时，在旋转器上的管。 </li><li>固定后在原位杂交用脑的wholemount如前所述（Swain 等人，1994）。从控制或假手术动物的大脑，应始终平行于从受伤的动物的大脑运行。该wholemounted幼虫脑可以通过原位杂交，因为也由于没有髓鞘10，这使得大脑相当半透明其平而薄的形状并且被研究。 </li></ol>

<ol>
	<li>Rodicio, M. C., Barreiro-Iglesias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lampreys+as+an+animal+model+in+regeneration+studies+after+spinal+cord+injury.">Lampreys as an animal model in regeneration studies after spinal cord injury.</a> Rev Neurol. 55, 157-166 (2012).</li><li>Davis, G. R., McClellan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+and+time+course+of+restoration+of+descending+brainstem+projections+in+spinalcord-transected+lamprey.">Extent and time course of restoration of descending brainstem projections in spinalcord-transected lamprey.</a> J Comp Neurol. 344, 65-82 (1994).</li><li>Jacobs, A. J., Swain, G. P., Snedeker, J. A., Pijak, D. S., Gladstone, L. J., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+neurofilament+expression+selectively+in+regenerating+reticulospinal+neurons.">Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.</a> J Neurosci. 17, 5206-5220 (1997).</li><li>Shifman, M. I., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+netrin+receptor+UNC-5+in+lamprey+brain+modulation+by+spinal+cord+transection.">Expression of the netrin receptor UNC-5 in lamprey brain modulation by spinal cord transection.</a> Neurorehabil Neural Repair. 14, 49-58 (2000).</li><li>Barreiro-Iglesias, A., Laramore, C., Shifman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sea+lamprey+UNC5+receptors+cDNA+cloning,+phylogenetic+analysis+and+expression+in+reticulospinal+neurons+at+larval+and+adult+stages+of+development.">The sea lamprey UNC5 receptors cDNA cloning, phylogenetic analysis and expression in reticulospinal neurons at larval and adult stages of development.</a> J Comp Neurol. 520, 4141-4156 (2012).</li><li>Shifman, M. I., Yumu, l. R. E., Laramore, C., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+repulsive+guidance+molecule+RGM+and+its+receptor+neogenin+after+spinal+cord+injury+in+sea+lamprey.">Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey.</a> Exp Neurol. 217, 242-251 (2009).</li><li>Busch, D. J., Morgan, 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=Synuclein+accumulation+is+associated+with+cell-specific+neuronal+death+after+spinal+cord+injury.">Synuclein accumulation is associated with cell-specific neuronal death after spinal cord injury.</a> J Comp Neurol. 520, 1751-1771 (2012).</li><li>Shifman, M. I., Zhang, G., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+death+of+identified+reticulospinal+neurons+after+spinal+cord+injury+in+lampreys.">Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys.</a> J Comp Neurol. 510, 269-282 (2008).</li><li>Swain, G. P., Jacobs, A. J., Frei, E., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+in+situ+hybridization+in+wholemounted+lamprey+brain+neurofilament+expression+in+larvae+and+adults.">A method for in situ hybridization in wholemounted lamprey brain neurofilament expression in larvae and adults.</a> Exp. Neurol. 126, 256-269 (1994).</li><li>Bullock, T. H., Moore, J. K., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+myelin+sheaths:+both+lamprey+and+hagfish+lack+myelin.">Evolution of myelin sheaths: both lamprey and hagfish lack myelin.</a> Neurosci Lett. 48, 145-148 (1984).</li><li>Cohen, A. H., Kiemel, T., Pate, V., Blinder, J., Guan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+can+alter+the+function+outcome+of+spinal+cord+regeneration+in+larval+lampreys.">Temperature can alter the function outcome of spinal cord regeneration in larval lampreys.</a> Neuroscience. 90, 957-965 (1999).</li></ol>

The authors declare that they have no competing financial interests.

在这里，我们提出了一个详细的协议来执行一个完整的脊髓横断，后脑部解剖幼虫海七鳃鳗。这个程序允许通过全贴脑原位杂交的方法在脊髓损伤后可识别的脊髓神经元投射之间的基因表达差异分析。在该过程中的关键步骤是一个完整的脊髓横断的正确性能，从而可以通过观察stereomicrocope下脊髓的切割端24小时后，轻轻触摸幼虫的口鼻部具有一对被控制和确认钳子，以查看是否有任何移动尾椎到损伤部位。它也是用于动物的是，用于外科手术的剪刀不钝的恢复重要的，一个单一的和干净的切割是为了完全横切脊髓。 动物受伤后的生存高度通过保持阿尼姆改善在外科手术和用于使伤口风干，然后在冰冷的水中进行的第一个24小时的时间期间，在冰上ALS。在此期间后伤口闭合和动物可以在室温下已经被转移到容器用淡水。 幼虫七鳃鳗被允许恢复在室温下，而不是在他们一贯的冷温度，因为更大的大部分动物的伤害在较高温度下恢复后，完全自发的行为功能。11运动的正常模式的恢复是观察8〜10周后脊髓完全横断。 在选定的时间点幼虫大脑可以不仅处理执行完整脊髓横断和大脑解剖执行后如下所示整个安装原位杂交（ 图2），但也可用于其它组织学方法，如免疫组织化学（ 例如:7 </s向上&gt;），神经元示踪剂的检测，可以在横断的时间被加在脊髓损伤部位（ 例如 8）或活化的胱天蛋白酶通过使用荧光染料标记的胱天蛋白酶抑制剂的检测。5

在哺乳动物脊髓损伤（SCI）是一种破坏性的条件，导致的损伤部位以下功能永久性丧失，因为受伤的轴突不需要通过创伤区再生和重新连接到适当的目标。相反，哺乳动物，七鳃鳗恢复完全性脊髓损伤后运动。1有趣的是，七鳃鳗有一套36脊髓投射神经元是单独识别整个安装，因为他们的大尺寸2,3（ 图1）脑准备。所有这些脊髓投射神经元是由一个高层次的脊髓完全横断金黄地鼠。我们的组和其他的以前的研究已经表明，即使在功能恢复脊髓损伤后某些神经元表现出非常低的再生能力的存在（它们被认为是"坏的蓄热室"），而其他通常通过损伤部位再生的轴突（他们被认为是"GOOD再生"）。2,3这种特性使得七鳃鳗有趣脊椎动物模型来研究在好的和坏的再生脊髓投射神经元，这反过来会导致神经元的企图的固有再生能力的差异之间基因表达的差异再生的轴突在同一外在环境。1 利用该模型，我们以前曾表明，脊髓投射神经元的轴突与导向分子受体如UNC5 4,5和neogenin，6分别介导netrin诱导和RGM的抑制作用，低蓄热能力秀的表情。此外，通过使用这个方法，我们小组还表明，只有在良好的蓄热表明神经丝的表达的恢复的损伤后和在再生过程。最近，布希和摩根7表明，免疫荧光，坏再生器显示的increas突触核蛋白的表达主编受伤后，已经涉及了作者这一事实，即"坏再生"脊髓投射神经元脊髓完全横断5,7,8后慢慢死去。因此，一个完整的脊髓损伤的七鳃鳗的模型已经成为一个非常有用的模型，以了解是什么造就了脊髓神经元投射一个"坏再生"切断后。 进行我们的研究，我们在期望的时间点执行一个完整的脊髓横断手术协议和后脑部解剖损伤后原位杂交执行wholemount。在本方法的文章中，我们提出了一个详细的协议为完全性脊髓损伤手术在七鳃鳗幼体的正常性能，后续维护动物和最终大脑解剖和准备的大脑原位杂交wholemount。一份详细的协议，PErform原位杂交的wholemount在幼虫七鳃鳗的大脑已经被报告过。9另外，该协议用 ​​于脊髓损伤和脑解剖也可用于再加工的大脑用于免疫组化分析或其他组织学方法。

<table border="0" cellpadding="0" cellspacing="0" width="280">
	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Name of the reagent</td>
			<td style="width:83px;">Company</td>
			<td style="width:64px;">Catalogue number</td>
			<td style="width:71px;">Comments (optional)</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:64px;">Tricaine methane sulfonate</td>
			<td style="width:83px;">Spectrum</td>
			<td style="width:64px;">TR108</td>
			<td style="width:71px;">Benzocaine saturated solution in PBS for sacrifice</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Scalpel #3</td>
			<td style="width:83px;">Fine Science Tools (FST)</td>
			<td style="width:64px;">10003-12</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Blades for scalpel: #11</td>
			<td style="width:83px;">Fine Science Tools&#160;</td>
			<td style="width:64px;">10011-00</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Castroviejo scissors #8</td>
			<td style="width:83px;">Fine Science Tools&#160;</td>
			<td style="width:64px;">15002-08</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:64px;">Forceps #4 &#38; #5</td>
			<td style="width:83px;">Dumont, Switzerland</td>
			<td style="width:64px;">Roboz RS4955</td>
			<td style="width:71px;">#4 for dissection of Spinal cord; #5 for stripping menninges</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Dissecting Microscope</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:64px;">SZ51</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="42">
			<td height="63" rowspan="2" style="height:63px;width:64px;">Sylgard</td>
			<td rowspan="2" style="width:83px;">Dow Corning Co.</td>
			<td rowspan="2" style="width:64px;">184</td>
			<td rowspan="2" style="width:71px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="168">
			<td height="189" rowspan="2" style="height:189px;width:64px;">Insect pins 0.15, 0.20 mm</td>
			<td rowspan="2" style="width:83px;">Austerlitz</td>
			<td rowspan="2" style="width:64px;">No catalogue #</td>
			<td rowspan="2" style="width:71px;">0.15 mm for pinning brain and spinal cord; 0.20 mm for the body</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:64px;">7 ml HDPE Scintillation Tubes with Caps</td>
			<td style="width:83px;">Fisher Scientific</td>
			<td style="width:64px;">03-337-1</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:64px;">Paraformaldehyde 16%</td>
			<td style="width:83px;">Electron Microscopy Science (EMS)</td>
			<td style="width:64px;">19210</td>
			<td style="width:71px;">Dilute to 4% in PBS</td>
		</tr>
	</tbody>
</table>

complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey

经过完全性脊髓损伤，海七鳃鳗在第一瘫痪横断以下的水平。然而，它们恢复运动数周之后，与此相伴的propriospinal轴突和从脑干脊髓伸出的轴突的短距离再生（几个毫米）。其中36个大型识别脊髓投射神经元，有些是很好的再生等是坏的再生。这些神经元可最容易在wholemount CNS制剂来鉴定。为了了解在有利于神经元内在机制或在脊椎动物中枢神经系统损伤后抑制轴突再生，我们确定好坏再生器之间的差异的基因表达，以及如何表达由脊髓横断影响。本文阐述了在淡水舱的技术外壳幼虫和近期改造成海七鳃鳗，产下的显微视觉完全性脊髓横断，并准备BR泉和脊髓wholemounts用于原位杂交。简要地，将动物保持在16 &#160; °C和麻醉的1％苯佐卡因的七鳃鳗铃声。脊髓经由背侧入横断与虹膜切除术剪刀和该动物被允许在淡水罐在23℃下进行恢复。用于原位杂交，将动物再次麻醉，并在大脑和脊髓经由背侧的方法除去。

作为可使用这种方法时得到的结果的一个例子，wholemounted大脑的表示控制的可识别的脊髓投射神经元和2周neogenin转录物的表达的代表性图像受损后幼虫海七鳃鳗示于图2。的读者可以参考先前的研究报告6完全脊髓横断和识别脊髓投射海七鳃鳗的神经元的全部细节再生能力后neogenin的表达之间的关系。简要地说，结果表明，在受伤后有变化，在识别脊髓投射神经元的neogenin受体的表达。一个完整的脊髓横断后neogenin受体是优先表达在恶劣的蓄热，如M1，I1，I2，I4，或第M个神经元（参见图2B），这些结果证明了该结果两周的协议和表明可参与这些细胞的再生失败的neogenin受体。 <img alt="图1" fo:content-width="3in" src="/files/ftp_upload/51494/51494fig1highres.jpg" width="300" /> 图1:海七鳃鳗的大脑识别出神经元网状脊髓的位置的背视图的示意图 。从巴雷罗-伊格莱西亚斯和Shifman。5转载有关缩写，见下表。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51494/51494fig2highres.jpg" width="500" /> 图2显微照片的成熟幼虫海七鳃鳗的脑背示出了neogenin成绩单的对照组（A）的表达，2周后损伤（B）的动物。注意在B中受伤的后neogenin转录物是优先表达在已知是不良再生神经元（ 例如 ，M1，I1，I2，I4和第M个神经元）。此外，请注意所使用的保持大脑（箭头）的销离开孔的存在。的读者可以参考以前的研究Shifman和同事。6对于缩写，见下表。 缩略语 <table border="0" cellpadding="0" cellspacing="0" width="606"><tbody><tr height="20"><td height="20" style="height:20px;width:123px;"> B（B1-B6） </td><td style="width:484px;">延髓区域Müller细胞（中rhombencephalic网状核） </td></tr><tr height="20"><td height="20" style="height:20px;"> hab.-PED。 TR。 </td><td> habenulopeduncular道</td></tr><tr height="20"><td height="20" style="height:20px;"> I1-I6 </td><td>峡区Müller细胞</td></tr><tr height="20"><td height="20" style="height:20px;"> í点¯x </td><td>舌咽运动核</td></tr><tr height="20"><td height="20" style="height:20px;"> inf文件。 </td><td>漏斗</td></tr><tr height="20"><td height="20" style="height:20px;"> ISTH。网状细胞。 </td><td>峡部裂网状结构</td></tr><tr height="20"><td height="20" style="height:20px;"> M1-M4 </td><td> Müller细胞1至4 </td></tr><tr height="20"><td height="20" style="height:20px;">个月</td><td>的Mauthner细胞</td></tr><tr height="20"><td height="20" style="height:20px;"> MTH" </td><td>辅助的Mauthner细胞</td></tr><tr height="20"><td height="20" style="height:20px;"> SMI </td><td>沟medianus劣</td></tr><tr height="20"><td height="20" style="height:20px;"> VM </td><td>三叉神经运动核</td></tr><tr height="20"><td height="20" style="height:20px;">点¯x </td><td>迷走神经运动核</td></tr></tbody></table>

Watch this Scientific Journal Video about Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey at JoVE.com

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

七鳃鳗完整的脊髓损伤后恢复运动。然而，一些脊髓投射神经元是很好的再生和其他人都没有。本文说明了技术的外壳海七鳃鳗幼体（最近转化成人），产生完全性脊髓横断和原位杂交准备wholemount大脑和脊髓的。

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

complete-spinal-cord-injury-brain-dissection-protocol-for-subsequent

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

9.8K 次观看.  University of Edinburgh. 七鳃鳗完整的脊髓损伤后恢复运动。然而，一些脊髓投射神经元是很好的再生和其他人都没有。本文说明了技术的外壳海七鳃鳗幼体（最近转化成人），产生完全性脊髓横断和原位杂交准备wholemount大脑和脊髓的。 

视频: Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

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 ...

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, ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

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 ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

在体内挫伤脊髓损伤后影像血管动力学的双色法

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

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 ...

完全性脊髓损伤和脑解剖协议后续Wholemount

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

双荧光原位杂交食脑切片

侧向液压冲击:创伤性脑损伤小鼠模型

鸡胚胎脊髓片培养协议

在斑马鱼幼虫脊髓横断

一个

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

在体内挫伤脊髓损伤后影像血管动力学的双色法

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

完全性脊髓损伤和脑解剖协议后续Wholemount

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

双荧光原位杂交食脑切片

侧向液压冲击:创伤性脑损伤小鼠模型

鸡胚胎脊髓片培养协议

在斑马鱼幼虫脊髓横断

一个

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

在体内挫伤脊髓损伤后影像血管动力学的双色法

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