这项工作是由R01 NS052532（HLH），穆迪基金会，麻醉科。我们感谢劳加粗，恭Courteau编辑援助，巴特勒和克里斯蒂·佩里和克里斯蒂·佩里的所有图形，表格和艺术品的布局和精良的生产。

1。外科手术和闭合性脑外伤<ol><li>所有的动物实验是第一个被批准的实验动物护理和使用委员会的德克萨斯州医学科，加尔维斯顿，德克萨斯州和国家机构的健康指南大学实验动物护理和使用（第8版，美国国家研究委员会）。 </li><li>大鼠（成年雄性SD大鼠，400〜500克获得供应商查尔斯河，波特兰，缅因州），每笼都装了两个，并提供食物和水在动物饲养，自由采食这些不变的条件下：光周期（600小时到1800小时），温度（21°C至23°C）和湿度（40％〜50％）一个星期后才能使用。 </li><li>麻醉大鼠用4％的异氟醚，插管，机械通风（NEMI科学;新英格兰医学仪器，梅德韦，MA）1.5-2.0％异氟醚氧的作用：空气（70:30），并准备为矢状窦旁液压冲击损伤如先前所述。810 </li><li>在损伤后的适当的时间点取决于实验设计牺牲大鼠。快速消除大脑，立即冻结干冰，储存在-80°C在50毫升的试管或立即着手嵌入在OCT冰冻切片。 </li></ol> 2。大鼠脑切片和染色<ol><li>脑组织中，不立即使用，可以在-80℃下保持长达1个月如果保持在一个恒定的温度。大脑，冷冻于-80℃，然后解冻至-20℃的切片，然后再冻结不产生高质量的RNA后的第二解冻。一旦大脑解冻，OCT安装在切片，幻灯片应当在24小时之内染色的染色30分钟至一小时内用于LCM。这将确保优良的品质RNA。 LCM，LCM帽捕获细胞可以被存储在裂解缓冲液在-80°C至一个星期，但RNA应及时隔离，以确保最高的质量。/ P&gt; <li>在此之前的大脑切片，抹用RNase-ZAP和恒温器清洁刷ETOH（取代一次性刀片的大脑之间）。 </li><li>从-80℃的冰箱中，在50毫升的试管中的找回大脑，将其放置到低温恒温器中，在温度为-22℃，约10分钟，管解冻。消除大脑管和地方搬上了舞台上的纱布，腹部的一面朝上。 </li><li>用剃刀刀片，大脑切片取出大脑延髓小脑和前部的视交叉后部。填充一个cryomold用OCT安装介质（组织康），并与前侧向下放置到模具中的脑。让脑组织冻结在安装介质中，直到它变成白色（约10分钟）。 </li><li>冻结的样品盘（组织TEK）到大脑与华侨城。的大脑从模具中取出。将大脑标本头，拧紧螺丝。 </li><li>将一个disposa中BLE，低调刀片（Fisher Scientific）的进刀持有人，并拧紧杆向下。 </li><li>开始20μm的大脑切片除去外层的OCT。一旦海马区，设置转盘至10微米。收集冠状连续切片，置于载玻片上加载玻片上的组织切片（Fisher Scientific）的。的幻灯片被保存在-20°C，在一个无RNA酶染色架上，直到切片是完整的。 </li><li>要删除所有玻璃器皿核糖核酸酶从组织切片处理，擦拭所有染色容器和​​量筒E​​liminase（Fisher Scientific）的Milli Q水冲洗。无RNase水和过滤甲酚紫（Sigma-Aldrich公司）和氟翡翠台（组织化）染色用0.2微米过滤器在使用前准备好所有的解决方案。 </li><li>解冻后的脑切片在室温，持续30秒，并固定在75％乙醇中（1分钟）。 </li><li>后固定受伤的单神经元的LCM，冲洗幻灯片中无RNA酶的水（1分钟），染液用1％的甲酚紫（15-20秒），无RNase的水（2×30秒）中冲洗，染色剂与氟翡翠台（4分钟），冲洗在无RNase的水（3×1分钟），脱水95％ETOH由无RNase的水（30秒），100％的乙醇中（30秒），和二甲苯（2×3分钟）。 </li><li> LCM固定后神经元的大片，冲洗幻灯片中无RNA酶的水（1分钟），用1％的甲酚紫（1分钟）染色，在无RNase水冲洗（3×1分钟），脱水95％ ETOH（2×30秒），100％的乙醇中（2×30秒），和二甲苯（2×3分钟）。 </li><li>风干15分钟前，LCM在通风橱中的所有部分。幻灯片可以存储，节双方起来，在一个滑动箱内衬干燥剂，如果他们需要从一个房间到另一个房间。然而，最佳的结果得到的，如果LCM后立即进行部分干燥。 LCM应限于从30分钟至1小时。在接下来的部分，我们首先描述如何捕捉连续的细胞，这是EA的大片LTD制，SEAST对于那些正在学习这个技术的掌握。然后，我们将介绍如何精确地捕捉单个神经元。在我们的程序中，我们确定的亲和力氟玉的一个标志退化的神经元死亡的神经元。氟玉阴性细胞被推定为神经元存活。 </li></ol> 3。激光捕获显微切割（LCM） LCM丰富的海马神经元群体的大片<ol><li> LCM使用PixCell IIe的显微镜用红外二极管激光器（Life Technologies公司）进行。 </li><li>调整中心“操纵杆垂直位置。旋转和位置锁定目标到4倍。 </li><li>在显微镜载物台的中心放置的滑动，并手动调整的海马，直到区域处于视图。使用粗微调焦调整，使图像成为关注的焦点。 </li><li>按真空控制器上的按钮激活的真空吸盘。旋转并锁定目标为PL 10倍王牌。我们再关注一下粗，细调整。 </li><li>加载的CAPSURE宏LCM上限（Life Technologies公司）的CAPSURE盒模块和位置，盖在载重线的位置。旋转各地的帽帽放置手臂和位置。提高上限配售臂，到删除的CAPSURE的上限从纸盒模块。 </li><li>旋转帽放置手臂的幻灯片，并降低在滑动的手臂，这将放置在幻灯片上的帽。调整微调对焦和移动操纵杆检查，如果细胞里面的黑色圆帽上。所有的细胞捕获在这个黑圈。 </li><li>设定了激光器的参数。首先，按激光使控制器上的按钮。激光目标点，将显示在计算机监视器上的视图中的字段，为粉红色圆点。 </li><li>设置激光光斑尺寸小（7.5微米），聚焦激光和调整的功率和持续时间为65 - 75毫瓦，如所需的最佳的细胞捕获为1.0-2.0毫秒。 </li><li>到TEST的激光，使用操纵杆移动的激光光斑面积有没有回升的细胞。火焰使用拇指开关激光。使用操纵杆移动的激光光斑从熔融的聚合物现货和检查，看看如果一个可见的暗环围绕着一个明确的区域，在激光发射。 </li><li>要捕获细胞，使用操纵杆移动的激光光斑区域的细胞捕捉。火焰使用拇指开关激光。发射激光时，捕捉到了大面积的细胞移动操纵杆。当激光发射时，它熔化的热塑性聚合物膜的靶细胞的表面上的帽。该聚合物吸收的激光辐射，从而不改变的RNA，DNA或蛋白质为未来的分子的应用确保样品的完整性。 </li><li>烧成后所有细胞，抬起上限配售手臂。捕获细胞从组织切片中分离出来，和坚持到CAPSURE帽。将剩余的组织和失踪细胞对的视场IBLE在。帽上的细胞也可以被看作通过将一个空的滑动部的上限。 </li><li>提起盖放置手臂，将其旋转到上限卸载站。放下帽注入站和旋转盖放置手臂回到它以前的位置。 </li><li>沿平台上的帽将CAPSURE插入工具。提起工具的平台。该上限将保持连接。轻轻触碰帽到的CAPSURE的清洁垫，清理掉不需要的组织。 </li><li>放置到0.5毫升的无RNase的填充有从RNAqueous微分离试剂盒得到的100μl的裂解缓冲液的管帽。立即涡流的上限为30秒，以溶解细胞。孵育样品在42℃下30分钟，并冷冻在-80℃下，直到RNA分离。 </li></ol> LCM单FJ +海马神经元<ol start="15"><li>为了捕捉单个细胞，加载CAPSURE HS LCM帽的CAPSURE盒模块和positi在载重线位置的上限。旋转瓶盖放置手臂，并把它周围的上限。提高上限配售臂，到删除的CAPSURE的上限从纸盒模块。 </li><li>设定了激光器的光点尺寸小（7.5微米）的激光聚焦，并调整激光功率和持续时间至65 -75毫瓦为0.45〜0.50毫秒为单细胞的最佳的细胞捕获。 </li><li>旋转帽放置手臂的幻灯片和降低手臂向下的幻灯片。这将会把盖到幻灯片上。在LCM，坐在提升CAPSURE HS盖表面的样品。 </li><li>使用操纵杆移动的激光单FJ +细胞。所有被捕获的细胞必须是里面的小黑色上的套环。火焰使用拇指开关激光。 </li><li>当所有细胞内的黑圈面积已发射的激光，抬起上限配售手臂。捕获细胞从组织切片中分离出来，坚持以的CAPSURE HS帽。 </li><li>将另一张幻灯片上的微应对阶段，并收集FJ +细胞，直到HS盖全。帽放置到0.5毫升的无RNase的管40-50微升的裂解缓冲液充满。立即涡流的上限为30秒，以溶解细胞。孵育样品在42℃下30分钟，并冷冻在-80℃下，直到RNA分离。 </li></ol> 4。 LCM样品的总RNA提取（这部分只是在视频演示） <ol><li>从细胞中分离总RNA，使用RNAqueous Micro试剂盒（Ambion公司），按照制造商的协议。本试剂盒中的所有试剂和清洗解决方案。预湿的微过滤器盒组件，通过添加30微升溶液的裂解缓冲液的过滤器。 5分钟后，以10,000×g，30秒，离心处理，以使过滤器。 </li><li> LCM添加剂的样品裂解液中加入3μl，吸液混合，和离心机。 </li><li>加入52μl的100％乙醇中（1/2体积）的样品裂解液;移液管混合，溶胞产物转移到过滤器列。 </li><li>以10,000×g离心过滤器柱1分钟，绑定到列的RNA。如果有一个样本的多个上限，旋转每个溶胞产物分别通过相同的列。 </li><li>完成RNA分离过程中所描述的RNAqueous微型套件。 </li><li>执行DNase处理和DNase失活的LCM RNA的试剂盒中所描述的样品，将RNA转移到无RNase的管和存储在-80℃。 </li></ol> 5。 RNA的定量和下游应用 RNA的质量和量化评估的安捷伦生物分析仪用试剂由Pico套件（安捷伦科技公司，加利福尼亚州，圣克拉拉市）按照制造商的协议。 <ol><li>在Agilent 2100生物分析仪（Agilent Technologies）中的总RNA进行分析。该软件的计算结果的浓度和通过分配的RNA完整性号（RIN），向每个样品范围f的RNA样品的完整性rom的1至10个。 1-5表示降解的RNA和7-10表示质量好的RNA。 </li><li>非退化的完整RNA将被用于实时荧光定量PCR;个人分析或RT2分析器的阵列，使用SYBR绿色化学。的RNA也可以用于mRNA的微阵列分析和microRNA微阵列分析。 </li></ol>

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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=The+potential+use+of+laser+capture+microdissection+to+selectively+obtain+distinct+populations+of+cells+for+proteomic+analysis--+preliminary+findings.">The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis-- preliminary findings.</a> Electrophoresis. 20 (4-5), 689-700 (1999).</li><li>Fend, F., Emmert-Buck, M. 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=Immuno-LCM:+laser+capture+microdissection+of+immunostained+frozen+sections+for+mRNA+analysis.">Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.</a> Am. J. Pathol. 154 (1), 61-66 (1999).</li></ol>

作者宣称，他们有没有利益冲突。

LCM技术使我们能够理解的分子机制TBI显着的进步。8,10,11,12,13，我们是第一个利用这种技术的调查表明，不同次区域的大鼠海马基因的表达谱，与选择性的脆弱性损伤。我们的研究表明，我们可以量化基因的表达，通过qPCR，从少至10个激光捕获的细胞，并且我们可以执行从少600捕获的细胞的全基因组微阵列分析。 LCM将是一个宝贵的工具，在被牵连在不同的神经学和神经精神障碍，被称为大脑的其他地区的类似的研究。例如，使用LCM的研究已经揭示出在，14的黑质多巴胺神经元，帕金森氏病的发病机制，并资助的全基因组图谱研究牵连s中的奖赏回路中的伏隔核，这是涉及ubstance滥用障碍。 神经细胞的异质性是体现在基因组水平16，并可能导致缺乏成功的实验性治疗脑外伤的临床试验。因此，我们的目标是利用这种技术，研究创伤性脑损伤后神经元的存活影响的关键要素。我们最近的全基因组分析研究死亡和相邻的海马神经元存活的创伤性脑损伤后颅脑外伤后，细胞变阻器，反映了细胞死亡基因的表达水平的细胞存活比率调节细胞的命运。这些正在进行的研究将有助于设计和开发的药物治疗策略，可以产生积极的影响细胞的存活变阻器。此外，我们目前使用的LCM进行跟踪和监视的潜在治疗药物治疗创伤性脑损伤后的海马神经元的影响。因此，我们的研究表明，审慎无RNA酶处理技术和一些修改现有的协议，它是可以从LCM准确的定量基因表达分析，获得高品质的RNA样品。 然而，也有带LCM技术相关联的几个陷阱。例如，只收集神经细胞，没有任何小胶质细胞的污染几乎是不可能的。在海马锥体细胞层中，它已被估计有95％的细胞是神经元与锥体细胞和10％的interneurons这一人口的90％，留下一个小胶质和其它类型的细胞的百分比。8,17， 18在我们的研究中，我们使用氟玉的荧光染料，标签只受伤的神经元在大脑组织。一个不同的染色是一个标记为GFAP将是必要的小胶质细胞进行染色。19收集只有氟玉染色的单神经元机构确保更均匀的人口的神经元。另一个常见的​​问题，可能需要排除故障是一个圆圈是不T定义当激光发射。如果发生这种情况，它是必要的检查激光设置和必要的调整的功率和持续时间。还有一点很重要，以确保帽平放在对组织和正确地放置在手臂上。在谈到的LCM技术手册或致电技术支持有时是必要的。 LCM和RNA分离的RNA的质量应该始终使用生物分析仪之前，任何基因表达分析评估。 目前市场上几种类型的激光捕获工具包括激光切割（Life Technologies公司，徕卡显微系统）和激光弹射（蔡司）的仪器。我们的LCM系统的工作原理，以及用于捕捉小的细胞数。其他高吞吐量和更自动化的系统可能是更适合用于获得较大数量的细胞的基因组，特别是蛋白质组分析。事实上，LCM使用这些自动化系统具有很大的潜力进行分析的公关otein在确定细胞或富集的细胞群中的表达。20此外，液晶显示模块可以方便immunolabeled细胞，21允许我们调查定义的类型的细胞中的基因表达的复杂性无关的最异类的组织中的基因表达分析的。因此，LCM是单一或富集的细胞群的尖端分子生物学研究的一个很好的工具。

异质性组织的基因表达分析一直是有问题的，这是特别真实的哺乳动物大脑中，其中有大约5000种不同类型的细胞。激光捕获显微切割（LCM）技术，基因组学研究的影响TBI的发展前在体内在混合组成脑细胞的人口，不仅不同的神经细胞类型，也支持神经胶质细胞和免疫调节细胞基因表达分析的基础上。由此产生的复杂从这些异构组织获得的基因的表达谱，和损伤诱导的细胞信号，经常相互冲突的模式，可照射。5在临床前研究中证明是有效的治疗策略在人体临床试验的失败是一个解释为了得到清晰的了解弱势群体的神经元损伤诱导的基因表达在大鼠髋部pocampus，我们采用的LCM技术，埃默特降压等人首次报道。随后，我们修改和优化，这丰富的神经元和单个神经元的基因分析，采用实时定量PCR和基因芯片分析人口有效地捕获显微切割技术。为了保持完整的mRNA在脑组织冰冻切片进行基因组分析，我们修改了现有的协议从冷冻鼠脑切片神经元的固定，染色和快速捕获。对于受伤和死亡的海马神经细胞的识别和分离，我们还优化了氟玉染色技术LCM。氟玉不区分细胞凋亡和坏死的细胞死亡。因此，所有类型的退化的神经元可以检测到这个污点6,7。 在这里，我们描述了协议，用于在我们的实验室获得单一的死亡或存活的神经元池以及大片的丰富populati附加不同的神经细胞类型（ 即 CA1-CA3神经元的基因表达分析，TBI后）。岛村等人 ，8中详细描述在我们的实验室中执行的程序的流体冲击脑损伤是非常相似的横向的流体冲击损伤协议JOVE阿尔德等发表在小鼠。9由于LCM技术具有被证明具有极小或没有影响，对DNA的完整性，在组织中的RNA和蛋白质，这是一个极好的工具定义的类型的细胞的分子和蛋白质分析。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td></td>
 </tr>
 <tr>
 <td>PixCell IIe Laser Capture Microscope</td>
 <td>Life Technologies (Arcturus)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Agilent 2100 Bioanalyzer</td>
 <td>Agilent Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>RNAqueous Micro- Kit</td>
 <td>Life Technologies (Ambion)</td>
 <td>AM1931 </td>
 <td></td>
 </tr>
 <tr>
 <td>Agilent 6000 Pico Kit</td>
 <td>Agilent Technologies</td>
 <td>5067-1513</td>
 <td></td>
 </tr>
 <tr>
 <td>Capsure LCM caps</td>
 <td>Applied Biosystems</td>
 <td>LCM0211/LCM0214</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluoro-Jade</td>
 <td>Histo-chem Inc.</td>
 <td>#1FJ</td>
 <td></td>
 </tr>
 <tr>
 <td>Cresyl Violet Acetate</td>
 <td>Sigma-aldrich</td>
 <td>C5042-10G</td>
 <td></td>
 </tr>
 <tr>
 <td>Xylene (Histological grade)</td>
 <td>Fisher Scientific</td>
 <td>X3P-1GAL</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol 200 proof (Histological grade)</td>
 <td>UTMB pharmacy</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>RNase free barrier pipette tips</td>
 <td>Fisher Scientific</td>
 <td>2123635, 212364, 212361, 21-236-2A</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td><a href="http://tools.invitrogen.com/content/sfs/brochures/cms_086283.pdf" target="_blank">Arcturus Laser Capture Microdissection system (Life Technologies)</a> 
 <a href="http://microscopy.zeiss.com/microscopy/en_de/downloads/brochure-downloads.html?catalog=laser_microdissection" target="_blank">Zeiss Laser Microdissection The PALM family </a> 
 <a href="http://www.leica-microsystems.com/fileadmin/downloads/Leica DM6000 B/Brochures/Leica_DM4000-6000_B-Brochure_en.pdf" target="_blank">Leica Microsystems</a></td>
 </tr>
 </tbody>
</table>

laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury

长期创伤性脑损伤后认知障碍与海马区域在内侧颞叶是至关重要的学习，记忆和执行功能的损伤引起的神经退行性疾病。1,2因此，我们的研究集中于特定的神经元的基因表达分析人口在不同次区域的海马状突起。埃默特降压，在1996年推出的激光捕获显微切割技术（LCM） 等 。，允许单细胞基因表达分析的显着进步，丰富了细胞的异质性组织，如哺乳动物的大脑包含的种群数以千计的功能的细胞类型。我们使用LCM和模型的建立大鼠创伤性脑损伤（TBI）调查所依据的脑外伤发病的分子机制。液压冲击脑外伤，大脑在预先确定的时间伤后取出，立即用干冰冷冻，并准备切片在低温恒温器。老鼠的大脑可以嵌入立即在OCT和切片，或存储的数个月在-80°C激光捕获显微切割前切片。此外，我们使用LCM研究TBI昼夜节律的影响。对于这一点，我们捕捉包含的主时钟的哺乳动物的大脑的视交叉上核神经元。在这里，我们演示了如何使用实时PCR和/ LCM获得单确定神经元（受伤和退化，氟玉阳性，或没有受伤，氟玉负）和丰富的人口为后续基因表达分析的海马神经元全基因组芯片。这些LCM功能的研究表明，解剖学上的不同区域的大鼠海马神经元选择性的脆弱性体现在不同的基因表达图谱LCM从这些不同的区域不同人群的神经元。我们的单细胞研究，其中的结果我们比较死亡和相邻的生存海马神经元的转录图谱表明，存在着调节细胞死亡和生存，创伤性脑损伤后的细胞存活变阻器的。

在我们的第一个LCM研究，我们能够表明，功能不同的次区域（大鼠海马CA1和CA3）基因的表达谱，反映他们的脆弱性TBI。 图 1A-1C激光捕获海马锥体神经元（CA1 CA3），齿状回神经元和SCN神经元前，后LCM。清洁捕获的锥体，颗粒剂或SCN神经元，分别，示上宏上限。 图2A-2B说明了死亡，氟翡翠台阳性锥体神经元存活，氟翡翠台负锥体神经元之前和之后LCM。通过查看捕获的细胞的LCM帽上的清洁捕获的死亡或存活的神经元。 LCM后，总RNA可用于多样化的分子生物学研究，包括基因表达的实时定量PCR分析使用TaqMan或SYBR绿色化学品（ 图3A），小focused的PCR阵列与SYBR绿色探头（ 图3B）或全基因组微阵列技术的研究（ 图3C）。 <img alt="图1" fo:content-width="3in" fo:src="/files/ftp_upload/50308/50308fig1large.jpg" src="/files/ftp_upload/50308/50308fig1.jpg" /> 图1。激光捕获显微切割的大片定义的大鼠脑细胞群。乙醇固定在冷冻10微米的大鼠脑冠状切片，染色，用尼氏染色（甲酚紫）和LCM准备。图中显示的图片前后LCM捕获的细胞可视化的宏上限。A.从CA1-CA3子域的大鼠海马锥体神经元。海马齿状回颗粒细胞的大鼠海马和视交叉上核。 C.双边视交叉上核位于任一侧上的第三脑室和位于视交叉上方。 <a href="https://www.jove.com/files/ftp_upload/50308/50308fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="3in" fo:src="/files/ftp_upload/50308/50308fig2large.jpg" src="/files/ftp_upload/50308/50308fig2.jpg" /> 图2。激光捕获显微切割的大鼠海马神经元单显示的是A.退化，氟玉（彩色）海马CA3区神经元B.尚存，氟玉阴性（未染色）前后LCM CA3神经元。捕获细胞的可视化。 <a href="https://www.jove.com/files/ftp_upload/50308/50308fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3" fo:content-width="6.5in" fo:src="/files/ftp_upload/50308/50308fig3large.jpg" src=“机构/ files/ftp_upload/50308/50308fig3.jpg”/&gt; 图3。激光捕获的神经元的总RNA的基因表达分析。A.定量实时PCR的生物钟基因表达数据的SCN。在死亡和生存的海马神经元凋亡相关基因的表达，使用集中的PCR阵列基因表达B.热图C.安捷伦全基因组芯片分析基因表达的海马CA1-CA3神经元的大鼠接受假损伤，脑外伤或脑外伤，加上重组腺相关设计的siRNA病毒引起的脑损伤（神经型一氧化氮敲除基因的表达合成酶和谷胱甘肽过氧化物酶1）。 <a href="https://www.jove.com/files/ftp_upload/50308/50308fig3large.jpg" target="_blank">点击此处查看大图</a> 。

Watch this Scientific Journal Video about Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury at JoVE.com

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

我们描述了如何使用激光捕获显微切割（LCM），以获得丰富的海马神经元群体或单个神经元，随后采用实时定量PCR和/或整个基因组微阵列基因表达分析的损伤大鼠的大脑冰冻切片。

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

laser-capture-microdissection-enriched-populations-neurons-or-single

Research

JoVE Journal

Neuroscience

21.2K 次观看.  University of Texas Medical Branch. 我们描述了如何使用激光捕获显微切割（LCM），以获得丰富的海马神经元群体或单个神经元，随后采用实时定量PCR和/或整个基因组微阵列基因表达分析的损伤大鼠的大脑冰冻切片。

视频: Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

通过鼻腔活检获得联合激光捕获显微切割的嗅觉神经元：一种可能的方法来研究治疗反应的精神障碍

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.