我们要感谢伊丽莎白的帮助编辑这份手稿。该项目的资金部分由穆迪项目的转化性颅脑损伤研究和 RO1NS052532 HLH。

注: 所有动物实验均由德克萨斯大学医疗科的机构动物保育和使用委员会批准, 由美国国立卫生研究院指导, 负责护理和使用实验动物 (第八版, 国家研究委员会). 
 1. 组织集合、区域标识和切片 <ol> <li> 主题成人, 男性, 大鼠, 大约六周老和 250-300 g, 实验性液体撞击伤, 如岛 et al. 所述 4 . </li> <li> 在实验性脑外伤后二十四小时, 人道地安乐动物, 把它们放在一个有异氟醚浓度4% 的房间里, 直到被麻醉。通过斩首确保死亡, 切除大脑, 并立即冻结在粉状干冰。将冷冻大脑储存在-80 #176; C 冷冻直到切片. </li>
	<li> 在任何 LCM 程序之前, 包括转录分析, 清洁所有表面与核糖核酸消除洗涤剂, 以减轻污染和 RNA 降解的风险. 
	注意: 此清洗包括用于染色的区域、组织切片的低温以及 LCM 设备周围的区域. </li>
	<li> 将脑组织从储存和放置到低温冷却到-20 和 #176; c. 允许大脑平衡低温室的温度为 ~ 10 分钟. </li>
	<li> 将大脑腹侧放在低温舞台顶部的纱布片上。用干净的, 无核糖核酸刀片, 切断后部只是侧的小脑 (可以保存或丢弃) 和部分只是前面的光学交叉 (och). </li>
	<li> 将少量的最佳切削温度 (OCT) 介质放入两个单独的 cryomolds。将大脑 (包括前额组织皮质 (FrA) 和伏隔核 (AcbC)) 置于 cryomolds 前侧。添加足够的 OCT 介质以覆盖组织。
	<ol> <li> 将此步骤与包含海马 (Hp) 和交叉核 (SCN) 的脑组织重复. </li>
		<li> 允许 OCT 介质在低温中完全冻结20分钟. </li>
	</ol> </li> <li> 组织和 oct 介质完全冻结后, 将少量的 oct 挤压到安装头上, 然后将含有组织的冷冻 cryomolds 压在安装头上。允许 OCT 完全冻结, 使包含组织的模具牢固地连接到头部. </li>
	<li> 将头部附件固定到切片安装上, 拧紧螺钉。调整切削角度与低温刀片的调整杠杆, 以确保节被削减均匀. </li>
	<li> 将截面厚度设置为30和 #181; m。当切片的组织块含有 FrA 和 AcbC, 首先开始切片, 直到大脑皮层是明显的, 然后开始收集. </li>
	<li> 参考鼠脑图谱 12 , 切片和收集大脑部分通过轻轻地放置室温聚乙烯 napthalate (钢笔) 膜幻灯片上的切片组织的顶部的 FrA, 直到二级马达皮质 (M2) 到达在 Bregma 5.16 毫米。
	通过检查组织和 OCT 是否完全融化到幻灯片上, <ol> <li> 直观地确保了对幻灯片的坚持。将所有带有组织切片的幻灯片存储在低温中直到染色. </li>
	</ol> </li> <li> 继续剖切, 直到前合 (aca) 变为可见, 并在现在明显的侧脑室 (LV) 下的一个完整合 Bregma 1.80 mm. </li>
	<li> 收集部分, 直到 lv 看起来与 aca 在 Bregma 0.84 毫米连接. </li>
	<li> 一旦切片为 FrA 和 AcbC 已经完成, 删除安装的组织块, 并替换为包含 HP 和 SCN 的块. </li>
	<li> 节直到 och 在 Bregma-0.48 毫米被夷为平地, 当第三脑室 (3V) 变得明显. </li>
	<li> 从该点收集 SCN 的部分直到 Bregma-0.72 mm, 当视交叉 (sox) 开始时. 
	注意: 可能需要在整个 och 中收集更多的组织切片, 因为 SCN 很容易通过. </li>
	<li> 对于 HP 收集, 节直到颗粒细胞层齿状回 (GrDG) 的角明显在 Bregma-3.00 毫米. </li>
	<li> 收集部分, 直到海马 CA 子在 Bregma-4.78 毫米的冠状部分熔化。这一细节允许完整收集的海马下开颅和损伤现场. 
	注: 如本实验室以前的出版物所示, 大多数受伤的细胞在这个范围内都是明显的, 它适合于基因或 rna 表达分析. </li>
</ol> 2。染色协议 <ol> <li> 在染色前, 用核糖核酸清除洗涤剂清洗所有餐具和化学油烟罩中的染色区域。这种制剂减少了因污染而导致 RNA 降解的风险. </li>
	<li> 准备所有染色试剂和溶液核酸自由水. </li>
	<li> 在低温中放置一张存储在通风橱中的幻灯片。允许幻灯片在三十年代预热. 将它们放入染色溶液 (用核酸自由水配制) 如下: 75% 乙醇1分钟, 核酸自由水为1分钟, 1% 甲酚紫色为1分钟, 核酸免费水为三十年代, 核酸免费水三十年代, 95% 乙醇为三十年代, 100% 乙醇为三十年代, 二甲苯为3分钟, 二甲苯为3分钟. </li>
	<li> 允许机架在通风柜的室温下风干不超过10分钟。或者, 将衣架放入无核糖核酸的真空干燥中, 以便更快地干燥。一旦干燥, 立即进行 LCM. </li>
</ol> 3。采用 lcm 系统的立体定向激光捕获显微切割术 <ol> <li> 在开始任何用于 RNA 分析的 lcm 程序之前, 用核糖核酸消除洗涤剂和100% 乙醇清除该设备周围的收集区域和区域. </li>
	在打开显微镜底座之前, <li> 在 IR 激光发电机组上翻转电源开关, 并允许系统在从桌面启动软件之前进行初始化. 
	注: 如果未按此顺序完成, 软件将无法识别显微镜. </li>
	<li> 一旦软件启动并运行其初始化步骤, 请按 "#34;P" #34 "和" 软件和 #34 "中的按钮; 安装面板. #34; 模块化阶段将移动到可以加载和卸货 LCM 宏盖帽和幻灯片的位置. </li>
	<li> 将钢笔膜滑入持纸者 (一次可达三次), 并将其磨砂边缘朝向向右. 
	注: 如果放置在持卡人错误的方向, 软件可能无法正确识别所需的 IR 或 UV 切割区域. </li>
	<li> 单击和 #34; #34; #34; 盖帽和滑动处理区和 #34; 在各自的幻灯片 ( 即 、a、B 或 C) 旁边的复选框。这一步表示, 如果幻灯片是一个膜玻璃幻灯片。然后单击要首先捕获的所需幻灯片. </li>
	<li> 要使照相机以平铺的图像为组织定位和定位, 选择和 #34; 图像和 #34; 在工具栏上选择和 #34; 获取概述. #34; 
	注意: 如果用户熟悉所收集的组织, 则可以使用手动滚动球来定位和 #34, 从而省略此步骤; 区域中心和 #34; 用于收集. </li>
	<li> 将光标放在平铺图像上的区域 (或没有平铺图像的相对位置) 上, 右键单击并选择 #34;P 区域中心的花边帽. #34; 软件将自动在所选位置放置一个盖. </li>
	<li> 选择一个位置. 
	注意: 要确保 IR 激光器正确对齐, 并且只在继续操作之前, 将只拾取该软件所放置的斑点的区域。
	<ol>
	<li> 移动到不存在组织的 cap 内的区域。单击和 #34; #34; 在和 #34; Microdissect 工具窗格和 #34; 选择 #34; IR 定位和 #34; 选项卡。从该窗格中, 发射红外测试镜头, 以评估红外激光发射的位置和光斑的大小. </li>
		<li> 使用光标, 右键单击 ir 点的中间, 然后选择和 #34; 定位到 ir 点. #34; 
		注: 通过手动改变 #34;P 功率、直径或持续时间和 #34, 可以改变红外光束的大小和强度; 在每个点大小指示符下或在对话框底部移动滑动刻度。为了确保适当的光斑大小, 可能需要发射几张试射。每次更换盖帽的位置或切换幻灯片时, 红外激光器必须重新. </li>
		<li> 用于 uv 切割, 通过选择和 #34 的 uv 激光定位; 在对话框中的 "uv 定位和 #34;" 选项卡. 
		注: 由于 uv 激光器和红外激光器一致移动, 因此无需在每次更换位置时连续重新 uv 激光器. </li>
	</ol> </li> <li> 选择 #34; 手绘绘图和 #34; 选项 #34; 选择工具窗格和 #34;。使用触摸屏触笔, 在感兴趣区域周围绘制, 同时确保触摸屏和触笔头之间不会失去联系。确保将开始和 end-points 连接在一起. 
	注: IR 斑点将通过软件自动铺设, 但用户可以选择放置额外的斑点, 以确保在紫外线切割完成后, 组织被附着在瓶盖上. </li>
	<li> 对于 FrA 采集, 在 M2 皮层出现在 Bregma 5.16 毫米时, 执行对皮质组织的总激光捕获. 
	注: 此详细信息可以修改, 以便仅收集 FrA 或特定单元格类型的特定部分. </li>
	<li> 对于 AcbC 集合, 激光捕获靠近 aca 的区域. 
	AcbC 的核心和外壳执行不同的功能, 在生理学上是独一无二的。因此, 重要的是要尽可能密切地跟踪大脑图谱。由于两个分区域之间的联系过于密切, 建议从不再 bregma 0.84 毫米的距离收集. </li>
	<li> 对于 Hp 采集, 激光捕获 CA 1、2和3的海马子, 从 Bregma 3.00 mm 开始, 并使用完全成形的 GrDG 作为形态学识别的物理标志. 
	注意: 为了本研究的目的, 不要收集过去 Bregma-4.78 毫米, 这可以在视觉上被标定为点时, Hp 子是融合和点腹。这个区域被选择, 因为多数受伤的神经元可以被发现直接地在损伤站点之下 6 . </li>
	<li> 对于 SCN 收集, 首先在视觉上识别 och 的密被填充的原子核, 然后再收集. </li>
	<li> 在区域集合 (上面列出) 之后, 将 LCM 宏上限移动到和 #34; QC 职位和 #34; 通过视觉上确保 UV 切割组织附着在膜上, 检查完全组织粘附。快速放置 ontoan 核糖核酸0.5 毫升薄壁反应管, 内含100和 #181; 细胞裂解缓冲液. </li>
	<li> 涡流样品, 以确保完全溶解和储存在-80 和 #176;在这一点上, LCM 可以暂停和样品存储, 直到用于下游基因组分析 6 . </li>
</ol>

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J., Che, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+levels+assessed+by+CA1+pyramidal+neuron+and+regional+hippocampal+dissections+in+Alzheimer's+disease.">Gene expression levels assessed by CA1 pyramidal neuron and regional hippocampal dissections in Alzheimer's disease.</a> Neurobiol.Dis. 45 (1), 99-107 (2012).</li><li>Elstner, M., 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=Expression+analysis+of+dopaminergic+neurons+in+Parkinson's+disease+and+aging+links+transcriptional+dysregulation+of+energy+metabolism+to+cell+death.">Expression analysis of dopaminergic neurons in Parkinson's disease and aging links transcriptional dysregulation of energy metabolism to cell death.</a> Acta Neuropathol. 122 (1), 75-86 (2011).</li><li>Wang, S., 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=Improvement+of+tissue+preparation+for+laser+capture+microdissection:+application+for+cell+type-specific+miRNA+expression+profiling+in+colorectal+tumors.">Improvement of tissue preparation for laser capture microdissection: application for cell type-specific miRNA expression profiling in colorectal tumors.</a> BMC.Genomics. 11, 163 (2010).</li></ol>

作者没有什么可透露的。

对于哺乳动物大脑的分子研究, LCM 已经成为一种必不可少的技术。本文论证了在 LCM 系统中使用 IR 和 UV 切割激光器的组合可以捕获哺乳动物大脑任何区域的基因组变化。这些区域包括那些与传统的 LCM 相容的污渍, 如甲酚紫罗兰或苏木精和曙红。激光捕捉过程的速度和能力执行 lcm 对厚30µm 节的钢笔膜幻灯片不仅可以获得足够数量的细胞样本, 而且还可以分离 RNA 从一个合适的质量 lcm 样品的所有类型的下游基因组分析;这些分析包括微阵列16, pcr 数组17, 定量 real-time pcr18。
我们的数据为使用 LCM 组织的研究提供了理论依据。我们发现, miR-15b 是上调在海马和皮质 (图 4), 但 downregulated 在伏隔核和可能是生物学相关的理解的差异效应的脑外伤。先前的一项研究表明, 皮质神经元易受伤害的增加是由于一些 rna 的过度表达而导致的, 如 Bcl-219, 对 pro-survival 基因有负调节作用。目标扫描分析显示 Bcl-2 也受 miR-15b;因此, 我们的数据提出了一个机械的解释, 为什么特定区域的大脑(即, FCx) 可能有选择性地易受创伤性脑损伤。重要的是要记住, 大多数基因是由多个 rna 和相关的变化, 任何一个 miRNA 的特定靶基因是困难的。此外, 这些数据表明, 基因和 rna 表达的某些变化可能被用作区域特定脑损伤的生物标志物。事实上, 我们目前正在使用这些数据, 研究新的抗抑郁药物化合物如何能在与神经精神紊乱相关的脑区中对基因和 rna 表达产生差异性影响。我们研究的一个限制是, 在 LCM 的幻灯片准备过程的多个步骤中, RNA 完整性可能会受到影响。本协议描述了减少 RNA 降解风险的必要步骤。另一个限制是用于统计计算的较小样本大小。在今后的研究中, 增加样本量应能减少个体动物之间基因和 miRNA 表达变化的影响。
利用人类疾病和病变组织动物模型进行转化基因组研究中的 LCM 的好处20,21,22,23。如果没有能力捕捉特定的细胞群, 不同脑区的转录谱将是许多细胞类型不可知和难以的混合。使用 LCM 方法的脑损伤研究已经导致目前的努力, 划定脑区的特定生物标记物, 并了解它们是如何与循环生物标志化合物的创伤。

哺乳动物的大脑是非常复杂和异构的, 有成百上千的细胞类型1。事实上, 人类研究表明, 在诸如额叶皮层, 白色和灰色物质的结构和功能上的差异反映在不同的和不同的转录配置文件2。大脑异质性一直是解释基因表达数据和脑损伤领域的主要障碍。临床前研究的这种含糊不清导致了数百例失败的治疗脑损伤的试验3。
我们使用激光捕获显微切割 (LCM) 方法研究大鼠脑外伤性脑损伤 (失调) 诱发的基因,4, 聚焦于海马体, 这是学习和记忆所必需的大脑区域5。激光捕获和分析基因表达的能力在死亡和幸存的神经元, 使我们更了解随机性在基因表达的作用, 确定的结果 (神经元生存) 后, 脑损伤6。LCM 技术也被证明是有用的探索的影响, 脑外伤的海马神经元时比较年轻和衰老小鼠7或大鼠8。
在最近的研究中, 我们检查了大鼠脑损伤后的其他区域, 重点放在大鼠和与执行功能相关的人颅脑外伤患者的区域 (即额叶皮质9) 和创伤性并存;这些并存包括抑郁症 (即伏隔核10) 和昼夜节律紊乱 (交叉核11)。在以前的研究中, Huusko 和 Pitkanen12和尔et al.13使用 LCM 检查丘脑和下丘脑的基因表达。我们的研究建立在这些先前的观察基础上, 包括四其他脑区。为了研究脑外伤后所诱导的区域特异分子的变化, 有必要利用 LCM 系统在这些地区识别和获取细胞类型方面获得专门知识。紫外线切割和红外线 (IR) 激光器允许精确的显微切割所需的大脑区域。在这里, 我们描述我们如何使用这个 LCM 系统, 在大鼠脑图集14的立体定位坐标下, 识别和激光捕获四大鼠脑区, 这是受实验性撞击脑损伤方法的差异影响4。

<table><tbody><tr><td>Arcturus Laser Capture Microdissection System</td><td>Applied Biosystems (Life Technologies)</td><td></td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2939AA</td><td></td></tr><tr><td>Agilent 6000 Pico Kit</td><td>Agilent Technologies</td><td>5067-1513</td><td></td></tr><tr><td>Arcturus PEN Membrane Glass Slides&amp;nbsp;</td><td>Applied Biosystems (Life Technologies)</td><td>LCM0522</td><td></td></tr><tr><td>Capsure LCM caps</td><td>Applied Biosystems (Life Technologies)</td><td>LCM0211/LCM0214</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>RNAqueous Micro- Kit</td><td>Life Technologies (Ambion)</td><td>AM1931</td><td></td></tr><tr><td>Taqman Gene Expression Primer/Probes</td><td>Applied Biosystems (Life Technologies)</td><td>4331182</td><td></td></tr><tr><td>Taqman microRNA Expression Primer/Probes</td><td>Applied Biosystems (Life Technologies)</td><td>A25576</td><td></td></tr><tr><td>Lightcycler 96 System</td><td>Roche</td><td></td><td></td></tr></tbody></table>

stereotactic atlas-guided laser capture microdissection of brain regions affected by traumatic injury

在不保留其空间分布的组织解除技术中, 能够分离出感兴趣的特定大脑区域的能力受到阻碍。这种技术也可能扭曲基因表达分析, 因为这个过程本身可以改变单个细胞的表达模式。在这里, 我们描述了激光捕获显微切割 (LCM) 方法, 以选择性地收集受创伤性脑损伤 (脑外伤) 的特定大脑区域, 使用改良的尼 (甲酚紫) 染色协议和指导的大鼠脑图谱。LCM 提供了进入他们的本地位置的大脑区域, 并有能力使用解剖地标来识别每个特定的区域。为此, LCM 已被用于研究脑外伤后脑区特异基因的表达。该协议允许检查在同一动物的不同脑区内, 创伤后诱发的基因改变和 rna 表达。本议定书的原则可以修改, 并适用于对其他疾病和/或动物模型中的基因组表达进行广泛研究。

在图 1中提供的示意图演示了 atlas 指导特定大脑区域的 LCM 的总体工作流程以及潜在的下游分析应用。这项研究集中于四大脑区域相关的脑外伤病理生理学和发展并存: FrA, AcbC, SCN, 和 Hp。在特定脑区的 LCM 中存在的一个局限性是, 解剖位置往往被缺乏定义的边界所遮蔽, 如图 2 (a、D、G、J)中所示。使用脑图谱来指导特定区域的切片和激光捕获, 可以减少与目标区域以外的脑区样本污染的可能性。在剖切和尼染色后, 当注意遵循组织标志时, LCM 技术可以提供一种高度一致的方法来获取离散的细胞、细胞核或区域15。这些研究的长期目标是识别基因和 rna, 它们可能成为脑区特定损伤的非侵入性生物标志物。该生物标志物开发管道的第一步是对实验性脑损伤后组织特异性转录变化的表征。这些数据可以与损伤引起的循环 biofluids 的变化相关。
为减少 rna 降解的风险, 本文提出的程序进行了优化, 以便在定量 real-time PCR (RT-qPCR) 之前通过总 LCM rna 的反向转录进行 rna 分析。总 rna (包括小和大 rna 种类) 被隔绝了使用列隔离方法在组织被激光被夺取从各自的脑子区域。为了评估 rna 的完整性, 质量和数量后的隔离程序, rna 样品被短暂变性在70° c 和运行的 rna 分析仪。定性测量包括分析18s 和 28s rRNA 波段的峰值振幅(图 3), 它可以代表整体退化, 并使用 "RNA 完整性数字" (林)。如果使用细致的无核糖核酸技术, 从 LCM 样品中分离出来的 RNA 通常会导致 RINs 的范围从6-8。较低的 rna 可能意味着 RNA 质量较差, 可能会降低基因和表达分析的准确性。反之, 更高的林能提高对 RNA 分析结果有效性的信心。
RT-qPCR 是使用入门/探针集的个别基因和 rna (图 4)。根据制造商的协议, 在 qPCR 之前, 总 RNA 的大约 1 ng 被反向转录成 cDNA 和 pre-amplified。本研究评估的损伤相关基因包括BCL2 相关的 X, 凋亡调控器 (Bax), B 细胞 CLL/淋巴瘤 2 (Bcl-2),酶 3, 凋亡相关的半胱氨酸肽(Casp3),脑源性神经营养因子(Bdnf) 和CAMP 应答元素结合蛋白 1 (Creb)。MiR-15b 被选中, 因为它已被证明是在实验性脑外伤后, 个别死亡神经元改变。它还具有实验验证和 bioinformatically 预测的基因目标与 pro-survival 功能 (未发布的数据)。采用ΔΔCt 法对脑外伤动物的基因和 rna 表达水平进行比较, 并分别对内源基因 (Gapdh) 或小 RNA (U6) 进行归一化。1以上的褶皱变化表明该基因或 rna 的整体上调, 反之, 低于1的褶皱变化表示下调。统计分析显示脑损伤与单纯性控制 (p ≤ 0.05) 之间的基因表达有显著的变化。在脑外伤与单纯性对照中, 未发现 miR-15b 表达的显著变化, 但在大脑区域依赖的流行趋势中, 出现了更高、更低的表达倾向。这些数据表明, 进一步优化是必要的, 以评估变化的 rna 表达。这也是可能的样本大小太小, 以获得统计的意义, 部分原因是固有的变化, 在表达。未来的研究将包括假操作动物, 以确保基因和 rna 表达变化归因于脑外伤, 而不是由于手术准备。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56134/56134fig1.jpg"> 
图1。用于下游基因组分析的阿特拉斯导向 LCM 工作流程.(A-F)从动物制剂到下游的 qPCR 分析: (A)成年, 雄性大大鼠 (~ 6 周龄和体重300克) 被麻醉, 受到液体撞击的创伤, 并在受伤后进行人道安乐死24小时。(B)新鲜冰冻大脑的连续切片 (30 µm) 是根据特定脑区 (FrA、AcbC、Hp、SCN) 的坐标从 Paxinos 和沃森的鼠脑图谱。(C)部分是固定的, 尼染色 (1% 甲酚紫色), 脱水和风干。(D). lcm 在已识别的大脑区域中使用 lcm 系统进行。(E) LCM 宏帽转移到一个无核糖核酸的0.5 毫升管上, 100 μ l 细胞裂解缓冲液, 并储存在-80 c, 直到 RNA 分离, 用于下游基因组分析。然后 RNA 可以是反向转录的基因或 rna RT qPCR 分析, 以检查差异表达的分子靶 (s) 后, 脑外伤和/或大脑区域之间的利益(F)。<a href="//ecsource.jove.com/files/ftp_upload/56134/56134fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56134/56134fig2.jpg"> 
图2。脑外伤的 LCM 影响大脑区域.在 LCM 系统(一-I)上, 用 IR 和 UV 激光功能在损伤部位从同侧采集组织切片的代表性图像。组织被切片在一个低温 (30 µm) 和收集在钢笔膜幻灯片。切片, 然后固定, 尼染色的甲酚紫 (1%), 和脱水, 以确定特定的大脑区域的基础上参考的解剖地标 Paxinos 和沃森的鼠脑图谱。(A-c)大脑前部组织皮质 (FrA) (D-F)的一个区域, 位于海马 (Hp) 的 CA1、CA2 和 CA3 锥体层的组成部分, 在齿状回 (GrDG) 的颗粒层的完全形成的角旁边。(G-I)伏隔核 co 的一个区域re (AcbC) 近端和侧到前合 (aca)。(J-K)交叉核 (SCN) 侧到视交叉 (och)。<a href="//ecsource.jove.com/files/ftp_upload/56134/56134fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56134/56134fig3.jpg"> 
图3。RNA 质量的代表性扫描.具有代表性的 electropherograms 和相关的凝胶图像的 RNA 从 LCM 收集的组织(A-D)。Electropherograms 和相关的凝胶图像显示完整的 RNA 的基础上出现18s 和 28s rRNA 峰和凝胶带。该 RNA 适用于所有下游应用, 包括基因组和蛋白质的分析。(A)额叶关联皮层 (FrA) RNA。凛6.1。(B)海马 (Hp) RNA。凛4.4。(C)伏隔核核 (AcbC) RNA。凛7.3。(D)交叉核 (SCN) RNA。凛7.6。<a href="//ecsource.jove.com/files/ftp_upload/56134/56134fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56134/56134fig4.jpg"> 
图4。脑区特异基因和 rna 表达的下游分析通过RT-qPCR.有兴趣基因的个体 RT qPCR (Bax, Bcl-2, Bdnf, Casp3, Creb) 和 rna 的兴趣 (miR-15b) 是在激光捕捉脑外伤后的大脑区域 (Hp 和 AcbC n = 5, FrA n = 4), 并与未受伤的幼稚动物 (n = 4) 进行比较。对 Hp、AcbC、FCx 进行了颅脑损伤相关基因的分析. 将数据作为归一化褶皱比值, 与单纯控制脑区 (与韦尔奇的修正± t 检验) 相比较; * p ≤ 0.05) (A)。miR-15b 在不同脑区的差异表达为归一化褶皱改变与单纯对照 (± SEM) (B)。来自 SCN (n=2) 的数据不包括在内。<a href="//ecsource.jove.com/files/ftp_upload/56134/56134fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury at JoVE.com

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

我们描述了利用激光捕获显微切割, 获得不同的细胞群体的样本, 从各个脑区的基因和 rna 分析。这项技术可以研究大鼠脑部特定区域创伤性脑损伤的差异效应。

立体定向激光捕获创伤性脑区显微解剖

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

stereotactic-atlas-guided-laser-capture-microdissection-brain-regions

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.1K 次观看.  University of Texas Medical Branch. 我们描述了利用激光捕获显微切割, 获得不同的细胞群体的样本, 从各个脑区的基因和 rna 分析。这项技术可以研究大鼠脑部特定区域创伤性脑损伤的差异效应。

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

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

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

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 ...

科研

神经科学

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

工业化、人工智能引导的激光显微切割，用于肿瘤微环境的微尺度蛋白质组学分析

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...

癌症研究

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

急性脑外伤小鼠其次是纵向双光子成像

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

医学

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

控制皮层影响模型创伤性脑损伤

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

一种基于微 ct 的小动物脑损伤和定位电极表征方法

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

大鼠运动皮层中的激光诱发脑损伤

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

激光捕获胶质瘤分区域的微切，用于肿瘤内异质性、肿瘤流和入侵的空间和分子表征

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes and are defined as the sudden blockage of a vessel supplying blood to the brain. Within minutes of the blockage, cells begin to die and result in irreparable tissue damage. Current therapeutic treatments focus on clot removal or lysis to allow for the reperfusion and prevent more severe brain damage. Although transient brain plasticity may salvage some of the damaged tissue over time, significant fractions of patients are left with neurological deficits that will never resolve. There is a lack of therapeutic options to treat neurological deficits caused by stroke, emphasizing the need to develop new strategies to treat this growing patient population. Injectable biomaterials are currently being designed to enhance brain plasticity and improve endogenous repair through the delivery of active agents or stem cells. One method to test these approaches is to utilize a rodent stroke model, inject the biomaterial into the stroke core, and assess repair. Knowing the precise location of the stroke core is imperative for the accurate treatment after stroke, therefore, a stroke model that results in a predictable stroke location is preferable to avoid the need for imaging prior to injection. The following protocol will cover how to induce a photothrombotic stroke, how to inject a hydrogel in a controlled and precise manner, and how to extract and cryosection the brain while keeping the biomaterial intact. In addition, we will highlight how these same hydrogel materials can be used for the co-delivery of stem cells. This protocol can be generalized to the use of other injectable biomaterials into the stroke core.

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

中风后向大脑注射水凝胶生物材料支架

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes ...

生物学

Cryopreserved Tissue Section Fixation and Staining: A Procedure to Process Frozen Brain Tissue Sections for Laser Microdissection

Source: Comba, A. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/60939/laser-capture-microdissection-glioma-subregions-for-spatial-molecular">Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion.</a>&#160;J. Vis. Exp.&#160;(2020)
This video presents the fixation and staining method to process frozen mouse brain tumor tissue. The processed tissue helps differentiate and obtain a specific population of cells during subsequent laser capture microdissection.

冷冻保存的组织切片固定和染色:一种处理冷冻脑组织切片以进行激光显微切割的程序

Source: Comba, A. et al. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, ...