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&#160;</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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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 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

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 ...

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 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 ...

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 ...

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 ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

一种新的脑外伤体损伤模型

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...