Name: microdissection of mouse brain into functionally and anatomically different regions
Uploaded: 2021-02-15T12:30:00
Description: Watch this Scientific Journal Video about Microdissection of Mouse Brain into Functionally and Anatomically Different Regions at JoVE.com

我们感谢塞什马利尼·斯里尼瓦桑女士、斯蒂芬·巴特勒先生和帕梅拉·斯佩尔曼女士的实验协助，并感谢达娜·优素福女士编辑这份手稿。非常感谢美国军事资源委员会的资助支持。日内瓦基金会为这项工作做出了贡献，并通过美国陆军研究办公室得到了军事和作战医学研究区第三局的资金支持。
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材料已由沃尔特·里德陆军研究所审查。不反对其提出和/或公布。此处包含的意见或断言是作者的私人观点，不得解释为官方观点，或反映陆军部或国防部的真实观点。研究是在AAAALAC认可的设施中根据批准的动物使用协议进行的，符合“动物福利法”和其他与动物和涉及动物的实验有关的联邦法规和法规，并遵守 “实验动物护理和使用指南”中规定的原则，NRC出版物，2011年版。

动物处理和实验程序是根据欧洲，国家和动物护理机构指南进行的。所有动物实验均由美国陆军环境卫生研究中心（现为沃尔特里德陆军研究所（WRAIR）的机构动物护理和使用委员会（IACUC）批准，并在实验室动物护理国际评估和认证协会（AAALAC）认可的设施中进行。 注意：该程序将在6至8周龄的C57BL / 6j菌株上进行，该菌株因宫颈脱位16而安乐死。我们的实验室没有进行灌注，...

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哺乳动物大脑是一个复杂的器官，由一系列形态上不同且功能独特的细胞组成，这些细胞具有不同的分子特征和执行特殊和离散功能的多个区域。这里报告的解剖程序可以有多个目标，具体取决于实验室的要求。在我们的实验室中，我们评估了从暴露于创伤后应激障碍（如应激16 ）的小鼠收集的多个大脑区域的转录。我们想进一步研究菌株遗传背景24 对多个大?...

大脑与脊髓和视网膜一起组成执行复杂行为的中枢神经系统，由整个身体中专门的，精确定位和相互作用的细胞类型控制1。大脑是一个复杂的器官，有数十亿个相互连接的神经元和神经胶质细胞，具有执行许多功能的精确电路。它是一种双侧结构，具有两个不同的叶和不同的细胞成分2。脊髓将大脑与外部世界连接起来，并受到骨骼，脑膜和脑脊液的保护，并将信息路由到大脑和从大脑路由信息2，3，4。大脑的表面，大脑皮层，是不平坦的，有不同的褶皱，称为回，凹槽，称为沟，将大脑分成功能中心5。大脑功能较弱的哺乳动物的大脑皮层为6，7。重要的是要表征和研究人脑的结构，以了解与不同大脑区域相关的疾病及其功能回路。近年来，神经科学研究不断扩大，各种实验方法被用于研究大脑的结构和功能。分子和系统级生物学领域的发展开创了探索大脑结构与分子功能之间复杂关系的新时代。此外，分子生物学，遗传学和表观遗传学正在迅速扩展，使我们能够提高我们对系统如何运作所涉及的潜在机制的知识。这些分析可以在更本地化的基础上进行，以帮助针对性地研究和开发更有效的治疗方法。
哺乳动物的大脑在结构上被定义为清晰可辨的离散区域;然而，这些离散结构的功能和分子复杂性尚未得到明确理解。脑组织的多维和多层次性质使得这种景观难以在功能水平上进行研究。此外，多个功能由相同的结构执行，反之亦然，这一事实使对大脑8的理解进一步复杂化。至关重要的是，为大脑区域的结构和功能表征而执行的实验方法使用精确的研究方法来实现将神经解剖学结构与功能相关联的采样的一致性。最近使用单细胞测序9，10 解释了大脑的复杂性，例如人脑的颞回，它由75种不同的细胞类型11组成。通过将这些数据与来自小鼠大脑类似区域的数据进行比较，该研究不仅揭示了其结构和细胞类型的相似性，而且还揭示了差异。因此，为了解开复杂的机制，必须完全精确地研究大脑的不同区域。人类和小鼠大脑之间的保守结构和功能使得能够使用小鼠作为阐明人脑功能和行为结果的初步替代品。
随着系统生物学方法的进步，从啮齿动物的离散大脑区域获取信息已成为神经科学研究的关键程序。虽然一些协议如激光捕获显微切割12 可能很昂贵，但机械协议是廉价的，并且使用常用的工具13，14执行。我们已经使用多个大脑区域进行转录组学测定15 ，并开发了一种动手和快速的程序，可以在短时间内逐步解剖感兴趣的小鼠大脑区域。解剖后，这些样品可以立即储存在寒冷的条件下，以保存这些组织的核酸和蛋白质。我们的方法可以更快地执行，从而实现高效率，并减少组织恶化的机会。这最终增加了使用脑组织产生高质量，可重复实验的机会。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brain Removal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deaver scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6762</td>
			<td>5.5&#34; straight sharp/sharp</td>
		</tr>
		<tr>
			<td>Deaver scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6763</td>
			<td>5.5&#34; curved sharp/sharp</td>
		</tr>
		<tr>
			<td>Delicate operating scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6703</td>
			<td>4.75&#34; curved sharp/sharp</td>
		</tr>
		<tr>
			<td>Delicate operating scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6702</td>
			<td>4.75&#34; straight sharp/sharp</td>
		</tr>
		<tr>
			<td>Light operating scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6753</td>
			<td>5&#34; curved Sharp/Sharp</td>
		</tr>
		<tr>
			<td>Micro spatula, radius and tapered flat ends</td>
			<td></td>
			<td></td>
			<td>stainless steel mirror finish</td>
		</tr>
		<tr>
			<td>Operating scissors 6.5&#34;</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6846</td>
			<td>curved sharp/sharp</td>
		</tr>
		<tr>
			<td>Tissue forceps</td>
			<td>Roboz Surgical Store</td>
			<td>RS-8160</td>
			<td>4.5&#8221; 1X2 teeth 2mm tip width</td>
		</tr>
		<tr>
			<td>Rongeur (optional)</td>
			<td>Roboz Surgical Store</td>
			<td>RS-8321 many styles to choose</td>
			<td>Lempert Rongeur 6.5&#34; 2X8mm</td>
		</tr>
		<tr>
			<td>Pituitary Dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Roboz Surgical Store</td>
			<td>RS-9843</td>
			<td>Scalpel Handle #3 Solid 4&#34;</td>
		</tr>
		<tr>
			<td>and blades</td>
			<td>Roboz Surgical Store</td>
			<td>RS-9801-11</td>
			<td>Sterile Scalpel Blades:#11 Box 100 40mm</td>
		</tr>
		<tr>
			<td>Super fine forceps Inox</td>
			<td>Roboz Surgical Store</td>
			<td>RS-4955</td>
			<td>tip size 0.025 X 0.005 mm</td>
		</tr>
		<tr>
			<td>Brain Dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A magnification visor</td>
			<td>Penn Tool Col</td>
			<td>40-178-6</td>
			<td>2.2x Outer and 3.3x Inner Lens Magnification, Rectangular Magnifier</td>
		</tr>
		<tr>
			<td>Dissection cold plate</td>
			<td>Cellpath.com</td>
			<td>JRI-0100-00A</td>
			<td>Iceberg cold plate &#38; base</td>
		</tr>
		<tr>
			<td>Graefe forceps, full curve extra delicate</td>
			<td>Roboz Surgical Store</td>
			<td>RS-5138</td>
			<td>0.5 mm Tip 4&#8221; (10 cm) long</td>
		</tr>
		<tr>
			<td>Light operating scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6753</td>
			<td>5&#34; curved sharp/sharp</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Roboz Surgical Store</td>
			<td>RS-9843 (repeated above)</td>
			<td>Scalpel Handle #3 Solid 4&#34;</td>
		</tr>
		<tr>
			<td>and blades (especially #11)</td>
			<td>Roboz Surgical Store</td>
			<td>RS-9801-11 (repeated above)</td>
			<td>Sterile Scalpel Blades:#11 Box 100 40mm</td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Amazon</td>
			<td>MS-SQRD9-4</td>
			<td>Double Ended Spatula Square AND Round End</td>
		</tr>
		<tr>
			<td>Tissue forceps</td>
			<td>Roboz Surgical Store</td>
			<td>RS-8160 (repeated above)</td>
			<td>4.5&#8221; 1X2 teeth</td>
		</tr>
	</tbody>
</table>

microdissection of mouse brain into functionally and anatomically different regions

大脑是哺乳动物神经系统的指挥中心，也是一个具有巨大结构复杂性的器官。大脑在颅骨内受到保护，由半球上的灰质外层覆盖物组成，称为大脑皮层。在这一层之下，还有许多其他专门的结构，这些结构对于对存在很重要的多种现象至关重要。获取特定大脑区域的样本需要快速而精确的解剖步骤。据了解，在微观层面上，存在许多子区域，并且可能跨越我们为了这种解剖而强加的任意区域边界。
小鼠模型通常用于研究人脑功能和疾病。基因表达模式的变化可能仅限于针对特定表型的特定大脑区域，具体取决于患病状态。因此，研究转录对其明确定义的结构组织的调节非常重要。要完全了解大脑，需要研究不同的大脑区域，定义连接，并确定每个大脑区域活动的关键差异。对这些不同区域中的每一个进行更全面的了解，可以为神经科学领域的新的和改进的治疗方法铺平道路。在这里，我们讨论了将小鼠大脑解剖成十六个不同区域的分步方法。在这个过程中，我们专注于雄性小鼠C57Bl / 6J（6-8周龄）大脑切除并解剖到多个区域，使用神经解剖学标志来识别和采样离散的功能相关和行为相关的大脑区域。这项工作将有助于在神经科学领域奠定坚实的基础，从而在更深入地了解大脑功能方面采取更有针对性的方法。

我们对复杂的大脑结构和功能的理解正在迅速发展和改善。大脑包含多个不同的区域，建立分子图谱可以帮助我们更好地了解大脑是如何工作的。在这种方法的论文中，我们讨论了将小鼠大脑解剖成多个不同的区域（表1）。在该协议中，根据关键地标识别结构，并通过保持其坚固性以立即解剖来用盐水溶液保持组织湿润来实现。该方法比其他报告21 覆盖更多的区域?...

Watch this Scientific Journal Video about Microdissection of Mouse Brain into Functionally and Anatomically Different Regions at JoVE.com

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

我们提出了一个动手的，一步一步的，快速的方案，用于从新鲜脑组织中去除小鼠大脑和解剖离散区域。获取用于分子分析的大脑区域已成为许多神经科学实验室的常规。这些大脑区域立即被冷冻，以获得高质量的转录组学数据，用于系统级分析。

将小鼠大脑显微切割成功能和解剖学上不同的区域

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

研究不同的大脑区域是理解大脑分子和结构复杂性的垫脚石。该协议描述了小鼠大脑的系统解剖以分离不同的大脑区域。从大脑解剖学的角度来看，这是一种易于复制的自上而下的方法。该协议的优点是双重的。首先，这种方法为理解小而关键的大脑区域的分子基础提供了机会。其次，整个解剖过程足够短，以确保保持分子完整性，这是背部和分子测定的关键先决条件。这个过程涉及练习和精细的处理。在这里，维护结构地标的时机非常关键，可以通过适当的培训和练习来实现。展示小鼠大脑切除过程的将是Seid Muhie，我们实验室的研究化学家和生物信息学家。进一步展示小鼠脑解剖程序的将是来自我们实验室的神经科学家James Meyerhoff。首先，清洁并去除皮肤以获得良好的抓地力。用止血器夹住被斩首头部的上颌骨，并用纱布将头皮反射到嘴部。将细弯曲的剪刀插入大孔中，以分离粘附的脑膜。然后将剪刀插入脊髓通过的颅底开口处，刀片垂直指向，但平行并压在基底板骨的内表面，刀片向左侧旋转 45 度，然后向右侧旋转。去除顶骨内枕骨以暴露小脑。将剪刀沿矢状中缝线向上推进到前膛，向上抬起剪裁，以避免大脑皮质撕裂伤。当顶骨被抬起时，识别并切断剩余的脑膜附着物。在矢状面上做两个平行的切口，去除额骨的碎片，避免撕裂脑表面。在切割脑膜附着物和颅神经时，倒置颅骨以允许重力帮助将大脑取出到预先装满盐水溶液的小杯子中。保持听大疱完整，以便识别垂体解剖结构。在固定垂体的膜帐篷的脊部两侧做一个极小的颌旁切口，并用超细镊子提起垂体的后叶。在最近的三叉神经垂体前叶的侧缘之间做一个矢状切口，然后抬起垂体前叶。将白光源引导到组织上。将大脑放在预冷的不锈钢块上，并通过在冰冷的盐溶液中用冰包围块来保持块冷。定期用冰冷的盐水溶液润湿组织以保存结构。将大脑定位为大脑皮质朝上。使用弯曲的小镊子，取出小脑。使用背侧方法，将小弯曲镊子的闭合刀片滑到胼胝体下方，并轻轻张开以双侧收缩新皮层，以保留关键突出的中线标志。最终面朝上，视交叉、下丘脑、嗅球、延髓、垂体和脑桥等结构将可见。尝试分隔半球。向上翻转大脑腹侧部位，从大脑半球嗅球之间的背部开始进行中矢状切口。去除髓质，然后轻轻分开两个半球。接下来，在池塘的内缘进行冠状切口，以获得后脑，并在池塘的后缘分离髓质。翻转脑组织以仔细分离两个大脑半球。在此步骤中取下嗅球。在胼胝体前方一毫米处做一个冠状切口，去除附属嗅球，然后将内侧和外侧前额叶皮层分开。从侧脑室前角下方的中线水平或冠状切开部分水平或冠状切开，以释放背侧隔核和腹侧纹状体。从侧表面去除少量皮层以去除屈服核和嗅结节。通过外囊外切的弯曲手术刀将纹状体的喙部与上覆的皮层分开。识别隔膜核或隔膜，并将其与该部分隔离。在此步骤中，进行弯曲切口以分离下丘脑。这将使用体后方的部分冠状切口来完成，该切口仅向下丘脑沟横向延伸。然后沿着下丘脑沟的长度用矢状旁切开下丘脑横截面。埃弗特侧脑室，以揭示剩余边缘系统组件中额外的边缘内连接。在内皮层的内侧，打开心室后观察到扇形结构。使用内嗅皮层中的扇形结构确定边缘以进行解剖，然后识别并去除杏仁核、内嗅皮层、后扣带皮层和海马体。通过观察丘脑背表面在中线皮质平面延伸的髓纹来确认丘脑的识别。通过冠状切口将丘脑与中脑完全分开。该方案可用于立即从颅骨中取出大脑，然后进行解剖。根据研究的要求，解剖的组织可以保存和稍后处理。从多个解剖组织中提取的RNA可以测定基因表达。代表性结果是使用收获的大脑获得的，这些大脑在RNA提取前在零下80摄氏度下储存数月。在研究创伤后应激障碍的侵略者暴露社会压力模型下收集了不同的大脑区域以及体重数据。此处显示了RNA浓度和分光光度读数。遵循此协议时，通过将大脑保持在冰钢块上并定期用冰盐水浇灌组织来确保组织始终冷却。一旦收集组织并立即冷冻，它们就可以在以后用于转录组学、代谢组学和蛋白质组学等测定。

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Anatomy and Physiology

Neuroscience

Unit 1

Anatomy of the Central and Peripheral Nervous System

SCIENTISTS IN ACTION

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

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  |

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 ...

科研

神经科学

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example

同时MEG / EEG和解剖约束的最小模估计皮质动态映射：听觉注意示例

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

在小鼠大脑的各个区域端粒酶活性：非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

设计，表面处理，电镀细胞和神经元的模块化网络功能组成跨接电路的培养

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...