Name: microdissection and whole mount scanning electron microscopy visualization of mouse choroid plexus
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这项工作得到了比利时阿尔茨海默氏症研究基金会（SAO;项目编号：20200032），法兰德斯研究基金会（FWO Vlaanderen;项目编号：1268823N，11D0520N，1195021N）和Baillet Latour基金的支持。我们感谢VIB生物成像核心的培训，支持和进入仪器园区。

本研究中描述的所有动物实验均根据国家（比利时法律14/08/1986和22/12/2003，比利时皇家法令06/04/2010）和欧洲立法（欧盟指令2010/63 / EU，86/609 / EEC）进行。所有关于小鼠和动物实验方案的实验均已获得根特大学伦理委员会的批准（许可证号LA1400091和EC 2017-026）。
1. 准备
<ol><li>麻醉药：准备终末麻醉药。例如，可以制备磷酸盐缓冲盐水（PBS）中的戊巴比妥钠（≥100mg...

<ol>
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(101), e52991 (2015).</li><li>Dani, N., 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=A+cellular+and+spatial+map+of+the+choroid+plexus+across+brain+ventricles+and+ages.">A cellular and spatial map of the choroid plexus across brain ventricles and ages.</a> Cell. 184 (11), 3056-3074 (2021).</li><li>Carloni, 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=Identification+of+a+choroid+plexus+vascular+barrier+closing+during+intestinal+inflammation.">Identification of a choroid plexus vascular barrier closing during intestinal inflammation.</a> Science. 374 (6566), 439-448 (2021).</li><li>Van Hoecke, L., 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=Involvement+of+the+choroid+plexus+in+the+pathogenesis+of+Niemann-Pick+disease+type.">Involvement of the choroid plexus in the pathogenesis of Niemann-Pick disease type.</a> C. Frontiers in Cell Neuroscience. 15, 757482 (2021).</li><li>Shipley, F. B., 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=Tracking+calcium+dynamics+and+immune+surveillance+at+the+choroid+plexus+blood-cerebrospinal+fluid+interface.">Tracking calcium dynamics and immune surveillance at the choroid plexus blood-cerebrospinal fluid interface.</a> Neuron. 108 (4), 623-639 (2020).</li><li>Guerin, C. J., Kremer, A., Borghgraef, P., Lippens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+studies+using+serial+block+face+and+focused+ion+beam+scan+electron+microscopy.">Targeted studies using serial block face and focused ion beam scan electron microscopy.</a> Journal of Visualized Experiments. 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这里描述了一种将脉络丛（CP）从小鼠大脑的侧脑室和第四脑室中分离出来的方法。CP 的整个安装方法有助于使用一系列技术进行进一步分析，以获得 CP 形态、细胞组成、转录组、蛋白质组和分泌组的完整视图。这种分析对于更好地了解从大脑脑室突出的这种显着结构至关重要。这些知识具有巨大的研究兴趣，因为越来越清楚的是，CP在健康和疾病中起着至关重要的作用9，10，<s...

紧密的屏障将中枢神经系统 （CNS） 与外围隔离开来，包括血脑屏障 （BBB） 和血脑脊液 （CSF） 屏障。这些屏障保护中枢神经系统免受外部侮辱，并确保平衡和受控的微环境1，2，3。虽然BBB随着时间的推移得到了广泛的研究，但位于脉络丛（CP）的血液 - 脑脊液屏障在过去十年中才获得了越来越多的研究兴趣。后一种屏障可以在大脑的四个脑室中找到（图1A，B），其特征在于围绕中央基质的单层脉络丛上皮（CPE）细胞，渗漏的毛细血管，成纤维细胞以及淋巴和骨髓细胞群（图1C）4，5，6.CPE细胞通过紧密的连接牢固地相互连接，从而防止从下面的开窗毛细血管渗漏到脑脊液和大脑中。此外，通过CPE细胞的运输受到许多向内和向外运输系统的调节，这些系统管理有益化合物（例如，营养物质和激素）从血液流入脑脊液以及有害分子（例如，代谢废物，过量的神经递质）在另一个方向的外排1，6。为了能够发挥其主动转运功能，CPE细胞在其细胞质中含有许多线粒体7。此外，CP是脑脊液的主要来源，并通过常驻炎症细胞的存在充当大脑的守门人1。由于其在血液和大脑之间的独特位置，CP也非常适合进行免疫监视8。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64733/64733fig01.jpg"> 图 1：脉络丛 （CP） 的位置和组成的示意图。 （A，B）CP组织存在于（A）人类和（B）小鼠大脑的两个侧脑室，第三脑室和第四脑室内。（C）CP组织由单层紧密连接的骰骨CP上皮（CPE）细胞组成，围绕开窗毛细血管，松散结缔组织以及淋巴和骨髓细胞，并形成血脑脊液屏障（根据参考文献23改编和修改）。用 Biorender.com 创建的图。<a href="https://www.jove.com/files/ftp_upload/64733/64733fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
在过去的十年中，越来越多的证据，包括我们研究小组的几份报告，表明CP在健康和疾病中起着核心作用9，10，11，12，13，14，15，16，17，18.例如，已知老化的血液-脑脊液屏障在细胞核、微绒毛和基底膜1，19 等中显示出形态改变。此外，在阿尔茨海默病的背景下，整体屏障完整性受到损害，所有这些与年龄相关的变化似乎更加明显1，8，20。除了形态变化外，CP的转录组，蛋白质组和分泌组在疾病12，21，22，23期间也发生了变化。因此，CP的先进知识对于更好地了解其在神经系统疾病中的作用并可能开发新的治疗策略至关重要。
一种将脑室CP准确显微解剖的有效方法是允许正确研究这种微小大脑结构的第一步。由于其高度血管化的性质（图2B），可以使用双目显微镜识别漂浮在脑室腔内的CP。然而，下游分析通常需要经心灌注，使CP组织的正确识别和分离复杂化（图2C）。如果进一步的处理步骤允许（例如，在RNA和蛋白质分析的情况下），可以通过溴酚蓝经心灌注 来 观察CP（图2A）。一些出版物已经描述了从大鼠24 和小鼠幼崽大脑25中分离CP。本文描述了一种显微解剖分离技术，用于从成年小鼠中分离CP。重要的是，这种分离技术保留了CP内细胞的活力，功能和结构。本文描述了漂浮在第四脑室和侧脑室中的CP的分离。简而言之，小鼠被终末麻醉，并在必要时经心灌注。但是，应该注意的是，灌注会损害CP内细胞的结构。因此，如果要使用透射电子显微镜（TEM），连续块面扫描电子显微镜（SBF-SEM）或聚焦离子束SEM（FIB-SEM）分析样品，则不应进行灌注。接下来，整个大脑被隔离，镊子被用来对大脑进行射手半切。从这里，可以识别和解剖漂浮在侧脑室中的CP，而来自第四脑室的CP可以从大脑的小脑侧分离出来。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64733/64733fig02.jpg"> 图 2：（A，D） 溴酚蓝灌注、（B，E） 无灌注和 （C，F） PBS/肝素灌注后的 （A-C） 第四和 （D-F） 侧脑室脉络丛 （CP） 的可视化。 图像是用体视显微镜（8x-32x放大倍率）拍摄的。<a href="https://www.jove.com/files/ftp_upload/64733/64733fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
一旦脑瘫从脑室中正确解剖出来，就可以应用一整套技术来进一步了解这种结构的功能。例如，可以进行流式细胞术或单细胞RNA测序，以量化和表型分析某些疾病条件下的浸润性炎症细胞26，27。除了细胞组成外，还可以 通过酶联 免疫吸附测定（ELISA）、免疫印迹或使用细胞因子珠阵列28同时分析多种细胞因子来分析CP的分子组成以评估细胞因子和趋化因子的存在。此外，转录组、血管、免疫细胞组织学和分泌组分析可以在显微解剖的 CP 外植体上进行 29。在这里，整个卡口CP上的扫描电子显微镜（SEM）用于获得CP结构的整体视图。SEM使用聚焦电子束扫描表面并创建表面形貌和成分的图像。由于电子的波长远小于光的波长，因此SEM的分辨率在纳米范围内，优于光学显微镜。因此，可以通过SEM 进行 亚细胞水平的形态学研究。 简而言之，将解剖的CP立即转移到含戊二醛的固定剂中进行过夜固定，然后进行渗透和乙酸铀酰染色。然后用天冬氨酸铅染色处理样品，脱水，最终包埋进行成像。
因此，该协议有助于从小鼠脑室中有效分离CP，可以使用各种下游技术进一步分析以研究其结构和功能。

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			<td>26G x 1/2 needle</td>
			<td>Henke Sass Wolf</td>
			<td>4710004512</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium specimen mounts</td>
			<td>EM Sciences</td>
			<td>75220</td>
			<td></td>
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			<td>Cacodylate buffer</td>
			<td>EM Sciences</td>
			<td>11652</td>
			<td></td>
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			<td>Carbon steel surgial blades</td>
			<td>Swann-Morton</td>
			<td>0210</td>
			<td>size: 0.45 mm x 12 mm</td>
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			<td>Carbon adhesive tabs -12 mm</td>
			<td>EM Sciences</td>
			<td>77825-12</td>
			<td></td>
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			<td>Critical point dryer&#160;</td>
			<td>Bal-Tec&#160;</td>
			<td>CPD030</td>
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			<td>Crossbeam 540</td>
			<td>Zeiss</td>
			<td></td>
			<td>SEM system</td>
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			<td>Forceps</td>
			<td>Fine Science Tools GmbH&#160;</td>
			<td>91197-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>EM Sciences</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H-3125</td>
			<td></td>
		</tr>
		<tr>
			<td>Ismatec Reglo ICC Digital Peristaltic pump 2-channel</td>
			<td>Metrohm Belgium N.V</td>
			<td>CPA-7800160</td>
			<td></td>
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			<td>Osmium Tetroxide&#160;</td>
			<td>EM Sciences</td>
			<td>19170</td>
			<td></td>
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			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
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			<td>Phosphate buffered saline (PBS)</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td></td>
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			<td>Platinum&#160;</td>
			<td>Quorum&#160;</td>
			<td>Q150T ES</td>
			<td>PBS without Ca++ Mg++ or phenol red; sterile filtered</td>
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			<td>Sodium pentobarbital</td>
			<td>Kela NV</td>
			<td>514</td>
			<td></td>
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			<td>Specimen Basket Stainless Steel</td>
			<td>EM Sciences</td>
			<td>70190-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi DV4 Stereo microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools GmbH&#160;</td>
			<td>91460-11</td>
			<td></td>
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microdissection and whole mount scanning electron microscopy visualization of mouse choroid plexus

脉络丛（CP）是一种突出到脑室的高度血管化结构，是神经科学中最缺乏研究的组织之一。随着越来越清楚的是，这种微小的结构在中枢神经系统（CNS）的健康和疾病中起着至关重要的作用，因此以允许下游处理的方式正确解剖脑室CP至关重要，从功能到结构分析。这里描述了侧脑室和第四脑室小鼠CP的分离，而无需专门的工具或设备。这种分离技术保留了CP内细胞的活力，功能和结构。由于其高血管化，可以使用双目显微镜可视化漂浮在大脑心室腔内的CP。然而，下游分析所需的经心灌注会使 CP 组织的鉴定复杂化。根据进一步的处理步骤（例如，RNA和蛋白质分析），这可以通过经心灌注溴酚蓝 可视化 CP来解决。分离后，可以使用多种技术（包括RNA，蛋白质或单细胞分析）处理CP，以进一步了解这种特殊大脑结构的功能。在这里，整个安装CP上的扫描电子显微镜（SEM）用于获得结构的整体视图。

所描述的方案有助于将CP与小鼠脑侧（图2A-C）和第四（图2D-F）心室有效分离。分离整个大脑后，使用镊子对大脑进行骨状半球化，并识别漂浮在侧脑室中的CP。来自第四脑室的CP可以从大脑的小脑侧分离出来。溴酚蓝灌注可用于可视化CP（图2A，D）;然而，当在进一步?...

Watch this Scientific Journal Video about Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus at JoVE.com

Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

脉络丛（CP）是神经科学中研究不足的组织，在中枢神经系统的健康和疾病中起着关键作用。该协议描述了用于分离CP的显微切割技术，并使用扫描电子显微镜来获得其细胞结构的整体视图。

小鼠脉络丛的显微切割和全安装扫描电子显微镜可视化

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in ...

为了更好地了解脉络丛的组成和结构，将脉络丛从脑室中分离出来的有效方法是宝贵的第一步。通过这种显微切割技术，可以将脉络丛从脑室中解剖出来，同时包含其功能、活力和结构，而不会用周围组织污染脉络丛。脉络丛分离的一个重大障碍是识别结构，因为它仍然漂浮在脑室中。用溴酚蓝染色脉络丛有助于分离。演示该程序的将是我们的实验室经理Elien Van Wonterghem。首先准备终末麻醉液、经心灌注溶液、扫描电子显微镜所需的溶液（SEM）以及文中提到的其他溶液。为了隔离小鼠大脑，将终末麻醉动物置于背褥位置，并通过将四肢固定在板上来固定小鼠。通过喷洒70%乙醇对胸部进行消毒，然后使用碳钢手术刀片在横膈膜下方切开约4厘米的切口。打开皮肤，用手术剪刀露出胸部。将横膈膜完全切开并分开胸部，露出肺部和跳动的心脏。使用灌注泵经心以每分钟4.5毫升的速度向小鼠灌注10毫升灌注溶液。在左心室插入 26 号针头，将溶液泵入全身回路。使用手术剪刀，切开右心房，使血液脱离循环。斩首后，通过从耳朵之间到眼睛上方的切口切开头皮。横向拉动皮肤以露出头骨。然后沿着鳞状缝合线向鼻部切开颅骨。完成后，将大脑放入冰冷的培养皿中，并向脑组织中添加一毫升冰冷的PBS。为了隔离漂浮在第四脑室中的脉络丛，用手术刀轻轻地切断小脑与大脑的联系。从小脑中取出剩余的脑干组织部分。旋转大脑，使切割线朝上，确保第四脑室腔在切片中间可见，脉络丛位于背侧。如有必要，用锋利的镊子拉动结缔组织来打开心室。使用微小锋利的镊子轻轻地将脉络丛从心室壁上撕下。为了将脉络丛与侧脑室隔离开来，使用微小锋利的镊子将大脑切成两个半球。旋转一个大脑半球，使切割线向上。为了露出侧脑室，轻轻地将皮层从丘脑中拉开。将海马体缩回矢状中部切割线。现在可以看到侧脑室，脉络丛位于心室底部。如有必要，用锋利的镊子拉开结缔组织，稍微打开心室。使用微小锋利的镊子轻轻地将脉络丛从心室壁上撕下。为了进行SEM的样品制备，将新鲜分离的脉络丛转移到新鲜的固定溶液中，并在4摄氏度下孵育过夜。完成后，使用三至五毫升0.1摩尔二甲胂酸钠缓冲液洗涤样品三次，每次洗涤五分钟。将样品固定在 3 至 5 毫升 2% 四氧化锇的 0.1 摩尔二甲胂酸钠缓冲液中 30 分钟。然后用三到五毫升的超纯水洗涤样品三次，每次五分钟。接下来，以增加的冰冷乙醇浓度系列使样品脱水，每乙醇溶液15分钟。使用临界点干燥器正确干燥样品。用碳贴纸将样品小心地放在试样支架上，并使用 SEM 可视化脉络丛样品。本协议有助于从小鼠脑侧脑室和第四脑室有效地分离脉络丛。溴酚蓝灌注导致脉络丛的可视化。当溴酚蓝不允许在进一步的处理步骤中用PBS-肝素灌注或不进行灌注进行处理时。脉络丛上皮或CPE细胞表面的SEM形态学分析清楚地显示了第四脑室的两臂结构和与侧脑室隔离的外侧脉络丛的典型C形形式。较高的SEM放大率揭示了CPE细胞顶端侧和微绒毛的囊泡样结构，表明可以使用SEM研究CPE细胞在疾病条件下的形态变化。重要的是只接触和取出脉络丛组织，以免周围组织污染样品。谨慎使用，以保持脉络丛的结构。各种下游技术可以应用于分离的脉络丛，以研究其结构和功能，从显微镜分析到转录组分析再到流式细胞术。将脉络丛从大脑中显微解剖，使我们能够更好地了解这种微小脑组织的结构和功能。这些知识对于更好地了解其在神经系统疾病中的作用至关重要。

Research

JoVE Journal

Neuroscience

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

聚焦离子束铣削和脑组织的扫描电子显微镜

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

从大鼠脑的解剖脑表面血管及相关脑膜和脉络丛的可视描述，

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

脉络丛上皮细胞的纤毛运动的观察<em&gt;离体</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

使用串行块面部扫描电子显微镜脑线粒体分析

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...