这项工作是由Labex MemoLife并通过ARSEP支持（倒基金会欧莱雅助手点菜RECHERCHE河畔拉sclérose恩斑）

1.解决方案和材料<ol><li>准备隔离器的解决方案:B1，加1.5毫升HEPES为1M至150ml的HBSS; B2，加3.6克葡聚糖到20ml B1的; B3，加1克的BSA至100毫升B1的。 </li><li>通过切割掉底的上旋拧部分修改过滤器保持器。 </li><li>制备免疫染色方案:固定溶液，在pH 7.4的PBS的4％多聚甲醛;透/封闭溶液，稀释山羊血清至5％和Triton X100至0.25％的pH 7.4的PBS。 注意:固定取决于所用第一抗体的类型。 </li></ol> 2.解剖注意:无菌条件不是必需的，除非船舶用于细胞培养的目的。 <ol><li>准备150毫升的烧杯用20毫升B1的。置于冰上，并用封口膜覆盖，避免空气污染。 </li><li>深深麻醉鼠标引擎盖下的小纸巾浸泡在1毫升纯异氟醚被加入Int澳笼。麻醉是由于缺乏反应到足尖捏验证。杀死小鼠颈椎脱位。 注意:这些步骤都完成符合国家和机构的规定。 </li><li>可选:用20毫升的PBS 1X中进行心内灌注，消除血液中的含量24。 </li><li>第皮肤从颈部到鼻子手术刀将其拉离。删除所有污染的毛发，用PBS 1X。 </li><li>要打开颅骨，先插入剪刀前方的嗅球，并打开剪刀破裂的头骨两部分。 </li><li>小心取出用脑铲大脑。解剖出脉络丛从 ​​侧脑室，因为它们会污染血管制剂38。 注:可选:最后的准备工作将包括实质和脑膜血管。如果不希望的，脑膜可剥离以下由38描述的过程。 </li><li> TRansfer脑入含冰B1液的烧杯中。多达8个的大脑可以一起处理。 </li></ol> 3.脑组织均质<ol><li>使用两个解剖刀，在B1的溶液随后获得小块的约2mm手动大力击败大脑。 </li><li>均质化制剂用automatized Dounce匀浆，执行20笔在400rpm。确保玻璃管被保持在冰中，该douncer的上部是在溶液中移动时上下，从而防止气穴的形成。如果几个样品制备，每间同质化离子水冲洗douncer。 </li></ol> 4.血管净化<ol><li>转移匀浆到50ml的塑料管，然后继续离心以2,000rpm离心10分钟，在4℃。一块巨大的白色接口（主要是髓鞘）将形成对血管颗粒（红色的顶部，如果未进行灌注）。 </li><li>弃去上清液。该船颗粒和白色的接口将保持连接在一起。加入20毫升冰冷的B2解决方案，手动和大力摇晃管1分钟。 </li><li>进入第二离心4400克15分钟，在4℃。髓鞘现在将形成致密的白色层在上清液的表面上。 </li><li>仔细保持所述管和缓慢转动它，以允许上清液通过沿着壁分离从管壁髓鞘层。丢弃髓鞘与上清。保持附着在管的底部含有血管沉淀。 </li><li>吸干管的内壁与吸收纸缠有5毫升的塑料吸管和除去所有的残余液体，避免触及容器沉淀。保持在一个吸收纸管倒置排出任何剩余的液体。 </li><li>移液器向上和向下低结合的技巧，keepi暂停1毫升冰冷的B3解决方案的颗粒纳克管在冰上，再加入另外的5毫升B3的解决方案。确保船只分散尽可能并且不形成聚集体。 </li></ol> 5.过滤<ol><li>准备在冰上的烧杯用30ml冰冷却的溶液B3。用封口膜覆盖，避免空气污染。 </li><li>放置在修改后的过滤器保持器20微米筛网过滤器上的贝克尔烧瓶的顶部并通过施加将10ml冰冷B3溶液平衡。 </li><li>倒在过滤器上的船只准备和冲洗容器用40毫升冰冷的B3的解决方案。 </li><li>用干净的镊子恢复的过滤器，并立即沉浸在包含B3溶液的烧杯中。通过轻轻晃动松脱，从过滤器的船只。 </li><li>倒在50ml塑料管中并离心烧杯内容在2000克5分钟，在4℃。 </li><li>注意:可替换地，大脑血管&#39;从步骤4.6的悬浮液可以过滤到一个100微米的网过滤器。在这种情况下，较大的血管优先保留在过滤器上，而流过的包含微血管（主要是毛细血管），其然后在20微米的网过滤器过​​滤如上。 </li><li>悬浮微血管的沉淀在1ml冰冷却的溶液B3和吹打它至1.5 ml Eppendorf管中传送它。离心机在2000克5分钟，在4℃。 </li></ol> 6.固定，透和阻塞<ol><li>转船吹打成0.2毫升含有PBS使用1.0毫升低结合尖端PCR管。注意不要触及容器，因为它们可能会附着在笔尖的外部分。执行双目显微镜下的所有执行下列步骤。 </li><li>对于以下每个介质改变，离开管在冰上直到船只已达到的底部，并吸取出来最采用长和东西凝胶加载枪头的液体。 </li><li>除去大部分的PBS，并添加200微升固定液。暂停船只通过轻轻摇动孵育20分钟，在室温。 </li><li>吸取出固定液，并将其用200μl的1×PBS（第一次洗涤）的替换，孵育5分钟，在室温，重复此步骤3次。每次，验证血管已正确沉到管和移液管出来的1×PBS的底部，确保船只未触及。 </li><li>在最后一次洗涤后，更换1×PBS中由透/封闭溶液孵育1小时，在室温，通过轻轻摇动不时重悬血管。 </li></ol> 7.免疫染色<ol><li>更换透/阻断原发性抗体（在这里看到的和稀释液用在材料模板表的抗体的引用）稀释，在通透/封闭液的混合溶液中，孵育4°CO / N。 </li><li>洗涤3次后在PBS中在RT下，孵育第二抗体的混合物在PBS中稀释2小时的船只在RT。 </li><li>注:强烈建议进行核染色，用Hoechst（1:2000）:为了区分可能在显微镜下污染的毛或灰尘船（2000一）或DAPI。 </li><li>洗涤3次后在PBS中，重悬船只于50μlPBS中。 </li></ol> 8.安装和观测<ol><li>准备一个尖端硅化玻璃毛细管:用手术刀切P200的枪头和内部调整的毛细血管。毛细管的近似体积为40微升。 </li><li>使用硅化玻璃毛细管转移血管载玻片并小心地用一块吸收纸除去液体。 </li><li>应用安装中的单滴到血管和保持一个盖玻片在45°使降沿滑的边缘扩散。让熄灭盖玻片，并允许媒体传播缓慢。待其干燥O / N在室温避光。船只可以在一个fluoresc观察耳鼻喉科共聚焦显微镜。 </li></ol>

The authors have nothing to disclose.

血 - 脑屏障调节生理物质在中枢神经系统中的进出的通道和保护其不受存在于血液中潜在的有害物质。它参与几个CNS病症，包括神经退行性疾病2和脑肿瘤28。血脑屏障的渗透率极低的也阻碍了靶向神经细胞和方法意欲可逆打开血脑屏障无有害的后果对大脑的研究3一个非常活跃的研究领域的发展的治疗剂的通过。已作出许多努力来开发有价值模型和技术允许细胞，分子和药...

中枢神经系统（CNS）的正常功能需要一个高度调节细胞外的环境中，以及相对于其他器官1其代谢需求是巨大的。中枢神经系统也是范围广泛的化学品，通常无害的外周器官，但它，神经毒性非常敏感。为确保正常运行，大部分的中枢神经系统"血管形成的血管内皮屏障;血-脑屏障（BBB），其控制分子和离子的流动，以及血液和大脑之间的免疫细胞的通路，从而保持适当的稳态2，也限制了治疗性药物的条目，从而妨碍治疗神经系统疾患3。在细胞水平，血脑屏障主要持续由内皮细胞，流出转运偏振光表达和非常低的胞转率4之间广泛紧密连接。性能及血脑屏障的功能大多是诱发NEighboring细胞4。具体地，周细胞发挥 ​​诱导和维持所述BBB 5,6中起重要作用。作为收缩细胞，周也调节血流量7做周围大血管的平滑肌细胞。最后，星形胶质细胞，脑的主要神经胶质细胞，发送名为endfeet围绕大型进程最大脑脉管8和调制血脑屏障完整性和免疫静止9，代谢物转移到神经元10，和诱导神经元活性之间的紧耦合和血流11,12。 研究脑血管系统的细胞和分子性质的能力是非常重要的特点更好其脑生理学和病理生理学的贡献。为了解决这个问题，已经发展战略，以隔离大脑的脑血管系统，该系统允许完整的脑血管片段的准备。脑血管purification被使用牛脑中13最初描述和改进，并适用于其它物种，特别是啮齿动物14。在这最后的研究中，使用不同大小的过滤器被引入到分离脑血管中，以级分富含不同直径的容器。有趣的是，在这些制剂，内皮细胞保持它们的代谢特性15，转运功能16和17的极化。在这里，我们详细描述了这个协议，并进一步证明了隔离容器保留大部分的原位结构。内皮细胞保持通过紧密连接相连，通过一个连续的基底层包围。周细胞和平滑肌细胞保持附着在内皮细胞层，以及血管周围星形胶质细胞膜。然而，星形胶质细胞，小神经胶质细胞，神经元和少突胶质细胞被消除。最后，我们描述了一个过程来执行免疫染色对离体脑血管。 </P&gt; 直至现在大多数关于脑血管系统的分子和细胞研究已经由解离纯化脑血管细胞进行细胞分选使用细胞特异性报道的小鼠品系或免疫染色基于程序18,19。尽管这些技术允许几乎纯脑血管的细胞群的分离，分离的细胞完全丧失其原位形态学和相互作用，这反过来又极大地影响它们的分子和细胞的特性。在这里描述的协议，从而允许整个脑血管片段无需特异性抗体或转基因小鼠的污渍的隔离，提供了一个很好的选择，作为分离的脑血管的总体结构是保守的，因此，减少它们的分子性能影响。隔离容器然后可以用于研究基因的活性，蛋白质合成和调节在所述BBB作为最近描述20,21 </suP&gt;。最后，相对于激光捕获显微切割22,23，本协议是廉价的，易于实施，并在任何实验室迅速适应。

<table><tbody><tr><td>Tissue Grinder Size C</td><td>Thomas scientific</td><td>3431E25</td><td></td></tr><tr><td>centrifuge 5415&amp;nbsp;R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>centrifuge 5810 R</td><td>Eppendorf</td><td>5811000320</td><td></td></tr><tr><td>High-performance, Modular Stereomicroscope</td><td>Leica</td><td>MZ6</td><td></td></tr><tr><td>Compact System Provides High Quality Leica LED1000</td><td>Leica</td><td>LED1000</td><td></td></tr><tr><td>low binding tips (P1000)</td><td>Sorenson BioScience</td><td>14200T</td><td></td></tr><tr><td>Swinnex 47 mm filter holder PP 8/Pk</td><td>Millipore</td><td>&lt;a href=&quot;http://www.millipore.com/catalogue/itemdetail.do?id=SX0004700&quot;&gt;SX0004700&lt;/a&gt;</td><td></td></tr><tr><td>Nylon net filter disc Hydrophilic 20 &amp;micro;m 47 mm 100/Pk</td><td>Millipore</td><td>&lt;a href=&quot;http://www.millipore.com/catalogue/itemdetail.do?id=NY2004700&quot;&gt;NY2004700&lt;/a&gt;</td><td></td></tr><tr><td>Nylon net filter disc Hydrophilic 100 &amp;micro;m 47 mm 100/Pk</td><td>Millipore</td><td>&lt;a href=&quot;http://www.millipore.com/catalogue/itemdetail.do?id=NY1H04700&quot;&gt;NY1H04700&lt;/a&gt;</td><td></td></tr><tr><td>Standard Wall Borosilicate Tubing</td><td>Sutter Instrument</td><td>B150-86-7.5</td><td></td></tr><tr><td>Microscope Slides</td><td>Thermo Scientific</td><td>1014356290F</td><td></td></tr><tr><td>Cover Slips, Thickness 1</td><td>Thermo Scientific</td><td>P10143263NR1</td><td></td></tr><tr><td>0.2 ml Thin-walled tubes and domed cap</td><td>Thermo Scientific</td><td>AB-0266</td><td></td></tr><tr><td>&amp;nbsp;PARAFILM M (roll size 4 in. &amp;times; 125 ft)</td><td>Sigma</td><td>P7793-1EA</td><td></td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>Life technology</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/14175129&quot;&gt;14175-129&lt;/a&gt;</td><td></td></tr><tr><td>HEPES (1M)</td><td>Life technology</td><td>15630056</td><td></td></tr><tr><td>Dextran from Leuconostoc spp. Mr ~70,000</td><td>Sigma</td><td>31390</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>10x PBS</td><td>Euromedex</td><td>ET330</td><td></td></tr><tr><td>16% Formaldehyde (w/v), Methanol-free&amp;nbsp;</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Triton&lt;sup&gt; &lt;/sup&gt; X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>bisBenzimide H 33342 trihydrochloride (Hoechst)</td><td>Sigma</td><td>14533</td><td></td></tr><tr><td>Mounting medium Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor 488 Conjugate; Dilution 1/100</td><td>Life technology</td><td>I21411</td><td></td></tr><tr><td>Agrin (rabbit) ; dilution 1/400</td><td>kindly provided by Dr Markus A Ruegg</td><td></td><td></td></tr><tr><td>Anti ZO-1 (mouse, clone 1A12)</td><td>Life technology</td><td>33-9100</td><td>dilution 1:500</td></tr><tr><td>Anti Smooth Muscle Actin (mouse, clone 1A4)</td><td>Sigma</td><td>A2547</td><td>&amp;nbsp;dilution 1:500</td></tr><tr><td>Anti GFAP (mouse, clone GA5)</td><td>Sigma</td><td>G3893</td><td>&amp;nbsp;dilution 1:500</td></tr><tr><td>Anti AQP4 (rabbit)</td><td>Sigma</td><td>A5971</td><td>&amp;nbsp;dilution 1:500</td></tr><tr><td>Anti Cx43 (mouse, Clone&amp;nbsp; 2)</td><td>BD Biosciences</td><td>610061</td><td>&amp;nbsp;dilution 1:500</td></tr><tr><td>Anti Olig2 (rabbit)</td><td>Millipore</td><td>AB9610</td><td>&amp;nbsp;dilution 1:200</td></tr><tr><td>Anti NF-M (mouse)</td><td>provided by Dr Beat M. Riederer, University of Lausanne, Switzerland.</td><td>&amp;nbsp;dilution 1:10</td><td></td></tr><tr><td>Anti Iba1 (rabbit)</td><td>Wako</td><td>019-19741</td><td>&amp;nbsp;dilution 1:400</td></tr><tr><td>Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody</td><td>Life technology</td><td>A11029</td><td>&amp;nbsp;dilution 1:2,000</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Life technology</td><td>A11034</td><td>&amp;nbsp;dilution 1:2,000</td></tr><tr><td>Alexa Fluor 555 Goat Anti-Mouse IgG (H+L) Antibody</td><td>Life technology</td><td>A21424</td><td>&amp;nbsp;dilution 1:2,000</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate</td><td>Life technology</td><td>A21429</td><td>&amp;nbsp;dilution 1:2,000</td></tr></tbody></table>

purification of mouse brain vessels

在大脑中，大部分的血管系统包括一个选择性屏障，血 - 脑屏障（BBB），调节脑和血液之间的分子和免疫细胞的交换。此外，庞大的神经细胞代谢的需求，需要一个时刻到时刻调节血流量。值得注意的是，这些规定的异常是最脑部病变的病因特点;包括成胶质细胞瘤，中风，水肿，癫痫症，退化性疾病（例如:帕金森氏病，阿尔茨海默氏病），脑肿瘤，以及炎症性疾病，如多发性硬化症，脑膜炎和脓毒症诱导的脑机能障碍。因此，了解的信号传导事件调制脑生理学是一个重大的挑战。多洞察构成该脑血管系统中的各种细胞类型的细胞和分子性质可以从原代培养物或细胞从新鲜分离的脑组织排序来获得。然而，如细胞极性，形态和细胞间的关系属性不保持在这样的准备。我们在这里描述的协议被设计成净化脑血管片段，同时保持结构的完整性。我们表明，隔离容器包括由是由一个连续的基底层包围紧密连接密封内皮细胞。周细胞，平滑肌细胞以及血管周围星形胶质细胞endfeet膜保持连接到内皮细胞层。最后，我们将介绍如何在纯化脑血管进行免疫染色实验。

在这里，我们描述了一种协议，允许为脑血管14的机械隔离。 图1概括了该技术的主要步骤。脑血管的结构是复杂的，并且包括几个细胞类型， 即，由紧密连接密封，并用周细胞，平滑肌细胞，和星形胶质细胞足突9包围内皮细胞。因此，下面的脑血管的隔离，我们的目的是通过如在我们的协议的第二部分中描述的免疫染色的纯化血管的结构表征。集聚蛋白，内?...

Watch this Scientific Journal Video about Purification of Mouse Brain Vessels at JoVE.com

Purification of Mouse Brain Vessels

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

23.3K 次观看.  Institut National de la Santé et de la Recherche Médicale. We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes. 

视频: Purification of Mouse Brain Vessels

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

成年鼠脑铸造三维成像和形态分析

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

科研

神经科学

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

代的永生化小鼠脑微血管内皮细胞株作为<em在体外</em血脑屏障型号

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

原代鼠脑微血管内皮细胞的分离

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

硬膜扩张症：血管性痴呆和阿尔茨海默病

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to enter and expel excessive amino acids or toxins from the brain. To model how the BBB can be compromised in diseases like vascular dementia (VaD) or Alzheimer's disease (AD), researchers developed novel methods to model vessel dilatation. A compromised BBB in these disease states can be detrimental and result in the dysregulation of the BBB leading to untoward and pathological consequences impacting brain function. We were able to modify an existing technique that enabled us to inject directly into the Cisterna magna (CM) to induce dilatation of blood vessels using elastase, and disrupt the tight junctions (TJ) of the BBB. With this method, we were able to see various metrics of success over previous techniques, including consistent blood vessel dilatation, reduced mortality or improved recovery, and improving the fill/opacifying agent, a silicone rubber compound, delivery for labeling blood vessels for dilatation analysis. This modified minimally invasive method has had promising results, with a 19%-32% increase in sustained dilatation of large blood vessels in mice from 2 weeks to 3 months post-injection. This improvement contrasts with previous studies, which showed increased dilatation only at the 2 week mark. Additional data suggests sustained expansion even after 9.5 months. This increase was confirmed by comparing the diameter of blood vessels of the elastase and the vehicle-injected group. Overall, this technique is valuable for studying pathological disorders that affect the central nervous system (CNS) using animal models.

作者聚焦：模拟转基因小鼠血管对阿尔茨海默病的影响

We demonstrate chemically inducing large blood vessel dilatation in mice as a model for investigating cerebrovascular dysfunction, which can be used for vascular dementia and Alzheimer's disease modeling. We also demonstrate visualizing the vasculature by injecting silicone rubber compound and providing clear visual guidance for measuring changes in blood vessel size.

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

生物学

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

一种从小鼠中分离大脑围取体的高输出方法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function&#8212;integral to the physiology and etiology of chronic degenerative diseases&#8212;requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.
In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial &#34;tubes&#34; (diameter, ~25 &#181;m) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 &#176;C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Intensive preparation of intact mouse cerebral endothelial &#34;tubes&#34; from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

小鼠脑实质小动脉内皮的分离及功能分析

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

Isolating Mouse Brain Vessels: A Protocol to Isolate Intact Vessels from Murine Brain Sample

Source: Boulay, A. C. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/53208" target="_blank">Purification of Mouse Brain Vessels.</a> J. Vis. Exp. (2015)
This video demonstrates the isolation of intact vessels from a mouse&#39;s brain that constitute the blood-brain barrier (BBB) along with neurons and glial cells. The isolated vessels can be used as an in vitro model to study their role in maintaining brain homeostasis.

分离小鼠脑血管:从小鼠脑样本中分离完整血管的方案

Source: Boulay, A. C. et al. Purification of Mouse Brain Vessels. J. Vis. Exp. (2015)
This video demonstrates the isolation of intact vessels from a ...

对小鼠脑血管净化

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此视频中的章节

成年鼠脑铸造三维成像和形态分析

代的永生化小鼠脑微血管内皮细胞株作为

原代鼠脑微血管内皮细胞的分离

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

一种从小鼠中分离大脑围取体的高输出方法

小鼠脑实质小动脉内皮的分离及功能分析

分离小鼠脑血管:从小鼠脑样本中分离完整血管的方案