Name: isolation of cerebral capillaries from fresh human brain tissue
Uploaded: 2018-09-12T13:00:00.000+00:00
Duration: 6 min 35 s
Description: Watch this Scientific Journal Video about Isolation of Cerebral Capillaries from Fresh Human Brain Tissue at JoVE.com

我们感谢并承认在英国 ADC 脑组织银行的彼得纳尔逊博士和索尼娅. 安德森提供了所有人脑组织样本 (NIH 赠款号: 国立老龄研究所的 P30 AG028383)。我们感谢马特哈兹达和汤姆. 杜兰, 信息技术服务, 学术技术和教师参与, 肯塔基大学的图形协助。该项目由国家神经系统疾病和中风 (1R01NS079507) 和1R01AG039621 国家老龄研究所的赠款编号 (a.m.s. H) 支持。内容完全是作者的责任, 不一定代表国家神经系统疾病和中风研究所或国家老龄研究所的官方意见。作者声明没有竞争的财政利益。

下面的信息是基于目前的安全和管理标准在肯塔基大学, 列克星敦, 肯塔基州, 美国。作为一种安全预防措施, 请参考该机构的生物安全计划和最新的法规和建议, 然后再与人体组织合作。
注意: 人体组织可以是血液传播病原体的来源, 包括人体免疫机能丧失病毒 (HIV)、乙肝病毒 (HBV)、丙型肝炎病毒 (HCV) 等。与人体组织一起工作会造成血液传播病原体感染的风险。因此, 在与人体组织合作保护实验室人员时, 一定要考虑到一些规章制度和安全问题。与美国的人体组织合作需要一个生物安全2级实验室以及安全预防措施和培训, 按照 NIH 科 IV-B-7, 1970年 5 (a) (1) 条的 OSHA 法案和用户的机构生物安全计划。一般而言, 必须在进行任何涉及人体材料 (组织、体液) 的研究之前获得机构生物安全委员会和/或机构审查委员会的批准。所有从事人体材料工作的人员都需要进行培训, 包括基本的实验室安全培训,例如化学卫生和实验室安全, 以及关于生物安全、危险废物和人类的具体培训。血液传播的病原体。所有使用人体材料的人员都强烈建议在使用人体材料之前获得乙肝疫苗接种。人员需要佩戴特定的个人防护设备, 同时使用人体材料, 例如, 袖口的实验室大衣和面罩, 并随时佩戴手套。所有工作都在生物安全柜 (2 类) 中进行。所有与人体材料接触的设备和从人体材料中产生的任何废料都应妥善处理, 以防止人员受到污染和/或感染。所有设备和表面清洗10% 漂白剂和75% 乙醇后, 每一个程序涉及人体材料。必须立即清理与人体材料的泄漏。玻璃器皿每次使用后都要蒸压。废物, 包括未固定的人体组织, 是收集在一个标签的生物危害废物袋和蒸压。锐器收集在一个穿刺和防漏容器标记为 biohazardous。所有从人体材料中产生的废料都是根据该机构的生物安全规定处置的。
注: 我们的实验室通过桑德斯-棕褐色中心 (IRB #B15-2602 米) 获得来自已故个体的新鲜额皮质样本。纳入标准是: 在英国的招生-ADC 纵向解剖队列研究和验尸间隔 (PMI) ≤4 h64。验尸遵循标准协议, 所有程序符合 NIH Biospecimen 最佳做法准则65。短的 PMI 低于4小时是最重要的, 以确保毛细血管存活后的隔离。新鲜和冰冻的组织都可以使用。如果需要冷冻, 新鲜获得的人脑组织应在液氮中休克冷冻, 储存在-80 摄氏度。新鲜或解冻的组织应存储在隔离缓冲 (见下文) 和处理迅速。我们发现, 10 克的新鲜人体组织产生约100毫克的脑毛细血管 (湿重)。
1. 设置
<ol><li>
缓冲准备 注意: 所需的缓冲量取决于组织的数量。以下协议中的所有缓冲卷都基于 10 g 的人脑皮质组织。<ol>
<li>L 隔离缓冲: 使用 Dulbecco 磷酸盐缓冲盐水的1.5 升 (DPBS; 2.7 毫米氯化钾, 1.47 毫米的2PO4, 136.9 毫米氯化钠, 8.1 毫米 Na2HPO4, 0.9 毫米 CaCl2, 0.49 毫米氯化镁2) 和补充与5毫米 d-葡萄糖 (1.35 g) 和1毫米丙酮酸钠 (0.165 克)。添加葡萄糖和丙酮酸后, 用氢氧化钠调节 pH 值7.4。冷却和存储缓冲区到4°c 之前使用。</li>
		<li>牛血清白蛋白 (bsa): 添加10克 BSA 粉到1升的隔离缓冲, 最后的 BSA 浓度为1%。搅拌缓慢, 以避免气泡, 调整到 pH 值 7.4, 并存储在4摄氏度过夜。立即使用前, 轻轻搅拌;避免形成气泡以避免白蛋白变性。</li>
		<li>密度梯度介质: 将浓度为18克的密度梯度介质放入玻璃瓶中, 加入一个磁搅拌杆。加入60毫升的隔离缓冲液, 用力摇动5分钟, 直到所有粉末悬浮。隔夜存放在4摄氏度, 允许密度梯度介质溶解。使用前先搅拌10分钟。</li>
		<li>将所有缓冲区存储在4摄氏度;在整个隔离过程中, 将所有工具和缓冲放在冰上。使用前请先搅拌所有缓冲器。</li>
	</ol>
</li>
	<li>
实验设置
	<ol>
<li>将波特-Elvehjem 组织磨床的杵装在电子架空搅拌器上。将波特-Elvehjem 组织磨床和 Dounce 均质机与杵在冰盖下。准备一个300µm 过滤网 (5 x 5 厘米2), 折叠它到一个锥, 并插入和附加到50毫升猎鹰管带胶带 (图 1A)。</li>
		<li>放置连接环和细胞应变过滤器 (孔隙大小:30 µm) 在50毫升猎鹰管。准备 biohazardous 垃圾袋。将所有必需的设备放在安全柜中 (见材料表)。</li>
	</ol>
</li>
</ol>2. 脑标本准备
注:图 1A显示了下面描述的整个隔离过程的工作流图表。人脑组织可以从皮层的任何部分中产生, 可以被新鲜或冰冻使用。冷冻脑组织可在室温下解冻 (无缓冲; ~ 30 分钟10克)。为了达到可比较的结果, 大脑组织应该从同一脑区获得每个实验。该协议是为新鲜的 (PMI < 4 小时) 的人大脑皮层没有被冻结的优化。
<ol><li>人脑组织的制备: 记录脑组织的重量。以下协议中的所有数字适用于10克新鲜人脑组织。将脑组织放置在100毫米培养皿中。小心地取出所有的脑膜与镊子。用手术刀切断白色物质。</li>
	<li>切碎的人脑组织: 小心地切断脑组织和碎它与手术刀。剁碎约5分钟 (2–3毫米片)。将脑组织转移到波特-Elvehjem 组织磨床上。添加30毫升的隔离缓冲。 注: 切碎的组织片很难看到, 因为大脑组织通过粉碎过程变成糊状。</li>
</ol>3. 同质化
<ol><li>波特-Elvehjem 组织磨床 (清除: 150–230µm): 融汇每个样品与100冲程在一个均质机速度 50 rpm。记录每25次笔画的时间和100笔画所需的总时间。见表 1所提议的均匀化协议;人额皮质均质10克的总时间约为22分钟。不要在空气中搅拌以防止气泡。</li>
	<li>Dounce 均质机 (游隙: 80–130µm): 将匀浆输送到冰上 Dounce 匀能机上。融汇20冲程 (~ 6 s/冲程, 总2分钟) 的悬浮。避免气泡。</li>
</ol>4. 离心
<ol><li>将脑匀浆均匀分布到四50毫升离心管中, 并记录匀浆的总容积。将50毫升的密度梯度缓冲器分配到离心管 (每管12.5 毫升)。使用10毫升的隔离缓冲液冲洗杵和均质体, 并分配到四离心管 (每管2.5 毫升)。</li>
	<li>用瓶盖紧紧地关闭离心机管。将匀浆、密度梯度介质和缓冲液混合在一起, 用力摇动管子。离心机在 5800 x g为15分钟在4°c (固定角度转子);选择中等减速速度以保持颗粒附着在管上。在2毫升的 1% BSA 中丢弃上清和并用重悬每个颗粒。</li>
</ol>5. 过滤
注意: 要将毛细血管与红细胞和其他细胞碎片分开, 需要采取一些过滤步骤。
<ol><li>300µm 网: 重新悬浮颗粒后, 通过300µm 网格过滤悬浮液。毛细血管通过网格过滤, 而较大的血管和更大的脑碎片留在网格上。仔细清洗网格, 多达50毫升 1% BSA。丢弃网格。 注: 这一过滤步骤清除了任何较大的血管或脑碎片大块的毛细管悬浮。</li>
	<li>
30µM 细胞应变过滤器 注意: 这个过滤步骤将毛细血管与红细胞和其他脑部碎片隔开。<ol>
<li>将毛细管滤液从步骤6.1 分布于五30µm 细胞应变过滤器 (每细胞应变过滤器约10毫升的毛细管滤液)。毛细血管被这种过滤器所控制, 而红细胞、其他单细胞和小的脑碎片通过过滤器并收集在滤液中。</li>
		<li>用25毫升 1% BSA 清洗每个过滤器。然后, 将所有滤液在第六过滤器上, 以增加产量。用50毫升 1% BSA 清洗每个过滤器;保持细胞应变过滤器含有毛细血管, 并丢弃滤液。</li>
	</ol>
</li>
</ol>6. 毛细管收集
<ol><li>将过滤器倒置, 用50毫升 1% BSA 清洗毛细血管, 将每个过滤器变成50毫升管。轻轻地将压力与5毫升 pipettor 的吸管尖端, 并移动它通过过滤器冲洗脑毛细血管。</li>
	<li>一定要洗掉所有的脑毛细血管, 特别是从过滤器的边缘。避免气泡, 因为这使得过滤过程更加困难, 增加了毛细血管损耗的机会。</li>
</ol>7. 洗涤
<ol><li>收集毛细血管后, 离心机所有样品在 1500 x g 3 分钟在4°c (摆动斗转子)。取出上清液并在大约3毫升的隔离缓冲器中重新悬浮颗粒。将所有悬浮颗粒从一个15毫升锥形管中的一个样品中组合, 然后用隔离缓冲器填充。离心机再在 1500 x g 3 分钟在4°c 和洗涤二次。</li>
	<li>用显微镜 (100X 放大) 和照相机 (图 1B) 记录毛细管纯度。 注:10 克人脑组织的脑毛细血管产量通常约为100毫克。分离的脑毛细血管现在可以用于实验, 处理 (e., 裂解, 膜分离), 或被闪光冷冻和储存在-80 °c 在 cryotubes 至少6–12月 (避免多个冻融循环)。</li>
</ol>

作者没有什么可透露的。

本议定书描述了从新鲜组织中分离完整和可行的人脑毛细血管。在本节中, 我们将详细讨论以下内容: 1) 对协议的修改, 2) 常见错误的疑难解答, 3) 技术的局限性, 4) 该模型对于现有的和替代的血脑屏障模型的意义, 以及 5)用于隔离人脑毛细血管的潜在应用。
这里描述的协议是优化 10 g 的新鲜人类额叶皮质组织。然而, 相对简单的修改这个程序为: 1) 或多或少超过10克的组织, 2) 冷...

血脑屏障是血液和大脑之间的紧密控制的界面, 它决定了大脑中的什么进入和出来。解剖学上, 内皮细胞构成血脑屏障, 形成一个复杂的连续毛细血管网络。从生理上来说, 这种毛细管网络为大脑提供氧气和营养, 同时处理二氧化碳和代谢废物。重要的是, 证据支持对屏障的改变会导致许多病症, 包括阿尔茨海默病、癫痫和中风1、2、3、4、5,6,7. 脑血管内皮细胞也可以阻断药物对大脑的吸收, 例如肿瘤切除术后化疗多形性胶质瘤,8,9, 10. 在这方面, 孤立的人脑毛细血管代表了一种独特的前体血脑屏障模型, 它与体内的屏障特性密切相关,可以对健康的屏障功能和功能障碍进行研究。和疾病。在这篇文章中, 我们提供了一个协议, 以保持良好的毛细血管质量和产量, 以研究血脑屏障的人脑毛细血管从人脑。
在 1969年, Siakotos 。11是第一个报告使用密度梯度离心和玻璃珠柱分离的牛和人脑组织的脑毛细血管分离。后来,等等。12通过添加多个过滤步骤, 减少了研究大鼠脑毛细血管所需的组织量, 同时保持葡萄糖转运的代谢活动, 改进了这种方法。从那时起, 研究人员对毛细血管隔离程序进行了多次优化, 改进了每次迭代13、14、15的方法和脑毛细管模型。例如, Pardridge等。16分离的牛毛细血管使用酶消化而不是机械均匀化, 然后通过一个毛细管悬浮通过一个210µm 网格过滤器和一个玻璃珠柱。这些修改改善了孤立脑毛细血管的台盼蓝排斥染色, 从而提高了内皮细胞的生存能力。二十世纪九十年代代初, 达赖尔. 17 glutamyl transpeptidase (γ-GTase) 和碱性磷酸酶的分离的牛和大鼠毛细血管, 其神经元污染明显, 维持代谢活性。在 2000年, 米勒和al。18、用分离的大鼠和猪脑毛细血管与共焦显微镜相结合, 显示输送基质在毛细血管腔内的积聚。随后, 我们的实验室继续优化脑毛细血管隔离程序, 我们已经建立了运输化验, 以确定 p-糖蛋白 (p gp)19,20,21, 乳腺癌抗性蛋白 (BCRP)22,23, 多药耐药性蛋白 2 (Mrp2)24运输活动。在 2004年, 我们发表了两份报告, 我们使用孤立的大鼠脑毛细血管调查各种信号通路。在 Hartz等。21, 我们发现肽 endothelin-1 通过内皮素受体 b (ETb) 受体、一氧化氮合酶 (NOS) 和蛋白激酶 C (PKC), 快速、可逆地减少脑毛细血管中的 P gp 转运功能。在鲍尔等。19, 我们显示核受体 pregnane X 受体 (PXR) 的表达, 并显示 PXR 调节的 P gp 表达和传输功能的脑毛细血管。在转基因人性化 PXR 小鼠实验中, 我们扩大了这一研究方向, 并通过 hPXR 活化25对植入 P gp在体内收紧屏障。在 2010年, Hartz 。26使用这种方法来恢复过度表达 tshr 开心的转基因人淀粉样蛋白 (开心) 小鼠的 p-gp 蛋白表达和转运活性。此外, 恢复开心小鼠 p-gp 显著降低淀粉样β (Aβ)40和 Aβ42的大脑水平。
除了研究信号通路, 孤立的脑毛细血管可以用来确定毛细血管通透性的变化, 我们称之为毛细血管渗漏。特别是, 德州红色泄漏检测是用来评估从毛细管腔内的荧光染料的泄漏, 从毛细血管流明, 然后这些数据被用来分析泄漏率。与控制毛细血管相比, 毛细血管渗漏率的增加表明血脑屏障2的物理完整性发生了变化。这是有价值的, 因为有许多疾病状态与障碍干扰, e., 癫痫, 多发性硬化症, 阿尔茨海默病, 创伤性脑损伤27,28,29, 30。其他组也利用孤立的毛细血管来辨别信号通路, 调节蛋白质表达和蛋白质31,32,33,34的传输活动, 35,36,37。最后, 我们继续优化这种方法, 以隔离人脑毛细血管和, 最近, 我们显示增加 P gp 表达在人血脑屏障的癫痫患者相比无癫痫控制个人38.结合起来, 这些发展表明, 孤立的脑毛细血管可以作为一个多功能模型来研究屏障功能。
各种体内、体外、内外血脑屏障模型已应用于基础研究和工业药物筛选, 主要目的是检测药物向大脑的传递39、40、41 ,42,43,44。除了离体脑毛细血管外, 目前的血脑屏障模型包括在硅模型中, 分离的脑毛细血管内皮细胞的离体细胞培养或永生化细胞系从各种人多潜能干细胞 (hPSC) 的体外培养, 分化为脑毛细血管内皮细胞和微流控芯片模型。
在以预测吸收、分布、新陈代谢和排泄 (ADME) 性质为基础的药物开发中, 在硅模型中最常用的方法是选择药物候选者。定量结构-属性关系 (QSPR) 模型和定量结构-活动关系模型是高吞吐量筛选的常用方法, 用于预测药物候选者的脑内渗透率。45,46. 这些模型对于屏蔽渗透特性的分子是有用的。
Betz等。47建立培养的脑毛细血管内皮细胞作为体外血脑屏障模型体系。体外细胞培养模型使用新鲜组织或永生化内皮细胞系如人脑微血管内皮细胞 (hCMECs) 可以是另一个高通量筛选工具的大脑渗透或机械研究。然而, 脑毛细血管内皮细胞培养模型缺乏毛细血管腔内血流的生理剪应力, 在整体生物学复杂性上受到限制, 并经历了重要屏障成分的表达和定位变化。如紧密连接蛋白, 表面受体, 转运体, 酶, 离子通道48,49,50。反之, 从 hPSCs 中提取的内皮细胞, 与 hCMEC/D3 培养相比具有低的蔗糖渗透性, 并含有某些血脑屏障转运体、黏附分子和紧密连接点51的极化表达,52. 然而, 这些细胞也受文化中变化的特性的改变, 必须对系统进行验证, 以确认其在体内屏障性质52中的重述。
血脑屏障研究的新趋势包括利用3D 组织培养系统制造人造毛细血管, 利用芯片上的器官技术产生微流控装置, 或利用中空纤维技术53,54,55. 然而, 人造毛细血管的直径 (100–200µm) 明显大于脑毛细血管 (3–7µm)。因此,体外剪切力不完全类似于体内的情况。这是在 "血脑屏障芯片" 微流控装置, 其中人工膜形成 "血液" 和 "大脑" 车厢和流体泵浦通过这些装置产生微流控剪切力。同样, 不同组合的内皮细胞与星形胶质细胞和血管平滑肌干细胞的联合培养也被应用于中空纤维技术, 以重现体内条件下的流变参数56 。,57,58. 然而, 目前还不清楚这个模型是如何反映血脑屏障的其他特性的, 如运输、新陈代谢、信号传递和其他。这些人造毛细管和切片模型适用于高通量筛选药物, 但用于生成这些模型的细胞在培养过程中也会发生变化。
冷冻和固定脑切片或原发性脑毛细血管内皮细胞培养是额外的模型, 可用于研究人类微血管5,59,60,61。例如, 固定脑组织的免疫组化被用来确定蛋白质的定位和健康的表达与患病的组织相比。
除了以上所述的组织切片和体外模型之外, 还可以利用新鲜的脑毛细血管来研究血脑屏障功能。这种孤立的毛细管模型的局限性包括难以获得新鲜的人脑组织, 缺乏星形胶质细胞和神经元, 以及一个相对耗时的隔离过程。分离的脑毛细管模型的优点是, 该模型与体内情况相似, 因此可以用来表征屏障功能和功能障碍。重要的是, 它也可以用来辨别信号机制使用大量的化验和分子技术3,19,62,63。
我们的实验室可以通过桑德斯-棕褐色中心 (IRB #B15-2602 米)64获得新鲜和冰冻的人脑组织。在这种情况下, 解剖遵循标准的协议, 大脑在 < 4 小时内获得, 所有程序符合 NIH Biospecimen 最佳做法准则65。考虑到人类大脑组织的这种独特的接触, 我们建立并优化了一项协议, 以隔离人脑组织中的毛细血管, 从而导致完整、可行的人脑毛细血管的高产。两个常见的端点的兴趣是确定的蛋白质表达和活动。在这方面, 我们和其他人已经建立了不同的化验方法, 可用于分离的脑毛细血管, 以研究蛋白质表达和活动水平。这些分析包括: 西方印迹, 简单的西方检测, 酶联免疫吸附试验 (ELISA), 反向转录聚合酶链反应 (rt-pcr), 定量聚合酶链反应 (qPCR), 酶谱法, 运输活动的检测, 和毛细管渗漏化验。这些化验可以让研究人员研究人体病理状况中屏障功能的变化, 确定控制蛋白表达和活性的途径, 并确定治疗血脑屏障的药理靶点。疾病。
结合起来, 新鲜的孤立的脑毛细血管可以作为一个强健的和可再生的血脑屏障模型。特别是, 该模型可以结合多种不同的检测方法, 确定广泛的端点来研究屏障功能。

<table><tbody><tr><td>&lt;strong&gt;Personal Protective Equipment (PPE)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Diamond Grip Plus Latex Gloves, Microflex Medium</td><td>VWR, Radnor, PA, USA</td><td>32916-636</td><td>PPE</td></tr><tr><td>Disposable Protective Labcoats</td><td>VWR, Radnor, PA, USA</td><td>470146-214</td><td>PPE; due to the nature of the human source material, the use of a disposable lab coat is recommended</td></tr><tr><td>Face Shield, disposable</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>19460102</td><td>PPE; due to the nature of the human source material, the use of a disposable face shield is recommended</td></tr><tr><td>Safety Materials</td><td></td><td></td><td></td></tr><tr><td>Clavies High-Temperature Autoclave Bags 8 x 12</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>01-815-6</td><td></td></tr><tr><td>Versi Dry Bench Paper 18&quot; x 20&quot;</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>14-206-32</td><td>to cover working areas</td></tr><tr><td>VWR Sharps Container Systems</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>75800-272</td><td>for used scalpels</td></tr><tr><td>Bleach 8.2% Clorox Germicidal 64 oz.</td><td>UK Supply Center, Lexington, KY, USA</td><td>323775</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4 &amp;deg;C Refrigerator</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>13-986-148</td><td></td></tr><tr><td>Accume BASIC AB15 pH Meter</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>AB15</td><td></td></tr><tr><td>Heidolph RZR 2102 Control</td><td>Heidolph, Elk Grove Village, IL, USA</td><td>501-21024-01-3</td><td></td></tr><tr><td>Sorvall LEGEND XTR Centrifuge</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>75004521</td><td></td></tr><tr><td>Leica L2 Dissecting Microscope</td><td>Leica Microsystems Inc, Buffalo Grove IL, USA</td><td></td><td>used to remove meninges</td></tr><tr><td>POLYTRON PT2500 Homogenizer</td><td>Kinematica AG, Luzern, Switzerland</td><td>9158168</td><td></td></tr><tr><td>Scale P-403</td><td>Denver Instrument, Bohemia, NY, USA</td><td>0191392</td><td></td></tr><tr><td>Standard mini Stir</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>1151050</td><td></td></tr><tr><td>Thermo-Flasks Liquid Nitrogen Dewar</td><td>Thermal Scientific, Mansfiled, TX, USA</td><td>11-670-4C</td><td>used to freeze the tissue?</td></tr><tr><td>Voyager Pro Analytical Balance</td><td>OHAUS, Parsippany, NJ, USA</td><td>VP214CN</td><td></td></tr><tr><td>ZEISS Axiovert Microcope</td><td>Carl Zeiss, Inc Thornwood, NY, USA</td><td></td><td>used to check isolated capillaries</td></tr><tr><td>&lt;strong&gt;Tools and Glassware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Finnpipette II Pipette 1-5 mL</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>21377823T1</td><td>wash capillaries off filter</td></tr><tr><td>Finnpipette II Pipette 100-1,000 &amp;micro;L</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>21377821T1</td><td>resuspend pellet in BSA</td></tr><tr><td>Pipet Boy</td><td>Integra, Hudson, NH, USA</td><td>739658</td><td></td></tr><tr><td>50 mL Falcon tubes 25/rack - 500/cs</td><td>VWR, Radnor, PA, USA</td><td>21008-951</td><td></td></tr><tr><td>EISCO Scalpel Blades</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>S95938C</td><td>to mince brain tissue</td></tr><tr><td>PARAFILM</td><td>VWR, Radnor, PA, USA</td><td>52858-000</td><td>to cover beaker and volumetric flask</td></tr><tr><td>Thermo Scientific Finntip Pipet Tips 5 mL</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>21-377-304</td><td>to wash capillaries off filter</td></tr><tr><td>60 mL syringe with Luer-Lok</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>BD309653</td><td>used with connector ring to filter capillaries</td></tr><tr><td>Scalpel Handle #4</td><td>Fine Science Tools, Foster City, CA, USA</td><td>10060-13</td><td>used for mincing</td></tr><tr><td>Dumont Forceps #5</td><td>Fine Science Tools, Foster City, CA, USA</td><td>11251-10</td><td>used to remove meninges</td></tr><tr><td>Potter-Elvehjem Tissue Grinder</td><td>Thomas Scientific, Swedesboro, NJ, USA</td><td>3431E25</td><td>50 mL volume, clearance: 150-230 &amp;mu;m</td></tr><tr><td>Dounce Homogenizer</td><td>VWR, Radnor PA USA</td><td>62400-642</td><td>15 mL volume, clearance: 80-130 &amp;mu;m</td></tr><tr><td>Spectra/Mesh Woven Filters (300 &amp;micro;m)</td><td>Spectrum Laboratories, Rancho Dominguez, CA, USA</td><td>146424</td><td>Used to filter capillary suspension to remove any meninges that may be left</td></tr><tr><td>pluriStrainers (pore size: 30 &amp;micro;m)</td><td>pluriSelect Life Science, Leipzig, Germany</td><td>43-50030-03</td><td></td></tr><tr><td>Connector Ring</td><td>pluriSelect Life Science, Leipzig, Germany</td><td>41-50000-03</td><td>reuse multiple time</td></tr><tr><td>1 L Volumetric Flask</td><td></td><td></td><td>for preparation of Isolation Buffer</td></tr><tr><td>1 L Beaker</td><td></td><td></td><td>for preparation of 1% BSA</td></tr><tr><td>Stir Bar</td><td></td><td></td><td>for preparation of 1% BSA and Ficoll&lt;sup&gt;&amp;reg;&lt;/sup&gt;</td></tr><tr><td>Schott Bottle (60 mL)</td><td></td><td></td><td>for preparation of Ficoll&lt;sup&gt;&amp;reg;&lt;/sup&gt;</td></tr><tr><td>Ice Bucket</td><td></td><td></td><td>to keep everything cold</td></tr><tr><td>100 mm Petri dish</td><td></td><td></td><td>for mincing of brain tissue</td></tr><tr><td>Tissue Culture Cell Scraper</td><td>VWR, Radnor, PA, USA</td><td>89260-222</td><td>to remove supernatant after centrifugation</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BSA Fraction V, A-9647</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>A9647-500g</td><td>prepare in DPBS with Ca&lt;sup&gt;2+&lt;/sup&gt; &amp;amp; Mg&lt;sup&gt;2+&lt;/sup&gt; the day before. Avoid bubbles during preparation. Store in the refrigerator. Slowly stir for 10 min before use.</td></tr><tr><td>DPBS with Ca&lt;sup&gt;2+&lt;/sup&gt; &amp;amp; Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Hyclone</td><td>SH30264.FS</td><td>DPBS - part of the Isolation Buffer</td></tr><tr><td>Ficoll PM400</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>F4375</td><td>Exact measurement is important here. Weigh out in bottle with stir bar. Shake vigurously after adding DPBS. Keep in the fridge O/N. It will be clear in the morning. Stir gently for 10-15 min before use. Keep on ice until use.</td></tr><tr><td>Glucose (D-(+) Dextrose)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>G7528</td><td>Glucose (D-(+) Dextrose) Concentration: 5 mM</td></tr><tr><td>Sodium Hydroxide Standard Solution</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>71474</td><td>to adjust pH of the DPBS</td></tr><tr><td>Sodium Pyruvate</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>P2256</td><td>Concentration: 1 mM</td></tr></tbody></table>

isolation of cerebral capillaries from fresh human brain tissue

在生理和病理生理学条件下了解血脑屏障功能对于制定新的治疗策略具有重要的作用, 这些战略有望加强脑药物的传递, 改善脑保护, 并治疗脑障碍。然而, 研究人体血脑屏障功能具有挑战性。因此, 迫切需要适当的模型。在这方面, 从人脑组织中分离出来的脑毛细血管代表了一种独特的工具, 可以在尽可能接近人类体内的情况下研究屏障功能。在这里, 我们描述了一个优化的协议, 以隔离毛细血管从人的大脑组织高产和一致的质量和纯度。毛细血管从新鲜的人脑组织中分离出来, 采用机械均匀化、密度梯度离心和过滤。隔离后, 人脑毛细血管可用于各种应用, 包括渗漏化验, 活细胞成像, 免疫分析, 以研究蛋白质的表达和功能, 酶活性, 或细胞内信号。孤立的人脑毛细血管是阐明人体血脑屏障功能调节的独特模型。该模型可为中枢神经系统 (cns) 发病机制提供深入的见解, 有助于发展治疗中枢性疾病的治疗策略。

隔离从人脑组织中产生的悬浮液富含人脑毛细血管 (图 1B), 少量较大的血管, 红细胞, 其他单细胞, 和一些细胞碎片。有些毛细血管是分枝的, 在某些情况下, 红血球被包裹在毛细血管流明。典型毛细管有3–7µm 直径和大约100-200 µm 长以开放流明;大多数毛细管末端都是折叠的。使用共焦显微镜, 孤立的人脑毛细血管揭示一个管状, 完整的结构和形态学。<s...

Watch this Scientific Journal Video about Isolation of Cerebral Capillaries from Fresh Human Brain Tissue at JoVE.com

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

新鲜人脑组织中毛细血管的分离

从人脑组织中分离出的脑毛细血管可作为研究生理和病理生理学条件下屏障功能的临床前模型。在这里, 我们提出了一个优化的协议, 以隔离脑毛细血管与新鲜的人脑组织。

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

isolation-of-cerebral-capillaries-from-fresh-human-brain-tissue

Research

JoVE Journal

Neuroscience

12.5K 次观看.  University of Kentucky. 从人脑组织中分离出的脑毛细血管可作为研究生理和病理生理学条件下屏障功能的临床前模型。在这里, 我们提出了一个优化的协议, 以隔离脑毛细血管与新鲜的人脑组织。

视频: Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Purification of Mouse Brain Vessels

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 immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified 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 ...

科研

神经科学

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

分离及脑实质小动脉插管

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

在五个脊椎动物群中可靠隔离中枢神经系统微血管

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

脑毛细管中培养细胞细胞钙测量和钙成像研究

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

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 of Cerebral Capillaries from Human Brain: An Optimized Method to Extract Cerebral Capillaries from Human Brain Tissue

Source: Hartz, A. M. S. et al. <a href="https://www.jove.com/video/57346" target="_blank">Isolation of Cerebral Capillaries from Fresh Human Brain Tissue.</a> J. Vis. Exp. (2018)
This video presents the isolation of cerebral capillaries from a fresh human brain tissue. The isolated capillaries can then be used to generate an ex vivo blood brain barrier model.

从人脑中分离脑毛细血管:从人脑组织中提取脑毛细血管的优化方法

Source: Hartz, A. M. S. et al. Isolation of Cerebral Capillaries from Fresh Human Brain Tissue. J. Vis. Exp. (2018)
This video presents the isolation of ...

JoVE Encyclopedia of Experiments

癌症研究

Cancers of the Nervous System

Ex vivo studies

Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination

Source: H&#248;rlyck, S. et al. <a href="https://www.jove.com/video/61253" target="_blank">Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.</a>&#160;J. Vis. Exp. (2020)
In this video, we demonstrate the procedure to isolate and culture pericytes from bovine brain capillaries without endothelial cell contamination.

原代脑周细胞的分离和培养:从牛脑毛细血管中分离和培养周细胞而不受内皮细胞污染的程序

Source: H&#248;rlyck, S. et al. Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.&#160;J. Vis. Exp. ...

In vitro studies

Purifying Capillaries From a Porcine Brain

Source: Nielsen, S. S. E., et al. <a href="https://app.jove.com/v/56277">Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells.</a> J. Vis. Exp. (2017).
This video demonstrates the isolation of capillaries from fresh porcine brains. The process involves removing the meninges, isolating the grey matter from the brain tissue, homogenizing and filtering the grey matter to isolate the capillaries, and finally using enzymatic digestion to purify the capillaries further.

从猪脑中纯化毛细血管

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Purification of Porcine Brain ...

Neuronal Culture Techniques

Advanced Neuronal Models

A Method for the Isolation of Neurovascular Units from Rat Brain

Source: Brzica, H., et al,. <a href="https://app.jove.com/v/57698">A simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels.</a> J. Vis. Exp. (2018).
This video demonstrates the process of isolating brain microvessels from euthanized rats. It includes the dissection of the brain, cleaning of the tissue, and mechanical digestion, followed by repeated centrifugation to isolate the pure fraction of microvessels.

一种从大鼠脑中分离神经血管单位的方法

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. The procedural flow for the protocol ...