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Neurosciences

新鲜人脑组织中毛细血管的分离

Published: September 12, 2018
doi:
10.3791/57346
, , , , , , , ,
1Sanders-Brown Center on Aging, Department of Pharmacology and Nutritional Sciences,University of Kentucky, 2Department of Pharmaceutical Sciences, College of Pharmacy,University of Kentucky, 3Department of Neuroscience,University of Kentucky

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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…

Representative Results

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…

Discussion

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…

Divulgations

The authors have nothing to disclose.

Acknowledgements

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。

Materials

Personal Protective Equipment (PPE)
Diamond Grip Plus Latex Gloves, Microflex Medium VWR, Radnor, PA, USA 32916-636 PPE
Disposable Protective Labcoats VWR, Radnor, PA, USA 470146-214 PPE; due to the nature of the human source material, the use of a disposable lab coat is recommended
Face Shield, disposable Thermo Fisher Scientific, Pittsburgh, PA, USA 19460102 PPE; due to the nature of the human source material, the use of a disposable face shield is recommended
Safety Materials
Clavies High-Temperature Autoclave Bags 8X12 Thermo Fisher Scientific, Pittsburgh, PA, USA 01-815-6
Versi Dry Bench Paper 18" x 20" Thermo Fisher Scientific, Pittsburgh, PA, USA 14-206-32 to cover working areas
VWR Sharps Container Systems Thermo Fisher Scientific, Pittsburgh, PA, USA 75800-272 for used scalpels
Bleach 8.2% Clorox Germicidal 64 oz UK Supply Center, Lexington, KY, USA 323775
Equipment
4°C Refrigerator Thermo Fisher Scientific, Pittsburgh, PA, USA 13-986-148
Accume BASIC AB15 pH Meter Thermo Fisher Scientific, Pittsburgh, PA, USA AB15
Heidolph RZR 2102 Control Heidolph, Elk Grove Village, IL, USA 501-21024-01-3
Sorvall LEGEND XTR Centrifuge Thermo Fisher Scientific, Pittsburgh, PA, USA 75004521
Leica L2 Dissecting Microscope Leica Microsystems Inc, Buffalo Grove IL, USA used to remove meninges
POLYTRON PT2500 Homogenizer Kinematica AG, Luzern, Switzerland 9158168
Scale P-403 Denver Instrument, Bohemia, NY, USA 0191392
Standard mini Stir Thermo Fisher Scientific, Pittsburgh, PA, USA 1151050
Thermo-Flasks Liquid Nitrogen Dewar Thermal Scientific, Mansfiled, TX, USA 11-670-4C used to freeze the tissue?
Voyager Pro Analytical Balance OHAUS, Parsippany, NJ, USA VP214CN
ZEISS Axiovert Microcope Carl Zeiss, Inc Thornwood, NY, USA used to check isolated capillaries
Tools and Glassware
Finnpipette II Pipette 1-5mL Thermo Fisher Scientific, Pittsburgh, PA, USA 21377823T1 wash capillaries off filter
Finnpipette II Pipette 100-1000 µL Thermo Fisher Scientific, Pittsburgh, PA, USA 21377821T1 resuspend pellet in BSA
Pipet Boy Integra, Hudson, NH, USA 739658
50mL Falcon tubes 25/rack – 500/cs VWR, Radnor, PA, USA 21008-951
EISCO Scalpel Blades Thermo Fisher Scientific, Pittsburgh, PA, USA S95938C to mince brain tissue
PARAFILM VWR, Radnor, PA, USA 52858-000 to cover beaker and volumetric flask
Thermo Scientific Finntip Pipet Tips 5 ml Thermo Fisher Scientific, Pittsburgh, PA, USA 21-377-304 to wash capillaries off filter
60 ml syringe with Luer-Lok Thermo Fisher Scientific, Pittsburgh, PA, USA BD309653 used with connector ring to filter capillaries
Scalpel Handle #4 Fine Science Tools, Foster City, CA, USA 10060-13 used for mincing
Dumont Forceps #5 Fine Science Tools, Foster City, CA, USA 11251-10 used to remove meninges
Potter-Elvehjem Tissue Grinder Thomas Scientific, Swedesboro, NJ, USA 3431E25 50 ml volume, clearance: 150-230 μm
Dounce Homogenizer VWR, Radnor PA USA 62400-642 15 ml volume, clearance: 80-130 μm
Spectra/Mesh Woven Filters (300 µm) Spectrum Laboratories, Rancho Dominguez, CA, USA 146424 Used to filter capillary suspension to remove any meninges that may be left
pluriStrainers (pore size: 30 µm) pluriSelect Life Science, Leipzig, Germany 43-50030-03
Connector Ring pluriSelect Life Science, Leipzig, Germany 41-50000-03 reuse multiple time
1 l Volumetric Flask for preparation of Isolation Buffer
1 l Beaker for preparation of 1% BSA
Stir Bar for preparation of 1% BSA and Ficoll®
Schott Bottle (60 ml) for preparation of Ficoll®
Ice Bucket to keep everything cold
100 mm Petri Dish for mincing of brain tissue
Tissue Culture Cell Scraper VWR, Radnor, PA, USA 89260-222 to remove supernatant after centrifugation
Chemicals
BSA Fraction V, A-9647 Sigma-Aldrich, St. Louis, MO, USA A9647-500g prepare in DPBS with Ca2+ & Mg2+ the day before. Avoid bubbles during preparation. Store in the refrigerator. Slowly stir for 10 min before use.
DPBS with Ca2+ & Mg2+ Hyclone SH30264.FS DPBS – part of the Isolation Buffer
Ficoll PM400 Sigma-Aldrich, St. Louis, MO, USA F4375 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.
Glucose (D-(+) Dextrose) Sigma-Aldrich, St. Louis, MO, USA G7528 Glucose (D-(+) Dextrose) Concentration: 5 mM
Sodium Hydroxide Standard Solution Sigma-Aldrich, St. Louis, MO, USA 71474 to adjust pH of the DPBS
Sodium Pyruvate Sigma-Aldrich, St. Louis, MO, USA P2256 Concentration: 1 mM
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Cerebral CapillariesBlood-brain BarrierIsolationHomogenizationFicoll Density GradientFiltrationCentrifugation
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Hartz, A. M., Schulz, J. A., Sokola, B. S., Edelmann, S. E., Shen, A. N., Rempe, R. G., Zhong, Y., Seblani, N. E., Bauer, B. Isolation of Cerebral Capillaries from Fresh Human Brain Tissue. J. Vis. Exp. (139), e57346, doi:10.3791/57346 (2018).
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