Ex Vivo 加压希马卡毛细管- 功能研究准备
Summary
本手稿详细介绍了如何从小鼠大脑中分离海马动脉和毛细血管，以及如何为压力的焦疗、免疫荧光、生物化学和分子研究加压它们。
Abstract
从微妙的行为改变到晚期痴呆症，血管认知障碍通常在脑缺血后发展。中风和心脏骤停是显性畸形疾病，都诱发脑缺血。然而，在理解血管认知障碍，然后开发性别特异性治疗方面的进展，在功能研究中受到研究小鼠模型大脑微循环的挑战部分限制。在这里，我们提出一种方法，以检查毛细管到动脉信号在从小鼠大脑前体海马毛细管毛细管动脉（HiCaPA）制备。我们描述了如何分离、增射和加压微循环，以测量动脉直径以响应毛细管刺激。我们展示了哪些适当的功能控制可用于验证HiCaPA制备的完整性，并显示典型结果，包括测试钾作为神经血管耦合剂和最近具有特征的抑制剂Kir2内矫正钾通道系列，ML133的效果。此外，我们比较了从雄性小鼠和雌性小鼠获得制剂的反应。虽然这些数据反映了功能调查，但我们的方法也可用于分子生物学、免疫化学和电生理学研究。
Introduction
大脑表面的皮环流一直是许多研究的对象，部分原因是它的实验可及性。然而，脑血管的拓扑结构创造了不同的区域。与富含麻醉剂的强健的皮球网络相比，脑内腹腔动脉（PAs）存在有限的辅助供应，每个细胞都渗透神经组织1、2的离散体积。这造成了对血流的瓶颈效应，结合独特的生理特征3，4，5，6，7，8，使脑动脉成为脑血流（CBF）调节9，10的关键部位。尽管PA的隔离和分离技术挑战固有的，但在过去十年中，使用加压容器11、12、13、14、15、16、17的体外功能研究的兴趣日益浓厚。引起这种兴趣增加的原因之一是，对神经血管耦合（NVC）进行了大量研究，这种机制支撑着大脑功能性高血症18。
区域方面，CBF在局部神经激活19后可迅速增加。控制NVC的蜂窝机制和信令特性尚未完全了解。然而，我们在NVC期间发现了一个以前未预料到的脑毛细血管作用，在感知神经活动并将其转化为超极化电信信号以稀释上游动脉20、21、22。作用电位23、24和大导导 Ca2+激活 K+ （BK） 通道在星形端脚25、26上增加间隙钾电浓度 [K]o，导致在毛细血管内膜中激活强的内整整流器 K+ （Kir） 通道。此通道由外部 K+激活，但也由超极化本身激活。通过间隙结扩散，超极化电流然后在相邻的毛细管内皮细胞中再生，并持续到动脉，导致肌细胞放松和CBF增加20，21。对该机制的研究导致我们开发出一种加压毛细管-皮伦皮毛毛（CaPA）制剂，以测量血管刺激期间用血管活性剂的动脉直径。CaPA 制剂由可口的脑内动脉段组成，具有完好的下游毛细管冲压。毛细管末端被微移液器压缩在室玻璃底部，从而遮挡并稳定整个血管形成20，21。
我们之前通过成像CaPA制剂从小鼠皮层20，21和动脉从大鼠杏仁核13和海马16，17进行仪器创新。由于海马血管因其对病理条件的易感性而受到更多的关注，因此，我们在此提供了一种从小鼠海马场（HiCaPA）制备CaPA的分步方法，该方法不仅可用于功能性NVC研究，还可用于分子生物学、免疫化学和电生理学。
Protocol
所有实验均获得科罗拉多大学安舒茨医学院机构动物护理和使用委员会（IACUC）的批准，并根据国家卫生研究院的指南进行。 1. 解决方案 使用MOPS缓冲盐水进行解剖，并在样品使用前保持4°C。不要为溶液加气。制备具有以下成分的 MOPS 缓冲盐水： 135 mM NaCl， 5 mM KCl， 1 mM KH2PO4， 1 mM MgSO4， 2.5 mM CaCl2，5 mM 葡萄糖， 3 mM MOPS， 0.02 mM EDTA， 2…
Representative Results
内皮小电导 （SK） 和中间导电 （IK） Ca2+- 敏感 K+通道对 PA 的直径产生拖延影响。浴液应用1μM NS309，一种合成IK和SK通道激动剂，引起接近最大扩张（图2A，B）。然而，毛细管内皮细胞缺乏IK和SK通道，没有超极化响应NS30920。因此，通过焦压喷射（20 s，5 psi）以1μM NS309刺激毛细管结束不会导致上游动脉?…
Discussion
本手稿中描述的加压 HiCaPA（海马毛细血管-皮毛动脉）制备是我们隔离、加压和研究眼皮动脉29的既定程序的延伸。我们最近报告说，脑毛细管内皮细胞中的Kir2.1通道感觉增加[K]o与神经激活相关，并产生一个上升的超极化信号，以向上动脉20。通过从皮质微循环20、21开发CaPA制剂，部分地揭示了毛细血?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者们感谢朱尔斯·莫林对手稿的有见地的评论。这项研究由CADASIL共同有希望的非营利组织、妇女健康与研究中心和NHLBI R01HL136636（FD）的奖项资助。
Materials
| 0.22µm Syringe Filters | CELLTREAT Scientific Products | 229751 | |
| 12-0 Nylon (12cm) Black | Microsurgery Instruments, Inc | S12-0 NYLON | |
| Automatic Temperature Controller | Warner Instruments | TC-324B | |
| Borosilicate Glass O.D.: 1.2 mm, I.D.: 0.68 mm | Sutter Instruments | B120-69-10 | |
| Bovine serum albumin | Sigma-Aldrich | A7030 | |
| CaCl2 dihydrate | Sigma-Aldrich | C3881 | |
| D-(+)-Glucose | Sigma-Aldrich | G5767 | |
| Dissection Scope | Olympus | SZ11 | |
| ECOLINE VC-MS/CA 4-12 — complete Pump with Drive and MS/CA 4-12 pump-head | Ismatec | ISM 1090 | |
| EGTA | Sigma-Aldrich | E4378 | |
| Fine Scissors – Sharp | Fine Science Tools | 14063-09 | |
| Inline Water Heater | Warner Instruments | SH-27B | |
| Integra™ Miltex™Tissue Forceps | Fisher Scientific | 12-460-117 | |
| KCl | Sigma-Aldrich | P9333 | |
| KH2PO4 | Sigma-Aldrich | P5379 | |
| Magnesium sulfate heptahydrate | Sigma-Aldrich | M1880 | |
| MgCl Anhydrous | Sigma-Aldrich | M8266 | |
| Micromanipulator | Narishige | MN-153 | |
| ML 133 hydrochloride | Tocris | 4549 | |
| MOPS | Sigma-Aldrich | M1254 | |
| NaCl | Sigma-Aldrich | S9625 | |
| NaH2PO4 | Sigma-Aldrich | S9638 | |
| NaHCO3 | Sigma-Aldrich | S8875 | |
| NS309 | Tocris | 3895 | |
| Picospritzer III – Intracellular Microinjection Dispense Systems, 2-channel | Parker Hannifin | 052-0500-900 | |
| Pressure Servo Controller with Peristaltic Pump | Living Systems Instrumentation | PS-200 | |
| Sodium pyruvate | Sigma-Aldrich | P3662 | |
| Super Fine Forceps | Fine Science Tools | 11252-20 | |
| Surgical Scissors – Sharp-Blunt | Fine Science Tools | 14001-13 | |
| Vertical Micropipette Puller | Narishige | PP-83 |
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Cite This Article
Rosehart, A. C., Johnson, A. C., Dabertrand, F. Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study. J. Vis. Exp. (154), e60676, doi:10.3791/60676 (2019).