小鼠脑实质小动脉内皮的分离及功能分析
Summary
来自脑实质小动脉的完整小鼠脑内皮“管”的强化制备用于研究脑血流调节。此外，我们证明了这种内皮研究模型在荧光成像和关键细胞信号通路的电生理学测量方面的实验优势，包括细胞内[Ca2 +]和膜电位的变化。
Abstract
脑血流通过血管阻力动脉和下游实质小动脉输送。稳态血管对血流的阻力随着从动脉到最终进入毛细血管的小动脉直径减小而增加。由于它们在实质中较小的尺寸和位置，小动脉的研究相对不足，并且与表皮动脉相比，其结果的可重复性较低。无论如何，小动脉内皮细胞结构和功能是慢性退行性疾病生理学和病因学不可或缺的一部分，需要广泛的研究。特别是，新出现的证据表明，内皮功能受损先于并加重认知障碍和痴呆。
在实质微循环中，内皮K+ 通道功能是精细控制血管舒张扩散以促进流向神经元活动区域血流的增加的最强大的刺激。本文说明了一种从小鼠脑实质小动脉中新鲜分离完整和电耦合的内皮“管”（直径，〜25μm）的改进方法。在生理条件下（37°C，pH 7.4）固定小动脉内皮管，以解析包含K + 通道功能及其调节的实验变量，包括细胞内Ca2 + 动力学，膜电位变化和膜脂质调节。与动脉内皮相比，一个明显的技术优势是增强了细胞和细胞器（例如线粒体）尺寸的形态学分辨率，这扩大了该技术的实用性。终生健康的脑灌注需要实质小动脉的强大内皮功能，直接将血液流动与大脑精确解剖区域的神经元和神经胶质活动提供燃料联系起来。因此，预计这种方法将显着推进有关健康和患病大脑的血管生理学和神经科学的一般知识。
Introduction
实质小动脉直接在整个大脑中输送必需的氧气和营养物质1.在与毛细血管连接时，高度血管活性的小动脉对由毛细血管离子通道启动的逆行信号作出反应，这些通道感知来自特定神经元区域的代谢信号2。由于脑实质历来接受了大部分研究，内皮功能障碍在阐明与痴呆症（例如缺血性卒中，阿尔茨海默病）相关的各种脑血管疾病（例如缺血性卒中，阿尔茨海默病）的病理机制方面已经出现的作用3，4，5，6.内皮是大脑灌注的组成部分，符合整个血管节段的遗传学，结构和功能的异质性7。Pial动脉由于其相对较大的尺寸，高节段血管阻力以及在向下层大脑的血流分布中的作用8，9，已被广泛研究。因此，更好地了解小动脉内皮机制可能会增强对健康和疾病中脑血流调节的理解，以开发新的治疗方案。
新出现的证据强调了研究实质小动脉与不同信号通路和疾病的关系的重要性8，10。然而，这种方法仅限于使用完整的加压小动脉11 和/或毛细血管 – 实质小动脉（CaPA）制剂12。尚未检查新鲜分离的天然脑小动脉内皮细胞，这可能是由于分离的技术困难。本文推进了先前的技术，突出了pial动脉内皮13 的分离，现在可靠且可重复地分离脑实质小动脉的内皮（宽度：〜25μm，长度：〜250μm）。该技术有助于在电耦合和化学耦合细胞的个体方向和细胞网络中实现最佳分辨率。
感兴趣的关键途径包括细胞内Ca2 +（[Ca2 + ]i）信号传导和膜电位（Vm）14，15的超极化 – 血管舒张16的组成部分 – 允许血液进入毛细血管并将氧气和营养物质输送到活动性实质17。这些制剂允许对离子通道进行实时电生理学记录，包括Ca2 +通透性，瞬时受体电位（TRP）和K +通道和/或在近生理条件下内皮细胞管内细胞器的荧光成像。对于对控制内皮细胞控制脑血流输送到脑实质的生理细胞机制感兴趣的研究人员来说，这是一种合适的技术。总而言之，这项技术将帮助研究人员更好地了解嵌入脑实质的小动脉的基本内皮信号通路和网络通信，同时解决与脑血管生理学和病理学相关的问题。
Protocol
实验人员应确保动物的指定使用和相关协议得到其机构动物护理和使用委员会（IACUC）的批准，并按照国家研究委员会的“实验动物护理和使用指南”（第 8版，2011年）和ARRARD指南进行。Loma Linda大学的IACUC和亚利桑那大学已经批准了用于C57BL / 6N和 3xTg-AD 小鼠（雄性和雌性;年龄范围：2-30个月）的所有手稿方案。参见 图1 作为从小鼠脑实质小动脉新鲜分离的小动…
Representative Results
方案的演示如图1所示，小动脉夹层和内皮管分离步骤分别如图2和图3所示。在这里，通过使用Fura-2光度测定法和锐电极电生理学（图4A）测量[Ca2 + ]i和Vm来评估内皮功能，以响应药理学剂[2-甲基硫代腺苷二磷酸（MTA），一种有效的嘌呤能受体（P2YR）激动剂]在37°C下。 在应用MTA（1μM）…
Discussion
越来越多的证据表明，脑血管疾病（CVD），衰老和阿尔茨海默病密切相关，并且是痴呆症研究的当前主题4，8，14，21。因此，很明显，对脑血管网络的研究将对健康产生广泛影响，同时需要在疾病条件下继续进行广泛的调查。作为脑灌注血管阻力的重要点，实质小动脉在CVD的病因和发展中的一般重要?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了美国国立卫生研究院（R00AG047198和R56AG062169）的资助。R00HL140106至PWP）和阿尔茨海默氏症协会（AZRGD-21-805835至PWP）。内容完全由作者负责，并不一定代表美国国立卫生研究院或阿尔茨海默氏症协会的官方观点。
Materials
| Amplifiers | Molecular Devices, Sunnyvale, CA, USA | Axoclamp 2B & Axoclamp 900A | |
| Audible baseline monitors | Ampol US LLC, Sarasota, FL, USA | BM-A-TM | |
| Bath Chiller (Isotemp 500LCU) | ThermoFisher Scientific | 13874647 | |
| Borosilicate glass capillaries (Pinning) | Warner Instruments | G150T-6 | |
| Borosilicate glass capillaries (Sharp Electrodes) | Warner Instruments | GC100F-10 | |
| Borosilicate glass capillaries (Trituration) | World Precision Instruments (WPI), Sarasota, FL, USA | 1B100-4 | |
| BSA: Bovine Serum Albumin | Sigma | A7906 | |
| CaCl2: Calcium Chloride | Sigma | 223506 | |
| Collagenase (Type H Blend) | Sigma | C8051 | |
| Cover Glass (2.4 × 5.0 cm) | ThermoFisher Scientific | 12-548-5M | |
| Data Acquision Digitizer | Molecular Devices, Sunnyvale, CA, USA | Digidata 1550A | |
| Dissection Dish (Glass Petri with Charcoal Sylgard bottom) | Living Systems Instrumentation, St. Albans City, VT, USA | DD-90-S-BLK | |
| Dithioerythritol | Sigma | D8255 | |
| DMSO: Dimethyl Sulfoxide | Sigma | D8418 | |
| Elastase (porcine pancreas) | Sigma | E7885 | |
| Endoplasmic Reticulum Tracker (ER-Tracker Red, BODIPY TR Glibenclamide) | ThermoFisher Scientific | E34250 | |
| Fiber optic light sources | Schott, Mainz, Germany & KL200, Zeiss | Fostec 8375 | |
| Flow Control Valve | Warner Instruments | FR-50 | |
| Fluorescence system interface, ARC lamp & power supply, hyperswitch and PMT | Molecular Devices, Sunnyvale, CA, USA | IonOptix Systems | |
| Forceps (Fine-tipped, sharpened) | FST | Dumont #5 & Dumont #55 | |
| Function Generator | EZ Digital, Seoul, South Korea | FG-8002 | |
| Fura-2 AM dye | Invitrogen, Carlsbad, CA, USA | F14185 | |
| Glucose | Sigma-Aldrich (St. Louis, MO, USA) | G7021 | |
| HCl: Hydrochloric Acid | ThermoFisher Scientific (Pittsburgh, PA, USA) | A466250 | |
| Headstages | Molecular Devices | HS-2A & HS-9A | |
| HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) | Sigma | H4034 | |
| Inline Solution Heater | Warner Instruments | SH-27B | |
| KCl: Potassium Chloride | Sigma | P9541 | |
| MgCl2: Magnesium Chloride | Sigma | M2670 | |
| Microforge | Narishige, East Meadow, NY, USA | MF-900 | |
| Micromanipulator | Siskiyou | MX10 | |
| Micropipette puller (digital) | Sutter Instruments, Novato, CA, USA | P-97 or P-1000 | |
| Microscope (Nikon-inverted) | Nikon Instruments Inc, Melville, NY, USA | Ts2 | |
| Microscope (Nikon-inverted) | Nikon Instruments Inc | Eclipse TS100 | |
| Microscope objectives | Nikon Instruments Inc | 20X (S-Fluor) and 40X (Plan Fluor) | |
| Microscope platform (anodized aluminum; diameter, 7.8 cm) | Warner Instruments | PM6 or PH6 | |
| Microscope Stage (Aluminum) | Siskiyou, Grants Pass, OR, USA | 8090P | |
| Microsyringe Pump Controller | World Precision Instruments (WPI), Sarasota, FL, USA | SYS-MICRO4 | |
| MTA: 2-Methylthioadenosine diphosphate trisodium salt | Tocris | 1624 | |
| NaCl: Sodium Chloride | Sigma | S7653 | |
| NaOH: Sodium Hydroxide | Sigma | S8045 | |
| Nuclear Stain (NucBlue Live ReadyProbes Reagent; Hoechst 33342) | ThermoFisher Scientific | R37605 | |
| Oscilloscope | Tektronix, Beaverton, Oregon, USA | TDS 2024B | |
| Papain | Sigma | P4762 | |
| Phase contrast objectives | Nikon Instruments Inc | (Ph1 DL; 10X & 20X) | |
| Plasma Membrane Stain (CellMask Deep Red) | ThermoFisher Scientific | C10046 | |
| Plexiglas superfusion chamber | Warner Instruments, Camden, CT, USA | RC-27 | |
| Scissors (3 mm & 7 mm blades) | Fine Science Tools (or FST), Foster City, CA, USA | Moria MC52 & 15000-00 | |
| Scissors (Vannas style; 9.5 mm & 3 mm blades) | World Precision Instruments | 555640S, 14364 | |
| Stereomicroscopes | Zeiss, NY, USA | Stemi 2000 & 2000-C | |
| Syringe filter (0.22 µm) | ThermoFisher Scientific | 722-2520 | |
| Temperature Controller (Dual Channel) | Warner Instruments | TC-344B or C | |
| Valve Control System | Warner Instruments | VC-6 | |
| Vibration Isolation Table | Technical Manufacturing, Peabody, MA, USA | Micro-g |
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Citar este artigo
Hakim, M. A., Pires, P. W., Behringer, E. J. Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma. J. Vis. Exp. (181), e63463, doi:10.3791/63463 (2022).