分离及脑实质小动脉插管
Summary
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.
Abstract
脑实质动脉（功率放大器），其包括实质小动脉，穿透动脉和预毛细管小动脉，是高电阻的血管从脑膜动脉和小动脉和潜水分支出来进入脑实质。个人PA灌注实质的离散圆柱形领土和其中所包含的神经元。这些小动脉脑血流量在全球（脑血管自动调节），并在本地（功能性充血）的调节中的球员。功率放大器是神经血管单元，一个相匹配的大脑内局部血流到代谢活性结构的一部分，并且还包括神经元，的interneurons，和星形胶质细胞。通过功率放大器灌注直接链接到该特定领土神经元和神经元代谢铅增加的活性，造成进料的PA的扩张局部灌注的增大。 PA的调控从更好的特点，不同软脑膜动脉。压力诱导的血管收缩更大的功率放大器和血管舒张机制有所不同。此外，功率放大器不会从血管周围神经受到外在的支配 – 神经支配是通过与星形胶质细胞endfeet内在联系和本质上是间接的。因此，通过使用软脑膜血管研究积累关于收缩调节数据并不直接转化为理解的PA的功能。此外，病理状态，如高血压，糖尿病，如何影响PA结构和反应性仍然未定。这方面的知识缺口部分的有关PA隔离和插管技术困难的结果。在这个手稿中，我们提出了隔离和啮齿动物功率放大器插管的协议。此外，我们显示，可以与这些小动脉，包括激动剂诱导收缩和肌原反应进行的实验的例子。虽然这个手稿的重点是PA插管和压力肌动描记，PA隔离s时，可以也可以用于生物化学，生物物理，分子，和成像研究。
Introduction
脑循环被唯一组织支持中枢神经元，即具有有限能量存储并因此在氧气压力和所需的营养供给的变化高度敏感的细胞的代谢需求。当执行特定任务特定的神经元亚群变得活跃，脉管促进灌注高度局部增加以防止局部缺氧和营养物1的耗竭。这是被称为神经血管耦合官能充血的一种形式，并且是依赖于血管神经单元的正确操作，活性神经元，星形胶质细胞和脑动脉2构成。脑实质动脉，一组的血管包含实质，穿透和预毛细管小动脉，是这种响应集中重要，这是再临界单独研究它们为了研究神经血管耦合3。
<p系列=“jove_content”>实质小动脉小（20 – 70微米内径）高电阻血管灌注在大脑中不同的神经元的人群。从表面上的软脑膜血管分支出来，实质动脉渗入脑实质在近90ᵒ角喂地下微循环（ 图1）。这些动脉在维持适当的灌注压，因为它们是最前端平滑含有肌肉血管保护的毛细管的关键作用。与此相反的表面软脑膜循环，实质动脉缺乏侧支和吻合，因此是脑循环4的“瓶颈”。其结果是，实质小动脉功能障碍有助于脑血管疾病的发展，如血管性认知障碍和小缺血性中风（也称为沉默或腔隙性脑梗塞）。研究indicatË是实质小动脉功能障碍可以通过原发性高血压5，慢性应激6诱导，并且是小血管病变的基因小鼠模型7的早期事件。大鼠单穿透动脉进一步，实验诱导的阻塞足以使小缺血性中风是圆柱形的，类似那些在年龄较大的人8观察。除了这些解剖优，调节收缩功能的机制脑膜动脉和实质动脉之间不同。生肌血管收缩是更大实质动脉9，可能是因为在细胞内Ca缺乏外在支配10，机械传导11的不同的模式，和差异2+在血管平滑肌细胞中的信号12,13。有证据表明，内皮依赖性血管舒张机制也是这些vascu之间不同与扩散因子如一氧化氮和前列环素14相比拉尔段，表现出对涉及钙机制的更大依赖实质激活的动脉血管壁内的K +通道和电紧张的沟通。因此，收集的数据使用的软脑膜动脉可能不一定适用于实质小动脉的实验，让我们的脑灌注的局部控制知识的差距。
尽管它们的重要性，实质小动脉有很大的不足的研究，主要是由于隔离和安装用于体外研究的技术挑战。在这个手稿，我们描述的方法来分离和导管插入脑实质动脉，其可以用于压力肌动描记，或为免疫标记，电生理学，分子生物学和生物化学分析隔离组织。
Protocol
Representative Results
Discussion
脑实质动脉是高抗动脉与灌注不同神经元的人口数吻合和分支。这些专门的血管是通过星形胶质细胞介导的血管扩张1脑血管自动调节和神经血管耦合中央的球员 。这些专业血管脑血管疾病的重要性已经知道了约50年的时候米勒费希尔博士的开创性工作，在高血压患者验尸 16的大脑描述结构变化的实质动脉腔隙性梗死的领土内。这些改变，耦合到功能障碍，可引起的深?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Funded by NHLBI R01HL091905 (SE), the United Leukodystrophy Foundation CADASIL research grant (FD) and AHA 15POST247200 (PWP). The authors would like to thank Samantha P. Ahchay for providing the image on Figure 1, and Dr. Gerry Herrera, Ph.D., for providing critical comments on the manuscript.
Materials
| artificial Cerebrospinal Fluid | |||
| NaCl | Fisher Scientific | S-640 | |
| KCl | Fisher Scientific | P217 | |
| MgCl Anhydrous | Sigma-Aldrich | M-8266 | |
| NaHCO3 | Fisher Scientific | S233 | |
| NaH2PO4 | Sigma-Aldrich | S9638 | |
| D-(+)-Glucose | Sigma-Aldrich | G2870 | |
| CaCl2 | Sigma-Aldrich | C4901 | |
| Bovine Serum Albumin | Sigma-Aldrich | A9647 | |
| Name | Company | Catalog Number | Comments |
| Isolation/ Cannulation | |||
| Stereo Microscope | Olympus | SZX7 | |
| Super Fine Forceps | Fine Science Tools | 11252-00 | |
| Vannas Spring Scissors | Fine Science Tools | 15000-00 | |
| Wiretrol 50 μL | VWR Scientific | 5-000-1050 | |
| 0.2 μm Sterile Syringe Filter | VWR Scientific | 28145-477 | |
| Micropipette Puller | Sutter Instruments | P-97 | |
| Borosilicate Glass O.D.: 1.2 mm, I.D.: 0.68 mm | Sutter Instruments | B120-69-10 | |
| Dark Green Nylon Thread | Living Systems Instrumentation | THR-G | |
| Linear Alignment Single Vessel Chamber | Living Systems Instrumentation | CH-1-LIN | |
| Pressure Servo Controller with Peristaltic Pump | Living Systems Instrumentation | PS-200 | |
| Video Dimension Analyzer | Living Systems Instrumentation | VDA-10 | |
| Four Channel Recorder with LabScribe 3 Recording and Analysis Software | Living Systems Instrumentation | DAQ-IWORX-404 | |
| Heating Unit | Warner Instruments | 64-0102 | |
| Automatic Temperature Controller | Warner Instruments | TC-324B |
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