Name: ex vivo pressurized hippocampal capillary-parenchymal arteriole preparation for functional study
Uploaded: 2019-12-18T14:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study at JoVE.com

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。

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
<ol><li>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...

<ol>
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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>0.22&#181;m Syringe Filters</td>
			<td>CELLTREAT Scientific Products</td>
			<td>229751</td>
			<td></td>
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			<td>TC-324B</td>
			<td></td>
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			<td>Sutter Instruments</td>
			<td>B120-69-10</td>
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			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td></td>
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			<td>CaCl2 dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td></td>
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			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td></td>
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			<td>Dissection Scope</td>
			<td>Olympus</td>
			<td>SZ11</td>
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			<td>ECOLINE VC-MS/CA 4-12 &#8212; complete Pump with Drive and MS/CA 4-12 pump-head</td>
			<td>Ismatec</td>
			<td>ISM 1090</td>
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			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E4378</td>
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			<td>Fine Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14063-09</td>
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			<td>Warner Instruments</td>
			<td>SH-27B</td>
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			<td>Fisher Scientific</td>
			<td>12-460-117</td>
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			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
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			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5379</td>
			<td></td>
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			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M1880</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl Anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
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			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-153</td>
			<td></td>
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			<td>ML 133 hydrochloride</td>
			<td>Tocris</td>
			<td>4549</td>
			<td></td>
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			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr>
			<td>NS309</td>
			<td>Tocris</td>
			<td>3895</td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer III - Intracellular Microinjection Dispense Systems, 2-channel</td>
			<td>Parker Hannifin</td>
			<td>052-0500-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Servo Controller with Peristaltic Pump</td>
			<td>Living Systems Instrumentation</td>
			<td>PS-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P3662</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>Fine Science Tools</td>
			<td>14001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Micropipette Puller</td>
			<td>Narishige</td>
			<td>PP-83</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo pressurized hippocampal capillary-parenchymal arteriole preparation for functional study

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。

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?...

Watch this Scientific Journal Video about Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study at JoVE.com

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

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。

Ex Vivo 加压希马卡毛细管- 功能研究准备

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

我们的方法允许研究位于结构中的血管，否则很难在Vivvo中成像，如海马。由于血管从脑组织中去除，因此可以研究毛细血管和动脉之间的信号，而不会对周围组织产生非靶效应。要分离海马，请使用小型解剖剪刀沿鼠标头部顶部的中线切割皮肤，然后将皮肤移到两侧。从头骨的头骨侧开始，沿着中线切割头骨，直到到达嗅觉球，取出头骨的部分，直到大脑暴露。从靠近鼠标的鼻子开始，慢慢取出大脑，用剪刀切开嗅觉球、颅神经和脊髓。将大脑、腹侧下潜于莫天斯缓冲盐水中，置于解剖板的中心，将板置于解剖显微镜下。握住与盘子底部平行的锋利边缘，用剃刀刀片一举将大脑沿着纵向裂缝切成两半。将一个半球放在板的中心，中线朝下，沿着横向裂缝将剃须刀刀片直推通过组织，以切除小脑和脑干。旋转半球，使中端朝上，并使用铲子将大脑保持到位。将第二个铲的尖端插入语料库的下方，在组织下方铲取，以从海马体中去除丘脑、隔膜和下丘脑。海马现在应该像大脑后侧附近的弯曲结构一样可见。用一个铲子将大脑保持到位，用第二个铲子将海马从脑中挖出来。然后将海马转移到一个新的解剖板，用新鲜的 MOP 溶液填充一半。对于海马动脉分离，在一个海马体的两端放置小针脚，以将海马动脉的样本固定向上。使用非常锋利的钳子，轻轻地伸展海马的小部分，以松开动脉周围的组织。并搜索通过后海马组织，以确定外部横向动脉。当动脉被定位时，从海马体中抓住外部横向动脉，然后慢慢将其从组织中拉开，以收集动脉和毛细血管。每个海马体的动脉被单独去除。当没有更多的容器要去除时，将样品放在冰上，丢弃剩余的海马组织。对于动脉的凝固，用一根以毛细血管结尾的分支定位和动脉，将动脉放入器官室中。小心地将管尖通过目标区域下方的动脉壁，以安装血管。然后，小心地将容器滑到管上，直到有足够的组织来系上领带。使用12-0尼龙缝合线，使一个松散的结，将适合在血管和管，并使用半钩结，以确保领带，拉两端拧紧结，以确保动脉到管。将另一条领带固定在动脉的另一端，以密封它。在腔室的对面，放下第二根管，直到管的点将细管端的领带固定下来。然后使用第三个管将毛细管分支固定在盖玻片上，将尖端靠近分支的末端，同时将毛细管的末端暴露在外。由于大脑微循环非常脆弱，一定要在割割过程中尽量减少血管的拉伸和处理，以确保动脉的生存。对于压力成像分析，使用记录软件将腔室转移到光学显微镜阶段，并连接进流管和流出管到腔室进行大量处理。以每分钟四毫升的流量以 37 摄氏度的人工脑脊液开始增压，并将压力管连接到与压力传感器配对的内向泵上。将内部压力带到 20 毫米的汞，然后启动记录软件。调整显微镜和图像设置，以实现尽可能清晰的图像。并使用边缘检测软件检查动脉在完全扩张时直径约为 15 到 30 微米。设置优化后，开始记录并增加容器内压力高达 40 毫米的汞，同时使用边缘检测软件记录动脉直径。然后将室中大量进行 15 至 20 分钟，以洗掉 MOP 溶液。为了测试容器的可行性，在浴池中应用一微摩尔NS309溶液，动脉部分应扩张，表现出30至40%的原生感。一旦建立了动脉的基线基调，使用玻璃拉拔器在一端用细点制作黄牛，并使用钳子打破每个动脉的尖端，使感兴趣的药物可以顺利地流过尖端，每平方英寸有五磅的压力。用感兴趣的药物填充管。将管管加载到连接到显微镜上的三轴微操纵器上。将压力喷射系统的管子连接到管，然后缓慢地将管子放入毛细管附近的浴缸中，注意不要撞到容器或腔室的任何部分。为了刺激毛细管，将风管降到毛细管旁边的盖玻片，并在所需的喷射时间激活压力喷射系统。在刺激结束时，稍微提高管，以避免进一步的刺激。为了确认只有毛细血管受到刺激，用NS309溶液的微摩尔填充新的细管，并刺激毛细血管，如所证明的。然后用无钙溶液沐浴制剂，获得最大的附庸。由于内皮中的钙敏感钾通道，一微摩尔NS309的沐浴应用导致动脉几乎最大扩张。然而，毛细管内皮细胞缺乏中小传导通道，不超极化，以回应激动剂，因此，刺激毛细管以NS309结束，不会引起上游动脉扩张，这表明NS309没有到达动脉，并可用作控制通过压力喷射施加在毛细管上的化合物的空间限制。如所证明，使用海马毛细管胶质制剂，将人工脑脊液与10毫摩尔钾补充到毛细管末端，导致上游动脉扩张，与雄性小鼠和雌性小鼠的制剂之间没有不同。此外，Kir2抑制剂ML 133的加入，几乎消除了毛细管诱导动脉扩张，以响应10毫摩尔钾制剂从男性和女性小鼠。安装血塞时，限制与血管的直接相互作用，以尽量减少对血管的损害，并增加血管的生存能力。一旦血管被分离，可以测试不同的药物化合物，或血管可以进一步处理，用于动物生物学、免疫化学或电生理学研究。

Research

JoVE Journal

Neuroscience

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

科研

神经科学

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

鼠标后脑<em&gt;前体内</em&gt;企业文化来研究面部Branchiomotor神经元迁移

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

富氧运动器械中慢性硅探针的植入及海马位细胞的记录

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

为电子生理记录准备急性人类河马切片

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

大鼠坐骨神经用于 离体 神经生理学的制备

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...

OCT-Based Multimodal Imaging of Human Donor Eyes for AMD Research

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration (Video) | JoVE 

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...