Name: isolation and functional analysis of arteriolar endothelium of mouse brain parenchyma
Uploaded: 2022-03-11T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma at JoVE.com

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。

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...

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+K+-sensing+initiates+retrograde+hyperpolarization+to+increase+local+cerebral+blood+flow.">Capillary K+-sensing initiates retrograde hyperpolarization to increase local cerebral blood flow.</a> Nature Neuroscience. 20 (5), 717-726 (2017).</li><li>Kelleher, R. J., Soiza, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+endothelial+dysfunction+in+the+development+of+Alzheimer's+disease:+Is+Alzheimer's+a+vascular+disorder.">Evidence of endothelial dysfunction in the development of Alzheimer's disease: Is Alzheimer's a vascular disorder.</a> American Journal of Cardiovascular Disease. 3 (4), 197-226 (2013).</li><li>Hakim, M. A., Behringer, E. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta+disrupts+unitary+calcium+entry+through+endothelial+NMDA+receptors+in+mouse+cerebral+arteries.">Amyloid-beta disrupts unitary calcium entry through endothelial NMDA receptors in mouse cerebral arteries.</a> Journal of Cerebral Blood Flow and Metabolism. , (2021).</li><li>De Silva, T. M., Modrick, M. L., Dabertrand, F., Faraci, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+cerebral+arteries+and+parenchymal+arterioles+with+aging:+Role+of+rho+kinase+2+and+impact+of+genetic+background.">Changes in cerebral arteries and parenchymal arterioles with aging: Role of rho kinase 2 and impact of genetic background.</a> Hypertension. 71 (5), 921-927 (2018).</li><li>Fontaine, J. T., Rosehart, A. C., Joutel, A., Dabertrand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HB-EGF+depolarizes+hippocampal+arterioles+to+restore+myogenic+tone+in+a+genetic+model+of+small+vessel+disease.">HB-EGF depolarizes hippocampal arterioles to restore myogenic tone in a genetic model of small vessel disease.</a> Mechanisms of Ageing and Development. 192, 111389 (2020).</li><li>Pires, P. W., Dabertrand, F., Earley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cannulation+of+cerebral+parenchymal+arterioles.">Isolation and cannulation of cerebral parenchymal arterioles.</a> Journal of Visualized Experiments: JoVE. (111), e53835 (2016).</li><li>Rosehart, A. C., Johnson, A. C., Dabertrand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+pressurized+hippocampal+capillary-parenchymal+arteriole+preparation+for+functional+study.">Ex vivo pressurized hippocampal capillary-parenchymal arteriole preparation for functional study.</a> Journal of Visualized Experiments: JoVE. (154), e60676 (2019).</li><li>Hakim, M. A., Behringer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurements+of+intracellular+calcium+and+membrane+potential+in+freshly+isolated+and+intact+mouse+cerebral+endothelium.">Simultaneous measurements of intracellular calcium and membrane potential in freshly isolated and intact mouse cerebral endothelium.</a> Journal of Visualized Experiments: JoVE. (143), e58832 (2019).</li><li>Hakim, M. A., Chum, P. P., Buchholz, J. N., Behringer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+alters+cerebrovascular+endothelial+GPCR+and+K++channel+function:+Divergent+role+of+biological+sex.">Aging alters cerebrovascular endothelial GPCR and K+ channel function: Divergent role of biological sex.</a> Journals of Gerontology, Series A: Biological Sciences and Medical Sciences. 75 (11), 2064-2073 (2020).</li><li>Behringer, E. J., Hakim, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interaction+among+KCa+and+TRP+channels+for+cardiovascular+physiology:+Modern+perspectives+on+aging+and+chronic+disease.">Functional interaction among KCa and TRP channels for cardiovascular physiology: Modern perspectives on aging and chronic disease.</a> International Journal of Molecular Sciences. 20 (6), 1380 (2019).</li><li>Marrelli, S. P., Eckmann, M. S., Hunte, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelial+intermediate+conductance+KCa+channels+in+cerebral+EDHF-mediated+dilations.">Role of endothelial intermediate conductance KCa channels in cerebral EDHF-mediated dilations.</a> American Journal of Physiology-Heart and Circulatory Physiology. 285 (4), 1590-1599 (2003).</li><li>Hannah, R. M., Dunn, K. M., Bonev, A. D., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+SKCa+and+IKCa+channels+regulate+brain+parenchymal+arteriolar+diameter+and+cortical+cerebral+blood+flow.">Endothelial SKCa and IKCa channels regulate brain parenchymal arteriolar diameter and cortical cerebral blood flow.</a> Journal of Cerebral Blood Flow and Metabolism. 31 (5), 1175-1186 (2011).</li><li>Hakim, M. A., Buchholz, J. N., Behringer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+dynamics+of+isolated+cerebral+and+skeletal+muscle+endothelial+tubes:+Differential+roles+of+G-protein-coupled+receptors+and+K++channels.">Electrical dynamics of isolated cerebral and skeletal muscle endothelial tubes: Differential roles of G-protein-coupled receptors and K+ channels.</a> Pharmacological Research and Perspectives. 6 (2), 00391 (2018).</li><li>Hakim, M. A., Behringer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methyl-beta-cyclodextrin+restores+KIR+channel+function+in+brain+endothelium+of+female+Alzheimer's+disease+Mice.">Methyl-beta-cyclodextrin restores KIR channel function in brain endothelium of female Alzheimer's disease Mice.</a> Journal of Alzheimers Disease Reports. 5 (1), 693-703 (2021).</li><li>Behringer, E. J., Shaw, R. L., Westcott, E. B., Socha, M. J., Segal, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+arterial+lesions+underlying+lacunes.">The arterial lesions underlying lacunes.</a> Acta Neuropathologica. 12 (1), 1-15 (1968).</li><li>Behringer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+electrical+signaling+in+arterial+endothelial+tubes:+New+insights+into+cellular+physiology+and+cardiovascular+function.">Calcium and electrical signaling in arterial endothelial tubes: New insights into cellular physiology and cardiovascular function.</a> Microcirculation. 24 (3), (2017).</li><li>Dunn, K. M., Nelson, M. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signal+profiles+in+vascular+endothelium+from+Cdh5-GCaMP8+and+Cx40-GCaMP2+mice.">Calcium signal profiles in vascular endothelium from Cdh5-GCaMP8 and Cx40-GCaMP2 mice.</a> Journal of Vascular Research. 58 (3), 159-171 (2021).</li><li>Bando, Y., Sakamoto, M., Kim, S., Ayzenshtat, I., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+genetically+encoded+voltage+indicators.">Comparative evaluation of genetically encoded voltage indicators.</a> Cell Reports. 26 (3), 802-813 (2019).</li><li>Pires, P. W., Sullivan, M. N., Pritchard, H. A., Robinson, J. 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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amplifiers</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>Axoclamp 2B &#38; Axoclamp 900A</td>
			<td></td>
		</tr>
		<tr>
			<td>Audible baseline monitors</td>
			<td>Ampol US LLC, Sarasota, FL, USA</td>
			<td>&#160;BM-A-TM</td>
			<td></td>
		</tr>
		<tr>
			<td>Bath Chiller (Isotemp 500LCU)</td>
			<td>ThermoFisher Scientific</td>
			<td>13874647</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries (Pinning)</td>
			<td>Warner Instruments</td>
			<td>G150T-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries (Sharp Electrodes)</td>
			<td>Warner Instruments</td>
			<td>GC100F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries (Trituration)</td>
			<td>World Precision Instruments (WPI), Sarasota, FL, USA</td>
			<td>1B100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA: Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2: Calcium Chloride</td>
			<td>Sigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase (Type H Blend)</td>
			<td>Sigma</td>
			<td>C8051</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass (2.4&#160;&#215;&#160;5.0&#160;cm)</td>
			<td>ThermoFisher Scientific</td>
			<td>12-548-5M</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquision Digitizer</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>Digidata 1550A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Dish (Glass Petri with Charcoal Sylgard bottom)</td>
			<td>Living Systems Instrumentation, St. Albans City, VT, USA</td>
			<td>DD-90-S-BLK</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithioerythritol</td>
			<td>Sigma</td>
			<td>D8255</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO: Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastase (porcine pancreas)</td>
			<td>Sigma</td>
			<td>E7885</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoplasmic Reticulum Tracker (ER-Tracker Red, BODIPY TR Glibenclamide)</td>
			<td>ThermoFisher Scientific</td>
			<td>E34250</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber optic light sources</td>
			<td>&#160;Schott, Mainz, Germany &#38; KL200, Zeiss</td>
			<td>Fostec 8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Control Valve</td>
			<td>Warner Instruments</td>
			<td>&#160;FR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence system interface, ARC lamp &#38; power supply, hyperswitch and PMT</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>IonOptix Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (Fine-tipped, sharpened)</td>
			<td>FST</td>
			<td>Dumont #5 &#38; Dumont #55</td>
			<td></td>
		</tr>
		<tr>
			<td>Function Generator</td>
			<td>EZ Digital, Seoul, South Korea</td>
			<td>FG-8002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 AM dye</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>F14185</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl: Hydrochloric Acid</td>
			<td>ThermoFisher Scientific (Pittsburgh, PA, USA)</td>
			<td>A466250</td>
			<td></td>
		</tr>
		<tr>
			<td>Headstages</td>
			<td>Molecular Devices</td>
			<td>HS-2A &#38; HS-9A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline Solution Heater</td>
			<td>Warner Instruments</td>
			<td>SH-27B</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl: Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2: Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige, East Meadow, NY, USA</td>
			<td>&#160;MF-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Siskiyou</td>
			<td>&#160;MX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller (digital)</td>
			<td>Sutter Instruments, Novato, CA, USA</td>
			<td>P-97 or P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Nikon-inverted)</td>
			<td>Nikon Instruments Inc, Melville, NY, USA</td>
			<td>Ts2</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Nikon-inverted)</td>
			<td>Nikon Instruments Inc</td>
			<td>Eclipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope objectives</td>
			<td>Nikon Instruments Inc</td>
			<td>20X (S-Fluor) and 40X (Plan Fluor)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope platform (anodized aluminum; diameter, 7.8&#160;cm)</td>
			<td>Warner Instruments</td>
			<td>PM6 or PH6</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Stage (Aluminum)</td>
			<td>Siskiyou, Grants Pass, OR, USA</td>
			<td>8090P</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsyringe Pump Controller</td>
			<td>World Precision Instruments (WPI), Sarasota, FL, USA</td>
			<td>SYS-MICRO4</td>
			<td></td>
		</tr>
		<tr>
			<td>MTA: 2-Methylthioadenosine diphosphate trisodium salt</td>
			<td>Tocris</td>
			<td>1624</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl: Sodium Chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH: Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear Stain (NucBlue Live ReadyProbes Reagent; Hoechst 33342)</td>
			<td>ThermoFisher Scientific</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix, Beaverton, Oregon, USA</td>
			<td>&#160;TDS 2024B</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma</td>
			<td>P4762</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast objectives</td>
			<td>Nikon Instruments Inc</td>
			<td>&#160;(Ph1 DL; 10X &#38; 20X)</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Membrane Stain (CellMask Deep Red)</td>
			<td>ThermoFisher Scientific</td>
			<td>C10046</td>
			<td></td>
		</tr>
		<tr>
			<td>Plexiglas superfusion chamber</td>
			<td>Warner Instruments, Camden, CT, USA</td>
			<td>RC-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors (3 mm &#38; 7 mm blades)</td>
			<td>Fine Science Tools (or FST), Foster City, CA, USA</td>
			<td>Moria MC52 &#38; 15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors (Vannas style; 9.5&#160;mm &#38; 3&#160;mm blades)</td>
			<td>World Precision Instruments</td>
			<td>555640S, 14364</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscopes</td>
			<td>Zeiss, NY, USA</td>
			<td>Stemi 2000 &#38; 2000-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter (0.22 &#181;m)</td>
			<td>ThermoFisher Scientific</td>
			<td>722-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller (Dual Channel)</td>
			<td>Warner Instruments</td>
			<td>TC-344B or C</td>
			<td></td>
		</tr>
		<tr>
			<td>Valve Control System</td>
			<td>Warner Instruments</td>
			<td>VC-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration Isolation Table</td>
			<td>Technical Manufacturing, Peabody, MA, USA</td>
			<td>&#160;Micro-g</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and functional analysis of arteriolar endothelium of mouse brain parenchyma

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。

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）...

Watch this Scientific Journal Video about Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma at JoVE.com

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

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。

小鼠脑实质小动脉内皮的分离及功能分析

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

这种方法将更好地了解实质小动脉内皮的功能，其直接将脑内血流与整个大脑神经元和神经胶质活动的燃料联系起来。与小动脉内皮相比，一个明显的技术优势是增强了单个脑内皮细胞及其各自细胞器之间的形态学尺寸以及钙和电信号传导的分辨率。最初，脑内皮的分离和检查将非常具有挑战性。但是，可以通过大量的练习以及耐心和奉献精神来掌握这种技术。在烧杯或培养皿中用冷解剖溶液清洗分离的大脑，以从大脑表面去除残留的血液。将腹侧脑侧朝上放在含有冷解剖溶液的腔室中，以隔离实质小动脉。为了分离实质小动脉，在含有超过50厘米深的木炭注入硅聚合物涂层的培养皿中，用钢针在冷解剖溶液中固定隔离的大脑。在MCA周围用锋利且对齐的解剖剪刀从两个半球切割一个矩形的脑组织，同时确保组织段的上部经过威利斯圆的分支点。将脑组织固定在培养皿中，MCA用钢针朝上。小心地在针脚附近做一个浅切口，并用小镊子取出pia，从一端轻轻剥离到另一端。用针在培养皿中小心地用从MCA分支的实质小动脉固定孤立的软脑膜，并小心地解剖实质小动脉。切断任何剩余的远端分支，并确保小动脉清洁，没有组织附着。使用这种干净完整的小动脉进行酶消化。或者，根据需要将每个小动脉切成两块进行酶消化，以制备内皮管。对于小动脉段的部分消化，将完整的小动脉段放入含有木瓜蛋白酶，二硫代赤藓糖醇，胶原酶和弹性蛋白酶的10毫升玻璃管中，放入一毫升解离溶液中。在34摄氏度下孵育小动脉段10至12分钟。为了分离小动脉内皮管，将与微型注射器注射器连接在腔室中的解离溶液中，并将其放置在靠近消化血管段一端的一端。在泵控制器上将速率设置为每秒 1 到 3 纳升的范围内，以便进行轻柔的研磨。在保持100X至200X放大倍率的同时，将小动脉段撤回移液管中，然后注射以解离外切和平滑肌细胞。对血管段进行研磨，直到所有平滑肌细胞解离，只有内皮细胞保持完整管状。使用微机械手，用硼硅酸盐玻璃固定移液管中玻璃盖玻片上的内皮管的每一端 要测量内皮膜电位，同时通过4X物镜观察，小心地将尖锐的电极尖端正好定位在小动脉内皮管的细胞上方，用微机械手进入流动的生理盐溶液中。逐渐将放大倍率增至400X，并根据需要重新定位电极尖端。使用微机械手，轻轻地将尖锐电极的尖端插入内皮管的一个细胞中，然后开始使用静电计记录VM。一旦内皮静息VM在零下30至零下40毫伏范围内稳定，根据实验目标应用所需的药理学药物。在37摄氏度下用质膜或所需细胞器的荧光跟踪器加载内皮管15至30分钟。用新鲜的超融合生理盐溶液洗涤细胞，并在显微镜下以相应染料的激发波长对活细胞进行成像。通过测量细胞内钙和膜电位来评估内皮功能，以响应药理学药物MTA，一种有效的嘌呤能受体激动剂在37摄氏度。在应用一个微摩尔MTA时，细胞内钙浓度随着伴随的超极化而迅速增加。此外，用荧光跟踪仪在37摄氏度下孵育完整的内皮，以进行可视化以检查细胞形态。活内皮细胞成像显示质膜和细胞核共染色。将质膜与内质网荧光染色一起染色，从而在质膜附近明显重叠的ER区域显示为橙色。实验者在解剖和隔离过程中应小心，以避免对小动脉的损伤，因为受损的小动脉肯定不会产生完整的内皮管。手术后，细胞可用于具有荧光抗体的固定免疫组织化学或细胞器和膜脂质的活细胞染色。该技术将产生新的信息，用于在生理学的基本水平上理解脑血流的潜在机制，并选择向病理学的过渡以进行治疗。

Research

JoVE Journal

Biology

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

微血管内皮生理E -选择素介导白细胞滚动分析

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

科研

生物学

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

分离翻译含肽- tRNA的核糖体的功能和结构分析

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

映射后的Theta突发刺激对人类听觉皮层功能成像

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...