Name: reliable isolation of central nervous system microvessels across five vertebrate groups
Uploaded: 2020-01-12T12:00:00
Description: Watch this Scientific Journal Video about Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups at JoVE.com

Cruz-Orengo博士得到了加州大学戴维斯分校兽医学院启动基金的支持。

本研究的所有程序均符合加州大学（UC）、戴维斯机构动物护理和使用委员会（IACUC）制定的指导方针。加州大学戴维斯分校的动物护理由多个独立资源监管，自1966年以来一直获得国际实验室动物护理评估与认证协会（AAALAC）的认证。猪中枢神经系统组织从UCD动物科学系，肉类科学实验室获得。来自河河猴的CNS组织从加州国家灵长类研究中心病理学部（NIH P51OD0111107）获得。实验室工作人员对猪和?...

<ol>
	<li>Bundgaard, M., Abbott, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+vertebrates+started+out+with+a+glial+blood-brain+barrier+4-500+million+years+ago.">All vertebrates started out with a glial blood-brain barrier 4-500 million years ago.</a> Glia. 56, 699-708 (2008).</li><li>Daneman, R., Prat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier.">The blood-brain barrier.</a> Cold Spring Harbor Perspectives in Biology. 7, a020412 (2015).</li><li>Obermeier, B., Verma, A., Ransohoff, R. 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+blood-brain+barrier.">The blood-brain barrier.</a> Handbook of Clinical Neurology. 133, 39-59 (2016).</li><li>Kealy, J., Greene, C., Campbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+regulation+in+psychiatric+disorders.">Blood-brain barrier regulation in psychiatric disorders.</a> Neuroscience Letters. , (2018).</li><li>Sweeney, M. D., Kisler, K., Montagne, A., Toga, A. W., Zlokovic, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+brain+vasculature+in+neurodegenerative+disorders.">The role of brain vasculature in neurodegenerative disorders.</a> Nature Neuroscience. 21, 1318-1331 (2018).</li><li>Sweeney, M. D., Zhao, Z., Montagne, A., Nelson, A. R., Zlokovic, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-Brain+Barrier:+From+Physiology+to+Disease+and+Back.">Blood-Brain Barrier: From Physiology to Disease and Back.</a> Physiological Reviews. 99, 21-78 (2019).</li><li>Wilhelm, I., Nyul-Toth, A., Suciu, M., Hermenean, A., Krizbai, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+the+blood-brain+barrier.">Heterogeneity of the blood-brain barrier.</a> Tissue Barriers. 4, e1143544 (2016).</li><li>O'Brown, N. M., Pfau, S. J., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridging+barriers:+a+comparative+look+at+the+blood-brain+barrier+across+organisms.">Bridging barriers: a comparative look at the blood-brain barrier across organisms.</a> Genes &amp; Development. 32, 466-478 (2018).</li><li>Cruz-Orengo, 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=CXCR7+influences+leukocyte+entry+into+the+CNS+parenchyma+by+controlling+abluminal+CXCL12+abundance+during+autoimmunity.">CXCR7 influences leukocyte entry into the CNS parenchyma by controlling abluminal CXCL12 abundance during autoimmunity.</a> Journal of Experimental Medicine. 208, 327-339 (2011).</li><li>Serres, S., 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=VCAM-1-targeted+magnetic+resonance+imaging+reveals+subclinical+disease+in+a+mouse+model+of+multiple+sclerosis.">VCAM-1-targeted magnetic resonance imaging reveals subclinical disease in a mouse model of multiple sclerosis.</a> FASEB Journal. 25, 4415-4422 (2011).</li><li>Tietz, S., Engelhardt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+barriers:+Crosstalk+between+complex+tight+junctions+and+adherens+junctions.">Brain barriers: Crosstalk between complex tight junctions and adherens junctions.</a> Journal of Cell Biology. 209, 493-506 (2015).</li><li>Sohet, F., 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=LSR/angulin-1+is+a+tricellular+tight+junction+protein+involved+in+blood-brain+barrier+formation.">LSR/angulin-1 is a tricellular tight junction protein involved in blood-brain barrier formation.</a> Journal of Cell Biology. 208, 703-711 (2015).</li><li>Cruz-Orengo, 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=Enhanced+sphingosine-1-phosphate+receptor+2+expression+underlies+female+CNS+autoimmunity+susceptibility.">Enhanced sphingosine-1-phosphate receptor 2 expression underlies female CNS autoimmunity susceptibility.</a> Journal of Clinical Investigation. 124, 2571-2584 (2014).</li><li>Dayton, J. R., Franke, M. C., Yuan, Y., Cruz-Orengo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Straightforward+method+for+singularized+and+region-specific+CNS+microvessels+isolation.">Straightforward method for singularized and region-specific CNS microvessels isolation.</a> Journal of Neuroscience Methods. 318, 17-33 (2019).</li><li>Smyth, L. C. D., 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=Markers+for+human+brain+pericytes+and+smooth+muscle+cells.">Markers for human brain pericytes and smooth muscle cells.</a> Journal of Chemical Neuroanatomy. 92, 48-60 (2018).</li><li>Granberg, T., 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=In+vivo+characterization+of+cortical+and+white+matter+neuroaxonal+pathology+in+early+multiple+sclerosis.">In vivo characterization of cortical and white matter neuroaxonal pathology in early multiple sclerosis.</a> Brain. 140, 2912-2926 (2017).</li><li>Datta, G., 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=Neuroinflammation+and+its+relationship+to+changes+in+brain+volume+and+white+matter+lesions+in+multiple+sclerosis.">Neuroinflammation and its relationship to changes in brain volume and white matter lesions in multiple sclerosis.</a> Brain. 140, 2927-2938 (2017).</li><li>Tommasin, S., Gianni, C., De Giglio, L., Pantano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroimaging+Techniques+to+Assess+Inflammation+in+Multiple+Sclerosis.">Neuroimaging Techniques to Assess Inflammation in Multiple Sclerosis.</a> Neuroscience. 403, 4-16 (2019).</li><li>Liebner, S., 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=Functional+morphology+of+the+blood-brain+barrier+in+health+and+disease.">Functional morphology of the blood-brain barrier in health and disease.</a> Acta Neuropathologica. 135, 311-336 (2018).</li><li>Cornford, E., Hyman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+brain+endothelial+luminal+and+abluminal+transporters+with+immunogold+electron+microscopy.">Localization of brain endothelial luminal and abluminal transporters with immunogold electron microscopy.</a> NeuroRx. 2, 27-43 (2005).</li><li>Boulay, A. C., Saubamea, B., Decleves, X., Cohen-Salmon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Mouse+Brain+Vessels.">Purification of Mouse Brain Vessels.</a> Journal of Visualized Experiments. , 53208 (2015).</li><li>Paul, D., Cowan, A. E., Ge, S., Pachter, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+3D+analysis+of+Claudin-5+reveals+significant+endothelial+heterogeneity+among+CNS+microvessels.">Novel 3D analysis of Claudin-5 reveals significant endothelial heterogeneity among CNS microvessels.</a> Microvascular Research. , (2012).</li><li>Munikoti, V. V., Hoang-Minh, L. B., Ormerod, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+digestion+improves+the+purity+of+harvested+cerebral+microvessels.">Enzymatic digestion improves the purity of harvested cerebral microvessels.</a> Journal of Neuroscience Methods. 207, 80-85 (2012).</li><li>Yousif, S., Marie-Claire, C., Roux, F., Scherrmann, J. M., Decleves, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+drug+transporters+at+the+blood-brain+barrier+using+an+optimized+isolated+rat+brain+microvessel+strategy.">Expression of drug transporters at the blood-brain barrier using an optimized isolated rat brain microvessel strategy.</a> Brain Research. 1134, 1-11 (2007).</li><li>Bourassa, P., Tremblay, C., Schneider, J. A., Bennett, D. A., Calon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-amyloid+pathology+in+human+brain+microvessel+extracts+from+the+parietal+cortex:+relation+with+cerebral+amyloid+angiopathy+and+Alzheimer's+disease.">Beta-amyloid pathology in human brain microvessel extracts from the parietal cortex: relation with cerebral amyloid angiopathy and Alzheimer's disease.</a> Acta Neuropathologica. 137, 801-823 (2019).</li><li>Porte, B., 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=Proteomic+and+transcriptomic+study+of+brain+microvessels+in+neonatal+and+adult+mice.">Proteomic and transcriptomic study of brain microvessels in neonatal and adult mice.</a> PLoS One. 12, e0171048 (2017).</li></ol>

BBB包括脑微血管内皮细胞的独特特性，加上复杂的结构，紧，粘附，"粘接"结，和粘附斑块关键CNS平衡2，3，19。内皮细胞的特性是由围细胞和周围的星体末端脚过程2，3，19诱导和维护。BBB微血管显示极性（即，在发光或腹皮细胞表面局部的蛋白质的不对称?...

我们的大脑是我们身体中最重要的器官。因此，保持大脑平衡，尽管外部因素，可能会触发偏离正常是一个优先事项。据一些学者说，大约4亿至5亿年前，脊椎动物发展了我们现在所知的血脑屏障（BBB）2，3。这种保护性"篱笆"通过严格调节血液和中枢神经系统之间的离子、分子和细胞的传输，对中枢神经系统 （CNS） 平衡和功能产生最大的影响。当BBB被打乱时，大脑变得易受毒性暴露、感染和炎症的影响。因此，BBB功能障碍与许多，如果不是全部，神经和神经发育障碍4，5，6相关。
BBB的精密功能归因于神经血管单元（NVU）2，3所符合的独特CNS微血管。高度专业化的内皮细胞、围细胞和星形端脚是NVU2，3的细胞成分。这些细胞产生的细胞外基质对NVU和BBB生理学也至关重要。虽然NVU的基本细胞和分子成分在脊椎动物中保存，但各阶和物种7、8之间报告存在异质性。然而，技术限制阻碍了我们在神经生物学、生物医学或转化研究方面充分考虑这些差异的能力。
因此，我们扩展了CNS区域特定的微容器隔离方法，使其适用于来自所有五个脊椎动物群的众多物种：鱼类、两栖动物、爬行动物、鸟类和哺乳动物。该协议被描述为用于小脑脑和大陀螺脊椎动物，包括具有翻译相关性的物种9。此外，我们包括中枢神经系统的其他地区以前没有在此范围内调查，但与神经生理学相关，具有巨大的临床影响：下丘脑，脑垂体和白质。最后，我们测试了这种分离方法作为识别沿NVU和/或BBB9、10、11的蛋白质表达变化的可靠工具的能力。作为概念验证，我们展示了如何使用免疫荧光之后的隔离方法确定 EAE 期间 VCAM-1 和 JAM-B 表达的变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10X PBS</td>
			<td>ThermoFisher</td>
			<td>BP39920</td>
			<td>Used for blocking and antibody diluent.</td>
		</tr>
		<tr>
			<td>20% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713-S</td>
			<td>Used as fixative (4% PFA)</td>
		</tr>
		<tr>
			<td>70,000 MW Dextran</td>
			<td>Millipore Sigma</td>
			<td>9004-54-0</td>
			<td>Used for MV-2 solution</td>
		</tr>
		<tr>
			<td>Adson Forceps</td>
			<td>Fine Science Tools (FST)</td>
			<td>11006-12</td>
			<td>Used for removal of muscle and skin</td>
		</tr>
		<tr>
			<td>Adson Forceps, student quality</td>
			<td>FST</td>
			<td>91106-12</td>
			<td>Same as above but cheaper</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Millipore Sigma</td>
			<td>A7906-100G</td>
			<td>Used for MV-3 solution, blocking and antibody diluent</td>
		</tr>
		<tr>
			<td>Corning 100 &#956;m Cell strainer</td>
			<td>Millipore Sigma</td>
			<td>CLS431752-50EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 70 &#956;m Cell strainer</td>
			<td>Millipore Sigma</td>
			<td>CLS431751-50EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Deskwork low-binding tips</td>
			<td>Millipore Sigma</td>
			<td>CLS4151</td>
			<td>Same as below but cheaper.</td>
		</tr>
		<tr>
			<td>Cultrex Poly-D-Lysine</td>
			<td>R&#38;D</td>
			<td>3439-100-01</td>
			<td>Used for slide coating</td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG-ALEXA 555</td>
			<td>Thermo</td>
			<td>A21432</td>
			<td>Used as secondary antibody. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG-ALEXA 488</td>
			<td>Thermo</td>
			<td>A21202</td>
			<td>Used as secondary antibody. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG-ALEXA 488</td>
			<td>Thermo</td>
			<td>A21206</td>
			<td>Used as secondary antibody. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG-ALEXA 647</td>
			<td>Thermo</td>
			<td>A31573</td>
			<td>Used as secondary antibody. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Donkey anti-Rat IgG-DyLight 650</td>
			<td>Thermo</td>
			<td>SA5-10029</td>
			<td>Used as secondary antibody. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Double-Pronged Tissue Pick</td>
			<td>FST</td>
			<td>18067-11</td>
			<td>Used for removal of meninges and choroid plexus</td>
		</tr>
		<tr>
			<td>Dumont #3c Forceps</td>
			<td>FST</td>
			<td>11231-20</td>
			<td>Used for more delicate and/or small CNS tissue handling (like pituitary)</td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps</td>
			<td>FST</td>
			<td>11274-20</td>
			<td>Used for CNS tisssue dissection and handling</td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps, student</td>
			<td>FST</td>
			<td>91197-00</td>
			<td>Same as above but cheaper</td>
		</tr>
		<tr>
			<td>ep Dualfilter T.I.P.S. LoRetention Tips</td>
			<td>Eppendorf</td>
			<td>22493008</td>
			<td>Better quality than the tips above (more expensive).</td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps, serrated</td>
			<td>FST</td>
			<td>11151-10</td>
			<td>Used for bone removal</td>
		</tr>
		<tr>
			<td>Fine Scissors, sharp</td>
			<td>FST</td>
			<td>14060-09</td>
			<td>Used for removal of pig and macaque dural sac</td>
		</tr>
		<tr>
			<td>Glass Pestle 1.5 mL Microcentrifuge Tube Tissue Grinder Homogenizer, Pack of 10</td>
			<td>Chang Bioscience Inc. (eBay)</td>
			<td>GP1.5_10</td>
			<td>Used for small vetebrate hypothalus and pituitary.</td>
		</tr>
		<tr>
			<td>Goat anti-CXCL12, biotinylated</td>
			<td>PeproTech</td>
			<td>500-P87BGBT</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended dilution: 1:20.</td>
		</tr>
		<tr>
			<td>Goat anti-JAM-B</td>
			<td>R&#38;D</td>
			<td>AF1074</td>
			<td>Used as primary antibody to assess neuroinflammation. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG-ALEXA 488</td>
			<td>Thermo</td>
			<td>A11001</td>
			<td>Used as secondary antibody. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG-ALEXA 555</td>
			<td>Thermo</td>
			<td>A21424</td>
			<td>Used as secondary antibody. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Goat anti-PDGFR&#946;</td>
			<td>R&#38;D</td>
			<td>AF1042</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG-ALEXA 555</td>
			<td>Thermo</td>
			<td>A21249</td>
			<td>Used as secondary antibody. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG-DyLight 488</td>
			<td>Thermo</td>
			<td>35552</td>
			<td>Used as secondary antibody. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Goat anti-Rat IgG-DyLight 650</td>
			<td>Thermo</td>
			<td>SA5-10021</td>
			<td>Used as secondary antibody. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Graefe Forceps, curved tip, 1X2 teeth</td>
			<td>FST</td>
			<td>11054-10</td>
			<td>Use for nylon filter net holding and shaking</td>
		</tr>
		<tr>
			<td>HBSS, 1X buffer with calcium and magnesium</td>
			<td>Corning</td>
			<td>21-022-CM</td>
			<td>Used for MV-1 solution</td>
		</tr>
		<tr>
			<td>HEPES, 1M liquid buffer</td>
			<td>Corning</td>
			<td>25-060-CI</td>
			<td>Used for MV-1 solution</td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4-Biotin-XX</td>
			<td>ThermoFisher Scientific (Thermo)</td>
			<td>I21414</td>
			<td>Glycoprotein isolated from legume Griffonia simplicifolia that binds D-galactosyl residues of endothelial cell glycocalysx. Used for avian and porcine CNS microvessels. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>LaGrange Scissors, serrated</td>
			<td>FST</td>
			<td>14173-12</td>
			<td>Used for skull dissection and laminectomy (except pig and macaque)</td>
		</tr>
		<tr>
			<td>Millicell EZ slide 8-well unit</td>
			<td>Millipore Sigma</td>
			<td>PEZGS0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-CLDN5</td>
			<td>Thermo</td>
			<td>35-2500</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Mouse anti-GGT1</td>
			<td>Abcam</td>
			<td>ab55138</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Mouse anti-Human CD31</td>
			<td>R&#38;D</td>
			<td>BBA7</td>
			<td>Used as primary antibody on primate CNS microvessels. Recommended concentration: 16.5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Mouse anti-NFM</td>
			<td>Thermo</td>
			<td>RMO-270</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;SMA</td>
			<td>Thermo</td>
			<td>MA5-11547</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Nylon Filter Net, roll</td>
			<td>Millipore Sigma</td>
			<td>NY6000010</td>
			<td>Laser-cut to 13 mm diameter filter net discs. Used for small vetebrate hypothalus and pituitary.</td>
		</tr>
		<tr>
			<td>Nylon Filter Nets, 25 mm</td>
			<td>Millipore Sigma</td>
			<td>NY2002500</td>
			<td>Used on most small vertebrates CNS tissues, except hypothalamus and pituitary. Used for macaque and pig hypothalamus and pituitary.</td>
		</tr>
		<tr>
			<td>Nylon Filter Nets, 47 mm</td>
			<td>Millipore Sigma</td>
			<td>NY2004700</td>
			<td>Used for macaque and pig CNS tissues, except hypothalamus and pituitary.</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI</td>
			<td>ThermoFisher</td>
			<td>P36935</td>
			<td>Used to coverslip slides.</td>
		</tr>
		<tr>
			<td>Rabbit anti-AQP4</td>
			<td>Millipore Sigma</td>
			<td>A5971</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Rabbit anti-LSR</td>
			<td>Millipore Sigma</td>
			<td>SAB2107967</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Rabbit anti-NG2</td>
			<td>Millipore Sigma</td>
			<td>AB5320</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Rabbit anti-OSP</td>
			<td>Abcam</td>
			<td>ab53041</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 1 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Rabbit anti-VE-Cadherin</td>
			<td>Abcam</td>
			<td>ab33168</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Rabbit anti-ZO-1</td>
			<td>Thermo</td>
			<td>61-7300</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Rat anti-CD31</td>
			<td>Becton Dickinson</td>
			<td>BD 550274</td>
			<td>Used as primary antibody for murine CNS microvessels. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Rat anti-GFAP</td>
			<td>Thermo</td>
			<td>13-0300</td>
			<td>Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200.</td>
		</tr>
		<tr>
			<td>Rat anti-VCAM-1</td>
			<td>Becton Dickinson</td>
			<td>BD 553329</td>
			<td>Used as primary antibody to assess neuroinflammation. Recommended concentration: 5 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Sterile Ringer&#39;s Solution, Frog</td>
			<td>Aldon Corporation</td>
			<td>IS5066</td>
			<td>Used for amfibian anesthesia</td>
		</tr>
		<tr>
			<td>Streptavidin-ALEXA 555</td>
			<td>Thermo</td>
			<td>S32355</td>
			<td>Used as secondary antibody to label biotinylated primary antibodies. Recommended dilution of 1:500.</td>
		</tr>
		<tr>
			<td>Streptavidin-ALEXA 647</td>
			<td>Thermo</td>
			<td>S32357</td>
			<td>Used as secondary antibody to label biotinylated primary antibodies. Recommended dilution of 1:500.</td>
		</tr>
		<tr>
			<td>Surgical Scissors, sharp</td>
			<td>FST</td>
			<td>14002-12</td>
			<td>Used for removal of muscle and skin</td>
		</tr>
		<tr>
			<td>Surgical Scissors, sharp-blunt</td>
			<td>FST</td>
			<td>14001-16</td>
			<td>Used for decapitation (except pig and macaque)</td>
		</tr>
		<tr>
			<td>Swinnex Filter Holder, 13 mm</td>
			<td>Millipore Sigma</td>
			<td>SX0001300</td>
			<td>Modified by laser-cut. Used for small vetebrate hypothalus and pituitary.</td>
		</tr>
		<tr>
			<td>Swinnex Filter Holder, 25 mm</td>
			<td>Millipore Sigma</td>
			<td>SX0002500</td>
			<td>Modified by laser-cut. Used on most small vertebrates CNS tissues, except hypothalamus and pituitary. Used for macaque and pig hypothalamus and pituitary.</td>
		</tr>
		<tr>
			<td>Swinnex Filter Holder, 47 mm</td>
			<td>Millipore Sigma</td>
			<td>SX0004700</td>
			<td>Modified by laser-cut. Used for macaque and pig CNS tissues, except hypothalamus and pituitary.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher</td>
			<td>50-165-7277</td>
			<td>Used for blocking and antibody diluent.</td>
		</tr>
		<tr>
			<td>Wheaton 120 Vac Overhead Stirrer</td>
			<td>VWR (Supplier DWK Life Sciences)</td>
			<td>62400-904 (DWK #903475)</td>
			<td>Used for macaque and pig CNS tissues with 55 mL tissue grinder, except hypothalamus and pituitary.</td>
		</tr>
		<tr>
			<td>Wheaton Potter-Elvehjem tissue grinder with PTFE pestle, 10 mL</td>
			<td>VWR (Supplier DWK Life Sciences)</td>
			<td>14231-384 (DWK #357979)</td>
			<td>Used on most small vertebrates CNS tissues, except hypothalamus and pituitary. Used for macaque and pig hypothalamus and pituitary.</td>
		</tr>
		<tr>
			<td>Wheaton Potter-Elvehjem tissue grinder with PTFE pestle, 55 mL</td>
			<td>VWR (Supplier DWK Life Sciences)</td>
			<td>14231-372 (DWK #357994)</td>
			<td>Used for macaque and pig CNS tissues, except hypothalamus and pituitary.</td>
		</tr>
	</tbody>
</table>

reliable isolation of central nervous system microvessels across five vertebrate groups

从中枢神经系统 （CNS） 分离微血管通常通过结合多只动物（最常见的是啮齿动物）的皮质组织来执行。这种方法将血脑屏障（BBB）特性的询问限制在皮层，不允许进行个体比较。该项目的重点是开发一种隔离方法，允许比较来自多个中枢神经系统区域的神经血管单元（NVU）：皮层、小脑、视叶、下丘脑、脑干和脊髓。此外，最初为鼠类样本开发的这个方案被成功应用于中小脊椎动物的CNS组织，从中我们也能够从大脑半球白质中分离微血管。当与免疫标记配对时，该方法允许对蛋白质表达进行定量，并在个体、组织类型或治疗之间进行统计比较。通过评估实验性自身免疫性脑脊髓炎 （EAE） 期间蛋白质表达的变化，我们证明了这种适用性，这是神经炎疾病多发性硬化症的一种小鼠模型。此外，通过这种方法分离的微容器可用于下游应用，如qPCR、RNA-seq和西体斑点等。尽管这不是第一次尝试在不使用超离心或酶分离的情况下分离CNS微容器，但它在单一个体和多个CNS区域的比较方面的独特之处。因此，它允许调查一系列差异，否则可能仍然模糊： 中枢神经系统部分 （皮质， 小脑， 视叶， 脑干， 下丘脑， 脑垂体， 和脊髓）， CNS 组织类型 （灰色或白色物质）， 个人，实验性治疗组和物种。

从鼠中枢分离的微血管显示神经血管单元2，3的所有内在细胞成分。使用血小板内皮细胞粘附分子-1（PECAM，也称为CD31）或同化素IB4（一种结合内皮细胞的糖蛋白，用于内皮细胞， 血小板衍生的生长因子-β（PDGFR®）或神经元胶质抗原2（NG2）用于围细胞和水孢素-4（AQP4），用于星体端脚（图3A，B），</...

Watch this Scientific Journal Video about Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups at JoVE.com

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

该协议的目的是从脑血管和陀螺脊椎动物的中枢神经系统的多个区域分离微血管。

在五个脊椎动物群中可靠隔离中枢神经系统微血管

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...

该协议是重要的，因为它允许比较不同中枢神经系统区域之间，以及不同个体之间的微血管。这种微维塞尔分离技术可以在一天内完成，并且消除了超离心和酶分离的需要。去除毛虫和胆小毛症可能很困难。一定要缓慢而小心地工作，以确保每个组织完全去除。对于从小利森脑脊椎动物标本中解剖的CNS组织，将收获的大脑放入含有MV-1溶液的15毫升锥形管中，并使用钳子从头骨的脑骨中取回垂体。将垂体放在1.7毫升的MV-1溶液的微离心管中，取出皮肤和肌肉以暴露椎柱。切除四肢、肋骨和内脏器官后，使用装有18针的10毫升注射器，用新鲜的MV-1溶液冲洗椎柱，在整个腰椎前肢方向。然后将脊髓放在含有新鲜 MV-1 溶液的 5 毫升管中。接下来，将大脑放入解剖显微镜下的培养皿中，用双管齐下刺去除脑筋。请务必检查脊髓，以确保没有剩余的脑膜。使用钳子分离下丘脑、小脑、脑干和皮层。必要时，使用钳子、虹膜和弹簧剪刀切除嗅觉球茎和丘脑，并使用双管齐下刺从所有脑心室中去除所有胆管丛。对于从大型陀螺脊椎动物标本中解剖的 CNS 组织，使用解剖显微镜和双管齐下刺、钳子、虹膜和弹簧剪刀从每个 CNS 组织中去除脑膜，注意从脑心室取出任何胆结丛。为了使收获的 CNS 组织均质化，将每个 CNS 区域切碎为一到两毫米的单个培养皿中。要使小脊椎动物标本中的CNS组织均质化，使用转移移液器添加一毫升MV-1溶液，以悬浮切碎的组织。然后使用相同的转移移液器将皮层、小脑、脑干、视叶和脊髓片分发到单独的 10 毫升玻璃组织研磨机中，并使用 PTFE 害虫和 5 毫升 MV-1 溶液研磨每组组织约 10 次。将每个组织浆料转移到冰上的单个 15 毫升圆锥管中，并使用钳子将下丘脑和垂体放入 MV-1 溶液的 100 微升中，放入单独的 1.7 毫升管中。然后，小心地用玻璃微虫使每个组织均质。要使大型脊椎动物标本均质化，请使用斜面切割转移移液器将切碎的组织转移到 55 毫升玻璃组织研磨机中，然后将研磨机连接到架空搅拌器上。根据表中所示的均质化的特定 CNS 部分添加推荐 MV-1 溶液体积的一半，并将头顶搅拌器打开至每分钟约 150 次旋转。小心上下移动玻璃管约 30 秒，然后关闭头顶搅拌器，以添加更多 MV-1 溶液，实现额外的均质化，直到获得均质浆。然后将均质组织转移到冰上50毫升锥形管中。对于微维塞尔纯化，通过离心使CNS组织均匀，并丢弃超钠。对于小型脊椎动物标本微维塞尔分离，移液皮层、小脑、脑干、视叶和脊髓颗粒，每样本5毫升新鲜、冰冷的MV-2溶液，约10倍，然后向每个管中再添加5毫升冰冷MV-2溶液。小心地翻转管子进行混合，并在一毫升的MV-2溶液中重新悬浮下丘脑和垂体颗粒。对于大型脊椎动物标本微韦塞尔分离，在皮层、小脑、脑干和脊髓微韦塞尔颗粒中加入20毫升冰冷的MV-2溶液，以每分钟40次旋转的速度将管左轮手枪中的颗粒重新悬浮约5分钟。在重新悬浮结束时，通过离心分离CNS组织，并缓慢旋转每个管，使上清液沿壁小心地从管壁上分离液体界面上厚而密的麦林层。丢弃米林和液体接口，用用低绒纸擦拭包裹的铲子对每管的内壁进行污点。然后，使用扭曲的低绒纸擦拭吸收多余的液体，并使用低结合提示在一毫升的MV-3溶液中重新悬浮每个颗粒。对于微韦塞洗脱和过滤，将几毫升新鲜 MV-3 溶液混合到每个 CNS 组织微韦塞尔浆中，在混合以避免聚合时，通过普鲁伊特过滤器将每个悬浮液应变到单独的 50 毫升锥形管中。将一个 20 微米尼龙网过滤器放在每个 CNS 区域的一个经过修改的过滤器支架上。将滤芯支架转移到 50 毫升锥形管上，用 5 毫升冰冷的 MV-3 溶液将过滤器湿润，确保缓冲器向下塞片架倒入锥形管中。将洗涤的微纤维转移到每个 20 微米尼龙网过滤器上，然后用 5 到 10 毫升的冰冷 MV-3 溶液冲洗微维。使用钳子将每个过滤器转移到包含冰冷 MV-3 溶液的单个烧杯中，轻轻摇动每个过滤器约 30 秒以分离微纤维。转移到15毫升锥形管后，通过离心收集微壁体，并使用低结合移液器将每粒颗粒重新悬浮在一毫升的冰冷的MV-3溶液中。然后，将小脊椎动物标本的微维塞尔悬浮液转移到1.7毫升的微离心管中，在4摄氏度的G下以2万倍G的2万倍进行离心5分钟。对于大型脊椎动物标本，将悬浮液转移到单独的五毫升离心管中，并加入四毫升 MV-3 溶液，在摄氏四度下以 2000 倍 G 进行离心 5 分钟。神经血管单元的内在成分可以通过CD31、PDGFR-beta和水波林四种的免疫标记在微壁体中检测，如证明，包括皮质、小脑、垂体、下丘脑、脑干和脊柱微维塞尔。同样，粘附蛋白-结蛋白 VE-cadherin、紧密结蛋白CLDN5和Zonula Occlens-1、三细胞结蛋白阿古林-1和基底标记化疗基体配体CXCL图案12和伽马-谷氨基转移酶-1的表达可以可视化。此外，大多数微韦塞尔没有阿尔法平滑肌肉行为素的表达，表明这种隔离协议有选择地针对小口径微韦塞尔。从其他小型利森脑脊椎动物（如鸟类、利德、青蛙和鱼类）获得的微鱼具有一些形态特征，这表明这种方法对于进一步描述物种间神经血管单位的差异是有用的。蛋白质表达变化的定量显示血管细胞粘附分子一和交结粘附分子B沿脊髓微囊，在实验自身免疫性脑脊髓炎的高峰期增加。然而，血管细胞粘附分子一在垂体微壁体中也显著增加，在下丘脑和脑干微纤维中减少。此外，在所有中枢神经系统组织中，血管细胞粘附分子一种表达中观察到变化，在下丘脑和垂体微囊中，结粘附分子B表达也观察到变化。定量分析，如西方印迹，PCR和免疫组织化学，可以按照这个程序进行，以揭示对血脑屏障内基因和蛋白质表达模式的见解。甲醛是一种剧毒的试剂，在佩戴适当的 PPE 时应始终在烟罩下处理。

Research

JoVE Journal

Neuroscience

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

全身和局部给药治疗的中枢神经系统疾病的啮齿动物模型

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

科研

神经科学

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

视神经横断：一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...