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

Dr. Cruz-Orengo blev støttet af University of California, Davis, School of Veterinary Medicine opstart midler.

Alle procedurer i denne undersøgelse er i overensstemmelse med de retningslinjer, der er fastsat af University of California (UC), Davis institutionel dyrepleje og brug udvalg (IACUC). Animal Care på UC Davis er reguleret af flere uafhængige ressourcer og er blevet fuldt akkrediteret af foreningen for vurdering og akkreditering af laboratorium Animal Care International (AAALAC) siden 1966. Porcin CNS væv blev opnået fra UCD Department of Animal Sciences, kød Sciences laboratorium. CNS-væv fra rhesus macaques blev ...

<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 omfatter de unikke egenskaber af hjernen mikrovaskulaturen endotelceller kombineret med en sofistikeret arkitektur af stramme, klæber-, "peg-socket"-vejkryds, og vedhæftning plaques kritisk for CNS homøostase2,3,19. Endotelcellernes egenskaber induceres og vedligeholdes af pericytes og de omgivende astroglia-endefods processer2,3,19</s...

Vores hjerne er det vigtigste organ i vores krop. Af denne grund, at holde hjernen homøostase trods eksterne faktorer, der kan udløse en afvigelse fra normalitet er en prioritet. Ifølge nogle lærde, omkring 400-500 millioner år siden1, hvirveldyr udviklet, hvad vi nu kender som blod-hjerne barrieren (BBB)2,3. Denne beskyttende "hegn" udøver den største indflydelse på centralnervesystemet (CNS) homøostase og funktioner ved stramt regulere transport af ioner, molekyler, og celler mellem blod og CNS parenchyma. Når BBB er forstyrret, hjernen bliver modtagelig for giftig eksponering, infektion, og inflammation. Derfor, BBB dysfunktion er forbundet med mange, hvis ikke alle, neurologiske og neuroudviklingsmæssige lidelser4,5,6.
Den sofistikerede funktion af BBB tilskrives den unikke CNS mikrovaskulaturen konforme af neurovaskulære enhed (Nvu)2,3. Højt specialiserede endotelceller, pericytes og astrocytiske endefødder er de cellulære komponenter i Nvu2,3. Den ekstracellulære matrix genereret af disse celler er også afgørende for Nvu og BBB fysiologi2,3. Selvom de essentielle cellulære og molekylære bestanddele i Nvu bevares blandt hvirveldyr, rapporteres heterogenitet blandt ordrer og arter7,8. Men, tekniske begrænsninger hindrer vores evne til fuldt ud at overveje disse forskelle i neurobiologi, biomedicinsk, eller Translationel forskning.
På grund af dette, vi udvidet en CNS region-specifikke Micro Vessel-isolation metode til at gøre det gældende for mange arter fra alle fem hvirveldyr grupper: fisk, padder, krybdyr, fugle, og pattedyr. Protokollen er beskrevet til brug på små-lissencephalic-og store-gyrencephalic-hvirveldyr, herunder arter med translationel relevans9. Derudover omfatter vi andre regioner i CNS ikke undersøgt før i denne sammenhæng, men relevant for Neuro fysiologi og med enorme kliniske konsekvenser: hypothalamus, hypofyse, og hvide stof. Endelig testede vi kapaciteten af denne isolationsmetode som et pålideligt værktøj til at identificere ændringer i protein ekspression langs Nvu og/eller BBB9,10,11. Som en proof-of-koncept, viste vi, hvordan man kan bestemme ændringer i VCAM-1 og JAM-B udtryk under EAE ved hjælp af isolations metoden efterfulgt af immunofluorescence.

<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

Isolering af mikrobeholdere fra centralnervesystemet (CNS) udføres almindeligvis ved at kombinere kortikale væv fra flere dyr, oftest gnavere. Denne fremgangsmåde begrænser forhør af blod-hjerne barrieren (BBB) egenskaber til cortex og tillader ikke individuel sammenligning. Dette projekt fokuserer på udvikling af en isolationsmetode, der gør det muligt at sammenligne den neurovaskulære enhed (NVU) fra flere CNS-regioner: cortex, cerebellum, optisk lap, hypothalamus, hypofyse, hjernestammen og rygmarven. Desuden blev denne protokol, der oprindeligt blev udviklet til murine prøver, med succes tilpasset til brug på CNS-væv fra små og store hvirveldyr, hvorfra vi også er i stand til at isolere mikrofartøjer fra hjernen halvkugle hvid stof. Denne metode, når parret med immunolabeling, giver mulighed for kvantitation af protein ekspression og statistisk sammenligning mellem individer, vævstype, eller behandling. Vi beviste denne anvendelighed ved at evaluere ændringer i protein ekspression under eksperimentel autoimmun hesteencephalitis (EAE), en murine model af en Neuro inflammatorisk sygdom, multipel sklerose. Desuden kan mikrofartøjer isoleret ved denne metode anvendes til downstream-applikationer som qPCR, RNA-SEQ, og Western blot, blandt andre. Selv om dette ikke er det første forsøg på at isolere CNS-mikrofartøjer uden brug af ultracentrifugering eller enzymatisk dissociation, er det unikt i sin adepthed for sammenligning af enkeltpersoner og flere CNS-regioner. Derfor, det giver mulighed for undersøgelse af en række forskelle, som ellers kan forblive uklar: CNS portioner (cortex, cerebellum, optiske lap, hjernestammen, hypothalamus, hypofyse, og rygmarv), CNS vævstype (grå eller hvide stof), individer, eksperimentelle behandlingsgrupper og arter.

Mikrofartøjer isoleret fra murine CNS viste alle iboende cellulære komponenter i den neurovaskulære enhed2,3. Ved hjælp af enten trombocyttal endotel celle adhæsions molekyle-1 (pecam, også kendt som CD31) eller isolectin IB4 (et glykoprotein, der binder endotel celle glycocalyx) for endotelceller, trombocytafledt vækstfaktor-β (pdgfrβ) eller neuron-glial antigen 2 (NG2) for pericytes og aquaporin-4 (AQP4) for astrocytisk...

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

Målet med denne protokol er at isolere mikrofartøjer fra flere regioner i centralnervesystemet af lissencephalic og gyrencephalic hvirveldyr.

Pålidelig isolering af Mikrofartøjer i central nervesystemet på tværs af fem hvirveldyr grupper

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

Denne protokol er betydelig, fordi det giver mulighed for sammenligning af mikrovasculatur mellem forskellige centralnervesystemet regioner, samt mellem forskellige individer. Denne mikrosøg isolation teknik kan afsluttes inden for en enkelt dag, og det eliminerer behovet for ultracentrifugering og enzymatisk dissociation. Fjernelse af meninges og choroid plexus kan være svært.Sørg for at arbejde langsomt og forsigtigt for at sikre fuldstændig fjernelse af hvert væv. For CNS væv dissektion fra en lille lissencephalic hvirveldyr prøve, placere den høstede hjerne i en 15 milliliter konisk rør, der indeholder MV-1 opløsning på is, og bruge pincet til at hente hypofysen fra sella turcica af kraniet. Placer hypofysen i en 1,7 milliliter mikrocentrifuge rør MV-1 opløsning på is, og fjern hud og muskler til at udsætte rygsøjlen.Efter fjernelse af lemmer, brystkasse og indre organer anvendes en 10 millilitersprøjte udstyret med en 18 gauge nål til at skylle rygsøjlen med frisk MV-1 opløsning i en caudal-til-rostral retning i hele lændehvirvelsøjlen foramen. Derefter anbringes rygmarven i et 5 milliliterrør med frisk MV-1 opløsning. Dernæst placere hjernen i en petriskål under dissekerende mikroskop, og fjern meninges med dobbelt-bens.Sørg for at kontrollere rygmarven for at sikre, at ingen stykker af meninges er tilbage. Brug spint til at adskille hypothalamus, lillehjernen, hjernestammen og cortex. Når det er nødvendigt, bruge tang og iris og foråret saks til punktafgifter olfaktoriske pærer og thalamus, og bruge dobbelt-pronged til at fjerne alle de choroid plexus fra alle hjernen ventrikler.For CNS væv dissektion fra en stor gyrencephalic hvirveldyr prøve, bruge en dissekere mikroskop og en dobbelt-striger prik, tang, og iris og foråret saks til at fjerne meninges fra hver CNS væv, idet man skal passe på at fjerne alle stykker af choroid plexus fra hjernen ventrikler. Til homogenisering af det høstede CNS-væv hakkes hver CNS-region i en til to millimeter stykker i individuelle petriskåle. For at homogenisere CNS væv fra små hvirveldyr prøver, bruge en overførselpipette til at tilføje en milliliter MV-1 opløsning til at suspendere hakket væv.Brug derefter den samme overførselpipette til at dispensere cortex, cerebellum, hjernestamme, optisk lap og rygmarvsstykker i individuelle 10 milliliter glasvævsslibere, og brug en PTFE-støder og fem milliliter MV-1-opløsning til at male hvert sæt væv i omkring 10 slag. Hver vævsslam i individuelle 15 milliliter koniske rør på is, og brug kraftbefængdere til at placere hypothalamus og hypofysen i 100 mikroliter MV-1 opløsning i individuelle 1,7 milliliterrør. Derefter omhyggeligt homogenisere hvert væv med et glas mikropestle.For at homogenisere en stor hvirveldyr prøve, bruge en skrå skåret overførsel pipette til at overføre hakket væv til en 55 milliliter glas vævsliber, og fastgør kværnen til en overhead omrører. Der tilsættes halvdelen af den anbefalede MV-1-opløsning i henhold til den specifikke CNS-del, der homogeniseres som angivet i tabellen, og omrøreren tændes for ca. 150 omdrejninger i minuttet. Flyt forsigtigt glasrøret op og ned i ca. 30 sekunder, før du slukker for omrøreren for at tilføje mere MV-1-opløsning for yderligere homogenisering, indtil der opnås en homogen gylle.Overfør derefter det homogeniserede væv til et konisk rør på 50 milliliter på is. Til rensning af mikrovesøg pellets cns-væv homogeniseringen ved centrifugering, og supernatanterne kasseres. For en lille hvirveldyr prøve mikrovessel isolation, pipette cortex, lillehjernen, hjernestamme, optisk lap og rygmarv pellets, i fem milliliter frisk, iskold MV-2 opløsning pr prøve, omkring 10 gange, før du tilføjer fem flere milliliter iskold MV-2 opløsning til hvert rør.Forsigtigt flip rørene til at blande, og re-suspendere hypothalamus og hypofyse pellets i en milliliter MV-2 opløsning. For store hvirveldyr prøve mikrovessel isolation, tilsæt 20 milliliter iskold MV-2 opløsning til cortex, lillehjernen, hjernestammen, og rygmarvsmikrovessel pellets, og re-suspendere pellets i et rør revolver ved 40 rotationer i minuttet i ca 5 minutter. Ved afslutningen af re-suspension, adskille CNS væv lycates ved centrifugering, og langsomt rotere hvert rør, så supernatanten til at passere langs væggen for omhyggeligt at løsne den tykke og tætte myelin lag på væsken interface fra røret vægge.Kassér myelin og flydende grænseflader, og skamplet den indvendige væg af hvert rør med en spatel indpakket med en lav-fnug papir tørre. Brug derefter en snoet lav-fnug papir tørre til at absorbere overskydende væske, og bruge lav-bindende tips til at re-suspendere hver pellet i en milliliter MV-3 opløsning. For mikrovessel edation og filtrering, bland et par milliliter frisk MV-3 opløsning til hver CNS væv mikrovessel gylle, og mens blanding for at undgå aggregater, stamme hver suspension gennem en Pruitt si i individuelle 50 milliliter koniske rør.Der skal være et 20 mikrometersnylonnetfilter på én modificeret filterholder pr. CNS-område. Filterholderen overføres til et konisk rør på 50 milliliter, og filteret skal være vådt med fem milliliter iskold MV-3-opløsning, hvor sørg for, at bufferen hælder filterholderen ned i det koniske rør. De eluterede mikrolæller overføres til hvert 20 mikrometeret nylonnetfilter, og mikrovældret med fem til 10 milliliter iskold MV-3-opløsning.Brug vicepenp til at overføre hvert filter til individuelle bægerglas, der indeholder den iskolde MV-3-opløsning, og ryst forsigtigt hvert filter i ca. 30 sekunder for at tage mikrovæsnerne af. Efter overførsel til et 15 milliliter konisk rør opsamler mikrovæsnerne ved centrifugering, og der anvendes en lavbindingspipette til at re-suspendere hver pellet i en milliliter iskold MV-3 opløsning. Derefter overføres mikrovesselsuspensioner fra små hvirveldyrprøver til et 1,7 milliliter mikrocentrifugerør til centrifugering i fem minutter ved 20.000 gange G i fire grader celsius.For store hvirveldyrsprøver overføres suspensionen til individuelle fem millilitercentrifugerør, og der tilsættes fire milliliter MV-3-opløsning til centrifugering i fem minutter ved 2.000 gange G i fire grader celsius. De iboende komponenter i den neurovaskulære enhed kan påvises i mikrovessels, isoleret som påvist, ved immunmærkning til CD31, PDGFR-beta, og aquaporin fire, herunder kortikale, cerebellar, hypofyse, hypothalamic, hjernestamme, og spinal mikrovessels. Ligeledes udtryk for klæbende-krydset protein VE-cadherin, stramme kryds proteiner CLDN5 og Zonula Occludens-1, trekantede krydset protein angulin-1, og basal markes chemokine ligand CXCL motiv 12, og gamma-glutamyltransferase-1, kan visualiseres.Desuden er størstedelen af mikrovessels blottet for udtrykket af alpha glat muskel actin, hvilket indikerer, at denne isolation protokol selektivt rettet mod små kaliber mikrovessels. Mikrovessels fremstillet af andre små lissencephalic hvirveldyr, som fugl, løgner, frø og fisk, deler nogle morfologiske træk, hvilket tyder på, at denne metode er nyttig for yderligere karakterisering af forskelle i neurovaskulære enheder mellem arter. Kvantificering af ændringerne i proteinudtryk afslører en stigning i vaskulær celle vedhæftning molekyle en, og junctional vedhæftning molekyle B langs rygmarven mikrovessels, under toppen af eksperimentelle autoimmune encephalomyelitis.Men, vaskulære celle vedhæftning molekyle man er også signifikant øget i hypofyse mikrovessels, og faldt i hypothalamus og hjernestammen mikrovessels. Desuden observeres ændringer i vaskulær celle vedhæftning molekyle et udtryk under kronisk eksperimentel autoimmun encephalomyelitis i alle CNS væv, og i junctional vedhæftning molekyle B udtryk i hypothalamus og hypofyse mikrovessels. Kvantitative analyser, såsom western blot, PCR og immunhisemi, kan udføres efter denne procedure for at afsløre indsigt i genet og proteinudfoldelsesmønstre i blod - hjerne barrieren.Paraformaldehyd er et akut giftigt reagens, og bør altid håndteres under røghætten, mens iført korrekt PPE.

Research

JoVE Journal

Neuroscience

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

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

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

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

Multimodal Imaging af stamcelle implantation i det centrale nervesystem mus

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

Produktion af lentivirusvektorer til transduktion af celler fra det centrale nervesystem

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

Mærkning af enkeltceller i centralnervesystemet hos<em&gt; Drosophila melanogaster</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

En skade Paradigm at undersøge centrale nervesystem reparation i<em&gt; Drosophila</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

Direkte Intraventrikulær Delivery of Drugs til Rodent centrale nervesystem

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

In vivo Afhøring af centralnervesystemet Translatome af Polyribosome Fraktionering

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

In vivo optogenetic Stimulering af Rodent centralnervesystemet

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