Dr. Cruz-Orengo was supported by the University of California, Davis, School of Veterinary Medicine Start Up Funds.

All procedures of the present study are in accordance with the guidelines set by the University of California (UC), Davis Institutional Animal Care and Use Committee (IACUC). Animal care at UC Davis is regulated by several independent resources and has been fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) since 1966. Porcine CNS tissues were obtained from UCD Department of Animal Sciences, Meat Sciences Laboratory. CNS tissues from rhesus macaques were obtained from the California National Primate Research Center Pathology Department (NIH P51OD011107). No anesthesia, euthanasia, or necropsy was performed by laboratory staff on pigs and macaques. Therefore, there are no particular recommendations regarding these matters.
NOTE: This method was validated for multiple species, but the provided protocol corresponds more directly to mouse and porcine tissues. Details pertaining to the optic lobe do not apply when using mammalian specimens. All biohazardous materials must be handled in an appropriate biosafety level (BSL) facility. All acutely toxic materials must be handled underneath a fume hood. All biohazardous medical waste and acutely toxic waste must be disposed of properly.
1. Preparation
<ol>
	<li>Prepare solutions (Table 1) at least one day ahead. 
	NOTE: It is very difficult to dissolve 70 kDa molecular weight (MW) dextran. It is more efficient to let the solution stir overnight covered with either paraffin film or foil. The protocol requires the use two different MV-2 solutions depending on the specimen under investigation. The 18% dextran efficiently separates the myelin from the microvessel pellet when performing the protocol on small lissencephalic specimens. However, it develops a smear of myelin within the interface of CNS tissues from large gyrencephalic specimens, as well as the hypothalamus and the pituitary from small specimens. This smear is prevented by using 20% dextran.</li>
	<li>Prepare well-slides by loading 50 &#181;L per well of poly-D-lysine and let dry for 2 h at room temperature (RT), preferably in a biological safety cabinet-class 2 (BSC-2). Do not overdry. Rinse twice with phosphate-buffered saline (PBS) and keep in the fridge until ready to use.</li>
</ol>
2. CNS Tissue Dissection from Small Lissencephalic Vertebrate Specimen
NOTE: The article demonstrates the application of the protocol on a C57BL6/J, 10 week-old, ~25 g, male mouse.
<ol>
	<li>Prepare two 15 mL conical tubes and a 1.7 mL microcentrifuge tube with 5 mL and 1 mL, respectively, of MV-1 solution per specimen. Keep tubes on ice.</li>
	<li>Anesthetization
	<ol>
		<li>Anesthetize mice by intraperitoneal injection of a ketamine, xylazine, and acepromazine anesthetic cocktail at 100/10/3 mg per kg body weight and spray with 70% ethanol. Confirm anesthesia by assessing the absence of palpebral and pedal reflexes by touching an eye and pinching a foot, respectively.</li>
		<li>Anesthetize pigeons by inhalation of 5% isoflurane using an induction anesthesia box.</li>
		<li>Anesthetize fish and frogs in 1% Tricaine in either a fish holding water tank or amphibian Ringer&#39;s solution (Table of Materials), respectively.</li>
		<li>Anesthetize lizards by cooling at 4 &#176;C prior to euthanasia.</li>
	</ol>
	</li>
	<li>Decapitate the animal with surgical scissors, peel away the skin with forceps to expose the skull, and cut with LaGrange scissors through the foramen magnum (Figure 1A).</li>
	<li>Dissect the brain out with a spatula and transfer to a 15 mL conical tube with MV-1 solution. Keep tube on ice.</li>
	<li>Retrieve the pituitary from the skull&#39;s sella turcica with forceps and transfer to a 1.7 mL microcentrifuge tube with MV-1 solution. Keep tube on ice.</li>
	<li>Remove the skin and muscle to expose the vertebral column. Cut out the limbs, rib cage, and internal organs (Figure 1B).</li>
	<li>Dissect the spinal cord by one of the two methods described below. Then, transfer to a 5 mL conical tube with MV-1 solution. Keep tube on ice.
	<ol>
		<li>Perform laminectomy by cutting in a rostral to caudal direction with LaGrange scissors, starting through the cervical vertebral foramen, until reaching the lumbar cord.</li>
		<li>Perform flushing in a caudal to rostral direction throughout the lumbar vertebral foramen with an 18 G needle and 10 mL syringe loaded with MV-1 solution (Figure 1C,D). 
		NOTE: This method only works with very small specimens, mostly rodents (&#60;100 g).</li>
	</ol>
	</li>
	<li>Using a dissecting scope, cut the dural sac from the spinal cord with spring scissors and remove the remaining meninges with a double-pronged pick (Figure 2).</li>
	<li>Transfer the brain to a Petri dish and, using a dissecting scope, remove the meninges with a double-pronged pick (Figure 2A&#8211;C).</li>
	<li>Excise the olfactory bulbs and thalamus with forceps, iris scissors, and spring scissors. Using the double-pronged pick, remove the choroid plexus from all the brain ventricles. Make sure to remove all the choroid plexus. 
	NOTE: The choroid plexus and olfactory bulbs are a massive source of contamination. In contrast to the BBB, these capillaries are highly fenestrated and the results of the subsequent experiments will be compromised if they are not removed.</li>
	<li>Using forceps, separate the distinct CNS regions: cortex, cerebellum, brainstem, optic lobe (not on mammalian specimens), and hypothalamus.</li>
</ol>
3. CNS Tissue Dissection from a Large Gyrencephalic Vertebrate Specimen
NOTE: This protocol uses porcine CNS tissues obtained from an abattoir. Therefore, no anesthesia, euthanasia, or necropsy is described or shown here.
<ol>
	<li>Transport porcine CNS tissues in a 0.946 L (32 oz) histology container loaded with MV-1 solution inside an ice chest/bucket.</li>
	<li>Using a dissecting scope, remove the meninges with a double-pronged pick, forceps, iris scissors, and spring scissors from each CNS tissue. Make sure to remove any pieces of choroid plexus from the brain ventricles.</li>
</ol>
4. CNS Tissue Homogenization
NOTE: It is more efficient when two investigators engage in the homogenization process: one dissecting the meninges under the stereoscope and the other homogenizing the minced tissues. This way, the tissues are quickly returned to the ice bucket and kept cold.
<ol>
	<li>Place each CNS region on a Petri dish with ~1 mL of MV-1 solution. Using a single-edge blade, mince the tissue to obtain 1&#8211;2 mm pieces.
	<ol>
		<li>For a small vertebrate specimen, use a 100 mm x 20 mm Petri dish.</li>
		<li>For a large vertebrate specimen, use a 150 mm x 15 mm Petri dish.</li>
	</ol>
	</li>
	<li>Homogenization of a small vertebrate specimen (Table 2 and Table 3)
	<ol>
		<li>Transfer the cortex, cerebellum, brainstem, optic lobe, and spinal cord to ~1 mL of MV-1 solution in an individual 10 mL Potter-Elvehjem glass tissue grinder with a PTFE pestle using a transfer pipette. Add 5 mL of MV-1 solution and homogenize each tissue (~10 strokes). Transfer to individual 15 mL conical tubes. Keep tubes on ice. 
		NOTE: For a small vertebrate, 5 or 4, with or without optic lobe, respectively, 10 mL Potter-Elvehjem grinders and 15 mL conical tubes are needed.</li>
		<li>Transfer the hypothalamus and pituitary with forceps to ~100 &#181;L of MV-1 solution in an individual 1.7 mL tube and carefully homogenize with a glass micropestle. Rinse each micropestle with ~1 mL of MV-1 solution. Keep tube on ice. 
		NOTE: For a small vertebrate, 2 glass micropestles and 1.7 mL tubes are needed.</li>
	</ol>
	</li>
	<li>Homogenization of a large vertebrate specimen (Table 4 and Table 5)
	<ol>
		<li>Using a transfer pipette, transfer the minced tissue to a 55 mL Potter-Elvehjem glass tissue grinder with a PTFE pestle and attach to overhead stirrer. Add half of the recommended volume of MV-1 solution, according to the specific CNS portion being homogenized (Table 5).</li>
		<li>Turn on the overhead stirrer at very low speed (~150 rpm) and carefully move the glass tube up and down for about 30 s.</li>
		<li>Turn off overhead stirrer, add more MV-1 solution, and repeat step 4.3.2 until obtaining a homogenous slurry.</li>
		<li>Transfer to a 50 mL conical tube. Keep tube on ice.</li>
		<li>Wash the grinder with deionized water between each CNS tissue homogenization. 
		NOTE: For a large vertebrate 5 or 4, with or without white matter, respectively, 50 mL conical tubes are needed. Likewise, two (2) 15 mL conical tubes are needed for the hypothalamus and pituitary.</li>
	</ol>
	</li>
</ol>
5. Microvessel Purification
<ol>
	<li>Centrifuge tissue homogenates resulting from step 4.2.1, 4.2.2, or 4.3.4 at 2,000 x g for 10 min at 4 &#176;C. A large white interface of myelin will form on the top of the reddish microvessels&#39; pellets. Discard the supernatants.
	<ol>
		<li>Small vertebrate specimen (Table 2 and Table 3)
		<ol>
			<li>Resuspend the cortex, cerebellum, brainstem, optic lobe, and spinal cord pellets in 5 mL of ice-cold MV-2 solution with a 5 mL serological pipette (~10 flushes). Add 5 mL of ice-cold MV-2 solution to each suspension and carefully mix by flipping the tubes.</li>
			<li>Resuspend the hypothalamus and pituitary pellets with 1 mL of MV-2 solution.</li>
		</ol>
		</li>
		<li>Large vertebrate specimen (Table 4 and Table 5)
		<ol>
			<li>Add 20 mL of ice-cold MV-2 solution to the cortex, cerebellum, brainstem, and spinal cord microvessel pellets. Resuspend the pellets by mixing on a tube revolver shaker, stirring at 40 rpm for ~5 min. Balance the conical tubes&#39; volumes of the cortex, cerebellum, brainstem, and spinal cord suspensions by adding more MV-2 solution.</li>
			<li>Resuspend the hypothalamus and pituitary microvessel pellets in 5 mL of MV-2 solution with a 5 mL serological pipette (~10 flushes). Add 5 mL of ice-cold MV-2 solution to the suspensions and carefully mix by flipping the tube.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Centrifuge at 4,400 x g for 15 min at 4 &#176;C.</li>
	<li>Carefully detach the thick and dense myelin layer on the liquid interface from each tube&#39;s walls by slowly rotating the tubes and allowing the supernatant to pass along the walls.</li>
	<li>Discard the myelin and liquid interface and blot the inside wall of each tube with a spatula wrapped with a low-lint paper wipe. For small vertebrate specimens, remove myelin layer from each hypothalamus and pituitary tube by suctioning carefully with a transfer pipette.</li>
	<li>Wipe out excess liquid with a twisted low-lint paper wipe. Resuspend each pellet with 1 mL of MV-3 solution using low-binding tips. Keep tubes on ice.</li>
</ol>
6. Microvessel Elution and Filtration
<ol>
	<li>Pour ice-cold MV-3 solution in individual beakers for each CNS region. Keep at 4 &#176;C.
	<ol>
		<li>For small vertebrate specimens, use 10 mL of MV-3 solution per 50 mL beaker. A total of 6&#8211;7 beakers is necessary depending on whether or not the optic lobe is included.</li>
		<li>For large vertebrate specimens, use 30 mL of MV-3 solution per 100 mL beaker. A total of 6&#8211;7 beakers is necessary depending on whether or not white matter is included.</li>
	</ol>
	</li>
	<li>Place a cell strainer on top of a 50 mL conical tube. Use one per CNS region. Use a 100 &#181;m strainer for the cortex, brainstem, optic lobe, spinal cord, and pituitary. Use a 70 &#181;m cell strainer for the cerebellum and hypothalamus.</li>
	<li>Wet each strainer with 1 mL of ice-cold MV-3 solution.</li>
	<li>Add more MV-3 solution to the suspensions prepared in step 5.5 with a serological pipette while mixing to avoid aggregates. Carefully load microvessels on top of the strainer. Rinse with ice-cold MV-3 solution.
	<ol>
		<li>For small vertebrate specimens (Table 2 and Table 3), add 10&#8211;15 mL of MV-3 solution with a serological pipette and rinse with 5 mL of MV-3 solution.</li>
		<li>For large vertebrate specimens, add 20 mL of MV-3 solution with a serological pipette and rinse with 10 mL of MV-3 solution.</li>
	</ol>
	</li>
	<li>Assemble the filtration unit by placing a 20 &#181;m nylon net filter on a modified filter holder (Figure 2D), one per CNS region.
	<ol>
		<li>For small vertebrate specimens (Table 2 and Table 3), use 25 mm modified filter holders for the cortex, cerebellum, brainstem, optic lobe, and spinal cord. Use 13 mm modified filter holders for the hypothalamus and pituitary.</li>
		<li>For large vertebrate specimens (Table 4 and Table 5), use 47 mm modified filter holders for the cortex, cerebellum, brainstem, and spinal cord. Use 25 mm for the hypothalamus and pituitary.</li>
	</ol>
	</li>
	<li>Place filter on top of a 50 mL conical tube and wet filter with 5 mL of ice-cold MV-3 solution making sure buffer pours down the filter holder to the conical tube.</li>
	<li>Transfer the eluted microvessels (from step 6.4) on top of the 20 &#181;m nylon net filter and rinse the microvessels with 5&#8211;10 mL of ice-cold MV-3 solution.</li>
	<li>Recover the filter using clean forceps and immerse it in the beaker containing the ice-cold MV-3 solution prepared in step 6.1.</li>
	<li>Detach the microvessels from the filter by shaking it gently for about 30 s. For small vertebrate specimens, pour the beaker content in a 15 mL conical tube. For large vertebrate specimens, pour the beaker content in a 50 mL conical tube.</li>
	<li>Centrifuge at 2,000 x g for 5 min at 4 &#176;C and resuspend the pellet in 1 mL of ice-cold MV-3 solution using a low-binding pipette tip.
	<ol>
		<li>For small vertebrate specimens, transfer the suspension (from step 6.10) into a 1.7 mL microcentrifuge tube and centrifuge at 20,000 x g for 5 min at 4 &#176;C. 
		NOTE: This spin is performed on a benchtop centrifuge at maximum speed (~20,000 x g) to ensure a robust yield.</li>
		<li>For large vertebrate specimens, transfer the suspension from step 6.10 into a 5.0 mL microcentrifuge tube, add 4 mL of MV-3 solution, and centrifuge at 2,000 x g for 5 min at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
7. Immunostaining
NOTE: Hematoxylin and eosin (H&#38;E) staining was performed on reptile, amphibian, and fish specimens as a proof-of-concept of the protocol feasibility. Therefore, there is no recommendation for immunolabeling for these specimens.
<ol>
	<li>Remove the supernatant from the microcentrifuge tubes.</li>
	<li>Resuspend the pellet in 1x sterile PBS using a low-binding pipette tip (Table of Materials). Keep the microvessels from forming aggregates by pipetting multiple times. For small vertebrate specimens (Table 3), use ~100 &#181;L&#8211;2,000 &#181;L according to the pellet size. For large vertebrate specimens (Table 5), use ~1,000 &#181;L&#8211;4,000 &#181;L according to the pellet size.</li>
	<li>Using a low-binding pipette tip, transfer the microvessels to well slides, being careful to add them to the center of each well, avoiding the sidewalls.</li>
	<li>Set the well slides, uncovered, inside a BSC-2, and let dry for 20 to 30 min at RT. 
	NOTE: A relatively high volume of 1x PBS is used to avoid aggregate formation. Because of this, it is necessary to let the slides dry prior to fixation to make sure the microvessels are being held by the poly-D-lysine coating.</li>
	<li>Remove residual 1x PBS by pipetting out with a transfer pipette, add 200 &#181;L of 4% paraformaldehyde (PFA) and incubate for 30 min at RT.</li>
	<li>Pipette out the fixative solution and wash 3x with 200 &#181;L of 1x PBS for 5 min at RT. 
	NOTE: H&#38;E staining on fish, amphibian, and reptile CNS microvessels was performed at this point.</li>
	<li>Add 200 &#181;L of blocking buffer to the well slide and incubate for 60 min at 37 &#176;C.</li>
	<li>Remove the blocking buffer with a transfer pipette and add 200 &#181;L of the primary antibody cocktail in antibody diluent (Table of Materials). Incubate at 4 &#176;C overnight.</li>
	<li>Pipette out the primary antibody cocktail and wash 3x with 200 &#181;L of 1x PBS for 5 min at RT.</li>
	<li>Load the secondary antibody cocktail (Table of Materials) diluted in 1x PBS and incubate for 2 h at RT, protected from light.</li>
	<li>Pipette out the secondary antibody cocktail and wash 3x with 200 &#181;L of 1x PBS for 5 min at RT, protected from light. After the last wash detach the well-slide&#39;s frame and blot-dry any excess 1x PBS with a low lint paper wipe.</li>
	<li>Add a coverslip, liquid antifade mountant medium, and 4&#8242;,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Let dry overnight at RT protected from light.</li>
	<li>Once dry, keep protected from light on a slide box at 4 &#176;C until ready for confocal microscopy and data acquisition.</li>
</ol>

<ol><li>Bundgaard, M., Abbott, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((All+vertebrates+started+out+with+a+glial+blood-brain+barrier+4-500+million+years+ago%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((The+blood-brain+barrier%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Perspectives+in+Biology%22%5BJournal%5D)">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/pubmed?term=((The+blood-brain+barrier%5BTitle%5D)+AND+%22Handbook+of+Clinical+Neurology%22%5BJournal%5D)">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/pubmed?term=((Blood-brain+barrier+regulation+in+psychiatric+disorders%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">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/pubmed?term=((The+role+of+brain+vasculature+in+neurodegenerative+disorders%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Blood-Brain+Barrier%3A+From+Physiology+to+Disease+and+Back%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">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/pubmed?term=((Heterogeneity+of+the+blood-brain+barrier%5BTitle%5D)+AND+%22Tissue+Barriers%22%5BJournal%5D)">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/pubmed?term=((Bridging+barriers%3A+a+comparative+look+at+the+blood-brain+barrier+across+organisms%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">Bridging barriers: a comparative look at the blood-brain barrier across organisms.</a> Genes & Development. 32, 466-478 (2018).</li>
<li>Cruz-Orengo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CXCR7+influences+leukocyte+entry+into+the+CNS+parenchyma+by+controlling+abluminal+CXCL12+abundance+during+autoimmunity%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">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/pubmed?term=((VCAM-1-targeted+magnetic+resonance+imaging+reveals+subclinical+disease+in+a+mouse+model+of+multiple+sclerosis%5BTitle%5D)+AND+%22FASEB+Journal%22%5BJournal%5D)">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/pubmed?term=((Brain+barriers%3A+Crosstalk+between+complex+tight+junctions+and+adherens+junctions%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((LSR%2Fangulin-1+is+a+tricellular+tight+junction+protein+involved+in+blood-brain+barrier+formation%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Enhanced+sphingosine-1-phosphate+receptor+2+expression+underlies+female+CNS+autoimmunity+susceptibility%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">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/pubmed?term=((Straightforward+method+for+singularized+and+region-specific+CNS+microvessels+isolation%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">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/pubmed?term=((Markers+for+human+brain+pericytes+and+smooth+muscle+cells%5BTitle%5D)+AND+%22Journal+of+Chemical+Neuroanatomy%22%5BJournal%5D)">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/pubmed?term=((In+vivo+characterization+of+cortical+and+white+matter+neuroaxonal+pathology+in+early+multiple+sclerosis%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">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/pubmed?term=((Neuroinflammation+and+its+relationship+to+changes+in+brain+volume+and+white+matter+lesions+in+multiple+sclerosis%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">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/pubmed?term=((Neuroimaging+Techniques+to+Assess+Inflammation+in+Multiple+Sclerosis%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Functional+morphology+of+the+blood-brain+barrier+in+health+and+disease%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">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/pubmed?term=((Localization+of+brain+endothelial+luminal+and+abluminal+transporters+with+immunogold+electron+microscopy%5BTitle%5D)+AND+%22NeuroRx%22%5BJournal%5D)">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/pubmed?term=((Purification+of+Mouse+Brain+Vessels%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Novel+3D+analysis+of+Claudin-5+reveals+significant+endothelial+heterogeneity+among+CNS+microvessels%5BTitle%5D)+AND+%22Microvascular+Research%22%5BJournal%5D)">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/pubmed?term=((Enzymatic+digestion+improves+the+purity+of+harvested+cerebral+microvessels%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">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/pubmed?term=((Expression+of+drug+transporters+at+the+blood-brain+barrier+using+an+optimized+isolated+rat+brain+microvessel+strategy%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">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/pubmed?term=((Beta-amyloid+pathology+in+human+brain+microvessel+extracts+from+the+parietal+cortex%3A+relation+with+cerebral+amyloid+angiopathy+and+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">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/pubmed?term=((Proteomic+and+transcriptomic+study+of+brain+microvessels+in+neonatal+and+adult+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Proteomic and transcriptomic study of brain microvessels in neonatal and adult mice.</a> PLoS One. 12, e0171048(2017).</li>
</ol>

The authors have nothing to disclose.

The BBB includes the unique properties of the brain microvasculature endothelial cells coupled by a sophisticated architecture of tight-, adherens-, &#34;peg-socket&#34;- junctions, and adhesion plaques critical for CNS homeostasis2,3,19. Endothelial cells properties are induced and maintained by pericytes and the surrounding astroglia end-foot processes2,3,19. BBB microvasculature displays polarity (i.e., the asymmetrical expression pattern of proteins localized on luminal or abluminal endothelial cell surfaces)20. While this may briefly define the BBB, in reality it remains one of the most enigmatic notions in neuroscience. For example, regional, sex, and age differences within the NVU may explain CNS regional vulnerabilities to trauma, toxicity, infection, and inflammation, which can have major clinical consequences3,5,6. Another obstacle in the understanding of the BBB is the differences observed among multiple taxa7,8. One of the major hurdles of understanding and investigating the BBB is precisely the difficulty related to the isolation of the NVU that encompasses the BBB, while still keeping its barrier-specific properties14.
In an attempt to accelerate our understanding of the BBB, CNS-regional differences, as well as species differences, we adapted previously published methods. We successfully adapted these, in particular Boulay et al.21, to obtain microvessels from single cortical, cerebellar, hypothalamic, pituitary, brainstem, and spinal tissues on a myriad of lissencephalic small vertebrates: fish, amphibians, reptiles, birds, and mammals (Figure 3, Figure 4, Figure 5, and Figure 6). One advantage of this method is that its adaptations are based on the overall size of CNS tissue: the researcher chooses the amount of solution, size of tissue grinder, conical tube, filter holders, etc. according to the wet tissue, regardless of species, sex, and age. In our experience with immunolabeling, a ~20 g specimen yields enough microvessels for an 8 well slide per cortex, cerebellum, optic lobe, brainstem, and spinal cord and half an 8 well slide per hypothalamus and pituitary. However, yield of cortical and optic lobe is much higher in comparison to cerebellum, brainstem, and spinal cord.
As shown, we performed the necessary adaptations for isolation and immunolabeling of CNS microvessels of pig and macaque, larger gyrencephalic mammals that are more relevant for translational research (Figure 7). Notably, we were able to include periventricular white matter on these species only. Since the CNS in these animals is larger, we collected enough white matter to allow us to separate a microvessel pellet from the myelin layer during the second centrifugation (step 5.3, MV-2 with 20% dextran). We speculate that a critical mass is needed to be able to achive this separation and we are actively seeking how to obtain a similar result with murine CNS tissue.
Although omitted for the sake of simplicity, we did perform immunolabeling beyond the NVU canonical markers on avian, porcine, and macaque microvessels. Notably, all species shared cellular and molecular markers previously identified on murine samples as relevant for BBB function (junctional proteins VE-cadherin, CLDN5, ZO-1, and LSR and apicobasal markers CXCL12 and GGT1) or in proximity to NVU (NFM, OSP, and GFAP). Again, these findings encourage the use of this method on other species to further identify NVU orthology and divergence among species. It also opens the opportunity for further investigation into NVU strain, sex, and age differences within the same species and the feasibility of using other organisms for BBB biomedical research. We also show evidence of a successful use of this microvessel-isolation method to quantitate changes in protein expression levels during neuroinflammation (Figure 8). Even though we performed this experiment as a proof-of-concept, the approach used here is currently extensively exploited in our laboratory. We favor this approach over other quantitative methods (e.g., Western blot) because we want to focus not only on the expression level but relative protein abundance, relocation of CXCL12 and other apicobasal proteins, linkage of junctional proteins, etc., within the complicated appearance of CNS microvessels9,13,22. Likewise, we are presently troubleshooting our method for other applications, such as further isolation of NVU cellular components (endothelial cells and pericytes), RNA-seq, and proteomics23,24,25,26.

Our brain is the most important organ in our body. For this reason, keeping brain homeostasis despite external factors that may trigger a deviation from normalcy is a priority. According to some scholars, about 400&#8211;500 million years ago1, vertebrate animals developed what we now know as the blood-brain barrier (BBB)2,3. This protective &#34;fence&#34; exerts the greatest influence over central nervous system (CNS) homeostasis and functions by tightly regulating the transport of ions, molecules, and cells between blood and CNS parenchyma. When the BBB is disrupted, the brain becomes susceptible to toxic exposure, infection, and inflammation. Therefore, BBB dysfunction is associated with many, if not all, neurological and neurodevelopmental disorders4,5,6.
The sophisticated function of the BBB is attributed to the unique CNS microvasculature conformed by the neurovascular unit (NVU)2,3. Highly specialized endothelial cells, pericytes, and astrocytic end-feet are the cellular components of the NVU2,3. The extracellular matrix generated by these cells is also essential to the NVU and BBB physiology2,3. Although essential cellular and molecular components of the NVU are conserved among vertebrates, heterogeneity is reported among orders and species7,8. However, technical limitations impede our ability to fully consider these differences in neurobiology, biomedical, or translational research.
Because of this, we expanded a CNS region-specific microvessel-isolation method to make it applicable to numerous species from all five vertebrate groups: fish, amphibians, reptiles, birds, and mammals. The protocol is described for use on small-lissencephalic and large-gyrencephalic vertebrates, including species with translational relevance9. Additionally, we include other regions of the CNS not investigated before within this context, but relevant to neurophysiology and with tremendous clinical implications: the hypothalamus, pituitary, and white matter. Lastly, we tested the capacity of this isolation method as a reliable tool to identify changes in protein expression along the NVU and/or BBB9,10,11. As a proof-of-concept, we showed how to determine changes in VCAM-1 and JAM-B expression during EAE using the isolation method followed by immunofluorescence.

<table><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 &amp;mu;m Cell strainer</td><td>Millipore Sigma</td><td>CLS431752-50EA</td><td></td></tr><tr><td>Corning 70 &amp;mu;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&amp;amp;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&amp;amp;D</td><td>AF1074</td><td>Used as primary antibody to assess neuroinflammation. Recommended concentration: 5 &amp;mu;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&amp;beta;</td><td>R&amp;amp;D</td><td>AF1042</td><td>Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 &amp;mu;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 &amp;mu;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 &amp;mu;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 &amp;mu;g/mL.</td></tr><tr><td>Mouse anti-Human CD31</td><td>R&amp;amp;D</td><td>BBA7</td><td>Used as primary antibody on primate CNS microvessels. Recommended concentration: 16.5 &amp;mu;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 &amp;mu;g/mL.</td></tr><tr><td>Mouse anti-&amp;alpha;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 &amp;mu;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 &amp;mu;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 &amp;mu;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 &amp;mu;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 &amp;mu;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 &amp;mu;g/mL.</td></tr><tr><td>Sterile Ringer&#039;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

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

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

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

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

reliable-isolation-central-nervous-system-microvessels-across-five

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.5K Views.  University of California Davis. The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

Video: Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Medicine

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Immunology and Infection

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Isolating All CNS Resident Cell Types Simultaneously from Autoimmune Mice

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

A Method for the Isolation of Neurovascular Units from Rat Brain

Source: Brzica, H., et al,. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57698">A simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels.</a> J. Vis. Exp. (2018).
This video demonstrates the process of isolating brain microvessels from euthanized rats. It includes the dissection of the brain, cleaning of the tissue, and mechanical digestion, followed by repeated centrifugation to isolate the pure fraction of microvessels.

Metoda izolacji jednostek nerwowo-naczyniowych z mózgu szczura

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. The procedural flow for the protocol ...