Name: Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups (Video) | JoVE Journal
Uploaded: 2020-01-12T12:00:00.000+00:00
Duration: 10 min 35 s
Description: Learn a reliable method to isolate CNS microvessels from lissencephalic and gyrencephalic vertebrates without ultracentrifugation. This technique enables comparative vascular studies across species in a single day.

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5 minutes

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>

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<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,<s...

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

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.

Microvessels isolated from murine CNS showed all intrinsic cellular components of the neurovascular unit2,3. Using either platelet endothelial cell adhesion molecule-1 (PECAM, also known as CD31) or isolectin IB4 (a glycoprotein that binds the endothelial cell glycocalyx) for endothelial cells, platelet derived growth factor-β (PDGFRβ) or neuron-glial antigen 2 (NG2) for pericytes and aquaporin-4 (AQP4) for astrocytic end-feet (<s...

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

Research

JoVE Journal

Neuroscience

This video demonstrates the isolation of intact vessels from a mouse's brain that constitute the blood-brain barrier (BBB) along with neurons and glial cells. The isolated vessels can be used as an in vitro model to study their role in maintaining brain homeostasis.

Isolating Mouse Brain Vessels: A Protocol to Isolate Intact Vessels from Murine Brain Sample

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.

A Method for the Isolation of Neurovascular Units from Rat Brain

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.

Isolation of Primary Murine Brain Microvascular Endothelial Cells

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.

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

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.

Purification of Mouse Brain Vessels

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.

Isolation and Cannulation of Cerebral Parenchymal Arterioles

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.

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

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.

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

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.

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

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.

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

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 naïve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

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

Learn a reliable method to isolate CNS microvessels from lissencephalic and gyrencephalic vertebrates without ultracentrifugation. This technique enables comparative vascular studies across species in a single day.

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

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