Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

This protocol is significant because it allows for the comparison of microvasculature between different central nervous system regions, as well as between different individuals. This microvessel isolation technique can be completed within a single day, and it eliminates the need for ultracentrifugation and enzymatic dissociation. Removing the meninges and the choroid plexus can be difficult.

Be sure to work slowly and carefully to ensure the complete removal of each tissue. For CNS tissue dissection from a small lissencephalic vertebrate specimen, place the harvested brain into a 15 milliliter conical tube containing MV-1 solution on ice, and use forceps to retrieve the pituitary from the sella turcica of the skull. Place the pituitary in a 1.7 milliliter microcentrifuge tube of MV-1 solution on ice, and remove the skin and muscle to expose the vertebral column.

After removing the limbs, ribcage and internal organs, use a 10 milliliter syringe equipped with an 18 gauge needle to flush the vertebral column with fresh MV-1 solution, in a caudal-to-rostral direction throughout the lumbar vertebral foramen. Then place the spinal cord in a 5 milliliter tube containing fresh MV-1 solution. Next, place the brain into a Petri dish under the dissecting microscope, and remove the meninges with double-pronged prick.

Be sure to check the spinal cord to ensure that no pieces of meninges are remaining. Use the forceps to separate the hypothalamus, cerebellum, brain stem, and cortex. When necessary, use forceps and iris and spring scissors to excise the olfactory bulbs and thalamus, and use the double-pronged prick to remove all of the choroid plexus from all the brain ventricles.

For CNS tissue dissection from a large gyrencephalic vertebrate specimen, use a dissecting microscope and a double-pronged prick, forceps, and iris and spring scissors to remove the meninges from each CNS tissue, taking care to remove any pieces of choroid plexus from the brain ventricles. For homogenization of the harvested CNS tissues, mince each CNS region into one to two millimeter pieces within individual Petri dishes. To homogenize CNS tissues from small vertebrate specimens, use a transfer pipette to add one milliliter of MV-1 solution to suspend the minced tissue.

Then use the same transfer pipette to dispense the cortex, cerebellum, brain stem, optic lobe, and spinal cord pieces into individual 10 milliliter glass tissue grinders, and use a PTFE pestle and five milliliters of MV-1 solution to grind each set of tissues for about 10 strokes. Transfer each tissue slurry into individual 15 milliliter conical tubes on ice, and use forceps to place the hypothalamus and pituitary in 100 microliters of MV-1 solution, in individual 1.7 milliliter tubes. Then, carefully homogenize each tissue with a glass micropestle.

To homogenize a large vertebrate specimen, use a beveled cut transfer pipette to transfer the minced tissue to a 55 milliliter glass tissue grinder, and attach the grinder to an overhead stirrer. Add half of the recommended volume of MV-1 solution according to the specific CNS portion being homogenized as indicated in the table, and turn on the overhead stirrer to approximately 150 rotations per minute. Carefully move the glass tube up and down for about 30 seconds before turning off the overhead stirrer to add more MV-1 solution for additional homogenization, until a homogenous slurry is obtained.

Then transfer the homogenized tissue to a 50 milliliter conical tube on ice. For microvessel purification, pellet the CNS tissue homogenates by centrifugation, and discard the supernatants. For a small vertebrate specimen microvessel isolation, pipette the cortex, cerebellum, brain stem, optic lobe and spinal cord pellets, in five milliliters of fresh, ice-cold MV-2 solution per sample, about 10 times, before adding five more milliliters of ice-cold MV-2 solution to each tube.

Carefully flip the tubes to mix, and re-suspend the hypothalamus and pituitary pellets in one milliliter of MV-2 solution. For large vertebrate specimen microvessel isolation, add 20 milliliters of ice-cold MV-2 solution to the cortex, cerebellum, brain stem, and spinal cord microvessel pellets, and re-suspend the pellets in a tube revolver at 40 rotations per minute for about 5 minutes. At the end of re-suspension, separate the CNS tissue lycates by centrifugation, and slowly rotate each tube to allow the supernatant to pass along the wall to carefully detach the thick and dense myelin layer on the liquid interface from the tube walls.

Discard the myelin and liquid interfaces, and blot the inside wall of each tube with a spatula wrapped with a low-lint paper wipe. Then, use a twisted low-lint paper wipe to absorb excess liquid, and use low-binding tips to re-suspend each pellet in one milliliter of MV-3 solution. For microvessel elution and filtration, mix a few milliliters of fresh MV-3 solution to each CNS tissue microvessel slurry, and while mixing to avoid aggregates, strain each suspension through a Pruitt strainer into individual 50 milliliter conical tubes.

Place one 20 micrometer nylon net filter onto one modified filter holder per CNS region. Transfer the filter holder onto a 50 milliliter conical tube, and wet the filter with five milliliters of ice-cold MV-3 solution, making sure that the buffer pours down the filter holder, into the conical tube. Transfer the eluted microvessels onto each 20 micrometer nylon net filter, and rinse the microvessels with five to 10 milliliters of ice-cold MV-3 solution.

Use forceps to transfer each filter into individual beakers containing the ice-cold MV-3 solution, and gently shake each filter for about 30 seconds to detach the microvessels. After transferring to a 15 milliliter conical tube, collect the microvessels by centrifugation, and use a low-binding pipette to re-suspend each pellet in one milliliter of ice-cold MV-3 solution. Then, transfer microvessel suspensions from small vertebrate specimens into a 1.7 milliliter microcentrifuge tube for centrifugation for five minutes at 20, 000 times G, in four degrees celsius.

For large vertebrate specimens, transfer the suspension into individual five milliliter centrifuge tubes, and add four milliliters of MV-3 solution for centrifugation for five minutes at 2, 000 times G in four degrees celsius. The intrinsic components of the neurovascular unit can be detected within microvessels, isolated as demonstrated, by immunolabeling for CD31, PDGFR-beta, and aquaporin four, including the cortical, cerebellar, pituitary, hypothalamic, brain stem, and spinal microvessels. Likewise, the expression of adherens-junction protein VE-cadherin, tight junction proteins CLDN5 and Zonula Occludens-1, tricellular junction protein angulin-1, and basal markes chemokine ligand CXCL motif 12, and gamma-glutamyltransferase-1, can be visualized.

Moreover, the majority of the microvessels are devoid of the expression of alpha smooth muscle actin, indicating that this isolation protocol selectively targets small-caliber microvessels. Microvessels obtained from other small lissencephalic vertebrates, like bird, liard, frog, and fish, share some morphological features, suggesting that this method is useful for further characterization of differences in neurovascular units between species. Quantification of the changes in protein expression reveals an increase in vascular cell adhesion molecule one, and junctional adhesion molecule B along spinal cord microvessels, during the peak of experimental autoimmune encephalomyelitis.

However, vascular cell adhesion molecule one is also significantly increased in pituitary microvessels, and decreased in the hypothalamus and brainstem microvessels. In addition, changes are observed in vascular cell adhesion molecule one expression during chronic experimental autoimmune encephalomyelitis in all CNS tissues, and in junctional adhesion molecule B expression in the hypothalamus and pituitary microvessels. Quantitative analyses, such as western blot, PCR and immunohistochemistry, can be performed following this procedure to reveal insights into gene and protein expression patterns within the blood-brain barrier.

Paraformaldehyde is an acutely toxic reagent, and should always be handled under the fume hood while wearing proper PPE.