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

Brain vessels are surrounded by a layer of tightly connected endothelial cells. These cells have a basement membrane around them along with cellular components such as pericytes and astrocytes.

To isolate brain vessels from surrounding neurons and glial cells, take a beaker containing mouse brain submerged in a homogenization buffer. Mince it into small pieces. Homogenize the brain tissue to dissociate it, releasing neurons, glial cells, and brain vessels. This treatment also disrupts myelin sheath surrounding the neurons.

Transfer the homogenate to a tube and centrifuge to pelletize the vessels. The lighter components such as neurons and glial cells remain in the supernatant while myelin forms a dense interface layer surrounding the pellet.

Discard the supernatant and resuspend the vessel pellet with the attached myelin layer in a suitable density gradient medium. Centrifuge to pellet the vessels and obtain a band of fat-rich myelin floating at the top of the supernatant. Remove the myelin band along with spent media and resuspend the pellet in a fresh buffer.

Strain the crude vessel preparation using a filter assembly containing a filter small enough that the vessels cannot pass through it. Transfer the filter into a fresh beaker containing buffer solution. Gently scrape the filter paper to detach the vessels and store them for further analysis.

Use a scalpel to incise the skin from the neck of the mouse to the nose and pull the skin back. Wash the skull with PBS to remove any contaminating hairs and then insert a pair of scissors into the skull, anterior to the olfactory bulbs. Open the scissors to rupture the skull in two parts and remove the brain with a spatula.

Next, dissect out the choroid plexus from the lateral ventricles and transfer the brain into a 150-milliliter beaker containing 20 milliliters of B1 solution on ice. Then, use two scalpels to vigorously chop the brain into 2-millimeter fragments of tissue and use an automated Dounce homogenizer to homogenize the tissue for 20 strokes at 400 rpm on ice.

To purify the vessels, transfer the homogenate into a 50-milliliter plastic tube and spin down the tissue slurry in a swinging rotor centrifuge. A large white interface consisting mostly of myelin will form on the top of the vessel pellet. Discard the supernatant.

Add 20 milliliters of ice-cold B2 solution and shake the tube vigorously for 1 minute. After a second centrifugation, the myelin will form a dense white layer at the surface of the supernatant. Slowly rotate the tube to allow the B2 solution to run along the wall as it is poured out and discard the myelin with the supernatant.

Then, use a plastic pipette wrapped in absorbent paper to remove any residual fluid from the walls of the tube, taking care not to touch the vessel pellet, and turn the tube upside down on ice. Next, resuspend the pellet in 1 milliliter of ice-cold B3 solution, followed by the addition of another 5 milliliters of B3 solution, taking care that the vessels do not form aggregates.

Now, place a 20-micron nylon mesh filter on top of a Becker flask and equilibrate the filter with 10 milliliters of ice-cold B3 solution. Pour the vessel preparation onto the filter and carefully rinse the vessels with another 40 milliliters of ice-cold B3 solution.

Then, use clean forceps to immediately transfer the filter into a beaker containing 30 milliliters of fresh B3 solution and attach the vessels from the filter with gentle shaking. Pour the beaker contents into a 50-milliliter plastic tube. Then, after a centrifugation, resuspend the pellet in 1 milliliter of ice-cold B3 solution.