isolating mouse brain vessels: a protocol to isolate intact vessels from murine brain sample

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

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.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Dissection

NOTE: Sterile conditions are not required unless vessels are used for cell culture purposes. Prepare isolation vessel solutions: B1, add 1.5 mL of HEPES 1M to 150 mL of HBSS; B2, add 3.6 g of Dextran to 20 mL of B1; B3, add 1 g of BSA to 100 mL of B1.

<ol>
	<li>Prepare a 150 mL beaker with 20 mL of B1. Keep on ice and cover with parafilm to avoid air contamination.</li>
	<li>Deeply anesthetize the mouse under a hood with a small paper towel soaked in 1 mL of pure Isoflurane that is added into the cage. Anesthesia is verified by a lack of reaction to a toe pinch. Kill the mouse by cervical dislocation. 
	NOTE: These steps are accomplished in compliance with national and institutional regulations.</li>
	<li value="3">Optional: Perform intracardiac perfusion with 20 mL of PBS 1x to eliminate the blood content.</li>
	<li>Section the skin with a scalpel from the neck to the nose and pull it away. Remove all contaminating hairs with PBS 1x.</li>
	<li>To open the skull, first, insert scissors anteriorly to the olfactory bulb, and open the scissors to rupture the skull in two parts.</li>
	<li>Carefully remove the brain using a brain spatula. Dissect out the choroid plexus from the lateral ventricles as they would contaminate the blood vessel preparation. 
	NOTE: Optional: The final preparation will contain parenchymal and meninges vessels. If not desired, meninges can be peeled off.</li>
	<li value="7">Transfer the brain into the beaker containing B1 solution on ice. Up to 8 brains can be treated together.</li>
</ol>

2. Brain Tissue Homogenization
<ol>
	<li>Using two scalpels, manually and vigorously beat the brain in the B1 solution subsequently obtaining small pieces of about 2 mm.</li>
	<li>Homogenize the preparation with an automatized Dounce homogenizer, performing 20 strokes at 400 rpm. Ensure that the glass tube is maintained in ice and that the upper part of the douncer is in solution when moving up and down, so as to prevent the formation of air pockets. If several samples are prepared, wash the douncer with ionized water between each homogenization.</li>
</ol>

3. Vessel Purification

<ol>
	<li>Transfer the homogenate into a 50 mL plastic tube and proceed to the centrifugation at 2,000 x g for 10 min at 4 &#176;C. A large white interface (mostly myelin) will form on the top of the vessel pellet (red if no perfusion was performed).</li>
	<li>Discard the supernatant. The vessel pellet and the white interface will remain attached together. Add 20 mL of ice-cold B2 solution and shake the tube manually and vigorously for 1 min.</li>
	<li>Proceed to the second centrifugation at 4,400 x g for 15 min at 4 &#176;C. The myelin will now form a dense white layer at the surface of the supernatant.</li>
	<li>Carefully detach the myelin layer from the tube walls by holding the tube and slowly rotating it to allow the supernatant to pass along the walls. Discard myelin with the supernatant. The pellet containing the vessels remains attached at the bottom of the tube.</li>
	<li>Blot the inside wall of the tube with an absorbent paper wrapped around a 5 mL plastic pipette and remove all residual fluids, avoid touching the vessel pellet. Keep the tube upside-down on an absorbent paper to drain any remaining liquid.</li>
	<li>Suspend the pellet in 1 mL of ice-cold B3 solution by pipetting up and down with low-binding tips, keeping the tube on ice, then add another 5 mL of B3 solution. Make sure that vessels are dispersed as much as possible and do not form aggregates.</li>
</ol>

4. Filtration

<ol>
	<li>Prepare a beaker on ice with 30 mL of ice-cold B3 solution. Cover with parafilm to avoid air contamination.</li>
	<li>Place a 20 &#956;m-mesh filter on a modified filter holder on the top of a becker flask and equilibrate by applying 10 mL of ice-cold B3 solution.</li>
	<li>Pour the vessel preparation on the filter and rinse the vessels with 40 mL of ice-cold B3 solution.</li>
	<li>Recover the filter using clean forceps and immediately immerse it in the beaker containing the B3 solution. Detach the vessels from the filter by shaking it gently.</li>
	<li>Pour the beaker content in a 50 mL plastic tube and centrifuge at 2,000 x g for 5 min at 4&#176;C. 
	NOTE: Alternatively, the brain vessels&#8217; suspension from step 3.6 can be filtered onto a 100 &#956;m-mesh filter. In this case, larger vessels are preferentially retained on the filter while the flow-through contains microvessels (mainly capillaries), which are then filtered on a 20 &#956;m-mesh filter as above.</li>
	<li value="6">Resuspend the pellet of microvessels in 1 mL of ice-cold B3 solution and transfer it by pipetting it into a 1.5 mL Eppendorf tube. Centrifuge at 2,000 x g for 5 min at 4 &#176;C.</li>
</ol>

<table><tbody><tr><td>Tissue Grinder Size C&amp;nbsp;</td><td>Thomas scientific&amp;nbsp;</td><td>3431E25</td><td></td></tr><tr><td>Centrifuge 5415 R Eppendorf</td><td></td><td></td><td></td></tr><tr><td>Centrifuge 5810 R Eppendorf 5811000320</td><td></td><td></td><td></td></tr><tr><td>High-performance, Modular Stereomicroscope</td><td>Leica MZ6</td><td></td><td></td></tr><tr><td>Low binding tips (P1000) Sorenson BioScience 14200T</td><td></td><td></td><td></td></tr><tr><td>Swinnex 47 mm filter holder PP 8/Pk</td><td>Millipore&amp;nbsp;</td><td>SX0004700</td><td></td></tr><tr><td>Nylon net filter disc Hydrophilic 20 &amp;mu;m 47 mm 100/Pk</td><td>Millipore&amp;nbsp;</td><td>NY2004700</td><td></td></tr><tr><td>Nylon net filter disc Hydrophilic 100 &amp;mu;m 47 mm 100/Pk</td><td>Millipore&amp;nbsp;</td><td>NY1H04700</td><td></td></tr><tr><td>PARAFILM M (roll size 4 in. &amp;times; 125 ft)</td><td>Sigma</td><td>P7793-1EA</td><td></td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>Life technology</td><td>14175-129</td><td></td></tr><tr><td>HEPES (1M)&amp;nbsp;</td><td>Life technology</td><td>15630056</td><td></td></tr><tr><td>Dextran from Leuconostoc spp. Mr ~70,000</td><td>Sigma&amp;nbsp;</td><td>31390</td><td></td></tr><tr><td>Bovine serum albumin&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>A2153</td><td></td></tr><tr><td>10x PBS</td><td>Euromedex</td><td>ET330</td><td></td></tr></tbody></table>

Source: Boulay, A. C. et al. <a href="https://www.jove.com/video/53208" target="_blank">Purification of Mouse Brain Vessels.</a> J. Vis. Exp. (2015)
This video demonstrates the isolation of intact vessels from a mouse&#39;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.

Source: Boulay, A. C. et al. Purification of Mouse Brain Vessels. J. Vis. Exp. (2015)
This video demonstrates the isolation of intact vessels from a ...

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.

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

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