Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

The overall goal of this procedure is to mimic ischemic stroke in mice. First, the common carotid artery is temporarily ligated, whereas the external carotid artery is permanently ligated as distally as possible. Another temporary suture slightly tight is placed on the ECA above the bifurcation.

The second step is to clamp the internal carotid artery using reverse action tweezers to avoid bleeding. Then cut the ECA between the permanent and temporary ligatures. Next, the silicone coated monofilament is introduced into the ECA.

Then the ECA is inverted to introduce the monofilament into the ICA until it includes the base of the middle cerebral artery. One hour later, the blood flow is restored by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic claw in humans. Ultimately, the neurological deficit, which reflects the success of T-M-C-A-O and the severity of the infarct can be evaluated just after reperfusion and a different times post reperfusion.

Cyan image software from NIH is used to calculate the infarct volume on representative brain slices stained by crestal Violet. One of the main advantages of the transient middle cerebral artery occlusion using the intraluminal filament technique is that it mimics ischemic stroke in patient with the restoration of blood flow and the development of this procedure in mice offers the possibility of using a label genetics tool to identify the role of a target protein. In stroke Transient middle cerebral artery occlusion is performed on two to three month old male C 57 black six mice weighing between 22 and 28 grams.

This protocol was approved by the IRCM Bioethics Care Committee to increase the reproducibility in the infarct volume. We are using reusable 12 millimeter long, six oh silicon coated monofilament from the Doco Corporation. Two hours before surgery.

Inject a mouse intraperitoneal with buprenorphine at a dose of 0.03 milligrams per kilogram. To begin the procedure, anesthetize mice with 5%isof fluorine and maintain anesthesia at 2.5%ISO fluorine use a heating pad to keep the mouse body temperature constant during surgery, disinfect the fern skin of the mouse with 70%ethanol or Betadine. Then make a midline incision in the neck and pull apart the soft tissues under a stereo microscope.

Bluntly dissect the mouse neck in order to expose the trachea. Next, retract the muscles to locate the carotid artery. Carefully dissect the left common carotid artery from the surrounding tissue.

Place a temporary five oh silk suture, cut into 20 millimeter segments around the artery. Take care not to damage the vagal nerve. Next, separate the bifurcation of the left internal common carotid artery or ICA and external common carotid artery or ECA place a permanent suture around the ECA as distally as possible.

And another temporary suture on the ECA above the bifurcation. Then clip the left ICA using reverse action tweezers to avoid bleeding. Again, be very careful not to harm the vagal nerve.

Cut a small hole in the ECA before the bifurcation to the ECA and the ICA and between the permanent and temporary sutures placed earlier. Next, introduce a 12 millimeter six oh silicon coated monofilament suture into the ECA completely cut the ECA and invert the monofilament into the ICA. Tie the suture tightly to prevent bleeding and remove the reverse action tweezers.

Then introduce the monofilament into the circle of Willis until the monofilament occludes the base of the MCA stop. Its insertion about nine to 10 millimeters beyond the bifurcation of the ECA and CCA. Generally, the monofilament will be blocked and cannot move anymore.

Be careful not to penetrate the tego palatine artery. Tie the suture on the ECA tightly in order to fix the filament into position. Now close the skin with an auto clip wound closing system.

Inject one milliliter of saline solutions subcutaneously and place the mice under an infrared heating lamp during the post occlusion period, which is 60 minutes. Anesthetize the mice as before and remove the auto clips slightly open the suture on the ECA to allow the monofilament to withdraw and the blood to reperfuse. Then permanently tie off the temporary suture on the ECA to prevent blood loss.

One hour post stroke, withdraw the monofilament and keep it for reuse. Next, remove the temporary suture on the CCA to allow blood to recirculate. Close the wound and subcutaneously.

Inject another one milliliter of saline solution into the mice. After making sure the animal regains mobility, put it back into its cage and place half of the cage on the heating pad to allow mice to choose their environment. 12 hours post surgery, mice receive another dose of buprenorphine for all sham operations.

All procedures are identical except that the monofilament is not inserted after surgery. The neurological deficit is evaluated to confirm the success of the T-M-C-A-O and determine its efficiency score the neurological deficits of the mice at 1 24 48 and 72 hours Post perfusion on a six point scale as follows, zero equals normal, one equals mild, turning with or without inconsistent curling when picked up by the tail. More than 50%attempts to curl to the contralateral side.

Two equals mild consistent curling over 50%attempts to curl to the contralateral side. Three equals consistent, strong and immediate curling. The mouse holds a curled position for more than one to two seconds.

With its nose almost reaching its tail. Four equals severe curling. Progressing into barreling loss of walking or writing reflex five equals comatose or mound.

After perfusion with PBS solution, breeze the brains quickly in isop pentane and store them at minus 80 degrees Celsius. Note that mouse brains may also be perfused with 4%paraform aldehyde depending on the planned immunohistochemistry studies. Next, use a cryostat to cut 17 micron coronal brain sections using 30 slides.

Place one section of every 30 on the same slide. Then stain the first and the 15th slides with crestal violet. For a more accurate injury volume estimation, use an image analysis system such as Scion image from NIH to evaluate the lesion deli, the infarct area which appears white in the left and right brain hemispheres.

Repeat this D limitation on all brain slices. Calculate the infarct volume in arbitrary units and express them as a percentage of the contralateral non lesioned area for each section. Keep the other slides at minus 80 degrees Celsius for immunohistochemistry studies.

The neuro score evaluation confirms the success of T-M-C-A-O in my subjected to the intraluminal procedure. As seen here, consistent, strong and intermediate curling is observed and the mouse holds a curled position for more than one to two seconds with its nose almost reaching its tail. In our experiment, all mice presented at least a mild consistent curling, which corresponds to a neuro score of two throughout the test period.

Kressel violet staining was done in order to evaluate the extent of brain infarct. Following T-M-C-A-O shown here a representative coronal sections of mouse brain stained with crest violet after 72 hours post reperfusion. The infarct area, mainly the striatum and cortex and adjacent brain areas, which are labeled appears in white as it is unstained by crest.

Violet shown here is the injury volume, which is the white part of the right hemisphere expressed as a percentage of the contralateral non lesioned area on the left hemisphere. The infarct volume in C 57 black six male mice following ischemic stroke represents around 70%of the non leashing hemisphere. This procedure provides a strong lesion reproducibility.

After watching this video, you should have a good understanding of how to perform transient ischemic stroke in mice. In addition, the quiz viol approach to measure your brain in fact volume, offers you the great advantage of having a lot of materials to test relevant markers by immunochemistry. Thanks for watching and good luck with your experiment.