Hi, my Name is Terrence Jam. My name is Hai Joe. Today we'll show you how to perform a mouse model of middle al artery occlusion.
So let's get started. This experiment starts off with the preparation of the suture. Begin by cutting the suture into 20 millimeter pieces.
Next round the tip by bringing the suture towards a cauterizer. Afterwards, measure the tip of the suture. Now we are ready to begin the surgery with the mask placed on its back.
Make an incision along the midline. Use forceps to expose the ECA. The external carotid artery along the ECA are many branches.
One of these branches is the STA, the superior thyroid artery. The STA needs to be coagulated with bipolar forceps. Then the STA needs to be cut to allow the ECA to better be isolated.
Next, the ECA is coagulated and in size as far away from the CCA As possible. At this point, you may want to isolate the ECA For better access. Then loosely tie two sutures around the ECAA few millimeters apart.
Apply the clamp at the branching of the CCA to ECA and ICA. Then proceed to clip the tip of the ECA stump. Next, reposition the ECA to align with the ICA.
This allows the suture to have continuous entry from the ECA to the ICA. Measure the total length of suture before insertion. Insert the suture into the opening made by clipping the ECA stump.
As the suture passes the distal knot, secure the knot by tightening it. Next, remove the clamp and insert the suture further into the ECA. As the suture passes the proximal knot, secure the knot by tightening it.
After securing both knots, carefully insert the suture along the artery until nine to 10 millimeters of suture is passed. The bifurcation point, this can be done by subtracting the length of suture up to the bifurcation point from the total length of suture. As the suture reaches the origin of the MCA.
The MCA is occluded. After 24 hours, The masses sacrifice and the brain isolated. For TTC staining, the isolated brain is placed in a mold and sliced with a razor blade.
Then the brain is placed in 2%TTC for 20 minutes at room temperature. Finally, the brain is fixed, overnight and neutral. Buffered formin.
Suture preparation is a delicate process. Here are some helpful tips. Position the suture and cauterizer like so.
Then while keeping the cauterizer stationary, slowly bring the suture closer. Next, measure the suture to make sure it is of the right size. The size of the suture we use in this experiment is around 0.21 to 0.22 Millimeters.
To begin the surgery, Place the mask on its back and induce anesthesia with 30%oxygen and 70%nitrogen. Make an incision along the midline and then apply retractors to open access to the area. It should look like this.
Separate the Tissue with the forceps carefully. Then expose and isolate the ECA with the forceps in the process of isolating the ECA, the STA needs to be coagulated and cut. This allows a longer Piece of ECA to be isolated.
Next, the ECA needs to be coagulated with the help of bipolar forceps After coagulation, the ECA needs to be cut. Next, Isolate the ECA and tie two sutures loosely around the ECAA Few millimeters apart. The result should look something like this.
Now apply the clamp at the bifurcation of CCA to ICA and ECA. Make sure the Clam completely obstructs blood flow to the ECA. Then clip the end of the ECA stump.
Measure the suture Before insertion after an opening is made on the ECA. Insert the suture and the opening past the distal Knot. After the suture is passed the distal knot, tighten the knot.
Next, remove the clamp. After verifying the Distal knot is secure, insert the suture further along the ECA Past the proximal knot. Next, tighten the proximal knot.
Now carefully insert the suture along the ECA into the ICA. Continue inserting the Suture along the ICA until nine to 10 millimeters that the suture is past the bifurcation point. This can be done by subtracting the length of suture up to the bifurcation point from the total length of suture.
Next, trim the suture such that it does not injure the surrounding tissue. Then suture up the incision and allow the Mouse to recover. Prepare for TTC staining 24 hours after the surgery.
Begin by sacrificing the mouse with cervical dislocation. Then isolate the brain from the skull and place in a mold. Then slice with razor blade, stain the brain in 2%TTC for 20 minutes at room temperature.
Then fix overnight and 10%neutral buffered for melin. So that's it. Thanks for watching and good luck with your experiments.
