Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

The overall goal of this procedure is to develop a model of embolic stroke in the rat. This is accomplished by first ligating, the common carotid artery temporarily with a slip knot and the external carotid artery as tight as possible. Then place a loose suture around the ECA above the bifurcation.

The second step is to isolate the tego palatine artery and the internal carotid artery and temporarily ligate them with slip knots. Then cut a small hole into the ECA between the tight and loose ligatures. Next, the modified P 50 tube containing a blood clot is introduced into the ECA.

Then the catheter is inverted into the ICA and the tube is gently advanced until the tip of the catheter reaches the origin of the MCA. The final step is to inject the blood clot through the catheter along with saline, and then withdraw the catheter from the ECA five minutes later. Ultimately, 24 hours after stroke, TTC staining is used to show the infarct areas in the brain slices.

The main advantage of this technique, our existing stroke model, like MC model, is that this embolic stroke model closely mimic human is stroke and use, suitable for pre study of SLI therapy for ischemic stroke, demonstrating the procedure will be Dr.A port failure from my laboratory. Dr.Gene has more than 10 years of experience performing red circuit models. Visual demonstration of this method is critical steps is difficulty to naming.

To prepare the PE 50 tube gradually soften and stretch a 30 centimeter long PE 50 tube above a gas fire. Select a point on the stretch tube using a digital caliper. Then cut it into a 25 centimeter long segment with a one centimeter long tip and an outside diameter between 0.30 and 0.34 millimeters.

Next, collect the femoral artery blood from an anesthetized rat with a 20 centimeter long PE 50 tube. Place the tube at room temperature for two hours for the blood to clot. Then retain the tube for 22 hours at four degrees Celsius.

Cut the PE 50 tube into 50 millimeter segments and flush out the clot from the tube into a sterile Petri dish containing normal saline. Transfer the 50 millimeter long clot into a 60 millimeter long PE 10 tube. Then connect each end of the PE 10 tube to a 20 centimeter long PE 50 tube, which is connected to a one milliliter syringe containing normal saline with a 23 gauge needle to remove the blood cells without disrupting the fibrin core, move the clot from one syringe to the other and vice versa continuously for five minutes after that, cut the clot into a 40 millimeter long segment and transfer the clot segment into a modified PE 50 catheter.

In this procedure, sterilize all the surgical tools by autoclaving and sanitize the surgery table and the associated surgical equipment with 70%ethanol. Next, anesthetize the rat with iso fluorine and ensure the depth of anesthesia by performing a toe pinch on the rear feet. Apply a small amount of ointment on the eyes to prevent dryness.

Then place the rat in the supine position on a heating pad. After that, shave the fur on the ventral neck region with the electric clipper disinfect the skin and the surrounding fur with 70%ethanol. Insert a rectal probe and maintain the body temperature at around 37 degrees Celsius using a homeo themic blanket control unit.

Then under a dissecting microscope, make a two centimeter long midline incision on the neck. Use the retractors to expose the surgical field. Dissect the CCA free from the surrounding nerves without harming the vagal nerve, and place a sterile five zero silk suture under the artery.

Tie a slip knot and pull the suture toward the body. Next, dissect the ECA and its two branches, the OA and the STA coagulate the two branches using a veterinary electrosurgical unit, then place two pieces of sterile six zero silk suture under the ECA, separate the two silk sutures placed under the artery, one piece towards the head and the other towards the body. Tie a tight ligature on the side closest to the head.

Grasp the silk suture with small hemostats and pull toward the head. Prepare a loose knot with the other silk suture for later use. Now dissect the ICA and PPA from the surrounding nerves without harming the vagal nerve.

Tie the PPA with a six zero suture. Then make a slipknot around the ICA or clip the ICA using a microvascular clip. Next, cut a small hole into the ECA between the tight and loose ligatures with Venice style spring scissors.

Insert the modified PE 50 tube containing the blood clot into the incision and advance to the bifurcation of the CCA. Tighten the loose ligature around the lumen just enough to securely preserve the mobility of the indwelling tube. Then cut off the ECA at the site of the small hole to free the stump.

Position the stump below the bifurcation of the ECA and ICA. This will allow the modified tube to easily slide into the ICA. Open the ICA and gently advance the tube from the lumen of the ECA into the ICA until the tip of the catheter reaches the origin of the MCA.

Now inject the clot through the modified P 50 catheter along with 10 microliters of saline over 10 seconds using a 100 microliter Hamilton syringe. Five minutes later, withdraw the catheter from the ECA, tie the ECA and reopen the ECA afterward, suture the incision on the neck. In this procedure, make a 1.2 centimeter long midline incision in the scalp of an anesthetized rat to expose the skull bone.

Next drill, a 1.5 millimeter diameter bur hole located at two millimeters posterior and five millimeters lateral to the bgma using a 0.7 millimeter spherical stainless steel burr. Place the probe 0.5 millimeters above the dura surface. Measure the RCBF using a blood flow meter at 0 5 15, 30, 60 90, and 120 minutes after the embolization at two hours of embolization.

Treat the rat with TPA and continually monitor the blood flow at 5 15, 30, 45, and 60 minutes after TPA treatment after the surgery, inject 2.5 milliliters of saline solution in the animal subcutaneously to prevent dehydration and inject Bren at 0.05 milligrams per kilogram subcutaneously immediately after the surgery and every six to 12 hours as needed for pain relief. Stop isof fluoride anesthesia. Place the rat in a 37 degree Celsius veterinary recovery chamber and keep monitoring it.

After about 10 minutes, place the animal in a sterilized cage with some wet food in a Petri dish before returning the cage to the animal sterilization room. Two hours after embolization, the neurological score is assessed using the BESON score. Rats showing no deficit are excluded from further study.

This figure shows the RCBF measurement using a laser doppler flow meter. The clot injection led to more than 70%RCBF reduction of the baseline value. TPA treatment at two hours after the clot for 30 minutes restored.

RCBF close to baseline. Here the representative pictures showing the blood clots in the origin of the MCA and a CA 24 hours after stroke. And these are the representative images of the TTC stained brain slices.

24 hours after stroke Once mastered This technique should be done in 30 minutes.