A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

This study develops a rat model for middle cerebral artery occlusion or reperfusion that maintains cerebral vessel integrate. It focuses on introducing a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. The project uses microsurgical techniques and the filament induced ischemia.

It introduces a method to induce ischemia without damaging the common carotid artery or external carotid system. Contrary to techniques that may permanently impair blood flow or damage the cerebral vascular anatomy, this protocol maintains several vessel anatomical integrity. It provides a more accurate, less harmful approach to studying ischemic stroke.

To begin, place the anesthetized rat on an operating platform. After shaving the fur around the throat and left neck area, disinfect the surgical site three times with Betadine and 70%ethanol. Make a 1.5 centimeter long midline incision on the ventral side of the neck and open the superficial fascia.

Perform blunt dissection to expose the external carotid artery, internal carotid artery and common carotid artery. Place three 4-0 silk sutures around the common carotid artery and another silk suture through the external carotid artery near the internal carotid artery bifurcation. Tie a releasable slip knot on the proximal common carotid artery.

Tighten the suture on the distal common carotid artery using a hemostat to block blood flow. Loosely tie another slip knot around the common carotid artery during suturing to ensure the vessel is not occluded. Now, using scissors, make a 0.5 millimeter incision between the proximal silk suture and the middle suture on the common carotid artery.

Introduce a three centimeter long 4-0 filament into the common carotid artery. Tighten the silk sutures around the common carotid artery to secure the intraluminal filament and prevent bleeding. Then remove the suture from the distal common carotid artery.

Continue to insert the filament into the lumen of the middle cerebral artery controlling the sutures around the external carotid artery. Start a timer and record the time when occlusion begins. Suture the neck incision with 3-0 silk sutures.

Carefully place the animal in a recovery cage. After ischemic modeling, reopen the neck incision from the anesthetized rat. Under a dissecting microscope, gently withdraw the occluding filament while placing the micro clamp horizontally near the intraluminal suture knot.

Continue withdrawing until the white tip is clearly visible near the common carotid artery incision. Then completely remove the filament and release the secured intraluminal suture knot. Place another micro clamp near the heart-end side of the proximal common carotid artery knot.

Subsequently, remove the suture knot around the proximal common carotid artery. Rinse the incision site with saline. Dry the area with a sterile cotton ball to remove the blood and achieve a clear surgical field.

Now at 4x magnification, suture the common carotid artery incision using 11-0 microsurgical sutures in an interrupted pattern. After suturing, remove the micro clamps from both ends of the common carotid artery to restore blood flow. Apply pressure with a sterile cotton ball to the common carotid artery suture site for 30 seconds.

Observe the common carotid artery for two minutes to ensure no bleeding occurs. Suture the neck incision and place the rat in a 35 degrees Celsius care box for anesthesia recovery. Three days following ischemia reperfusion, remove the brain from the euthanized rat.

Freeze the brain at minus 20 degrees Celsius for 20 minutes. After freezing, use a scalpel to cut the brain into two millimeter thick coronal sections. Incubate the brain sections with 2%TTC for 20 minutes at 37 degrees Celsius.

After incubation, transfer the brain sections into a small Petri dish and discard 2%TTC. Then fix the brain sections with 10%formalin for 30 minutes at room temperature. Discard the formalin and rinse the brain sections with PBS.

Capture digital photographs of consecutive slices. Using ImageJ software, quantitatively measure the infarction areas. After TTC staining, the infarcts resulting from cerebral ischemia were observed in both the striatum and the cortex.

At three days post ischemic stroke, the total infarcted percentage was measured at approximately 30%of the hemisphere with a mortality rate of 16.7%after surgery.