Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

This two-vessel occlusion mouse model of cerebral ischemia-reperfusion can investigate the pathophysiology of stroke. This model will induce a stable infarct volume of bulk of brain infiltrating immune cells in the ischemic brain and the behavioral deficit after cerebral ischemia-reperfusion. In this model, an infarct is caused in the cortical areas and the mortality rate is low.

Because the ligation MCAO reperfusion model requires craniotomy to expose the size of distal MCA, the size can be easily confirmed, and the exam whether the blood flow in the distal MCAO is disrupted during the procedure is straightforward. Provide the mice with free access to water and food until the surgery. Autoclave the surgical tools, and use 70%ethanol to sanitize the surgery table and equipment.

After anesthetizing an eight to 12-week-old mouse, make sure that it does not have a pedal reflex when tested using a firm toe pinch. Use vet ointment to prevent eye dryness while the mouse is under anesthesia. To monitor the blood pressure, first remove the hair, and then use a non-invasive blood pressure system and a physiological monitoring system to monitor rectal temperature and arterial blood gasses.

Inject the mouse subcutaneously with a prophylactic antibiotic. Next, place the mouse in the supine position on the heating pad. To expose the skin, use electric clippers to shave the fur on the ventral neck region and in the region between the right eye and right ear.

Clear the fur from the animal's body with epilating cream, and disinfect the surgical site with 70%ethanol. Then cut a one-centimeter-long midline incision at the neck using iris scissors. With iris forceps, carefully dissect the common carotid artery free from the vagus nerves without causing physical injury, and use 5-0 silk sutures to isolate the artery.

Next, use iris scissors to make a 0.3-centimeter incision in the scalp at the midpoint between the right eye and right ear. Using micro scissors, cut the temporalis muscle to expose the zygomatic and squamosal bone. Place the mouse under a stereo dissecting microscope, and use a micro drill to create a two milliliter hole directly over the right-side distal middle cerebral artery.

Use a 10-0 suture to ligate the trunk of the right-side distal MCA, and use a nontraumatic aneurism clip to occlude the right-side CCA. After desired duration of ischemia, remove the suture and aneurism clip to restore blood flow to the MCA and CCA. Use a suture clip to seal the skin incision on the head and seal the cervical skin incisions with surgical glue.

Inject buprenorphine subcutaneously for pain relief. Maintain the animal's body temperature at 36.5 plus or minus 0.5 degrees Celsius on the heating pad, and do not leave unattended or return it to the company of other animals until it is fully recovered from the anesthesia. Finally, place the mouse into the autoclave cage so that it can freely access water and food after it has fully recovered.

Use iris scissors to make an incision in the skin of the head of the anesthetized and decapitated mouse to expose the skull. Then use operating scissors to cut the anterior of the frontal bone, and with iris scissors, cut the skull along the sagittal suture. With a bone rongeur, push aside the frontal and parietal bone, and expose the brain.

Remove the brain, and place it in a mouse brain matrix. Use razor blades to obtain two-milliliter coronal slices, and place them in 50-milliliter tubes filled with five milliliters of 2%TTC. When using mouse brain matrix to obtain coronal slices, the cutting size should be same in different brains.

Cutting size affect the size of brain slices, which also affect the calculation of infarct size. Stain the brain slices with 2%TTC and 1X PBS for 10 minutes at 37 degrees Celsius, and then rinse two times with 10%formalin. Fix the brain slices in 10%formalin at room temperature for 24 hours.

After drying the brain slices on the tissue paper to absorb excess formalin, arrange them on a clean plastic slide, orienting them from rostral to caudal, and cover them with another clean plastic slide. Scan the slide using a scanner. Flip the slide over, and scan the reverse side.

Use ImageJ software to calculate the infarction area of each section, by first opening the image file and setting up the scale for the image. Then use freehand selection to select the infarct area, and use the regions of interest ROI manager to measure the area of interest. Finally, sum the infarct areas for each section, and multiply the result by the section thickness to estimate the total infarction volume, and then complete statistical analysis as described in the manuscript.

Increase in ischemia duration during this MCAO reperfusion procedure produced different degrees of ischemia-induced infarct volume and neuronal loss in the cerebral cortex of the right MCA area. Furthermore, the ischemia decreased the animal's locomotor activity at 48 hours after the MCAO reperfusion. After the cerebral ischemia-reperfusion, a bulk of peripheral immune cells infiltrated the ipsilateral hemisphere of the ischemic brain.

In addition, when this two-vessel occlusion model was compared with the MCAO model, their infarct volumes were not significantly different. The micro drill must be carefully used when creating a hole in the skull. With inappropriate action, easy causing bleeding from the MCA.

The MCA should not be damaged, and the bleeding must be avoided before and after the ligation procedure. The MCA occlusion and the reperfusion status can be checked by the laser Doppler. This method can confirm the condition of the blood flow of MCA.