Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

The overall goal of this surgical procedure is to officially and reliably induce Ischemic stroke while leaving the external cerebral artery intact. This method can help answer key questions in the field of stroke research, such as the impact of treatment on stroke severity. The main advantage of this technique is that following stroke induction, the external cerebral artery remains available to infuse treatment directly into the affected hemisphere.

Place a mouse anesthetized with isoflurane onto the prepared surgery surface and fit a nose cone to administer isoflurane mixed with oxygen. Ensure that the respiratory rate is around one to two respirations per second without gasping. In addition, ensure that the animal does not exhibit a reaction to whisker stimulation or the pedal reflex.

Put a drop of ophthalmic lubricant on each eye, using a steril swab to prevent them from drying during the procedure. After shaving the incision area, thoroughly disinfect the surgery area using 70%ethanol and a chlorhexidine-based solution. Keep the animal on a warm surgical surface under a stereo microscope.

Next, using surgical scissors and forceps, perform a shallow midline incision in the neck from above the breast bone to below the jaw. Using forceps, carefully separate fatty and connective tissues to expose the trachea. The tissues should easily and naturally separate to both sides.

Then place a pillow or other round object of about 0.5 centimeters in diameter under the back of the neck to extend the neck and further expose the area for surgery. Open the incision using either a tissue retractor or hooks. Then on the left side of the trachea, carefully tweeze apart the connective tissue to expose the left common carotid artery without damaging nearby nerves and major veins.

Next, insert the curved forceps under the CCA to pierce the connective tissue and then slowly allow them to open to release the CCA. Detach the CCA from the underlying tissue and expose the top y branching of internal cerebral artery and external cerebral artery. Once the CCA is exposed, insert three segments of nylon suture under the CCA.

Ensure that the CCA is not twisted, as this would drastically complicate the insertion of the suture. Then at the lowest point possible, close the bottom suture using a permanent knot. Tie the top suture just below the branching of the ICA and ECA using a removable slipknot.

Next, tie the middle suture using a removable slipknot, but keep it wide open. Then, using micro dissection spring scissors, create a 0.2 millimeter in the CCA between the bottom and middle sutures closer to the bottom suture. Using forceps, insert the MCAO suture into the CCA incision, guiding it towards the top suture.

Then gently tighten the slipknot of the middle suture on the CCA so that it restricts blood flow around the MCAO suture, but is loose enough to allow the MCAO suture to move freely. Now carefully undo the top suture, making sure that the MCAO suture does not slip out. Insert the MCAO suture a couple of millimeters into the ICA and then reclose the top suture with a slipknot as before.

Next, guide the MCAO suture to the occlusion area, first by routing it into the ICA past the ECA, then by ensuring by ensuring that the suture stays in the ICA, bypassing the pterygopalatine artery. The insertion is successful if there is little blood backflow from the CCA incision and if the silver nine millimeter mark is situated between the CCA incision and the bifurcation of the ICA and ECA. After successful occlusion, tie down the middle and top suture tightly then tuck the sutures into the incision area.

After removing the retractor or tissue hooks and the pillow, clean the suture area using sterile saline on a cotton swab. Then close the skin with nylon suture. Place the mouse in a clean cage situated on a heated pad to prevent hypothermia and allow ad libitum access to water and softened food.

Monitor the animal for signs of stroke for at least five to 10 minutes. Depending on the ischemia reperfusion protocol, leave the MCAO suture in place from 30 minutes to 120 minutes or more. After anesthetizing the animal as before and removing the wound closing sutures, use forceps and tissue separators to reopen the incision and expose the CCA.

Then carefully remove the top suture and gently pull on the MCAO suture until the silicon-coated part is situated at the middle knot. Redo the top knot suture to prevent blood from flowing pass the MCAO suture. Then carefully undo the middle knot and pull the MCAO suture past the top knot, keeping it inside the CCA.

Tightly close the top knot to block artery blood flow. Next, completely pull out the MCAO suture and tightly close the middle knot. Close the wound and administer post operative care as before.

This schematic shows the occlusion area that results from the procedure demonstrated in this video in blue. The surgery area is located at the bottom of the figure, indicated by the oval shape, and suture placement is indicated by black lines. The following images show TTC standing of brain sections.

This image represents typical staining following sham insertion. The image is representative of the tissue damage caused by a 60 minute MCAO, follow by 90 minutes of reperfusion. Greater tissue damage is seen after 24 hours of reperfusion, following a 60 minute MCAO.

Immunofluorescent staining for the neuronal marker MAP2 allows neuronal loss from 60 minutes of occlusion, followed by 23 hours of reperfusion to be clearly visualized in the image on the right. Astrogliosis is visible after immunofluorescent staining with GFAP. In the picture on the right, the astroglia are concentrated in the area adjacent to tissue damage in the ischemia reperfusion brain, whereas a diffused patter of staining is seen in the shame operated control.

While attempting this procedure, it's important to monitor animal well-being and occlusion time. Following this procedure, other techniques such as odd behavior analysis and immunofluorescence can be performed to answer additional questions on tissue injury and recovery.