Generating a Middle Cerebral Artery Occlusion in a Rat Pup to Induce Local Brain Ischemia

Begin with a secured anesthetized rat pup on a heating pad.

Sterilize the cervical area alternately with ethanol and povidone-iodine. Administer local anesthesia at the incision site. 

Make a cervical incision, exposing the common carotid artery.

Place retractors to ensure clear access.

Identify the internal carotid artery or ICA and remove surrounding tissue.

Tie a ligature around the base of the ICA and retract it to temporarily halt blood flow. 

Place a loose second ligature on the ICA.

Make an incision between the ligatures.

Take a silicone-coated nylon occlusion suture and insert it through the incision until it reaches the middle cerebral artery or MCA.

This blocks the blood flow to the MCA, inducing local ischemia in the brain.

Tie the ligature around the occlusion suture to secure it.

Close the incision site. Allow the pup to recover before further studies.

After anesthetizing the pup using inhaled isoflurane according to approved protocols, ensure that there is no response to a foot pinch. Maintain body surface temperature with the use of a heating pad under the surgical stage. Secure the animal in a supine position with tape across the shoulder regions.

Use sterile cotton swabs to swab the anterior cervical area with 70% ethanol, followed by a swab of povidone iodine solution. Next, locally infiltrate 0.25% bupivacaine into the planned incision site. Then, while working through a stereoscope, make a midline 5 to 7 millimeters anterior cervical incision to expose the common carotid artery.

Place 2 to 4 retractors to keep the cavity open and the artery exposed. Next, locate the internal carotid artery, occipital artery, and external carotid artery. Remove connective tissue and fat from the arteries to get a clear view.

After cutting 1.5 centimeters of 6-0 silk braided suture thread. Unbraid the suture, pulling out single strands. Make sure that the single strands are neat and not frayed.

While holding the suture strand with 45-degree forceps, sweep the forceps in an arc under the ICA so that the tips emerge between the ICA and OA. Next, grab the end of the suture strand and pull it so that the end is easy to access. Release the strand from the forceps and back the forceps out from under the ICA, reversing the prior motion.

Tie a temporary ligature around the ICA at the base closest to where it separates from the CCA. Carefully retract the ICA laterally, then use a clip to secure the retracting strand to excess skin near the axilla region on the side opposite to the incision. Ensure that the retracting strand is taut enough to stop blood flow before proceeding to the next step.

The artery should be flat and pale. Use a 45-degree forceps to grasp another unbraided suture strand, and loop it under and around the ICA as before. Position this strand lateral to the retraction strand.

Then cut a 0.2 millimeter arteriotomy, midway between the tied and untied ligatures, are in closer to the tied ligature. Now, using a metric ruler, measure 10 millimeters of silicone-coated nylon suture as the occluding suture. Cut the suture with an extra allowance of 2 to 3 millimeters for removal during reperfusion.

Hold the occluding suture with 45-degree forceps, and use straight forceps to create a bend, marking the stopping point of advancement. Feed the occluding suture into the arteriotomy, aiming it parallel to the ECA and towards the head. Smoothly advance the suture until the bend is reached. Then use the strand positioned lateral to the retraction strand to tie a temporary ligature to secure the occlusion suture. Remove the retractor clip. Then trim the strands of both temporary ligatures, leaving the strand that will be used to remove the knot longer than the strand used to tighten it. Remove the retractors. And close the cavity using 6-0 braided silk to create three or four interrupted sutures.

Remove the pup from anesthesia and place it on a heating pad in room air. Monitor the pup until it has regained sufficient consciousness to maintain sternal recumbency, and make sure that it is fully recovered before returning it to the dam.