Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

The overall goal of transient middle cerebral artery occlusion in neonatal rats is to provide a translational model of stroke in full-term neonates. This method can help answer key questions in the field of neonatal ischemic brain injury by understanding the mechanisms of injury and repair in the developing brain, and how we can modify these with different therapeutic strategies. The main advantage of this technique is that is provides a physiologically-accurate model of focal arterial ischemic stroke in neonates, and it also includes a reperfusion component, which is an important part of injury progression in human neonates.

Visual demonstration of this model is critical, because the steps of this surgery can be very difficult to learn. Two-dimensional representations of the arterial anatomy do not always adequately demonstrate the complex nature of 3-D branching in the rat. Begin by weighing the pup to ensure that it is between 19 and 21 grams.

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 five to seven millimeter anterior cervical incision to expose the common carotid artery.

Place two to four 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 within 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, erring closer to the tied ligature. Now, using a metric ruler, measure 10 millimeters of silicon-coated nylon suture as the occluding suture.

Cut the suture with an extra allowance of two to three 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.

During occlusion, diffusion-weighted magnetic resonance imaging can be used to verify the appropriate induction of injury. These images show ongoing ischemic injury, indicated by the arrow, detected by DW-MRI during transient MCAO. Approximately two hours and 50 minutes after occlusion of the MCA, prepare the surgical materials, and then begin the reperfusion procedure by anesthetizing as before.

Remove the interrupted sutures, and locate the junction, which is marked by the two ligatures and the tail end of the occluding suture. Carefully untie the most lateral knot of the temporary ligature by pulling the longer strand, and remove the suture strand from the cavity. At exactly three hours following MCA occlusion, slowly back the occluding suture out of the artery.

There should be no resistance. Use forceps to apply pressure to the arteriotomy, and then carefully untie the medial knot to restore blood flow. After removing the suture strand from the body, apply a hemostatic agent to the arteriotomy to stop bleeding.

The return of the artery's original shape and red color confirms that ICA blood flow has been restored. Remove the retractors, and close the cavity using 6-0 braided silk to created three or four interrupted sutures. Remove the pup from anesthesia, and allow it to recover with monitoring as before before returning it to the home cage.

Inspect the pups daily for five days. Record their weights, and inspect the incision sites closely for appropriate healing. These posterior-to-anterior 50-micron cresyl violet-stained coronal brain sections harvested from P-38 animals demonstrate fairly severe injury following a three-hour TMCAO at P-10.

The arrow indicates ipsilateral cyst formation and reduced cortical and striatal volume. The round hole on the left side represents a contralateral hemisphere identifier. While attempting this procedure, it's important to remember that while this procedure is conceptually simple, success and mastery may take hours of optimization and practice.

Once mastered, occlusion can be completed in less than 10 minutes, while reperfusion can be completed in less than five minutes.