Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

In the filament model described here, complete restoration of blood flow and it's recording, are requirements to ensure a producible influx among mice, especially in translational stroke studies. This model has two main advantages compared to other previously described stroke models. No craniotomy is required, and a complete re-perfusion of the blood vessel is achieved.

The filament model is a complex surgical intervention involving the process manipulation of different arteries. The visualization and explanation of all these are small but important steps, might accelerate the learning period of neurosurgeons. Connect the heat blanket to maintain the temperature of the operation area and the mouse body temperature during anesthesia at 37 degrees Celsius.

Prepare autoclave scissors and forceps, 70%ethanol solution and dexpanthenol eye ointment, and keep several pieces of cotton and Five O coated braided polyester suture, ready to use. Prepare a 0.9%saline solution in a one milliliter syringe without a needle to keep the incision site hydrated. Prepare a holder for the laser Doppler probe by cutting the tip of a 10 microliter pipette tip.

Once the mouse is completely anesthetized, place it in a prone position in the operation area. Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. After disinfecting the surgical site, cut this scalp between the left ear and the eye to expose the skull bone.

Next, cut and retire the temporal muscle to visualize the multiple cerebral arteries or MCA beneath the skull. Fix the outer part of the tip holding the laser Doppler probe or fiber on top of the left MCA with glue and close the skin so that the skin is glued as well. Apply two to three drops of hardener glue to speed up the process.

Make sure that the laser Doppler fiber is not glued and can be easily removed from the tip holder at any time. Turn the mouse to the supine position, then put the snout into the anesthesia cone and fix the paws with tape. Disinfect the skin and hair surrounding the chest and make a two centimeter long midline incision in the neck.

Use forceps to pull the skin submandibular gland and sternomastoid muscle apart. Use retractors to expose the surgical field and find the left common carotid artery or CCA. Dissect the CCA free from connective tissue and surrounding nerves without harming the vagal nerve and perform a transient ligation before the bifurcation.

Dissect the external carotid artery or ECA and tie a permanent knot at the most distal visible part. Place another suture under the ECA and close to the bifurcation, then prepare a loose knot to be used later. Dissect the internal carotid artery or ICA and place a microvascular clip on it about five millimeters over the bifurcation.

Make sure not to damage the vagal nerve. Then cut a small hole into the ECA between the tight and the loose ligations, ensuring not to cut the entire ECA. Introduce the filament and advance it towards the CCA.

Tighten the loose ligation in the ECA around the lumen to momentarily secure the filament in that position. Removing the microvascular clip and prevent bleeding. After removing the microvascular clip, insert the filament through the ICA until the origin of the MCA is reached.

Fix the filament in this position by further tightening the knot around the ECA. Record laser Doppler values before and after filament insertion. Remove the retractor and relocate the sternomastoid muscle in the submandibular gland before suturing the wound.

Remove the laser Doppler probe and place the animal in a recovery chamber at 37 degrees Celsius for what one hour. Place the mouse in a prone position in the operation area with its snout in the anesthesia mask, then fix the animal's paws with tape. Insert the laser Doppler probe into the probe holder.

Remove the wound suture and use forceps to pull the skin, the submandibular gland and the sternomastoid muscle apart. Use retractors to expose the surgical field. Loosen the ECA suture that tightens the filament and gently pull the filament.

Avoid damaging the silicone rubber coating of the filament during the removal. Record the laser Doppler values before and after filament removal. Tightly tie the ECA suture and confirm the increase in the cerebral blood flow in the laser Doppler device.

Open the transient ligation before the bifurcation from the CCA to restore the complete blood flow. Remove the retractor and relocate the sternomastoid muscle in the submandibular gland before suturing the wound. Then, place the animal in a recovery chamber at 37 degrees Celsius for one hour to recover from anesthesia.

After recovery return the mice to their cages in a temperature controlled room and take care of the animals by adding wet food pellets and hydrogel in small Petri dishes on the cage floor and inject analgesia every 12 hours until day three after the surgery. Once the blood flow in the CCA is restored after removing the filament, complete re-perfusion of the brain occurs, which is similar to the situation observed after successful mechanical thrombectomy in human patients. Stroke animals presented a significant change in the composite and focal neuro score, but not in the general neuro score when compared to sham animals.

Infarct volume retrieved was also performed using cresol violet staining of coronal serial brain sections 24 hours after stroke induction. The lesion area includes the somatosensory and motor cortex, as well as sub-cortical structures, such as the striatum. The infarct volume mean was 61 to 62 millimeters square, representing 48%of the effected brain hemisphere.

It is important to perform an adequate dissection of the CCA, ECA and RCA without causing damage to the adjacent tissue, especially the vagus nerve, to have a good visualization of all structures, and to be able to insert the filament and occlude the MCA origin.