This method can help answer key questions in the experimental stroke field, such as how to reduce associated with these models, and how to improve reproducibility. The main advantage of this technique is that it allows re-perfusion via the common carotid artery, and therefore is not reliant on the circle of Willis, which can vary anatomically between animals, and therefore may contribute to the variability seen in experimental outcome measures. Visual demonstration of this method is critical, as the CCA vessel repair steps are difficult to learn due to the technical difficulty involved when using the sealant to ensure that the CCA is fully patent, following repair.
To begin, prepare and anesthetize the mouse according to the test protocol. Then use a number 15 scalpel blade to make a 1.5cm midline incision on the exposed ventral neck. Using blunt dissection techniques, gently retract the salivary glands to the side, to expose the trachea.
Then dissect the common carotid artery, or CCA, free from the surrounding tissue and vagal nerve. After this, pass two small sections of a non-dissolvable 6-0 suture below the CCA, dorsal to the CCA, and ventral to the vagal nerve. Draw one silk tie closer to the surgeon, and tie it loosely around the CCA.
Then loosely tie a second silk tie toward the bifurcation of the internal and external carotid arteries. Next, apply a microvascular clip just above the distal tie, ensuring the bifurcation is not obstructed. Use micro venous scissors to make a small hole in the CCA.
Insert a 7-0 silicone-coated micro filament into the CCA, and advance it toward the microvascular clip. Then tighten the distal tie to fasten the filament in place. And use clip holders to remove the microvascular clip.
Next, advance the filament into the internal carotid artery, or ICA. Once the filament head passes the distal tie, pull the tie tighter, to prevent blood loss. Using the distal silk tie, lift and pull the CCA to the outer side of the mouse's body, and steer the filament to the bend, past the opening of the pterygopalatine artery, or PPA.
Once the filament is in place, secure it by tightening the distal silk tie further. At the end of the MCAO period, retract the filament until the white filament head is clearly visible. Then loosen the distal CCA tie just enough to remove the filament head most of the way, out of the vessel.
Next, fully remove the filament head from the vessel. After this, place a microvascular clip in the horizontal position, toward the CCA bifurcation, and next to the distal tie. Then place another clip below the proximal CCA tie, toward the surgeon.
Carefully use Dumont 5 forceps to remove both of the silk ties. And dry the area using sterile cotton buds. Then add Fibrinogen and Thrombin sealant solutions one and two, to a sterile Petri dish.
Using micro venous scissors and blunt dissection technique, obtain a thin ventral slice of sternocleidomastoid muscle. Then, using Dumont 5 forceps, take the tissue pad and mix it evenly across the Fibrinogen and Thrombin sealant solutions. Here it is important to ensure the tissue pad is large enough to cover the CCA incision.
And once it is mixed across the two solutions, it must be immediately placed over the incision, to prevent over-coagulation and poor adhesion. Once mixed between the two solutions, remove the tissue pad to the CCA incision. Place the tissue pad flat down on the incision with medium pressure, and open forceps.
Swiftly remove the distal microvascular clip, while holding the tissue pad in place. Next, slowly alleviate the pressure from the tissue pad, allowing blood to flow under the incision area. Now, slowly and gently release the pressure from the proximal microvascular clip, then remove completely.
Removal of the top clip allows a small amount of blood to activate the sealant. A steady release of the distal clip allows a gradual pressure increase at the incision site, activating further and helping to prevent a failed repair. Finally, once the vessel is sealed, suture the wound with dissolvable 6-0 sutures.
In this protocol, middle cerebral artery occlusion, or MCAO, was performed on 24 adult male mice. An avoidance of ECA ligation and the addition of analgesia showed a trend toward reduced weight loss at 48 hours post MCAO. Five minutes after filament removal, the regional cerebral blood flow significantly increased in the brain region of the MCA.
The perfusion was maintained up to the vessel repair, with an increase in perfusion to the MCA territory, after CCA vessel repair. This suggests that the CCA repair allowed increased blood perfusion to the ischemic territory, compared to reliance on the circle of Willis alone. A T2 weighted MRI was used to determine total LV and DTI scans were used to determine core LV at 48 hours after MCAO.
There was no significant difference between these two groups. Interestingly, that a variability for total and core LV was significantly reduced in the CCA repair group. Our analysis indicated that fewer animals per treatment group would be required to demonstrate a 30%reduction in LV, following MCAO, using CCA repair, versus typical CCA ligated procedure.
While attempting this procedure, it's important to remember to ensure the tissue pack is of sufficient size to cover the CCA incision. Also, the speed is important, to prevent the sealant over-coagulating, prior to placing the pad on the CCA.
