The overall goal of this procedure is to mimic the human condition of restenosis after revascularization therapy in mice to study the molecular mechanisms involved in the injury response. The main advantages of these techniques are that it is highly reproducible and easily performed, enabling researcher with even minimal experience to apply the method in small animal models. Demonstrating the procedure will be Adelina Curaj, a postdoc for my laboratory.
To induce hyperlipidemia, feed six to eight-week-old 18 to 20 gram Apoe knockout mice an atherogenic diet one week prior to the surgical procedure and until the atherosclerotic plaque analysis. On the day of the surgery, after confirming a lack of response to toe pinch, shave the anesthetized mouse in the ventral neck region and disinfect the exposed skin with Betadine. Make a one centimeter skin incision in the median region of the neck over the trachea and separate the two fat bodies to obtain a complete view of the tracheal region.
Using one retractor to hold the muscle layer open, expose the carotid artery and use sharp curved forceps to separate the artery from the vagus nerve and jugular vein until the bifurcation area of the internal and external carotid artery is visible. Loosely, place a seven centimeter long 0-5 silk suture under the carotid artery proximal to the aortic arch, followed by one loosely placed 1.5 centimeter long 0-7 silk suture around the external carotid artery close to the bifurcation point, and one loosely placed 1.5 centimeter long 0-7 silk suture loop as distal from the bifurcation as possible. Place another open 1.5 centimeter long 0-7 silk suture under the internal carotid artery.
Then position the animal with the head at the six o'clock position. Immediately, tightly close the suture loops on the internal carotid artery and the distal suture on the external carotid artery. Use hemostat forceps to pull on the ends of the 0-5 suture to stop the blood flow through the common carotid artery.
Using small scissors, make a small arteriotomy of half of the vessel diameter distal to the external carotid artery between the two loops and insert a 14 inch alcohol polished flexible guidewire moistened with a drop of 0.9%sodium chloride into the common carotid artery via the incision. To avoid arterial eruption during Y-incision, use a carefully polished guidewire. Obtain endothelial denudation by passing the wire along the vessel while rotating three times, then tightly close the proximal loop on the external carotid artery and cut the sutures around the common and internal carotid arteries to restore the blood flow.
Next, remove the retractors and return the muscle layer and the two fat bodies to the physiological position. Close the skin with metallic clips, then allow the mouse to recover on its left side under an infrared light with monitoring until it is fully recumbent. The neointima formation can be assessed using Movat staining.
The total plaque size, which can vary between 70, 000 and 100, 000 micron squared, depending on the skill of the surgeon, can then be calculated for each sample using the appropriate software. The developed plaque resembles instant restenosis, which predominantly consists of proliferated and migrated smooth muscle cells from the medium. The smooth muscle cell content represents approximately 30 to 40%of the plaque while the macrophages found within the neointima of the injured vessel make up 15 to 25%of the total cell number.
The re-endothelialization typically reaches 80 to 90%after three weeks and should be almost complete after four weeks. To track the plaque growth during its development, the analysis can be repeated at multiple time points after the wire injury depending on the experimental question. Once mastered, this technique can be completed in 15 minutes if it is performed properly.
While attempting this procedure, it is important to remember that the surgical area should be constantly hydrated to prevent drying and to make the wire manipulation easier.
