The overall goal of this Rat Carotid Balloon Injury procedure is to mimic clinical angioplasty and its potential consequences. This message can help answer key questions in the cardiovascular research field, about the cardiovascular surgical injury elicited neointimal hyperplasia which can significantly compromise the benefits of the surgery. The main advantage of this technique is that the result can be obtained within two weeks, allowing a quick evaluation of the therapeutic reagents administered during the treatment period.
Demonstrating the procedure will be Dr.Natalia Petrasheskatya from the laboratory of cardiovascular sciences and at the National Institute on Aging. Before beginning the surgery, autoclave all of the surgical instruments and use 70%ethanol to disinfect the operating platform surface. Put on the proper personal protective equipment, placing a box of sterilized gloves near the surgical platform for additional use.
Next, fill a 1 milliliter syringe with sterile water and attach the syringe to a sterile, water-filled, two-way stopcock. Gently push the water through the stopcock to fill the lure-lock portion of the balloon catheter, and remove any air bubbled from the opening of the catheter. Then, test the balloon inflation to confirm that the balloon can be inflated with 0.02 milliliters of water.
Now, check the pedal withdrawal reflex of an anesthetized 400 to 450 gram male Wistar rat. If the rat does not withdraw its foot, transfer the animal to the surgical platform. Cover the rat with a sterile surgical sheet, and cut the sheet to expose the neck region.
Then adjust the animal into the supine position on a headed pad with the head toward the surgeon and tape the limbs into place. To isolate the left carotid artery, us a number 14 scalpel to make a straight incision from below the chin to the top of the sternum, just below the ribcage. Using a 7S forceps, blunt dissect under the skin and through the salivary gland tissues to expose the underlying muscle layer.
Separate the muscle tissues to gain access to the carotid vasculature and the vagus nerve in the neck. Continue to blunt dissect the tissues surrounding the carotid artery, carefully separating the vagus nerve and the vascular fascia without damaging either tissue, until the bifurcation of the common carotid artery and the internal and external carotid branches are clearly visible. When the left common carotid artery has been exposed all the way to the sternum, place a 4-0 silk stay suture behind the artery as close to the sternum as possible.
Permanently ligate the smaller arteries with the sutures, along the external carotid artery, including the ascending pharyngeal, the occipital, and the superior thyroid to avoid arterial leakage. Place another 4-0 silk stay suture immediately distal to the bifurcation, behind the external carotid artery as far from the bifurcation as possible. Then gently retract the overlying carotid artery to the right to visualize the internal carotid artery and place a 4-0 silk stay suture around the artery to avoid significant retrograde blood-loss.
To introduce the balloon catheter, retract the proximal suture on the common carotid artery, and temporarily stop the blood flow with an arterial clamp. Make a small incision on the external carotid artery as distal to the suture as possible, and gently insert the uninflated balloon catheter into the incision. Advance the catheter into the lumen until it is close to the arterial clamp on the common carotid artery.
Then remove the clamp and advance the catheter to the aortic arch, approximately 35 to 40 millimeters from the incision. Now use the syringe of sterile water to manually inflate the catheter to a 0.02 milliliter volume, and turn the stopcock to the locked position. Slowly withdraw the catheter while rotating to produce the injury.
When the instrument is close to the arterial incision, deflate the balloon and retract the catheter back to its original position. After repeating the balloon injury procedure three times, carefully remove the catheter from the arterial lumen and close the arteriotomy with size 5-0 silk stay sutures. Release the internal carotid artery to restore the blood flow and check for arterial leakage.
Finally, remove the stay sutures and clamps and close the glandular tissue with 6-0 silk sutures. Then, close the skin and monitor the animal until it is fully recovered. Two weeks after the balloon injury, the carotid arteries can be isolated for histomorphological analysis.
The balloon injury elicits neointimal growth, or thickening of the vessel wall. If left untreated or treated with a saline control, compared to a non-operated vessel section from the same animal. By contrast, the vessel section from a rat that received a therapeutic reagent that blocks neointimal growth demonstrates a significantly reduced thickening in the vessel wall.
The effects of the balloon injury and therapeutic treatment can also be evaluated in vivo, by ultrasound sonography. And these data collaborate well with the results from histomorphological analysis. When mastered, this techniques can be completed in 30 minutes, if it is performed properly.
While attempting this procedure, it is important to remember to prepare and test the balloon catheter for leaks prior to the surgery. After watching this video, you should have a good understanding of how to perform the rat carotid balloon injury procedure, for evaluating tracks and therapeutic reagents that block neointimal growth and to study the mag-ni-sen of injury elicited neointimal growth. Don't forget that working with animals requires an approved protocol by your institutional AICUC.
You should also consult with your institutional laboratory animal veterinarians to establish the best practices for post-surgical care, and monitoring of the animals.
