Annual models are emerging as an important tool to understand the pathologic mechanisms underlying aortic valve stenosis because of the lack of access to reliable sources of diseased human aortic valves. Traditional aortic valve stenosis rabbit models require long-term dietary supplementation and genetic manipulation to induce significant stenosis in the aortic valve, limiting their use in experimental studies. This technique describes a new procedure for inducing aortic valve stenosis in rabbits through a direct ballon injury to the aortic valve, which can accurately mimic the condition of a human with aortic stenosis.
Demonstrating the procedure will be Eun-Hye Park, Jin-Moo Kim, and Eunmi Lee, research specialists from my regulatory. Begin preparing the dilation balloon catheter set by connecting the in deflation device filled with the one-to-one mixture of saline and commercially available contrast medium to the Luer lock part of the balloon catheter. After filling the balloon with an inflation solution, remove the air from the balloon catheter.
Begin the surgery procedure by inserting a 24 gauge intravenous or IV catheter into the marginal auricular vein of the anesthetized rabbit. Connect an infusion set with 100 units per kilogram of heparin heparinized saline. Next, connect the rabbit with a multi-parameter veterinary monitor to continuously monitor the vital signs such as the oxygen saturation signal or SpO2 temperature and blood pressure.
After placing the rabbit in a supine position on an operating table equipped with a C-arm fluoroscopy, remove the hair from the ventral neck area using animal hair clippers. Sterilize the incision area with iodine and cover the rabbit with surgical towels. To position the rabbit's heart, turn on the C-arm and select the fluoroscopic mode for cardiac imaging.
Adjust the rabbit's position to ensure the heart is at the center of the imaging field. Once the position is adjusted, make a longitudinal incision of approximately three centimeter in the skin of the neck and use surgical scissors to cut the fascia and fat tissue. Then carefully separate the muscles until approximately 3 to 3.5 centimeters of the left common carotid artery, or LCCA, is exposed.
Ligate the LCCA with a 3-0 silk suture at the top and end of the exposed LCCA to stop the blood flow. Insert a 22 gauge IV catheter into the LCCA. Then introduce a 0.016 inch guide wire into the left ventricle through the IV catheter, ensuring that the tip of the catheter is properly positioned in the imaging field of the C-arm.
Next, withdraw the IV catheter, leaving the guide wire before placing a 4 French sheath over the guide wire into the LCCA to introduce the balloon catheter. Carefully insert the eight millimeter balloon catheter over the guide wire into the aortic valve under C-arm fluoroscopic guidance. Place the balloon catheter tip approximately one to two centimeters distal to the aortic valve and inflate the balloon by purging the inflation solution with a pressure inflator at six atmospheres.
Next, advance the balloon into the left ventricle or LV apex and pull it back into the LV outlet. Repeat this procedure five times before deflating the balloon. Withdraw the balloon catheter and guide wire, then slowly remove the sheath from the LCCA and immediately tie the LCCA with the suture on the way down to the aortic valve.
Clean the incision area with saline to remove the blood clots. Inspect the punctured site for arterial bleeding before closing the muscle and skin with a 3-0 non-absorbable suture. Once done, sterilize the wound using iodine.
After the surgery, remove the monitoring patches and clips and keep the rabbit in an intensive care incubator. After eight weeks of the balloon injury, place the anesthetized rabbit on an echo table in the supine position. Apply ultrasound transducer gel to the chest after shaving the chest area.
Adjust the transducer to obtain the peristernal long-axis view and the peristernal short-axis view of the aortic valve. Use mode imaging to record images of the aortic valve and save the images for later analysis. The structural change assessment in the aortic valve revealed that eight weeks after the aortic valve injury, the cusps were thickened and the motion was restricted in the injured rabbits fed with the high cholesterol and vitamin D2 diet compared to the control or wild type rabbits, and the rabbits fed the high cholesterol and vitamin D2 diet without valve injury.
The rabbits were sacrificed and the hearts were excised eight weeks after the injury for histological analysis of the aortic valve. The aortic valve stained with Masson's trichrome showed increased thickness of the aortic valve cusps in the injured group compared to the control in high cholesterol and vitamin-D2-diet-induced groups. Alizarin Red staining and Von Kossa staining were performed to compare the degree of valvular calcium deposits indicated negligible calcium deposits in the valvular leaflets in the high cholesterol and vitamin-D2-diet induced group.
In contrast, significant calcific deposits were observed in the balloon injured group. Successful technique for inducing arctic valve stenosis, in the rabbit model is demonstrated here with a step-by-step explanation of a direct balloon injury to the aortic valve. This simple and reproducible model offers a promising approach for studying the initiation and progression of aortic valve stenosis, and provides a valuable tool for investigating the underlying pathological mechanisms of aortic valve stenosis.
