Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.
The overall goal of this procedure is to induce the formation of atherosclerotic plaques in rabbits by surgically disrupting the endothelium of their left iliac arteries, and feeding them an atherogenic diet. By lone injury in rabbits is often performed in the aorta to study plaque composition. However, the iliac artery is very similar to the human coronary artery, making it an attractive place for modeling atherosclerotic plaques.
The muscular content uniform lesions, eye tissue factor activity, and consistent vessel dimensions of the rabbit iliac artery are all comparable to the human coronary artery. Ultimately, this allows for the evaluation of commercially manufactured devices to morphometric and angiographic endpoints. Demonstrating the procedure will be Aurelien Frobert, a collaborator from my laboratory.
Be certain to sterilize all the surgical tools using, for example, a glass bead sterilizer. Next, prepare and check the two balloon catheter assemblies. Onto each one, attach a one-milliliter Luer lock syringe filled with normal saline, and then check for trapped embliques using the balloon's inflation mechanism.
Next, set the thermal pad at the surgical platform to 37 degree celsius. Then weigh the rabbit. Next, provide an injection of buprenorphine and anesthetize the rabbit using 5%isofluorane at five liters per minute for 10 to 15 minutes.
Now, place the anesthetized rabbit on the heating pad on a surgical platform. Attach sensors to monitor the rabbit's temperature and respiration and to collect electrocardiograms. Next, attach a face mask to deliver 4%isofluorane at 2.5 liters per minute.
Then confirm a proper level of anesthesia by testing for a lost of gag and petty reflexes. A universally relaxed muscle tone is also indicator of deep anesthesia. Next, apply abtonic ointment to both eyes.
Next, remove the hair from the ventral area just below the knee joints using animal hair clippers. Then, remove the loose hair and clean the exposed skin with three alternating scrubs of 70%ethanol and betadine and drape the animal leaving only the lower limb exposed. To begin the surgery, locate the saphenous artery and make a 1.5 centimeter incision.
Then using small curved forceps carefully expose a small portion of saphenous artery. Be careful to avoid damaging the femoral vein or femoral nerve. Cleaning the artery thoroughly is very important during this exposure.
Next, place two loose ligature loops beneath the saphenous artery and tie one ligature loop near the distal end of the artery. Then place some micro vascular clamp along the artery above the ligature and close the clamp to stop the blood flow. Immediately apply a drop of papaverine to dilate the artery and prevent phase spasms.
Next, using the ligature, lift the saphenous artery and to make a small arteriotomy incision using a 24 gauge needle. Then lift the incision flaps and slowly insert a vein pick or a guiding needle into the lumen of the artery. Now, carefully insert an arterial embolectomy catheter into the saphenous artery.
Be gentle to avoid making ruptures. Then remove the vein pick and release the micro vascular clamp on the artery. To create the injury, first advance the balloon catheter to the third mark.
This should put a roughly two to five centimeters above the iliac bifurcation. Next, inflate the balloon with 0.1 milliliters of normal saline. Now, clip the catheter and pull it back six centimeters while rotating it to damage the tissue.
Once retreated, deflate the balloon. And to ensure complete endothelium dilatation, repeat the process twice more. Then remove the catheter and immediately tie off the ligature loop just above the arteriotomy site to stop the bleeding.
Next, apply a suitable antiseptic around the perforate of the wound and swab away the blood clots. Lastly, close the skin incision and disinfect the surgery site with povidone-iodine solution. Then repeat the entire surgical procedure on the contralateral iliac artery using the second catheter.
After the surgery, first clean the abtonic ointment from the rabbit's sides. Then administer sulfadoxine and trimethoprim. Then transfer the rabbit with the heading pad into a clean recovery cage and a monitoring patch and clips.
Once the animal regains external recumbency and is ambulating, return it to its home cage. For pain management, provide buprenorphine. Now, start feeding the animal on an atherogenic diet for as long as needed.
To harvest early thin flax, two or four weeks post surgery, first anesthetize the rabbit and then euthanize the rabbit with an intercardinal incision. Next, open the abdomen and expose the retroperitoneum. Then trace the aorta towards the iliac bifurcation and tie it above the bifurcation.
Follow by carefully removing the surrounding tissues and isolating both iliac arteries. Now dissect out both arteries and immerse them in ice cold PBS. Therein remove the clots using forceps and divide each artery into 46 segments.
Then immediately embed the segments in a mold containing OCT compound and snap freeze tissues in liquid nitrogen. Store the tissues at minus 80 degree Celsius and later stain five micron thick sections using standard methodology. Balloon injury of the iliac arteries was performed successfully without complication in under an hour.
Rabbit's recovered within one hour and/or appeared healthy without significant weight loss. No post operative complications were encountered. And after four weeks of atherogenic diet, the rabbits all had hypercholesterolemia.
The combination of a balloon injury and the cholesterol diet resulted in structural changes in the vessel wall leading to the development of atheroschlorotic plax in two weeks. Uninjured and balloon injured iliac arteries were isolated from the same animal and compared. Flax were characterized by lipid infiltration as well as smooth muscle sound migration and proliferation.
The flax continue to grow in the injured vessels while the lumen size continue to shrink. The resulting changes were very pronounced by four weeks. After two and four weeks, morphometric quantification revealed an increased intima media thickness, an increased plaque area, a decreased lumen area, and finally an increased in lipid accumulation.
Staining with alpha smooth muscle actin anti body reacts with muscle actin, thus identifying vascular smooth muscle cells. While staining with more 11 monochlonal antibody reveals the cytoplasm of macrophages. Together the stains reveal the extent to which both smooth muscle cells and macrophage proliferate into the atherosclerotic plaque in the injured vessels.
After watching this video, you should have a good understanding of auto safety and easily perform a violent injury on the rabbit's iliac arteries. This injury with atherogenic diet results in plaque's developing of the iliac arteries in homogenous on time fashion.
