This protocol describes the calcium phosphate induced abdominal aortic aneurysm triple A mouse model to study the pathological features in the molecular mechanisms of abdominal aortic aneurysm. The calcium phosphate induced abdominal aortic aneurysm model is a rapid and the cost-effective model compared to the angiotensin tube and the elastase induced abdominal aortic aneurysm models. Demonstrating the procedure will be Zeyu Cai, post-doctor here, a fellow from our laboratory.
To begin cut 0.3 centimeter wide strips of powder free rubber gloves and gauze. Then purchase eight to ten week old C57BL6J Mayo mice, and house the animals in an air conditioned environment with a 12 hour light dark cycle and free access to food and water. Next, autoclave the gauze, cotton swabs, scissors, and forceps prior to surgery, and obtain Betadine, 70%ethanol, and antiseptic hand wash.
Shave the abdominal hair of the anesthetized mouse using an electric clipper or hair removal cream. Then swab and wipe the shaved area with Betadine, followed by 70%ethanol several times in a circular motion. Then make an approximately 1.5 centimeter incision at the lower abdomen along the midline of the abdomen using scissors.
Using a sterile cotton swab moistened with normal saline, carefully remove the intestine until the infrarenal aorta is visible. Next, dissect the connective tissue and fat from the infrarenal aorta for an approximately 0.5 centimeter section while avoiding any tearing in the small vessels to the dorsal side. Then pack a piece of the saline soaked rubber glove strip under the abdominal aorta and abdominal main vein while wiping off excess liquid using a cotton swab.
Afterward, pack a piece of gauze soaked with 0.5 molar calcium chloride on the adventitia of the infrarenal abdominal vasculature for 10 minutes. Remove the gauze and pack another piece of gauze soaked with PBS solution for five minutes to generate calcium ortho phosphate crystals in C2 in the adventitia of the aorta. Then carefully remove the rubber glove strip and gauze.
After resetting the intestinal tract of the mouse, suture the abdominal incision and skin with a five zero suture. Next, place the mouse on a heating pad until the mouse regains consciousness. House the mouse for another 14 days and monitor the mouse closely after the surgery and observe at least every day subsequently.
After 14 days of the surgery, cut open the euthanized mouse thoracic and abdominal cavities ventrally and cut open the right atrium. Profuse the mice with PBS buffer through the left ventricle of the heart to remove blood in the aorta and then perfuse with 4%paraformaldehyde as described in the manuscript. Remove adventitial tissue and fat carefully under a stereo microscope.
Then harvest the aorta and place it in a tube containing five milliliters of 4%paraformaldehyde for 48 hours. Then pin the aorta on a black wax plate with insect needles and proceed to acquire aortic images. After 14 days of calcium orthophosphate application the morphology of the aortas was imaged to visualize abdominal aortic aneurysm formation.
It was observed that calcium orthophosphate led to dilation of the infrarenal abdominal aorta and resulted in a dramatic degradation of elastic fibers as illustrated by elastin breaks. Using a sterile cotton swab moistened with normal saline to remove the intestine until the infrarenal aorta is visible and then extracting the connective tissue and the fat from the infrarenal aorta. Overall, the calcium phosphorate induced mice abdominal aortic aneurysm model is a rapid and cost effective approach to explore molecular mechanisms and the therapeutic strategies of abdominal aortic aneurysm and could be applied in parallel with the other models to fully imitate the features of human abdominal aortic aneurysm.
