Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

The overall goal of this procedure is to study the pathological features and molecular mechanisms of aortic aneurysms in mice. This is accomplished by first preparing an osmotic pump with angiotensin two solution. A pouch is then made in the skin of the animal's back into which the pump is inserted.

Ultimately, the dilation of the aorta in the thoracic and abdominal aortic regions can be assessed by ex vivo imaging. So we first implemented this technique cause we really wanted to define the role of blood pressure when we were infusing angiotensin to promote the development of atherosclerosis. Demonstrating the procedure will be Deborah Hart and Angio banan, both members of the Soha Cardiovascular Research Center To fill the osmotic pumps.

Begin by recording the lot number of the pumps and flow moderators included with the main body of the pump and the flow. Moderator wearing gloves to avoid transferring hand oil to the exterior casing of the pump. Next open only the number of pumps and flow moderators needed for the study.

Then weigh each pump, including both the main body and the flow moderator, noting the weight to four decimal places. This weight termed the pump weight empty in the template will be used to calculate the filled ratio when all of the pumps have been weighed. Attach the pump filling needle to a sterile one cc syringe and carefully fill the syringe with freshly prepared angiotensin two solution from the appropriately numbered plastic tube.

Carefully remove all air bubbles from the syringe with the needle positioned downwards. Then keeping the syringe in this position to prevent the introduction of bubbles into the pump. Gently insert the filling needle into the pump body.

When the needle is in place, advance the tip into the pump until it just touches the bottom of the pump. Then slowly depress the plunger to dispense approximately 246 microliters of the solution. The dark shadow inside the pump can be used to monitor the fill level.

The pump is full. When a bead of fluid rises out of the pump, carefully remove the needle and insert the flow moderator into the pump through the hole on top of the pump body until no gap is seen between the head of the flow moderator and the top of the pump body. Carefully blot away any extra fluid leakage that results from the insertion.

Then weigh the filled pump and record the weight in the appropriate pump, weight filled cell in the template. The filling ratio can then be calculated using the formula, and this data can be entered into the filled ratio cell in the template. Ideally, the filling ratio should be equal to or greater than 95%When the pump is filled, place it into an appropriate corresponding sterile four milliliter plastic tube with the moderator head facing upward, and add a sufficient volume of sterile saline to cover the pump.

Then when all of the pumps have been stored, place the tubes in a 37 degree Celsius incubator for at least 12 hours to allow partial priming of the pumps on the day of the implantation. Wearing a gown mask and gloves, shave a quarter sized area over the shoulder of an anesthetized mouse and transfer the animal to a laminar hood. After placing ointment on the animal's eyes and confirming a lack of response to toe, pinch, swab and wipe the shaved area with Betadine, followed by three wipes with 70%ethanol.

Next, use an autoclave sterilized surgical scalpel to make a one centimeter incision in the skin perpendicular to the tail and behind the ear over the shoulder blade of the front leg. Then with forceps in one hand, open the incision using a hemostat. In the other hand, make a subcutaneous tunnel under the skin advancing the hemostat tip toward the tail.

Carefully open the jaws of the hemostat to open up a pouch for the pump. Then replace the hemostat with the appropriate corresponding pump with the moderator head positioned to the rear of the mouse, and gently push the pump completely into the pocket. There should be enough free space to close the wound with no tension or stretching of the skin.

Once the pump is in position, firmly pinch and straighten both sides of the incision so that the edges meet and close the skin with one to two wound clips. Inspect the incision site to ensure that there is complete closure of the wound and that the pump is not pressing directly on the incision. Then use a clean cotton swab to apply topical lidocaine cream to the area.

Finally, transfer the mouse to a heating pad until it regains consciousness and then return the animal to its cage. In this experiment, after four weeks of angiotensin, two infusion from pumps implanted as just demonstrated, these aortas were harvested from low density lipoprotein receptor knockout mice for visualization of their aortic dilations. As observed in the images, the aortas exhibited several different characteristics, including the expansion of the Supra renal region, the expansion of the ascending region, or the expansion of both regions with one mouse exhibiting a grossly normal morphology.

Dilation of the abdominal aorta was then quantified by measuring the ex vivo maximal width of the senal region as illustrated by the red line to measure the ascending aortic dilation. The aorta were then opened and pinned as shown. The intimal surface area was measured in the ascending aortic region with the rulers in each of these and the proceeding images included to ensure standardization of the measurements Once mastered.

This procedure should take no more than five minutes to complete per mass.