This Surgical procedure used to create an abdominal aortic aneurysm begins with a midline abdominal incision. The abdominal contents are moved aside and all of the aortic branches are ligated. The proximal and distal portions of the aorta are tied off.
The heart is punctured with a needle and a profusion of elastase is delivered through a heat tapered polyethylene tube. When the profusion is complete, the AOR autotomy is closed. The ligatures are removed, the aorta fills with blood and the abdominal cavity is closed.
The mouse is then allowed to recover and the aneurysm will be completely dilated in 14 days. Hi, I'm Julian Asma from the laboratory of Fetal Cell in the Department of Cardiovascular Medicine at Stanford University. Today I'll show you the procedure for injecting new experimental abdominal aortic aneurysm with elastase.
We use this procedure in your laboratory to study the PEs of abdominal art S.So let's get Started. To begin your experiments, select a mouse of interest, for instance, a C 57 black six mouse, and anesthetize it with inhaled iso fluorine until the mouse is unconscious. Please be aware that different mouse strains will vary in the rate of aneurysm growth.
Remove the anesthetized mouse from the chamber at an adequately illuminated preparation station. Shaved the fur from the abdomen from the end of the cosal bone to the root of the legs. Use hair remover cream to remove the excess hair.
Scrub the bare skin with 70%alcohol until all the furs cleared away from the shaved region. Then scrub the skin a minimum of three times with Betadine swabbing with alcohol between scrubs. Now, place the mouse supine on the operative field to keep the mouse unconscious.
During surgery, place a tube on its nose that delivers iso fluorine at the same flow rate used to induce unconsciousness. The elastase is kept on ice and extracted into a syringe. The syringe is then clamped and placed next to the operative field and the catheter is attached.
It is important that the tip of the catheter remains sterile, which is done by placing the tip in a sterile vial. Before cutting open the mouse, ensure that sterile technique will be used. Begin by donning sterile gloves.
The surgical instruments, scissors, needle holders, and forceps must be sanitized prior to the surgery. Take a moment to be conscious of your preparations and try and remedy any other potential sources of contamination during the surgery. If sterile technique is broken during the procedure, re sterilize your instrument in a hot bead sterilizer.
Once everything is prepared, begin the surgery with a long midline abdominal incision inside the abdominal cavity. Move aside the abdominal contents into free abdominal space or retract them outside the abdomen with sterile gauze to expose the abdominal aorta. Now use forceps to carefully isolate the abdominal aorta from the level of the left renal vein to the bifurcation.
In the case shown, there is a single branch which is above the site where we ligate the aorta and thus it does not need to be tied. If there are aortic branches between the sites of the distal and proximal aortic ligations then ligate the branches within one centimeter of the bifurcation with 10 aut sutures at the proximal and distal portions of the aorta place. Temporary 6.0 silk ligatures to stop the blood flow.
Now perform an atomy at the bifurcation. Begin by puncturing the bifurcation with a 30 gauge needle. Heat tapered polyethylene tubing can be introduced through the AOR autotomy and secured with a knot.
Use the forceps to carefully and gently insert the tube into the aorta. Be careful to not denude the endothelial layer using a pressure of 100 milliliters. Mercury perfused the aorta with elastase from a saline bag hung at the height of 136 centimeters.
Make sure there is no air in the tube otherwise the pressure will not be exact. The saline contains 4.5 units per milliliter. Type one porcine pancreatic elastase, and should fill the aorta in five to 15 minutes.
During the perfusion, the aorta typically dilates to about 140%of its original diameter. When completed, remove the perfusion and close the aor autotomy with a 10 aught suture to avoid constriction. After the atomy is closed, the distal ligature must also be removed.
If there is little or no bleeding from the repaired atomy, the proximal ligature is removed. If there is bleeding place, additional temporary 6.0 soak ligatures at the proximal and distal portions of the aorta and add one more suture to close the AOR autotomy completely. The expanded aorta will fill with blood immediately after removal of the proximal ligature.
Once the procedure to the aorta has been finished, return the intestines to the abdomen. If it was retracted, the intestines will slip in as a large unit and there was no need to worry about their geometry. With all the organs in place, the external wound can now be closed in two layers with six aut nylon, and polypropylene sutures.
Now provide the mouse a local anesthetic such as one drop of 0.25%boop of ICAN per centimeter of incision. Also give one dose of the anti-inflammatory Carin at five milligrams per kilogram by subcutaneous injection. The surgery is now complete and the mouse should be placed on a warm area where it is allowed to return to consciousness.
Recovery usually occurs within 15 minutes. When the mouse regains consciousness, it is returned to a solo cage and monitored periodically through its recovery. After the procedure, the aorta typically dilates to about 140 to 160%of its original diameter.
This initial dilation is observed with stabilization up to day seven. Rapid secondary dilation occurs between day seven and day 14. At day 14, the aneurysm can be observed in infrarenal aorta.
I've just shown you how to indect abdominal antic aneurysm with elastase in mice. When doing this procedure, it's important to avoid creating any blood clots inside the ata. So that's it.
Thank you for watching and good luck with your experiment.
