The overall goal of this mouse based model system is to study the development of myointimal hyperplasia after aortic balloon injury. This model system can help answer key questions about myointimal hyperplasia such as what factors contribute to its development. By studying the development of myointimal hyperplasia in transgenic mice we hope to gain a better understanding of the pathomechanism of the disease.
Another advantage of this animal model, is that the disease progresses quickly. In just weeks, we're studied in mice. First, prepare the required catheter.
Remove it from the holder and pull the guide wire out through the lumen through the distal port. Next put in a drop of cyanoacrylate on the distal end of the catheter and feed the guide wire through the RX port, then through the lumen and out the distal port. Next pull the guide wire back slightly to leave about five millimieters sticking out and let the adhesive dry.
Later fill a three milliliter syringe with 1.5 milliliters of 0.9%saline and connect it to the ballon inflation port of the catheter. Then push the plunger to test the balloon inflation. Leave about 0.6 milliliters of saline in the syringe.
Next, prepare the mouse for surgery. Anesthetize it in an isoflurane chamber and later, during surgery, deliver isoflurane via a nose cone. Before proceeding, confirm a sufficient depth of anesthesia by pinching the hind feet and tail to verify an absence of reflexes.
Then remove the abdominal hair using a hair trimmer and fix the position of the hind legs using tape. Lastly, disinfect the abdominal area using three alternating scrubs of povidone iodine followed by 80%ethanol. Begin with dissecting open the skin and muscle layers along the linea alba to expose the abdominal organs.
Then move aside the intestines, wrap them in a saline soaked gauze to keep them moist. Now using two fine forceps to remove the fatty tissue above the abdominal aorta. Then from an insulin syringe, inject about 12 units of heparin sulfate in to the IVC and wait three minutes for systemic distribution.
Next use forceps to dissect the infrarenal aorta down to the bifurcation and to reveal the outgoing branches. Then using cauterization, ligate the side vessels along the infrarenal aorta within the area that will be clamped. Then, stop the blood flow by clamping the vessel directly beneath the renal arteries and clamping at a distal position just above the aortic bifurcation.
Now perform a small horizontal incision along the vessel at the midpoint between the clamps. The incision should be about 1/3 the circumference of the vessel. Then, using an insulin syringe, flush the aorta with heparin solution as before.
Next place 10-0 sutures with single knots on each side of the incision. To proceed, dilate the aorta. Place a vessel dilator in to the incision and spread the vessel slightly.
Do this two or three times. Then prepare the balloon catheter by wetting it with saline. Then insert the balloon catheter deflated in to the aorta and advance it towards the proximal clamp.
Carefully open the proximal clamp and inflate the balloon to prevent blood leakage by injecting about 0.6 milliliters of saline. Next, advance the catheter retrograde about two centimeters. Pull the catheter back while deflating the balloon slightly.
When the catheter reaches the incision of the aorta, reattach the proximal clamp. Then, deflate the balloon completely and remove it. Now rinse the aorta with heparin solution.
Then proceed by closing the aortic incision using interrupted 10-0 sutures on each lateral side. Followed by one or two stitches on the ventral side. Next open the distal clamp, check for bleeding and place additional sutures as needed.
Once there's no bleeding, carefully open the proximal clamp. Use swabs on the sutures for support and to slow bleeding. Then place absorbable hemostats on the sutures to sustain them.
With both clamps opened, an aortic pulse should be visible distally from the incision. Now place the intestines back in to the abdomen and rinse the abdominal cavity with saline warmed to 37 degrees Celsius. Next close the abdominal muscle layer using 6-0 running sutures.
And then close the skin with 5-0 running sutures. Next immediately inject an analgesic subcutaneously before the animal wakes up. Now monitor the mouse until it has gained consciousness and can maintain sternal recumbency.
Once conscious, house the animal alone until it has completely recovered. Provide Metamizole to the drinking water for three days. Be sure to check on the mouse every day to ensure a good recovery.
Balloon denudation is a suitable method to study the development of myointimal hyperplasia in mice. Animals recovered well from the surgery and were in excellent physical condition. Less than 3%died in over 50 surgeries.
Myointimal hyperplasia developed progressively in the graft. Histological staining with Masson's trichrome showed the myointima formation inside the internal elastic lamina. The myointimal lesions consisted mainly of cellular components positive for SM22 indicating smooth muscle cells and ECM cells like myofibroblasts also shown in red.
After watching this video you should have a good understanding how to perform the balloon injuries in mice. Once mastered, this technique can be done in 20 minutes if it's performed properly.
