A regenerative treatment for peripheral arterial disease would be extremely beneficial and this animal model may be able to better test whether experimental treatments could work in human patients. Our procedure aims to put forth an improved model. One that more accurately replicates the therapeutic resistance encountered in patients with diabetes or hyperlipidemia.
This animal model can be used to test new therapies for peripheral ischemia and provides a pre-clinical model that has compromised neovascularization to ischemia. After confirming a lack of response to hind paw pinch in an anesthetized four to six month old diabetic and hyperlipidemic rabbit, use a scalpel with a number 15 blade to make a four to five centimeter long incision just lateral to the right side of the trachea and use blunt dissection to expose the right common carotid artery. Use small Weitlaner retractors to open the incision and carefully isolate the carotid artery from the jugular vein and vagus nerve.
When the carotid artery has been fully separated from the nerve and jugular vein, place a 4-O silk suture at the proximal and distal ends of the exposed artery and tie off the distal end of the carotid with a surgeon's knot and four square knots. On the proximal end, use a vascular silicone tie to allow for tightening or loosening of the vessel, as needed. Next, apply approximately 0.5 milliliters of 1%lidocaine along the exposed carotid artery and nerve to promote vasodilation and reduce nerve irritation.
Administer 500 international units of heparin IV.Place a four-inch wire insertion tool into the artery. Feed a 0.014 inch by 185 centimeter guidewire through the insertion tool to the aortic bifurcation at the iliac crest in the descending aorta, and advance a three French pigtail angiographic catheter over the wire. Advance the pigtail catheter to two centimeters proximal to the aortic bifurcation at the iliac crest in the descending aorta, positioning the tip of the catheter between the seventh lumbar and the first sacral vertebra.
Test the location of the catheter by manually injecting two to four milliliters of contrast agent. Once correct positioning is confirmed, administer an intraarterial injection of 100 micrograms of nitroglycerin through the catheter to increase vasodilation. Then administer 0.8 to one milliliter of 1%lidocaine through the catheter to assist with vasodilation and flush the catheter with heparinized saline.
Attach the tubing for the automated angiographic injector to the catheter, being sure to remove any air from the line. Use the automated angiographic injector to deliver nine milliliters of contrast medium through the catheter at three milliliters per second and perform digital subtraction angiography at six frames per second. Select the serial images and alter the image of the angiogram using minus 40%setting to minimize the appearance of bone and capture a complete picture of the vessel perfusion with contrast.
To isolate the femoral artery, use a number 15 scalpel blade to make a longitudinal skin incision over the right femoral artery, making sure that the incision extends inferiorly from the inguinal ligament and ends at the area just proximal to the patella. Use blunt dissection to expose the femoral artery and open the incision with Weitlaner retractors. Apply approximately 0.5 milliliters of 1%lidocaine locally to reduce nerve irritation and promote vasodilation.
Continue the blunt dissection of the tissues to free the entire length of the femoral artery along with all of its branches, including the inferior epigastric, deep femoral, lateral circumflex and superficial epigastric arteries. Dissect along the popliteal and saphenous arteries, as well as the external iliac artery, periodically hydrating the area with saline to protect against tissue damage. Carefully separate the femoral artery from the femoral vein and nerve and place two 4-O silk sutures on the femoral artery with just enough space between the sutures to allow the artery to be severed.
Then use small Metzenbaum scissors to cut between the two ties of the ligated artery. Excise the femoral artery from where the artery bifurcates to form the saphenous and popliteal arteries, working proximately, tying off any side branches to excise the rest of the femoral artery to just below the epigastric artery. For repeat angiography, use a silastic sheet with a mounted three millimeter stainless steel ball.
To attach the stainless steel ball into the upper part of the quadriceps muscle with 4-O silk sutures. Pull the skin over the ball when it is in place. Administer an intraarterial injection of 100 micrograms of nitroglycerin through the catheter to increase vasodilation.
If needed, administer another 0.8 to one milliliter of 1%lidocaine through the catheter to assist with vasodilation and flush with heparinized saline. Use the automated angiographic injector at the same settings to inject another nine milliliters of contrast medium. Then perform another angiogram, as demonstrated.
At the end of the procedure, remove the catheter from the right artery and tie off the artery using the 4-O silk suture already in place around the artery. Then close the muscle and subcuticular layers with 4-O polydioxanone or 3-O polyglactin 910 sutures on a taper needle in a continuous suture pattern and close the skin using 4-O polydioxanone or 4-O polyglactin 910 sutures on a reverse cutting needle in a buried continuous subcuticular suture pattern. In non-diabetic animals, even with higher cholesterol, there is an increased recovery of the blood pressure in the ischemic limb and vascularity in the angiograms at the final time point compared to diabetic animals.
Histologically, changes in the muscle structure consistent with edema and ischemic damage are observed as a loss or disruption of the muscle fibers. Further, immunostaining for PECAM one and alpha smooth muscle actin can be used to identify the number and size of the vessels in the tissue sections. Other imaging methods, such as Doppler ultrasound, laser Doppler imaging, infrared thermography, and MRI can be added for further monitoring of the ischemic limb reperfusion.
With this incorporation of therapeutic resistance, our model more accurately mimics ischemia in a diseased state, advancing the assessment of new therapies for peripheral arterial disease.
