Methods for Acute and Subacute Murine Hindlimb Ischemia

The overall goal of this protocol is to model human peripheral artery disease by the placement of sutures or ameroid constrictors on the mouse femoral artery for the induction of acute or subacute hindlimb ischemia, respectively. Peripheral artery disease is an important clinical problem, and these techniques using animal models have been developed to better represent the human disease. The primary advantage of these techniques is that the severity and timing of the onset of limb ischemia can be controlled by the investigator.

Demonstrating the procedure will be Mike Padgett, a technician from my laboratory. One to two days before the procedure, confirm that the mouse is fully sedated by lack of response to toe pinch, and apply eye ointment. Next, use a small electric trimmer to shave the hair from both hindlimbs.

When all of the hair has been trimmed, apply pre-warmed hair removal cream. After one minute, gently wipe away the cream with a moistened gauze pad. Then place the mouse in an empty paper towel lined recovery cage until it is able to maintain sternal recumbency.

On the day of the surgery, place the fully sedated mouse under a technic stereo dissection microscope in the supine position, and secure both legs with surgical tape. If a temperature controlled heating pad is used, attach the temperature probe, and secure the probe to the base of the surgical platform with surgical tape. Next, use three alternating povidone iodine and alcohol wipes to clean the surgical site, and cover the animal with a sterile surgical drape with a hole at the incision site.

Using a scalpel, make an initial incision along the center of the medial thigh from the knee toward the abdomen. Then use fine scissors to lengthen the incision to approximately one centimeter. Using forceps, open the incision to expose the membrane covering the inguinal fat tissue.

Then use closed forceps to pierce through the membrane, into the space between the fat tissue and the abdomen. Gently release the pressure on the forceps to separate the fat tissue from the abdominal muscles, exposing the neurovascular bundle. It is crucial that the major blood vessels and their branches are properly identified prior to the dissection of the neurovascular bundle.

Variations in the location of the constrictors or the sites of ligation can significantly alter the severity of the ischemia, and lead to a substantial variability in the outcomes. Insert a retractor into the incision, and pull the abdominal tissue toward the animal's head to expose the proximal ameroid constrictor site, which is just proximal to the lateral circumflex femoral artery. Then insert one medial and one lateral retractor into the distal part of the incision to widen the surgical field, pulling the inguinal fat tissue toward the foot and away from the surgical site.

The proximal and superficial caudal branches of the femoral artery should be easily observed. The lateral circumflex femoral artery is located about five millimeters proximal to the caudal branches, and may be obscured by membrane. Now gently slide the tip of one forceps under the membrane that binds the vessels together to insert 1/2 of the tip between the vein and the artery.

Then closet he forceps and gently tear away the membrane. When all of the membrane has been removed, insert the tip of the closed forceps between the vein and the artery, and release the pressure on the forceps to create a gap between the vessels. For subacute limb ischemia, slide the tip of the forceps under the femoral artery to isolate the vessel from the neurovascular bundle.

Use a second set of angled forceps to grip the edge of the constrictor, and guide the constrictor under the femoral artery. Then lay the artery into the constrictor slot. Next, position the distal constrictor on the femoral artery immediately proximal to where the artery bifurcates into the popliteal and saphenous arteries, and place the femoral artery into the constrictor slot.

For acute limb ischemia, thread a 7-0 suture under the artery at the same position as the proximal constrictor, and ligate the artery. Then tie a second ligature about one millimeter distal to the first, and use spring scissors to transect the artery between the two ligatures. For the distal arterial transection, place the ligatures about one millimeter apart, just proximal to where the femoral artery bifurcates into the popliteal and saphenous arteries.

Then close the incision with interrupted five-0 vicryl sutures, and move the mouse to a 37 degree Celsius heating pad beneath a laser doppler perfusion imager. Now turn on the imager, and launch the image capture software. Click the New Single Image icon to open the scanner setup window, and set the scan size to large, and the scan speed to four milliseconds per pixel.

Change the X and Y values under the Scan Area units panel to the appropriate values, then click the Video and Distance tab, and arrange both of the hindlimbs within the scan area. Click Auto Distance, then click Next to open the Subject Details window, and enter the subject information. Click Next to move on to the scanning window.

Then click the Start Measurement button to open the Confirm or Override Scan Distance dialog box, and click OK to begin the scanning process. When the scanning is complete, move the mouse to an empty recovery cage with monitoring until it is fully recovered. In addition to the variations inherent in animal studies, other factors can introduce variability within the laser doppler perfusion imaging, including the body temperature, and the position of the animal.

The subacute hindlimb ischemia model is dependent on the quality of the ameroid constrictors, which can vary widely within a given batch. Laser doppler perfusion imaging should be performed immediately after the procedure while the animal is still anesthetized to demonstrate the effects of the surgery, and to establish a baseline post-operative level of perfusion. A complete loss of hindlimb perfusion should be observed after the induction of acute limb ischemia, whereas only a mild decrease in perfusion is typically observed after the ameroid constrictor placement in the subacute model.

These procedures allow exploration of the effects of varying degrees of ischemia on vascular remodeling and skeletal muscle regeneration in the mouse as a model of human peripheral artery disease. Once mastered, these procedures can be completed in about 15 minutes if they are performed correctly. While attempting these surgeries, it is important to remember to continually monitor the body temperature and the adequacy of the anesthesia in the animal.

By adjusting the placement of the ligatures or the constrictors, the severity of the ischemic injury can be modified, which can be an important variable depending on the animal's genetic background, or the presence of cardiovascular risk factors. After watching this video, you should have a good understanding of how to induce both acute and subacute hindlimb ischemia in the mouse, and to define the vascular anatomy to ensure a proper outcome and reproducibility of the procedure.