Murine Model of Hindlimb Ischemia

In the United States peripheral arterial disease, PAD affects about 10 million individuals in particular, smokers, diabetics, and people over the age of 70 years. The overall goal of this surgery is to induce hind limb ischemia by ligating the proximal and distal segments of the femoral artery. The procedure begins with anesthetizing shaving and positioning the mouse on the surgical table in the supine position.

Then an incision of the skin is made from the knee towards the medial thigh, exposing the underlying femoral artery. Next, the femoral artery is then carefully separated from the femoral vein. Double knots are made at the distal ligation site as well as the proximal ligation site.

The femoral artery segment is then excised. The incision site is closed by sutures and the animal is allowed to recover from anesthesia. To confirm the induction of hind limb ischemia, the anesthetized mouse is placed in the supine position.

Then both hind limbs are scanned by the laser doppler. The image is then saved and assessed for perfusion. Hi, I'm John Cook.

I'm professor of medicine here in the division of cardiovascular medicine at Stanford University. Our laboratory is interested in angiogenesis and vascular regeneration. In this video, two of my postdoctoral fellows, Dr.Nan Wong and Hir Niyama, will introduce you to the Muren hind limb Ischemia preparation, which is an important model for peripheral arterial disease and frequently used in our laboratory to assess the effects of growth factors and cell therapy on angiogenesis.

We use these procedures in our laboratory to study the effect of stem cell derived endothelial cells for vascular repair. So let's get start. The surgical tools needed for this operation include fine pointed forceps, pointed forceps, spring scissors, surgical scissors, needle holder, and retractor.

We make our own retractor using a paperclip because it is smaller than commercially available. Retractors sterilize these tools prior to surgery with a hot bead sterilizer, a cautery tool and sterile fine pointed cotton swabs will also be needed for this surgery. When the tools are ready, place the mouse into the anesthesia induction chamber containing one to 3%iso fluorine in a hundred percent oxygen at a flow rate of one liter per minute.

Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber. Then place the animal in the supine position onto the pre-operation table and connect it to a continuous flow of isoflurane.

Using an electric shaver, remove the hair from the hind limb. Apply hair removal cream to thoroughly remove hair. After shaving the mouse, extend and secure the hind limb with a piece of tape.

Once the hind limb is secure, wipe the skin with three alternating Betadine and alcohol scrubs. Using fine forceps and surgical scissors, make an incision of the skin approximately one centimeter long from the knee towards the medial thigh using phosphate buffered saline. PBS moist and defined pointed cotton swabs.

Gently brush away subcutaneous fat tissue surrounding the thigh muscle. Next, apply the cautery transversely to in sizes and dissect through the subcutaneous fat tissue to reveal the underlying femoral artery. Next, use a retractor to open the wound and to obtain a better view of the lower extremity vasculature.

Using fine forceps in a fine pointed cotton swab, gently pierced through the membranous femoral sheath to expose the neurovascular bundle. Then using a clean set of fine forceps and cotton swab, dissect and separate the femoral artery from the femoral vein and nerve at the proximal location near the groin. After the dissection pass a strand of seven oh silk suture underneath the proximal end of the femoral artery.

Occlude the proximal femoral artery using double knots. Place the tie on the vessel as proximal in the wound as possible in order to leave length for the second tie and an intervening segment that will be transected. Separate the femoral artery from the femoral vein at the distal location close to the knee.

Pass a strand of seven oh suture underneath the distal end of the femoral artery proximal to the popliteal artery. Occlude the vessel using double knots. Occlude the distal femoral artery with a second set of double knots just proximal to the first set of knots.

This second set of sutures will be used for gripping the artery during the transaction procedure. Similarly, for gripping purposes, occlude the proximal fal artery with the second set of double knots just distal to the first set of knots. Transect the segment of femoral artery between the distal and proximal knots with a fine pointed cotton swab and a pair of spring scissors.

Use caution not to pierce the femoral vein wall. Remove the retractor and close the incision using five oh quil sutures. Once the incision is closed, return the animal to the recovery cage and monitor until awake After the animal has recovered for one hour, proceed with the laser doppler blood perfusion.

Step in order to confirm the ischemia induction. To begin the laser doppler perfusion step, place the mouse into the anesthesia induction chamber containing one to 3%iso fluorine in a hundred percent oxygen at a flow rate of one liter per minute. Leave the mouse in the induction chamber until it is unresponsive to external stimuli.

Then remove the animal from the induction chamber and place it on the pre-operation table connected to continuous flow of isof fluorine. Then remove the hair from the hind limb using an electric shaver followed by hair removal cream as necessary. After removing the hair, place the animal on a 37 degree Celsius heated surface for five minutes under continuous flow of iso fluorine.

After five minutes, place the animal in the supine position on a non-reflective light absorbing surface, such as green colored cloth connected to a continuous flow of iso fluorine. Extend the hind limbs. Next, turn on the laser doppler imager and the acquisition software and initialize the software.

Specify the size of the field of view and resolution. It is best to keep the field of view and pixel density consistent between animals. In order to make future analysis easier, open a new file.

Press start to begin acquiring the image data. When prompted, specify the distance of the animal to the imager. The laser will show the borders of the field of view and then start acquiring data.

After acquisition is complete, the image will begin to show a range of colors that are indicative of the level of blood profusion to the legs. The colors can be set to a specific profusion range. To better compare data between animals when the data acquisition is complete, save the file, then return the animal to the recovery cage and monitor the animal continuously until awake.

To analyze the data, use the threshold adjustment in the analysis software to subtract any background noise. A threshold of 0.2 is usually a reasonable value. Select two regions of interest or ROIs that cover each hind limb area.

A variety of landmarks can be used to standardize the ROI between limbs and animals, then determine the mean perfusion and variability in the ROI. The perfusion difference and perfusion ratio between the ischemic limb and control limb can then be easily determined. This procedure can be repeated to follow changes in hind limb perfusion.

Over time at a desired time point, the animal can be euthanized for assessment of tissue function. In comparison to perfusion image data, a representative diagram of the hind limb after femoral artery explanation is shown here. The sutures mark the locations of the proximal and distal ligation sites.

When the surgery is performed carefully, there is minimal bleeding and the surgery can be completed in less than one hour. And here are results of the laser doppler perfusion image analysis. The image on the left shows the profusion of both limbs prior to ischemic induction.

While the image on the right shows the profusion of both limbs. After the left hin limb has undergone ischemic induction. The color intensity in the image indicates the amount of blood flow.

In comparison to the control limb, the ischemic limb shows very little color, which suggests a dramatic reduction in blood flow. Using this data, the software can quantify the mean perfusion in ischemic and control limb to determine the perfusion difference and perfusion ratio. Therefore, these results confirm the induction of ischemia to the hind limb.

We've just shown you how to induce unilateral hind limb ischemia in the mouse model and confirm the induction of ischemia by laser Doppler blood profusion. The hind limb ischemia model with laser doppler imaging analysis is an excellent system for studying vascular pathologies and for assessing therapeutic candidates. This procedure can be followed by the transplantation and non-invasive tracking of cells, which is demonstrated in another jve video by Huang et al.

When doing this procedures, it's important to remember to use caution not to pierce the femoral vein wall when excising the femoral artery with sprain scissors. Also use similar ligation site for all animals in the experiment for consistency of the highly ischemia model. So that's it.

Thanks for watching and good luck with your experiments.