High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

This protocol presents how to create a mouse foot pad gangrene model using administration of mass inhibitor and femoral artery coagulation. DiI perfusion then allows for high-resolution visualization of the foot pad vasculature. This model is clinically relevant and can be used for preclinical drug testing and to study limb ischemia.

High-resolution visualization of vasculature offers a technically facile means of assessing neovascularization. This foot pad gangrene model has important applications in preclinical research of peripheral arterial disease and critical limb ischemia. DiI perfusion is a useful tool to study neovascularization in animal models.

Demonstrating the procedure will be Antoine Ribieras, of surgical resident, and Yulexi Ortiz, a research associate from my laboratory. First, make DiI working solution by adding 200 microliters of DiI stock solution to 10 milliliters of the working diluent solution immediately before use. Shake by hand to mix well.

Connect two or three three-way stopcocks and a 25 gauge butterfly needle in series. Prepare 10 milliliter syringes with four milliliters of PBS, 10 milliliters of DiI solution, and 10 milliliters of 10%neutral buffered formalin. Connect the syringe with formalin to the proximal inflow port and inject the solution to flush air from the line.

Then turn the stopcock to close the port. Repeat the same procedure, connecting the syringes with DiI and then PBS to the middle and distal inflow ports respectively. Post-euthanasia, place the animal in a supine position on an absorbent pad and secure axillae and lower extremities with needles.

Using scissors, make a transverse incision to open the abdominal cavity, then expose and divide the left and right diaphragm to access the thoracic cavity. Cut the chest wall on either side of the sternum from the lower ribs to the first or second ribs. Use a hemostat to grab the lower end of the sternum and reflect it towards the animal's head to expose the thoracic cavity.

Identify the left ventricle, which appears lighter in color than the right. Gently grasp the heart with blunt forceps and insert the butterfly needle into the left ventricle. Use scissors or an 18 gauge needle to puncture the right atrium, allowing blood and perfusion solutions to return to the heart to drain.

Open the port of the syringe with PBS and manually inject two to four milliliters at a rate of one to two milliliters per minute for one to two minutes to flush blood from the vascular system. Ensure successful perfusion by observing bleeding from the right atrium. After injection, close the port of the PBS syringe.

Open the port of the syringe with DiI and inject five to 10 milliliters at a rate of one to two milliliters per minute for five minutes. After injection, close the port of the DiI syringe and wait for two minutes to allow incorporation of the dye before injection of fixative. Open the port of the syringe with formalin and inject five to 10 milliliters at a rate of one to two milliliters per minute for five minutes.

After injection, remove the needle from the left ventricle. Using heavy scissors, dislocate the tibia at the ankle, completely separating the left and right feet from the lower legs. Place harvested feet in a 12 well plate with one to two milliliters of 10%formalin solution.

Wrap the plate with foil and store at four degrees Celsius overnight. The next day, replace the fixative solution in a 12 well plate with one to two milliliters of PBS per well. Make a longitudinal incision with a scalpel on the plantar and dorsal part of the foot.

Then using toothed forceps and a small hemostat, carefully remove all skin from the foot and digits, not damaging the underlying soft tissues. To mount tissues, individually place feet between two glass microscope slides with a foam biopsy pad folded over itself at each end. Use two small binder clips to compress the glass slides together at each end.

Alternatively, return the foot pad to a 12 well plate with one to two milliliters of PBS. After covering it with foil, store it at four degrees Celsius to maintain fluorescence for up to one month. Turn on the imaging system, start the acquisition software, and use a low magnification or low numeric aperture objective to capture images.

Click on yes to the activate stage dialog box. Activate the 561 nanometer laser in the configuration tab. On the main screen, activate a visible beam path by clicking on the visible button.

Set a detector to the 570 to 600 nanometer range by clicking on the corresponding active checkbox. Select the tile scan icon in the acquisition tab and set the desired resolution. Position dry mounted tissue sample compressed between glass slides on the microscope stage and bring tissue into focus.

Navigate to one corner of the sample. Under the tile scan menu, click on the mark position button. Navigate to the opposite corner and click on the mark position button again setting the scanning boundaries.

To set the depth of the Z-stack, click on the live button. Navigate to the center of the sample and use the z-axis knob to scroll through to the bottom of the sample. In the acquisition tab under the Z-stack menu, click on the begin button.

Scroll through to the top of the sample and click on the end button. Click on the z-step size and set to the desired value. Then click on start to begin image acquisition.

The figure shows the variation in tissue loss that can be expected from this model one week after surgery with FABER scores recorded in the bottom right-hand corner of each image. Reconstruction microscopy images reveal normal vascular anatomy in non-ligated control foot pad compared with severely diminished perfusion to the foot pad of ligated hindlimb five days after surgery. 20 days after surgery, perfusion to the foot pad has significantly improved, although not to the extent of non-ligated control.

The video shows that surface rendering was then created using the animation functionality. Flush all air bubbles from the perfusion assembly. If the lungs expand and turn pink during perfusion, the needle has penetrated the right ventricle and should be retracted slightly.

Limb tissues can be used for immunostaining to assess muscle necrosis, tissue regeneration, vessel density, and recruitment of progenitor cells, and other tissue repair cells. The technique of DiI perfusion in this murine gangrene model has been used in studies of gene and cell therapies. The research aims to improve perfusion in limbs of patients with critical limb ischemia.