A Modified Surgical Model of Hind Limb Ischemia in ApoE^-/- Mice using a Miniature Incision

What we introduced today is a simplified modified and surgically efficient method, to establish hind limb Ischemia in ApoE mice. This method can be implemented in any animal facility. The main advantage of synovial method is a low invasiveness, using an incision of less than five millimeters.

The method has a learning hof. We therefore, advise to practice on microsurgical models before working on the animals, which are going to be included in the final analysis. The method will be demonstrated by Kaixuan Yan, a doctor of medicine and the student from my lab.

Begin by preparing the required equipment and tools for the surgery. After administering the anesthesia to the mouse, apply that ointment on the eyes to prevent dryness. Then place the mouse on a heating pad to keep the core body temperature at approximately 37 degrees Celsius.

Using a cotton swab and hair removal cream, carefully remove hair from the hind limb skin on the right side. Lay the mouse in the supine position on the heating pad under a dissecting microscope. Use alcohol to disinfect the skin and wipe with the cotton away from the plant side of incision.

Using the pointed forceps and surgical scissors make an incision approximately three to four millimeters in the middle of the inguinal region. Carefully remove the subcutaneous fat tissue with the help of fine pointed forceps to expose the proximal femoral neurovascular bundle. Using a cotton swab moistened with saline, carefully move the femoral artery away from the femoral nerve and femoral vein.

Then using the tip of a 7.0 suture, carefully pierce the membrane of the femoral sheath. Then pass to 7-0 absorbable sutures through the proximal femoral artery and make double knots using the springs scissors. Afterwards transect the femoral artery between the two ties.

Pass the 6-0 absorbable suture through the lower edge of the incision and gently drag the incision to the region of the right side of the knee of the hind limb to expose the distal femoral artery. Carefully move the subcutaneous tissue aside to expose the neurovascular bundle. Dissect the femoral artery from the femoral vein and femoral nerve, and use cotton to protect the nerve.

Then using the tip of a 7.0 suture, pierce the membrane of the femoral sheath. Pass two 7-0 absorbable sutures through the distal femoral artery and make double knots using the spring scissors to transect the femoral artery between two ties. Afterward, stitch the incision using the 6-0 absorbable sutures.

Place the mouse on a heating pad in a clean cage and continue to monitor its vital parameters until recovery. Determine the success of double ligation of the femoral artery or DLFA on the next day via MRI. Before the DLFA procedure, weights of mice ranged from 29.6 to 38.0 grams.

Seven days after the DLFA surgery, the weights ranged from 26.5 to 34.1 grams, significantly lower than the pre-DLFA weights. The proximal and distal regions of the right FA showed no perfusion. The Tarlov scale results were significantly decreased one day after the surgery.

Although they slowly increased over the following days, they were still lower than baseline until day seven. The paws of the ischemic hind limbs in seven mice were unable to stretch naturally compared to the contralateral side. In addition, four of the mice exhibited slight discoloration of the paws compared to the contralateral side.

H&E staining of the right GM muscle revealed myofibers that exhibited irregular ischemic necrosis, where proliferating satellite cells had replaced the necrotic myofibers and were distributed in a mass or with a regular dispersion. Myofibers exhibiting inflammatory infiltration by multinucleated macrophages had lost their normal morphological characteristics, with very few regenerated myofibers being present. Transverse sections of these regenerated myofibers were round.

The cytoplasm was stained red and one small nucleus or multiple nuclei were located at the center. However, this kind of inflammatory pattern was not observed in the left GM.CD31 antibody staining performed to observe microvascular density, indicated that ischemic hind limbs exhibited significantly more microvascular density than the non ischemic side. The most important thing is dissection of femoral artery.

The operator needs to be experienced in microsurgical techniques, as well as be familiar with mouse hindlimb anatomy. In principle, this method could also be used to study Ischemia-reperfusion injury in the leg. Hereby, artery can be occluded with a soft vessel clamp before allowing reperfusion.