The overall goal of this experiment, our hindlimb ischemia model is to light the experiment a foundation for a comprehensive assessment of the functional, histologic and molecular effects of putative angiogenic agents. This protocol fields a standard for the testing of possible angiogenic therapies in a small animal model with the potential for future applications in the clinical context. As revascularization is not suitable in twenty to thirty percent of critical ischemia patients Therapeutic angiogenesis has emerged as a novel strategy for improving blood profusion at the ischemic site.
This experimental protocol could provide a comprehensive understanding of the mechanisms by which angiogenic therapies exert their effects in the measure of their efficacy at each of their outcomes. After confirming a lack of response to toe pinch in a 22 week old C57 black six female mouse, shave the hair from the right hindlimb and secure the animal in the dorsal to cubitus position on a heated pad. Place the animal under a dissecting microscope.
And use a number eleven surgical scalpel blade to make a one centimeter skin incision over the thigh of the right hindlimb. Blunt dissect the subcutaneous fat to expose the neurovascular bundle. And use pointed forceps, spring scissors, and an ophthalmic needle holder to open the membraneous ileofemoral sheath to expose the distal external iliac and femoral vessels.
After dissecting and separating the artery and vein from the nerve, use seven O nonabsorbable polypropylene sutures to ligate the distal femoral artery and vein and distal external iliac artery and vein. The identification of the distal external iliac artery and the ligation and excision of the distal external iliac and femoral artery and veins are critical for the success of the procedure. Use the spring scissors to transect the segment of the iliofemoral artery and veins between the distal and proximal knots.
And close the incision with an absorbable suture. Then, inter-peritoneally deliver antisedative solution into the animal and return the mouse to its home cage with monitoring until full recovery. To harvest the gastrocnemius muscles, remove the skin from both hindlimbs.
And use the cotton swab to separate the skin from the muscles. Holding the achilles tendon, use the spring scissors to separate the muscle from the tibia and fibula up to the knee joint. And separate the biceps femoris from the gastrocnemius muscle.
Use the spring scissors to cut the insertions of the gastrocnemius muscle at the knee joint level. And use 10%trigasanth to position the harvested muscles in a transverse orientation on a small cork disk. Then, snap freeze the specimens in liquid nitrogen cooled isopentane for minus 80 degrees Celsius storage.
For capillary microdissection laser capture, after obtaining 12 micrometer sections of the muscle specimens, fix the tissues to the slides in 50 milliliters of cooled pure acetone for five minutes at minus twenty degrees Celsius. After air drying and circle each specimen with a hydrophobic pen. And rehydrate the samples with two molar sodium chloride in PBS at four degrees Celsius.
At the end of the re-hydration, label the samples with the appropriate primary antibody of interest at four degrees Celsius overnight. Followed by two three minute washes in fresh two molar sodium chloride in PBS per wash. After the second wash, label the sections with an appropriate secondary antibody for thirty minutes at four degrees celsius followed by two washes in sodium chloride in PBS as demonstrated.
Next, label the samples with avidin conjugated peroxidase complex for thirty minutes at four degrees celsius. Followed by a wash and five minutes of labeling with DAB peroxidase substrate. At the end of the incubation, dehydrate the tissues in sequential ice cold 90%ethanol and 100%ethanol emersions.
Then, use a later microdissection system, equipped with a pulse to solid state 355 nanometer laser to dissect 10, 000 capillaries per mouse and catapult the dissect capillaries into a microfuge tube adhesive cap. As illustrated, a complete loss of hindlimb perfusion is observed immediately after hindlimb ischemia induction. Quantitative evaluation of blood flow, expressed as a ratio of ISC to NISC limb, demonstrated significantly enhanced limb blood perfusion in mice exposed to the pro-angiogenic stimulus at days 15 and 45 post-HLI.
The quantification of CD31 positive capillaries in histological sections of gastrocnemius muscle tissue sections shows that the capillary density is greater in the ischemic versus the non-ischemic limb. As expected. But this effect is further amplified after treatment with the pro-angiogenic agent.
The collateral vessel density also consistently increases in the ischemic limb and this increase is significantly higher after exposure with the pro-angiogenic stimulus. Reverse transcriptase polymerase chain reaction analysis of pro-angiogenic gene expression in CD31 positive endothelial cells demonstrates a clear variation in gene expression between mice exposed or not to the pro-angiogenic agent. It is important to use an ophthalmic needle older to open the membraneous iliofemoral sheath to avoid tearing of the artery and vein during the vessel dissection.
The vessel responds to hindlimb ischemia in the readily evaluated by laser doppler based perfusion measurements and the histological quantification of the capillary vessel density allows us to evaluate the angiogenesis.
