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Home > Medical Professionals > Research Initiatives Conference > Poster Abstracts Accepted > Tihonov Murine Hindlimb Ischemia Model

Murine Hindlimb Ischemia Model for Studying Angiogenesis: Calf vs. Thigh, It Makes a Difference

Nikita Tihonov, Sherwin Park, Mark J Hobeika, Luis Chiriboga, Herman Yee, Elizabeth R Gagne, Peter Brooks, Paul J Gagne
New York University, New York, NY

Objectives: Chronic limb threatening ischemia is marked by variable collateral (i.e. arteriogenesis) formation and inadequate angiogenesis. The murine hindlimb ischemia model (MHIM) is frequently used for studying angiogenesis. After high ligation and division of the hindlimb femoral artery, angiogenesis occurs in the calf in response to tissue ischemia and arteriogenesis occurs in the thigh in response to altered intravascular pressure and shear stress. Surprisingly, investigators report harvesting muscle indiscriminately from calf and/or thigh for experiments aimed at studying angiogenesis. These experiments were designed to evaluate the hypothesis that significant physiologic, biochemical, and histologic differences exist between calf and thigh skeletal muscle in the MHIM that may affect experiments aimed at studying angiogenesis.
Methods: Wild-type mice (SvEv129) underwent proximal, unilateral femoral artery ligation/division. Laser Doppler imaging (LDI) (physiologic assessment) was used to evaluate thigh and calf reperfusion (n=5). Mice were sacrificed on day 0 (pre-ischemia) and days 3, 7, and 14 (n=10/day) (post-ischemia). Calf and thigh muscle from ischemic and nonischemic hindlimbs was harvested and analyzed. The type-IV collagenase, MMP-2, was measured using ELISA (n=10) (biochemical assessment). Immunohistochemical staining for the cryptic collagen epitope, HU177, and the endothelial cell marker, CD-31, was digitally recorded and quantitated (n=6 animals/day) (histologic assessment).
Results: LDI (Fig. 1) revealed significant regional differences (p<0.05) in blood flow in the ischemic limb at all time points. Severe and prolonged ischemia occured in the calf but not the thigh. Total MMP-2 levels were increased (p<0.05) in the thigh and calf after ischemia (day 3), but remain elevated thereafter only in the calf (day 7, p<0.05). Though active MMP-2 levels were elevated in the calf and thigh at day 3 (p<0.05), the calf levels were 3 fold higher than the thigh levels (p<0.05). HU177 exposure was 2 fold higher in the calf than the thigh at day 7 (p=0.04) and remained higher (day 14, p<0.05) (Fig. 2). Endothelial cell counts increased slightly and equally in the thigh and calf at day 3, but become markedly increased in the calf (2.5 fold) at day 7 and 14 (p<0.05) (Fig.3).
Conclusion: Ischemia in the thigh is less severe than in the calf, and is largely resolved within 3 days. Revascularization is largely through arteriogenesis, as reported in the literature, and as suggested by the minimal observed increase in endothelial cells. In contrast, in the calf where significant ischemia persists, MMP-2 levels and HU177 exposure remain elevated and are associated with elevated endothelial cells counts, indicative of angiogenesis. These differences suggest that with the MHIM, muscle harvested from the calf is most appropriate for studying angiogenesis.