Abstract 475: Novel Gene Therapy Facilitates Critical Limb Ischemia Reperfusion in Mouse Hindlimb Gangrene Model

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Lack of a reliable animal hindlimb gangrene model limits molecular investigation of critical limb ischemia. We sought to develop a mouse hindlimb gangrene model, used to assess the efficacy of novel gene therapy. We hypothesized that priming ischemic hindlimb tissue with E-selectin/adeno-associated virus (AAV) enhances therapeutic angiogenesis and attenuates gangrene. We tested two methods to induce hindlimb gangrene. First, FVB mice underwent femoral artery ligation (FAL) to achieve critical limb ischemia. Second, FVB underwent combined FAL and administration of NG-nitro-L-arginine methyl ester (L-NAME), nitric oxide synthase inhibitor, which further reduces hindlimb perfusion. Prior to FAL and L-NAME use, gangrene-induced mice were intramuscularly administered E-selectin/AAV (treatment) or LacZ/AAV (control) to the hindlimb. Gangrene was assessed with a standardized ischemia score ranging from 0 (no gangrene) to 11 (forefoot gangrene), recorded on postoperative day (POD)’s 2,7,14. Hindlimb reperfusion using Laser doppler imaging was quantified by mean perfusion of ligated:non-ligated limb on same POD’s. Live animal Dil perfusion plus laser scanning confocal microscopy quantified limb neovascularization. Most FVB did not develop gangrene with FAL-only (n=2/8, 25% gangrene incidence). Combined FAL and L-NAME consistently induced hindlimb gangrene (n=14/14, 100% gangrene incidence). Laser doppler imaging score on POD 7 for E-selectin/AAV (n=7) and LacZ/AAV (n=7) was 0.41 vs 0.27 (P=0.071) and on POD 14 was 0.54 vs 0.29 (P=0.017). Dil perfused ligated hindlimb in E-selectin/AAV and LacZ/AAV revealed significantly higher mean neovascularization intensity score of 44 vs 21 (P=0.037). Dil perfused non-ligated limb in respective mice demonstrated mean intensity score of 50 vs 25 (P=0.006). Mean limb ischemia score on POD 2,7,14 for E-selectin/AAV and LacZ/AAV was 1.9, 2.9, 3.7 vs 2.7, 3.9, 5.3 (P=0.104). We developed a highly reliable mouse hindlimb gangrene model where E-selectin-based novel gene therapy improved limb reperfusion and neovascuclarization in critical limb ischemia. This hindlimb gangrene model can be used to further understand Redox pathways contributing to gangrene, facilitating future translational studies.