Toward a mouse model of hind limb ischemia to test therapeutic angiogenesis

IntroductionSeveral clinical trials are currently evaluating stem cell therapy for patients with critical limb ischemia that have no other surgical or endovascular options for revascularization. However, these trials are conducted with different protocols, including use of different stem cell populations and different injection protocols, providing little means to compare trials and guide therapy. Accordingly, we developed a murine model of severe ischemia to allow methodic testing of relevant clinical parameters.MethodsHigh femoral artery ligation and total excision of the superficial femoral artery was performed on C57BL/6 mice. Mononuclear cells (MNCs) were isolated from the bone marrow of donor mice, characterized using fluorescence-activated cell sorting, and injected (5 × 105 to 2 × 106) into the semimembranosus (proximal) or gastrocnemius (distal) muscle. Vascular and functional outcomes were measured using invasive Doppler imaging, laser Doppler perfusion imaging, and the Tarlov and ischemia scores. Histologic analysis included quantification of muscle fiber area and number as well as capillary density.ResultsBlood flow and functional outcomes were improved in MNC-treated mice compared with controls over 28 days (flow: P < .0001; Tarlov: P = .0004; ischemia score: P = .0002). MNC-treated mice also showed greater gastrocnemius fiber area (P = .0053) and increased capillary density (P = .0004). Dose–response analysis showed increased angiogenesis and gastrocnemius fiber area but no changes in macroscopic vascular flow or functional scores. Overall functional outcomes in mice injected proximally to the ischemic area were similar to mice injected more distally, but muscle flow, capillary density, and gastrocnemius fiber area were increased (P < .05).ConclusionsHigh femoral ligation with complete excision of the superficial femoral artery is a reliable model of severe hind limb ischemia in C57BL/6 mice that shows a response to MNC treatment for functional and vascular outcomes. A dose response to the injection of MNCs appears to be present, at least microscopically, suggesting that an optimal cell number for stem cell therapy exists and that preclinical testing needs to be performed to optimally guide human trials. Injection of MNCs proximal to the site of ischemia may provide different outcomes compared with distal injection and warrants additional study.

Clinical RelevanceDespite advances in surgical and endovascular technique, the options for many patients with critical limb ischemia remain limited. The injection of bone marrow-derived stem cells into ischemic tissue has been identified as a potential therapeutic alternative. Trial results have been consistently positive, yet the methods under which cell therapy is administered remain highly variable. The rationale for our study was to design a murine hind limb ischemia model capable of testing cell therapy parameters, such as optimal cell population, site of administration, and dose, and to use this preclinical knowledge to better guide and compare human trials.