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A Novel Model of Hind Limb Ischemia to Test Human Therapeutic Angiogenesis
*Robert A Brenes1,2, *Mackenzie Bear2, *Caroline Jadlowiec2, *Peter Hashim2, Juan A Sanchez1, Alan Dardik2,3
1The Stanley J. Dudrick Department of Surgery, Saint Mary's Hospital, Waterbury, CT;2Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, New Haven, CT;3Department of Surgery, Yale University School of Medicine, New Haven, CT

To develop a model of limb ischemia in mice and answer clinical questions with regards to stem cell based therapy in treating critical limb ischemia in humans.
C57BL/6 male mice (age 6-8 weeks) underwent unilateral high femoral artery ligation and excision; the contralateral leg was the control limb. Mice were divided into two groups, control and bone marrow mononuclear cell (BM-MNC) injected.
Yale University School of Medicine, Interdepartmental Program in Vascular Biology and Therapeutics
BM-MNC were isolated from donor mice and characterized using fluorescence activated cell sorting and injected into the gastrocnemius muscle in one group of mice. The other group (control) was injected with an equal volume of medium without cells.
Main Outcome Measures
Muscle blood flow was measured with “deep probe” laser Doppler. Functional Tarlov and ischemia scales were recorded at intervals between preoperatively and 4 weeks postoperatively. Mice were sacrificed at 1, 2 and 4 weeks for histological analysis.
Blood flow was significantly higher in MNC injected mice as compared to control (p<0.0001). Tarlov scores were statistically higher throughout the first week postoperatively in MNC mice (p=0.0064). Ischemia scores were significantly higher in MNC mice (p=0.0269). Average number of muscle fibers was lower, and fiber area was higher in MNC mice at all time points (p<0.01, n=3).
High femoral ligation and excision is a reproducible model of limb ischemia in C57BL/6 mice that shows response to BM-MNC injection. These studies suggest several parameters of human trials can be tested in a small animal, cost-effective manner, allowing optimization of human trial parameters.

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