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Objective
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.
Design
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.
Setting
Yale University School of Medicine, Interdepartmental Program in Vascular Biology and Therapeutics
Interventions
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.
Results
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).
Conclusions
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.