Establishment of a two-stage limb ischemia in diabetic rats

Background: This study aimed to establish a clinically relevant animal model for peripheral arterial disease (PAD) that better replicates the complexity observed in human patients. Materials and Methods: Thirty male rats were randomly assigned into the sham (SM), femoral artery resection (FE), constrictor-induced ischemia (CI), two-stage ischemia (TS), or diabetic two-stage ischemia (DT) groups. In the FE group, rats underwent femoral artery resection, whereas the SM group had sham surgery. The CI group received progressive ischemia using two ameroid constrictors, and the TS and DT groups underwent a two-stage ischemia procedure involving initial gradual narrowing with two ameroid constrictors and subsequent femoral artery resection in healthy and diabetic rats, respectively. Perfusion evaluation and functional assessment were conducted at postoperative days 14, 28, and 42. On day 42, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) protein expression were measured, along with histological examination and immunofluorescence analysis. Results: Motor function deficits and reduced limb reperfusion were most prominent in the TS and DT groups on days 28 and 42 (P < 0.05), exacerbated by type 2 diabetes. Gastrocnemius exhibited upregulated HIF-1α and VEGF protein expression, as well as increased capillary density in response to ischemia. However, the DT group showed significantly lower protein expression and capillary density, along with more severe structural damage compared to other groups (P < 0.05). Conclusion: A clinically relevant rat model of PAD was established by implementing a two-stage ischemia procedure involving initial progressive narrowing and subsequent femoral artery excision in the context of diabetes.