Development and characterization of rat hindlimb ischemia models mimicking peripheral arterial disease severity

Peripheral arterial disease (PAD) is a progressive atherosclerotic disorder that reduces blood flow to the lower extremities, leading to skeletal muscle degeneration, functional decline, and limb loss in advanced stages. To enable rigorous preclinical evaluation of regenerative therapies across disease stages, we developed and quantitatively characterized three rat hindlimb ischemia models representing graded arterial insufficiency: iliac artery ligation (Iliac), femoral artery ligation and excision (Femoral), and combined femoral plus popliteal artery ligation and excision (Fem/Pop). Laser Doppler perfusion imaging demonstrated an immediate reduction to approximately 60–65% of baseline in Iliac, 40–50% in Femoral, and less than 30% in Fem/Pop limbs. Perfusion recovery occurred earliest in the Iliac model, with delayed recovery in the Femoral and Fem/Pop models, while overall temporal recovery trends were comparable by mixed-effects analysis. Histological and quantitative morphometric analyses at the chronic endpoint revealed progressively severe muscle remodeling with increasing ischemic burden, including increased centrally nucleated fibers and fibrosis, most pronounced in the Fem/Pop model. Oxidative stress markers trended higher with increasing ischemia severity but did not differ significantly among models. Among the three models, Fem/Pop produced the most sustained clinical ischemia and structural remodeling, while regenerative, inflammatory, and biochemical endpoints exhibit heterogeneity at chronic stages, enabling stage-appropriate evaluation of vascular and regenerative therapeutic strategies.