Age and Sex Influence in a Murine Hindlimb Ischemia Model: Implications for Cell-Based Therapies Targeting Critical Limb Ischemia

Purpose: Critical limb ischemia (CLI), a severe complication of peripheral artery disease, presents a significant clinical challenge due to limited treatment options. Existing preclinical models of CLI frequently neglect biological variables such as age and sex, limiting their capacity to effectively assess novel therapies. This study seeks to address the deficiencies in the widely used murine hindlimb ischemia model for CLI by examining the influence of age and sex on recovery outcomes. Methods: Foxn1nu mice with varying age and biological sex were subjected to femoral artery ligation to establish unilateral hindlimb ischemia. Hindlimb perfusion was measured on multiple post-surgery days using Laser Doppler Imaging. Motor function and tissue damage were recorded through gross examination. Gastrocnemius muscles, collected on postsurgical day 36, underwent vascular and muscular histological analysis. All parameters were compared across age and sex groups. Results: Mature adult mice (12–18 weeks old) consistently exhibited significantly heightened motor function impairment and tissue loss, with significantly slower perfusion recovery, in comparison to their younger counterparts (6–12 weeks old). Sexual dimorphism manifested, with females experiencing more pronounced symptoms than males. Histological analysis revealed skeletal muscle pathology in mature adults, similar to the clinical pathology of CLI, including muscle fiber loss, fibrosis, and adipose-like tissue infiltration. Animals of older age and female biological sex exhibited slower vascular regeneration. Conclusion: Age and biological sex significantly influence tissue recovery in the murine hindlimb ischemia model. To better evaluate pro-regenerative therapeutics for CLI, the utilization of older, mixed-sex mice in preclinical studies is recommended. Lay Summary: This study underscores the critical impact of age and biological sex on tissue response in the murine hindlimb ischemia model. When evaluating both male and female mice across two distinct age groups, the older female group is significantly associated with worse outcomes in blood perfusion recovery, ambulatory function, tissue loss, and muscular and vascular characteristics. These findings strongly advocate for the use of mature adult mice with mixed biological sex in preclinical investigations. This comprehensive characterization and validation of a widely employed preclinical model significantly enhances the outcome’s credibility while evaluating pro-regenerative therapies for the treatment of critical limb ischemia.