Optimized Murine Hindlimb Ischemia Model to Assess Vascular Regeneration in Peripheral Artery Disease

Introduction:: Lower extremity peripheral arterial disease (PAD), characterized by atherosclerotic obstructions of blood flow in the lower limbs, currently affects 200 million patients worldwide. Ischemia in PAD causes lower extremity skeletal muscle pathology, including reduced muscle area, increased fatty infiltration and fibrosis, and metabolic and cellular abnormalities, leading to impaired walking performance and loss of mobility. To address this, stem cell-based therapeutics have been proposed as a potential strategy to regenerate vascular tissues and restore blood flow. A commonly used animal model for PAD to evaluate these therapeutics is the murine acute hindlimb ischemia model. However, inconsistent outcomes in clinical trials of cellular therapeutics developed based on the commonly used model may indicate limitations in its ability to accurately reflect the clinical condition of PAD. This study aims to optimize the murine hindlimb ischemia model by examining the impact of variables such as age and sex on tissue regeneration, while also evaluating vascular and muscle pathologies in the model. By accounting for these factors, we hope to improve the translational potential of rodent findings to preclinical evaluations of cell-based regenerative therapies.


Materials and Methods:: Animal work was approved by Northwestern University Institutional Animal Care and Use Committee (NU-IACUC). Nude mice (male/female, young/old (6.5/12.5 weeks old), n=6) underwent femoral artery double knotted ligation at proximal and distal locations to induce acute ischemia. Lack of perfusion was confirmed via Laser Doppler Perfusion Imaging (LDPI) and contralateral limbs were used as controls. Blood perfusion, body weight, motor function and tissue loss were recorded weekly until euthanasia (day 36). Animals were given a score based on motor function; 0 for normal walking, 1 for limping, or 2 for dragging the limb. Tissue loss was also scored based on the extent of the necrotic tissue (0 for no apparent damage, 1 for evident necrosis on nails, 2 for digits and 3 if the damaged tissue reached the paw). LDPI analysis was modified to account for lost tissue in the old mice groups in order to not overestimate the perfusion ratio. After euthanasia on day 36, lower limb muscles from the ischemic and contralateral limbs were processed, sectioned and stained with histology and immunohistochemistry. Multiple Masson’s Trichrome images (n=3) per animal were taken, in proximity of the femoral artery. Preliminary data (n=3, per experimental group) was analyzed regarding average muscle fiber cross sectional area, percentage of centralized nuclei, and fibrosis. Statistical analysis was done via 2-way ANOVA, 1-way ANOVA with Tukey correction, and Mann–Whitney rank-sum tests; for all evaluations, a p-value of ≤ 0.05 was considered as statistically significant.


Results, Conclusions, and Discussions:: The functional assessments revealed significant differences in blood perfusion rates among all experimental groups, from week two until the end of the observational period (Figure 1. A). Notably, the young male group demonstrated the fastest blood perfusion recovery and highest perfusion score, while the old female group showed the worst performance. Motor function was equally impaired following surgery, with the young mice returning to normal walking within one week, while the older groups took an additional week to regain normal walking motion (Figure 1. B). Tissue loss assessments showed that none of the young mice exhibited necrosis, while the older cohort experienced more frequent and severe injuries in females than their male counterparts (Figure 1. C). These findings align with well-documented clinical observations, which indicate that female PAD patients tend to experience more complications than males.



Histological analysis of the muscle tissue revealed age- and sex-related differences in muscle pathology. Specifically, old mice, particularly the females, exhibited increased muscle atrophy compared to younger mice. These findings were supported by changes in the muscle fiber size and nuclei distribution. When compared to younger mice, the older mice had a smaller average muscle fiber cross sectional area (Figure 2. A), irregular shape of muscle fibers, a significantly higher percentage of muscle fibers with centralized nuclei (Figure 2. B), a higher percentage of fibrotic tissue (Figure 2. C) and an increased accumulation of adipose-like tissue (Figure 2. D). These findings are consistent with calf muscle pathology in human PAD patients. Notably, younger mice exhibited increased blood perfusion, likely correlated to an increased angiogenesis, which may have contributed to reduced muscle atrophy, resulting in less tissue remodeling, increased motor function and lack of tissue necrosis.



Our study shows that the age and sex of mice significantly influence recovery from hindlimb ischemia induction. These findings suggest that age and sex should be taken into consideration when evaluating the effectiveness of regenerative therapies for limb salvage and long-term vascular regeneration in PAD patients. Further studies investigating the underlying mechanisms responsible for these age and sex-related differences are warranted.


Acknowledgements (Optional): : This work is supported by the American Heart Association (19TPA34890008).

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