Development of Local Bleomycin-Induced Hindlimb Model Recapitulating Skin Fibrosis and Vasculopathy of Systemic Sclerosis

Background: Systemic sclerosis (SSc) features microvascular injury and progressive fibrosis, often culminating in peripheral ischemia, digital ulcers, and tissue loss. Existing animal models typically capture either fibrosis (e.g., bleomycin skin models) or ischemia (surgical hindlimb ligation) but rarely both simultaneously in an extremity. Methods: We established a non-surgical, localized model by administering subcutaneous bleomycin (BLM; 300 µg per day) to four peri-ankle sites of the right hindlimb in BALB/c and C57BL/6 mice for 14 consecutive days, followed by a 7-day rest. Outcomes included clinical scoring (edema, joint stiffness, skin thickening), laser Doppler perfusion imaging (LDPI; ischemic/contralateral perfusion ratio), histology (H&E; dermal thickness; inflammation; collagen deposition), quantitative RT-PCR (RT-qPCR) for fibrotic and vascular markers, and immunohistochemical (IHC) staining for COL1A1, α-SMA, CD31, and CD34 with ImageJ-based quantification. Results: BLM induced persistent hindlimb edema and contracture with significantly reduced perfusion measured by LDPI compared with the contralateral limb, persisting after the rest period. Histology revealed marked dermal thickening with dense collagen and leukocyte infiltration. RT-qPCR demonstrated dose-dependent upregulation of α-SMA and TGF-β mRNA, and IHC confirmed increased COL1A1- and α-SMA–positive areas alongside reduced CD31/CD34 capillary density in BLM-treated limbs. The protocol was technically simple and showed reproducible results. Conclusions: Local peri-ankle BLM produces a chronic fibrotic phenotype accompanied by microvascular alteration in an accessible limb, complementing classic SSc models and surgical hindlimb ischemia. This platform enables functional perfusion readouts alongside fibrosis endpoints and is well-suited for preclinical screening of pro-angiogenic or anti-fibrotic strategies of interest to reconstructive surgery.