Abstract
INTRODUCTION: Interstitial lung diseases (ILDs) are a heterogeneous group of pulmonary disorders characterized by variable inflammation and fibrosis, leading to progressive impairment of gas exchange. Current therapeutic strategies rely on immunosuppressants and antifibrotics, but early diagnosis and treatment remain crucial to improve long-term outcomes. The development of targeted therapies requires accurate preclinical models that reproduce the initial disease stages. METHODS: Here, we propose an in vivo model based on chronic administration of low-dose subcutaneous bleomycin (BLM, 30 U/kg) to induce early lung injury. To detect early fibrotic alterations, we implemented a micro-CT segmentation strategy, dividing the lung parenchyma into ten aeration-based regions. RESULTS: This approach enabled the identification of subtle changes in aeration on day 14 after BLM exposure. Further molecular characterization by Western blot analysis revealed, at day 21, significant activation of pro-inflammatory and pro-fibrotic pathways, including upregulation of the TGF-β/Smad 2-3 signaling cascade and induction of the NLRP3 inflammasome pathway. These findings confirm that low-dose BLM is sufficient to initiate pathogenetic mechanisms relevant to ILD. DISCUSSION: Overall, our study establishes a novel preclinical protocol integrating reduced BLM dosage with refined micro-CT analysis, providing a promising platform for investigating early ILD pathogenesis and facilitating translational research aimed at optimizing therapeutic intervention windows.