Optimization of intranasal bleomycin dose for effective pulmonary fibrosis induction in mice with minimal animal distress.

Background: Pulmonary fibrosis is a progressive interstitial lung disease driven by aberrant alveolar epithelial repair and dysregulated crosstalk with mesenchymal cells, leading to myofibroblast activation and excessive extracellular matrix deposition. Current mouse models for studying its pathogenesis often use invasive methods like intratracheal bleomycin administration, which can cause significant animal distress. This study aims to refine the bleomycin model via intranasal instillation, reducing animal suffering while improving reproducibility and translational value. Methods: The MALDI-TOF MS analysis allowed for the identification of the spatial distribution of bleomycin directly on the lung tissue of C57BL/6J mice after intranasal administration to demonstrate the effectiveness of this route. Different doses (1, 3, 5 mg/kg) of bleomycin were tested in mice. Non-invasive whole-body plethysmography enabled the monitoring of the disease progression and the assessment of pulmonary function in conscious mice. Histopathological analyses were performed to follow fibrosis progression. Results: This method resulted in impaired respiratory function, with a similar pattern observed for both 5 and 3 mg/kg doses. Histopathological evaluations revealed rapid acute inflammation followed by chronic fibrosis in both dosage groups, similar to human IPF. Key molecular markers (CD68 and COL 1a1) were upregulated in response to the treatment, with similar expression patterns between the two doses, suggesting an inflammatory response and established fibrosis. 1 mg/kg dosage did not lead to any relevant clinical changes and was therefore excluded from further analyses. Conclusions: Intranasal administration proved to be an efficient technique, delivering a uniform bleomycin distribution in the lungs. Bleomycin instillation induced lung injury and fibrosis comparable to those achieved with the conventional intratracheal protocol, both histologically and functionally, while reducing invasiveness and animal distress. The optimal bleomycin dosage was determined to be 3 mg/kg, which induced a similar level of respiratory distress, but with less weight loss and reduced risk of mortality. This approach provides a reproducible model for preclinical studies of pulmonary fibrosis. This strategy ensures consistent and reproducible scientific data, pivotal for bridging the gap between benchside and bedside.