Comparative analysis of collagen quantification in the bleomycin mouse model of lung fibrosis

Background: Evaluation of fibrosis and its severity is the commonest parameter to screen potential new medicines in rodents treated with bleomycin (BLM). Results are mainly reported by histological analysis and indirect measurement of collagen through hydroxyproline (HYP) quantification. Objectives: In the present study we performed a comparative analysis of different methods to assess collagens induced by BLM in the mouse lung. Methods: BLM (0.02 U/kg) was administered by oropharyngeal aspiration to male C57BL/6 mice on day 0 and day 4. Oral Nintedanib (60 mg/kg/day) started from day 7. Three and four weeks after the first BLM administration, histopathological examination and collagen genes expression analysis were applied. Moreover, HYP and collagen isoforms (LC-MS) quantification in lung homogenates were assessed. Results: Ashcroft score and automated measure of lung fibrosis revealed significant fibrotic lesions in BLM-treated animals. Specific collagen genes appeared up-regulated in BLM group. LC-MS quantification of collagen isoforms correlated with HYP levels although, interestingly, showed a higher therapeutic window between control and BLM group (1.5 vs. 0.5 fold, respectively). Nintedanib significantly affected all the evaluated readouts. Conclusions: Our study provides a comprehensive analysis of changes in collagen genes and proteins following BLM treatment in mice and the effect of Nintedanib. Importantly, our data indicate for the first time LC-MS as a sensitive and reliable method for collagen quantification in lung homogenate. This approach might implement the most employed HYP assay to achieve reliable predictions of efficacy for therapeutics in lung fibrosis.