Quantitation of hydroxyproline in a mouse model of bleomycin-induced lung fibrosis: comparison with histological analysis

Le résumé fourni par la source

Biochemical evaluation of collagen content by hydroxyproline assay is one of the standard endpoints in the preclinical assessment of therapeutics for IPF. The presence of many steps in the hydroxyproline assay workflow could contribute to low assay reproducibility and impact on the detection of a pharmacologic response. The aim of this work was to evaluate the quality of hydroxyproline data obtained using a colorimetric assay kit and determine the correlation to histopathological scores using the modified Ashcroft scale in the same animals. Lung samples were obtained from an in vivo model in which bleomycin (BLM; 0.1U/mouse) is administered twice to male C57BL/6J mice. Extensive assay validation was performed with analysis of sample stability, including freeze/thaw cycles, and linearity. Precision was assessed by assaying identical replicates within a single experiment (intra-assay) or in independent experiments (inter-assay). Hydroxyproline data showed good assay performance, with high precision for collagen content. In an in vivo time course study, BLM-treatment induced a reproducible ~2-fold increase in hydroxyproline compared to controls at both 21 and 28 days. Comparison with modified Ashcroft scores showed a good correlation between the two readouts (R2: 0.68 and 0.77 at 21 and 28 days respectively) in the same animals. In conclusion, our data confirm that the hydroxyproline assay can be used as a reliable method for fibrosis quantitation, it correlates with histology data and contributes to the preclinical assessment of potential therapeutics for IPF.

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