AIM: The purpose of this study was to develop an improved method for collagen and protein assessment of fibrotic lungs while decreasing animal use. METHODS: 8-10 week old, male C57BL/6 mice were given a single intratracheal instillation of crocidolite asbestos or control titanium dioxide. Lungs were collected on day 14 and dried as whole lung, or homogenized in CHAPS buffer, for hydroxyproline analysis. Insoluble and salt-soluble collagen content was also determined in lung homogenates using a modified Sirius red colorimetric 96-well plate assay. RESULTS: The hydroxyproline assay showed significant increases in collagen content in the lungs of asbestos-treated mice. Identical results were present between collagen content determined on dried whole lung or whole lung homogenates. The Sirius red plate assay showed a significant increase in collagen content in lung homogenates however, this assay grossly over-estimated the total amount of collagen and underestimated changes between control and fibrotic lungs, conclusions: The proposed method provides accurate quantification of collagen content in whole lungs and additional homogenate samples for biochemical analysis from a single animal. The Sirius-red colorimetric plate assay provides a complementary method for determination of the relative changes in lung collagen but the values tend to overestimate absolute values obtained by the gold standard hydroxyproline assay and underestimate the overall fibrotic injury.
AIM: The purpose of this study was to develop an improved method for collagen and protein assessment of fibrotic lungs while decreasing animal use. METHODS: 8-10 week old, male C57BL/6 mice were given a single intratracheal instillation of crocidolite asbestos or control titanium dioxide. Lungs were collected on day 14 and dried as whole lung, or homogenized in CHAPS buffer, for hydroxyproline analysis. Insoluble and salt-soluble collagen content was also determined in lung homogenates using a modified Sirius red colorimetric 96-well plate assay. RESULTS: The hydroxyproline assay showed significant increases in collagen content in the lungs of asbestos-treated mice. Identical results were present between collagen content determined on dried whole lung or whole lung homogenates. The Sirius red plate assay showed a significant increase in collagen content in lung homogenates however, this assay grossly over-estimated the total amount of collagen and underestimated changes between control and fibrotic lungs, conclusions: The proposed method provides accurate quantification of collagen content in whole lungs and additional homogenate samples for biochemical analysis from a single animal. The Sirius-red colorimetric plate assay provides a complementary method for determination of the relative changes in lung collagen but the values tend to overestimate absolute values obtained by the gold standard hydroxyproline assay and underestimate the overall fibrotic injury.
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