The lung provides a unique challenge for fibrosis investigations, in that many stimuli for fibrosis are administered intratracheally and create a heterogeneic pattern of injury in the lung.
Evaluating only one lung for fibrosis or protein analysis likely introduces sampling error, as one lung may contain more fibrosis than the other. This study presents a simplified method of evaluating lung fibrosis that eliminates this error and provides additional information about insoluble collagen (mature fibers) and soluble collagen burdens (immature, newly synthesized fibers). Additionally, proteomic analysis can be performed on the remaining 1.5 ml of lung homogenate.
Homogenization of both lungs and then sampling only a portion of the homogenate, prior to the hydroxyproline assay results in comparable data to performing hydroxyproline analysis on whole lungs (). The amounts of collagen determined for the TiO2-treated mice through this assay are very close to previous reports suggesting that the lung of a normal 20-g mouse contains approximately 1.2 mg of collagen (~15-20% of the dry lung weight). This data suggests that this method provides equivalent results and efficiency allowing sampling from a single mouse. This homogenization method could be utilized to quantify fibrosis in other tissues where protein homogenates are also needed.
Sirius red staining of collagen has been used for many years. The present colorimetric plate assay allows for rapid assessment of collagen content. One major problem with the Sirius red assay is that it appears to over-estimate the amount of collagen in a sample. Only 1-5% of collagen in the adult lung is believed to be salt soluble [1
]. The assay utilizes only a small fraction of the original sample (10ml of a an original samples volume of 3 ml) and small variations inherent to the staining procedure are amplified with subsequent calculations to determine the total collagen content. This data suggests that the Sirius red plate assay may be an overestimate of the true collagen burden of the lung. However, the assay does provide information about the relative changes of immature precursor collagen during fibrosis development. In addition, this assay under-estimates the relative increase in fibrosis between control and injured animals, further suggesting that there is nonspecific binding of the Sirius red dye to non-collagen proteins. Thus, the use of the Sirius red assay may not reflect significant changes in fibrosis that would be seen with the hydroxyproline assay.
There are several important advantages to this new method: 1) Only one mouse is required to attain fibrosis and protein/biochemical data compared to twice the number of animals with the previous method. 2) The homogenized tissue samples require less drying time during the hydroxyproline assay, which can shorten the procedure by 24 hours. 3) The assay provides an accurate quantification of total lung hydroxyproline and collagen through analysis of both lungs, not a single lung or lobe. 4) The Sirius red colorimetric plate assay can be utilized as a complementary assay to evaluate the relative changes in salt-soluble collagens, which are the precursors to mature collagen. One disadvantage to the new procedure is that is requires careful homogenization of tissues in identical volumes to ensure that error is not introduced through this step.
In summary, we provide an improved method for the assessment of collagen and biochemical analysis from a single mouse. This allows for a significant reduction in the number of animals used while producing accurate data on the fibrotic environment of the lung. Additionally, this homogenization technique would likely work for other pulmonary disease models, in which injury is induced by intratracheal instillation of a stimuli and which require multiple methods of lung tissue analysis.