With laser scanning confocal microscopy (LSCM), it is possible to simultaneously quantify lung fibrosis and distinguish pathological lesions of intact lung tissue. Lucifer yellow (LY) has been shown to be an ideal fluorescent probe to stain connective tissue matrix macromolecules selectively. It was the objective of the present work to evaluate the use of LSCM in quantifying fibrosis after exposure to amiodarone (AD), a highly effective anti-arrhythmic drug known to cause debilitating lung fibrosis in humans. Male F344 rats were dosed intratracheally with 6.25 mg/kg AD (3.125 mg/L solution in sterile water) or with the sterile water vehicle on day 0, and again on day 2. On day 28, the left lung was dissolved in 6 M HCl and assayed for hydroxyproline, a biochemical measure of collagen. The right lung was fixed, sectioned into blocks, dehydrated, stained with LY (0.1 mg/ml), and embedded in Spurr plastic. The area of LY-stained matrix in tissue sections was quantified by LSCM. The LY-stained connective tissue matrix of the AD group appeared as bright linear bands in the alveolar septae, and was significantly elevated (p<0.05) as measured by image analysis when compared with the control group. A fibrotic response in the AD group was further confirmed as evidenced by a significant increase (p<0.05) above control values in total left lung hydroxyproline. Histopathologic assessment of trichromestained sections revealed a minimal to mild, multifocal, interstitial pulmonary fibrosis in all AD-treated rats which was absent in control animals. LSCM, with its advanced image analysis system and 3-D capabilities, is an alternative method to quantify fibrotic lung disease quickly and may prove useful in examining human lung-biopsy samples where biochemical analysis is impossible.
American Journal of Respiratory and Critical Care Medicine. Abstracts of the American Thoracic Society 2001 International Conference, May 18-23, 2001, San Francisco, California