Search

Amiodarone (AD) has been shown to produce a transient pulmonary fibrosis in hamsters after intratracheal (i.t.) instillation. The goal of this study was to examine bronchoalveolar lavage (BAL) parameters during the development of fibrosis after i.t. AD in hamsters and to examine the responses to oral AD in hamsters for comparison to responses to i.t. AD in an effort to explore the roles of inflammation, phospholipidosis, and lung drug burden in AD-induced pulmonary disease. Two i.t. instillations on Days 0 and 7 of AD in hamsters produced fibrosis as characterized by elevated lung hydroxyproline content and variable increases in lavage macrophage, neutrophil, and eosinophil number through Day 28. Intratracheal AD also increased the permeability of the alveolar-capillary barrier as evidenced by an increase in BAL fluid albumin only on Day 8. Pulmonary phospholipidosis was not induced by i.t. AD and only small amounts of AD and its metabolite desethyl-AD (dAD) were detected in lung tissue through Day 10 after instillation on Days 0 and 7. The repeated oral administration of AD did not result in pulmonary fibrosis during the 35-day course of this study. Oral AD did cause a sustained increase in BAL fluid neutrophil number; other BAL cells were only slightly affected. Oral AD did not increase BAL fluid albumin content but a prominent BAL cell phospholipidosis was noted. Measurement of AD and dAD in lung tissue demonstrated a substantial accumulation of drug and metabolite after oral treatment with AD. The results of this study indicate that lung drug burden, pulmonary phospholipidosis, and lung neutrophil influx are not crucial factors in the development of AD-induced pulmonary fibrosis in hamsters. This study supports the possible involvement of physical damage to the lung and/or pulmonary eosinophilia in the generation of AD-induced pulmonary fibrosis in hamsters.

Laser scanning confocal microscopy (LSCM) allows us to simultaneously quantitate the degree of lung fibrosis and distinguish various pathological lesions of intact lung tissue. Lucifer Yellow has been shown an ideal fluorescent stain to examine the connective tissue matrix components of embedded lung tissue with LSCM. We evaluated the use of LSCM in quantitating lung fibrosis and compared this procedure with the more traditional method of assessing fibrosis by measuring hydroxyproline, a biochemical assay of collagen. CD/VAF rats were intratracheally dosed with silica (highly fibrogenic), Fe2O3 (non-fibrogenic), and saline (vehicle control) at a high dose of 10-mg/100 g body weight. At 60 days post-instillation, the left lung was dissolved in 6 M HCl and assayed for hydroxyproline. Silica induced increases of 58% and 94% in hydroxyproline content over the Fe2O3 and control groups, respectively. The right lung lobes were fixed, sectioned into blocks, dehydrated, stained with Lucifer Yellow (0.1 mg/ml), and embedded in Spurr plastic. Using LSCM and ImageSpace software, the tissue areas of ten random scans from ten blocks of tissue for each of the three groups were measured, and three-dimensional reconstructions of random areas of lung were generated. The silica group showed increases of 57% and 60% in the lung areas stained by Lucifer Yellow over the Fe2O3 and control groups, respectively. Regression analysis of hydroxyproline vs. lung tissue area demonstrated a significant positive correlation (p < 0.05) with a correlation coefficient of 0.91. Histological analysis of right lung tissue revealed a marked degree of granulomatous interstitial pneumonitis for the silica group, which was absent in the Fe2O3 and control groups. No significant differences (p < 0.05) in hydroxyproline content and measured tissue area were observed between the Fe2O3 and control groups. LSCM, and its associated advanced image analysis and three-dimensional capabilities, is an alternative method to both quickly quantitate and examine fibrotic lung disease without physical disruption of the tissue specimen.