Application of laser scanning confocal microscopy in the analysis of particle-induced pulmonary fibrosis

Laser scanning confocal microscopy (LSCM) allows us to simultaneously quant itate the degree of lung fibrosis and distinguish various pathological lesi ons of intact lung tissue. Lucifer Yellow has been shown an ideal fluoresce nt stain to examine the connective tissue matrix components of embedded lun g tissue with LSCM. We evaluated the use of LSCM in quantitating lung fibro sis and compared this procedure with the more traditional method of assessi ng fibrosis by measuring hydroxyproline, a biochemical assay of collagen. C D/VAF rats were intratracheally dosed with silica (highly fibrogenic), Fe2O 3 (non-fibrogenic), and saline (vehicle control) at a high dose of 10-mg/10 0 g body weight. At 60 days post-instillation, the left lung was dissolved in 6 M HCI and assayed for hydroxyproline. Silica induced increases of 58% and 94% in hydroxyproline content over the Fe2O3 and control groups, respec tively. The right lung lobes were fixed, sectioned into blocks, dehydrated, stained with Lucifer Yellow (0.1 mg/ml), and embedded in Spurr plastic. Us ing 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 si lica group showed increases of 57% and 60% in the lung areas stained by Luc ifer Yellow over the Fe2O3 and control groups, respectively. Regression ana lysis of hydroxyproline vs. lung tissue area demonstrated a significant pos itive correlation (p < 0.05) with a correlation coefficient of 0.91. Histol ogical analysis of right lung tissue revealed a marked degree of granulomat ous interstitial pneumonitis for the silica group, which was absent in the Fe2O3 and control groups. No significant differences (p < 0.05) in hydroxyp roline 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 quantit ate and examine fibrotic lung disease without physical disruption of the ti ssue specimen.