Three-dimensional mapping of ozone-induced acute cytotoxicity in tracheobronchial airways of isolated perfused rat lung

Acute lung injury induced by reactive oxygen gases such as ozone (O-3) is f ocal and site-selective. To define patterns of acute epithelial injury alon g intrapulmonary airways, we developed a new analytic approach incorporatin g labeling of permeable cells, airway microdissection, and laser scanning c onfocal microscopy, and applied it to isolated perfused rat lungs where ven tilation and breathing pattern could be controlled. After exposure to O-3 ( 0, 0.25, 0.5, or 1.0 ppm), lungs were lavaged to assess lactate dehydrogena se (LDH) and protein, or infused with the permeability marker ethidium homo dimer-l (EthD-1) via tracheal cannula, gently lavaged, and fixed by airway infusion. The airway tree of the right middle lobe was exposed by microdiss ection of the axial pathway down to the terminal bronchioles; the dissectio n was incubated with a second nuclear dye, YOPRO-1, to label all nuclei; an d whole mounts were examined by confocal microscopy. Abundance of EthD-1-po sitive (injured) cells was estimated as the number per epithelial volume us ing stereology on Z-series of projected images. For ozone concentrations of 1.0 ppm, lavage fluid LDH and total protein did not increase over controls . Exposure produced a concentration-dependent but nonhomogeneous increase i n the abundance of EthD-1-labeled cells in proximal and distal conducting a irways both in the main pathway, including terminal bronchioles, and in sid e branches. Overall, the highest EthD-1 labeling occurred in the side branc hes of the most proximal part of the airway tree at 1 ppm with the adjacent axial pathway airway having approximately one-third the labeling density. Density of EthD-1-labeled cells was lowest in terminal bronchioles at all 0 , doses. For the model we used, identification of injured epithelial cells by differential permeability and laser confocal microscopy appeared to be h ighly sensitive and permitted mapping of acute cytotoxicity throughout the airway tree and quantitative comparisons of sites with different branching histories and potential dosimetry rates.