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

Abstract：

:Acute lung injury induced by reactive oxygen gases such as ozone (O(3)) is focal and site-selective. To define patterns of acute epithelial injury along intrapulmonary airways, we developed a new analytic approach incorporating labeling of permeable cells, airway microdissection, and laser scanning confocal microscopy, and applied it to isolated perfused rat lungs where ventilation 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 dehydrogenase (LDH) and protein, or infused with the permeability marker ethidium homodimer-1 (EthD-1) via tracheal cannula, gently lavaged, and fixed by airway infusion. The airway tree of the right middle lobe was exposed by microdissection of the axial pathway down to the terminal bronchioles; the dissection was incubated with a second nuclear dye, YOPRO-1, to label all nuclei; and whole mounts were examined by confocal microscopy. Abundance of EthD-1-positive (injured) cells was estimated as the number per epithelial volume using 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 in the abundance of EthD-1-labeled cells in proximal and distal conducting airways both in the main pathway, including terminal bronchioles, and in side branches. Overall, the highest EthD-1 labeling occurred in the side branches 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 O(3) doses. For the model we used, identification of injured epithelial cells by differential permeability and laser confocal microscopy appeared to be highly sensitive and permitted mapping of acute cytotoxicity throughout the airway tree and quantitative comparisons of sites with different branching histories and potential dosimetry rates.

journal_name

Am J Respir Cell Mol Biol

journal_title

American journal of respiratory cell and molecular biology

authors

Postlethwait EM,Joad JP,Hyde DM,Schelegle ES,Bric JM,Weir AJ,Putney LF,Wong VJ,Velsor LW,Plopper CG

doi

10.1165/ajrcmb.22.2.3674

keywords:

subject

Has Abstract

pub_date

2000-02-01 00:00:00

pages

191-9

issue

eissn

1044-1549

issn

1535-4989

journal_volume

pub_type

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