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.

Keratinocyte growth factor protects against Clara cell injury induced by naphthalene

“…Previous studies have adapted EH-1 for use on microdissected airways and demonstrated it to be a reliable probe for cells undergoing necrosis [43]. Intraperitoneal injection of NA resulted in a marked increase in EH-1-permeable Clara cells by 6 h after injection in Swiss Webster mice [29], and 12 h after injection in C57BL/6 mice in the present study, indicating that NA-induced exfoliation of Clara cells was due to necrosis.…”

Design-Based Sampling and Quantitation of the Respiratory Airways

“…In microscopy, a "disector" is used as a volume probe (Sterio, 1984). The disector approach can be used with thick sections and viewed with a brightfield or laser confocal microscopes that optically focus through the section (Figure 7) (Postlethwait et al, 2000). The usual section thickness is 30 to 50 µm and a short depth of focus (usually about 1 µm) is essential to optically section the tissue.…”

Asthma: a comparison of animal models using stereological methods

“…More efficient design-based methods for estimation of cell numbers are now available using a ''disector'' [107]. These are used widely to provide accurate estimates of cell numbers during injury and repair of conducting and distal airways [108,109]. These methods are critical because, as illustrated in table 6, profile counts are not equivalent to number counts in three-dimensional volume and should be avoided as an endpoint because of their inherent bias.…”

“…These methods are critical because, as illustrated in table 6, profile counts are not equivalent to number counts in three-dimensional volume and should be avoided as an endpoint because of their inherent bias. The disector approach can be used with thick sections and viewed with a brightfield or laser confocal microscope that optically focuses through the section [109]. The usual section thickness is 30-50 mm and a short depth of focus (usually ,1 mm) is essential to optically section the tissue using a light microscope.…”