Fluorescence microtopography of oxidative stress in lung ischemia-reperfusion

Generation of oxidizing radicals in lung ischemia-reperfusion (I/R) was evaluated with the oxidation-dependent fluorogen 2'-7' dichlorofluorescin diacetate (DCF-DA).

Isolated, artificial media perfused, oxygen ventilated rat lungs were subjected to 1 hour of ischemia followed by 1 hour of reperfusion. DCF-DA (5 microM) present in 40 ml of recirculating perfusate served as a source of subsequent intracellular fluorogen. Fluorescence was measured in lung homogenate, and in histologic sections of lung tissue using a confocal laser scanning microscope. Control experiments were 2 hours of continuous perfusion, and 1 hour of perfusion with 1 mM tertiary butyl hydroperoxide as oxidative challenge. Dye distribution was evaluated with the oxidation-independent fluorogen analog 5'- (and 6') carboxy 2'-7' dichlorofluorescin diacetate. Dependence of oxidant generation on the presence of oxygen was studied by substituting oxygen with nitrogen in the ventilation gas.

Perfusion with 5'-(and 6') carboxy 2'-7' dichlorofluorescein diacetate resulted in even distribution of fluorescence, indicating uptake and esterolytic conversion of this dye by all cell types. Perfusion with an exogenous oxidant, tertiary butyl hydroperoxide, plus DCF-DA resulted in a marked generalized increase in fluorescence indicating the presence of the peroxidases necessary to produce the fluorogen from the precursor. Lung I/R, in the presence of oxygen in the ventilation gas, also resulted in marked increase in DCF-DA-associated fluorescence, indicating generation of oxidants. Tissue distribution of fluorescence was nonhomogenous; significant increases were associated with the arteries, veins and bronchial epithelium and at the alveolar level. Of the alveolar cells, the greatest increase in fluorescence was noted in cuboidal epithelium, capillary endothelium, and macrophages. One class of bronchial cells (tentatively identified as goblet cells) took up the dye but showed no fluorescence change with I/R. Ventilation with nitrogen (plus 5% CO2) virtually abolished the I/R-induced fluorescence increase. Preperfusion with catalase before I/R markedly attenuated the increase in fluorescence, indicating the diffusion of hydrogen peroxide from its sites of production.

The results indicate generation of oxidizing species in lungs as a result of I/R, and show predominant localization of oxidants in endothelial, type II, Clara, and ciliated cells and in macrophages.