FLUORESCENCE MICROTOPOGRAPHY OF OXIDATIVE STRESS IN LUNG ISCHEMIA-REPERFUSION

BACKGROUND: Generation of oxidizing radicals in lung ischemia-reperfus ion (I/R) was evaluated with the oxidation-dependent fluorogen 2'-7' d ichlorofluorescin diacetate (DCF-DA). EXPERIMENTAL DESIGN: Isolated, a rtificial media perfused, oxygen ventilated rat lungs were subjected t o 1 hour of ischemia followed by 1 hour of reperfusion. DCF-DA (5 mu M ) present in 40 mi of recirculating perfusate served as a source of su bsequent intracellular fluorogen. Fluorescence was measured in lung ho mogenate, and in histologic sections of lung tissue using a confocal l aser scanning microscope. Control experiments were 2 hours of continuo us perfusion, and 1 hour of perfusion with 1 mM tertiary butyl hydrope roxide as oxidative challenge. Dye distribution was evaluated with the oxidation-independent fluorogen analog 5'- (and 6') carboxy 2'-7' dic hlorofluorescin diacetate. Dependence of oxidant generation on the pre sence of oxygen was studied by substituting oxygen with nitrogen in th e ventilation gas. RESULTS: Perfusion with 5'-(and 6') carboxy 2'-7' d ichlorofluorescein diacetate resulted in even distribution of fluoresc ence, indicating uptake and esterolytic conversion of this dye by all cell types. Perfusion with an exogenous oxidant, tertiary butyl hydrop eroxide, plus DCF-DA resulted in a marked generalized increase in fluo rescence indicating the presence of the peroxidases necessary to produ ce the fluorogen from the precursor. Lung I/R, in the presence of oxyg en in the ventilation gas, also resulted in marked increase in DCF-DA- associated fluorescence, indicating generation of oxidants. Tissue dis tribution of fluorescence was nonhomogenous; significant increases wer e associated with the arteries, veins and bronchial epithelium and at the alveolar level. Of the alveolar cells, the greatest increase in fl uorescence was noted in cuboidal epithelium, capillary endothelium, an d 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 dif fusion of hydrogen peroxide from its sites of production. CONCLUSIONS: The results indicate generation of oxidizing species in lungs as a re sult of I/R, and show predominant localization of oxidants in endothel ial, type II, Clara, and ciliated cells and in macrophages.