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.