Fluorophotometric detection of intravitreal peroxides after panretinal laser photocoagulation.

PURPOSE To develop a highly sensitive method for in vivo quantitation of intravitreal peroxides by vitreous fluorophotometry with 2',7'-dichlorofluorescin (DCFH), a hydrogen peroxide (H2O2)-sensitive fluorescent dye, and to measure peroxides in the vitreous humor after panretinal laser photocoagulation (PRP). METHODS In the presence of H2O2 and lipid hydroperoxides, nonfluorescent DCFH was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF; excitation, 495 nm; emission, 520 nm), which is detectable by vitreous fluorophotometry. Reactions of DCFH, including hematin with various concentrations of H2O2, were investigated in vivo. Fluorophotometry with DCFH was performed 1, 7, 14, and 28 days and 2 months after argon laser PRP. Untreated eyes served as the controls. RESULTS Exogenously applied H2O2 oxidized DCFH to DCF in a dose-dependent manner, ranging from 6 x 10(-8) mol/l to 6 x 10(-5) mol/l in concentration in vivo. Intravitreal DCF concentration was 83.7 +/- 6.8 nmol/l in control eyes. A significant increase of DCF was detected 1 day after PRP (330.7 +/- 123.8 nmol/l, P < 0.002). The increase peaked on day 7 (352.4 +/- 239.5 nmol/l, P < 0.002) and remained elevated at 2 months after PRP (161.8 +/- 51.4 nmol/l, P < 0.01). CONCLUSIONS This method allowed a highly sensitive quantitation of intravitreal peroxides in vivo. The authors' findings indicated that PRP induces increased production of peroxides in rabbit vitreous for 2 months. The data suggested that persistently high levels of peroxides in the vitreous humor affect the development of vitreous liquefaction after PRP.