Fluorophotometric quantitation of oxidative stress in the retina in vivo.
OBJECTIVE
To establish a new fluorophotometric method to quantitate oxidative stress in the retina in vivo with a hydrogen peroxide (H2O2)-sensitive fluorescent dye.


METHODS
For in vitro fluorophotometric study, nonfluorescent 2',7'-dichlorofluorescein (DCFH) was incubated with H2O2 (10 pM to 100 nM), and the production of fluorescent 2',7'-dichlorofluorescein (DCF) was measured with fluorophotometric analysis. The inhibitory effect of catalase was also examined. For in vivo fluorophotometric study, rabbit eyes received vitrectomy and were perfused with 5 microM 2',7'-dichlorofluorescein diacetate (DCF-DA) or 2',7'-dichlorofluorescein diacetate (DCFH-DA). For oxidative stress, 300 microM H2O2 was infused after perfusion of DCFH-DA. Fluorophotometric measurements of the chorioretinal peak were performed. The eyes were enucleated for fluorescent microscopic examination to determine the localization of DCF fluorescence.


RESULTS
H2O2 converted DCFH to DCF in a dose-dependent manner, which was inhibited by catalase dose dependently. In vivo fluorophotometric study showed DCF-DA and DCFH-DA caused production of 2006 +/- 274 picomole/ml (mean +/- SD, n = 5) and 8.35 +/- 1.11 picomole/ml (n = 5), respectively, in the chorioretinal peak. DCFH-DA with stimulation by H2O2 induced 30.7 +/- 13.1 (n = 4) picomole/ml DCF. Fluorescent microscopy showed DCF production in the retina was significant in the eye treated with DCF-DA and minimal in the eye treated with DCFH-DA. Moderate DCF production in the nerve fiber layer was observed in the eye treated with DCFH-DA and H2O2.


CONCLUSIONS
This new fluorophotometric method with DCFH-DA may be useful in quantitatively evaluating oxidative stress in the retina in vivo.
Takanashi
T
T
Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, Japan.
Ogura
Y
Y
Taguchi
H
H
Hashizoe
M
M
Honda
Y
Y
D000818:Animals; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes; D015590:Fluorophotometry; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D011817:Rabbits; D012160:Retina / Q000187:drug effects / Q000378:metabolism*
D005452:Fluoresceins; D005456:Fluorescent Dyes; C029569:diacetyldichlorofluorescein; C037631:2',7'-dichlorofluorescein; D006861:Hydrogen Peroxide