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Abstract #10238 Published in IGR 6-1
Peroxynitrite-induced apoptosis in photoreceptor cells
Ito S; Wu GS; Kimoto T; Hisatomi T; Ishibashi T; Rao NACurrent Eye Research 2004; 28: 17-24
PURPOSE: To study the mechanisms of peroxynitrite-induced photoreceptor cell damage, using retinal cultures and a peroxynitrite donor, 3-morpholinosydnonimine (SIN-1). METHODS: Retinal explants obtained from 20-day-old Lewis rat pups were exposed to SIN-1 for varying lengths of time at varying concentrations. Apoptosis in the photoreceptor cells was detected using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method and a DNA fragmentation assay. Selected retinal samples were processed for an ultrastructural analysis to confirm apoptosis. The retinas exposed to SIN-1 were tested for the expression of caspase-3 by immunohistochemistry and a Western blot analysis. The retinas were also evaluated for the prevention of apoptosis in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk). RESULTS: The retinal explants exposed to SIN-1 showed a significant increase in the presence of TUNEL-positive photoreceptor cells. Similarly lineal increases in TUNEL-positive cells were seen in the presence of increasing concentrations of SIN-1. DNA ladder formation was seen with the exposure of SIN-1. Ultrastructurally, SIN-1 exposed retinas revealed typical apoptotic changes in the photoreceptor cell nuclei. The retinas preincubated with urate for six hours and exposed to SIN-1 for 16 hours showed significantly fewer TUNEL-positive cells compared to the retinas exposed to SIN-1 alone (p < 0.05). Moreover, the retinas exposed to SIN-1 showed the expression of caspase-3. This expression, as well as the number of apoptotic photoreceptors, significantly decreased in the presence of Z-VAD-fmk. CONCLUSIONS: These results show that peroxynitrite induces apoptosis in photoreceptor cells and that such retinal damage appears to be mediated by caspase-3. The apoptotic process can be minimized by peroxynitrite scavenger urate, as well as by the caspase inhibitor Z-VAD-fmk.
Dr. S. Ito, Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
Classification:
2.13 Retina and retinal nerve fibre layer (Part of: 2 Anatomical structures in glaucoma)
3.3 Immunohistochemistry (Part of: 3 Laboratory methods)
