Abstract
Abstract: :
Purpose: Phenyl–N–tert–butylnitrone (PBN) is a radical scavenger that is frequently used as a spin trap agent. Our lab reported that PBN protects rats from light–induced retinal degeneration. However, the mechanism is unclear because PBN has a variety of pharmacological effects. Methods: Sprague Dawley (SD) rats (5 – 6 weeks old) raised in dim (5 lux) cyclic light (12 hr On / Off) from birth were injected intraperitonealy with PBN or water 30 minutes prior to exposure to 2700 lux light for 0, 3, 6, 12 and 24 hours. For immunohistochemistry, eyes were fixed with 4% paraformaldehyde at 4 °C and 10 µm thickness cryo–sections were cut. TUNEL staining and DNA laddering were performed as apoptosis markers. Total RNA was extracted to perform RNase protection assays with apoptosis related probes. AP–1 activity was investigated by using the Electrophoretic Mobility Shift assay. The level of c–fos in the nuclear fractions was analyzed by the western blotting. Results: Typical apoptotic features were seen in 24 hours of continuous light exposure of control, but not in PBN–injected rats. RNase protection assay revealed a time–dependent up–regulation of apoptosis–related genes by continuous light exposure. The expression of caspase–3 was markedly inhibited by the injection of PBN, as was the activity of AP–1. The western blots showed that c–fos increase in the nuclear fraction at 6 hours of light exposure was inhibited by the injection of PBN. Discussion: PBN inhibits up–regulation of genes or gene products previously found to be relevant in light–induced apoptosis. Grants: NIH (EY04149, EY00871, EY12190, RR17703), Research to Prevent Blindness, Inc., and the Foundation Fighting Blindness.
Keywords: photoreceptors • apoptosis/cell death • retinal degenerations: cell biology