Role Of Non–feminizing Estrogen Analogues In Neuroprotection Of Rat Retinal Ganglion Cells Against glutamate–induced Cytotoxicity

Abstract

Abstract: : Purpose: To compare and characterize the neuroprotective effects of 17ß–estradiol and novel 2–adamantyl estrogen analogues against glutamate induced cytotoxicity of rat retinal ganglion cells (RGC–5 cells). Methods: RT–PCR, PCR–Southern Blot, and immunocytochemistry were used to determine the expression of estrogen receptors a and b in RGC–5 cells. RGC–5 cells, 17ß –estradiol, ICI estrogen receptor antagonist, three novel 2–adamantyl estrogen analogues (ZYC 1, ZYC 3, and ZYC 10), and L–glutamic acid were used for these studies. RGC–5 cells were pretreated with 17ß–estradiol or one of the estrogen analogues followed by an insult with L–glutamic acid (5 mM). Cell viability was assessed using the neutral red dye uptake assay. ICI compound was used, as an antagonist of estrogen receptors, to assess their involvement in neuroprotection. Levels of γ–glutamylcysteinsynthetase in cells pretreated with estrogen analogue ZYC3, followed by glutamate challenge, were monitored by immunoblot analysis. Results: RGC–5 cells were shown to express both α and ß estrogen receptors. Glutamate treatment resulted in 50% RGC–5 cell death. 17ß–estradiol and the three estrogen analogues protected the RGC–5 cells against glutamate cytotoxicity in a dose dependent manner that was minimally inhibited by the estrogen receptor antagonist ICI. The efficacy of neuroprotection by the estrogen analogues was as follows: ZYC 3 >ZYC 10 > ZYC 1. In RGC–5 cells preincubated with ZYC3 followed by a glutamate challenge, the protein levels of the catalytic and regulatory subunits of γ –glutamylcysteinsynthetase were increased. Conclusins: 17ß–estradiol and non–feminizing estrogen analogues ZYC 3, ZYC 10, and ZYC 1 protect RGC–5 cells against glutamate cytotoxicity. These compounds appear to affect their neuroprotection via an antioxidant pathway. The data support the hypothesis that estrogen analogues may be useful in the neuroprotection of retinal ganglion cells in ocular pathologies such as glaucoma.

Keywords: neuroprotection • oxidation/oxidative or free radical damage • ganglion cells