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Yong H Park, Brett H Mueller, Nolan McGrady, Adnan Dibas, Thomas Yorio; Retinal Ganglion Cells are Resistant to AMPA Receptor Mediated Excitotoxicity. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1904.
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© ARVO (1962-2015); The Authors (2016-present)
The ionotropic glutamate receptors (iGluR) have been hypothesized to play a role in glaucoma pathogenesis by mediating excitotoxic death of retinal ganglion cells (RGC). Previous studies on iGluR in the retina have been focused on two broad classes of receptors: NMDA and non-NMDA receptors including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and Kainate receptor. In this study, we examined the specific excitotoxic effects of activation of the AMPAR in RGCs in-vitro.
Purified rat RGCs were isolated from P3-P5 Sprague-Dawley rats by a double immunopanning technique using an antibody to Thy 1.1. RGCs were cultured for 7 days before s-AMPA (100μM) treatments. s-AMPA excitotoxicity was determined by Caspase3/7 luciferase activity assay, immunoblot analysis for α-fodrin and Live (calcein AM)/Dead (ethidium homodimer-1) assay. Gap-43 expression was assessed by immunocytochemistry.
Treatment of cultured RGCs with s-AMPA (100μM) for 24, 48 and 72 hr, both in the presence and absence of trophic factors (BDNF and CNTF), did not alter caspase 3/7 activity and cleavage of α-fodrin (neuronal apoptosis marker), compared to untreated controls. A significantly higher (p<0.05) cell survival of RGCs (85.3±1.5% alive cells) was observed after a 72h treatment with 100μM s-AMPA compared to control untreated RGCs (74.8±3.1% alive cells). Quantification of s-AMPA (100μM) - mediated excitotoxicity in purified RGCs incubated for 24h in an oxygen/glucose deprived (0.5% oxygen) medium demonstrated no statistically significant differences in cell survival compared to control RGCs maintained under either normoxia or hypoxia. Additionally, immunocytochemical analysis showed increased GAP-43 staining in RGCs after 24h of treatment with s-AMPA (100μM).
These results indicate that purified RGCs in-vitro are not susceptible to AMPA excitotoxicity as previously hypothesized. Activation of AMPAR increased GAP-43 expression, suggesting AMPAR could possibly increase neurite outgrowth. The ability of AMPA receptors to promote neuroprotection of RGCs remains to be confirmed.
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