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Yong H Park, Nolan McGrady, Thomas Yorio; Oxygen Glucose Deprivation Produced Increased Susceptibility of Retinal Ganglion Cells to AMPA Receptor-Mediated Cell Death. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4960.
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© ARVO (1962-2015); The Authors (2016-present)
Excessive AMPA receptor (AMPAR) stimulation has been implicated in producing excitotoxicity in many neurodegenerative diseases such as glaucoma. The purpose of this study was to investigate if AMPAR desensitization attenuates excitotoxicity in purified retinal ganglion cells (RGCs) under normoxic and hypoxic conditions.
Purified RGCs were cultured for 7 days in vitro before series of AMPAR agonists (100µM s-AMPA (desensitizing), 100µM kainic acid (non-desensitizing)), an AMPAR modulator (100µM cyclothiazide), AMPAR antagonist (50µM CFM-2), and Kainate receptor antagonist (50µM UBP301) for 72h in RGC defined medium. To determine if excitotoxicity occurs following hypoxic injury, RGCs were incubated in 0.5% O2 with DMEM/without glucose (OGD) for 4h, following which the cells were treated with s-AMPA in OGD for an additional 4h. A Live-Dead Assay determined cell viability.
Significantly enhanced viability was found in RGCs treated with 100µM s-AMPA (84 ± 1% viable) compared to vehicle (0.1% DMSO) group (71 ± 4% viable) alone (p<0.05). Treatments with s-AMPA in combination with cyclothiazide or kainic acid significantly reduced cell viability to 50±3% and 54±2%, respectively (p<0.01). Blocking AMPARs with a non-competitive antagonist, CFM-2, was able to protect RGCs from kainic acid’s excitotoxic effects (77 ± 1% viable), but kainate receptor antagonism with UBP301 was not protective (58 ± 2% viable) against kainic acid. A marked decrease in RGC survival was observed with cells treated with s-AMPA following OGD injury compared to normoxic glucose deprivation condition. Pretreatment with the AMPAR antagonist, CFM-2, was able to protect RGCs from OGD-injury/s-AMPA mediated cell death. Additionally, no significant decrease in RGC survival was observed when OGD was carried out for 8h in the presence of s-AMPA in the cell culture medium.
Desensitization of AMPAR is a key determinant of s-AMPA-mediated excitotoxicity, whereby blocking the desensitization of AMPAR induces cell death. Following OGD, the desensitization effect of AMPA is compromised and increased s-AMPA-mediated cell death is observed in RGCs. Future studies will determine if AMPAR subunits with greater ion current influx possibly mediate the increased sensitivity to excitotoxicity following injury.
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