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A.F. Ambrosio, A.R. Santiago, M.J. Garrido, P.F. Santos, A.J. Cristovao, J.G. Cunha-Vaz; Neurotoxic Effect Caused by Hyperglycemia in Cultured Rat Retinal Neurons: Possible Involvement of Glutamate . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3881.
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Purpose: It has been shown that retinal neurons may undergo apoptosis in the early stages of diabetic retinopathy. Moreover, it was hypothesized that neural degeneration may involve persistent glutamate toxicity. The purpose of this study was to investigate whether hyperglycemia causes toxicity in primary cultured retinal neurons and if the release of glutamate is altered. Methods: Rat retinal neurons were cultured in DMEM with 10% FCS, and the cells were incubated either with 25 mM D-glucose (final concentration 30 mM) or with 25 mM D-mannitol, for 7 or 12 days. Cell viability was determined with the MTT assay. The release of [3H]-D-aspartate was performed under continuous perfusion and the radioactivity was measured in a liquid scintillation analyzer. Results: Exposure of retinal neurons to hyperglycemia for 7 days did not cause a significant toxic effect, but the incubation with 30 mM glucose for 12 days, without changing medium, significantly decreased the MTT reduction to 91.9±3.4% of the control. Moreover, when the culture medium was changed at culture day 7, the MTT reduction decreased to 74.1±3.1% of the control. Incubation of neurons with mannitol did not cause any significant changes comparatively to the control. Extracellular levels of glucose remained elevated even after 14 days in vitro without changing culture medium. The release of [3H]-D-aspartate evoked by 50 mM KCl was significantly enhanced (148.4±14.0% of the control) in neurons exposed to high levels of glucose for 7 days. Contrarily, the release of [3H]-D-aspartate decreased in cells exposed for 12 to hyperglycemia. Incubation with mannitol did not cause significant changes in [3H]-D-aspartate release as compared to control. Conclusions: These results clearly show that long-term hyperglycemia causes neurodegeneration in cultured rat retinal neurons. In addition, we also showed that the release of glutamate might be altered, supporting a role for glutamate at the early stages of diabetic retinopathy. Support: FCT, Portugal, POCTI/CBO/38545/01.
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