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A.F. Ambrosio, A. Santiago, S.C. Rosa, A.J. Cristovão, P.F. Santos; High Glucose Causes Caspase–3–Independent Apoptosis in Retinal Neural Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):419.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: It has been shown that retinal neurons may undergo apoptosis in the early stages of diabetic retinopathy, which may ultimately lead to vision loss. We previously showed that high glucose causes a significant decrease in retinal neural cells viability, assessed with the MTT assay. In this work, we investigated the effect of elevated glucose on apoptosis in the same in vitro model. Methods: Rat retinal neural cells were cultured with 30 mM glucose or 25 mM mannitol (osmotic control) for 7 days. Nuclei morphology was evaluated by fluorescence microscopy using Hoechst 33342. Apoptotic cells were identified with TUNEL assay. Caspase–3–like enzymes activity was determined with z–DEVD–AFC, a fluorescent substrate for caspase–3–like enzymes. Results: Elevated glucose induced a significant increase in the number of cells with condensed nuclei, as compared to the control. Similarly, the number of TUNEL–positive cells significantly increased in glucose–treated cells. However, the activity of caspase–3–like enzymes measured in glucose–treated cells and in control cells did not differ significantly. Mannitol, which was used as osmotic control in all experiments, did not cause significant differences compared to the control. Conclusions: These results show that elevated glucose increases retinal cell apoptosis, supporting previous findings showing that neurodegeneration occurs in diabetic retinas. However, in this in vitro model, apoptosis was independent of caspase–3 activation.
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