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Alexander Wurm, Richard H. Funk, Monika Valtink, Cora Roehlecke, Marius Ader, Lilla Knels; Effects Of The Advanced Glycation End Products-Inductor Glyoxal On Mouse Retinal Explants And Neuroprotection By Memantine And Erythropoietin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4456.
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Accumulation of AGEs can augment the risk of AMD or diabetic retinopathy. We were interested to determine which cell types are predominantly affected, which pathways of cell death are involved, and if memantine or erythropoietin (Epo) can counteract damages triggered by AGEs.
C57/BL6 wild type retinal explants were cultured on well inserts with the photoreceptors facing the membrane. Medium (DMEM/F12 with 10% FCS, 2% B27 and 1% P/S) was applied from the bottom side only. Explants were incubated with 1000µM glyoxal to induce AGE formation. Some were co-incubated with 10µM memantine or 0,2U/ml Epo and cultured for up to 5 days. Explants cultured only in medium or in medium with memantine or Epo served as control. Samples were analysed by western blotting for Nε-carboxymethyllysine (CML), apoptosis inducing factor (AIF), and Bcl2-associated protein X (Bax), and reverse transcription PCR (RT-PCR) for heme oxygenase-1 (HO-1), Bax, AIF, receptor for AGEs (RAGE), and immunofluorescence staining of paraffin sections for CML and Bax.
Mouse retinal explants maintained their architecture during the cultivation period. Treatment with glyoxal induced CML formation and increased AIF and Bax protein level, as detected by western blotting. RT-PCR revealed up-regulation of AIF, Bax and HO-1 upon Glyoxal incubation, while RAGE expression remained constant. Immunohistochemical staining revealed strong CML formation mainly in the outer nuclear layer (ONL). Bax positive cells could be seen in the ONL, inner nuclear layer and ganglion cell layer (GCL), with most Bax positive cells in the ONL and fewest in the GCL. Memantine-treated cells showed decreased Bax protein levels in western blots but no modified amount of CML. Epo exerted no effect on Bax or CML.
Glyoxal leads to formation of AGEs and thereby to an up-regulation of the tested mitochondria-associated apoptosis markers in retinal explants. While memantine may attenuate apoptotic reactions, Epo does not exert a neuroprotective effect in the retinal model of age-related oxidative stress and can not counteract eventual apoptotic events.
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