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J. Sanderson, P. Sidaway, N. Niyadurupola, D. C. Broadway; Ischaemia-Mediated Retinal Ganglion Cell Death in Human Organotypic Retinal Cultures: Potential Role of the P2X7 Receptor. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4708.
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© ARVO (1962-2015); The Authors (2016-present)
Ischaemia is proposed to play a role in the pathogenesis of glaucoma. In these experiments, the effects of ischaemia on retinal ganglion cell (RGC) death were investigated in human organotypic retinal cultures (HORCs).
HORCs were dissected from donor human globes within 24 hours post mortem. Ischaemia was simulated using the oxygen/glucose deprivation (OGD) model in which HORCs were cultured for 1 hour in glucose-free DMEM in a modular incubator gassed with 95%N2/5%CO2. They were then cultured for a further 24 hours in control medium (serum-free DMEM). RGC number was assessed by immunohistochemistry using the RGC marker, NeuN, and real time quantitative (Taqman) RT-PCR for the RGC marker,Thy-1. Apoptosis in retinal sections was assessed by TUNEL assay.
A 1 hour period of OGD (simulated ischaemia) caused a 40% decrease in the number of NeuN-positive cells in the HORCs at 24 hours compared to control. Dual-labelling for TUNEL-positive (apoptotic) nuclei showed an approximate 4-fold increase in the number of NeuN-labelled TUNEL-positive cells. In addition, there was a corresponding decrease (44%) in expression of the RGC-marker Thy-1 as a result of the simulated ischaemia. Incubation with the P2X7 antagonist BBG (1µM) almost totally inhibited the OGD-induced loss of RGCs assessed by NeuN-labelling and Thy-1 expression. In addition, the P2X7 agonist BzATP (100µM) caused a decrease in RGC number at the 24 hour time point (51% decrease in NeuN-positive cells; 36% decrease in Thy-1 mRNA). This was also inhibited by BBG (1µM).
These experiments indicate that there is a loss of RGC viability in response to ischaemia in human organotypic retinal cultures and that this may be mediated by activation of P2X7 receptors. This supports evidence suggesting a potential role for the P2X7 receptor in RGC degeneration in glaucoma.
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