Abstract
Purpose: :
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).
Methods: :
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.
Results: :
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).
Conclusions: :
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.
Keywords: retinal culture • ischemia • neuroprotection