April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Ischaemia-Mediated Retinal Ganglion Cell Death in Human Organotypic Retinal Cultures: Potential Role of the P2X7 Receptor
Author Affiliations & Notes
  • J. Sanderson
    School of Pharmacy, University of East Anglia, Norwich, United Kingdom
  • P. Sidaway
    School of Pharmacy, University of East Anglia, Norwich, United Kingdom
  • N. Niyadurupola
    Ophthalmology, Norfolk and Norwich University Hospital, Norwich, United Kingdom
  • D. C. Broadway
    Ophthalmology, Norfolk & Norwich Univ Hospital, Norwich, United Kingdom
  • Footnotes
    Commercial Relationships  J. Sanderson, None; P. Sidaway, None; N. Niyadurupola, None; D.C. Broadway, None.
  • Footnotes
    Support  Humane Research Trust; Norwich Glaucoma Research Fund
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 4708. doi:
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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)

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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 
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