Purchase this article with an account.
Farzana Rahman, Shereen Nizari, Joana Galvao, Miles Parnell, ABUBAKAR HABIB, Eduardo Normando, M Francesca Cordeiro; Reversal of Apoptosis in Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):434.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Early detection of retinal ganglion cell (RGC) apoptosis and its prevention holds promise as a novel and effective treatment for glaucoma. Reversible apoptosis, during which there is exposure of phosphatidylserine (PS) on to the outer leaflet of the plasma membrane, has recently been described, using Galectin-1(Gal-1). The aim of this study was to determine whether Gal-1 induces apoptosis in RGCs, and to investigate whether this apoptosis is reversible.
RGCs as part of a mixed cell culture with Muller cells were treated with human Gal-1 at concentrations of 2.5μM, 1.25 μM and 0.63μM. Hoechst staining was used to determine cell viability and apoptosis following 30 minutes of treatment with Gal-1. Live cell imaging with Annexin-V was then used to assess PS exposure following Gal-1 treatment. Gal-1 was combined with Annexin-V (2.5µl), binding buffer (10µl) and Hoechst stain (0.1µl) resulting in concentrations of 5.27µM, 2.64μM, 2.00µM and 1.32μM. Live cell imaging was performed for 30 minutes on cells in Dulbecco's modified eagle medium (DMEM) alone initially. After 30 minutes, DMEM was replaced with Gal-1 treatments for 30 minutes and then replaced with DMEM again. Live cell imaging continued for 2 hours.
A one-way ANOVA showed significant reduction in the viability of mixed cells following treatment with 1.25μM Gal-1 (p<0.05) and 2.50μM Gal-1 (p<0.001), compared to the control (DMEM set as 100% viability). A 23.31% decrease in total cells was observed with 1.25μM Gal-1 compared to the control (DMEM). A 38.34% decrease in total cells was observed with 2.5μM Gal-1 compared to the control. When analysing the dose-response relationship, there was 97.78% certainty that the reduction in cell viability was due to the action of Gal-1 (Pearson r = -0.9777, p = 0.0223). Live cell imaging demonstrated increased Annexin-V binding following treatment with Gal-1 at concentrations of 1.32µM, 2.00µM, 2.64µM and 5.27µM. This suggested that Gal-1 at these concentrations induced PS exposure. Removal of Gal-1 after 30 minutes caused a reduction and redistribution in Annexin-V staining.
These results provide a mechanistic insight into RGC apoptosis and suggest potential for its reversal. This would be of great benefit clinically for the early management of glaucoma and other pathologies such as diabetic retinopathy. A process once considered an irreversible pathway leading to cell death now holds hope as a therapeutic target.
This PDF is available to Subscribers Only