Abstract
Purpose :
Previous studies have shown that the ocular generation of ceramides contributes to retinal degeneration associated with ischemic and ocular hypertensive stress. In the eye, acid and neutral sphingomyelinase are the major enzymes responsible for the stress-induced generation of ceramides, and studies have shown that these enzymes are expressed in the optic nerve head. The goal of this study is to evaluate the effects of ceramides on astrocyte viability, the secretion of cytotoxic cytokines and the impact these events have on retinal ganglion cell (RGC) viability.
Methods :
Optic nerve head astrocytes were cultured and purified from human donor eyes. Differentiated human RGCs were generated from human pluripotent stem cells and enriched to over 80% purity. Astrocyte cultures were treated with C2-ceramide (0.1 to 10 μM) for 24 hours. Media were then collected and used to treat RGCs cultures or for TNF-α ELISA. Effects on cell viability was determined by manual cell counts. Selected cultures were processed for immunohistochemistry.
Results :
Immunohistochemical analysis demonstrated that both acid and neutral sphingomyelinases are expressed by human astrocytes. The addition of C2-ceramide, at concentration up to 10 μM, did not significantly affect astrocyte viability. However, the treatment of RGCs with conditioned media from C2-ceramide (10 μM) treated astrocytes significantly reduced cell number by 24.7± 4.8%. The cytotoxic actions of conditioned astrocyte media was significantly reduced by the addition of neutralizing antibody against TNF-α. Analysis of conditioned media demonstrated that C2-ceramide produced a concentration-dependent increase in TNF-α levels (EC50 = 0.59 µM). The addition of C2-ceraimde (10 μM) directly to RGC cultures for 24 hrs significantly reduced cell number by 45.6 ± 6.0% when compared to vehicle-treated RGCs.
Conclusions :
These results demonstrate that ceramides can affect RGCs viability both directly through apoptotic actions and indirectly via the secretion of cytokines from optic nerve astrocytes. These actions of ceramides support the idea that the generation of bioactive lipids play multiple roles in the pathogenesis of several retinal disorders.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.