Abstract
Purpose:
During oxidative stress, phagocytosis of outer segments, or neurotrophin addition, RPE cells up regulate the synthesis of 10,17(S)-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid (neuroprotectin D1; NPD1) from DHA, which is endowed with potent neuroprotective bioactivity. Preconditioning is sub-lethal stress which exerts protection from a subsequent lethal stress. Inhibition of 15-lipoxygenase-1 (15-LO1) has been shown to eliminate the protection bestowed by ischemic preconditioning in rat cardiac muscle. It has been theorized that this loss of protection is due to the loss of 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HETE) (Murphy et al. Circ Res 1995). However, NPD1 is also synthesized via 15-LO1. Therefore, it is the purpose of this study to determine if NPD1 acts as a preconditioning trigger to initiate protective events prior to lethal oxidative challenge.
Methods:
ARPE-19 cells were plated at ~10,000 cells/well in 10% FBS DMEM\D12 1:1 medium; and grown for 72 hours to reach confluencey. After 18 hours in 0.5% FBS medium, cells were treated with ethanol or NPD1 (100nM) for 6 hours. After 6 hours, treatment medium was removed; cells were allowed to rest for 24 hours in 0.5% FBS medium, and subsequently stressed with 300-450 µM H2O2 plus 10ng/ml TNF-α. Nuclear pyknosis was monitored via morphonuclear analysis imaging method (MAIM) to determine cell survival (D. Stark and N. Bazan J. Neuros. 2011).
Results:
Control cells display a survival rate of 97 ± 3%. Cells treated with vehicle followed by H2O2 plus TNF-α for 16 hours undergo nuclear condensation and demonstrate a cell survival rate of 51± 4 %. Cells treated with 100 nM NPD1 for 6 hours prior to stress exhibit protection with a survival rate of 69 ± 9%.
Conclusions:
We have previously shown that NPD1 protects RPE cells when administered during an oxidative insult. Present results demonstrate that NPD1 ameliorates oxidative damage- induced cell death with preventative treatment suggesting that this novel therapeutic tool may be useful in the prevention, attenuation, or early progression of retinal degenerations.
Keywords: 615 neuroprotection •
701 retinal pigment epithelium •
634 oxidation/oxidative or free radical damage