June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Docosahexaenoic Acid Bolsters Mild Oxidative Stress Preconditioning in Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • Nicolas Bazan
    Ophthal & Neuroscience, LSU Health Sciences Center, New Orleans, LA
  • William Gordon
    Ophthal & Neuroscience, LSU Health Sciences Center, New Orleans, LA
  • Eric Knott
    Ophthal & Neuroscience, LSU Health Sciences Center, New Orleans, LA
  • Footnotes
    Commercial Relationships Nicolas Bazan, None; William Gordon, None; Eric Knott, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1794. doi:
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      Nicolas Bazan, William Gordon, Eric Knott; Docosahexaenoic Acid Bolsters Mild Oxidative Stress Preconditioning in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1794.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Preconditioning is an acquired protection to a lethal stress induced by a previous sub-lethal stress. Others have theorized that phospholipase A2 becomes activated during mild ischemia, arachidonic acid is released, and arachidonic acid cascade modulates cell signaling for protection. However, docosahexaenoic acid (DHA), also released via phospholipase A2, is a substrate of lipoxygenases. When released during ischemia or oxidative stress, DHA is converted to the potent mediator neuroprotectin D1 (NPD1). Our lab has found that DHA and NPD1 protect RPE cells when added during and prior to oxidative stress. Thus, the purpose of this study was to determine if DHA could enhance oxidative preconditioning.

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 50µM H2O2 and/or ethanol, or DHA 200nM, 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). Media and cells were collected for lipidomic analysis via LC-ESI-MS\MS.

Results: Preconditioning with 50µM H2O2 for 6 hours resulted in protection of ARPE-19 cells when challenged with subsequent lethal oxidative stress. MS lipidomic analysis demonstrated that DHA is released during this preconditioning and 200nM DHA administration during oxidative preconditioning bolstered the protective effect elicited by preconditioning alone.

Conclusions: This data suggests that the DHA released during preconditioning is neuroprotective through synthesis of the docosanoid NPD1. This supports the idea that the application of DHA/NPD1 could prevent, attenuate, or downregulate retinal damage in pathological conditions, including early stages of retinal degenerative disease.

Keywords: 615 neuroprotection • 701 retinal pigment epithelium • 583 lipids  
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