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S. Hong, W. C. Gordon, Y. Lu, V. L. Marcheselli, N. G. Bazan; Lipidomic Approach to Define Pro- and Anti-Inflammatory Mediators in Laser-Induce Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2007;48(13):6014.
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Choroidal neovascularization (CNV) leads to loss of vision in exudative forms of age-related macular degeneration. We have explored using a lipidomic approach pathways leading to the accumulation of mediators of inflammation during the initiation and progression of experimental CNV.
Laser retinal photo-coagulations (100 mW, 100 mS, 50 µm diameter) were performed on mice. Retinas were collected throughout a time course up to 20 days post-flash. Whole retinas were isolated, for lipidomic analysis, and immunohistochemistry to localize markers for inflammation and neovascularization by deconvolution microscopy. Retina lipid extracts were analyzed via liquid chromatography-linear trap tandem mass spectrometry (LC-MS/MS).
Immunohistolocalization revealed that by 2-4 h inflammatory markers (e.g. COX-2) were localized to the burn site, followed by appearance of markers for neovascularization (e.g. VEGF) at 4 h with an increase within the scar region, and retina-vitreous interface, throughout the time course. Neuroprotectin D1, 4,16-dihydroxy-docosahexaenoic acid (4,16-diHDHA), 16-hydroxy docosahexaenoic acid (16-HDHA), 20-HDHA, resolvin E1, lipoxin B4, PGE3, and PGE2, and leukotriene B4 were found to increase at 2 hrs after the laser injury, then returned to the near control levels after 18 to 24 hrs (Fig. 1). Additionally resolvin D1, D4, and D5 (7,17-diHDHA) were found in the retina 4 days after injury. NPD1, resolvins, and LXB4 are known to be anti-inflammatory and promote resolution. However, LTB4 and PGE2 are proinflammatory. Docosahexaenoic acid is the precursor of NPD1, resolvin D series, and 4,16-diHDHA. 16-HDHA is an intermediate for the biosynthesis of 4,16-diHDHA. Free arachidonic acid the substrate for COX-2 shows a transient increase after 2 hours of injury, then drop below control levels by 18 to 24 hrs, but raises again up to more than 3 folds at 8 days after injury. Similar pattern is shown by free DHA although its rates of increase goes above 10 fold.
Immunohistochemistry shows that a rapid inflammatory response is followed by initiation of endothelial growth, typical of the laser-induced CNV. Moreover the laser injury triggered the formation of a proinflammatory and anti-inflammatory lipid mediators. Novel mediators resolvins D1, D4, D5, and E1, as well as NPD1 are generated during CNV. Our results have uncovered cascades and networks of lipid mediators during the initiation and progression of CNV. These observations will allow to study the mediators bioactivity and their potential use to manage CNV.
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