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Karen Joos, Raymond Mernaugh, Ratna Prasad, Pengcheng Lu, Lyman Roberts; Lipid Peroxidation in the Rat Retina after Elevated Intraocular Pressure. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3053.
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Evidence of oxidative stress has been demonstrated in tissues with glaucoma damage. Oxidative stress may lead to lipid peroxidation including formation of highly-reactive γ-ketoaldehydes (γ-KA) which rapidly form covalent adducts to proteins, phospholipids, or DNA. In the current study, we determined whether there is increased γ-KA protein adducts within the eye following early moderate intraocular pressure (IOP) elevation.
The study was approved by the Vanderbilt IACUC and conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. IOP was transiently elevated for 1 hour with an adjustable lasso around the right topically anesthetized eye of Sprague-Dawley rats. IOP was measured before, immediately after, at the end of 1 hour treatment, and 1 hour after rest using TonoLab tonometry (ICare, Finland). Rats were euthanized and retinas immediately harvested for SDS-PAGE/Western blot analysis, or rats were euthanized and perfused transcardially with phosphate buffered saline followed by 3% formaldehyde, 0.1% glutaraldehyde (v/v) and 0.2% saturated picric acid (v/v), in 0.1M phosphate buffer. Ocular sagittal sections were prepared for immunohistochemistry (IHC) with a single-chain antibody that recognizes γ-KA protein adducts on all proteins. Intensity of immunoreactivity was assessed semi-quantitatively using the MetaMorph® Microscopy Automation & Image Analysis 7.6 Software (Molecular Services, Sunnyvale, CA).
Mean baseline IOP of 19.6 ± 1.4 mm Hg increased to 43.6 ± 1.1 mm Hg (P < 0.01) during 1-hour treatments and returned to 20.4 ± 2.3 mm Hg (P = 0.75) 1 hour after completion. The final IOP also was not significantly different from the final control eye IOP of 20.4 ± 0.9 mm Hg (P = 0.75). Elevated γ-KA protein adducts were found in the Western Blot retinal lysate, and in the inner plexiform layer of the IHC experimental eyes compared to the paired contralateral control eyes (n =10) with an OD/OS ratio = 1.55 ± 0.23, (P = 0.006, Wilcoxon Rank Sum Test).
One hour of moderate elevation in IOP increased the presence of γ-KA adducts within the inner retina in the rat model. Lipid peroxidation appears present within the eye following early IOP elevation.
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