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B.H. Jeng, K.G. Shadrach, D.M. Meisler, J.G. Hollyfield, T. Koeck, K.S. Aulak, D.J. Stuehr; Immunohistochemical Evaluation of the Inhibition of Protein Nitration in Human Corneal Epithelial Cells During Cold Storage . Invest. Ophthalmol. Vis. Sci. 2005;46(13):868.
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Purpose: We have previously demonstrated that nitric oxide (NO) production occurs in corneal tissue during storage prior to transplantation and that this production, which leads to protein nitration, can be inhibited with the addition of a nitric oxide synthase inhibitor to the corneal storage media. The aim of this study was to determine if the inhibition of NO production would lead to a decrease in immunohistochemical staining for nitrated protein in corneal tissue during storage. Methods: Paired human corneas stored in Optisol–GS corneal storage media were obtained from the Cleveland Eye Bank. NG–monomethylene–L–arginine (LMMA), a non–selective nitric oxide synthase inhibitor, was added to 20 ml of storage media of one cornea from each pair of corneas to a final concentration of 2mM. Paired corneas were stored for 3 days and then bisected. One half of each cornea was placed in formalin fixative, and the remaining halves of each cornea were returned to their original storage media and stored for 4 additional days. The remaining tissue was then subjected to the same treatment as the other halves. Mouse polyclonal antibody to nitrotyrosine was used as the primary antibody, and goat anti–mouse biotin–labeled antibody was used as a secondary antibody. The slides were then treated with a peroxidase ABC kit and photographed with a digital camera system. Results: Less immunohistochemical staining for nitrated protein was observed in epithelial cells which were exposed to LMMA than in those of the control group at day 7 of storage. No substantial differences in immunohistochemical staining occurred at day 3 between the inhibited and control groups. Conclusions: The addition of a nitric oxide synthase inhibitor to corneal storage media seems to decrease the amount of immunohistochemical staining for nitrated protein in human corneal epithelial cells during storage. This may correlate clinically to less damage to corneal cellular elements during storage due to the toxic effects of protein nitration.
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