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ORIGINAL RESEARCH ARTICLE

Corneal Protein Nitration in Experimental Uveitis

Mutay Aslan^*,1, clal Yucel, Akif Ciftcioglu, Berna Sava, Yusuf Akar, Gultekin Yucel^* and Salih Sanlioglu^||

^* Departments of Biochemistry, Ophthalmology, and Pathology, Akdeniz University School of Medicine, Antalya 07070, Turkey; Department of Pathology, Ankara University Medical School, Ankara 06100, Turkey; and ^|| Human Gene Therapy Unit, Akdeniz Unversity School of Medicine, Antalya 07070, Turkey

To whom requests for reprints should be addressed at ¹ Akdeniz University Medical School, Department of Biochemistry, 07070 Antalya, Turkey. E-mail: mutayaslan{at}akdeniz.edu.tr

Increased expression of inducible nitric oxide synthase (NOS-2) in inflammatory diseases like uveitis suggests that it contributes to the observed pathological state. The aim of this study was to evaluate corneal expression of NOS-2 and corneal protein nitration in a rat model of uveitis. A single injection of intravitreal lipopolysaccharide was used to induce uveitis. Corneal proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by Coomassie blue staining. Expression of NOS-2 and nitrotyrosine (NO₂Tyr) formation were determined via immunohistochemistry and Western blot analysis. Total nitrate/nitrite levels in the vitreous were measured by spectral analysis via the Griess reagent. Immunohistochemical analysis revealed increased corneal NOS-2 and NO₂Tyr immunoreactivity in rats with uveitis compared with controls. NOS-2 and NO₂Tyr immunoreactivity was observed in and around basal cells in the corneal epithelium. Western blot analysis of corneal lysates showed multiple nitrated protein bands in uveitic rats. Spectrophotometric measurement of total nitrate/nitrite levels in the vitreous affirmed significantly increased levels of nitric oxide generation in uveitis (126 ±2.63 µM/mg protein) compared with controls (65 ±6.57 µM/mg protein). The presented data suggests that extensive formation of protein nitration and reactive nitrogen species in the cornea contributes to tissue destruction in uveitis. Hence, selective inhibition of NOS-2 may prevent long-term complications and lead to an improvement in the management of uveitis.

Key Words: uveitis • cornea • nitration • inducible nitric oxide synthase