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W.J. O'Brien, C.L. Krema, B. Qian, T.G. Heimann; Superoxide Production in HSV-Infected Corneas/Anterior Segments of Rabbits . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4626.
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Purpose:Herpes simplex virus(HSV) infections of the cornea induce an inflammatory response in the tissues of the anterior segment that may result in impaired vision. The mechanisms responsible for the inflammation are not understood in adequate detail to permit the design of highly selective anti-inflammatory drugs. These studies were designed to investigate the pathways of superoxide production in the anterior segments of rabbits with herpetic stromal keratitis(HSK). Methods:Rabbits were infected with HSV-1 by injection of 10^5 pfu of HSV-1(RE) at the midline of the alveolar mucosa of the mandible. After 30 days the corneas of seroconverted but asymptomatic rabbits were infected by intrastromal injection of 10^3 pfu of HSV-1(RE) or by topical application of 10^5 pfu of HSV-1 McKrae or Shealy. Herpetic eye disease was evaluated by slit-lamp examination and a subjective scoring system. Corneal thickness was measured using an ultrasonic pachymeter. Animals were killed at various times during development of disease and the anterior segments were taken for analysis by RT-PCR, immunohistochemistry, and western blotting. Results:RT-PCR using primers specific for the components of the NADPH oxidase complex indicated that gp91 phox, p22 phox, p67 phox , p47 phox and p40 phox were constitutively expressed in central cornea, limbal regions and iris/ciliary body of both uninfected and HSV-1 infected rabbits. In addition a cell specific isoform of gp91 phox, NOX 1 was also expressed. Sequencing of the PCR products confirmed 97%-100% base sequence identity to the appropriate published rabbit sequences. Incubation of frozen sections with 10 uM dihydroethidine suggested that cells of the anterior segment and infiltrating macrophages and PMNs constitutively produced superoxide, albeit in differing amounts. Immunohistochemistry(IHC) documented the expression of gp91 phox in infiltrating macrophages and PMNs. IHC also showed the expression of NOX 1 and the remaining subunits of the NADPH oxidase complex in both infiltrating cells and some resident tissue cells. Cultures of corneal epithelial and stromal cells also constitutively expressed mRNAs of the component genes of the NADPH oxidase complex and produced superoxide as measured by the reduction of cytochrome C. Conclusions: Our results document that resident tissue cells are capable of producing superoxide via a NADPH oxidase complex similar to that existing in PMNs and macrophages. Further studies will be required to determine the role of superoxide in the pathogenesis of HSK.
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