Ultrastructural localization of hydrogen peroxide in experimental autoimmune uveitis.

One of the most prominent features of S-antigen induced uveitis is the massive infiltration of polymorphonuclear leukocytes (PMNs) and mononuclear cells in the ocular tissues and fluids. These inflammatory cells generate reactive oxygen metabolites as microbicidal agents and release these oxidants into the surrounding tissues. Using the cerium perhydroxide method, we have localized subcellular hydrogen peroxide in various inflamed ocular tissues. Most notably, the positive electron-dense granules were seen in the plasma membranes of PMNs that were infiltrating in the retina and uvea. These deposits were noted also in PMNs located within the extravascular spaces. For the intravascular PMNs, the positive reaction products were seen in much lower concentrations. A direct demonstration of substantial concentrations of hydrogen peroxide in experimental autoimmune uveitis, therefore, suggests the possibility that this reactive metabolite is an inflammatory mediator in this condition.

Chemotactic activity of the peroxidized retinal membrane lipids in experimental autoimmune uveitis.

We investigated the mechanism for amplification of intraocular inflammation in rats with experimental autoimmune uveitis by examining the chemotaxis potentials of peroxidized lipids extracted from the retinas. Utilizing thin layer chromatography, we found that the peroxidized products isolated from the inflamed retinas were fatty acid hydroperoxides that corresponded to the autooxidized products from commercial methyl docosahexaenoate, with Rf values ranging from 0.30 to 0.37. These were not demonstrated in similar preparations from normal retinas or in unoxidized docosahexaenoate. Boyden chamber assay revealed that the hydroperoxides isolated from inflamed eyes and the products of oxidized methyl docosahexaenoate possessed significantly higher chemotactic activity than did the retinal lipids isolated from normal eyes (P less than 0.01). These findings may help to explain the mechanism of inflammatory amplification induced by peroxidized retinal lipids that is seen in this animal model of uveitis.

Histochemical localization of superoxide production in experimental autoimmune uveitis.

Although the presence and role of oxygen reactive species in uveal inflammation is the subject of intense investigation, there is little direct evidence that oxygen metabolites are present at the site of inflammation. We used the nitroblue tetrazolium test for superoxide to determine production of this oxygen reactive species in experimental autoimmune uveitis (EAU). The choroidal tissues of animals with this disease contained intracellular, blue-staining granules. Most of the positive staining cells appeared to be polymorphonuclear leukocytes. This localization of superoxide in EAU is further evidence of the generation of oxygen reactive species in uveal inflammation.

Immunohistochemical localization of peroxidative enzymes in ocular tissue.

Localization of the peroxidative enzymes catalase and glutathione peroxidase in rabbit ocular tissue was investigated by immunohistochemical methods. We raised antisera to both enzymes in rabbits using commercially available purified enzymes. Immunoreactive catalase and glutathione peroxidase were found in the corneal epithelium and endothelium, the choroid, the inner segment of photoreceptors, and the retinal pigmented epithelium.

Immunohistochemical localization of glutathione peroxidase in ocular tissue.

In this study enzyme glutathione peroxidase was localized in ocular tissue of Lewis rats using the peroxidase antiperoxidase immunohistochemical technique. Antisera to glutathione peroxidase was raised in a rabbit. Immunoreactive glutathione peroxidase was found to be distributed predominantly in the corneal epithelium and endothelium, choroid, inner segment of photoreceptors and retinal pigmented epithelium. Its presence in these sites suggests an adaptation to oxidative stress where this antioxidant enzyme along with other antioxidant systems serves to prevent damage.

Hydrogen peroxide localization in ocular tissue: an electron microscopic cytochemical study.

A reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent hydrogen peroxide-generating activity of rat ocular tissue was investigated cytochemically utilizing the cerium method. NADPH or NADH oxidation resulted in electron-dense fine granular deposits on the corneal epithelial and endothelial plasma membranes, around the photoreceptors of the retina, and on the processes of the retinal pigmented epithelium. Control specimens incubated in a substrate-free medium did not show any such deposits. These results suggest that, under normal conditions, there is generation of oxidants, such as hydrogen peroxide, in the ocular structures, and that such oxidants can be visualized by cytochemical techniques.

Demonstration of cytomegalovirus retinitis by in situ DNA hybridization.

Five cases of the acquired immune deficiency syndrome (AIDS), each exhibiting retinitis, were studied by DNA hybridization technique to detect viral infection. Five involved eyes were obtained at the time of autopsy from three patients who had received no treatment for the retinitis. Retinal biopsy specimens were obtained at the time of surgery from two other patients who developed retinal detachments after being treated with ganciclovir (dihydroxy propoxymethyl guanine). DNA hybridization revealed cytomegalovirus in retina and retinal pigment epithelium in all five specimens from patients who had not been treated with ganciclovir. No hybridization occurred in the two retinal biopsy specimens obtained from the ganciclovir-treated patients. These results suggest that in situ DNA hybridization is a highly specific and easily interpretable means of establishing the tissue diagnosis of viral retinitis.