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The goal of this study is the characterization of the strong yellow fluorescence of oxidized melanin in the retinal pigment epithelium (RPE) and the choroid.Naturally occurring melanin in the human retina and choroid was oxidized by exposing fixed and plastic-embedded sections of a human eye to light and hydrogen peroxide. Synthetic melanin was also oxidized in vitro by exposure to light and hydrogen peroxide. The fluorescence of oxidized melanin was examined by absorption spectroscopy, fluorescence spectroscopy, and fluorescence microscopy.Naturally occurring melanin oxidized in situ exhibited a lipofuscin-like yellow fluorescence. Oxidation of melanin in vitro degraded the melanin polymer, resulting in a fluorescent solution. Fluorescence spectroscopy gave an excitation maximum at approximately 470 nm and an emission maximum at approximately 540 nm for both natural and synthetic melanin. Increasing the time of exposure to light or hydrogen peroxide increased melanin fluorescence.The results indicate that the strong yellow fluorescence of melanin in the RPE and choroid in situ is a property of oxidized melanin and is not due to contamination of the melanin by proteinaceous or lipid materials. The data presented allow a reinterpretation of the results obtained from fluorescence investigations of melanin-containing tissue and suggest a link between melanin degradation and lipofuscin formation.

Our study was performed to test the hypothesis that subretinally injected protein can induce melanogenesis in the retinal pigment epithelium (RPE). Rod outer segments (ROS) were isolated from cattle eyes and injected into the subretinal space of Long Evans rats. Five days after surgery, the injected eyes were investigated by electron microscopy. The number of early-stage melanosomes and small melanin granules was compared in injected and noninjected eyes. It was found that the injected ROS were phagocytized by the RPE cells, and the number of early-stage melanosomes in the RPE was significantly increased in injected eyes compared to eyes without injection. The ROS-containing endosomes fused with melanolysosomes in which melanogenesis took place. The increased number of early-stage melanosomes indicates new formation of melanin.

Localization of light-induced lipid peroxides in the rat retina at an ultrastructural level as benzidine-reactive substances.Long-Evans rats with nondilated pupils were exposed to intense light of 6000 lux for 12 or 24 hours. Control animals were kept under physiological light conditions. Rats with dilated pupils were exposed to a light intensity of 50 lux or 150,000 lux for 1 hour. For ultrastructural localization the enucleated eyes were fixed in a 0.1-M cacodylate buffer (pH 7.4) containing 2% glutaraldehyde for 2 hours. Pieces of the superior part of the central eyecup were incubated overnight with tetramethylbenzidine (TMB; pH 3.0) at 4 degrees C, postfixed with 1.5% OSO4, and embedded for electron microscopy.In animals exposed to intense light, electron-dense structures appeared exclusively throughout the rod outer segments after an irradiation of 6000 lux for 24 hours or 150,000 lux for 1 hour and were absent in animals with nondilated pupils kept at physiological light conditions. Dilation of the pupils leads to the appearance of electron-dense structures after just 1 hour of 50 lux, whereas rats with nondilated pupils withstand even a 12-hour irradiation with 6000 lux. No electron-dense structures were found when no TMB was used in incubation.The appearance of electron-dense structures in the rod outer segments depends on the incubation with TMB and intensive light exposure of the rat. Dilation of the pupils lowers the threshold for the emergence of electron-dense structures significantly. This strongly supports the view that light-induced lipid peroxides in the rat retina are localized at an ultrastructural level as benzidine-reactive substances. This protocol presents a tool for the generation and ultrastructural localization of lipid peroxides in rat retinas.

Lipid peroxidation is considered a prominent feature of age-related retinal degeneration. It is known that lipid peroxides can oxidize benzidine. This property was used to localize lipid peroxides ultrastructurally in the retina.(1) Lipid peroxides were formed by incubation of linoleic acid with lipoxygenase from soybean, separated by thin layer chromatography and incubated with tetramethylbenzidine. (2) Lipid peroxides were formed by incubation of porcine retinae with soybean lipoxygenase in an oxygensaturated atmosphere. For ultrastructural localization, isolated retinae with and without enzymatically synthesized lipid peroxides were fixed with 2% glutaraldehyde, incubated with 0.5 mg/ml tetramethylbenzidine and embedded for electron microscopy. (3) Eye cups from Syrian golden hamsters were treated in the same way except for incubation with lipoxygenase. The hamsters were kept under constant illumination (1000 lux) for 12 h to enhance lipid peroxidation.(1) Tetramethylbenzidine was oxidized by linoleic acid peroxides. (2) In the isolated retinae of pigs lipid peroxides became visible as electron-dense structures in the rod outer segments (ROS) after treatment with lipoxygenase and were lacking in the other parts of the retina. Without treatment with lipoxygenase lipid peroxides were only infrequently seen in ROS. (3) In the eyes of light-exposed hamsters, electron-dense reaction products of lipid peroxides were particularly prominent between the basal infoldings of the RPE and within the apical parts of the ROS.Light or enzymatically induced lipid peroxides can be localized ultrastructurally due to their ability to react with tetramethylbenzidine and osmium in the absence of H2O2 to an electron-dense reaction product. Lipid peroxides seem to be removed from the RPE via Bruch's membrane and blood vessels. Disturbance of this pathway may enhance lipofuscin or drusen formation.