Purpose: One of the most prominent changes that occurs in the retinal pigment epithelium during senescence is the progressive accumulation of the autofluorescent pigment lipofuscin. Experiments were conducted to evaluate the role of nonenzymatic oxidation of photoreceptor outer segments in retinal pigment epithelium lipofuscin formation.
Methods: Albino Fischer rats were given intravitreal injections of ferrous sulfate, a catalyst that promotes nonenzymatic lipid oxidation. At 2 hours, 24 hours, and 7 days after ferrous sulfate administration, the retinas were examined with fluorescence microscopy to assess the formation of fluorescent products. At these same time intervals, organic solvent extracts of the retinas and retinal pigment epithelium-choroid complexes were prepared. The extracts were analyzed with thin layer chromatography to assay for the presence of soluble fluorophores. The ultrastructural appearances of the retinas were examined at the same time points.
Results: At both 2 hours and 24 hours after the ferrous sulfate treatment, the photoreceptor outer segments displayed a yellow-green fluorescence emission that was not present in untreated eyes. Associated with this in situ fluorescence were a number of blue-green emitting fluorophores in organic solvent extracts that did not correspond to any of the fluorophores extracted from the retinal pigment epithelium of old animals. One week after the ferrous sulfate treatment, the photoreceptor cells had degenerated and the retinal pigment epithelium contained large amounts of an autofluorescent pigment with a golden-yellow emission typical of lipofuscin. The iron-induced fluorophores could not be extracted from this pigment into either chloroform or dichloromethane.
Conclusions: The initial fluorophores that were formed as a result of nonenzymatic oxidation of outer segment components did not appear to be the same as those responsible for retinal pigment epithelium lipofuscin fluorescence. However, after the oxidized outer segments were phagocytosed by the retinal pigment epithelium, the latter cells became filled with a yellow-emitting fluorescent pigment that was similar in its fluorescence properties to lipofuscin. These observations suggest that lipofuscin fluorophores are not direct products of nonenzymatic lipid oxidation. However, some of these oxidation products may be modified after uptake by the retinal pigment epithelium to form insoluble lipofuscin fluorophores.