Iron-induced fluorescence in the retina: dependence on vitamin A.

PURPOSE

Intravitreal iron injection induces fluorophore formation in the photoreceptor outer segments, followed by an accumulation of inclusions with lipofuscin-like fluorescence in the retinal pigment epithelium (RPE). The accumulation of RPE lipofuscin during aging is dependent on vitamin A availability. Experiments were conducted to determine whether iron-induced fluorophore formation in the outer segments and in RPE is also dependent on vitamin A, and thus whether oxidation promotes the participation of vitamin A in lipofuscin formation.

METHODS

For 23 weeks, beginning at weaning, albino Fischer rats were fed diets containing vitamin A either in the form of retinyl palmitate (+A), which can be metabolically converted into the retinoids involved in vision, or retinoic acid (-A), which does not support visual function. After 23 weeks, when rhodopsin levels had decreased more than 90% in the -A rats, some animals in this group were given an intramuscular injection of all-trans retinol and were allowed to recover from retinoid deficiency for 7 days (-A+A). Animals in all three treatment groups were then given an intravitreal injection of ferrous sulfate. Both 1 day and 7 days after the iron injections, the retinas and RPEs were examined for fluorophores with excitation and emission properties similar to those of RPE lipofuscin fluorophores.

RESULTS

In retina sections examined with fluorescence microscopy 24 hours after the ferrous sulfate treatment, the photoreceptor outer segments of rats in all of the treatment groups displayed a fluorescence with a blue emission maximum. This outer-segment fluorescence was not present in untreated eyes. The in situ outer-segment fluorescence was correlated with the appearance of blue-emitting fluorophores in organic solvent extracts of the retinas. One week after the iron injections, the RPE cells of the +A animals became filled with inclusions that displayed a golden-yellow fluorescence emission when excited by blue light. Very little of this lipofuscin-like fluorescence was observed in the RPE of the -A rats 1 week after iron treatment. However, in the -A rats that had been repleted with vitamin A, the ability of iron to induce the RPE fluorescence was restored. Several orange-emitting fluorophores were present in organic solvent extracts of the RPE-choroids of the +A rats. The amounts of these fluorophores were not appreciably affected by the iron treatment. These orange-emitting compounds were not observed in extracts of any eyes in the -A or -A+A groups.

CONCLUSIONS

The results of this study suggest that oxidation of the photoreceptor outer-segment lipids generates blue-emitting fluorophores that are not directly involved in RPE lipofuscin fluorophore formation. The findings also indicate that retinoids are direct precursors of RPE lipofuscin fluorophores, and that oxidative stress to the retina promotes participation of vitamin A in the formation of some of the compounds responsible for RPE lipofuscin fluorescence.