Katz M L, Christianson J S, Gao C L, Handelman G J
Mason Institute of Ophthalmology, University of Missouri School of Medicine, Columbia 65212.
Invest Ophthalmol Vis Sci. 1994 Sep;35(10):3613-24.
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.
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.
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.
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.
玻璃体内注射铁可诱导光感受器外段形成荧光团，随后视网膜色素上皮（RPE）中出现具有脂褐素样荧光的内含物积聚。衰老过程中RPE脂褐素的积聚取决于维生素A的供应情况。进行实验以确定铁诱导外段和RPE中荧光团形成是否也依赖于维生素A，从而确定氧化是否促进维生素A参与脂褐素形成。
从断奶开始，对白化费希尔大鼠喂食含维生素A的饲料23周，维生素A的形式为棕榈酸视黄酯（+A），其可代谢转化为参与视觉的类视黄醇，或视黄酸（-A），其不支持视觉功能。23周后，当-A组大鼠视紫红质水平下降超过90%时，该组中的一些动物接受肌肉注射全反式视黄醇，并从类视黄醇缺乏中恢复7天（-A+A）。然后对所有三个治疗组的动物进行玻璃体内注射硫酸亚铁。在铁注射后1天和7天，检查视网膜和RPE中具有与RPE脂褐素荧光团相似激发和发射特性的荧光团。
在硫酸亚铁处理后24小时用荧光显微镜检查的视网膜切片中，所有治疗组大鼠的光感受器外段均显示出最大发射波长为蓝色的荧光。未处理的眼睛中不存在这种外段荧光。原位外段荧光与视网膜有机溶剂提取物中发射蓝光的荧光团的出现相关。铁注射后1周，+A组动物的RPE细胞充满内含物，当用蓝光激发时显示出金黄色荧光发射。铁处理后1周，在-A组大鼠的RPE中很少观察到这种脂褐素样荧光。然而，在补充了维生素A的-A组大鼠中，铁诱导RPE荧光的能力得以恢复。+A组大鼠的RPE-脉络膜有机溶剂提取物中存在几种发射橙色荧光的荧光团。这些荧光团的量未受到铁处理的明显影响。在-A组或-A+A组任何眼睛的提取物中均未观察到这些发射橙色荧光的化合物。
本研究结果表明，光感受器外段脂质的氧化产生发射蓝光的荧光团，其不直接参与RPE脂褐素荧光团的形成。研究结果还表明，类视黄醇是RPE脂褐素荧光团的直接前体，并且视网膜的氧化应激促进维生素A参与形成一些导致RPE脂褐素荧光的化合物。
