Abstract
Abstract: :
Purpose: Lipofuscin, which is accumulated in the cytoplasm of retinal pigment epithelial (RPE) cells in aging eyes, is supposed to derive from oxidation products in outer segment of photoreceptor cells. Because of high supply of oxygen and glucose around RPE cells, such oxidation products may be further modified through glycoxidation process. In the previous study, we reported a new drusen model in rabbits implanted with advanced glycation (glycoxidation) end products (AGEs). The objective of this study was to investigate the impact of AGEs on RPE cells in vitro and in vivo. Methods: Highly glycoxidized albumin microspheres were prepared with incubation of rabbit serum albumin (RSA) with glycolaldehyde and bound to rhodamine B isothiocyanate, used as MS(AGER). Control microspheres [MS(RSAF)] were made by use of glutaraldehyde and fluorescein isothiocyanate. After each or both microspheres were added in the culture medium of human RPE cells, cell viability, adhesion activity, and proliferation activity were assessed by cell counting. Phagocytosed microspheres were observed for 4 weeks by fluorescein microscopy. In rabbit eyes, 250 µg of MS(RSAF) and/or MS(AGER) were injected into the subretinal space. Eyes were enucleated at week 2 or 4. After eyecups were made, the implanted lesion was observed by fluorescein microscopy. Results: When RPE cells were challenged with 100 microspheres per cell, all cells phagocytosed them. In these groups, 1-day incubation did not impair the cell viability of RPE cells. Detached cells adhered again as well as in control. Proliferation was minimally attenuated on days 3 and 5 with no difference between two groups of MS(RSAF) and MS(AGER). At week 4, while most phagocytosed MS(RSAF) were degraded or deposited, some MS(AGER) were still stagnated in the cytoplasm of RPE cells. In the eyes with MS(RSAF), microspheres decreased at week 4. On the other hand, MS(AGER) were sustained even at week 4. In the eyes with both microspheres, only MS(RSAF) decreased at week 4. In eyes with MS(AGER), drusen was formed at week 4 significantly more frequently than in those with MS(RSAF). Conclusions: Advanced glycoxidation products tended to stagnate in the cytoplasm of RPE cells after phagocytosis with no acute impairment of cell viability and adhesion activity and with slight proliferation activity loss, resulting in the formation of drusen. These results suggest that the overload of hardly digestible lipofuscin itself may play a role in drusen biogenesis.
Keywords: age-related macular degeneration • retinal pigment epithelium • drusen