Lipofuscin formation in cultured retinal pigment epithelial cells exposed to photoreceptor outer segment material under different oxygen concentrations

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Abstract
Lipofuscin accumulates in the course of time in the acidic vacuolar apparatus of retinal pigment epithelial (RPE) cells and may influence their metabolic functions. In order to study the effect of oxidative stress on lipofuscin accumulation, rabbit RPE cell cultures were kept at an ambient oxygen concentration of either 8% or 40%. To simulate the normal phagocytic function of RPE cells, bovine photoreceptor outer segments (POS) were added daily. The lipofuscin-specific autofluorescence was measured after 1, 2 and 3 weeks. RPE cells cultured under normobaric hyperoxic conditions (40% oxygen) showed significantly higher levels of lipofuscin-like autofluorescence than those kept under normobaric and probably normoxic conditions (8% oxygen) after 1 (p = 0.0050), 2 (p = 0.0001) as well as 3 (p = 0.0077) weeks. At both oxygen concentrations, the lipofuscin accumulation level was increased after 2 weeks of POS exposure (40% p = 0.0001; 8% p = 0.0037) and even further after 3 weeks (40% p = 0.0541; 8% p = 0.0377). The results suggest an involvement of oxidative mechanisms in the formation of lipofuscin from phagocytized POS by RPE cells. The autofluorescence of control cells, not exposed to POS, was significantly (40%: 1 week p = 0.0011, 2 weeks p = < 0.0001, 3 weeks p = 0.0001; 8%: 1 week p = 0.0036, 2 weeks p = 0.0063, 3 weeks p = 0.0066) lower than that of the POS-fed cells. The autofluorescence increased significantly (40% p = 0.0059; 8% p = 0.0034) between week 1 and week 3 in the control cells. This finding may reflect a contribution to lipofuscin formation by autophagocytized intracellular material. The present model seems to be useful for further studies on the mechanisms behind lipofuscinogenesis of RPE cells as well as the possible effects of lipofuscin accumulation on cell functions and viability.