X-RAY-MICROANALYSIS AND PHAGOCYTOTIC ACTIVITY OF CULTURED RETINAL-PIGMENT EPITHELIAL-CELLS IN HYPOXIA

Purpose: To investigate the influence of the functional and morphologi cal changes induced in retinal pigment epithelial (RPE) cells by retin al ischemia, we evaluated the phagocytotic activity, the concentration of various elements, and ultrastructure in cultured RPE cells in hypo xia. Methods: The concentrations of oxygen in incubators were adjusted to 20, 10, and 1% by the addition of nitrogen for 72 hr. To observe p hagocytotic activity and its relationship to actin filaments, the fila ments of RPE cells incubated with fluoresbrite carboxylate YG microsph eres were stained with rhodamine phalloidin. Some of the specimens wer e subjected to X-ray microanalysis by scanning electron microscope aft er being fixed, freeze-dried, and coated with carbon to investigate th e cytoplasmic concentration of elements. A part of the latter specimen s was also observed by transmission electron microscope after being em bedded in epon and cut into ultrathin sections to see the ultrastructu ral changes inside cell. Results: Lowering oxygen concentrations from 20% to 1% swelled RPE cells and decreased the number of fluoresbrite c arboxylate YG microspheres phagocytized by RPE cells. Phagocytosis of a large amount of latex beads (30 mu l) for 24 hr in 1% oxygen caused a disruption of RPE cells. Na, S, and P were detected in RPE cells cul tured in 20% oxygen, Reducing the oxygen concentration from 20 to 10 o r 1% significantly decreased Na and increased S. Mitochondria were obs erved in RPE cells in 20 and 10% oxygen, but many vacuoles were observ ed in the cytoplasm in 1% oxygen. Conclusion: Hypoxia as low as 1% oxy gen induced malfunction of phagocytosis and the fragility of RPE cells . We could speculate the imbalance of the electrolytes such as Na or a decrease of antioxidants such as glutathione containing S as a reason of disturbance of cell viability.