Abstract
Purpose: :
To investigate the effects of indocyanine green (ICG) on human retinal pigment epithelial (RPE) cells and photosensitization of ICG by continuous illumination of visible rays.
Methods: :
RPE cells were cultured in the dark for 48 hours in a medium containing 10 µg/mL ICG and propidium iodide (PI) to detect dead cells. After fixation and nuclear staining with DAPI, the cells were microscopically observed. In another experiment, the cells were cultured in a colorless medium containing ICG (0 to10 µg/mL) for 24 hours in the dark or under continuous illumination of fluorescent lamp (daylight, 2000 Lux). After microscopic observation, cell viability was assayed by MTS method and cell death was evaluated by measuring LDH released from dead cells. TUNEL was done to detect apoptotic cells. Absorbance spectra of 10 µg/mL ICG in the colorless medium were measured after 24-hour incubation with or without the cells in the dark or under the illumination.
Results: :
As well as the culture without ICG, almost all cells maintained normal flattened morphology and PI-positive cells were rare in ICG-containing medium after 48 hours. The cells stained both with ICG and PI usually had condensed nuclei. Although there were few, ICG-stained cells without PI staining were present, implying incorporation of ICG into the cells preceding cell death. Some of them had condensed nuclei. After 24-hour culture under the illumination, the cells changed their morphology to be shrunken and oval shape, cell viability decreased, and dead cells increased dependently on ICG concentration while not in the dark. There were more TUNEL-positive cells in the culture under the illumination than in the dark at each ICG concentration. Absorbance spectrum of ICG showed two peaks at about 725 nm and 785 nm, and fell a half after incubation in the dark without cells. When incubated in the dark with cells, the two peaks were lower than without cells, but clearly recognized. Illuminated incubation dramatically decreased the absorbance of ICG between 590 nm and 900 nm, and the relative absorbance at 725 nm was lower than in the dark. The presence of cells lowered the absorbance of ICG in incubation under the illumination as well as in the dark.
Conclusions: :
ICG was incorporated into RPE cells and induced apoptosis. Continuous illumination of visible rays changed ICG to be another strong cytotoxic agent with different spectrum.
Keywords: retinal culture • apoptosis/cell death • drug toxicity/drug effects