Blue LED light exposure develops intracellular reactive oxygen species, lipid peroxidation, and subsequent cellular injuries in cultured bovine retinal pigment epithelial cells.

The effects of blue light emitter diode (LED) light exposure on retinal pigment epithelial cells (RPE cells) were examined to detect cellular damage or change and to clarify its mechanisms. The RPE cells were cultured and exposed by blue (470 nm) LED at 4.8 mW/cm(2). The cellular viability was determined by XTT assay and cellular injury was determined by the lactate dehydrogenase activity in medium. Intracellular reactive oxygen species (ROS) generation was determined by confocal laser microscope image analysis using dihydrorhodamine 123 and lipid peroxidation was determined by 4-hydroxy-2-nonenal protein-adducts immunofluorescent staining (HNE). At 24 h after 50 J/cm(2) exposures, cellular viability was significantly decreased to 74% and cellular injury was significantly increased to 365% of control. Immediately after the light exposure, ROS generation was significantly increased to 154%, 177%, and 395% of control and HNE intensity was increased to 211%, 359%, and 746% of control by 1, 10, and 50 J/cm(2), respectively. These results suggest, at least in part, that oxidative stress is an early step leading to cellular damage by blue LED exposure and cellular oxidative damage would be caused by the blue light exposure at even lower dose (1, 10 J/cm(2)).

Local fundus response to blue (LED and laser) and infrared (LED and laser) sources.

Light damage research began during the early years of laser light exploration. There is a clear and significant literature that identifies an easily demonstrated retina-pigment epithelium pathology which is associated with short wavelength exposures below 520 nm. Recent interest has expanded because of the growing evidence for a blue light contribution to the retina aging process by way of a poorly understood chemical process(es) that involve circulation, oxidative reactions and the spectral absorption properties of the pigment epithelium. New powerful sources of relatively inexpensive blue energy have become available as a family of light emitting diodes. In this experiment, we examined funduscopic, angiographic and scanning laser tomographic measures of the retinal-pigment epithelium response to LED and laser spectral blue and infrared emissions closely matched in wavelengths and delivered under carefully matched circumstances. Ten retinas in normal young rhesus monkeys were locally exposed to various energy density values at 458 nm (Argon laser) ranging from 5 to 54 J cm(-2). Eight rhesus eyes were exposed to LED irradiation with a peak wavelength of 460 nm ranging from 9 to 62 J cm(-2). Similarly, a matched infrared (IR) laser and IR LED pair were used to expose an additional ten eyes for comparison of the long wavelengths. IR irradiance ranged from 21 to 306 J cm(-2). There was no response to IR exposure in any of the eyes. Blue light exposure results were measured from the color fundus photographs, scanning laser tomographs and early- and late-phase fluorescein angiogram responses at 2 and 30 days after the exposure. Results scores were accumulated for the four measures at the two time periods. The resulting lesion scores when plotted against the exposure in J cm(-2)showed no demonstrable effect at irradiance lower than 10 J cm(-2)and near 100% effectiveness for irradiance greater than 30 J cm(-2). The most sensitive and enduring indicator of change was the late fluorescein angiograms. Nonparametric statistical analysis of the scores from the two samples support the conclusion that there is no difference in the consequences of LED and laser light exposures under these matched conditions.

In vitro testing of antioxidants and biochemical end-points in bovine retinal tissue.

Lipid peroxidation in aliquots of bovine retina (without rod outer segments, ROS), purified ROS and retinal pigment epithelium (RPE) was initiated with 5 mM ferric iron and 80 mM ADP. After 30 min of oxidation at 37 degrees C, the concentration of thiobarbituric-acid-reacting substances (TBARS) which approximates lipid hydroperoxide (LHP), increased in the ROS from 2.0 +/- 3.6 to 90.2 +/- 34.5 nmol malondialdehyde (MDA)/mg protein and in the RPE from 0.54 +/- 0.2 to 51.5 +/- 15.8 nmol MDA/mg protein. Sixteen lipid and aqueous antioxidants (AOX) from natural or synthetic sources, including five flavonoids, were evaluated for their ability to inhibit the oxidative reaction. Palm-oil-derived vitamin E showed significant protection in retina, ROS and RPE (64, 68 and 74%), respectively. Of the flavonoids tested, good protection in the retina was found at 10(-5) M for epigallocatechin gallate (50%) and at 50 ng/ml for pycnogenol (61%) and catechin (52%). When catechin and palm oil vitamin E, catechin and coenzyme Q(10) or coenzyme Q(10) and pycnogenol were combined, the individual effect was enhanced. TBARS as an indirect measure of LHP level and hemoglobin-methylene blue determination for direct LHP were used as alternative end-point determinations of lipid peroxidation. These measure different aspects of AOX reactions. The results demonstrate the usefulness of an in vitro model system that can rapidly and accurately determine the capacity of a single AOX against lipid peroxidation or be used to show synergistic efficacy.