Abstract
Purpose: :
In vivo exposure of primate RPE to bright light causes an immediate reduction in autofluorescence (AF) followed by complete recovery within about 4 hours. AF reduction can be observed at light levels that are two orders of magnitude below the ANSI photochemical maximum permissible exposure. We developed an in vitro model of AF reduction to clarify its photochemical basis and its implications for light safety.
Methods: :
ARPE-19 cells that had accumulated the bisretinoid A2E (m/z 592) were exposed to 0.3 - 0.5 J/cm2 of 480 or 430 nm light. Analysis was by fluorescence microscopy or UPLC (ultraperformance liquid chromatography) with monitoring by mass spectrometry, absorbance and fluorescence. Irradiated cell-free A2E was also studied.
Results: :
AF reduction and recovery were observed in the in vitro models; AF reduction occurred at light levels lower than those associated with the in vivo response. AF reduction was associated with A2E consumption (measured as absorbance) and accompanying increases in some oxidized forms of A2E (oxo-A2E) even under conditions that permitted fluorescence recovery as detected by UPLC. Recovery from AF reduction was dependent on irradiation dose and was facilitated by pre-treatment with the antioxidant vitamin E. Photoisomers of A2E exhibiting similar or greater fluorescence efficiency formed immediately upon irradiation. At the light levels utilized thus far, AF recovery was not observed in the cell-free assay.
Conclusions: :
A2E is at least one of the bisretinoids exhibiting bleaching and recovery that is qualitatively similar to the AF reduction observed in vivo. Exposing A2E to light leads to both photoisomerization and photooxidation. Whether the photoproducts produced by light exposures lower than those that lead to the structural disruption of RPE cells have deleterious consequences in vivo is not known. Protective mechanisms may help mitigate harmful effects of light exposure.
Keywords: retinal pigment epithelium • ipofuscin • imaging/image analysis: non-clinical