Blue Light Damage To Retinal Pigment Epithelial Cells And Protection Of Blue Light-filtering Intraocular Lens

Blue light-induced retinal damage in ratsObjective To evaluate blue light-induced retinal damage in rats.Methods Albino rats were exposed to diffuse, cool blue light for short time periods ofup to 20 or 60 minutes. Animals were killed after different light exposure. The eyeswere enucleated and the lower central retina was processed for light and electronmicroscopy. DNA fragmentation was analysed in situ by TdT-mediated d-UTP nickend labeling (TUNEL).Results We observed that the timing of apoptosis in the pigment epithelial cells andphotoreceptors was remarkably different, the photoreceptors showing a distinct delaybefore the onset of apoptosis. Blue light-damage was found most prominently in theretinal pigment epithelium.Part 2 The characteristic of cultured human retinal pigment epithelial cellsObjective To culture human retinal pigment epithelial (RPE) cells and observe their characteristic in vitro. To provide basis for study of blue-light induced RPE cell damage.Methods Six human donor eyes were obtained from eye bank of Eye & ENT Hospital of Fudan University. From eyes ranging in age from 24 to 35 years (male, died of accidents, and no significant systemic and ophthalmologic history), the RPE cells were isolated with trypsin and cultured in vitro. The growth, morphological features and division of RPE cells were observed under phase contrast microscope. The growth curve was drawn based on growth data. The cultured RPE cells were identified by immunochemistry method. The RPE cells were also observed with transmission electron microscope.Results Human RPE cells grew exuberantly in vitro. RPE cells attached 1 day after seeding, circular shape, with affluent melanin granules intracellular. After 3-4 days, they became thin and flat. With increased division times, intracellular melanin granules decreased gradually. Cells were confluent at about 7 days. Withimmunochemistry method, cell plasma was stained brown. Under transmission electron microscope, there were abundant mitochondria and melanin granules, microvilli, basal membrane folds and intercellular junction complexes.Part 3The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigment epithelial cellsObjective To optimizing the in vitro synthesis of lipofuscin fluorophore A2E derived from all-trans-retinal and ethanolamine, observe the characterization of A2E and intemalization of A2E by RPE cells; To determine whether A2E participates in blue light-induced damage to RPE cells and compare protection effects of blue light-filtering intraocular lens (IOL) and ultraviolet filtering IOL against blue light damage. Methods1. A mixture of all-trans-retinal (100mg) and ethanolamine (9.5mg) was used to produce A2E in one step. A solution of HPLC-purified A2E in methanol (=40uM) was subjected to room temperature and room light. The extent of isomerization was analyzed by RP-HPLC. A2E granules were delivered to cultured RPE cells from 25 uM concentrations in media for internalization.2. Human RPE cells accumulated A2E from 25, 50, 75 and 100uM concentrations in media. Confluent cultures were subsequently exposed to blue or green light for 20min. Phototoxicity was quantified at 12, 18, or 24 hours after exposure by CCK-8 kit of viable cells, by fluorescence staining of the nuclei of membrane-compromised cells, and by flow cytometer of apoptotic cells.3. Cultured human RPE cells that had accumulated A2E were exposed to blue, green, or white light with and without an IOL in the light path.Results1. The reaction of all-trans-retinal (100mg) and ethanolamine (9.5mg) produced more than 50mg A2E in one step. Exposure of A2E to light gives rise to 4:1 A2E:iso-A2E equilibrium mixtures.When A2E was delivered to RPE cells in culture, it accumulated intracellularly. Internalized A2E presented as autofluorescent granules having a perinuclear distribution.2. Nonviable cells were located in blue light-exposed zones of A2E-containing RPE cells, whereas cells situated outside the illuminated areas remained viable. As shownby CCK-8 viable cell counting, by fluorescence labeling of the nuclei of membrane-damaged cells , by the analysis of flow cytometer, the numbers of nonviable cells increased with duration after light exposure and as a function of the concentration of A2E used to load the cells before illumination. Conversely, blue light-exposed RPE cells that did not contain A2E remained viable. In addition, illumination with green light resulted in the appearance of substantially fewer nonviable cells.3. The blue light-filtering IOL was associated with significant reduction in the death of A2E-laden RPE that were exposed to blue, white, and green light. While the protection effect of other ultraviolet-filtering IOL were much smaller.Part 4Clinical results after implantation with UV-blocking and blue-light Altering intraocular lensObjective To research the difference in subjective sensation ,best corrected visual acuity(BCVA), contrast sensitivity function(CSF)and color vision after UV-blocking(AcrySof)and blue-light filtering(AcrySof Natural) intraocular lens(IOL) implantation.Methods 60 senile cataract patients (62 eyes) were divided into two groups in random. AcrySof IOLs were implanted in 32 eyes and AcrySof Natural IOLs in 30 eyes. Subjective sensation ,BCVA,CSF and FM - 100 color vision were recorded at 1 day,3 months and 6 months after operation,respectively.Results The subjective sensation after AcrySof Natural IOLs implantation was better than AcrySof IOLs. BCVA and CSF improved in all cases postoperatively, and there was very significant difference ( P < 0. 01.). Both CSF and color vision were similar between AcrySof and AcrySof NaturallOLs implantation postoperatively.Conclusions1. Rat retinal pigment epithelium is more sensitive to damage by exposure to blue light than the neural retina. Apoptosis is one of the important mechanisms in blueligbt-induced RPE cells damage.2. Cultured human RPE cells provided basis for future study of physical characteristic and phototoxicity of RPE cells.3. One step preparation of A2E in vitro derived from all-trans-retinal and...