Influence of blue light on photoreceptors in a live retinal explant system

Abstract

Purpose: The present study was performed to investigate the early effects of blue light irradiation of photoreceptors in retinal explant cultures.

Methods: Murine retinal explant cultures were irradiated with visible blue light (405 nm) with an output power of 1 mW/cm2. Dihydroethidium was used to determine the production of reactive oxygen species. Morphological alterations of photoreceptor outer segments were determined by live imaging microscopy with mitochondrial dye JC-1. Transmission and scanning electron microscopy were used for ultrastructural evaluations. Cell death in the retina was assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay method.

Results: Live retinal explants displayed an increase in reactive oxygen species production, as revealed by fluorescent dihydroethidium products in photoreceptor cells after 30 min of blue light exposure. After 3 h of exposure, blue light caused disorganization of the normally neatly stacked outer segments of living photoreceptors. Ultrastructural analysis revealed breaks in the cell membrane surrounding the outer segments, especially in the middle section. The outer segments appeared tortuous, and the lamellar structures had been disrupted. TUNEL-staining revealed that long-term blue light exposure induced photoreceptor cell death.

Conclusions: In vitro blue light irradiation of retinal explants is a suitable model system for investigating early ultrastructural changes, as well as damage that leads to cell death in photoreceptor cells.

Figure 1

Production of intracellular reactive oxygen species is increased by blue light damage. In part A, representative images of reactive oxygen species production by live imaging fluorescence microscopy in photoreceptor cells of retinal explants are presented. After 1 h of blue light exposure, irradiated explants and respective non-irradiated explants (controls) were loaded with 10 µM dihydroethidium. The scale bar represents 50 µm. In part B, quantitative analysis of reactive oxygen species production in photoreceptor cells of retinal explants is demonstrated. Retinas were exposed to visible blue light for 0.5 h, 1 h, and 3 h. The graph displays the mean fluorescence intensity ratios of irradiated photoreceptor cell layers versus non-irradiated time-matched controls, which are normalized to 1. Bars represent the mean±standard error of mean (SEM) from n=5 independent experiments (*p<0.05).

Figure 2

Blue light damage caused disorganization of photoreceptor outer segments. Confocal laser scanning microscope images of 5,5′,6,6’-tetrachloro-1,1’,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1) fluorescence in outer segments in a living murine retinal explant by 3-D reconstruction comprising serial confocal sections are presented of an undulating portion of the retina. Representative images of photoreceptor outer segments are provided for a three-hour untreated control (A) and a sample (B) that was irradiated for three hours with blue light with an output power of 1 mW/cm2. All units are 8.8 µm.

Figure 3

Disorganization of photoreceptor disks is caused by light damage after 12 h of blue light exposure. Scanning electron microscopy images of photoreceptor outer segments from untreated (A, C) and irradiated retinal explants (B, D) are presented. After irradiation with blue light, the outer segments of the retinas lost their shape (B, D) and were no longer as precisely stacked as the controls (A, C). The contacts between disks were lost, and the lamellar structures became disrupted (D). Scale bars are 1 µm.

Figure 4

Blue light damage induced tortuous photoreceptor outer segments with disrupted lamellar structures after 24 h of blue light exposure. Transmission electron microscopy images of photoreceptors from untreated (A, C) and irradiated eyes (B, D) are presented. In part A, an overview of precisely stacked photoreceptor outer segments and the adjacent retinal pigment epithelium in the upper right part of the image is demonstrated. In part B, an overview of light-damaged photoreceptor outer segments and their disrupted lamellar structures is presented. C: The enveloping membrane of the outer segment continuously enclosed the disks in the control retina. D: The enveloping membrane was frequently interrupted or completely lost in the middle section of the outer segment, and the disks started to shift. Scale bars are 1 µm in A and B, and 500 nm in C and D.

Figure 5

Membrane breaks in photoreceptor outer segments are caused by blue light damage. Quantitative analysis of breaks in the enveloping membrane of photoreceptor outer segments was performed. Error bars represent the mean±standard deviation (SD) from n=100 different outer segments. A: Eyeballs were exposed to blue light for 3 h, 6 h, and 24 h. The graph displays the number of breaks in the middle section of the outer segments. During the prolonged incubation time, the number of breaks increased in treated eyes but was consistently significantly higher than that observed in time-matched controls (*p<0.05). B: Eyeballs were exposed to blue light for 6 h. The graph displays the number of breaks in the proximal, middle, and distal sections of the outer segments. The number of breaks was significantly higher only in the middle section, compared to that in the controls (*p<0.05).

Figure 6

Blue light damage induced cell death in the outer nuclear layer of retinal explants. Photoreceptor cell death was quantified using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay. All vibratome sections are counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Red represents cells with DNA strand breaks labeled with tetramethylrhodamine (TMR)-dUTP, and blue represents DAPI-stained nuclei. The figure shows retinal sections with their layers (outer segments [OS], outer nuclear layer [ONL], outer plexiform layer [OPL], inner nuclear layer [INL], inner plexiform layer [IPL], ganglion cells [GC]). A: The untreated retina (0 h) was incubated with complete TUNEL reaction mixture (negative control). B: Retinas were treated with DNase I before labeling (positive control). Retinas were not treated (C) or were irradiated (D) with visible blue light for 6 h. Cell death initiation was detected in the ONL after 6 h. Cell death initiation was detected, especially in the outer nuclear layer (ONL). Retinas were not treated (E) or were irradiated (F) with visible blue light for 18 h. The number of TUNEL-positive cells significantly increased in blue light-damaged retinas after 18 h, although some TUNEL-positive cells were detected in control explants due to the culture conditions. The scale bar represents 50 µm. In part G, quantitative analysis of cell death in the ONL of retinal explants after blue light exposure for 6 h and 18 h is presented. The graph displays the number of TUNEL-positive cells in the ONL of 100 µm sections from middle and peripheral parts of the retina. The mean values of at least eight different counted sections per time point and treatment are shown. TUNEL-positive cells appear initially at 6 h and increase during prolonged incubation. At 18 h, the TUNEL-positive cells in the ONL of blue light-treated eyes are significantly more numerous than the cells observed in the time-matched controls (*p<0.05). Error bars represent SD.