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

Chemicals and antibodies

We obtained the following reagents: propidium iodide, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and 2’7’-dihydro ethidium (DHE; Sigma-Aldrich, München, Germany); Dulbecco’s modified eagle medium (DMEM)/F12 and 50X B-27 supplement (Invitrogen, Darmstadt, Germany); 5,5′,6,6’-tetrachloro-1,1’,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1; Molecular Probes, Leiden, The Netherlands); penicillin-streptomycin and glutamine (Biochrom, Berlin, Germany); agarose (Roth, Karlsruhe, Germany); and paraformaldehyde (PFA; Merck, Darmstadt, Germany); sodium cacodylate trihydrate, osmic acid, and glutaraldehyde (SERVA Electrophoresis, Heidelberg, Germany), and EMbed 812 resin (Electron Microscopy Sciences, Hatfield, PA).

Preparation of eyes

On postnatal day 24±4 days (shortly after weaning), C57BL/6 mice of either sex were sacrificed by cervical dislocation. Their eyes were immediately enucleated and transferred into phosphate buffered saline (PBS; components of Dulbecco’s PBS: potassium chloride, potassium dihydrogen phosphate, sodium chloride, di-sodium hydrogen). The eyeballs were punctured with a needle to create a small hole, which enabled fluid exchange, and were transferred into DMEM/F12 medium containing 10% fetal calf serum, 2% B-27 supplement, 1% penicillin-streptomycin, and 2 mM glutamine.

All experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of Animals in Ophthalmic and Vision Research and in strict compliance with EU and German law (Tierschutzgesetz).

Irradiation with blue light

The eyeballs were cultivated in almost their original form in the medium at 37 °C, with a CO₂ level of about 5% in a cell culture incubator for different lengths of time.

Illumination was produced by an LED-based system (# LZ1–00UA05 BIN U8; LedEngin, Santa Clara, CA) that generated 405 nm blue light with an output power of 1 mW/cm2. The eyes were positioned so their corneas faced the blue light diodes. Non-irradiated eyes were used as the controls.

Reactive oxygen species production

DHE was used to evaluate ROS production. For this purpose, eyes were irradiated with blue light for 0.5 h, 1 h, 3 h, 6 h, and 24 h at 37 °C in a cell culture incubator. After cultivation, each eyeball was cut in half through the equator to permit removal of the anterior segment and vitreous body. For ROS production imaging, the retina was gently detached from the retinal pigment epithelium and transferred to a dish (ibidi, Martinsried, Germany) for live-cell analysis in fresh medium. Next, the retinal explants were loaded with 10 µM DHE for 1 h at 37 °C in a cell culture incubator. After incubation, the samples were rinsed once in PBS and transferred to fresh medium. Live-imaging microscopy was performed at 37 °C. The images of the retinal explants were acquired using a fluorescence microscope (IX81; Olympus, Hamburg, Germany). The same detection settings were used for each set of treatments to allow direct comparison of retinal explants treated with blue light and the time-matched cultivated controls. Fluorescence images were collected using a single rapid scan. The mean fluorescence intensity was determined from the digital pictures using Cell R software from Olympus. The ratio between irradiated retinas (numerator) and non-irradiated, time-matched control retinas (denominator) was calculated to determine increases in ROS production in specific treatment groups.

Imaging of outer segments of living photoreceptor cells

Following blue light exposure for 3 h, each eyeball was cut in half through the equator to permit removal of the anterior segment and vitreous body. Each eyecup (retina still attached to retinal pigment epithelium) was transferred to a dish for live cell analysis in fresh medium and was loaded with the conventional mitochondrial dye JC-1 (7.5 µM) for 15 min at 37 °C. The samples were then viewed using confocal laser scanning microscope imaging at 23 °C. Measurements were acquired with a TCS SP5 inverted confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) with 405 nm, 458 nm, 477 nm, 488 nm, 514 nm, 561 nm, and 633 nm laser lines. Specimens were examined with an HCX PL APO 63X/1.2 NA water immersion objective with a working distance of 0.22 mm. Retinas stained with JC-1 were excited at 488 nm. The resulting images were acquired, stored, and visualized using Leica confocal software (Wetzlar, Germany). Image elaboration, analysis, and 3D rendering were performed using Velocity software (Perkin Elmer, Rodgau, Germany). Confocal microscopy imaging of live explants enabled differentiation between the cells present in the outer segment layer.

Transmission electron microscopy

After blue light exposure for 3 h, 6 h, and 24 h at 37 °C in a cell culture incubator, each eyeball was cut in half through the equator to permit removal of the anterior segment and vitreous body. Next, the eyecup (retina still attached to retinal pigment epithelium) was fixed overnight at 4 °C in Karnovsky’s fixative (components: 2,5% glutaraldehyde+2,5% PFA) in 0.1 M cacodylate buffer (CB; pH 7.4).

Fixed eyecups were quartered. The tissue pieces were washed and post-fixed in 1% osmic acid prepared in 0.1 M CB (pH 7.4) for 1 h at room temperature and washed in CB. For dehydration of the samples, an ethanol series with ascending concentrations was used. The sample was infiltrated with an EMBed 812 resin overnight and then placed in an embedding mold and polymerized in an oven at 60 °C for 48 h. Ultrathin sections (65 nm) were applied to a grid.

Ultrastructural studies were conducted using an EM 906 electron microscope (Carl Zeiss, Oberkochen, Germany).

Quantification of breaks in the surrounding membrane of outer segments

The ultrathin retina sections of at least three animals were analyzed, and around 100 photoreceptor outer segments were counted per time point and treatment. We carefully selected only rod outer segments. The counted photoreceptor outer segments were randomly distributed over the retina cross-section, but it was confirmed that the number of counted photoreceptors in the periphery was roughly the same as that of the counted photoreceptors in the middle part of the section. Every clear disruption in the surrounding membrane of the outer segment was counted as a break, was limited in size to one-third of the outer membrane, and was assigned to a respective part. Breaks were counted in three equal parts of the outer segment—in a proximal third, in a middle third, and in a distal third.

Scanning electron microscopy

Following blue light exposure for 12 h, each eyeball was cut in half through the equator to permit removal of the anterior segment and vitreous body. The retina was gently detached from the retinal pigment epithelium and fixed overnight at 4 °C in Karnovsky’s fixative in 0.1 M CB (pH 7.4). The fixed eyecups were sectioned into strips. The tissue pieces were washed and post-fixed in 1% osmic acid prepared in 0.1 M CB (pH 7.4) for 1 h at room temperature, and washed in CB. Subsequently, they were dehydrated in ascending concentrations of acetone and were critical point-dried in liquid CO₂. The parts of the dried retina were gold-coated (20 nm in thickness) in a sputter coater and then examined and photographed using a JSM-7500F field emission scanning electron microscope (JEOL, Eching, Germany).

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay

The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay was used to detect fragmented DNA in photoreceptor cells following light exposure. After blue light exposure for 6 and 18 h, each eyeball was cut in half through the equator to permit removal of the anterior segment and vitreous body. The retina was gently detached from the retinal pigment epithelium and fixed overnight at 4 °C in 4% PFA. The fixed retina was cut into two parts and embedded in 4% agarose. Sections (30 µm) were obtained using a vibratome (VT1200 S; Leica Microsystems, Wetzlar, Germany) and then mounted and stained on glass slides. The TUNEL assay was performed according to the instruction manual of the In Situ Cell Death Detection Kit (TMR red; Roche Diagnostics, Mannheim, Germany). Subsequently, DAPI staining was conducted for 10 min. Fluorescence images were obtained using an Axio Imager Z1 microscope (Carl Zeiss). Digital pictures were acquired, stored, and visualized with Axio Vision 4.7 Software (Carl Zeiss).

Each image was acquired as a z-stack of approximately 7 µm thickness and merged for the counting of TUNEL-positive cells in the ONL. The middle of each retina was determined by using the length of the undisturbed ganglion cell layer. Sections of 100 µm were counted in central parts of the retina (ca. 200 µm from the middle) and in peripheral parts of the retina (ca. 1,000 µm from the middle) of different animals. At least eight different sections were counted per time point and treatment, and the mean value was determined.

Statistical analysis

Values are presented as the mean±standard deviation (SD), and n represents the number of independent experiments. Unless stated otherwise in the figure legends, all assays were performed at least five times. Statistical analysis was performed by one-way ANOVA (ANOVA) with Bonferroni correction for post hoc multiple comparisons using the Statistical Package for the Social Sciences (SPSS, v. 12.0; Chicago, IL). Significance was accepted at p<0.05.

Production of reactive oxygen species in photoreceptor cells after short-term exposure to blue light

To test for blue light-induced ROS formation in our system, we treated live retinal explants with blue light for 0.5 h, 1 h, 3 h, 6 h, and 24 h. Intracellular ROS production was measured by incubating the cells with DHE, which is commonly used to measure intracellular superoxide generation. In our system, the retinal explants displayed an increase in fluorescent products of DHE in photoreceptors following exposure to blue light. We discovered that 1 h of exposure to blue light stimulated the greatest amount of ROS formation, as evidenced by the oxidation of DHE (Figure 1). The blue light exposure induced a 1.7 fold increase in ROS production in the outer segments and ONL after 0.5 h, a 3.4 fold increase after 1 h, and a 1.5 fold increase after 3 h. After this time, the blue light exposure led to ROS production comparable to that observed in the controls (data not shown).

Shape and structural changes in the outer segments

To image live photoreceptor cells ex vivo, we used the mitochondrial dye JC-1 according to the technique described by Bianchini et al. [28]. Briefly, the eyecup was incubated for about 20 min with JC-1 until the rods spontaneously detached from the retinal pigment epithelium. Then the retina was mounted on coverslip glass for confocal microscopy measurements with a TCS SP5 inverted confocal laser scanning microscope (Leica Microsystems). This technique resulted in staining of not only the retinal mitochondria but also the outer segments of photoreceptor cells, which are devoid of mitochondria. This additional JC-1 staining was not observed in any other cells throughout the entire living retina, as first described in Bianchini et al. [28].

Images of living retina tissue were obtained using confocal microscopy. Figure 2 shows that the fluorescence was distributed in the outer segments of photoreceptor cells. The morphology of the outer segments was sufficiently observable to allow unambiguous identification of its structure and organization. Blue light irradiation for 3 h caused disorganization (Figure 2B) of the normally neatly stacked outer segments (OS; Figure 2A).

Scanning electron microscopy showed disorganization of the disks in the photoreceptor outer segments of irradiated retinal explants after 12 h of blue light exposure (Figure 3B,D). The outer segments lost their shape and were no longer precisely stacked, and the contacts between disks were abrogated. The outer segments appeared tortuous, and the lamellar structures became disrupted, as visualized by transmission electron microscopy (Figure 4B,D). Interestingly, this method also allowed the observation of defects in the enveloping cell membrane of outer segments (Figure 4D). An increase in cell membrane defects as the cultivation time increased was also detected in the controls, but the defects in the treated samples were significantly increased following irradiation for 3 h, 6 h, and 24 h, compared with the time-matched controls (Figure 5A). Surprisingly, most of the breaks occurred in the middle section of the outer segments after exposure to blue light (Figure 5B). Both the distal and proximal ends of the outer segments demonstrated a lower defect ratio for the surrounding cell membrane, compared to the middle section. No significant effects on cell membrane breaks in the distal and proximal ends of the outer segments were found, compared to the controls. No differences in mitochondria were detected in the inner segments (data not shown).

Cell death in the outer nuclear layer

The TUNEL assay method was used to analyze cell death in retina layers following exposure to blue light. Cell death increased during an extended blue light treatment, especially in the ONL (Figure 6). The initiation of cell death was observed after 6 h, and this process progressed during extended blue light irradiation. Although some TUNEL-positive cells were also detected in the control samples due to the culture conditions, a significant increase in blue light-damaged retinas was observed after 18 h of irradiation. No significant changes were detected in the thickness of the ONL, compared to the control retinas.