Expression of SOD-1 was increased in OS after 12 h of blue light.

<p><b>A,</b> Retina paraffin section after 12 h of cultivation. <b>B,</b> Retina paraffin section after 12 h of blue light exposure. Under both conditions the expression of SOD-1 was higher in OS than in IS. The irradiated sample showed an increase of the protein in the OS. <b>A–B</b>, scale bar 50 µm; images are representative of n = 3 experiments <b>OS</b>: outer segments; <b>IS</b>: inner segments; <b>ONL</b>: outer nuclear layer; <b>OPL</b>: outer plexiform layer; <b>INL</b>: inner nuclear layer; <b>IPL</b>: inner plexiform layer; <b>GCL</b>: ganglion cell layer.</p

Reactive oxygen species (ROS) production is increased in outer retinal layers after blue light damage, particularly in outer segments.

<p><b>A.</b> Confocal laser scanning microscopy images of 40 µm vibratome sections of retinas are presented. After 0.5 h of blue light exposure, irradiated explants and respective non-irradiated explants (controls) were loaded with 25 µM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA; ROS indicator). Irradiated explants showed higher fluorescence intensity in OS and IS compared to time-matched controls (same microscope settings). Scale bar represents 10 µm; images are representative of 3 experiments <b>B.</b> Quantitative analysis of ROS production in outer and inner segments of retinal explants. Retinas were exposed to visible blue light for 0.5 h and 1 h. The graph displays the mean fluorescence intensity ratios of irradiated photoreceptor cell layers versus non-irradiated time-matched controls, the IS of the controls are normalized to 1 (determined by Image J software). Bars represent the mean ± standard error of mean (SEM) from n = 10 different equal regions of interest (ROIs; * shows significance compared to IS-control; *p<0.05 determined by ANOVA, post hoc Bonferroni test). <b>C:</b> Magnification of confocal laser scanning microscopy images of the outer retinal layers of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071570#pone-0071570-g001" target="_blank">figure 1</a> after 0.5 h of blue light exposure. The ROS production in the outer segments of the photoreceptors is increased more than in the inner segments. This is indicated by a more intense fluorescence of CM-H₂DCFDA in the outer segments than in the inner segments after irradiation periods with blue light. <b>OS</b>: outer segments; <b>IS</b>: inner segments; <b>ONL</b>: outer nuclear layer; <b>OPL</b>: outer plexiform layer; <b>INL</b>: inner nuclear layer.</p

Effect of blue light on Nox-2 and Nox-4 protein expression.

<p><b>A,</b>Western blot analysis showing increased Nox-2 and Nox-4 protein expression in OS following 1 h of blue light exposure (+) or in controls. The blots were first exposed to anti-Nox-2 or anti-Nox-4, respectively and then to anti-beta-Actin antibody as loading control. Images are representative of 5 experiments. <b>B,</b> Bar chart of densitometric analysis of Nox-2 and Nox-4 expression after 1 h and 12 h compared to control beta Actin. Bars represent the mean ± SEM from n = 5 experiments (* shows significance compared to control; *p<0.05 determined by ANOVA, post hoc Bonferroni test).</p

Decrease of TMRE products after 6 h and 12 h of blue light irradiation in OS.

<p>Images of the outer segment layer of whole mounts stained with 20 nM TMRE after 6 h (<b>A</b>, <b>B</b>) and 12 h cultivation (<b>C</b>, <b>D</b>). The blue light irradiated samples (<b>B</b>, <b>D</b>) showed a distinct decrease of TMRE products in the outer segment layer compared to the time-matched controls (<b>A</b>, <b>C</b>). The images are representative of 3 experiments. The arrows point to leftovers of the RPE.</p

Immunofluorescence intensity of Nox-2 and Nox-4 proteins increased after 12 h of blue light exposure.

<p><b>A, C,</b> Paraffin sections of retinas after 12 h of cultivation. <b>B, D,</b> Paraffin sections of retinas after 12 h of blue light exposure. Nox-2 was increased in the OS (<b>B</b>) while Nox-4 immunofluorescence intensity appeared slightly increased compared to the control (<b>C, D</b>). <b>A–D</b>, scale bar 50 µm; images are representative of n = 3 experiments <b>OS</b>: outer segments; <b>IS</b>: inner segments; <b>ONL</b>: outer nuclear layer; <b>OPL</b>: outer plexiform layer; <b>INL</b>: inner nuclear layer; <b>IPL</b>: inner plexiform layer; <b>GCL</b>: ganglion cell layer.</p

Short-term cultivation with Mitochondrial membrane potential (MMP)-dyes TMRE and JC-1.

<p>Images of RPE-OS-layer of whole mounts directly after preparation of the retina during live staining with TMRE (<b>A</b>) and JC-1 (<b>B</b>), respectively. The color of JC-1 slightly changes from orange-yellow (t = 2 min) to yellow (t = 10 min) and then to yellow-green (t = 20 min) in the pictures of merged fluorescence channels and DIC-images, which could indicate a change of a extra-mitochondrial membrane (<b>B</b>). TMRE does not show such distinctive changes in intensity after short-term cultivation (<b>A</b>). <b>A–B</b>, images are representative of three experiments; scale bar 20 µm.</p

Effect of blue light on MDA adducts.

<p>Western blot analysis of MDA adducts after 12 h blue light exposure and in controls. The blot was first exposed to anti-MDA and then to anti-ß-Actin antibody as loading control. From the retina that was irradiated with blue light (+) or not (−), were different fractions loaded on a gel – isolated outer segments (OS), cytosolic proteins in the pellet and other proteins (remaining fractions). The expression of different MDA adducts changed with blue light damage, especially of the proteins with sizes of 70 kDa and 38 kDa, marked with asterisks.</p

Shift of JC-1 staining after blue light irradiation in OS.

<p>Images of the outer segment layer of whole mounts stained with 10 µg/ml JC-1 after cultivation. The eyes were irradiated with blue light for 6 h (<b>B</b>) and 12 h (<b>D</b>), the time-matched controls cultivated for 6 h (<b>A</b>) and 12 h (<b>C</b>), respectively. An increase of green JC-1 monomers (sign for MMP collapse) was detected in both irradiated retinas to a greater extent than in the controls. Some fluorescent red J-aggregates (seen in healthy cells) were still visible after 12 h irradiation (<b>D</b>). In the controls the aggregates were observed as spots additionally to the staining of the outer segments in the whole mount (<b>B</b>, <b>D</b>). Images are representative of three experiments.</p

Expression of malondialdehyde (MDA) and 4-hydroxy-nonenal (4-HNE) increased after 12 h of blue light exposure in outer segments.

<p><b>A, C,</b> Paraffin sections of retinas after 12 h of cultivation. <b>B, D,</b> Paraffin sections of retinas after 12 h of blue light exposure. Oxidative stress caused by blue light exposure led amongst others to lipid peroxidation and end-products like MDA and 4-HNE increased considerably in the OS (<b>B, D</b>). The respective time-matched controls showed only a weak autofluorescence in the OS (<b>A, C</b>). <b>A–D</b>, scale bar 50 µm; <b>OS</b>: outer segments; <b>IS</b>: inner segments; <b>ONL</b>: outer nuclear layer; <b>OPL</b>: outer plexiform layer; <b>INL</b>: inner nuclear layer; <b>IPL</b>: inner plexiform layer; <b>GCL</b>: ganglion cell layer.</p

ROS production is reduced by the Nox inhibitor apocynin.

<p>Merged CM-H₂DCFDA fluorescence and bright field microscopy images of 40 µm vibratome sections of retinas are presented. After 1 h of blue light exposure, irradiated explants and respective non-irradiated explants (controls) were loaded with 25 µM CM-H₂DCFDA. In some cases, explants were pretreated with 4 mM apocynin during blue light exposure. The Nox inhibitor apocynin effectively reduced the levels of ROS production in the photoreceptors. The arrowheads mark the assumed border between IS and OS. The images are representative of 3 experiments. OS: outer segments; IS: inner segments; ONL: outer nuclear.</p