The Involvement of the Oxidative Stress in Murine Blue LED Light-Induced Retinal Damage Model

Abstract

The aim of study was to establish a mouse model of blue light emitting diode (LED) light-induced retinal damage and to evaluate the effects of the antioxidant N-acetylcysteine (NAC). Mice were exposed to 400 or 800 lx blue LED light for 2 h, and were evaluated for retinal damage 5 d later by electroretinogram amplitude and outer nuclear layer (ONL) thickness. Additionally, we investigated the effect of blue LED light exposure on shorts-wave-sensitive opsin (S-opsin), and rhodopsin expression by immunohistochemistry. Blue LED light induced light intensity dependent retinal damage and led to collapse of S-opsin and altered rhodopsin localization from inner and outer segments to ONL. Conversely, NAC administered at 100 or 250 mg/kg intraperitoneally twice a day, before dark adaptation and before light exposure. NAC protected the blue LED light-induced retinal damage in a dose-dependent manner. Further, blue LED light-induced decreasing of S-opsin levels and altered rhodopsin localization, which were suppressed by NAC. We established a mouse model of blue LED light-induced retinal damage and these findings indicated that oxidative stress was partially involved in blue LED light-induced retinal damage.

Light emitting diodes (LEDs) are long-lived, low heat-generating light sources that require little electricity, leading to the increasing widespread use of blue LEDs on liquid crystal displays of electronic devices such as smartphones, computers, and others. Therefore, penetration of LED is increasing. However, the intensity of blue light emitted by current LEDs is much higher than that emitted by filament lamps, fluorescent, and natural light.¹⁾ While blue light is essential for retinal physiology, due to its role in controlling the circadian rhythm,^2,3) excessive exposure of the retina to blue light can have an adverse impact. Blue light (400–500 nm) is the shortest wavelength within the visible light range (360–830 nm), and production of reactive oxygen species increases with decreasing wavelength.⁴⁾ In rats, blue light exposure irreversibly inhibits the expression of the mitochondrial enzyme, cytochrome oxidase, in both the retina and the retinal pigment epithelium (RPE) with consequent retinal damage.⁵⁾ Blue light-induced oxidative stress was also demonstrated to induce death of photoreceptors⁶⁾ and RPE cells.⁷⁾ Blue light exposure increased cell death rate and reactive oxygen species generation which was caused by apoptosis in ARPE-19 cells.⁸⁾ Therefore, blue light is considered a risk factor of age-related macular degeneration (AMD).⁹⁾ Moreover, oxidative stress-induced accumulation of free radicals aggravates lysosome function, increasing byproducts such as lipofuscin. N-Retinylidene-N-retinylethanolamine (A2E), a major fluorophore component of lipofuscin in RPE, impairs the function of RPE cells, decreasing their membrane integrity, increasing susceptibility to phototoxicity,¹⁰⁾ and leading to apoptosis.^11,12) Accumulation of lipofuscin is RPE cells with advancing age increases the risk of AMD development. However, there are no acute rodent models to investigate the role of blue LED in retinal damage. Therefore, in the present study, we established the murine blue LED light-induced retinal damage model and evaluated the effects of the antioxidant N-acetylcysteine (NAC).

MATERIALS AND METHODS

Animals

All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and they were approved and monitored by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University. Eight weeks old male adult albino ddY mice purchased from Japan SLC (Hamamatsu, Japan) were used in this study. All animals were housed under controlled lighting conditions (12 h : 12 h light/dark).

Exposure to Light

After dark adaptation for 24 h, the pupils were dilated with 1% cyclopentolate hydrochloride eye drops (Santen Pharmaceuticals, Osaka, Japan) at 30 min before exposure to light. Blue LED system (456 nm, Cree; Durham, NC, U.S.A.) was used. Non-anesthetized mice were exposed to 400 or 800 lx blue LED light for 2 h in mirrored device separated individually by clear board for the reflection of blue LED light. Mice were awakened by every 10 min. Ambient temperature during the light exposure was maintained at 25±1.5°C. After exposure to light, all mice were placed in the dark for 24 h, followed by return to the normal light/dark cycle.

Treatment with N-Acetylcysteine

NAC (100 or 250 mg/kg) or vehicle [phosphate buffered saline (PBS)] was administered intraperitoneally twice a day, both before dark adaptation and before light exposure based on previous studies showing that light-induced retinal damage was acute.^13,14)

Electroretinogram

Electroretinogram (ERG) was recorded on 5 d after blue LED light exposure. Mice were housed in a completely dark room for 24 h, followed by anesthesia with ketamine (120 mg/kg, intraperitoneally (i.p.); Daiichi-Sankyo, Tokyo, Japan) and xylazine (6 mg/kg, i.p.; Bayer Health Care, Tokyo, Japan). The pupils were dilated with 1% tropicamide and 2.5% phenylephrine (Santen). Flash ERG was recorded in the left eyes of the dark-adapted mice by placing a gold ring electrode (Mayo, Aichi, Japan) in contact with the cornea and a reference electrode (Nihon Kohden, Tokyo, Japan) on the tongue. A neutral electrode (Nihon Kohden) was inserted subcutaneously near the tail. High pass filtering at 0.3 Hz and low pass filtering at 500 Hz were provided. All procedures were performed under dim red light, and mice were kept on heating pads (Mycoal, Tochigi, Japan) to maintain a constant body temperature during ERG recordings. The amplitude of the a-wave was measured from the baseline to the maximum a-wave peak, and the b-wave was measured from the maximum a-wave peak to the maximum b-wave peak.

Histological Analysis

After cervical-spine dislocation, each left eye was enucleated and kept immersed for at least 24 h at 4°C in a fixative solution containing 4% paraformaldehyde. Six paraffin-embedded sections (thickness, 5 µm) were cut through the optic disc of each eye, prepared in the standard manner, and stained with hematoxylin and eosin. Light-induced damage was evaluated using six sections from each eye by morphometric analysis described below. Images were captured by light microscopy, and the thickness of the outer nuclear layer (ONL) was measured at 240 µm intervals from the optic disc on the photographs. Data from three sections (selected randomly from a total of six sections) were averaged for each eye.

Immunohistochemistry

Immunohistochemical staining was performed in accordance with the following protocol. The eyes were enucleated, fixed overnight in 4% paraformaldehyde, and immersed for two days in 25% sucrose with PBS. The eyes were then embedded in a supporting medium for frozen tissue specimens (OCT compound; Tissue-Tek, IL, U.S.A.), and stored at −80°C. Ten micrometer thick retinal sections were cut on a cryostat at −20°C and stored at −80°C until staining. Briefly, tissue sections were washed in 0.01 M of PBS for 10 min, followed by preincubation with normal horse serum in 0.01 M PBS for 1 h. For staining with antibodies derived from mouse, M. O. M blocking reagent (Vector Laboratories, CA, U.S.A.) was used instead of normal horse serum. Next, specimens were incubated at 4°C overnight with primary antibodies in 0.01 M PBS containing 0.3 (v/v) Triton X-100 and 10% horse serum. For mouse-derived primary antibodies, M. O. M protein concentrate (Vector Laboratories, CA, U.S.A.) was used. After washing with a mixture of secondary antibody. Finally, specimens were stained with Hoechest33342 (1 : 1000) for 10 min. Primary antibodies used in this study were anti-goat OPN1SW (1 : 50 dilution; Santa Cruz Biothechnology, TX, U.S.A.), anti-mouse rhodopsin and anti-mouse glutamine synthetase (1 : 1000 dilution; Millipore, MA, U.S.A.). Secondary antibodies used, were Alexa Fluor^®546 goat anti-mouse Immunoglobulin G (IgG), Alexa Fluor^®488 donkey anti-goat IgG and Alexa Fluor^®488 goat anti-mouse IgG (Invitrogen, CA, U.S.A.). Positive staining was confirmed by comparison with the negative control. Immunofluorescence images were captured using a microscope (FV10i; Olympus, Tokyo, Japan).

Quantification of S-Opsin and Rhodopsin Intensity

The ratio of S-opsin and rhodopsin intensity in inner and outer segments (IS/OS) was further divided by the S-opsin and rhodopsin intensity in outer nuclear layer (ONL). The intensity was measured using ImageJ (National Institutes of Health).

Statistical Analysis

Data were presented as the mean±standard error of the mean (S.E.M.). Statistical comparisons were performed using Dunnett’s test or Student’s t-test using SPSS statistical software (IBM, NY, U.S.A.). p<0.05 was considered statistically significant for all analysis performed.

RESULTS

Decreased of Visual Function by Blue LED Exposure

We first determined the optimum illuminance in mice by evaluating the effects of exposure to 400 or 800 lx blue LED light for 2 h (Fig. 1A). Functional damage was assessed by electrophysiological analysis. The a-wave amplitudes indicate photoreceptor function, whereas the b-wave amplitudes reflect the function of bipolar and Müller cells. Consequently, decreases in a- and/or b-wave amplitudes indicates retinal dysfunction. In this study, we observed that, compared with the non-exposed control group, there were significant reductions in the amplitudes of a- and b-waves in both groups exposed to LED light. Furthermore, the reductions in a- and b-wave amplitudes were more severe in the group exposed to 800 lx blue LED light than those exposed to 400 blue LED light (Figs. 1B–D).

Fig. 1. Measurement of the Dark-Adapted Electretinogram Amplitudes 5 d after Blue Light Emitting Diode Light Exposure (400 or 800 lx) in the Mouse Retina

(A) Protocol of the preliminary experiment to select an optimal illumination range in mice. (B) Typical traces of dark-adapted electroretinogram (ERG) responses measured 5 d after exposure to blue light emitting diode (LED) light. Stimulus flashes betweeen −2.92 and 0.98 log cds/m² were used. (C and D) Amplitudes of a- and b-waves in response to light exposure (400 or 800 lx). Data are shown as means±S.E.M., n=8 to 10. ^# p<0.05, ^## p<0.01 vs. the non-exposed group (Student’s t-test).

Atrophy of Outer Nuclear Layer by Blue LED Light Exposure

Next, we examined blue LED light-induced retinal damage by histological analysis. Representative retinal images from optic nerves were taken 5 d after exposed to 400 or 800 lx blue LED light (Figs. 2B–D). The ONL was significantly thinner in the blue LED light-treated group (Figs. 2C, D) than the non-exposed control group (Fig. 2B). The thickness of the ONL was measured in 240-µm intervals, as depicted in Fig. 2D. And also the damage to IS/OS was increasing in dependence upon light intensity. We chose 800 lx blue LED light exposure, for this analysis as the degree of blue light-induced retinal damage at 800 lx was approximately equal to that of white fluorescent light-induced retinal damage (8000 lx for 3 h) at our laboratory.¹⁵⁾

Fig. 2. Measurement of the Outer Nuclear Layer 5 d after Blue LED Light Exposure (400 or 800 lx) in the Mouse Retina

(A–C) Retinal cross-sections collected from eyes of mice 5 d after light exposure in mice: (A) non-exposed, (B) exposed to 400 lx blue LED light, and (C) exposed to 800 lx blue light. (D) Measurement of the thickness in the outer nuclear layer 5 d after blue LED light exposure. Data are shown as means±S.E.M., n=9 or 10. ^# p<0.05, ^## p<0.01 vs. the non-exposed group (Student’s t-test). The scale bar represents 50 µm.

Protective Effects of N-Acetylcysteine against Blue LED Light-Induced Retinal Damage

We next investigated whether the condition of blue LED light exposure (800 lx) was able to assess drug efficiency.

We examined whether antioxidant, NAC, could block blue LED light-induced retinal damage by assessing ERG amplitudes and ONL thickness in group exposed to 800 lx blue LED light (Fig. 3A). The blue LED light-induced retinal damage was significantly suppressed by intraperitoneal injection of NAC in a dose-dependent manner. Treatment with NAC at both 100 and 250 mg/kg doses significantly prevented the decreases both a- and b-wave amplitudes, in comparison with the vehicle-treated group (Figs. 3B–D). We also examined the effect of NAC on blue LED light-induced retinal damage by histological measurement of ONL thickness. Representative retinal images were captured at 5 d after blue LED light exposure (Figs. 4B–E). The ONL in the vehicle-treated group was significantly thinner than that in the non-exposed control group (Figs. 4B, C). Importantly, systemic NAC administration significantly reduced the blue LED light-induced photic damage observed in the vehicle-treated group (Figs. 4C–E). The ONL thickness was measured in 240-µm intervals in Fig. 4E.

Fig. 3. N-Acetylcysteine Reduces Retinal Damage Induced by Blue LED Light in Mice

Measurement of dark-adapted ERG amplitudes at 5 d after exposure to blue LED light in the mouse retina. (A) Protocol of an experiment to assess drug efficiency against blue LED light-induced retinal damage. (B) Typical traces of dark-adapted ERG responses measured 5 d after exposure to light. Stimulus flashes between −2.92 and 0.98 log cds/m² were used. (C and D) Amplitudes of a- and b-waves in response to light exposure (800 lx) in the vehicle-treated group versus those in response to light exposure in the NAC treated groups (100 or 250 mg/kg, i.p.). Data are shown as means±S.E.M., n=7 to 11. * p<0.05, ** p<0.01 vs. blue LED light exposed in the vehicle-treated group (Dunnett’s test). ^# p<0.05, ^## p<0.01 vs. the non-exposed group (Student’s t-test).

Fig. 4. Effect of N-Acetylcysteine against Retinal Damage Induced by Blue LED Light

(A–C) Retinal cross-sections collected from eyes of mice 5 d after light exposure in mice: (A) non-exposed, (B) blue LED light exposure (800 lx) in PBS-treated group, and (C) blue LED light exposure (800 lx) in NAC-treated group (100 mg/kg, i.p.) and (D) blue LED light exposure (800 lx) in NAC-treated group (250 mg/kg, i.p.). (F) Measurement of ONL thickness 5 d after light exposure. Data are shown as means±S.E.M., n=9 or 10. * p<0.05, ** p<0.01 vs. blue LED light exposure in the vehicle-treated group (Dunnett’s test). ^# p<0.05, ^## p<0.01 vs. the normal group (Student’s t-test). The scale bar represents 50 µm.

N-Acetylcysteine Reverses Blue LED Light-Induced Alterations in Retinal Protein Expression

We investigated the effect of blue LED light on expression levels of S-opsin and rhodopsin after 5 d of exposure. S-Opsin, used as a marker for cone photoreceptor cells, is a visual pigment that is more sensitive to light in short wavelength of the visible spectrum, including blue light. Conversely, rhodopsin is a marker of rod photoreceptor cells. Excessive light activates rhodopsin, and triggers photoreceptor cell damage.^16,17) In our novel in vivo model of blue LED light-induced retinal damage, light exposure led to the collapse of S-opsin and induced a change in the localization of rhodopsin from the inner and outer segment to the ONL. Importantly, NAC administration reversed the S-opsin collapse and mislocalization of rhodopsin (Figs. 5A–C). Moreover, we investigated whether Müller glia was impaired by blue LED exposure after 5 d of exposure. The exposure to blue LED caused a decrease of glutamine synthetase (GS) expression in INL containing nucleus of Müller glia and NAC was suppressed the damage of Müller glia (Fig. 5D). It was reported that the retinal light damage decreased the expression of the GS.¹⁸⁾ These results suggest that the Müller glia was impaired by blue LED exposure and NAC was suppressed the damage of Müller glia.

Fig. 5. Immunofluoresence Analysis of Retina after Exposure Blue LED Light

(A) Immunohistochemistry for S-opsin (magenta) and rhodopsin (green) expression 500 µm form the optic nerve. (B) Quantification of S-opsin fluorescence intensity. The ratio of S-opsin intensity in inner and outer segments (IS/OS) was further divided by the S-opsin intensity in outer nuclear layer (ONL). Data are shown as means±S.E.M., n=6. ** p<0.01 vs. blue LED light exposure in the vehicle-treated group (Dunnett’s test). ^## p<0.01 vs. the normal group (Student’s t-test). (C) Quantification of rhodopsin intensity. The ratio of S-opsin intensity in inner and outer segments (IS/OS) was further divided by the S-opsin intensity in outer nuclear layer (ONL). Data are shown as means±S.E.M., n=6. ** p<0.01 vs. blue LED light exposure in the vehicle-treated group (Dunnett’s test). ^## p<0.01 vs. the non-exposed group (Student’s t-test). The scale bar represents 50 µm. (D) Immunohistochemistry for GS (green) expression 500 µm form the optic nerve. Müller glia impaired by blue LED exposure, and NAC was suppressed damage of müller glia in a dose-dependent manner. We performed the same experiment (n=5) and all samples were the same results. Scale bar is 50 µm.

DISCUSSION

In the present study, we established an in vivo model of blue LED light-induced retinal damage in mice to investigate blue LED light damage to retina. Retinal damage caused by 2 h exposure to 800 lx blue LED light was comparable to that induced by white fluorescent light (8000 lx, for 3 h) at our laboratory. Therefore, we need the 800 lx of blue LED exposure for the drug evaluation. Unlike the currently utilized light-induced retinal damage models, the model used in this study should be useful in assessing potential protective effects of drugs specifically against blue LED light-induced retinal damage.

In previous reports, very strong blue light exposure (30 min, 8 mW/cm²), which is 12 times stronger than that used in this study, caused degenerative the changes in inner retina and an almost complete disappearance of photoreceptor cells in rats.¹⁹⁾ Short exposure to blue light was shown to induce deleterious changes in retinal morphology, and blue light-induced lesions were mediated by rhodopsin in rats.²⁰⁾ Thus, blue light-induced retinal damage in previously published models might be too strong and these models are not suitable for drug evaluation. Additionally, irradiation intensity of blue light used in previous models is not practical, as recently developed fluorescent lamps utilize blue LEDs. For example, blue LED light exposure (750 lx, 12 h/d, for 28 d) was previously demonstrated to induce retinal damage after dark adaption for 14 d in rats. They showed that decrease of visual function, accompanied with ONL atrophy, and increased markers of oxidative stress in response to blue LED light exposure for day 9.²¹⁾ In mice, blue LED light exposure (1000 lx, 12 h/d, 1 week) induced phospholipid oxidation and upregulated monocyte chemoattactant protein-1, whereas blue LED light exposure (500 lx, 12 h every 2 d, for 4 or 6 month) induced angiogenesis in another study.²²⁾ These models are not adequate to assess drug efficacy as the timelines are long, whereas the model presented in this study, which demonstrated retinal injury 5 d after exposure may be appropriate for investigating the mechanism of retinal damage as well as the potential interventions to reverse or alleviate damage induced by blue LED light. Previous blue LED-induced retinal damage model caused the damage by directly exposure of blue LED to eye under ketamine–xylazine anesthesia or exposure in cage that mice were moving freely.^19–22) Previous report showed that the light-induced retinal damage was decreased under ketamine–xylazine anesthesia.²³⁾ We used the mirrored device separated individually by clear board for the reflection of blue LED light in non-anesthesia. Mice were awakened by every 10 min. Therefore, we succeeded the induction of the retinal damage by 12 times lower intensity and 6 times shorter hours of blue LED exposure than the previous model.^19,21,22)

While several studies reported that NAC protected against light-induced retinal damage,^13,14) no studies exist that that showed its protective effect in an in vivo model of LED light-induced retinal damage. In the present study, NAC was protective against murine blue LED light-induced retinal damage. In the retina, under physiological conditions, protection against reactive oxygen species and free radicals are mediated by vitamins C and E, superoxide dismutase, and the glutathione (GSH), and thioredoxin (TRX) systems.^24–28) NAC, as a precursor of the antioxidant GSH, alleviates light-induced oxidative stress by modulating TRX and GSH levels,¹⁴⁾ removing free radicals by reducing their binding to proteins by disulfide bonds,²⁹⁾ and suppressing translocation of nuclear factor-kappaB (NF-κB) and NF-κB activation.³⁰⁾ Importantly, we previously showed that NAC protected against blue LED light-induced damage via suppression of NF-κB activation in a cone photoreceptor-derived cell line,³¹⁾ suggesting that its protective effect might directly involve photoreceptor cells in the mouse model presented here. Our results also indicated that blue LED light exposure altered localization of rhodopsin. Rhodopsin activated by excessive light induces phototransduction in rod cell.¹⁶⁾ However, prolonged rhodopsin signaling is a key factor that triggers photoreceptor damage¹⁷⁾ Therefore, prolonged rhodopsin activation in the retina of blue LED light-exposed mice might induce the photoreceptor cell apoptosis. Our findings showing that NAC prevented the mislocalization of rhodopsin suggested that NAC prevented rhodopsin signaling that initiated apoptosis in rod photoreceptor cells. Moreover, blue LED causes the collapse of S-opsin. This alteration may be caused by the apoptosis of cone photoreceptor cells. We investigated whether blue LED light exposure induced apoptosis of cone photoreceptor-derived cell line (661W). AnnexinV (a marker of apoptosis) and propidim iodide (PI) double positive cells were increased by blue LED light exposure in cone photoreceptor-derived cell line (not data shown). These results suggested blue LED light exposure induced apoptosis of cone photoreceptor cells. In a previous report, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL)-positive cell increased by blue LED exposure in genetically engineered mice which have all cone photoreceptor cells.³²⁾ Therefore, the apoptosis of cone photoreceptor cells may also be detected in blue LED light-induced retinal damage model.

In conclusion, we established a mouse model of blue LED light-induced retinal damage, and this model may be appropriate to evaluate the drug evaluation. Particularly, NAC clearly showed the protection effect against blue LED light-induced retinal damage in present study. These findings indicate that oxidative stress is partly involved in the blue LED light-induced retinal damage.

Conflict of Interest

The authors declare no conflict of interest.

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