Excessive visible light exposure can induce damage to retinal cells, especially to photoreceptor and retinal pigment epithelial (RPE) cells; such exposure may also contribute to the development or progression of age-related macular degeneration (AMD) and retinitis pigmentosa (RP) [1
]. AMD is the leading cause of irreversible central vision loss in the elderly population in the world [3
]. Prevalence data suggest that 1.75 million people are affected by AMD and 7 million people are at risk of developing AMD in the United States [4
]. The pathogenesis of AMD is not well understood, but it is thought that a high oxygen concentration and high levels of polyunsaturated fatty acids in the retina, increasing sensitivity to light exposure and contribute to oxidative stress and inflammation, which injure the RPE, resulting in loss of photoreceptor cells [5
Photochemical damage occurs after exposure to high energy radiation with a wavelength within the visible spectrum of light [7
]. At the retinal level, exposure to light increases phagocytosis of the outer segment (OS) of photoreceptors and induces the formation of superoxide anions by RPE cells [8
]. An imbalance develops between light-induced reactive oxygen species (ROS) and endogenous antioxidative systems, thereby resulting in photo-oxidative stress in the retina [9
]. Additionally, impaired retinal antioxidant status leads to overexpression of proinflammatory and angiogenic parameters, which are primarily responsible for injured retinal microvasculature [10
ROS expedite oxidative stress-induced damage of photoreceptor and RPE cells, and nutritional supplements such as polyphenol and xanthophyll that scavenge ROS can prevent or delay progression of early AMD [11
]. Bilberries (Vaccinium myrtillus
L.) are particularly rich in anthocyanins, such as delphinidin, malvidin, petunidin, cyanidin, and peonidin [13
]. Various mechanisms of action have been proposed for anthocyanins, such as antioxidation, anti-inflammation, as well as effects on anti-apoptotic pathways and gene expression [14
]. Anthocyanins have been shown to act directly as antioxidants to neutralize ROS by donating hydrogen ions and modulating cell signaling pathways [15
]. A mouse model of endotoxin-induced retinal inflammation shows that an impairment in visual function, which has been improved after anthocyanin intervention [16
]. Anthocyanins facilitate rhodopsin regeneration or interact directly with the rhodopsin molecule [17
]. However, the possible mechanisms of protective effects of bilberry anthocyanins in visible light-induced retinal degeneration in vivo
are still limited and controversial.
Pigmented rabbits were chosen because of melanin in RPE cells, which were similar to the structure of the human retina; moreover, their eye size is more accessible and allows for performing experimental operation [19
]. In addition, fluorescent white light having an emission spectrum similar to daylight, which was chosen to mimic excessive exposure to daylight. In the present study, we generated a pigmented rabbit model of visible light-induced retinal damage that exhibits retinal cell oxidative stress, inflammation, angiogenesis, and apoptosis, which possesses several pathophysiological characteristics of AMD and can be used to investigate the mechanisms of retinal degeneration.
This study also aimed to elucidate the related mechanisms of the protective effects of bilberry anthocyanin extract (BAE) against visible light-induced retinal degeneration in this model. Scotopic electroretinogram (ERG), photopic ERG, and maximal response ERG (max-ERG) were performed at 1 day before and 1, 3, and 7 days after light exposure (18,000 lx for 2 h). Retinal damage was quantified by measuring the thickness of the outer nuclear layer (ONL) and OS length at 7 days after light-induced damage. Experiments tested the expression of apoptotic proteins (Bax, Bcl-2, and caspase-3) in retinal cells. We determined the changes in indices associated with oxidative stress metabolism, namely, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), total antioxidant capacity (TAOC), and malondialdehyde (MDA), in the retinal tissues. We also investigated the levels of proinflammatory cytokines (interleukin-1β (IL-1β)) and angiogenic parameters (vascular endothelial growth factor (VEGF)) in the retina.
3. Experimental Section
3.1. Chemicals and Reagents
BAE purchased from JF-NATURAL Chemical Co. (Tianjin, China) was suspended in PBS at a concentration of 250 mg/mL. Methyl cyanide was of chromatographic grade and purchased from Merck (Darmstadt, Germany). Formic acid was purchased from Sigma (St. Louis, MO, USA). Deionized water was produced using a Milli-Q water-purification system (Millipore, Billerica, MA, USA). Tropicamide eye drops were purchased from Xingqi Pharmaceuticals Co., Ltd. (Shenyang, China). Sumianxin was purchased from Shengda Pharmaceuticals Co., Ltd. (Dunhua, China). Anti-Bax (ADI-AAS-040) was purchased from Enzo Life Sciences (New York, NY, USA). Anti-Bcl-2 (PAB19562) was purchased from Abnova (Taipei, Taiwan). Anti-caspase-3 (ab82585) was purchased from Abcam (Cambridge, UK). Anti-β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). RIPA buffer and bicinchoninic acid (BCA) protein assay kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). All other chemicals and reagents were purchased from Sigma-Aldrich.
3.2. Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS) Analysis of Bilberry Anthocyanin Extract (BAE)
LC-ESI-MS/MS analysis was performed using an Agilent 1260 series HPLC combined with an Agilent 6460 Series Triple Quad LC/MS equipped with a Jet Stream ESI source (Agilent Technologies, Santa Clara, CA, USA). MS was operated in the positive ion mode. Nitrogen was used as a collision gas. The analytical column was a 250 mm × 4.6 mm i.d. Agilent Zorbax SB-C18 column (Agilent, Palo Alto, CA, USA), which was maintained at 25 °C. Prior to analysis, all samples were filtered through a 0.45 μm membrane filter. The injection volume was 20 μL. The elution solvents, namely, (A) methyl cyanides with 2% formic acid and (B) H2O with 2% formic acid, were applied as follows: isocratic 3% A for 3 min, from 3% to 15% A for 12 min, from 15% to 25% A for 40 min, from 25% to 3% A for 3 min, and isocratic 3% A for 7 min. The flow rate was 0.5 mL/min, and detection was performed at 520 nm. The detailed MS conditions were as follows: collision energy, 15 eV; gas temperature, 300 °C; gas flow, 7 L/min; nebulizer pressure, 35 psi; sheath gas temperature, 300 °C; sheath gas flow, 11 L/min; capillary voltage, 3.5 kV; and nozzle voltage, 500 V.
3.3. Animal Care
A total of 40 healthy pigmented rabbits weighing 2.5–3.0 kg were purchased from the Animal Center of the Beijing Kaiyuan Co. (Beijing, China). All procedures were performed according to the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research. The procedures were approved by the Ethical Committee for Animal Experimentation of the First Hospital Affiliated to General Hospital of the Chinese People’s Liberation Army. All rabbits were housed at a 12 h light-dark cycle for one week at 22–25 °C and 55%–60% humidity. All rabbits were freely fed a standard breeding diet (Beijing Ke Ao Xie Li Co., Beijing, China).
3.4. Treatment with Bilberry Anthocyanin Extract (BAE) and Exposure to Light
After a week-long adaptation period, the rabbits were randomly divided into five groups: control group (no light exposure and vehicle administration; CG) (n = 8), low light-induced retinal damage model group (11,000 lx light exposure and vehicle administration; LLMG) (n = 8), high light-induced retinal damage model group (18,000 lx light exposure and vehicle administration; HLMG) (n = 8), treatment group 1 (18,000 lx light exposure and administration of a low dosage of BAE group, 250 mg/kg/day; LBAG) (n = 8), and treatment group 2 (18,000 lx light exposure and administration of a high dosage of BAE group, 500 mg/kg/d; HBAG) (n = 8). The prescribed dosages of BAE were intragastrically administered to the rabbits. Administration began two weeks before light exposure and continued for one more week thereafter.
After dark adaptation (60–100 lx) for 24 h, the pupils were dilated with tropicamide eye drops at 20 min before light exposure. Non-anesthetized rabbits were then exposed to 11,000 ± 1000 or 18,000 ± 1000 lx (illumination meter: TES-1332A; TES Electrical Electronic Corporation, Taipei, Taiwan) of diffused cool-white fluorescent light (Zhongcheng, Guangdong, China) for 2 h in cages with reflective interiors (50 cm × 45 cm × 35 cm). The temperature during exposure to light was maintained at 25 ± 1.5 °C. After exposure to light, all the rabbits were placed in the dark for 24 h before they were returned to the normal light/dark cycle.
3.5. Electroretinograms (ERGs)
ERGs were recorded by a visual electrophysiology system (APS-2000AER, Kanghua Rui Ming Technology Co., Ltd., Chongqing, China) to measure retinal function. To allow the electrodes to stick to the rabbits’ skin, body hair on the forehead and bitemporal region was scraped off with a scalpel. ERGs were simultaneously recorded from both eyes by two golden-ring electrodes, two forehead reference electrodes, and one ground electrode in the bitemporal region. The standard ERG signals were recorded at 1 day before, as well as 1, 3, 7, and 14 days after light exposure, according to previously described methods [54
]. After dark adaptation for more than 1 h, the rabbits were anesthetized with an intramuscular injection of sumianxin (0.2 mL/kg). Pupils were fully dilated with tropicamide eye drops. After anesthesia, methylcellulose ophthalmic solution was dripped on the left eye, and the right eye was covered with gauze and a black eyeshade. According to the standards set by the International Society for Clinical Electrophysiology of Vision, ERGs of the left eye were recorded before those of the right eye. All procedures were performed under dim red light.
3.6. Hematoxylin and Eosin (HE) Staining
After the ERG was recorded, the rabbits were sacrificed. After marking the 12 o’clock position of the cornea with a silk suture, the eyeballs were quickly enucleated and the eyeball was immersed for 48 h in a fixative solution containing 2.5% glutaraldehyde and 2% paraformaldehyde. Dehydration was achieved via successive baths of ethanol at increasing concentrations, followed by clearing with xylene in an automatic tissue processor. Samples were embedded in paraffin taking into account the sample orientation, and 4 μm slides were obtained with a microtome. Six paraffin-embedded sections were cut through the optic nerve head (ONH) of each eye. These sections were prepared with the standard procedure, stained with HE, and observed under a light microscope (Leica, Heidelberg, Germany).
3.7. Measurement of the Outer Nuclear Layer (ONL) Thickness and Outer Segment (OS) Length
For ONL thickness and OS length quantification, light micrographs were photographed. The ONL thicknesses were counted within 250–2750 μm (counted at every other 500 μm interval) of the superior and inferior edges to the ONH based on photographs of HE-stained sections by personnel blinded to the study groups. The mean ONL thickness was calculated from three sections (randomly selected from the six sections) for each retina. The OS length was measured 1 mm from the ONH in the superior and inferior retina (one eye of each of eight rabbits).
3.8. Western Blot Analysis
Whole-cell lysates from the retinal extracts were prepared for western blot analysis by sonication in RIPA buffer containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA), followed by centrifugation at 15,000× g for 30 min at 4 °C to collect the supernatant. After the protein concentrations were determined with a BCA protein assay kit, equal aliquots (20–30 μg) of protein samples were applied to 10% sodium dodecyl sulfate polyacrylamide gels (Invitrogen) and electrophoretically separated. Resolved proteins were electrophoretically transferred to nitrocellulose membrane (Millipore) and blocked with 5% nonfat dry milk for 1 h at room temperature. The membranes were incubated with Bax (1:1000), Bcl-2 (1:20), or β-actin (1:5000) antibodies for 2 h at room temperature, after which they were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The signals were visualized by enhanced chemiluminescence (Fisher/Pierce, Rockford, IL, USA) and recorded on X-ray films (Estman Kodak Company, Rochester, NY, USA). The intensities of the protein bands were determined with ImageJ software (1.32j, National Institutes of Health, Bethesda, MD, USA).
3.9. Immunohistochemistry for Caspase-3
Sections of 4 μm thickness were prepared for immunohistological staining. Endogenous peroxidase was quenched by freshly prepared 3% H2O2 with 0.1% sodium azide. The treated sections were placed in antigen retrieval solution (0.01 mol/L citrate buffer, pH 6.0) for 15 min in a microwave oven at 100 °C and 600 W. The samples were blocked in 10% fetal bovine serum in PBS and incubated at 4 °C overnight in primary antibody solution of anti-caspase-3 (ab2171, 1:50). After washing with 0.01 M PBS buffer, the samples were incubated with horseradish peroxidase-conjugated secondary antibodies (1:200, Dako, Glostrup, Denmark) for 60 min at room temperature, developed with 3,3′-diaminobenzide tetrahydrochloride, counterstained with hematoxylin, dehydrated, and mounted. Consistent negative controls were obtained by replacing primary antibody with PBS. In case of discrepancies, a final score was established by reassessment on a double-headed microscope. During the scoring of caspase-3 expression, the extent and intensity of immunopositivity were both considered. The staining intensity was scored as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. Positivity was quantified according to the percentage of positive cells: 0, <5%; 1, >5%–25%; 2, >25%–50%; 3, >50%–75%; 4, >75%. The final score was determined by multiplying the intensity and quantity scores, which yielded a range from 0 to 12.
3.10. Determination of the Activities of SOD, GSH-Px and CAT, Levels of MDA and TAOC
For biochemical analysis purposes, the retina was homogenized with an Ultra-Turrax apparatus (IKA T10basic; Staufen, Germany). The TAOC and MDA levels, as well as the activities of SOD, GSH-Px, and CAT, in the retinal homogenate were determined with assay kits purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China) following the manufacturer’s instructions. The protein concentrations were determined with the BCA protein assay kit.
3.11. Determination of the IL-1β and VEGF Levels
IL-1β and VEGF levels in retina were estimated using a commercially available ELISA kit from Keyingmei Biotechnology and Science Inc. (Beijing, China) following the manufacturer’s instructions. The protein concentrations were determined with the BCA protein assay kit.
3.12. Statistical Analysis
Values were expressed as the mean ± standard deviation. Data shown in the study were obtained from at least three independent experiments. Statistical comparisons between different groups were determined using one-way ANOVA followed by Tukey’s test. Values with p < 0.05 were regarded as significant.