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Retina  |   June 2013
Role of Heparin-Binding Epidermal Growth Factor–Like Growth Factor in Light-Induced Photoreceptor Degeneration in Mouse Retina
Author Notes
  • Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan 
  • Correspondence: Hideaki Hara, Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan; hidehara@gifu-pu.ac.jp
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3815-3829. doi:10.1167/iovs.12-11236
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      Yuki Inoue, Kazuhiro Tsuruma, Tomohiro Nakanishi, Atsushi Oyagi, Yuta Ohno, Tomohiro Otsuka, Masamitsu Shimazawa, Hideaki Hara; Role of Heparin-Binding Epidermal Growth Factor–Like Growth Factor in Light-Induced Photoreceptor Degeneration in Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(6):3815-3829. doi: 10.1167/iovs.12-11236.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Although heparin-binding epidermal growth factor–like growth factor (HB-EGF) has been reported to have protective effects against various neuronal cell damage, its role in the retina has not been elucidated. Here, we investigated its role in light-induced photoreceptor degeneration using retinas and ventral forebrain–specific Hb-egf knockout (KO) mice.

Methods.: Disruption of Hb-egf was confirmed by β-galactosidase (LacZ) staining and RT-PCR. Time-dependent changes in retinal HB-EGF were measured using quantitative RT-PCR and Western blotting. Retinal damage was induced by exposure to light. Recombinant human HB-EGF was injected intravitreally. Electroretinogram (ERG) and histological analyses were performed. To evaluate the effect of HB-EGF against light irradiation–induced cell death, 661W cells, a transformed mouse cone cell line, were used.

Results.: LacZ-positive cells were observed and Hb-egf deletion was confirmed in the retinas of Hb-egf KO mice. Hb-egf and pro-HB-EGF levels were increased after light exposure in wild-type (WT) mice. Exposure to light reduced the a- and b-wave amplitudes of the dark-adapted ERG, and also outer nuclear layer (ONL) thickness, in Hb-egf KO mice versus WT mice. Treatment with HB-EGF improved both the a- and b-wave amplitudes and the thickness of the ONL. The 661W cell death induced by light irradiation was exacerbated by Hb-egf knockdown. HB-EGF also protected against light-induced cell death and reduced reactive oxygen species (ROS) production in 661W cells. HB-EGF treatment improved the a-wave amplitudes and the thickness of the ONL in Hb-egf KO mice.

Conclusions.: These data suggest that HB-EGF plays a pivotal role in light-induced photoreceptor degeneration. It therefore warrants investigation as a potential therapeutic target for such light-induced retinal diseases as age-related macular degeneration.

Introduction
Age-related macular degeneration (AMD) is characterized by progressive degeneration of the macula, usually bilaterally, leading to a severe decrease in vision and a central scotoma. AMD is one of the main causes of blindness, but its pathogenesis is still unclear. The decrease in vision results either from retinal degeneration, which is termed geographic atrophy (i.e., the dry type of AMD), or from the secondary effects of choroidal neovascularization (i.e., the wet type of AMD). 1 Light-induced retinal degeneration is a well-known model of the dry type of AMD. 2 Light energy is important for visual function, but in excessive amounts it produces photochemical damage to retinal neurosensory cells. Loss of photoreceptors and thinning of the outer nuclear layer (ONL) have been shown to occur in albino rats and mice after they have been exposed to constant light. 2,3 During such exposure to light, intracellular calcium levels are increased in the photoreceptors, and reactive oxygen species (ROS) are generated. 3,4 Many researchers have reported the efficacy of antioxidants such as ascorbate 5 and curcumin 6 against light-induced retinal damage. In addition, we previously described both the mechanism of disease progression and the efficacies of drugs such as antioxidants (edaravone, 7,8 crocetin, 9 anthocyanin 10 ) and a calpain inhibitor 11 against light-induced retinal damage. Thus, oxidative stress is likely to be involved in the pathogenesis of light-induced retinal degeneration. 
Many neutrophic factors have been reported to protect against retinal damage 1217 ; and during light-induced retinal degeneration, microglia–Müller glia interaction controls the production of neurotrophic factors. 18 In Müller glial cells light reared in microglia-conditioned medium, nerve growth factor (NGF), neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), and glia cell line–derived neurotrophic factor (GDNF) have been found to be upregulated compared with their levels in control cells, 18 whereas basic fibroblast growth factor (bFGF) was downregulated. 18 Importantly, it has been demonstrated that injection of neurotrophic factors, cytokines, or growth factors (such as brain-derived neurotrophic factor [BDNF], basic fibroblast growth factor [bFGF], acidic fibroblast growth factor [aFGF], and interleukin 1β [IL-1β]) has protective effects against light-induced photoreceptor degeneration. 19  
Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family. 20 In the central nervous system, HB-EGF is widely expressed within neurons and neuroglia throughout the brain, 21 and it is also expressed in the retina. 22 Membrane-bound pro-HB-EGF is proteolytically cleaved by “a disintegrin and metalloprotease” (ADAM) 9, 12, 10, or 17 to release a soluble form of HB-EGF. 2327 HB-EGF binds to, and activates, the EGF receptor (EGF receptor/ErbB1), 20 and it also binds to ErbB4, 28 which has been implicated in neuronal survival and glia/stem cell proliferation. 2931 Moreover, after intestinal ischemia/reperfusion injury, HB-EGF induces decreases in oxygen free radical production, nitric oxide synthase, and nitric oxide production. 32,33 In the retina, HB-EGF is upregulated both after ischemia-reperfusion 22 and in proliferative vitreoretinopathy (PVR). 34 HB-EGF inhibits the swelling of glial cells, 22 and it is both necessary and sufficient for Müller glia dedifferentiation and retinal regeneration in zebrafish. 35 To judge from the above observations, HB-EGF may play a critical role in neural development and in neuroprotection. 
In the present study, we investigated the role of endogenous HB-EGF in light-induced retinal degeneration and the effects of HB-EGF treatment. 
Materials and Methods
Animals
All experiments were performed in accordance with the 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. Male albino ddY, C57BL/6J mice (Japan SLC, Hamamatsu, Japan) and male and female HB-EGF knockout mice, aged 8 to 12 weeks, were used in this study. HB-EGF KO mice were generated using the Cre-lox–mediated conditional gene KO approach with a Six3 promoter as described previously. 36 Because they are associated with cardiac enlargement, HB-EGF KO mice died. 37 Six3 plays an important role in vertebrate visual system development. 38 Therefore, we used the Six3 promoter to make conditional knockout mice. This gene encodes a member of the sine oculis homeobox transcription factor family. The encoded protein plays a role in eye development. 39 The background strain of HB-EGF KO mice was C57BL/6J. The mice were kept under controlled lighting conditions (12 hours/12 hours light/dark). 
Exposure to Light
After dark adaptation for 24 hours, the pupils were dilated with 1% cyclopentolate hydrochloride eye drops (Santen Pharmaceuticals Co. Ltd., Osaka, Japan) at 30 minutes before exposure to light. Pigmented rodents tolerate light better than albino rodents 2 ; therefore, nonanesthetized mice were exposed for 3 hours to 8000 (ddY mice; albino) or 14,000 lux (C57BL/6J mice; pigmented) of white fluorescent light (Toshiba, Tokyo, Japan) in cages with reflective interiors, according to our previous report. 40  
The ambient temperature during the exposure to light was maintained at 25 ± 1.5°C. After the exposure, all mice were placed in the dark for 24 hours and then returned to the previous light/dark cycle (see above). 
RNA Isolation
To examine the time-dependent changes in Hb-egf gene expression after light exposure, nontreated and light-exposed retinas were obtained immediately or at 6, 12, or 24 hours after the end of the 3-hour light exposure. Mice were euthanized by cervical spine dislocation, and the eyeballs were quickly removed. The retinas were carefully separated from the eyeballs and rapidly frozen in liquid nitrogen. RNA was isolated from retinas with the aid of a High Pure RNA Isolation kit (Roche Diagnostics, Tokyo, Japan). RNA concentrations were determined spectrophotometrically at 260 nm. First-strand cDNA was synthesized in a 10 μL reaction volume using a PrimeScript RT reagent kit (Perfect Real Time; Takara, Shiga, Japan). 
Real-Time PCR
The Hb-egf gene expression was quantified by means of real-time PCR, performed using SYBER Premix Ex Taq (Takara) and a TP 8000 Thermal Cycler Dice Real Time system (Takara). The PCR primer sequences used were as follows: HB-EGF, 5′-AAGTGSSGTTGGGCGTGGCTA-3′ (forward) and 5′-CGTGTAACGAACCACTGTCTCAGAA-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-TGTGTCCGTCGTGGATCTGA-3′ (forward), 5′-TTGCTGTTGAAGTCGCAGGAG-3′ (reverse). 
Immunoblotting
Mice were euthanized by cervical dislocation and their eyeballs quickly removed. The retinas were immediately separated from the eyeballs and rapidly frozen in liquid nitrogen. For protein extraction, the tissue was homogenized in cell lysis buffer using a homogenizer (Physcotron; Microtec Co. Ltd., Funabashi, Japan). The lysate was centrifuged at 12,000g for 20 minutes, and the supernatant was used for this study. The protein concentration was measured by comparison with a known concentration of bovine serum albumin using a BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA). Lysates were solubilized in sodium dodecyl sulfate sample buffer, separated on 15% sodium dodecyl sulfate–polyacrylamide gradient gels, and transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore Corp., Billerica, MA). Transfers were blocked for 30 minutes at room temperature with 5% Block One-P (Nakarai Tesque, Inc., Kyoto, Japan) in 10 mM Tris-buffered saline with 0.05% Tween 20, then incubated overnight at 4°C with the primary antibody. The transfers were rinsed with Tris-buffered saline with 0.05% Tween 20 and incubated for 1 hour at room temperature in horseradish peroxidase goat antimouse (Thermo Fisher Scientific) diluted 1:2000 or goat antirat (Funakoshi, Tokyo, Japan). The immunoblots were developed using chemiluminescence (ImmunoSter LD; Wako, Osaka, Japan) and visualized with the aid of a digital imaging system (LAS-4000UVmini; Fujifilm, Tokyo, Japan). The primary antibodies used were as follows: rat monoclonal anti-HB-EGF (6B41E) diluted 1:1000 and mouse monoclonal anti-β-actin (Sigma-Aldrich, St. Louis, MO) diluted 1:20,000. 
Quantitative Reverse Transcription–Polymerase Chain Reaction (RT-PCR) Analysis
HB-EGF gene disruption in the retina was confirmed by RT-PCR analysis. Total RNA was isolated from the retinas of Hb-egf KO and wild-type (WT) mice using an Aurum RNA Fatty and Fibrous Tissue kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. RT-PCR was performed using a RT-PCR Kit (Qiagen, Venlo, The Netherlands) and Thermal Cycler Dice Real Time System (Takara). The reverse transcription reaction was performed at 50°C for 15 minutes. The target cDNA was amplified by 32 cycles of PCR using the following primers: HB-EGF forward primer, 5′-GGA ATTCTGGAGCGGCTTCGGAGAG-3′; HB-EGF reverse primer, 5′-CAAGCTTTGCAAGAGGGAGTACGGAACT-3′. 
β-galactosidase (LacZ) Staining
Eyes were removed and then fixed in 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde (PFA) for 24 hours at 4°C. Eyeballs were equilibrated in 25% sucrose and frozen in Optimal Cutting Temperature (OCT) compound (SakuraFineTech, Torrance, CA). After fixation with 0.2% glutaraldehyde and 1% formalin (Active Motif, Carlsbad, CA), tissues were stained with 5-bromo-4-chloro-3-indolyl β-galactoside (X-gal) (Active Motif) at 37°C for 24 hours. 
Electroretinogram Analysis
Electroretinograms (ERG) were recorded at 5 or 28 days after light exposure. Mice were maintained in a completely dark room for 24 hours. They were then intraperitoneally anesthetized either with a mixture of ketamine (120 mg/kg; Daiichi-Sankyo, Tokyo, Japan) and xylazine (6 mg/kg; Bayer Health Care, Tokyo, Japan) or with 50 mg/kg intraperitoneal sodium pentobarbital. The pupils were dilated with 1% tropicamide and 2.5% phenylephrine (Santen Pharmaceutical Co. Ltd.). A flash ERG was recorded from the left eye of each dark-adapted mouse by placing a golden ring electrode (Mayo, Aichi, Japan) in contact with the cornea and a reference electrode (Nihon Kohden, Tokyo, Japan) through the tongue. A neutral electrode (Nihon Kohden) was inserted subcutaneously near the tail. All procedures were performed under dim red light, and the mice were kept on a heating pad to maintain a constant body temperature during the ERG recording. The amplitude of the a-wave was measured from the baseline to the highest a-wave peak, while the b-wave was measured from the highest a-wave peak to the highest b-wave peak. 
Histological Analysis
After cervical spine dislocation, each eye was enucleated and kept immersed for at least 24 hours 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. The damage induced by light exposure was then evaluated, with six sections from each eye being used for the morphometric analysis described below. Light microscope images were photographed, and the thickness of the ONL was measured at 240 μm intervals from the optic disc on the photographs. Data from three sections (selected randomly from the six sections) were averaged for each eye. 
HB-EGF Injection
Human recombinant HB-EGF (R&D Systems, Minneapolis, MN) was injected into the vitreous space by puncturing the eye at the corneal–scleral junction using a syringe equipped with a 33-gauge needle. This was done at 2 hours before light exposure under isoflurane anesthesia. For buffer control experiments, mice were injected with the same volume of PBS. The injection volume was 2 μL in all cases. 
Cell Culture
661W cells, a transformed mouse cone cell line derived from mouse retinal tumors, were a kind gift from Muayyad R. Al-Ubaidi (University of Oklahoma Health Sciences Center, Oklahoma City, OK). The 661W cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin (Meiji Seika Kaisha Ltd., Tokyo, Japan), and 100 μg/mL streptomycin (Meiji Seika Kaisha Ltd.) under a humidified atmosphere of 95% air and 5% CO2 at 37°C. Cells were passaged by trypsinization every 2 to 3 days. 
Light Irradiation–Induced Cell Death Assay
To examine the effects of HB-EGF on the cell death induced by light exposure, 661W cells were seeded at 3 × 103 cells/well in 96-well plates and then incubated for 24 hours. The entire medium was then replaced with fresh medium containing 1% FBS. Next, HB-EGF was added and the cells were then exposed to 2500 lux of white fluorescent light (Nikon, Tokyo, Japan) for 24 hours at 37°C. HB-EGF was dissolved in PBS and diluted with DMEM containing 1% FBS. 
Nuclear staining assays were carried out at the end of the light exposure treatment. Hoechst 33342 (λex = 360 nm, λem > 490 nm) and propidium iodide (PI) (λex = 535 nm, λem > 617 nm) were added to the culture medium for 15 minutes at final concentrations of 8.1 μM and 1.5 μM, respectively. Hoechst 33342 freely enters living cells and then stains the nuclei of viable cells, as well as those that have suffered apoptosis or necrosis. PI is a membrane-impermeable dye that is generally excluded from viable cells. Images were collected using an Olympus IX70 inverted epifluorescence microscope (Olympus, Tokyo, Japan). The total number of cells was counted and the percentage of PI-positive cells calculated. 
RNA Interference
For mouse HB-EGF and negative control small interfering RNAs (siRNA), we used duplexes (HB-EGF RNAi [Nippon Gene Material, Toyama, Japan] and RNAi Negative Control Duplex [Nippon Gene Material], respectively). The sense and antisense strands of mouse HB-EGF siRNA were as follows: sequence 1, 5′-CUU GGA AAG UGA AGA GAA A-3′ (sense) and 5′-UUU CUC UUC ACU UUC CAA G-3′ (antisense); sequence 2, 5′-UCG UGG GAC UUC UCA UGU U-3′ (sense) and 5′-AAC AUG AGA AGU CCC ACG A-3′ (antisense). 
Transfection With siRNA In Vitro
661W cells were seeded at a density of 1000 cells/well into 96-well plates (for cell death assay) or at a density of 5000 cells/well into 24-well plates (for assessment of the effects of HB-EGF silencing) containing cultured antibiotic-free normal medium at 37°C for 24 hours. The 661W cells were transfected with 50 nM siRNA using Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA) and Opti-MEN (Invitrogen) according to the manufacturer's protocol. After 24-hour transfection, the medium containing siRNA and complex (Lipofectamine RNAiMAX Reagent; Invitrogen) was replaced by DMEM supplemented with 1% FBS. One hour after the change of medium, cells were irradiated with visible light (2500 lux) for 24 hours at 37°C, or real-time RT-PCR was performed. 
Measurement of Reactive Oxygen Species (ROS) Production in 661W Cells
Intracellular radical activation in 661W cells was measured using CM-H2DCFDA (Invitrogen). After 24-hour light irradiation, CM-H2DCFDA was added to the culture medium and incubated at 37°C for 1 hour for a final concentration of 10 μM. The 96-well plate was loaded into a plate holder in a fluorescence spectrophotometer (Varioskan Flash; Thermo Fisher Scientific). The reaction was carried out at 37°C, and fluorescence was measured at 488 nm excitation and 525 nm emission at the end of light irradiation and 1 hour after light irradiation. The number of cells was measured by Hoechst 33342 staining and used to calculate ROS production per cell. 
Statistical Analysis
Data are presented as the means ± SEM. Statistical comparisons were made by way of a Tukey test, Dunnett's test, or Student's t-test using statistical analysis software (StatView version 5.0; SAS Institute, Cary, NC). P < 0.05 was considered to indicate statistical significance. 
Results
Changes in Hb-egf mRNA and HB-EGF Protein Levels After Light Exposure
To investigate the changes in Hb-egf mRNA and HB-EGF protein levels after exposure to light, real-time RT-PCR was performed (Fig. 1A). Just after light exposure, Hb-egf mRNA was increased approximately 1.4-fold (versus the control group) in C57BL/6J WT mice. Moreover, pro-HB-EGF was significantly increased at 6 hours after exposure to light (Fig. 1B). 
Figure 1
 
Expression of Hb-egf mRNA after light exposure. (A) Hb-egf mRNA was increased just after exposure to light (14,000 lux). Data are shown as mean ± SEM (n = 8–10). *P < 0.05 versus normal group (Dunnett's test). (B) Representative band images show immunoreactivities against pro HB-EGF. Pro-HB-EGF was significantly increased at 6 hours after light exposure. The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 6). *P < 0.05 versus normal group (Dunnett's test).
Figure 1
 
Expression of Hb-egf mRNA after light exposure. (A) Hb-egf mRNA was increased just after exposure to light (14,000 lux). Data are shown as mean ± SEM (n = 8–10). *P < 0.05 versus normal group (Dunnett's test). (B) Representative band images show immunoreactivities against pro HB-EGF. Pro-HB-EGF was significantly increased at 6 hours after light exposure. The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 6). *P < 0.05 versus normal group (Dunnett's test).
Confirmation of the HB-EGF Knockdown in HB-EGF KO Murine Retina
To confirm the disruption of HB-EGF, Hb-egf mRNA was measured by RT-PCR. In the HB-EGF KO murine retina, expression of Hb-egf mRNA was reduced compared with that in the WT mouse (Fig. 2A). Furthermore, LacZ staining revealed disruption of the Hb-egf gene in the ONL, inner nuclear layer (INL), and ganglion cell layer (GCL) (Fig. 2B). This indicates that retinal HB-EGF expression was reduced in HB-EGF KO mice. Embryologically, retina is differentiated from the forebrain. In Hb-egf knockout mice, brain-derived HB-EGF in the eye was disrupted because of the Six3 promoter, and weak Hb-egf expression derived from blood vessels may have been present (Fig. 2A). 
Figure 2
 
Role of HB-EGF in light-induced retinal morphological change, and confirmation of Hb-egf knockdown in HB-EGF KO mouse retina. (A) RT-PCR analysis revealed that Hb-egf mRNA expression was decreased in HB-EGF KO mice (versus WT mice). (B) In HB-EGF KO mice, LacZ-positive cells were observed in the retina. Scale bars: 50 μm (upper); 15 μm (lower). (C) Representative photographs showing hematoxylin and eosin staining of retinal section from WT mice and HB-EGF KO mice, and from light-exposed (14,000 lux) WT mice and light-exposed (14,000 lux) HB-EGF KO mice (retinas obtained at 5 days after light exposure). The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. Data are shown as mean ± SEM (n = 4–7). *P < 0.05, **P < 0.01 versus control WT; #P < 0.05, ##P < 0.01 versus light-exposed WT group (Tukey test).
Figure 2
 
Role of HB-EGF in light-induced retinal morphological change, and confirmation of Hb-egf knockdown in HB-EGF KO mouse retina. (A) RT-PCR analysis revealed that Hb-egf mRNA expression was decreased in HB-EGF KO mice (versus WT mice). (B) In HB-EGF KO mice, LacZ-positive cells were observed in the retina. Scale bars: 50 μm (upper); 15 μm (lower). (C) Representative photographs showing hematoxylin and eosin staining of retinal section from WT mice and HB-EGF KO mice, and from light-exposed (14,000 lux) WT mice and light-exposed (14,000 lux) HB-EGF KO mice (retinas obtained at 5 days after light exposure). The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. Data are shown as mean ± SEM (n = 4–7). *P < 0.05, **P < 0.01 versus control WT; #P < 0.05, ##P < 0.01 versus light-exposed WT group (Tukey test).
Role of HB-EGF in Light-Induced Retinal Degeneration
To explore the role of HB-EGF in light-induced retinal damage, histological analysis was performed. Figure 2C shows representative retinal images taken between 380 and 610 μm from the optic nerve in the superior retina at 5 days after exposure to light (14,000 lux). The thickness of ONL was decreased at 5 days after light exposure compared with the corresponding control (Fig. 2D). HB-EGF knockdown exacerbated the light-induced thinning of ONL by 1.4-fold (versus the light-exposed WT group). 
Role of HB-EGF in Light-Induced Retinal Function
To evaluate the role of HB-EGF in the light-induced changes in retinal function, ERG analysis was performed. The a-wave relates to the function of photoreceptors, while the b-wave reflects bipolar cell and Müller cell functions. In HB-EGF KO mice, both the a- and b-wave amplitudes were decreased at 5 days after exposure to 14,000-lux white fluorescent light compared with those in the WT group (Figs. 3A–C). In particular, at 0.98 log cds/m2, the a- and b-wave amplitudes were reduced by 37% and 40%, respectively, versus the corresponding WT values. 
Figure 3
 
Role of HB-EGF in light-induced retinal functional damage. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 4–7). *P < 0.05, **P < 0.01 versus control WT; #P < 0.05, ##P < 0.01 versus light-exposed WT (Tukey test).
Figure 3
 
Role of HB-EGF in light-induced retinal functional damage. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 4–7). *P < 0.05, **P < 0.01 versus control WT; #P < 0.05, ##P < 0.01 versus light-exposed WT (Tukey test).
Effects of Hb-egf siRNA on Light Irradiation–Induced Changes in 661W Cells
The groups treated with Hb-egf siRNAs containing sequence 1 or 2 showed significantly less Hb-egf mRNA (76% and 46% less, respectively) compared with negative siRNA-transfected cells (negative control) (Fig. 4A). The negative siRNA-transfected group did not change the expression of Hb-egf. To investigate the effects of Hb-egf siRNA on the viability of 661W cells following light exposure, cells were stained with Hoechst 33342 and PI (typical images are shown in Fig. 4B). All the dark control groups showed limited cell death, indicating that Hb-egf knockdown itself did not cause any toxicity (Fig. 4C). However, exposure to light significantly induced photoreceptor cell death. Both siRNAs 1 and 2 increased the cell death induced by light irradiation (58% and 38% increase, respectively, compared with the negative control siRNA with light irradiation) (Fig. 4C). 
Figure 4
 
Effect of HB-EGF siRNA on response of 661W cell to light irradiation. (A) Expression of Hb-egf mRNA after transfection with Hb-egf or negative control siRNA. Gapdh mRNA was used as the internal control. Data are shown as mean ± SEM (n = 5). *P < 0.05, **P < 0.01 versus negative control siRNA group (t-test). (B) Fluorescence microscopy of Hoechst 33342 and propidium iodide (PI) staining at 24 hours after light irradiation. (C) The number of cells exhibiting PI fluorescence was counted, and positive cells were expressed as the percentage of PI-stained cells in relation to Hoechst 33342-stained cells. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus no siRNA group (t-test); ##P < 0.01 versus light-exposed negative control siRNA group (t-test). Scale bars: 50 μm.
Figure 4
 
Effect of HB-EGF siRNA on response of 661W cell to light irradiation. (A) Expression of Hb-egf mRNA after transfection with Hb-egf or negative control siRNA. Gapdh mRNA was used as the internal control. Data are shown as mean ± SEM (n = 5). *P < 0.05, **P < 0.01 versus negative control siRNA group (t-test). (B) Fluorescence microscopy of Hoechst 33342 and propidium iodide (PI) staining at 24 hours after light irradiation. (C) The number of cells exhibiting PI fluorescence was counted, and positive cells were expressed as the percentage of PI-stained cells in relation to Hoechst 33342-stained cells. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus no siRNA group (t-test); ##P < 0.01 versus light-exposed negative control siRNA group (t-test). Scale bars: 50 μm.
Protective Effects of HB-EGF Against Light-Induced Photoreceptor Degeneration
To investigate the effects of HB-EGF treatment against light-induced photoreceptor degeneration, histological analysis was performed. The representative retinal images in Figure 5A show the superior retina between 380 and 720 μm from the optic nerve at 5 days after exposure to light (8000 lux). ONL thickness was decreased in the vehicle-treated retina compared with that in the nontreated one (Fig. 5A), while the HB-EGF–treated retina showed significantly less photic damage than the vehicle-treated one (Fig. 5A). Group data revealed that treatment with HB-EGF suppressed the maximum thinning of ONL by 34% compared with vehicle treatment (Fig. 5B). 
Figure 5
 
Protective effects of HB-EGF against light-induced morphological changes in the retina. (A) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (B) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. The strain of mice was ddY. Data are shown as mean ± SEM (n = 7–9). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test). (C) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 28 days after light exposure. The strain of mice was ddY. Data are shown as mean ± SEM (n = 8–11). **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Figure 5
 
Protective effects of HB-EGF against light-induced morphological changes in the retina. (A) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (B) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. The strain of mice was ddY. Data are shown as mean ± SEM (n = 7–9). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test). (C) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 28 days after light exposure. The strain of mice was ddY. Data are shown as mean ± SEM (n = 8–11). **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Protective Effects of HB-EGF Against Light-Induced Retinal Functional Damage
The functional effects of HB-EGF were evaluated by recording ERG responses. Both the a- and b-wave amplitudes were significantly decreased at 5 or 28 days after exposure to 8000-lux white fluorescent light. Intravitreal injection of HB-EGF reduced these effects of exposure to light (Figs. 6B–E). In particular, at 5 days after light exposure, the a- and b-wave amplitudes recorded at −0.02 log cds/m2 were improved by 18% and 28%, respectively, compared with those in the vehicle-treated group. Moreover, at 28 days after exposure to light, the a- and b-wave amplitudes recorded at 0.98 log cds/m2 were improved by 25% and 32%, respectively, compared with those in the vehicle-treated group. 
Figure 6
 
Protective effects of HB-EGF against light-induced retinal damage in mice. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (8000 lux). The strain of mice was ddY. Data are shown as mean ± SEM (n = 7–9). **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test). (D) Typical dark-adapted ERG traces recorded at 28 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (E, F) Amplitudes of a- and b-waves at 5 days after light exposure (8000 lux). The strain of mice was ddY. Data are shown as mean ± SEM (n = 10 or 13). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Figure 6
 
Protective effects of HB-EGF against light-induced retinal damage in mice. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (8000 lux). The strain of mice was ddY. Data are shown as mean ± SEM (n = 7–9). **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test). (D) Typical dark-adapted ERG traces recorded at 28 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (E, F) Amplitudes of a- and b-waves at 5 days after light exposure (8000 lux). The strain of mice was ddY. Data are shown as mean ± SEM (n = 10 or 13). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Protective Effects of HB-EGF Against Light-Induced Damage in 661W Cells
To investigate whether HB-EGF might prevent the cell death induced by light exposure, 661W cells were treated with HB-EGF, and then, starting 1 hour later, exposed to light (2500 lux) for 24 hours. Recombinant human HB-EGF was added to 661W cells at concentrations from 10 to 100 nM, based on previous reports. 22,34 Typical images of Hoechst 33342 and PI staining are shown in Figure 7A. Light exposure induced significant cell death, and treatment with HB-EGF significantly inhibited this cell death at concentrations of 50 and 100 nM, as also did treatment with Trolox (30 μM; Enzo Life Science, Farmingdale, NY) (Fig. 7B). 
Figure 7
 
Protective effect of HB-EGF against 661W cell death after light exposure. In this in vitro study, HB-EGF reduced the cell damage induced by light exposure (2500 lux) in 661W cells. (A) Fluorescence microscopy of Hoechst 33342 and propidium iodide (PI) staining at 24 hours after light exposure. Scale bars: 50 μm. (B) Number of PI-positive cells (expressed as the percentage of PI-positive in relation to Hoechst 33342-positive cells). Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus light-exposed vehicle group (Dunnett's test). (C) Effects of HB-EGF on light-induced ROS production. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control group; ##P < 0.01 versus light-exposed vehicle group (Dunnett's test).
Figure 7
 
Protective effect of HB-EGF against 661W cell death after light exposure. In this in vitro study, HB-EGF reduced the cell damage induced by light exposure (2500 lux) in 661W cells. (A) Fluorescence microscopy of Hoechst 33342 and propidium iodide (PI) staining at 24 hours after light exposure. Scale bars: 50 μm. (B) Number of PI-positive cells (expressed as the percentage of PI-positive in relation to Hoechst 33342-positive cells). Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus light-exposed vehicle group (Dunnett's test). (C) Effects of HB-EGF on light-induced ROS production. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control group; ##P < 0.01 versus light-exposed vehicle group (Dunnett's test).
Effects of HB-EGF on Light-Induced ROS Production in 661W Cells
Treatment with HB-EGF at 50 and 100 nM inhibited the light-induced radical activity in 661W cells, as also did treatment with Trolox (30 μM; Enzo Life Science) (Fig. 7C). Data revealed that treatment with HB-EGF (100 nM) suppressed the ROS formation by 19% compared with vehicle treatment. 
Protective Effects of HB-EGF on Light-Induced Retinal Degeneration on HB-EGF KO Mice
To investigate the effects of HB-EGF treatment against light-induced photoreceptor degeneration, histological analysis was performed. The representative retinal images in Figure 8A show the superior retina between 200 and 520 μm from the optic nerve at 5 and 28 days after exposure to light (14,000 lux). ONL thickness was decreased in the vehicle-treated retina compared with that in the nontreated one (Figs. 8A, 8C), while the HB-EGF–treated retina showed significantly less photic damage than the vehicle-treated one (Figs. 8A, 8C). Group data revealed that treatment with HB-EGF suppressed the maximum thinning of ONL by 54% and 68%, respectively, after 5 and 28 days following light irradiation compared with vehicle treatment (Fig. 8B). 
Figure 8
 
Protective effects of HB-EGF against light-induced morphological changes in the retina of HB-EGF KO mouse. (A) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB EGF treated). Scale bar: 50 μm. (B) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05 versus vehicle group (t-test). (C) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 28 days after light exposure. The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Figure 8
 
Protective effects of HB-EGF against light-induced morphological changes in the retina of HB-EGF KO mouse. (A) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB EGF treated). Scale bar: 50 μm. (B) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05 versus vehicle group (t-test). (C) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 28 days after light exposure. The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Protective Effects of HB-EGF Against Light-Induced Retinal Functional Damage on HB-EGF KO Mice
The functional effects of HB-EGF were evaluated by recording ERG responses. Both the a- and b-wave amplitudes were significantly decreased at 5 and 28 days after exposure to 14,000-lux white fluorescent light. Intravitreal injection of HB-EGF reduced these effects of exposure to light on a-wave amplitude (Figs. 9B, 9E). In particular, at 5 days after light exposure, the a-wave amplitudes recorded at −0.02 log cds/m2 were improved by 31% compared with those in the vehicle-treated group. Moreover, at 28 days after exposure to light, the a-wave amplitudes recorded at 0.98 log cds/m2 were improved by 47% compared with those in the vehicle-treated group. However, intravitreal injection of HB-EGF did not influence these effects on b-wave amplitudes either at 5 or 28 days after light exposure (Figs. 9C, 9F). 
Figure 9
 
Protective effects of HB-EGF against light-induced functional changes in the retina of HB-EGF KO mouse. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05 versus normal group; #P < 0.05 versus vehicle group (t-test). (D) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (E, F) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05 versus vehicle group (t-test).
Figure 9
 
Protective effects of HB-EGF against light-induced functional changes in the retina of HB-EGF KO mouse. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05 versus normal group; #P < 0.05 versus vehicle group (t-test). (D) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (E, F) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05 versus vehicle group (t-test).
Discussion
In the present study, several important findings were made concerning the effects and the possible role of HB-EGF in light-induced retinal degeneration. First, after exposure to light, Hb-egf mRNA and protein levels were increased in the retina. Second, in Hb-egf KO mice, retinal function was decreased after light irradiation (versus that in WT mice). Moreover, in 661W cells, the cone photoreceptor cell death induced by irradiation with light was increased by Hb-egf knockdown in vitro. Third, treatment with HB-EGF protected against the damaging effects of light exposure both in vivo and in vitro, and reduced ROS production. Fourth, treatment with HB-EGF protected against light-induced photoreceptor degeneration in Hb-egf KO mice. The putative mechanism of HB-EGF against light-induced photoreceptor degeneration is shown in Figure 10
Figure 10
 
Putative mechanism of HB-EGF against light-induced photoreceptor degeneration. Exposure to light induced oxidative stress and it led to the expression of impaired factors (i.e., p38 and JNK) and survival factors (i.e., ERK 1/2 and Akt). Exogenous HB-EGF decreased ROS production, and may activate EGF receptors and the downstream signaling (ERK 1/2 and Akt pathway). These effects may be protective against light-induced photoreceptor degeneration. Pro-HB-EGF and the carboxy-terminal fragment of pro-HB-EGF (HB-EGF-C) may play some roles in cell survival and/or cell proliferation. However, further experiments will be needed to confirm the roles of pro-HB-EGF and the carboxyl-terminal fragment. The balance between cell death and cell survival may decide “death” or “survival” in the cells.
Figure 10
 
Putative mechanism of HB-EGF against light-induced photoreceptor degeneration. Exposure to light induced oxidative stress and it led to the expression of impaired factors (i.e., p38 and JNK) and survival factors (i.e., ERK 1/2 and Akt). Exogenous HB-EGF decreased ROS production, and may activate EGF receptors and the downstream signaling (ERK 1/2 and Akt pathway). These effects may be protective against light-induced photoreceptor degeneration. Pro-HB-EGF and the carboxy-terminal fragment of pro-HB-EGF (HB-EGF-C) may play some roles in cell survival and/or cell proliferation. However, further experiments will be needed to confirm the roles of pro-HB-EGF and the carboxyl-terminal fragment. The balance between cell death and cell survival may decide “death” or “survival” in the cells.
The expression of various receptors, growth factors, and neurotrophic factors undergoes alterations due to light irradiation. For example, exposure to light causes increased expressions of EGF receptors (EGFR). 41 In vascular smooth muscle cells (VSMC), Hb-egf mRNA is increased by bFGF and PDGF, and also by HB-EGF itself. 42 Moreover, HB-EGF can induce an increase in Cntf mRNA in VSMC. 43 These data indicate whether HB-EGF is upstream or downstream of each of those factors and how it affects their expressions of these factors. To judge from the above observations, HB-EGF may play a pivotal role in the pathogenesis of light-induced retinal degeneration. 
Cleavage of pro-HB-EGF is necessary for EGFR transactivation by G protein–coupled receptor signaling, 44 and the carboxy-terminal fragment of pro-HB-EGF (HB-EGF-C) is translocated from plasma membrane to nucleus after ectodomain shedding of pro-HB-EGF. 45 Extracellular signaling–regulated kinase (ERK) and protein kinase B (Akt) are known to be downstream of HB-EGF signaling, 31 and ERK and Akt activations are thought to have protective effects against light-induced retinal damage. 7,46 Thus, ERK and Akt signaling may be downregulated, and their protective effects eliminated, by Hb-egf knockdown. HB-EGF-C causes nuclear export of promyelocytic leukemia zinc finger protein, one of the transcriptional repressors, and activates a proliferative suppression abreaction. 45 Moreover, pro-HB-EGF itself has also some roles. 47,48 These functions may be disrupted by Hb-egf knockdown. Since the protective effect of recombinant HB-EGF (i.e., secretory HB-EGF) was limited (Figs. 5, 6, 8, 9), the carboxy-terminal fragment of pro-HB-EGF and/or pro-HB-EGF itself may also be important for its protective effect. Furthermore, HB-EGF may be involved in ROS production (Fig. 7C) and antioxidative stress. 32,33 In fact, postischemic administration of HB-EGF protects against focal cerebral ischemia in the rat, 49 while Hb-egf KO mice exhibit exacerbated ischemia and reperfusion injury in the brain. 50 On the other hand, intravitreal injection of EGF does not exert protective effects against light-induced photoreceptor degeneration, 19 even though HB-EGF binds to and activates the EGF receptor (EGF receptor/ErbB1), 20 as does EGF. These differences indicate that other receptors may be associated with the protective effects of HB-EGF. Possibly, HB-EGF may act not only upon EGF receptors in the outer retina, but also upon those in Müller glia cells. In the zebrafish retina, HB-EGF is both necessary and sufficient for Müller glia dedifferentiation and retinal regeneration, 35 Furthermore, microglia–Müller glia cell interactions control neutrophic factor production during light-induced retinal degeneration. 18 In fact, as shown in Figures 9C and 9F, b-wave amplitude was not improved by the HB-EGF injection in HB-EGF KO mice. These data suggest that intracellular HB-EGF may play an important role on Müller cells. To judge from these data, HB-EGF may protect against light-induced photoreceptor degeneration not only via EGF signaling, but also via Müller glia dedifferentiation and antioxidative stress. 
In conclusion, we have found evidence that HB-EGF may play a pivotal role in light-induced retinal degeneration, and that it may represent a potential therapeutic target in the dry type of AMD. 
Acknowledgments
We thank Shigeki Higashiyama (Ehime University, Ehime, Japan) for the gift of HB-EGF antibody and Yuta Inokuchi and Kazuki Ojino (Gifu Pharmaceutical University, Gifu, Japan) for technical support. 
Disclosure: Y. Inoue, None; K. Tsuruma, None; T. Nakanishi, None; A. Oyagi, None; Y. Ohno, None; T. Otsuka, None; M. Shimazawa, None; H. Hara, None 
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Figure 1
 
Expression of Hb-egf mRNA after light exposure. (A) Hb-egf mRNA was increased just after exposure to light (14,000 lux). Data are shown as mean ± SEM (n = 8–10). *P < 0.05 versus normal group (Dunnett's test). (B) Representative band images show immunoreactivities against pro HB-EGF. Pro-HB-EGF was significantly increased at 6 hours after light exposure. The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 6). *P < 0.05 versus normal group (Dunnett's test).
Figure 1
 
Expression of Hb-egf mRNA after light exposure. (A) Hb-egf mRNA was increased just after exposure to light (14,000 lux). Data are shown as mean ± SEM (n = 8–10). *P < 0.05 versus normal group (Dunnett's test). (B) Representative band images show immunoreactivities against pro HB-EGF. Pro-HB-EGF was significantly increased at 6 hours after light exposure. The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 6). *P < 0.05 versus normal group (Dunnett's test).
Figure 2
 
Role of HB-EGF in light-induced retinal morphological change, and confirmation of Hb-egf knockdown in HB-EGF KO mouse retina. (A) RT-PCR analysis revealed that Hb-egf mRNA expression was decreased in HB-EGF KO mice (versus WT mice). (B) In HB-EGF KO mice, LacZ-positive cells were observed in the retina. Scale bars: 50 μm (upper); 15 μm (lower). (C) Representative photographs showing hematoxylin and eosin staining of retinal section from WT mice and HB-EGF KO mice, and from light-exposed (14,000 lux) WT mice and light-exposed (14,000 lux) HB-EGF KO mice (retinas obtained at 5 days after light exposure). The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. Data are shown as mean ± SEM (n = 4–7). *P < 0.05, **P < 0.01 versus control WT; #P < 0.05, ##P < 0.01 versus light-exposed WT group (Tukey test).
Figure 2
 
Role of HB-EGF in light-induced retinal morphological change, and confirmation of Hb-egf knockdown in HB-EGF KO mouse retina. (A) RT-PCR analysis revealed that Hb-egf mRNA expression was decreased in HB-EGF KO mice (versus WT mice). (B) In HB-EGF KO mice, LacZ-positive cells were observed in the retina. Scale bars: 50 μm (upper); 15 μm (lower). (C) Representative photographs showing hematoxylin and eosin staining of retinal section from WT mice and HB-EGF KO mice, and from light-exposed (14,000 lux) WT mice and light-exposed (14,000 lux) HB-EGF KO mice (retinas obtained at 5 days after light exposure). The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. Data are shown as mean ± SEM (n = 4–7). *P < 0.05, **P < 0.01 versus control WT; #P < 0.05, ##P < 0.01 versus light-exposed WT group (Tukey test).
Figure 3
 
Role of HB-EGF in light-induced retinal functional damage. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 4–7). *P < 0.05, **P < 0.01 versus control WT; #P < 0.05, ##P < 0.01 versus light-exposed WT (Tukey test).
Figure 3
 
Role of HB-EGF in light-induced retinal functional damage. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 4–7). *P < 0.05, **P < 0.01 versus control WT; #P < 0.05, ##P < 0.01 versus light-exposed WT (Tukey test).
Figure 4
 
Effect of HB-EGF siRNA on response of 661W cell to light irradiation. (A) Expression of Hb-egf mRNA after transfection with Hb-egf or negative control siRNA. Gapdh mRNA was used as the internal control. Data are shown as mean ± SEM (n = 5). *P < 0.05, **P < 0.01 versus negative control siRNA group (t-test). (B) Fluorescence microscopy of Hoechst 33342 and propidium iodide (PI) staining at 24 hours after light irradiation. (C) The number of cells exhibiting PI fluorescence was counted, and positive cells were expressed as the percentage of PI-stained cells in relation to Hoechst 33342-stained cells. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus no siRNA group (t-test); ##P < 0.01 versus light-exposed negative control siRNA group (t-test). Scale bars: 50 μm.
Figure 4
 
Effect of HB-EGF siRNA on response of 661W cell to light irradiation. (A) Expression of Hb-egf mRNA after transfection with Hb-egf or negative control siRNA. Gapdh mRNA was used as the internal control. Data are shown as mean ± SEM (n = 5). *P < 0.05, **P < 0.01 versus negative control siRNA group (t-test). (B) Fluorescence microscopy of Hoechst 33342 and propidium iodide (PI) staining at 24 hours after light irradiation. (C) The number of cells exhibiting PI fluorescence was counted, and positive cells were expressed as the percentage of PI-stained cells in relation to Hoechst 33342-stained cells. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus no siRNA group (t-test); ##P < 0.01 versus light-exposed negative control siRNA group (t-test). Scale bars: 50 μm.
Figure 5
 
Protective effects of HB-EGF against light-induced morphological changes in the retina. (A) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (B) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. The strain of mice was ddY. Data are shown as mean ± SEM (n = 7–9). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test). (C) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 28 days after light exposure. The strain of mice was ddY. Data are shown as mean ± SEM (n = 8–11). **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Figure 5
 
Protective effects of HB-EGF against light-induced morphological changes in the retina. (A) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (B) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. The strain of mice was ddY. Data are shown as mean ± SEM (n = 7–9). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test). (C) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 28 days after light exposure. The strain of mice was ddY. Data are shown as mean ± SEM (n = 8–11). **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Figure 6
 
Protective effects of HB-EGF against light-induced retinal damage in mice. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (8000 lux). The strain of mice was ddY. Data are shown as mean ± SEM (n = 7–9). **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test). (D) Typical dark-adapted ERG traces recorded at 28 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (E, F) Amplitudes of a- and b-waves at 5 days after light exposure (8000 lux). The strain of mice was ddY. Data are shown as mean ± SEM (n = 10 or 13). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Figure 6
 
Protective effects of HB-EGF against light-induced retinal damage in mice. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (8000 lux). The strain of mice was ddY. Data are shown as mean ± SEM (n = 7–9). **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test). (D) Typical dark-adapted ERG traces recorded at 28 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (E, F) Amplitudes of a- and b-waves at 5 days after light exposure (8000 lux). The strain of mice was ddY. Data are shown as mean ± SEM (n = 10 or 13). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Figure 7
 
Protective effect of HB-EGF against 661W cell death after light exposure. In this in vitro study, HB-EGF reduced the cell damage induced by light exposure (2500 lux) in 661W cells. (A) Fluorescence microscopy of Hoechst 33342 and propidium iodide (PI) staining at 24 hours after light exposure. Scale bars: 50 μm. (B) Number of PI-positive cells (expressed as the percentage of PI-positive in relation to Hoechst 33342-positive cells). Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus light-exposed vehicle group (Dunnett's test). (C) Effects of HB-EGF on light-induced ROS production. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control group; ##P < 0.01 versus light-exposed vehicle group (Dunnett's test).
Figure 7
 
Protective effect of HB-EGF against 661W cell death after light exposure. In this in vitro study, HB-EGF reduced the cell damage induced by light exposure (2500 lux) in 661W cells. (A) Fluorescence microscopy of Hoechst 33342 and propidium iodide (PI) staining at 24 hours after light exposure. Scale bars: 50 μm. (B) Number of PI-positive cells (expressed as the percentage of PI-positive in relation to Hoechst 33342-positive cells). Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus light-exposed vehicle group (Dunnett's test). (C) Effects of HB-EGF on light-induced ROS production. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control group; ##P < 0.01 versus light-exposed vehicle group (Dunnett's test).
Figure 8
 
Protective effects of HB-EGF against light-induced morphological changes in the retina of HB-EGF KO mouse. (A) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB EGF treated). Scale bar: 50 μm. (B) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05 versus vehicle group (t-test). (C) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 28 days after light exposure. The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Figure 8
 
Protective effects of HB-EGF against light-induced morphological changes in the retina of HB-EGF KO mouse. (A) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB EGF treated). Scale bar: 50 μm. (B) Measurements of the thickness of the outer nuclear layer at 5 days after light exposure. The strain of WT mice used in the study and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05 versus vehicle group (t-test). (C) Representative photographs showing hematoxylin and eosin staining of retinal sections (normal, vehicle treated, and HB-EGF treated). Scale bar: 50 μm. (D) Measurements of the thickness of the outer nuclear layer at 28 days after light exposure. The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05, ##P < 0.01 versus vehicle group (t-test).
Figure 9
 
Protective effects of HB-EGF against light-induced functional changes in the retina of HB-EGF KO mouse. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05 versus normal group; #P < 0.05 versus vehicle group (t-test). (D) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (E, F) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05 versus vehicle group (t-test).
Figure 9
 
Protective effects of HB-EGF against light-induced functional changes in the retina of HB-EGF KO mouse. (A) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (B, C) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05 versus normal group; #P < 0.05 versus vehicle group (t-test). (D) Typical dark-adapted ERG traces recorded at 5 days after light exposure in response to stimulus flashes at 0.98 log cds/m2. (E, F) Amplitudes of a- and b-waves at 5 days after light exposure (14,000 lux). The strain of WT mice and the background of KO mice were C57BL/6J. Data are shown as mean ± SEM (n = 5 or 8). *P < 0.05, **P < 0.01 versus normal group; #P < 0.05 versus vehicle group (t-test).
Figure 10
 
Putative mechanism of HB-EGF against light-induced photoreceptor degeneration. Exposure to light induced oxidative stress and it led to the expression of impaired factors (i.e., p38 and JNK) and survival factors (i.e., ERK 1/2 and Akt). Exogenous HB-EGF decreased ROS production, and may activate EGF receptors and the downstream signaling (ERK 1/2 and Akt pathway). These effects may be protective against light-induced photoreceptor degeneration. Pro-HB-EGF and the carboxy-terminal fragment of pro-HB-EGF (HB-EGF-C) may play some roles in cell survival and/or cell proliferation. However, further experiments will be needed to confirm the roles of pro-HB-EGF and the carboxyl-terminal fragment. The balance between cell death and cell survival may decide “death” or “survival” in the cells.
Figure 10
 
Putative mechanism of HB-EGF against light-induced photoreceptor degeneration. Exposure to light induced oxidative stress and it led to the expression of impaired factors (i.e., p38 and JNK) and survival factors (i.e., ERK 1/2 and Akt). Exogenous HB-EGF decreased ROS production, and may activate EGF receptors and the downstream signaling (ERK 1/2 and Akt pathway). These effects may be protective against light-induced photoreceptor degeneration. Pro-HB-EGF and the carboxy-terminal fragment of pro-HB-EGF (HB-EGF-C) may play some roles in cell survival and/or cell proliferation. However, further experiments will be needed to confirm the roles of pro-HB-EGF and the carboxyl-terminal fragment. The balance between cell death and cell survival may decide “death” or “survival” in the cells.
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