2014 Volume 37 Issue 3 Pages 424-430
Although dry age-related macular degeneration (AMD) is one of the major causes of blindness, no effective therapies are developed. In this study, we investigated the effects of SUN N8075, a radical scavenger with neuroprotective properties, against light-induced retinal damage used as the model of dry AMD in mice. After dark adaption for 24 h, we exposed the mice at 8000 lx for 3 h. We evaluated the retinal damage by recording the electroretinagram (ERG) and measuring the thickness of outer nuclear layer (ONL) at 5 d after the light exposure. Retinal apoptotic cells were also detected by terminal deoxynucleotidyl transeferase mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining, and the expression of 8-hydroxy-2-deoxyguanosine (8-OHdG) as an index for oxidative stress at 48 h after exposure to light. In ERG measurement, the intraperitoneal administration of SUN N8075 at 30 mg/kg improved the retinal dysfunction induced by the excess light exposure. In the histological evaluation, SUN N8075 inhibited the reduction of ONL thickness. In addition, SUN N8075 decreased in both numbers of TUNEL- and 8-OHdG-positive cells in ONL. These findings suggest that the systemic administration of SUN N8075 has protective effects on excess light-induced photoreceptor degeneration, via inhibition of oxidative stress.
Age-related macular degeneration (AMD) is a degenerative disease of the retina and one of the major causes of blindness in the elderly of developed countries.1) The first stage of AMD is typified by the appearance of characteristic white or yellow deposits (drusen) in Bruch’s membrane, under the retinal pigment epithelial (RPE) layer and photoreceptor cells.2)
Two types of AMD are recognized in pathological classification: dry and wet. Dry AMD is characterized by retinal geographic atrophy; the pathological changes progress slowly and the vision is impaired as a result of the RPE and photoreceptor cell losses. Dry AMD can transform into wet AMD, which is characterized by choroidal neovascularization with leakage and bleeding.3) Intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents and photo-dynamic therapy are effective for wet AMD, but no available therapies are successful in the treatment of dry AMD. Thus, therapy for dry AMD is greatly desired all over the world. Excessive light exposure is considered a risk factor for AMD.4) Exposure to intense light causes irreversible injury to photoreceptor cells and results in narrowing of the visual field, which leads eventually to the associated vision loss. One model for dry AMD is the light-induced retinal damage model. Many morphological and molecular features of the light-induced retinal degeneration closely mimic hallmarks of dry AMD, for example photoreceptor cell death and RPE ablation.5) The process of light-induced retinal degeneration is thought to involve apoptosis,6) and oxidative stress plays a key role in the loss of photoreceptor cells.
SUN N8075, (2S)-1-(4-amino-2,3,5-trimethylphenoxy)-3-{4-[4-(4-fluorobenzyl) phenyl]-1-piperazinyl}-2-propanol dimethanesulfonate, a potent antioxidant, is a molecularly modified form of flunarizine, which is a double inhibitor for Na+ and T-type Ca2+ channels.7) Flunarizine, when used clinically, has a side effect of Parkinsonism, caused by its binding affinity for the dopamine D2 receptor, but SUN N8075 has no such effect.7) In our previous studies, we showed that SUN N8075 had protective effects against transient and permanent middle cerebral artery occlusion7,8) and Parkinson’s disease models.9) In the retina, SUN N8075 protected against the damage induced by intravitreous injection of N-methyl-D-aspartate (NMDA) and high-intraocular pressure, which are animal models of glaucoma.10) In the present study, we investigated the effects and action mechanism of SUN N8075 on the light-induced retinal damage.
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. Male albino ddY mice (Japan SLC, Hamamatsu, Japan), aged 8 weeks, were used in this study. They were kept under controlled lighting conditions (12 h : 12 h light/dark).
Exposure to LightAfter dark adaptation for 24 h, the pupils were dilated with 1% cyclopentolate hydrochloride eye drops (Santen Pharmaceuticals Co., Ltd., Osaka, Japan) at 30 min before exposure to light. Non-anesthetized mice were exposed to 8000 lx of white fluorescent light (rated lamp wattage; 30 W, color temperature; 4200 K) (Toshiba, Tokyo, Japan) for 3 h in cages with reflective interiors. The temperature during the exposure to light was maintained at 25±1.5°C. After the exposure to light, all mice were placed into the dark for 24 h and then returned to the normal light/dark cycle.
Treatment with SUN N8075SUN N8075 (30 mg/kg) or vehicle [6% captisol in 0.48% (w/v)] was administered intraperitoneally just before and just after exposure to light, because light-induced retinal damage model is an acute damage model, and following to our previous studies.10,11) SUN N8075 was kindly gifted from Asubio Pharma Co., Ltd. (Kobe, Japan).
Electroretinogram (ERG)ERG was recorded at 5 d after light exposure. In this experiment, 43 mice in total were used. Mice were maintained in a completely dark room for 24 h and then anesthetized by 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) through 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. The mice were kept on heating pad (Mycoal, Tochigi, Japan) to maintain a constant body temperature during the ERG recording. The amplitude of the a-wave was measured from the baseline to the trough of a-wave, and the b-wave was measured from the trough of the a-wave to the peak of the b-wave.
Histological AnalysisSixty-six mice in total were used in this experiment. The mice were euthanized by cervical dislocation under deep anesthesia, and each eye was enucleated. Eyes used for histological analysis were 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 parallel with the maximal circumference through the optic disc. These sections of each eye were 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 used for the morphometric analysis described below. Light-microscope images were photographed, and the thickness of the outer nuclear layer (ONL) from the optic disc was measured at 240 µm intervals on the photographs. The number of cells in GCL and the thickeness of IPL at a distance between 285 and 715 µm from the optic disc were measured in the two areas of retina and then averaged to give a single value. Data from three sections (selected randomly from the six sections) were averaged for each eye.
Terminal Deoxynucleotidyl Transferase Mediated Deoxyuridine Triphosphate Nick End Labeling (TUNEL) StainingThirty-five mice in total were used in this experiment. TUNEL staining were enucleated at 48 h after exposure to light for 3 h, fixed overnight in 4% paraformaldehyde, and immersed for 2 d in 25% sucrose with 0.01 M phosphate-buffered saline (PBS). The eyes were then embedded in a supporting medium for frozen-tissue specimens (OCT compound; Tissue-Tek, IL, U.S.A.). Retinal sections 10-µm thickness were cut parallel on a cryostat with the maximal circumference of the eye ball through the optic disc at −25°C and stored at −80°C until staining. TUNEL staining was performed according to the manufacturer’s protocols (In Situ Cell Death Detection kit; Roche Biochemicals, Mannheim, Germany) to detect the retinal cell death induced by exposure to light. After two washes with PBS, sections were incubated with TUNEL reaction mixture (10% terminal deoxyribonucleotidyl transferase (TdT) enzyme solution diluted in fluorescein–deoxyuridine triphosphate (dUTP) mixture solution) at 37°C for 1 h. The sections were washed three times in PBS for 5 min at room temperature. Fluorescent images were photographed, and labeled cells in the ONL at a distance between 285 and 715 µm from the optic disc were counted in the two area of the retina. The number of TUNEL-positive cells was averaged for these two areas.
ImmunohistochemistryThirty-five mice in total were used in this experiment. Immunohistochemical staining was performed in accordance with the following protocol 48 h after light-exposure. 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 kept at −80°C. Retinal sections were cut at 10-µm thickness 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 30 min, and then preincubated with 10% normal goat serum in 0.01 M PBS for 1 h. Then, they were incubated overnight at 4°C with 8-hydroxy-2-deoxyguanosine (8-OHdG) monoclonal antibody diluted 1 : 20 in a solution of 10% goat serum in 0.01 M PBS containing 0.3 (v/v) Triton X-100. After washing with a mixture of an Alexa Fluor 488 F(ab′)2 fragment of goat anti-rabbit immunoglobulin G (IgG) (H+L) (1 : 1000 dilution) (A11070; Invitrogen, Carlsbad, CA, U.S.A.). We confirmed the staining by comparison with the negative control. Immunofluorescence images were taken using a microscope (BX50; Olympus, Tokyo, Japan) with a cooled Charge-coupled device camera (DP30BP; Olympus) at 1360×1024 pixels via MetaMorph software (Molucular Devices, Sunnyvale, CA, U.S.A.), and the 8-OHdG-positive cells were counted in ONL at a distance of 285–715 µm from the optic disc on the images. Three eye section per eye (two images per one secion) were photographd and the average of the six images was used as the data per eye.
Statistical AnalysisData are presented as the means±S.E.M. Statistical comparisons were made using Dunnett’s test. A p<0.05 was considered to indicate statistical significance.
We first investigated the effects of SUN N8075 on light-induced functional damage using electrophysiological analysis (Fig. 1). The vehicle-treated group showed reduction in the amplitudes of a- and b-waves compared with the non-treated normal group; the rate of reduction of the a- and b-waves was 82% and 79%, respectively, at 0.98 log cds/m2. In contrast, intraperitoneal injection of SUN N8075 at 30 mg/kg significantly prevented the reduction of both a- and b-wave amplitudes when compared with the vehicle-treated group. The inhibition of a- and b-wave reductions in the SUN N8075-treated group was 20% and 27%, respectively, at 0.98 log cds/m2. We also investigated the effect of SUN N8075 without excess light exposure. There was no difference in a-wave or b-wave between vehicle-treated and SUN N8075-treated groups (Figs. 1D, E).
(A) Typical traces of dark-adapted ERG responses measured at 5 d after exposure to light. Stimulus flashes were used from −2.92 to 0.98 log cds/m2. (B and C) Amplitudes of a- and b-wave of light exposure (8000 lx) plus the vehicle-treated group versus light exposure plus the SUN N8075 treated group (30 mg/kg, i.p). (D and E) Amplitudes of a- and b-wave of normal light exposure plus the vehicle-treated group versus normal light exposure plus the SUN N8075 treated group (30 mg/kg, i.p). Data are shown as means±S.E.M., n=6 to 10. * p<0.05, ** p<0.01 vs. light exposure plus the vehicle treated group. ## p<0.01 vs. the normal group.
We investigated the absorbance of the SUN N8075 solution at a plasma concentration (3 µM) using distilled water as control. The solution of SUN N8075 did not have specific absorption spectrum same as the solvent, saline, at wavelength of visible ray (380 nm ca. 750 nm) (data not shown). Therefore, SUN N8075 by itself did not affect the amount of light-induced retinal damage.
Effects of SUN N8075 on Light-Induced Retinal Photoreceptor DegenerationWe also examined the effect of SUN N8075 on light-induced retinal damage by histological measurements of the thickness of the ONL. Figure 2 shows the representative retinal images between 285 µm and 715 µm from optic nerve head in the inferior area at 5 d after light exposure. In the vehicle-treated group, the ONL thickness was markedly thinner when compared to the non-treated group, but the systemic administration of SUN N8075 significantly reduced the photic damage when compared with that in the vehicle-treated group. The vehicle-treated group showed a 46% reduction of the ONL thickness versus the non-treated group, but SUN N8075 treated mice showed only a 27% reduction of the ONL thickness compared to the vehicle group. SUN N8075 did not affect ONL thickness by itself without excess light exposure (Fig. 2E).
Retinal cross sections at 5 d after light exposure from (A) non-treated, (B) light exposure (8000 lx) plus vehicle-treated, and (C) light exposure plus SUN N8075-treated (30 mg/kg, i.p.) (D) Measurement of thickness in the outer nuclear layer (ONL) at 5 d after light exposure and (E) without light exposure. Data are shown as mean±S.E.M., n=8 to 19. * p<0.05, ** p<0.01 vs. light exposure plus the vehicle treated group. # p<0.05, ## p<0.01 vs. the normal group. The scale bar represents 30 µm.
To show the effects of SUN N8075 against light-induced cell death, we investigated the expression of the TUNEL-positive cells, which indicate apoptotic cells, in the ONL at 48 h after excess light exposure. Nontreated retinas contained almost no TUNEL-positive cells (Figs. 3A, D). In contrast, the number of TUNEL-positive cells markedly increased in the vehicle-treated group (Figs. 3B, D) at 48 h after light exposure, and SUN N8075 significantly prevented this increase (Figs. 3C, D). When the mice were administrated the SUN N8075 without excess light exposure, there were few TUNEL-positive cells. This result indicates that SUN N8075 does not affect apoptosis by itself (Fig. 3E).
Retinal cross sections at 48 h after light exposure from (A) nontreated, (B) light exposure (8000 lx) plus vehicle-treated, and (C) light exposure plus SUN N8075-treated (30 mg/kg, i.p.) eyes. (D) Quantitative analysis of the number of TUNEL-positive cells in the outer nuclear layer (ONL) at 48 h after light exposure and (E) without light exposure. Data are shown as means±S.E.M., n=6 to 8. * p<0.05 vs. light exposure plus the vehicle treated group. ## p<0.01 vs. normal group. The scale bar represents 30 µm.
We also investigated the mechanism of cell death in the ONL by measuring the oxidative product, 8-OHdG, using immunostaining techniques. Few 8-OHdG-positive cells were found in non-treated retinas (Figs. 4A, D). However, 8-OHdG-positive cells were observed in the ONL at 48 h after light exposure in the vehicle-treated group (Figs. 4B, D), and SUN N8075 significantly reduced the number of these cells (Figs. 4C, D). SUN N8075 had no effect on oxidative stress by itself without excess light (Fig. 4E).
Retinal cross sections at 48 h after light exposure from (A) non-treated, (B) light exposure (8000 lx) plus vehicle-treated, and (C) light exposure plus SUN N8075-treated (30 mg/kg, i.p.) eyes. (D) Quantitative analysis of the number of 8-OHdG-positive cells in the outer nuclear layer (ONL) at 48 h after light exposure and (E) without light exposure. Data are shown as means±S.E.M., n=6 to 8. * p<0.05 vs. light exposure plus the vehicle treated group. ## p<0.01 vs. normal group. The scale bar represents 30 µm.
In the present study, we demonstrated the protective effects of SUN N8075 against light-induced retinal degeneration in mice. Light-induced retinal cell death was induced by many causes, such as increases in levels of intracellular calcium, nitric oxide (NO), and free radicals.12) Excessive light in the retina induces the formation of many reactive oxygen species (ROS), including free radicals, and ROS contribute to photoreceptor degeneration. In the present study, we examined the protective effects of SUN N8075 against light-induced retinal degeneration and focused the antioxidant effects. In our previous investigations, the retinal damage stabilizes at 5 d after light exposure. Therefore, we performed electrophysiological analysis and histological analysis at this time point. We administrated SUN N8075 at 30 mg/kg, i.p. in this study, because the dose of SUN N8075 showed the protective effects against NMDA- and high intraocular pressure-induced retinal damage.10) Five days after light exposure, the a-wave and b-wave amplitudes were reduced and the ONL was significantly thinned in the vehicle-treated group. The a-wave represents the function of the photoreceptors (mainly rods), while the b-wave arises from the Müller cells and is influenced by the rod bipolar cells, and the ONL consists of the cell bodies of the photoreceptors. Therefore, a reduction in the ONL thickness indicates photoreceptor cell death due to excessive light exposure, and the ONL thickness is known to correlation with the dark adapted a-wave amplitude. In ERG and histological analysis, SUN N8075 showed protective effects against light-induced retinal damage. These results suggest that SUN N8075 protected against loss of photoreceptors, which would lead to protection of retinal function.
Apoptosis is a main pathway of cell death in a light-induced retinal damage model.6) Nextly, we investigated the effects of SUN N8075 against the light-induced apoptosis of photoreceptor cells. In the present study, the number of TUNEL-positive cells remarkably increased at 48 h after light exposure in ONL, and treatment with SUN N8075 decreased the number of TUNEL-positive cells. As mentioned above, light-induced oxidative-stress leads photoreceptor to cell death. We also investigated the expression of 8-OHdG in ONL. At 48 h after light exposure, SUN N8075 reduced the number of the light-induced 8-OHdG-positive cells in ONL. This result indicates the SUN N8075 prevents light-induced retinal degeneration by the suppression of oxidative stress. However, the reduction rate of 8-OHdG expression in ONL was 23% between the vehicle-treated group and SUN N8075-treated group, nevertheless SUN N8075 reduced the number of TUNEL-positive cells by 47%. This result indicates a possibility that a mechanism other than anti-oxidant effect is also involved in the protective effects of SUN N8075. Membrane depolarization in photoreceptor cells is induced by excessive light, and this can activate Na+ channels to cause a subsequent Ca2+ overload as a result of reversal of the direction of the Na+/Ca2+ exchanger and voltage-dependent Ca2+ channels. Ca2+ overload then leads to apoptotic cell death. Flunarizine, a calcium overload blocker, protects against the light-induced retinal degeneration.13) We also found that SEA0400, a selective Na+/Ca2+ exchanger, reduced NMDA-induced retinal cell death.14) On the other hand, we demonstrated the protective effects of edaravone, a radical scavenger, in light-induced retinal degeneration.11) Furthermore, the administration of combination of flunarizine and dimethylthioura, a free radical scavenger, showed great protection against light-induced retinal degeneration compared with the individual administrations.15) These studies indicate that free radical formation and intracellular calcium overload are closely involved in light-induced retinal degeneration. SUN N8075 is a radical scavenger and also a double inhibitor for Na+ and T-type Ca2+ channels.7) Therefore, SUN N8075 may prevent the light-induced cell damage partly by scavenging the light-induced ROS and also by blocking Na+ and T-type Ca2+ channels.
In conclusion, we demonstrated that SUN N8075 showed protective effects on the light-induced retinal damage by suppressing oxidative stress. These findings indicate that SUN N8075 may be able to be one of candidates as therapeutic drugs for dry AMD.
We thank to Asubio Pharma Co., Ltd. (Kobe, Japan) for gifting SUN N8075 and Mr. Tomohiro Nakanishi and Mr. Masaki Nishiguchi (Gifu Pharmaceutical University, Gifu, Japan) for technical supports.
SUN N8075 was kindly gifted from Asubio Pharma Co., Ltd. (Kobe, Japan).
