Role of Oxidative Stress in Retinal Photoreceptor Cell Death in N-Methyl-N-nitrosourea–Treated Mice

J Pharmacol Sci 118, 351 – 362 (2012)

Journal of Pharmacological Sciences © The Japanese Pharmacological Society

Full Paper

Role of Oxidative Stress in Retinal Photoreceptor Cell Death in N-Methyl-N-nitrosourea–Treated Mice Kazuhiro Tsuruma1, Mika Yamauchi1, Yuta Inokuchi1, Sou Sugitani1, Masamitsu Shimazawa1, and Hideaki Hara1,* 1

Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan

Received June 22, 2011; Accepted January 11, 2012

Abstract. This study aimed to investigate whether oxidative stress contributes to retinal cell death in a mouse model of photoreceptor degeneration induced by N-methyl-N-nitrosourea (MNU). We measured in vitro MNU-induced radical production in retinal cell cultures of murine 661W photoreceptor–derived cells; RGC-5, a mouse ganglion cell line; and primary retinal cells. The addition of MNU induced oxidative radical generation in 661W and primary retinal cells, but not in RGC-5 cells. Edaravone, a free radical scavenger, at 1 μM reduced MNU-induced radical production in 661W and primary retinal cells. To induce in vivo retinal photoreceptor degeneration in mice, we administered 60 mg/kg MNU by intraperitoneal injection. We intravenously administered 1 mg/kg edaravone immediately and at 6 h after the MNU injection. Retinal photoreceptor degeneration was evaluated by measuring the thickness of the outer nuclear layer (ONL) by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and by oxidative stress markers. MNU caused photoreceptor cell loss at 7 days after administration. Edaravone inhibited ONL thinning and reduced TUNEL-positive cells and the oxidative stress markers. These findings indicate that MNU leads to selective photoreceptor degradation via oxidative stress in vitro and in vivo and may help to understand the pathogenic mechanism of retinitis pigmentosa. Keywords: edaravone, N-methyl-N-nitrosourea, oxidative stress, photoreceptor, retinitis pigmentosa Introduction

mation, and photoreceptor cell nuclei show apoptosis as evidenced by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. MNUinduced cell degeneration is accompanied by the upregulation of Bax protein, down-modulation of Bcl-2 protein, and activation of the caspase family of genes (5). Although the apoptosis cascade may be different in humans, retinitis pigmentosa is also characterized by a loss of photoreceptor cells via an apoptotic mechanism. Since MNU-induced retinal degeneration is caused by photoreceptor cell apoptosis, it is possible that a common mechanism exists between MNU-induced photoreceptor loss and retinitis pigmentosa. Oxidative stress can contribute to neuronal toxicity and appears to be involved in both acute injury and chronic neuropathological conditions (6). Reactive oxygen species (ROS) production in response to oxidative stress has been implicated as a stage in the final common pathway that leads to neurotoxicity in a variety of acute

Retinitis pigmentosa is a disease characterized by a loss of photoreceptor cells, especially rods, which leads to visual disturbance and, eventually, blindness. No therapeutic drugs have yet been discovered for retinitis pigmentosa; therefore, the development of new drugs for therapeutic intervention is of paramount importance. NMethyl-N-nitrosourea (MNU) is a direct-acting alkylating agent that induces retinal photoreceptor cell death by an apoptotic mechanism (1). In a range of animals including adult mice and rats, MNU has been reported to induce photoreceptor cell death at approximately 7 days after a single systemic administration (2 – 4). The cell death is attributed to the restriction of DNA adduct for*Corresponding author. [email protected] Published online in J-STAGE on February 23, 2012 (in advance) doi: 10.1254/jphs.11110FP

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and chronic neurologic eye diseases including glaucoma (7), age-related macular degeneration (8), and retinitis pigmentosa (9). An early and sustained increase in intracellular ROS precedes the disruption of mitochondrial transmembrane potential, nuclear condensation, DNA nicking, and cell shrinkage, all of which are well-characterized events of apoptotic photoreceptor cell death (10). MNU enhances these free radical-mediated processes (11), suggesting that oxidative stress may play a part in the underlying mechanism of MNU responses. Edaravone, a free radical scavenger, has been approved for the clinical treatment of acute cerebral infarction (12) and has been shown to exert protective effects against cerebral ischemia–reperfusion injuries in a variety of experimental animal models (13 – 15). In an experimental study using cerebrocortical slice cultures from neonatal rats, edaravone was found to reduce N-methyl-Daspartate (NMDA)-induced cytochrome c release and apoptosis (16). Edaravone has been shown to inhibit 1) the accumulation of oxidative by-products, such as 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-deoxyguanosine (8-OHdG) and 2) neuronal cell death in mice after transient focal cerebral ischemia (17). These protective effects of edaravone are thought to be attributed to its ROS scavenging ability. We reported that systemic administration of edaravone could attenuate NMDAinduced retinal damage (18). Therefore, the present study aimed to clarify the involvement of oxidative stress in MNU-induced retinal photoreceptor damage and evaluate the protective effects of edaravone against MNU-induced retinal cell damage. Materials and Methods Materials MNU was purchased from Sigma-Aldrich (St. Louis, MO, USA); edaravone (3-methyl-1-phenyl-2-pyrazolin5-one), from Wako (Osaka); and isoflurane, from Nissan Kagaku (Tokyo). Animals Male adult ddY mice (Japan SLC, Hamamatsu) were maintained under controlled lighting conditions (12 h : 12 h light/dark cycle). The ddY mice are an outbred albino strain and have been maintained as a closed colony. This strain has been widely used in trials for drug efficacy and various fields of research, including pharmacological, phamacokinetic, and toxicological studies. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved and monitored by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University.

661W photoreceptor cells and RGC-5 cultures The 661W cell line was derived from mouse retinal tumors and has been previously characterized and shown to be of the cone photoreceptor cell lineage (19). RGC-5 is a mouse ganglion cell line (20, 21). 661W and RGC-5 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), and 100 μg/mL streptomycin (Meiji Seika Kaisha) under a humidified atmosphere of 95% air and 5% CO2 at 37°C. The cells were passaged by trypsinization every 3 to 4 days. Primary retinal culture Retinas from P8 mice were dissected free of choroidal vessels and dissociated by activated papain for 15 min at 37°C, using the protocol of Yi et al. (22). Neurobasal medium (Invitrogen, Carlsbad, CA, USA) containing ovomucoid (Sigma-Aldrich) plus DNase (Invitrogen) was added, and the cells were centrifuged at 100 × g for 8 min at room temperature. The cell pellet was resuspended in neurobasal-Ovo medium without DNase, recentrifuged, and resuspended in neurobasal medium containing L-glutamine, B27 (Invitrogen), and antibiotics. Cells were plated onto poly-D-lysine/laminin–coated 96-well dishes at 2.5 × 105 cells/well. In vitro immunostaining The primary retinal cultures were plated onto glass chamber slides (Laboratory-Tek; Life Technologies, Gaithersburg, MD, USA), fixed with 4% fresh paraformaldehyde at room temperature, blocked in 3% goat serum and 0.2% Triton X-100 in PBS, and incubated overnight at 4°C with primary antibodies [mouse anti-rhodopsin (clone 4D2; Millipore, Bedford, MA, USA) and mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)] diluted in PBS. After being washed, the cells were incubated for 1 h with secondary antibodies (Alexa Fluor® 488 goat anti-mouse IgG and Alexa Fluor® 546 rabbit anti-mouse IgG, both from Invitrogen), washed, and counterstained with Hoechst 33342 (Invitrogen); images were captured using a BZ-9000 Biorevo all-inone fluorescence microscope (Keyence, Osaka). MNU was added at 100 μg/mL to the cultures. The total number of cells was determined in a blind manner (by M.Y.), and the percentages of rhodopsin- and GFAP-positive cells were calculated. Cell viability To evaluate cell survival, we examined the change in the fluorescence intensity after the cellular reduction of WST-8 to formazan. The experiment was performed in

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neurobasal medium containing B27 minus antioxidant at 37°C. Primary retinal cells were plated onto poly-Dlysine/laminin–coated 96-well dishes at 2.5 × 105 cells/ well. Cells were washed with neurobasal medium containing B27 minus antioxidant. MNU was added at 10, 100, and 1000 μg/mL, and the cells were incubated for 12 h. Cell viability was assessed by culturing the cells in a culture medium containing 10% WST-8 (Cell Counting Kit-8; Dojin Kagaku, Kumamoto) for 3 h at 37°C and then by scanning with a microplate reader (Varioskan Flash 2.4; Thermo Fisher Scientific, Waltham, MA, USA) at 492 nm. Absorbance was expressed as the percentage of the absorbance in control cells (cultured in Neurobasal medium containing B27 minus antioxidant) after subtracting background absorbance. Radical-scavenging capacity assay This assay measures the radicals induced by MNU in retinal cells. 661W and RGC-5 cells were seeded at a density of 2 × 103 cells/well into 96-well plates and then incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 24 h. Cells were washed with 1% FBSDMEM. After a 1-h pretreatment with edaravone or vehicle (PBS), 10 μM of 5-(and-6)-chloromethyl-2′,7′dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA, Invitrogen), the radical probe, was added to the cell culture and incubation was continued for an additional 20 min at 37°C; the cell culture medium was then replaced to remove any excess probe. The radical probe was converted to 2′,7′-dichlorodihydrofluorescein (DCFH, active for ROS) by the action of intracellular esterase. Intracellular DCFH (nonfluorescent) was oxidized to 2′,7′-dichlorfluorescein (DCF, fluorescent) by intracellular ROS. Then, MNU at 1 – 1000 μg/mL or H2O2 at 1 mM was added. H2O2 was treated as a positive control. Fluorescence was measured after H2O2 or MNU had been present for various time periods, using a Varioskan Flash 2.4 microplate reader (Thermo Fisher Scientific) at the excitation/emission wavelengths of 485/535 nm. Primary retinal cells were seeded at 2.5 × 105 cells/ well into 96-well plates. Cells were washed with neurobasal medium containing B27 minus antioxidant. Then, a radical assay was performed following the same protocol as described above. Radical integrals were calculated by integrating the area under the CM-H2DCFDA fluorescence intensity curve after a 20-min treatment with ROS-generating compounds. To detect the cell types that generate ROS in primary cells, we used fluorescence microscopy to analyze the cellular radicals induced by MNU. Primary retinal cells were seeded at 2.5 × 105 cells/well into 96-well plates.

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Cells were washed with neurobasal medium containing B27 minus antioxidant. After a 1-h pretreatment with edaravone or vehicle, 100 μg/mL MNU, CM-H2DCFDA, and Hoechst 33342 were added, and the cells were further incubated for 20 min. Images were collected using an Olympus CCD camera and microscope (IX-70; Olympus, Tokyo). The total number of cells was determined in a blind manner (by M.Y.), and the percent of cells exhibiting DCF fluorescence was calculated. MNU-induced retinal damage MNU-induced retinal damage in mice was induced by an i.p. injection of 60 mg/kg MNU dissolved in saline, as previously reported by Smith et al. (23). Edaravone (0.1 or 1 mg/kg) or vehicle (saline) was i.v. injected immediately and at 6 h after the injection of MNU. At 7 days after the injection, the eyeballs were enucleated for histological analysis. Histological analysis The mice were killed by an i.p. injection of a lethal dose of sodium pentobarbital (80 mg/kg), and each eye was enucleated. The eyes were kept immersed in a fixative solution containing 4% paraformaldehyde for at least 24 h at 4°C. Six paraffin-embedded sections (4-μm-thick) were cut parallel with the maximum circumference of the eyeball through the optic disc (as we extracted the eyeball, we identified the dorsal-most point by using the landmark of the optic axis). These sections were then stained with hematoxylin and eosin. The damage induced by MNU at 45, 60, and 75 mg/kg was evaluated by morphometric analysis of each eye. Light microscopic images were captured using a digital camera (CoolPix 4500; Nikon, Tokyo). The vertical lengths of the inner nuclear layer (INL) and outer nuclear layer (ONL) at a distance between 375 and 625 μm from the optic disc (nasal and temporal portions) were measured on the photomicrographs in a blinded manner by a single observer (Y.I.) and averaged to obtain a single value. The data were averaged for each eye. TUNEL staining TUNEL staining was performed according to the manufacturer’s protocols (In Situ Cell Death Detection Kit; Roche Biochemicals, Mannheim, Germany) to detect retinal cell death induced by MNU. The mice were killed by an i.p. injection of a lethal dose of pentobarbital sodium (80 mg/kg) at 12, 24, 36, 48, and 72 h after i.p. injection of 60 mg/kg MNU. The eyes were enucleated, fixed overnight in 4% paraformaldehyde, and immersed in 25% sucrose in PBS for 48 h at 4°C. The eyes were then embedded in a supporting medium for frozen tissue specimens (optimal cutting temperature compound,

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Tissue-Tek; Miles Laboratories, Naperville, IL, USA). Retinal sections of 10-μm thickness were cut at −25°C using a cryostat and stored at −80°C until staining. After being washed with PBS twice, the sections were incubated with fluorescein-labeled terminal deoxyribonucleotidyl transferase enzyme at 37°C for 1 h. The sections were washed 3 times in PBS at room temperature, each for 1 min, and then developed. Fluorescent microscopic images were acquired, and labeled cells in the ONL at a distance between 375 and 625 μm from the optic disc were obtained in 2 areas of the retina. Fluorescein-labeled TUNEL-positive retinal cell density in the ONL at a distance between 475 and 525 μm (50 × 50 μm) from the optic disc was evaluated by appropriately calibrated computerized image analysis, using Image J (Image Processing and Analysis in Java; National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA). The density of TUNELpositive cells in the ONL and the median volume were determined as an average of 2 areas. In vivo immunostaining The eyes were enucleated as described in the “Histological analysis” section, fixed in 4% paraformaldehyde overnight at 4°C, immersed in 25% sucrose for 48 h at 4°C, and embedded in optimal cutting temperature compound (Sakura Finetechnical Co., Ltd., Tokyo). Transverse cryostat sections of 10-μm thickness were cut and placed onto slides (MAS COAT; Matsunami Glass Ind., Ltd., Osaka). These tissue sections were washed in 0.01 mM PBS for 10 min, and then endogenous peroxidase was quenched by treating the sections with 3% hydrogen peroxide in absolute methanol for 10 min, followed by a pre-incubation with 10% normal goat serum. The primary antibodies used were 8-OHdG and 4-HNE mouse monoclonal antibodies (both from the Japan Institute for the Control of Aging, Shizuoka). These primary antibodies were diluted in PBS (1:1000) and then incubated with the tissue sections overnight at 4°C. The sections were washed and then incubated with biotinylated anti-rabbit IgG or anti-mouse IgG. They were subsequently incubated with the avidin-biotin-peroxidase complex for 30 min and then developed using diaminobenzidine (DAB) peroxidase as substrate for 1 min. We confirmed the staining by comparison with a negative control (not treated with the first antibody). Images were obtained using a digital camera (CoolPix 4500). 8-OHdG- and 4-HNE-positive cell expression To investigate 8-OHdG-positive cells, we evaluated the samples of DAB-labeled cells (for anti-8-OHdG) in the ONL at a distance between 375 and 625 μm from the optic disc. Retinal DAB-labeled cells were counted and

averaged for 2 areas. To investigate 4-HNE-positive cells, we evaluated samples in the DAB-labeled area (for anti-4-HNE) in photoreceptors at a distance between 475 and 525 μm (50 × 50 μm) from the optic disc. DAB-labeled retinal cell density, evaluated by appropriately calibrated computerized image analysis using Image J, and the median volume of the pixels in the image or selection were averaged for each area. Briefly, light micrographs of the measured areas were inverted in a gradation sequence using Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA, USA). The optical intensity was then evaluated using Image J. From the obtained score, we subtracted the score obtained for the negative control (not treated with the first antibody) as the background value. Statistical analysis Data are presented as the mean ± S.E.M. Statistical comparisons were made using a one-way ANOVA followed by a Dunnett’s test or Student’s t-test (using STAT VIEW version 5.0; SAS Institute, Cary, NC, USA). A P-value of < 0.05 was considered to indicate statistical significance. A P-value of < 0.01 was described as highly significant. Results Intracellular oxidation of DCFH induced by MNU in 661W and RGC-5 cells To investigate whether MNU induces radical production, we measured the intracellular ROS levels using the ROS-sensitive probe CM-H2DCFDA. The kinetics of ROS reactivity (monitored as fluorescence generation) is illustrated in Fig. 1, A – C. Treatment with MNU did not induce intracellular oxidation of DCFH in RGC-5 retinal ganglion cells (Fig. 1: B and E). In contrast, in 661W photoreceptor–derived cells, intracellular radicals were significantly generated by treatment with MNU at 100, 500, and 1000 μg/mL or with H2O2 at 1 mM compared with the controls (Fig. 1D). Edaravone at 1 – 20 μM scavenged radicals in a concentration-dependent manner (Fig. 1: C and F). Treatment with edaravone alone did not show any effects of ROS production in 661W cells without MNU (data not shown). Immunocytochemical identification of rod photoreceptors and retinal cell death induced by MNU in primary retinal cells To identify MNU-induced photoreceptor degeneration, we used primary dissociated retinal cultures from P8 mice. Immunostaining with antibodies against retinal cell–type markers was used to characterize the cell types in the retinal cultures. Representative photomicrographs

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of Hoechst 33342, rhodopsin, and GFAP stainings are shown in Fig. 2A. Figure 2A (a – d) and 2B show that the cultures contained rod photoreceptors at a proportion of 62.5%, and the rest were Müller glia, which showed GFAP positivity. We also performed rhodopsin and GFAP immunostaining after MNU treatment. Treatment with MNU at 100 μg/mL reduced the proportion of rod photoreceptors but increased that of Müller glia (Fig. 2: Ae – Ah and B). Although rhodopsin-positive cells were decreased by MNU, the total number of GFAP-positive cells did not change [vehicle-treated: 30.5 ± 3.62 cells/0.4 mm2 (1 area), MUN-treated: 27.3 ± 8.72 cells/0.4 mm2]. To investigate whether MNU induces primary retinal cell death, we examined the change in absorbance (at 450 nm) that followed the cellular reduction of WST-8 to

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Fig. 1. Kinetics and concentration–response of the intracellular oxidation of DCFH by MNU-induced ROS. ROS production was stimulated with 1 mM H2O2 or 1 – 1000 μg/mL MNU, and fluorescence was measured for various times in 661W photoreceptor–derived cells (A) and RGC5 retinal ganglion cells (B). C) The 661W cultures were treated with 0.1 to 20 μM edaravone for 1 h and then with 10 μM CM-H2DCFDA for 20 min. ROS production was stimulated with 100 μg/mL MNU, and fluorescence was measured for various times. D – F) Integrals of ROS production were calculated from the time–kinetics curves (see panels A – C). Data are compared with 1 considered the control value. Data are shown as mean ± S.E.M. (n = 8). F = 5.96 (D), 2.09 (E), and 5.93 (F). *P < 0.05, **P < 0.01 vs. control. #P < 0.05, ## P < 0.01 vs. cells treated with 100 μg/mL MNU alone (Dunnett’s test).

formazan. Treatment with MNU at 10 and 100 μg/mL significantly reduced cell viability. Edaravone at 10 μM inhibited cell death (Fig. 2C). Intracellular oxidation of DCFH induced by MNU in primary retinal cells We investigated the radical-generating capacity of MNU in whole primary retinal cultures and individual cells. The kinetics of ROS reactivity (monitored as fluorescence generation) are illustrated in Fig. 3, A and B. Intracellular radicals were significantly generated by treatment with MNU at 100 to 1000 μg/mL compared with the control (Fig. 3C). Edaravone at 0.1, 1, and 10 μM scavenged the radicals in a concentration-dependent manner (Fig. 3: B and D).

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We also evaluated MNU-induced intracellular radical generation in individual cells by fluorescence microscopy. Representative photomicrographs of Hoechst 33342 staining and DCF fluorescence are shown in Fig. 3E. Untreated control cells displayed low-intensity fluorescence (Fig. 3Ea). Treatment with 100 μg/mL MNU resulted in an increase in ROS production, as shown by the increased number of cells exhibiting DCF fluorescence (Fig. 3: Eb and F). Edaravone at 10 μM reduced the number of ROS-generating cells induced by MNU (Fig.

Fig. 2. MNU-induced primary rod photoreceptor cell death. A) Prominent staining with antibodies against rhodopsin (green, rod photoreceptors), GFAP (red, Müller glia), and Hoechst 33342 (blue, total cells). a – d, control; e – h, treatment with 100 μg/mL MNU for 12 h. B) Treatment with MNU induced a change in the ratio of rhodopsin- and GFAP-positive cells (n = 4). C) Cell viability was assessed by immersing the cells in WST-8 solution, with absorbance recorded at 492 nm. Data are shown as the mean ± S.E.M. (n = 6). F = 9.40 (C). **P < 0.01 vs. control (Dunnett’s test). ##P < 0.01 vs. cells treated with 1000 μg/mL MNU alone (Student’s t-test). Scale bar represents 20 μm.

3: Ec and F). Retinal damage induced by i.p. injection of MNU In the histological evaluation, representative retinal images were taken at 7 days after MNU injection (Fig. 4). No histological differences were apparent following the i.p. injection of 45 mg/kg MNU. MNU at 60 and 75 mg/kg decreased ONL thickness (vs. the untreated retina) drastically in a dose-dependent manner (Fig. 4: A and B). MNU at 75 mg/kg decreased the thickness of the INL

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Fig. 3. MNU-induced specific ROS production of rod photoreceptors in primary retinal cells. A) ROS production was stimulated with 1 mM H2O2 or 1 – 1000 μg/mL MNU, and fluorescence was measured for various times in primary retinal cells. B) The primary retinal cultures were treated with 0.1 to 10 μM edaravone for 1 h and then with 10 μM CM-H2DCFDA for 20 min. ROS production was stimulated with 100 μg/mL MNU, and fluorescence was measured for various times. C and D) Integrals of ROS production were calculated from the time–kinetics curves (see panels A and B). Data are compared with the control value of 1. E) Representative fluorescence micrographs of Hoechst 33342 (blue, total cells) and DCF fluorescence (red, ROS generating cells) obtained at 20 min after MNU treatment. F) Cells exhibiting DCF fluorescence was counted, and positive cells were expressed as the ratio of DCF- to Hoechst 33342-positive cells. Data are shown as the mean ± S.E.M. (n = 6). F = 9.47 (C) and 9.65 (D). *P < 0.05, **P < 0.01 vs. control [Dunnett’s test (C) and Student’s t-test (D and F)]. ##P < 0.01 vs. cells treated with MNU at 100 μg/mL alone [Dunnett’s test (D), Student’s t-test (F)].

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MNU-induced increase in the number of TUNEL-positive cells Fluorescein-labeled TUNEL staining revealed subtle intrinsic fluorescence in all retinal sections. TUNELpositive cells were observed in some parts of the ONL at 36, 48, and 72 h after the i.p. injection of 60 mg/kg MNU, but very few TUNEL-positive cells were detected in the untreated mice or at 12 and 24 h after MNU treatment (Fig. 5A). We evaluated the effect of edaravone by measurement of TUNEL-positive cell density at 36 h after injection of 60 mg/kg MNU (Fig. 5: B and C). Almost no fluoresceinlabeled TUNEL cells were detected in normal retinal sections. Substantial fluorescence intensity was observed in vehicle-treated retinas. Edaravone (1 mg/kg, i.v.) significantly reduced the fluorescence intensity of TUNELpositive cells (vs. the vehicle-treated retina) (Fig. 5C). MNU-induced expressions of 8-OHdG and 4-HNE Positive reactions for the oxidative stress biomarker 8-OHdG were observed in the ONL, beginning at 12 h after the i.p. injection of 60 mg/kg MNU, but almost none were seen in the normal retina (Fig. 6A). Edaravone at 1 mg/kg, i.v. significantly reduced the number of 8-OHdG-positive cells induced by MNU treatment (vs. the vehicle-treated retina, Fig. 6B). For the second biomarker, 4-HNE, immunostaining was observed in photoreceptors, beginning at 12 h after the i.p. injection of 60 mg/kg MNU, but almost no staining was seen in the untreated retina (Fig. 6C). Edaravone at 1 mg/kg, i.v. significantly reduced the expression of 4-HNE (vs. the vehicle-treated retina, Fig. 6D).

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Fig. 4. Edaravone reduced retinal degeneration in mice at 7 days after i.p. injection of MNU. A) Microscopy images of normal retina and retinas following injection of MNU at 45, 60, and 75 mg/kg. B) Retinal degeneration was evaluated by measuring the vertical length of the ONL relative to the INL after i.p. injection of MNU. C) Microscopy images of normal retina and retina at 7 days after treatment with 60 mg/kg MNU with vehicle, 0.3 mg/kg edaravone, or 1.0 mg/kg edaravone in vehicle. D) Vertical length of the ONL relative to the INL at 7 days after i.p. injection of MNU. Edaravone was i.v. administered immediately and at 6 h after MNU injection. Data are shown as the mean ± S.E.M. (n = 6 – 10). F = 90.8 (B) and 7.37 (D). **P < 0.01 vs. normal. ##P < 0.01 vs. the vehicle plus MNU–treated group (Dunnett’s test). Scale bar represents 25 μm.

(vs. the untreated retina). Edaravone at 1 mg/kg, i.v. but not at 0.3 mg/kg, i.v. significantly suppressed the reduction in ONL thickness (as compared with the vehicle) induced by MNU (60 mg/kg, i.p.; Fig. 4: C and D). There was no effect of the administration of edaravone alone on retinal histology (data not shown).

Discussion In the present study, MNU induced the generation of oxidative radicals in 661W photoreceptor cells, but not in RGC-5 cells. Edaravone treatment of 661W cells resulted in the scavenging of MNU-induced radicals. We also investigated the effects of MNU and edaravone in mouse primary retinal cultures. Primary retinal cells were isolated from postnatal day (P) 8 mice. Mouse retinal development is initiated at approximately embryonic day (E) 12 and involves proliferation, migration, and differentiation of neuronal precursor cells, a process that continues through final neuronal differentiation and maturation of neuronal synapses at approximately P8 (24). Therefore, we hypothesize that the primary retinal cultures represent the in vivo situation in the adult mouse retina. We confirmed that the primary retinal cultures contained 62.5% rod photoreceptors, and the rest were Müller glia. Moreover, MNU induced ROS production and decreased the number of rod photoreceptors. ROS

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production was observed in 53% of the cells, as shown in Fig. 3F. This value was similar to the proportion of rod photoreceptors. Therefore, MNU may induce ROS production at least in rod photoreceptor cells. Furthermore, treatment with MNU did not induce intracellular oxidation in the mouse RGC-5 retinal ganglion cell line. Hence, MNU-induced ROS production may be cell specific. The number of Müller glial cells was not affected by MNU treatment; therefore, Müller glia is not likely to be involved in cell death. This study is the first to report that MNU elevates ROS in rod and cone photoreceptor cells and induces photoreceptor cell death in vitro. Edaravone may inhibit MNU-induced photoreceptor cell degeneration by its ROS-scavenging activity. An extensive investigation in animals has revealed that MNU-induced photoreceptor cell loss is due to apoptosis with a decrease in Bcl-2 protein, increase in Bax protein, and nuclear factor κB and activation of caspases (1, 5). Previous studies have evaluated a caspase-3 inhibitor (1), nicotinamide (25), X-linked inhibitor of apoptosis protein (26), poly(ADP-ribose) polymerase inhibitor (27), and mutant of acidic fibroblast growth factor (28) as possible

Fig. 5. Edaravone reduced MNU-induced TUNEL-positive cell expression. A) TUNELstained sections of normal retina and retinas at 12, 24, 36, 48, and 72 h, respectively, after injection with 60 mg/kg MNU. B) Fluorescence images of normal retina and retina following i.p. injection of 60 mg/kg MNU plus i.v. injection of vehicle or 1 mg/kg edaravone in vehicle. C) Fluorescence intensity of TUNEL-positive cells in the ONL (Fold increase). Normal, n = 7; vehicle, n = 8; edaravone, n = 8. Edaravone (or vehicle) was i.v. administered immediately and at 6 h after the injection of MNU. Data are compared with the normal value of 1. Data are shown as the mean ± S.E.M. **P < 0.01 vs. the vehicle plus MNU–treated group (Student’s t-test). Scale bar represents 25 μm.

therapies for MNU-induced cell loss because they were found to prevent MNU-induced retinal damage and counteract photoreceptor cell loss. Oxidative stress caused by ROS induces the activation of the Bcl-2 family member proteins Bax/Bak, the opening of permeability transition pores, the disruption of the mitochondrial membrane potential, the release of cytochrome c, the activation of caspases, and cell death. Thus, the production of ROS by rod and cone photoreceptor cells might affect MNU-induced retinal degeneration in mice. Considering the specificity of MNU-induced retinal damage and its alkylation activity, MNU may induce the ROS production via protein alkylation, especially photoreceptor specific proteins such as rhodopsin, opsin, transducing, and allestin. When supplied in vivo in mice, MNU at 60 and 75 mg/ kg drastically decreased ONL thickness in a dose-dependent manner. In a previous study, a drastic decrease in ONL thickness was noticed at 7 days after MNU treatment, but the ganglion cell layer, inner plexiform layer, and INL were well preserved (29). However, in the present study, MNU at 75 mg/kg slightly decreased the

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Fig. 6. Edaravone suppressed expression of MNU-induced oxidative stress biomarkers. A) Expression of 8-OHdG in untreated normal retina and retinas following i.p. injection of 60 mg/kg MNU plus i.v. injection of vehicle or 1 mg/kg edaravone in vehicle. Upper panels show all layers of the retina and lower panels are details of the ONL. B) Number of 8-OHdG-positive cells in the GCL. Edaravone (or vehicle) was i.v. administered immediately and at 6 h after the injection of MNU. C) Expression of 4-HNE in untreated normal retina and retinas following i.p. injection of 60 mg/kg MNU plus i.v. injection of vehicle or 1 mg/kg edaravone in vehicle. Upper panels show all layers of the retina and lower panels are photoreceptors. D) Optical density analysis of the levels of 4-HNE protein expression in the photoreceptors at 12 h after i.p. MNU injection. Data are shown as the mean ± S.E.M. (n = 6 or 7). **P < 0.01 vs. the vehicle plus MNU–treated group (Student’s t-test). Scale bars represent 25 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer; PRC, photoreceptors.

thickness of the INL. This result shows that MNU at high doses may affect various retinal cell layers by mechanisms other than oxidative stress, such as an excessive alkylation. Edaravone protected against MNU-induced retinal cell degradation and decreased the number of cells showing positive TUNEL staining, which is a marker of DNA fragmentation. In our previous study, edaravone was found to prevent both retinal ganglion cell loss and TUNEL expression in retinal cells induced by intravitreally injected NMDA (18). In an in vitro study using retinal ganglion cells, edaravone showed stronger scavenging activity against the intracellular ·OH radical than the other examined radicals, O2·− and H2O2, whereas Trolox, a water-soluble vitamin E analogue, showed stronger

scavenging activity against O2·− and H2O2 than the ·OH radical (18). Edaravone also reduced the MNU-induced expressions of 8-OHdG and 4-HNE in the murine retinas. One of the major products of DNA damage induced by ROS is 8-OHdG, the level of which is a useful marker of oxidative damage due to aging, cancer, and other degenerative diseases (30 – 32). A second major compound, the aldehyde 4-HNE, is generated by a free-radical attack on ω-6 polyunsaturated fatty acids and is largely responsible for pathogenesis during oxidative stress (33, 34). The chemical nature of 4-HNE makes it highly reactive towards the free sulfhydryl groups of proteins, resulting in the formation of thioether adducts that undergo further cyclization to form hemiacetals (35). Both of these biomarkers are

MNU-Induced Photoreceptor Damage via ROS

therefore closely related to oxidative stress. Hence, edaravone would be expected to exert neuroprotective effects against retinal photoreceptor degradation induced by oxidative stress, most probably by scavenging the ·OH radicals. The prevalence of typical retinitis pigmentosa is reported to be approximately 1 in 4000, and no effective treatment yet exists. Retinitis pigmentosa originates from 1 of hundreds of mutations that result in a gene product that can damage the rods or from homozygous mutations that cause deficiency of a protein indispensable for rod survival. The death of rods is inevitably followed by cone cell death, caused mainly by oxidative damage (36, 37). Usui et al. reported that generation of excessive ROS caused oxidative damage to cones in the retinal degeneration-1 (rd1+/+) mouse, a model of retinitis pigmentosa (38). A mixture of antioxidants, including α-tocopherol, ascorbic acid, Mn (III) tetrakis (4-benzoic acid) porphyrin, and α-lipoic acid, reduced cone cell death in these rd1+/+ mice (36). Although the apoptosis cascade induced by MNU may differ from that in human retinitis pigmentosa, the present data indicate that MNU-induced retinal degeneration has features common to retinitis pigmentosa in that the degeneration is specific to photoreceptor cells and involves oxidative stress. In conclusion, we have demonstrated that MNU induces the generation of oxidative radicals and cell death specifically in retinal photoreceptor cells. In addition, systemic administration of edaravone, a free radical scavenger, inhibits retinal degeneration induced by MNU. These findings indicate that oxidative stress plays a pivotal role in MNU-induced retinal photoreceptor degradation. Although the mechanism of pathogenesis of MNU-induced photoreceptor cell damage differs from that of human retinitis pigmentosa, the results of our study may help us to understand the pathogenesis of retinitis pigmentosa.

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Acknowledgments The authors wish to express their gratitude to the Ministry of Education, Culture, Sports, Science and Technology, Japan for Grants-in-Aid for exploratory research and to Dr. Muayyad R. AlUbaidi (Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA) for the kind gift of 661W cells. The authors thank Shunsuke Imai, Masataka Nishiguchi, Mai Akane, and Tomohiro Nakanishi for technical assistance.

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References 1 Yoshizawa K, Yang J, Senzaki H, Uemura Y, Kiyozuka Y, Shikata N, et al. Caspase-3 inhibitor rescues N -methyl- N -nitrosoureainduced retinal degeneration in Sprague-Dawley rats. Exp Eye Res. 2000;71:629–635. 2 Nakajima M, Yuge K, Senzaki H, Shikata N, Miki H, Uyama M,

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et al. Photoreceptor apoptosis induced by a single systemic administration of N-methyl-N-nitrosourea in the rat retina. Am J Pathol. 1996;148:631–641. Nambu H, Yuge K, Nakajima M, Shikata N, Takahashi K, Miki H, et al. Morphologic characteristics of N-methyl-N-nitrosoureainduced retinal degeneration in C57BL mice. Pathol Int. 1997; 47:377–383. Yuge K, Nambu H, Senzaki H, Nakao I, Miki H, Uyama M, et al. N-methyl-N-nitrosourea-induced photoreceptor apoptosis in the mouse retina. In Vivo. 1996;10:483–488. Yoshizawa K, Nambu H, Yang J, Oishi Y, Senzaki H, Shikata N, et al. Mechanisms of photoreceptor cell apoptosis induced by Nmethyl-N-nitrosourea in Sprague-Dawley rats. Lab Invest. 1999; 79:1359–1367. Facchinetti F, Dawson VL, Dawson TM. Free radicals as mediators of neuronal injury. Cell Mol Neurobiol. 1998;18:667–682. Tezel G. Oxidative stress in glaucomatous neurodegeneration: mechanisms and consequences. Prog Retin Eye Res. 2006;25: 490–513. Kindzelskii AL, Elner VM, Elner SG, Yang D, Hughes BA, Petty HR. Human, but not bovine, photoreceptor outer segments prime human retinal pigment epithelial cells for metabolic activation and massive oxidant release in response to lipopolysaccharide and interferon-gamma. Exp Eye Res. 2004;79:431–435. Doonan F, Donovan M, Cotter TG. Caspase-independent photoreceptor apoptosis in mouse models of retinal degeneration. J Neurosci. 2003;23:5723–5731. Carmody RJ, McGowan AJ, Cotter TG. Reactive oxygen species as mediators of photoreceptor apoptosis in vitro. Exp Cell Res. 1999;248:520–530. Mabrouk GM, Moselhy SS, Zohny SF, Ali EM, Helal TE, Amin AA, et al. Inhibition of methylnitrosourea (MNU) induced oxidative stress and carcinogenesis by orally administered bee honey and Nigella grains in Sprague Dawely rats. J Exp Clin Cancer Res. 2002;21:341–346. Nishi H, Watanabe T, Sakurai H, Yuki S, Ishibashi A. Effect of MCI-186 on brain edema in rats. Stroke. 1989;20:1236–1240. Horiike O, Shimogori H, Ikeda T, Yamashita H. Protective effect of edaravone against streptomycin-induced vestibulotoxicity in the guinea pig. Eur J Pharmacol. 2003;464:75–78. Kono H, Asakawa M, Fujii H, Maki A, Amemiya H, Yamamoto M, et al. Edaravone, a novel free radical scavenger, prevents liver injury and mortality in rats administered endotoxin. J Pharmacol Exp Ther. 2003;307:74–82. Masaki Y, Kumano K, He N, Suyama I, Endo T. Protective effects of MCI-186 on cold kidney preservation/reperfusion injury in the rat. Transplant Proc. 1996;28:1885–1886. Nakano-Okuda Y, Hasegawa K, Hirai K, Kanai-Ochiai R, Morimoto M, Sugimoto T. Effects of edaravone on N-methyl-Daspartate (NMDA)-mediated cytochrome c release and apoptosis in neonatal rat cerebrocortical slices. Int J Dev Neurosci. 2006;24:349–356. Zhang N, Komine-Kobayashi M, Tanaka R, Liu M, Mizuno Y, Urabe T. Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain. Stroke. 2005;36:2220–2225. Inokuchi Y, Imai S, Nakajima Y, Shimazawa M, Aihara M, Araie M, et al. Edaravone, a free radical scavenger, protects against retinal damage in vitro and in vivo. J Pharmacol Exp Ther. 2009; 329:687–698.

362

K Tsuruma et al

19 Tan E, Ding XQ, Saadi A, Agarwal N, Naash MI, Al-Ubaidi MR. Expression of cone-photoreceptor-specific antigens in a cell line derived from retinal tumors in transgenic mice. Invest Ophthalmol Vis Sci. 2004;45:764–768. 20 Krishnamoorthy RR, Agarwal P, Prasanna G, Vopat K, Lambert W, Sheedlo HJ, et al. Characterization of a transformed rat retinal ganglion cell line. Brain Res Mol Brain Res. 2001;86:1–12. 21 Van Bergen NJ, Wood JP, Chidlow G, Trounce IA, Casson RJ, Ju WK, et al. Recharacterization of the RGC-5 retinal ganglion cell line. Invest Ophthalmol Vis Sci. 2009;50:4267–4272. 22 Yi H, Nakamura RE, Mohamed O, Dufort D, Hackam AS. Characterization of Wnt signaling during photoreceptor degeneration. Invest Ophthalmol Vis Sci. 2007;48:5733–5741. 23 Smith SB, Yielding KL. Retinal degeneration in the mouse. A model induced transplacentally by methylnitrosourea. Exp Eye Res. 1986;43:791–801. 24 Livesey FJ, Cepko CL. Vertebrate neural cell-fate determination: lessons from the retina. Nat Rev Neurosci. 2001;2:109–118. 25 Uehara N, Miki K, Tsukamoto R, Matsuoka Y, Tsubura A. Nicotinamide blocks N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis in rats through poly (ADP-ribose) polymerase activity and Jun N-terminal kinase/activator protein-1 pathway inhibition. Exp Eye Res. 2006;82:488–495. 26 Petrin D, Baker A, Brousseau J, Coupland S, Liston P, Hauswirth WW, et al. XIAP protects photoreceptors from n-methyl-nnitrosourea-induced retinal degeneration. Adv Exp Med Biol. 2003;533:385–393. 27 Miki K, Uehara N, Shikata N, Matsumura M, Tsubura A. Poly (ADP-ribose) polymerase inhibitor 3-aminobenzamide rescues N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis in Sprague-Dawley rats through preservation of nuclear factorkappaB activity. Exp Eye Res. 2007;84:285–292. 28 Xu H, Yang JN, Li XK, Zheng Q, Zhao W, Su ZJ, et al. Retina protective effect of acidic fibroblast growth factor after canceling its mitogenic activity. J Ocul Pharmacol Ther. 2008;24:445–451. 29 Nakajima M, Nambu H, Shikata N, Senzaki H, Miki H, Tsubura

30

31

32

33

34

35

36

37

38

A. Pigmentary degeneration induced by N-methyl-N-nitrosourea and the fate of pigment epithelial cells in the rat retina. Pathol Int. 1996;46:874–882. De Vizcaya-Ruiz A, Barbier O, Ruiz-Ramos R, Cebrian ME. Biomarkers of oxidative stress and damage in human populations exposed to arsenic. Mutat Res. 2009;674:85–92. Fraga CG, Onuki J, Lucesoli F, Bechara EJ, Di Mascio P. 5-Aminolevulinic acid mediates the in vivo and in vitro formation of 8-hydroxy-2′-deoxyguanosine in DNA. Carcinogenesis. 1994; 15:2241–2244. Kaneko T, Tahara S, Matsuo M. Non-linear accumulation of 8-hydroxy-2′-deoxyguanosine, a marker of oxidized DNA damage, during aging. Mutat Res. 1996;316:277–285. Gueraud F, Alary J, Costet P, Debrauwer L, Dolo L, Pineau T, et al. In vivo involvement of cytochrome P450 4A family in the oxidative metabolism of the lipid peroxidation product trans-4hydroxy-2-nonenal, using PPARalpha-deficient mice. J Lipid Res. 1999;40:152–159. Tanito M, Haniu H, Elliott MH, Singh AK, Matsumoto H, Anderson RE. Identification of 4-hydroxynonenal-modified retinal proteins induced by photooxidative stress prior to retinal degeneration. Free Radic Biol Med. 2006;41:1847–1859. Dogterom P, Mulder GJ, Nagelkerke JF. Lipid peroxidation-dependent and -independent protein thiol modifications in isolated rat hepatocytes: differential effects of vitamin E and disulfiram. Chem Biol Interact. 1989;71:291–306. Komeima K, Rogers BS, Lu L, Campochiaro PA. Antioxidants reduce cone cell death in a model of retinitis pigmentosa. Proc Natl Acad Sci U S A. 2006;103:11300–11305. Shen J, Yang X, Dong A, Petters RM, Peng YW, Wong F, et al. Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa. J Cell Physiol. 2005;203:457–464. Usui S, Oveson BC, Lee SY, Jo YJ, Yoshida T, Miki A, et al. NADPH oxidase plays a central role in cone cell death in retinitis pigmentosa. J Neurochem. 2009;110:1028–1037.