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Clinical and Experimental Ophthalmology 2015; ••: ••–•• doi: 10.1111/ceo.12525
Original Article Protective effect of molecular hydrogen against oxidative stress caused by peroxynitrite derived from nitric oxide in rat retina Takashi Yokota PhD,1 Naomi Kamimura PhD,1 Tsutomu Igarashi MD PhD,2 Hiroshi Takahashi MD PhD,2 Shigeo Ohta PhD1 and Hideaki Oharazawa MD PhD3 1
Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine and Department of Ophthalmology, Musashikosugi Hospital, Nippon Medical School, Kawasaki, Kanagawa, and 2Department of Ophthalmology, Nippon Medical School, Tokyo, Japan 3
ABSTRACT Background: Oxidative and nitrative processes have an important role in the pathogenesis of glaucomatous neurodegeneration. Oxidative stress occurs when cellular production of reactive oxygen species outweighs the protective capacity of antioxidant defences. Reactive oxygen species are generated as by-products of cellular metabolism, primarily in the mitochondria. Herein, we present a novel investigation of the effects of molecular hydrogen (H2) on retinal cells exposed to oxidative stress. Methods: We cultured adult rat retinal tissues in an organotypic culture system with a nitric oxide donor, S-nitroso-N-acetylpenicillamine, in the presence or absence of H2. Loss of mitochondrial membrane potential and apoptosis of retinal cells were analysed using a MitoTMRE detection kit and TdT-mediated dUTP nick end labeling (TUNEL) assay, respectively. Tyrosine nitration levels and oxidative stress damage in the retina were evaluated using immunohistochemical staining. Retinal damage was quantified by measuring the numbers of cells in the ganglion cell and inner nuclear layers and the thickness of the retina. Results: H2 suppressed loss of mitochondrial membrane potential and apoptosis in retinal cells. Moreover, H2 decreased the tyrosine nitration level and
suppressed oxidative stress damage in retinal cells. S-nitroso-N-acetylpenicillamine treatment decreased the cell numbers in the ganglion cell layer and inner nuclear layer, but the presence of H2 inhibited this reduction. These findings suggest that H2 has a neuroprotective effect against retinal cell oxidative damage, presumably by scavenging peroxynitrite. Conclusions: H2 reduces cellular peroxynitrite, a highly toxic reactive nitrogen species. Thus, H2 may be an effective and novel clinical tool for treating glaucoma and other oxidative stress-related diseases. Key words: antioxidant, molecular hydrogen, oxidative stress, retina.
INTRODUCTION Oxidative stress contributes to several steps in the pathophysiology of neurodegenerative diseases such as glaucomatous neurodegeneration. In various subcellular compartments in neuronal cells, oxidative stress causes molecular changes that contribute to the development of glaucomatous neurodegeneration, including oxidative protein modifications that are associated with neuronal damage and glial dysfunction. Reactive oxygen species (ROS) are generated through physiological metabolic processes mainly as by-products of the electron transport chain and oxidative phosphorylation, which take place in the
■ Correspondence: Dr Hideaki Oharazawa, Department of Ophthalmology, Musashikosugi Hospital, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki 211-8533, Kanagawa, Japan. E-mail: [email protected] Received 5 August 2014; accepted 17 January 2015. Conflict of interest: None. Funding sources: None. © 2015 Royal Australian and New Zealand College of Ophthalmologists
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mitochondria. Although ROS function as cellular signalling messengers, damage to cellular macromolecules such as DNA, proteins and lipids ensues when the cellular production of ROS overwhelms the intrinsic antioxidant capacity.1 Oxidative stress is caused by an imbalance between the production of ROS and the cellular antioxidant defensive system. Superoxide anion radicals (•O2−), highly reactive metabolites of O2, are formed during normal aerobic metabolism in mitochondria, and sustained levels of ROS cause severe mitochondrial damage.2 Accumulating evidence has demonstrated that mitochondrial dysfunction contributes to enhanced ROS production, and elevated ROS can further impair mitochondrial function and exacerbate oxidative stress.3 Moreover, recent evidence has shown that mitochondrial dysfunction is present in some patients with glaucoma.4 Nitric oxide (NO•) has been implicated as a pivotal contributor to neuronal damage. Neuronal production of NO• leads to N-methyl-D-aspartate receptor activation and neuronal cell death.5 However, several studies have revealed that the neurotoxic effects of NO• are caused by peroxynitrite (ONOO−), a highly toxic reaction product of NO• and •O2−.6 To investigate the role of NO• in neuronal cell death, a NO• generator, S-nitroso-Nacetylpenicillamine (SNAP), has been widely used.7 In neuronal or retinal ganglion cell culture, NO• is generated rapidly and reacts with •O2− to form ONOO− when cells are stimulated with SNAP.8 Several pharmacological studies have demonstrated the potential participation of ONOO− in SNAPinduced cell death.1 However, to our knowledge there is no report on the effects of molecular hydrogen (H2) on SNAP-induced damage of retinal cells. We previously reported that H2 has potential as a novel antioxidant in preventive and therapeutic applications.2 Furthermore, H2 was shown to react with strongly reactive oxygen/nitrogen species including hydroxyl radicals (•OH) and ONOO− in cell-free reactions and to protect cultured cells in a manner dependent upon the decrease of •OH.3 Subsequent and recent experiments have indicated that a small amount of H2 is also effective against various stimuli.2 When animal models consumed drinking water containing dissolved H2, a small amount of H2 was considerably effective.4,5 Furthermore, 0.8 mM H2-loaded eye drops were shown to be effective for treatment of retinal ischemia-reperfusion injury.6 However, it appears unlikely that direct reduction of •OH by a very small amount of H2 accounts for all the functions of H2. The saturated level of H2 is only 0.8 mM, and the dwell time of •OH in the body is very short.6 In fact, drinking as little as 0.04 or 0.08 mM H2 was shown to be effective.7,8 Although we have recently shown that H2 can accumulate with
hepatic glycogen, it is unlikely that this amount of H2 is sufficient to exhibit all of its functions.8 It remains unclear whether such regulations are the cause or consequence of its effects against oxidative stress. Moreover, the primary molecular target of H2 remains unknown. The rodent retina has become a useful bio resource for retinal and ophthalmological research. Recently, an in vitro whole-tissue culture system using adult rodent retinas and variable gene transfer was established.9 The explant tissue culture allows retention of the intact morphological retina structure for at least four days.9 Furthermore, it exhibits a variety of biological processes, including retinal development,10 central nervous system regeneration11–13 and neurodegeneration14,15 in response to various stimuli. This explant culture system is therefore a useful tool for assessment of biological responses to pathological conditions or screening potential disease therapies. Although this system does not exactly recapitulate in vivo homeostasis and histological structure, it allows direct examination of retinal responses of cells in situ and in contact with other cells in retinal tissue. Therefore, this whole-tissue rat retinal culture system was considered effective in its ability to verify the oxidative damage caused by ONOO−. Moreover, the viable retinal cells allow detailed real-time in situ analysis of mitochondrial membrane potential (MMP) in living retinal tissue and histological/morphological analysis of oxidative stress in vitro. Using this model, we observed damage to the mitochondrial membrane and oxidative damage to living retinal tissues under conditions that produce ONOO−. Accordingly, the study presented herein was designed to assess whether the presence of H2 has a neuroprotective effect against SNAP-induced retinal cell damage. We found that H2 inhibits protein nitration and mitochondrial dysfunction caused by oxidative stress damage in retinal cells and prevents the induction of apoptosis.
METHODS Animals Male Sprague–Dawley rats (10 weeks old) were purchased from Nippon SLC (Hamamatsu, Shizuoka, Japan). The care and use of laboratory animals were in accordance with the NIH guidelines. This study was approved by the Animal Care and Use Committee of Nippon Medical School (Kanagawa, Japan).
Organotypic tissue culture Retinas were isolated from rat eyes and placed ganglion cell side up on a 0.4-μm Millicell tissue culture insert (Millipore, Billerica, MA, USA). The quality of
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Protective effect of H2 against retinal damage
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the tissue after incubation depended on smooth attachment to the membrane via the application of gentle suction to the tissue for ∼30 s during mounting. Filter stands (2-cm diameter, 1-cm thickness) were cut from the 30 × 20 mm cell culture dish (Nunc, Rochester, NY, USA), and the Millicell filter was rested on a platform of four ‘stands’ upon placement into a 60 × 20 mm cell culture dish (Nunc, Rochester, NY, USA) (Fig. 1a). More than 20 mL of Ames’ medium (Sigma-Aldrich, St Louis, MO, USA) containing 0.192% sodium bicarbonate, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.292 mg/mL L-glutamine, 10% horse serum (Sigma-Aldrich) and 20 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) was added to the dish, such a
that the retina contacted the medium via the Millicell filter on the photoreceptor side and the incubator atmosphere (95% N2 or H2, 5% O2, 37 °C, humidified) on the ganglion cell side. All further manipulations were carried out with the retina attached to the Millicell filter. The retinas were incubated for 1–72 h in medium containing 100 μM SNAP (Cayman Chemical, Ann Arbor, MI, USA). As a positive control for staining, we used sections from retina treated with 1 mM 3-morpholinosydnonimine (SIN−1; Sigma-Aldrich), which generates both •O2− and NO• for spontaneous production of ONOO−. During culture in the incubator, dishes were agitated constantly at 55 rpm using an orbital shaker (MIR– S100C; SANYO, Tokyo, Japan).
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Figure 1. Rat retinal organotypic tissue culture and the effect of H2 on retinal mitochondrial membrane potential. (a) Photograph of retina culture. (b) Whole-mount cultured retinas were loaded with tetramethylrhodamine, ethyl ester (TMRE) dye for 20 min at 37 °C. Top to bottom: untreated normal retina, 100 μM S-nitroso-N-acetylpenicillamine (SNAP)-treated retina in the absence of H2 for 24 h at 37 °C and 100 μM SNAP-treated retina in the presence of H2 for 24 h at 37 °C. Scale bar: 1 mm. The boxes in the high-power filed shows mitochondria (red fluorescence; inset scale bar: 20 m). (c) The rate of membrane potential loss in retinas was measured by quantification of the TMRE-positive area using an image analysis programme, ImageJ. Data are the mean ± SD (n = 4). *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retinas. © 2015 Royal Australian and New Zealand College of Ophthalmologists
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H2 treatment We prepared H2-dissolved culture medium as described previously.3 Briefly, we dissolved H2 in the medium by bubbling H2 gas to a saturated level. We similarly prepared O2- and N2-saturated culture media by dissolving O2 or N2 gas, respectively. We combined these media to give a medium consisting of 5% O2 and either 95% H2 or 95% N2 (control medium) (v/v). We then cultured the retinas in a closed culture dish filled with the medium. The H2 concentration was maintained for 24–72 h, as described previously.8 We also prepared mixed gases consisting of 5% O2 and either 95% H2 or 95% N2 (v/v) for the incubator atmosphere as described previously.3
MMP assay MMP was assessed by measuring the potentialdependent accumulation of tetramethylrhodamine, ethyl ester (TMRE)16 which also apparently the membranes in retinal tissues. Reduction in TMRE retention is indicative of loss of MMP. Accurate assessment of the intact membrane potential in retinal tissues required rapid preparation of the living retinal tissues because time delay is known to lead to depolarization of mitochondria. Retinas on the Millicell tissue culture insert were cultured in the presence or absence of H2 for 24 h at 37 °C and then labelled with TMRE dye. TMRE was detected by MMP depolarization detection kits (Molecular Probes, Eugene, OR, USA) for 20 min at 37 °C. TMRE-positive cells in the whole retinal area were stained with red fluorescence and visualized with a confocal scanning laser microscope (FLUOVIEW FV300; Olympus, Tokyo, Japan) with excitation at 544 nm and emission at 590 nm. Changes in fluorescence levels after different treatments were normalized to the basal fluorescence, and the mean value after reaching the plateau was calculated. Images were analysed for membrane potential of individual mitochondria using the deviation of fluorescence intensities for the ratio of red fluorescence for several mitochondria within each cell. We measured the positive area (red fluorescence) against the whole retinal tissue area using ImageJ (version 1.4.1; National Institutes of Health, Bethesda, MD, USA).
Histological analysis Whole-mount cultured retinas were cultured in the presence or absence of H2 for 72 h at 37 °C, and harvested retinas were fixed in 4% paraformaldehyde and 1 M phosphate-buffered saline at 4 °C for
60 min. For vertical sections, each fixed retina was embedded in Tissue-Tek O.C.T. Compound (Sakura FineTechnical Co., Tokyo, Japan), frozen and sectioned to a thickness of 6 μm. These sections were then stained with hematoxylin and eosin (H.E.). The thickness is defined as the total width between the outer nuclear layer and the inner limiting membrane to the interface of the outer plexiform layer. In addition, the thickness and measurement of the numbers of cells in the ganglion cell layer (GCL) and the inner nuclear layer (INL) were made at 1–2 mm from the optical disc using light microscopy. Data from three sections (randomly selected from the five sections) were averaged for each cultured retina and used to evaluate the thickness and the number of cells in the GCL and the INL. These measurements were estimated using ImageJ from each section of the retina at a final magnification of ×200.
Apoptotic assay and immunohistochemical staining Whole-mount cultured retinas were cultured in the presence or absence of H2 for 24 h at 37 °C, and harvested retinas were fixed in 4% paraformaldehyde and 0.1 M phosphate-buffered saline at 4°C for 60 min. For vertical sections, each fixed retina was embedded in Tissue-Tek O.C.T. Compound (Sakura FineTechnical Co.), frozen and sectioned to a thickness of 6 μm. The apoptotic assay (TUNEL assay) was performed with the in situ Apoptosis Detection Kit according to the supplier’s instructions (Chemicon, Norcross, GA, USA). For immunohistochemical staining, the ABC kit was used according to the supplier’s instructions (Vector Laboratories, Burlingame, CA, USA). Primary antibodies against nitrotyrosine (1:100; Calbiochem, San Diego, CA, USA), 4-HNE (1:400; JaICA, Shizuoka, Japan) and 8-OHdG (1:200; JaICA) were used. The TUNEL assay and nitrotyrosine-stained sections were further counterstained for nuclei with methyl green (0.5%). The number of positive cells from each section of the retina was estimated using ImageJ at a final magnification of ×200.
Statistical analysis All results are expressed as mean ± standard deviation. The values were processed for statistical analyses (Tukey–Kramer test and Student’s t-test with Bonferroni’s correction). All statistical analyses were performed using Microsoft Excel statistics 2012 software (Microsoft Inc., Redmond, WA, USA), and differences were considered statistically significant at P < 0.05.
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Protective effect of H2 against retinal damage
RESULTS H2 protects retinal cells from mitochondrial dysfunction SNAP-induced changes in the MMP were detected on whole-mount cultured retinas in the presence or absence of H2 by confocal scanning laser microscopy using the potential-sensitive dye TMRE (Fig. 1b). Treatment with SNAP resulted in decreased TMRE bright red fluorescence intensity from mitochondria (high-power images) compared with that of untreated cells. In contrast, H2 significantly protected mitochondria from the loss of membrane potential caused by SNAP. Loss of MMP was estimated as the percentage of red fluorescence area (low-power images) against the total area of the whole retina (Fig. 1c). These findings indicate that H2 protects retinal cells from the loss of MMP caused by NO•derived stimuli.
H2 decreases SNAP-induced tyrosine nitration in retinal cells ONOO− is a strong modifier of protein nitration. To confirm that H2 decreased the SNAP-induced ONOO− levels derived from the NO•, we immunohistologically examined the levels of tyrosine nitration in retinal tissues. Treatment with SNAP increased the number of nitrotyrosinepositive cells, which were located almost exclusively in the GCL and the INL, while H2 suppressed this increase (Fig. 2a,b). Treatment with 100 μM SNAP for 24 h induced tyrosine nitration of cells in a timedependent manner, and H2 decreased the number of nitrotyrosine-positive cells at each time point (Fig. 2c). Thus, H2 likely decreases ONOO− derived from NO•.
H2 protects retinal cells from apoptosis and oxidative stress To confirm the antioxidant effects of H2 on the retina after SNAP treatment, apoptotic cell death and oxidative stress damage, indicated by 4-HNE and 8-OHdG levels, were measured using the TUNEL assay and immunohistochemical techniques, respectively. Treatment with 100 μM SNAP increased the number of TUNEL-positive cells and 4-HNEand 8-OHdG-positive cells, which were located almost exclusively in the GCL and the INL, whereas H2 significantly protected retinal cells from SNAP-induced apoptotic cell death and oxidative stress (Fig. 3a–f). These results indicate that the neuroprotective effects of H2 on SNAP-induced retinal damage could be mediated by a reduction in oxidative stress.
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H2 protected SNAP-induced cell loss in the GCL and the INL To confirm that the retinas were structurally intact after 72 h of culture, we examined the vertical organization of retinal layers and the horizontal whole-mount organization of the cultured specimens. Histologically, each retinal layer was clearly identified (Fig. 4a). For quantitative morphometry of the tissue survival impact of H2 on the retina after SNAP treatment ex vivo, tissue viability was measured using cell count and thickness analyses. The SNAP-free group showed a nearly normal structure with a thicker retina; however, the SNAP treatment group exhibited marked thinning and histological damage of the retina. Treatment with SNAP histologically damaged and reduced the number of cells in the GCL and the INL, while the presence of H2 suppressed this decrease. In contrast, no significant difference in the thickness was observed in the presence or absence of H2 (Fig. 4a,b). However, the effects of H2 on retinal tissue survival after SNAP treatment ex vivo indicate that histological damage of the retinal layers was decreased. Furthermore, treatment with 100 μM SNAP for 72 h reduced cell viability in a time-dependent manner, and H2 treatment partially restored the viability of SNAP-treated cells (Fig. 4c).
DISCUSSION Glutamate, a neurotransmitter released from photoreceptor and bipolar cells, is present at a high concentration in retinal ganglion cells, and glutamate-induced neurotoxicity has been shown to play an important role in glaucoma.17 Glutamate also stimulates the production of large quantities of NO• and produces •O2− in mitochondria.18 NO• reacts with •O2− to form ONOO−, which triggers cell death by apoptosis.19 The causal role of ONOO− in glutamate-induced retinal neurotoxicity in glaucoma, however, has not been determined. Overstimulation of N-methyl-D-aspartate receptors leads to excessive levels of intracellular calcium. This, in turn, leads to activation of NO• synthase and excess accumulation of •O2− and NO•, causing lipid peroxidation, mitochondrial dysfunction, deoxyribonucleic acid (DNA) damage, and eventual cell death.20,21 The reaction products of •O2−, NO• and ONOO− were identified in retinal ischemiareperfusion injury.21 In vitro and in vivo studies have demonstrated that ischemia-induced neurotoxicity can be reduced by antioxidants.22 Oxidative stress can contribute to neuronal toxicity and has been implicated in both acute injury and chronic neuropathological conditions.23 Previously, we found that H2 is an efficient antioxidant that
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Figure 2. Effect of H2 on S-nitroso-N-acetylpenicillamine (SNAP)-induced tyrosine nitration in retina. (a) Whole-mount cultured retinal tissues were incubated with 100 μM SNAP in the presence or absence of H2 for 24 h at 37 °C. Vertical tissue sections of retinal layers were stained with anti-nitrotyrosine antibody, visualized with DAB (3, 3’-diaminobenzidine) and counterstained for methyl green. Retinas incubated with 1 mM SIN-1 (3-Morpholinosydnonimine hydrochloride) and without SNAP were used for positive and negative staining controls, respectively. Positive cells are indicated by arrows. Scale bar: 50 m. (b) Numbers of nitrotyrosine-positive cells in the ganglion cell layer and the inner nuclear layer were calculated using an image analysis programme, ImageJ. Data are the mean ± SD (n = 5). *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retina. (c) Whole-mount cultured retinas were incubated with 100 μM SNAP for various times in the absence or presence of H2. Data are the mean ± SD (n = 5). *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retinas.
selectively reduces •OH and suppresses oxidative stress-induced injury in the retina.6 In this study, we have shown that treatment with H2 protected retinal cells from mitochondrial dysfunction, apoptotic cell death and oxidative stress induced by SNAP. SNAP is a donor of NO•; however, NO• has no strong toxicity itself, and H2 has no potential to reduce NO•. In addition, ONOO− levels are difficult to measure because of its instability. Therefore, the presence of ONOO− in pathophysiological processes is commonly demonstrated by the detection of tyrosine nitration.24 It should be noted that there are other potential sources of tyrosine nitration in addi-
tion to ONOO−.25 However, tyrosine nitration is considered to be a likely indicator of ONOO− under conditions of simultaneous production of NO• and •O2−.26 In the present study, using an organotypic culture system of adult rat retinas, we succeeded in maintaining intact retinal morphology and structure for 72 h. Most of these experiments used neuronal or retinal tissues from rodent retinas with neuronal cell degradation and histological damage, which are difficult to culture, and previous studies have generally been limited to incubations lasting less than 24 h. The TMRE assay for mitochondrial function detects the loss of orange-red fluorescence in
© 2015 Royal Australian and New Zealand College of Ophthalmologists
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Figure 3. Effect of H2 on S-nitroso-N-acetylpenicillamine (SNAP)-induced expression of 4-HNE- and 8-OHdG-positive cells in wholemount cultured retina. Vertical tissue sections of retinal layers were stained with TUNEL assay (a), anti-4-HNE (c) or anti-8-OHdG (e), visualized with DAB and counterstained for methyl green. Scale bar: 50 m. TUNEL assay and 4-HNE- or 8-OHdG-positive cells are indicated by arrows. Numbers of TUNEL-positive cells (b), 4-HNE-positive cells, (d) or 8-OHdG-positive cells (f) in the ganglion cell layer and the inner nuclear layer of retinas were determined using an image analysis programme, ImageJ. Data are the mean ± SD (n = 5). *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retinas. © 2015 Royal Australian and New Zealand College of Ophthalmologists
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Figure 4. Effect of H2 on S-nitroso-N-acetylpenicillamine (SNAP)-induced decline of cell viability in whole-mount cultured retinas. (a) Whole-mount cultured retinas were incubated with 100 μM SNAP in the presence or absence of H2 for 72 h at 37 °C. Left to right: untreated normal retina, 100 μM SNAP-treated retina in the absence of H2 for 24 h at 37 °C and 100 μM SNAP-treated retina in the presence of H2 for 24 h at 37 °C. Scale bar: 50 m. Vertical tissue sections of retinal layers were stained with hematoxylin and eosin. (b) Retinal damage was evaluated by retinal thickness and the number of cells in the ganglion cell layer and the inner nuclear layer. Data are the mean ± SD (n = 5). Ganglion cell layer (GCL) graph; *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retinas. Retinal thickness; *P < 0.05; untreated retinas versus 100 μM SNAP-treated retinas without H2. (c) Whole-mount retinas were incubated with 100 μM SNAP for various times in the absence or presence of H2. Data are the mean ± SD (n = 5). *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retinas.
TMRE-stained cells and provides a reliable assessment of apoptosis induction or oxidative stress-induced mitochondrial depolarization in experimental cell populations.27 Although the retina is advantages for studying the relationship between function and energy metabolism, it should be recognized that the retina is highly specialized. However,
the present results indicate that both MMP loss and retinal cell apoptosis may be responsible for the very high oxygen consumption of the retina. In addition, oxidative damage and protein nitration occur during high ONOO− production. Examination of living rat retinal tissues using this system revealed the contribution of ONOO− produced by NO• donation from
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Protective effect of H2 against retinal damage in vitro addition of SNAP to the damage induced in histological evaluation of retinal injury. Thus, we incubated retinas for 24 h at 37 °C in medium containing SNAP and found that the loss of MMP was reduced by H2 treatment in the medium. These results suggested that H2 suppressed the induction of retinal cell apoptosis by ONOO−. Moreover, we showed that the inclusion of H2 in culture medium suppressed tyrosine nitration of retinal cell proteins. Thus, we speculate that H2 protects SNAP-treated retinal cells by decreasing the production of ONOO− via reactions of NO• and •O2− in mitochondria. Histopathologically, H2 treatment decreased the numbers of TUNEL-, 4-HNE- and 8-OHdG-positive cells, indicating that H2 protected lipids from peroxidation or DNA from oxidation and reduced subsequent retinal cell death by ONOO− production. Thus, it is possible that even a very small amount of H2 exhibits antioxidant effects by reducing ONOO− in many situations. Potential preventive and therapeutic use of H2 in novel pharmacological strategies aimed at selective removal of ONOO− may represent a powerful approach to retinal and optic nerve ischemia in diseases. Recently, we showed that H2 exhibits cytoprotective effects and transcriptional alterations through reducing ONOO− derived from NO• in chondrocytes.28 Furthermore, the ex vivo culture of adult rodent retinas in the presence of H2 provided clear evidence that retinal cell viability and histological damage were maintained after 72 h in vitro through our interphase culture system despite high ONOO− production. In conclusion, this study elucidated that the functions of H2 in suppressing the loss of MMP and the increase in tyrosine nitration in retinal tissues are likely a result of reducing the levels of ONOO− derived from NO•. Accordingly, this neuroprotective antioxidant could offer a new therapeutic strategy to reduce retinal damage.
ACKNOWLEDGEMENT This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (No. 21592248 and 24500871).
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9 3. Ohsawa I, Ishikawa M, Takahashi K et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 2007; 13: 688– 94. 4. Itoh T, Fujita Y, Ito M et al. Molecular hydrogen suppresses FcepsilonRI-mediated signal transduction and prevents degranulation of mast cells. Biochem Biophys Res Commun 2009; 389: 651–6. 5. Nagata K, Nakashima-Kamimura N, Mikami T, Ohsawa I, Ohta S. Consumption of molecular hydrogen prevents the stress-induced impairments in hippocampus-dependent learning tasks during chronic physical restraint in mice. Neuropsychopharmacology 2009; 34: 501–8. 6. Oharazawa H, Igarashi T, Yokota T et al. Protection of the retina by rapid diffusion of hydrogen: administration of hydrogen-loaded eye drops in retinal ischemiareperfusion injury. Invest Ophthalmol Vis Sci 2010; 51: 487–92. 7. Fujita K, Seike T, Yutsudo N et al. Hydrogen in drinking water reduces dopaminergic neuronal loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease. PLoS ONE 2009; 4: e7247. 8. Kamimura N, Nishimaki K, Ohsawa I, Ohta S. Molecular hydrogen improves obesity and diabetes by inducing hepatic FGF21 and stimulating energy metabolism in db/db mice. Obesity (Silver Spring) 2011; 19: 1396–403. 9. Moritoh S, Tanaka KF, Jouhou H, Ikenaka K, Koizumi A. Organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro. PLoS ONE 2010; 5: e12917. 10. Zhang SS, Fu XY, Barnstable CJ. Tissue culture studies of retinal development. Methods 2002; 28: 439–47. 11. Bermel C, Tonges L, Planchamp V et al. Combined inhibition of Cdk5 and ROCK additively increase cell survival, but not the regenerative response in regenerating retinal ganglion cells. Mol Cell Neurosci 2009; 42: 427–37. 12. Feigenspan A, Dedek K, Schlich K, Weiler R, Thanos S. Expression and biophysical characterization of voltage-gated sodium channels in axons and growth cones of the regenerating optic nerve. Invest Ophthalmol Vis Sci 2010; 51: 1789–99. 13. Leaver SG, Harvey AR, Plant GW. Adult olfactory ensheathing glia promote the long-distance growth of adult retinal ganglion cell neurites in vitro. Glia 2006; 53: 467–76. 14. Manabe S, Kashii S, Honda Y, Yamamoto R, Katsuki H, Akaike A. Quantification of axotomized ganglion cell death by explant culture of the rat retina. Neurosci Lett 2002; 334: 33–6. 15. Xin H, Yannazzo JA, Duncan RS, Gregg EV, Singh M, Koulen P. A novel organotypic culture model of the postnatal mouse retina allows the study of glutamatemediated excitotoxicity. J Neurosci Methods 2007; 159: 35–42. 16. Scaduto RC Jr, Grotyohann LW. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 1999; 76: 469–77.
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17. Dreyer EB, Lipton SA. New perspectives on glaucoma. JAMA 1999; 281: 306–8. 18. Haefliger IO, Lietz A, Griesser SM et al. Modulation of Heidelberg Retinal Flowmeter parameter flow at the papilla of healthy subjects: effect of carbogen, oxygen, high intraocular pressure, and beta-blockers. Surv Ophthalmol 1999; 43 (Suppl. 1): S59–65. 19. Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA. Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci U S A 1995; 92: 7162–6. 20. Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders. Science 1993; 262: 689–95. 21. Dreyer EB, Zurakowski D, Schumer RA, Podos SM, Lipton SA. Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma. Arch Ophthalmol 1996; 114: 299–305. 22. Ciani E, Groneng L, Voltattorni M, Rolseth V, Contestabile A, Paulsen RE. Inhibition of free radical production or free radical scavenging protects from the excitotoxic cell death mediated by glutamate in cultures of cerebellar granule neurons. Brain Res 1996; 728: 1–6.
23. Pettmann B, Henderson CE. Neuronal cell death. Neuron 1998; 20: 633–47. 24. Misko TP, Highkin MK, Veenhuizen AW et al. Characterization of the cytoprotective action of peroxynitrite decomposition catalysts. J Biol Chem 1998; 273: 15646– 53. 25. Eiserich JP, Hristova M, Cross CE et al. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 1998; 391: 393–7. 26. Sawa T, Akaike T, Maeda H. Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase. J Biol Chem 2000; 275: 32467–74. 27. Jayaraman S. Flow cytometric determination of mitochondrial membrane potential changes during apoptosis of T lymphocytic and pancreatic beta cell lines: comparison of tetramethylrhodamineethylester (TMRE), chloromethyl-X-rosamine (H2-CMX-Ros) and MitoTracker Red 580 (MTR580). J Immunol Methods 2005; 306: 68–79. 28. Hanaoka T, Kamimura N, Yokota T, Takai S, Ohta S. Molecular hydrogen protects chondrocytes from oxidative stress and indirectly alters gene expressions through reducing peroxynitrite derived from nitric oxide. Med Gas Res 2011; 1: 18. doi:10.1186/2045-99121-18.
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Clinical and Experimental Ophthalmology 2015; ••: ••–•• doi: 10.1111/ceo.12525
Original Article Protective effect of molecular hydrogen against oxidative stress caused by peroxynitrite derived from nitric oxide in rat retina Takashi Yokota PhD,1 Naomi Kamimura PhD,1 Tsutomu Igarashi MD PhD,2 Hiroshi Takahashi MD PhD,2 Shigeo Ohta PhD1 and Hideaki Oharazawa MD PhD3 1
Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine and Department of Ophthalmology, Musashikosugi Hospital, Nippon Medical School, Kawasaki, Kanagawa, and 2Department of Ophthalmology, Nippon Medical School, Tokyo, Japan 3
ABSTRACT Background: Oxidative and nitrative processes have an important role in the pathogenesis of glaucomatous neurodegeneration. Oxidative stress occurs when cellular production of reactive oxygen species outweighs the protective capacity of antioxidant defences. Reactive oxygen species are generated as by-products of cellular metabolism, primarily in the mitochondria. Herein, we present a novel investigation of the effects of molecular hydrogen (H2) on retinal cells exposed to oxidative stress. Methods: We cultured adult rat retinal tissues in an organotypic culture system with a nitric oxide donor, S-nitroso-N-acetylpenicillamine, in the presence or absence of H2. Loss of mitochondrial membrane potential and apoptosis of retinal cells were analysed using a MitoTMRE detection kit and TdT-mediated dUTP nick end labeling (TUNEL) assay, respectively. Tyrosine nitration levels and oxidative stress damage in the retina were evaluated using immunohistochemical staining. Retinal damage was quantified by measuring the numbers of cells in the ganglion cell and inner nuclear layers and the thickness of the retina. Results: H2 suppressed loss of mitochondrial membrane potential and apoptosis in retinal cells. Moreover, H2 decreased the tyrosine nitration level and
suppressed oxidative stress damage in retinal cells. S-nitroso-N-acetylpenicillamine treatment decreased the cell numbers in the ganglion cell layer and inner nuclear layer, but the presence of H2 inhibited this reduction. These findings suggest that H2 has a neuroprotective effect against retinal cell oxidative damage, presumably by scavenging peroxynitrite. Conclusions: H2 reduces cellular peroxynitrite, a highly toxic reactive nitrogen species. Thus, H2 may be an effective and novel clinical tool for treating glaucoma and other oxidative stress-related diseases. Key words: antioxidant, molecular hydrogen, oxidative stress, retina.
INTRODUCTION Oxidative stress contributes to several steps in the pathophysiology of neurodegenerative diseases such as glaucomatous neurodegeneration. In various subcellular compartments in neuronal cells, oxidative stress causes molecular changes that contribute to the development of glaucomatous neurodegeneration, including oxidative protein modifications that are associated with neuronal damage and glial dysfunction. Reactive oxygen species (ROS) are generated through physiological metabolic processes mainly as by-products of the electron transport chain and oxidative phosphorylation, which take place in the
■ Correspondence: Dr Hideaki Oharazawa, Department of Ophthalmology, Musashikosugi Hospital, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki 211-8533, Kanagawa, Japan. E-mail: [email protected] Received 5 August 2014; accepted 17 January 2015. Conflict of interest: None. Funding sources: None. © 2015 Royal Australian and New Zealand College of Ophthalmologists
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mitochondria. Although ROS function as cellular signalling messengers, damage to cellular macromolecules such as DNA, proteins and lipids ensues when the cellular production of ROS overwhelms the intrinsic antioxidant capacity.1 Oxidative stress is caused by an imbalance between the production of ROS and the cellular antioxidant defensive system. Superoxide anion radicals (•O2−), highly reactive metabolites of O2, are formed during normal aerobic metabolism in mitochondria, and sustained levels of ROS cause severe mitochondrial damage.2 Accumulating evidence has demonstrated that mitochondrial dysfunction contributes to enhanced ROS production, and elevated ROS can further impair mitochondrial function and exacerbate oxidative stress.3 Moreover, recent evidence has shown that mitochondrial dysfunction is present in some patients with glaucoma.4 Nitric oxide (NO•) has been implicated as a pivotal contributor to neuronal damage. Neuronal production of NO• leads to N-methyl-D-aspartate receptor activation and neuronal cell death.5 However, several studies have revealed that the neurotoxic effects of NO• are caused by peroxynitrite (ONOO−), a highly toxic reaction product of NO• and •O2−.6 To investigate the role of NO• in neuronal cell death, a NO• generator, S-nitroso-Nacetylpenicillamine (SNAP), has been widely used.7 In neuronal or retinal ganglion cell culture, NO• is generated rapidly and reacts with •O2− to form ONOO− when cells are stimulated with SNAP.8 Several pharmacological studies have demonstrated the potential participation of ONOO− in SNAPinduced cell death.1 However, to our knowledge there is no report on the effects of molecular hydrogen (H2) on SNAP-induced damage of retinal cells. We previously reported that H2 has potential as a novel antioxidant in preventive and therapeutic applications.2 Furthermore, H2 was shown to react with strongly reactive oxygen/nitrogen species including hydroxyl radicals (•OH) and ONOO− in cell-free reactions and to protect cultured cells in a manner dependent upon the decrease of •OH.3 Subsequent and recent experiments have indicated that a small amount of H2 is also effective against various stimuli.2 When animal models consumed drinking water containing dissolved H2, a small amount of H2 was considerably effective.4,5 Furthermore, 0.8 mM H2-loaded eye drops were shown to be effective for treatment of retinal ischemia-reperfusion injury.6 However, it appears unlikely that direct reduction of •OH by a very small amount of H2 accounts for all the functions of H2. The saturated level of H2 is only 0.8 mM, and the dwell time of •OH in the body is very short.6 In fact, drinking as little as 0.04 or 0.08 mM H2 was shown to be effective.7,8 Although we have recently shown that H2 can accumulate with
hepatic glycogen, it is unlikely that this amount of H2 is sufficient to exhibit all of its functions.8 It remains unclear whether such regulations are the cause or consequence of its effects against oxidative stress. Moreover, the primary molecular target of H2 remains unknown. The rodent retina has become a useful bio resource for retinal and ophthalmological research. Recently, an in vitro whole-tissue culture system using adult rodent retinas and variable gene transfer was established.9 The explant tissue culture allows retention of the intact morphological retina structure for at least four days.9 Furthermore, it exhibits a variety of biological processes, including retinal development,10 central nervous system regeneration11–13 and neurodegeneration14,15 in response to various stimuli. This explant culture system is therefore a useful tool for assessment of biological responses to pathological conditions or screening potential disease therapies. Although this system does not exactly recapitulate in vivo homeostasis and histological structure, it allows direct examination of retinal responses of cells in situ and in contact with other cells in retinal tissue. Therefore, this whole-tissue rat retinal culture system was considered effective in its ability to verify the oxidative damage caused by ONOO−. Moreover, the viable retinal cells allow detailed real-time in situ analysis of mitochondrial membrane potential (MMP) in living retinal tissue and histological/morphological analysis of oxidative stress in vitro. Using this model, we observed damage to the mitochondrial membrane and oxidative damage to living retinal tissues under conditions that produce ONOO−. Accordingly, the study presented herein was designed to assess whether the presence of H2 has a neuroprotective effect against SNAP-induced retinal cell damage. We found that H2 inhibits protein nitration and mitochondrial dysfunction caused by oxidative stress damage in retinal cells and prevents the induction of apoptosis.
METHODS Animals Male Sprague–Dawley rats (10 weeks old) were purchased from Nippon SLC (Hamamatsu, Shizuoka, Japan). The care and use of laboratory animals were in accordance with the NIH guidelines. This study was approved by the Animal Care and Use Committee of Nippon Medical School (Kanagawa, Japan).
Organotypic tissue culture Retinas were isolated from rat eyes and placed ganglion cell side up on a 0.4-μm Millicell tissue culture insert (Millipore, Billerica, MA, USA). The quality of
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Protective effect of H2 against retinal damage
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the tissue after incubation depended on smooth attachment to the membrane via the application of gentle suction to the tissue for ∼30 s during mounting. Filter stands (2-cm diameter, 1-cm thickness) were cut from the 30 × 20 mm cell culture dish (Nunc, Rochester, NY, USA), and the Millicell filter was rested on a platform of four ‘stands’ upon placement into a 60 × 20 mm cell culture dish (Nunc, Rochester, NY, USA) (Fig. 1a). More than 20 mL of Ames’ medium (Sigma-Aldrich, St Louis, MO, USA) containing 0.192% sodium bicarbonate, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.292 mg/mL L-glutamine, 10% horse serum (Sigma-Aldrich) and 20 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) was added to the dish, such a
that the retina contacted the medium via the Millicell filter on the photoreceptor side and the incubator atmosphere (95% N2 or H2, 5% O2, 37 °C, humidified) on the ganglion cell side. All further manipulations were carried out with the retina attached to the Millicell filter. The retinas were incubated for 1–72 h in medium containing 100 μM SNAP (Cayman Chemical, Ann Arbor, MI, USA). As a positive control for staining, we used sections from retina treated with 1 mM 3-morpholinosydnonimine (SIN−1; Sigma-Aldrich), which generates both •O2− and NO• for spontaneous production of ONOO−. During culture in the incubator, dishes were agitated constantly at 55 rpm using an orbital shaker (MIR– S100C; SANYO, Tokyo, Japan).
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Figure 1. Rat retinal organotypic tissue culture and the effect of H2 on retinal mitochondrial membrane potential. (a) Photograph of retina culture. (b) Whole-mount cultured retinas were loaded with tetramethylrhodamine, ethyl ester (TMRE) dye for 20 min at 37 °C. Top to bottom: untreated normal retina, 100 μM S-nitroso-N-acetylpenicillamine (SNAP)-treated retina in the absence of H2 for 24 h at 37 °C and 100 μM SNAP-treated retina in the presence of H2 for 24 h at 37 °C. Scale bar: 1 mm. The boxes in the high-power filed shows mitochondria (red fluorescence; inset scale bar: 20 m). (c) The rate of membrane potential loss in retinas was measured by quantification of the TMRE-positive area using an image analysis programme, ImageJ. Data are the mean ± SD (n = 4). *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retinas. © 2015 Royal Australian and New Zealand College of Ophthalmologists
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H2 treatment We prepared H2-dissolved culture medium as described previously.3 Briefly, we dissolved H2 in the medium by bubbling H2 gas to a saturated level. We similarly prepared O2- and N2-saturated culture media by dissolving O2 or N2 gas, respectively. We combined these media to give a medium consisting of 5% O2 and either 95% H2 or 95% N2 (control medium) (v/v). We then cultured the retinas in a closed culture dish filled with the medium. The H2 concentration was maintained for 24–72 h, as described previously.8 We also prepared mixed gases consisting of 5% O2 and either 95% H2 or 95% N2 (v/v) for the incubator atmosphere as described previously.3
MMP assay MMP was assessed by measuring the potentialdependent accumulation of tetramethylrhodamine, ethyl ester (TMRE)16 which also apparently the membranes in retinal tissues. Reduction in TMRE retention is indicative of loss of MMP. Accurate assessment of the intact membrane potential in retinal tissues required rapid preparation of the living retinal tissues because time delay is known to lead to depolarization of mitochondria. Retinas on the Millicell tissue culture insert were cultured in the presence or absence of H2 for 24 h at 37 °C and then labelled with TMRE dye. TMRE was detected by MMP depolarization detection kits (Molecular Probes, Eugene, OR, USA) for 20 min at 37 °C. TMRE-positive cells in the whole retinal area were stained with red fluorescence and visualized with a confocal scanning laser microscope (FLUOVIEW FV300; Olympus, Tokyo, Japan) with excitation at 544 nm and emission at 590 nm. Changes in fluorescence levels after different treatments were normalized to the basal fluorescence, and the mean value after reaching the plateau was calculated. Images were analysed for membrane potential of individual mitochondria using the deviation of fluorescence intensities for the ratio of red fluorescence for several mitochondria within each cell. We measured the positive area (red fluorescence) against the whole retinal tissue area using ImageJ (version 1.4.1; National Institutes of Health, Bethesda, MD, USA).
Histological analysis Whole-mount cultured retinas were cultured in the presence or absence of H2 for 72 h at 37 °C, and harvested retinas were fixed in 4% paraformaldehyde and 1 M phosphate-buffered saline at 4 °C for
60 min. For vertical sections, each fixed retina was embedded in Tissue-Tek O.C.T. Compound (Sakura FineTechnical Co., Tokyo, Japan), frozen and sectioned to a thickness of 6 μm. These sections were then stained with hematoxylin and eosin (H.E.). The thickness is defined as the total width between the outer nuclear layer and the inner limiting membrane to the interface of the outer plexiform layer. In addition, the thickness and measurement of the numbers of cells in the ganglion cell layer (GCL) and the inner nuclear layer (INL) were made at 1–2 mm from the optical disc using light microscopy. Data from three sections (randomly selected from the five sections) were averaged for each cultured retina and used to evaluate the thickness and the number of cells in the GCL and the INL. These measurements were estimated using ImageJ from each section of the retina at a final magnification of ×200.
Apoptotic assay and immunohistochemical staining Whole-mount cultured retinas were cultured in the presence or absence of H2 for 24 h at 37 °C, and harvested retinas were fixed in 4% paraformaldehyde and 0.1 M phosphate-buffered saline at 4°C for 60 min. For vertical sections, each fixed retina was embedded in Tissue-Tek O.C.T. Compound (Sakura FineTechnical Co.), frozen and sectioned to a thickness of 6 μm. The apoptotic assay (TUNEL assay) was performed with the in situ Apoptosis Detection Kit according to the supplier’s instructions (Chemicon, Norcross, GA, USA). For immunohistochemical staining, the ABC kit was used according to the supplier’s instructions (Vector Laboratories, Burlingame, CA, USA). Primary antibodies against nitrotyrosine (1:100; Calbiochem, San Diego, CA, USA), 4-HNE (1:400; JaICA, Shizuoka, Japan) and 8-OHdG (1:200; JaICA) were used. The TUNEL assay and nitrotyrosine-stained sections were further counterstained for nuclei with methyl green (0.5%). The number of positive cells from each section of the retina was estimated using ImageJ at a final magnification of ×200.
Statistical analysis All results are expressed as mean ± standard deviation. The values were processed for statistical analyses (Tukey–Kramer test and Student’s t-test with Bonferroni’s correction). All statistical analyses were performed using Microsoft Excel statistics 2012 software (Microsoft Inc., Redmond, WA, USA), and differences were considered statistically significant at P < 0.05.
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Protective effect of H2 against retinal damage
RESULTS H2 protects retinal cells from mitochondrial dysfunction SNAP-induced changes in the MMP were detected on whole-mount cultured retinas in the presence or absence of H2 by confocal scanning laser microscopy using the potential-sensitive dye TMRE (Fig. 1b). Treatment with SNAP resulted in decreased TMRE bright red fluorescence intensity from mitochondria (high-power images) compared with that of untreated cells. In contrast, H2 significantly protected mitochondria from the loss of membrane potential caused by SNAP. Loss of MMP was estimated as the percentage of red fluorescence area (low-power images) against the total area of the whole retina (Fig. 1c). These findings indicate that H2 protects retinal cells from the loss of MMP caused by NO•derived stimuli.
H2 decreases SNAP-induced tyrosine nitration in retinal cells ONOO− is a strong modifier of protein nitration. To confirm that H2 decreased the SNAP-induced ONOO− levels derived from the NO•, we immunohistologically examined the levels of tyrosine nitration in retinal tissues. Treatment with SNAP increased the number of nitrotyrosinepositive cells, which were located almost exclusively in the GCL and the INL, while H2 suppressed this increase (Fig. 2a,b). Treatment with 100 μM SNAP for 24 h induced tyrosine nitration of cells in a timedependent manner, and H2 decreased the number of nitrotyrosine-positive cells at each time point (Fig. 2c). Thus, H2 likely decreases ONOO− derived from NO•.
H2 protects retinal cells from apoptosis and oxidative stress To confirm the antioxidant effects of H2 on the retina after SNAP treatment, apoptotic cell death and oxidative stress damage, indicated by 4-HNE and 8-OHdG levels, were measured using the TUNEL assay and immunohistochemical techniques, respectively. Treatment with 100 μM SNAP increased the number of TUNEL-positive cells and 4-HNEand 8-OHdG-positive cells, which were located almost exclusively in the GCL and the INL, whereas H2 significantly protected retinal cells from SNAP-induced apoptotic cell death and oxidative stress (Fig. 3a–f). These results indicate that the neuroprotective effects of H2 on SNAP-induced retinal damage could be mediated by a reduction in oxidative stress.
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H2 protected SNAP-induced cell loss in the GCL and the INL To confirm that the retinas were structurally intact after 72 h of culture, we examined the vertical organization of retinal layers and the horizontal whole-mount organization of the cultured specimens. Histologically, each retinal layer was clearly identified (Fig. 4a). For quantitative morphometry of the tissue survival impact of H2 on the retina after SNAP treatment ex vivo, tissue viability was measured using cell count and thickness analyses. The SNAP-free group showed a nearly normal structure with a thicker retina; however, the SNAP treatment group exhibited marked thinning and histological damage of the retina. Treatment with SNAP histologically damaged and reduced the number of cells in the GCL and the INL, while the presence of H2 suppressed this decrease. In contrast, no significant difference in the thickness was observed in the presence or absence of H2 (Fig. 4a,b). However, the effects of H2 on retinal tissue survival after SNAP treatment ex vivo indicate that histological damage of the retinal layers was decreased. Furthermore, treatment with 100 μM SNAP for 72 h reduced cell viability in a time-dependent manner, and H2 treatment partially restored the viability of SNAP-treated cells (Fig. 4c).
DISCUSSION Glutamate, a neurotransmitter released from photoreceptor and bipolar cells, is present at a high concentration in retinal ganglion cells, and glutamate-induced neurotoxicity has been shown to play an important role in glaucoma.17 Glutamate also stimulates the production of large quantities of NO• and produces •O2− in mitochondria.18 NO• reacts with •O2− to form ONOO−, which triggers cell death by apoptosis.19 The causal role of ONOO− in glutamate-induced retinal neurotoxicity in glaucoma, however, has not been determined. Overstimulation of N-methyl-D-aspartate receptors leads to excessive levels of intracellular calcium. This, in turn, leads to activation of NO• synthase and excess accumulation of •O2− and NO•, causing lipid peroxidation, mitochondrial dysfunction, deoxyribonucleic acid (DNA) damage, and eventual cell death.20,21 The reaction products of •O2−, NO• and ONOO− were identified in retinal ischemiareperfusion injury.21 In vitro and in vivo studies have demonstrated that ischemia-induced neurotoxicity can be reduced by antioxidants.22 Oxidative stress can contribute to neuronal toxicity and has been implicated in both acute injury and chronic neuropathological conditions.23 Previously, we found that H2 is an efficient antioxidant that
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Figure 2. Effect of H2 on S-nitroso-N-acetylpenicillamine (SNAP)-induced tyrosine nitration in retina. (a) Whole-mount cultured retinal tissues were incubated with 100 μM SNAP in the presence or absence of H2 for 24 h at 37 °C. Vertical tissue sections of retinal layers were stained with anti-nitrotyrosine antibody, visualized with DAB (3, 3’-diaminobenzidine) and counterstained for methyl green. Retinas incubated with 1 mM SIN-1 (3-Morpholinosydnonimine hydrochloride) and without SNAP were used for positive and negative staining controls, respectively. Positive cells are indicated by arrows. Scale bar: 50 m. (b) Numbers of nitrotyrosine-positive cells in the ganglion cell layer and the inner nuclear layer were calculated using an image analysis programme, ImageJ. Data are the mean ± SD (n = 5). *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retina. (c) Whole-mount cultured retinas were incubated with 100 μM SNAP for various times in the absence or presence of H2. Data are the mean ± SD (n = 5). *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retinas.
selectively reduces •OH and suppresses oxidative stress-induced injury in the retina.6 In this study, we have shown that treatment with H2 protected retinal cells from mitochondrial dysfunction, apoptotic cell death and oxidative stress induced by SNAP. SNAP is a donor of NO•; however, NO• has no strong toxicity itself, and H2 has no potential to reduce NO•. In addition, ONOO− levels are difficult to measure because of its instability. Therefore, the presence of ONOO− in pathophysiological processes is commonly demonstrated by the detection of tyrosine nitration.24 It should be noted that there are other potential sources of tyrosine nitration in addi-
tion to ONOO−.25 However, tyrosine nitration is considered to be a likely indicator of ONOO− under conditions of simultaneous production of NO• and •O2−.26 In the present study, using an organotypic culture system of adult rat retinas, we succeeded in maintaining intact retinal morphology and structure for 72 h. Most of these experiments used neuronal or retinal tissues from rodent retinas with neuronal cell degradation and histological damage, which are difficult to culture, and previous studies have generally been limited to incubations lasting less than 24 h. The TMRE assay for mitochondrial function detects the loss of orange-red fluorescence in
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Figure 3. Effect of H2 on S-nitroso-N-acetylpenicillamine (SNAP)-induced expression of 4-HNE- and 8-OHdG-positive cells in wholemount cultured retina. Vertical tissue sections of retinal layers were stained with TUNEL assay (a), anti-4-HNE (c) or anti-8-OHdG (e), visualized with DAB and counterstained for methyl green. Scale bar: 50 m. TUNEL assay and 4-HNE- or 8-OHdG-positive cells are indicated by arrows. Numbers of TUNEL-positive cells (b), 4-HNE-positive cells, (d) or 8-OHdG-positive cells (f) in the ganglion cell layer and the inner nuclear layer of retinas were determined using an image analysis programme, ImageJ. Data are the mean ± SD (n = 5). *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retinas. © 2015 Royal Australian and New Zealand College of Ophthalmologists
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Figure 4. Effect of H2 on S-nitroso-N-acetylpenicillamine (SNAP)-induced decline of cell viability in whole-mount cultured retinas. (a) Whole-mount cultured retinas were incubated with 100 μM SNAP in the presence or absence of H2 for 72 h at 37 °C. Left to right: untreated normal retina, 100 μM SNAP-treated retina in the absence of H2 for 24 h at 37 °C and 100 μM SNAP-treated retina in the presence of H2 for 24 h at 37 °C. Scale bar: 50 m. Vertical tissue sections of retinal layers were stained with hematoxylin and eosin. (b) Retinal damage was evaluated by retinal thickness and the number of cells in the ganglion cell layer and the inner nuclear layer. Data are the mean ± SD (n = 5). Ganglion cell layer (GCL) graph; *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retinas. Retinal thickness; *P < 0.05; untreated retinas versus 100 μM SNAP-treated retinas without H2. (c) Whole-mount retinas were incubated with 100 μM SNAP for various times in the absence or presence of H2. Data are the mean ± SD (n = 5). *P < 0.05; absence of H2 versus presence of H2 in 100 μM SNAP-treated retinas.
TMRE-stained cells and provides a reliable assessment of apoptosis induction or oxidative stress-induced mitochondrial depolarization in experimental cell populations.27 Although the retina is advantages for studying the relationship between function and energy metabolism, it should be recognized that the retina is highly specialized. However,
the present results indicate that both MMP loss and retinal cell apoptosis may be responsible for the very high oxygen consumption of the retina. In addition, oxidative damage and protein nitration occur during high ONOO− production. Examination of living rat retinal tissues using this system revealed the contribution of ONOO− produced by NO• donation from
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Protective effect of H2 against retinal damage in vitro addition of SNAP to the damage induced in histological evaluation of retinal injury. Thus, we incubated retinas for 24 h at 37 °C in medium containing SNAP and found that the loss of MMP was reduced by H2 treatment in the medium. These results suggested that H2 suppressed the induction of retinal cell apoptosis by ONOO−. Moreover, we showed that the inclusion of H2 in culture medium suppressed tyrosine nitration of retinal cell proteins. Thus, we speculate that H2 protects SNAP-treated retinal cells by decreasing the production of ONOO− via reactions of NO• and •O2− in mitochondria. Histopathologically, H2 treatment decreased the numbers of TUNEL-, 4-HNE- and 8-OHdG-positive cells, indicating that H2 protected lipids from peroxidation or DNA from oxidation and reduced subsequent retinal cell death by ONOO− production. Thus, it is possible that even a very small amount of H2 exhibits antioxidant effects by reducing ONOO− in many situations. Potential preventive and therapeutic use of H2 in novel pharmacological strategies aimed at selective removal of ONOO− may represent a powerful approach to retinal and optic nerve ischemia in diseases. Recently, we showed that H2 exhibits cytoprotective effects and transcriptional alterations through reducing ONOO− derived from NO• in chondrocytes.28 Furthermore, the ex vivo culture of adult rodent retinas in the presence of H2 provided clear evidence that retinal cell viability and histological damage were maintained after 72 h in vitro through our interphase culture system despite high ONOO− production. In conclusion, this study elucidated that the functions of H2 in suppressing the loss of MMP and the increase in tyrosine nitration in retinal tissues are likely a result of reducing the levels of ONOO− derived from NO•. Accordingly, this neuroprotective antioxidant could offer a new therapeutic strategy to reduce retinal damage.
ACKNOWLEDGEMENT This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (No. 21592248 and 24500871).
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