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Oxidative stress mediated by NMDA, AMPA/KA channels in acute hippocampal slices: Neuroprotective effect of resveratrol

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André Quincozes-Santos ⇑, Larissa Daniele Bobermin, Ana Carolina Tramontina, Krista Minéia Wartchow, Bárbara Tagliari, Diogo Onofre Souza, Angela T.S. Wyse, Carlos-Alberto Gonçalves

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Departamento de Bioquímica, Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

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a r t i c l e

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Article history: Received 27 October 2013 Accepted 28 December 2013 Available online xxxx

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Keywords: Resveratrol Glutamate toxicity Glutamate receptors Oxidative stress HO1

a b s t r a c t Glutamate is the major excitatory neurotransmitter in the brain and over-stimulation of the glutamate receptors, NMDA, AMPA and kainate (KA), may cause neuronal death in epilepsy, seizures and neurodegenerative diseases. Mitochondria have critical cellular functions that influence neuronal excitability, such as regulation of Ca2+ homeostasis and ATP production to maintain Na+K+-ATPase in the central nervous system (CNS). However, mitochondria are also the primary site of reactive oxygen species (ROS) production, and oxidative stress can induce cellular damage. Resveratrol, a polyphenol found in grapes and wines, presents antioxidant and neuroprotective effects on brain pathologies. This study sought to determine the neuroprotective effect of resveratrol against glutamate toxicity in acute hippocampal slices, using specific inhibitors of glutamate channels, and to investigate the targets of glutamate excitotoxicity, such as mitochondrial membrane potential (DWm), Na+K+-ATPase and glutamine synthetase (GS) activity. Resveratrol decreases intracellular ROS production, most likely by mechanisms involving NMDA, AMPA/KA, intracellular Ca2+ and the heme oxygenase 1 (HO1) pathway, and prevents mitochondrial dysfunction and impairments in Na+K+-ATPase and GS activity after glutamate activation. Taken together, these results show that resveratrol may exhibit an important neuroprotective mechanism against neuropsychiatric disorders, focusing on mitochondrial bioenergetics and oxidative stress, as well as inhibitory effects on ionic channels. Ó 2014 Elsevier Ltd. All rights reserved.

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1. Introduction Hippocampal slices have been widely used to investigate the electrophysiological and biochemical parameters of the Central Nervous System (CNS), due to partially preserved neuron-astrocyte circuitry with a suitable control of extracellular medium (Nardin et al., 2009; Thomazi et al., 2004, 2008). Morphological studies have confirmed the integrity of a large number of synaptic terminals, including glial cells (Nagy and Li, 2000). The use of brain slices does have some limitations that are inherent in the use of isolated neuronal and/or glial cells (Aitken et al., 1995; de Almeida et al., 2008; Nagy and Li, 2000; Nardin et al., 2009). Glutamate is the major excitatory neurotransmitter in the brain (Danbolt, 2001). Extracellular glutamate is normally kept at low levels by Na+-dependent transport into glia and neurons (Anderson

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⇑ Corresponding author. Address: Departamento de Bioquímica, Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600 – Anexo, Bairro Santa Cecília, 90035-003 Porto Alegre, RS, Brazil. Tel.: +55 51 3308 5559; fax: +55 51 3308 5535. E-mail address: [email protected] (A. Quincozes-Santos).

and Swanson, 2000; Danbolt, 2001). Excessive glutamate stimulation or persistent activation of glutamate-gated ion channels may cause neuronal degeneration in experimental models of epilepsy, seizures, ischemia, trauma and neurodegenerative diseases (Coyle and Puttfarcken, 1993; Friedman et al., 1994; Gupta et al., 2002; Maragakis and Rothstein, 2006). Ionotropic glutamate receptors, N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl4-isoxazole propionic acid (AMPA) and kainate (KA), are the predominant receptors at most excitatory synapses (Coyle and Puttfarcken, 1993). The overactivation of these receptors and disrupted glutamate homeostasis has been recognized to play a prominent role in the development and generation of neurological diseases (Lau and Tymianski, 2010). Furthermore, activation of an NMDA receptor under depolarizing conditions permits Ca2+ to flow through its channels, thus increasing the influx of Ca2+ and leading to neuronal degeneration (Carmignoto et al., 1998; Coyle and Puttfarcken, 1993; Ding et al., 2007). Oxidative stress and mitochondrial metabolic dysfunction are involved in neuronal death (Halliwell, 2006). Mitochondria have critical cellular functions that influence neuronal excitability, including production of adenosine triphosphate (ATP), fatty acid oxidation, neurotransmitter biosynthesis, and regulation of Ca2+

0887-2333/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.12.021

Please cite this article in press as: Quincozes-Santos, A., et al. Oxidative stress mediated by NMDA, AMPA/KA channels in acute hippocampal slices: Neuroprotective effect of resveratrol. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2013.12.021

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homeostasis (Halliwell, 2007; Rosenstock et al., 2004; Waldbaum and Patel, 2010). The majority of ATP produced by the mitochondria is utilized to maintain Na+K+-ATPase in the CNS (Lingrel, 1992; Waldbaum and Patel, 2010). However, mitochondria are the primary site of reactive oxygen species (ROS) production. ROS overproduction generates oxidative stress, and this condition can affect the functioning of cellular macromolecules, such as lipids, proteins and nucleic acids (Halliwell, 2006, 2007). Glutamate exposure may induce overactivation of NMDA-type glutamate receptors and may increase Ca2+ influx into neurons. This Ca2+ can be taken up by mitochondria, where it may stimulate the generation of oxidative/nitrosative species that damage the mitochondria and the cell (Nicholls, 2008). Resveratrol (3,5,40 -trihydroxy-trans-stilbene), a redox active compound, is a phytoalexin found in a wide range of dietary sources, including grapes, peanuts and wine (especially red wine); it exhibits antioxidant, anti-inflammatory, cardioprotective, estrogenic and antitumoral activities (Baur and Sinclair, 2006; Bobermin et al., 2012; Park et al., 2012; Pervaiz, 2003; Quincozes-Santos and Gottfried, 2011; Yang et al., 2013). Resveratrol also presents neuroprotective properties and has been investigated in several neurodegenerative models, such as epilepsy, stroke and Alzheimer’s and Parkinson’s diseases (Bastianetto and Quirion, 2010; Baur and Sinclair, 2006; Fukui et al., 2010; Kim et al., 2007; Kumar et al., 2007; Shetty, 2011). Although the neuroprotective role of resveratrol in the CNS is well established, the cellular mechanisms underlying resveratrol-induced neuroprotection must be better elucidated (Fukui et al., 2010; Lee et al., 2010). Classically, glutamate exposure induces activation of NMDA and AMPA/KA receptors, mitochondrial damage, and, consequently, oxidative stress (Coyle and Puttfarcken, 1993). These events are associated with neuropsychiatric disorders. Thus, in this study, the biochemical parameters and probable mechanisms by which resveratrol protects hippocampal slices from glutamate excitotoxicity were evaluated using specific inhibitors of glutamate-gated channels. Additionally, glutamate excitotoxicity targets were investigated, such as mitochondrial membrane potential (DWm), Na+K+-ATPase and glutamine synthetase (GS) activity.

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2. Materials and methods

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2.1. Animals

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Male Wistar rats (30 days old) were obtained from our breeding colony (Department of Biochemistry), maintained under controlled environment (12 h light/12 h dark cycle, 22 ± 1 °C, ad libitum access to food and water). All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Federal University of Rio Grande do Sul Animal Care and Use Committee (process number 21215).

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2.2. Materials

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Resveratrol, chemical reagents, anti-Na+K+-ATPase a-1 and a-3 subunits, anti-b-actin, MTT Formazan, 20 -70 -dichorofluorescein diacetate (DCFH) and c-glutamylhydroxamate were purchased from Sigma (St. Louis, MO, USA). JC-1 was purchased from Gibco/ Invitrogen (Carlsbad, CA, USA). Anti-HO1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from local commercial suppliers.

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2.3. Preparation and incubation of hippocampal slices

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Animals were killed by decapitation, the brains were removed and placed in cold saline medium with the following composition

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(in mM): 120 NaCl; 2 KCl; 1 CaCl2; 1 MgSO4; 25 HEPES; 1 KH2PO4 and 10 glucose, adjusted to pH 7.4 and previously oxygenated for 15 min with O2. The hippocampi were dissected and transverse slices of 0.3 mm were obtained using a Mcllwain Tissue Chopper. Slices were then transferred immediately into 24-well culture plates, each well containing 0.3 mL of physiological medium and only slice. The medium was changed every 15 min with fresh saline medium at room temperature (maintained at 25 °C). Following a 120 min equilibration period, the medium was removed and replaced with fresh saline containing, or not, 100 lM resveratrol for 30 min at 30 °C. After this period, the medium was maintained and 1 mM glutamate was added to the medium for 30 min at 30 °C in a water bath. For all analyzed parameters, the results obtained with vehicle (0.25% ethanol) were not different from those obtained under control conditions without ethanol.

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2.4. Membrane integrity and metabolic activity assays

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2.4.1. Lactate dehydrogenase assay The lactate dehydrogenase (LDH) assay was conducted in 50 lL of extracellular medium using a commercial colorimetric assay from Doles (Brazil). Results are expressed as percentages of the control value.

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2.4.2. Lactate release assay The lactate release assay was conducted in 100 lL of extracellular medium using a commercial colorimetric assay from Labtest (Brazil). Results are expressed as percentages of the control value.

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2.4.3. MTT reduction assay Slices were treated with 0.5 mg/mL of MTT for 30 min at 30 °C. The MTT formazan was dissolved in DMSO. Absorbance values were measured at 560 and 650 nm. Results are expressed as percentages of the control value.

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2.5. Intracellular ROS production

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DCFH oxidation was used to measure intracellular ROS production (Quincozes-Santos et al., 2009). DCFH-DA (20 -70 -dichlorofluorescein diacetate) is hydrolyzed by intracellular esterases to dichlorofluorescin (DCFH), which is trapped within the cell. This non-fluorescent molecule is then oxidized to fluorescent dichlorofluorescin (DCF) by action of cellular oxidants. To test whether NMDA and AMPA/KA activation, intracellular Ca2+ signaling, NO and heme oxygenase 1 (HO1) pathways were involved in the effect of resveratrol against glutamate-induced increase ROS production, we used the following specific inhibitors: Dizocilpine (MK801) – 10 lM, 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX) – 10 lM, 1,2-Bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) – 10 lM, Nx-nitro-L-arginine methyl ester (L-NAME) – 500 lM, and Zinc Protoporphyrin IX (ZnPP IX) – 10 lM, for 30 min added together with resveratrol. After glutamate incubation, slices were treated with DCFH-DA (10 lM) for 30 min at 30 °C. The fluorescence was measured in a plate reader (Spectra Max GEMINI XPS, Molecular Devices, USA) with excitation at 485 nm and emission at 520 nm (QuincozesSantos et al., 2009). The ROS production was calculated as Unit of Fluorescence – UF/mg protein and was expressed as percentage of control.

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2.6. Western blot analysis

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Slices were homogenate using lysis solution with 4% SDS, 2 mM EDTA, 50 mM Tris–HCl, pH 6.8. Equal amounts of proteins from each sample were boiled in sample buffer [62.5 mM Tris–HCl, pH 6.8, 2% (w/v) SDS, 5% b-mercaptoethanol, 10% (v/v) glycerol,

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0.002% (w/v) bromophenol blue] and submitted to electrophoresis in 10% (w/v) SDS–polyacrylamide gel (Souza et al., 2013). The separated proteins were blotted onto a nitrocellulose membrane. Equal loading of each sample was confirmed with Ponceau S staining (Sigma). The membranes were incubated with polyclonal antiHO1 (1:3000), anti-Na+K+-ATPase a-1 and a-3 (1:1000). b-actin was used as a loading control. After incubating overnight with the primary antibody at room temperature, membrane was washed and incubated with peroxidase-conjugated anti-rabbit immunoglobulin (IgG) at a dilution of 1:1000 for 1 h. The chemiluminescence signal was detected using an ECL (Amersham), after the films were scanned and bands were quantified using the Scion Image software.

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2.7. Thiobarbituric acid reactive substances (TBARS) measurement

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Thiobarbituric acid reactive substances assay evaluates the oxidative stress assayed for malondialdehyde (MDA), the last product of lipid breakdown caused by oxidative stress. The assay was performed as previously described (Esterbauer and Cheeseman, 1990). Briefly, slices were lysed in PBS with KCl (140 mM) and 100 lL of homogenates were added to 200 lL of cold 10% trichloroacetic acid (TCA) and 300 lL of 0.67% TBA in 7.1% sodium sulfate and put in a boiling water bath for 1 h. The mixture was placed in cold water for 3 min. Afterwards, 400 lL of butyl alcohol were added and samples were centrifuged at 5000g for 5 min. Pink-stained TBARS was determined in resulting supernatants in a spectrophotometric microtiter plate reader at 532 nm. A calibration curve was performed using 1,1,3,3-tetramethoxypropane. Results are expressed as percentages of the control condition.

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2.8. Mitochondrial membrane potencial – DWm (JC-1 assay)

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For determination of the DWm, after resveratrol and glutamate treatments, slices were incubated for 30 min with JC-1 (5,50 ,6,60 tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide, 2 lg/mL) (Reers et al., 1995). After, slices were homogenized and centrifuged, washed once with HBSS and transferred to a 96-well plate. Fluorescence was measured using excitation and emission wavelengths of 485 and 540/590 nm, respectively. The DWm was calculated using the ratio of 590 nm (red fluorescent J-aggregates)/540 nm (green monomeric). Results are expressed as percentages of the control condition.

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2.9. Na+K+-ATPase activity assay

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The reaction mixture for Na+K+-ATPase assay contained (in mM) 5 MgCl2, 80 NaCl, 20 KCl and 40 Tris–HCl, ph 7.4, in a final volume of 200 ll. The reaction was initiated by addition of ATP to a final concentration of 3 mM. Controls were carried out under the same conditions with the addition of 1 mM ouabain. Na+K+-ATPase activity was calculated by the difference between the two assays according to the method of Wyse et al. (2000). Released inorganic phosphate (Pi) was measured by the method of Chan et al. (1986). Specific activity of the enzyme was expressed as nmol Pi released per min per mg of protein.

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2.10. Glutamine synthetase (GS) activity

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The enzymatic assay was performed as previously described (dos Santos et al., 2006). Briefly, 0.1 mL homogenates solubilized in 140 mM KCl was added to 0.1 mL of the reaction mixture containing (in mM): 10 MgCl2, 50 L-glutamate, 100 imidazole-HCl buffer (pH 7.4), 10 2-mercaptoethanol, 50 hydroxylamine-HCl and 10 ATP, and incubated for 15 min (37 °C). The reaction was stopped by the addition of 0.4 mL of a solution containing (in mM): 370 ferric

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chloride, 670 HCl, and 200 TCA. After centrifugation, the absorbance of the supernatant was measured at 530 nm and compared to the absorbance generated using standard quantities of c-glutamylhydroxamate treated with a ferric chloride reagent. Results are expressed as percentages of the control condition.

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2.11. Protein assay

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Protein content was measured using Lowry’s method with bovine serum albumin as a standard (Lowry et al., 1951).

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2.12. Statistical analyses

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Data were statistically analyzed using two-way analysis of variance (ANOVA), followed by the Tukey’s test. P-values < 0.05 were considered significant. All analyses were performed using the Statistical Package for Social Sciences (SPSS) software version 15.0.

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3. Results

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3.1. Effects of resveratrol and glutamate on cellular viability

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The integrity and metabolic activities were evaluated by measuring LDH extracellular content, lactate release and MTT reduction. The treatment of hippocampal slices with 1 mM glutamate for 30 min did not cause loss of cellular integrity, as demonstrated by LDH extracellular content (Fig. 1A). However, glutamate exposure induced a decrease in lactate release (23%, P < 0.05) and MTT reduction (17%, P < 0.05) (Fig. 1B and C, respectively). Resveratrol was able to restore both parameters to their control values, with no effect per se.

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3.2. Resveratrol inhibits ROS accumulation and lipid peroxidation induced by glutamate

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Resveratrol alone decreased ROS production by approximately 14% (P < 0.05) compared to the control conditions (Table 1). Resveratrol was able to partially prevent the increased intracellular ROS accumulation that is typically induced by glutamate and also lowered DCFH levels from 127 ± 9% to 103 ± 9% (P < 0.01), indicating an antioxidant role (Table 1). Classically, glutamate induces oxidative stress and oxidative cell death through mechanisms that are dependent upon ROS production and/or excessive activation of glutamate receptors. Thus, the probable mechanisms by which resveratrol inhibited intracellular ROS production were tested. First, the hippocampal slices were treated with the following molecules: (i) MK801, (ii) CNQX, (iii) BAPTA-AM, (iv) L-NAME and (v) ZnPP IX. With the exception of ZnPP IX, an HO1 inhibitor, the molecules attenuated the glutamate-induced oxidative stress (Table 1). Moreover, the co-incubation of resveratrol with MK801, CNQX, BAPTAAM and L-NAME potentiated the attenuation of ROS accumulation induced by glutamate, indicating the involvement of NMDA and AMPA/KA activation and intracellular Ca2+ signaling and NO pathway in the neuroprotective effect of resveratrol (Table 1). Notably, the control, including inhibitors per se, is indicated as a percentage. Additionally, resveratrol seems to require HO1 for antioxidant activity. Furthermore, the expression of HO1 was investigated in these treatments, but changes in immunocontent were not observed (data not shown). Glutamate exposure also induced increased MDA levels (20%) in hippocampal slices (Fig. 2). Resveratrol completely prevented this effect (from 120 ± 12% to 101 ± 9%, P < 0.05; Fig. 2). Resveratrol alone also decreased lipid peroxidation (12%).

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Fig. 2. Effect of resveratrol on lipid peroxidation. Hippocampal slices were pretreated in either the absence or presence of 100 lM resveratrol (RSV) for 30 min at 30 °C, followed by the addition of 1 mM glutamate (GLU) for 30 min/30 °C. TBARS levels were measured as described in Section 2. Data are expressed as percentages of the control condition and represent the mean ± S.E.M of 4 independent experimental determinations performed in triplicate and analyzed statistically by two-way ANOVA, followed by the Tukey’s test. (a) Indicates significant differences from the control (P < 0.05) and (b) indicates significant differences from glutamate (P < 0.05).

Fig. 1. Effects of resveratrol on cellular viability. Hippocampal slices were pretreated in either the absence or presence of 100 lM resveratrol (RSV), for 30 min at 30 °C, followed by the addition of 1 mM glutamate (GLU) for 30 min/30 °C. LDH extracellular (A), lactate release (B) and MTT reduction (C) were measured as described in Section 2. Data are expressed as percentages of the control condition and represent the mean ± S.E.M of 4 independent experimental determinations performed in triplicate and analyzed statistically by two-way ANOVA, followed by the Tukey’s test. (a) Indicates significant differences from the control values (P < 0.05) and (b) indicates significant differences from glutamate (P < 0.05).

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3.3. Effect of resveratrol on DWm

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Mitochondria have emerged as a key regulator of oxidative stress and cytotoxicity. As expected, glutamate reduced DWm by approximately 35%, inducing mitochondrial dysfunction, and resveratrol completely prevented this effect (Fig. 3). Resveratrol per se did not affect DWm.

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3.4. Resveratrol protected against impaired Na+K+-ATPase activity typically induced by glutamate

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As shown in Fig. 4A, glutamate exposure significantly reduced Na+K+-ATPase activity in the hippocampal slices compared to the controls from 840 ± 64 to 613 ± 39 nmol Pi/min/mg of protein. Pre-treatment with resveratrol restored Na+K+-ATPase activity to values near the control levels. Resveratrol alone had no effect on Na+K+-ATPase. The a subunit is the catalytic subunit of Na+K+-ATPase in the CNS (Lingrel, 1992). Here, it is demonstrated that treatments of resveratrol and glutamate did not change the expression of the a-1 or a-3 subunits of Na+K+-ATPase protein (Fig. 4B).

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3.5. Resveratrol increased GS activity

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Once taken up by astrocytes, glutamate is converted into glutamine by GS (EC 6.3.1.2). Glutamate exposure decreased GS activity by about 25% compared to control levels. Resveratrol completely prevented this effect (from 0.75 ± 0.05 to 0.95 ± 0.8 lmol/mg protein/h, P < 0.05; Fig. 5). Resveratrol alone had no effect on GS activity.

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4. Discussion

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Two broad mechanisms (excessive activation of glutamate receptors and oxidative stress) converge and provide a final common pathway for cell vulnerability (Coyle and Puttfarcken, 1993). Furthermore, several pathologies, such as epilepsy, ischemia and Parkinson’s and Alzheimer’s diseases impair glutamate metabolism and antioxidant defenses through consequent cellular damage

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Table 1 Effects of resveratrol on ROS production.

Control RSV GLU RSV + GLU

NONE

MK801

CNQX

BAPTA-AM

L-NAME

ZnPP IX

100 ± 8 86 ± 7 a,b 127 ± 9 a 103 ± 9 b

100 ± 8 85 ± 7 a,b 107 ± 8 b 84 ± 7 a,b

100 ± 8 88 ± 9 a,b 104 ± 8 b 87 ± 7 a,b

100 ± 8 85 ± 9 a,b 106 ± 9 b 85 ± 7 a,b

100 ± 8 78 ± 8 a,b 109 ± 8 b 79 ± 8 a,b

100 ± 8 101 ± 7 120 ± 8a 117 ± 8a

Hippocampal slices were pre-treated for 30 min in either the absence or presence of 100 lM resveratrol (RSV) and/or 10 lM MK801, 10 lM CNQX, 10 lM BAPTA-AM, 500 lM L-NAME, 10 lM ZnPP IX for 30 min at 30 °C, followed by the addition of 1 mM glutamate (GLU) for 30 min/30 °C. Intracellular ROS production was measured as described in Section 2. Data are expressed as percentages of the control condition and represent the mean ± S.E.M of 4 independent experimental determinations performed in triplicate and analyzed statistically by two-way ANOVA, followed by the Tukey’s test. a Indicates significant differences from the control values (P < 0.05). b Indicates significant differences from glutamate (P < 0.05).

Please cite this article in press as: Quincozes-Santos, A., et al. Oxidative stress mediated by NMDA, AMPA/KA channels in acute hippocampal slices: Neuroprotective effect of resveratrol. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2013.12.021

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Fig. 3. Effect of resveratrol on DWm. Hippocampal slices were pre-treated in either the absence or presence of 100 lM resveratrol (RSV) for 30 min at 30 °C, followed by the addition of 1 mM glutamate (GLU) for 30 min/30 °C. DWm was measured as described in Section 2. Data are expressed as percentages of the control condition and represent the mean ± S.E.M of 4 independent experimental determinations performed in triplicate and analyzed statistically by two-way ANOVA, followed by the Tukey’s test. (a) Indicates significant differences from the control (P < 0.05) and (b) indicates significant differences from glutamate (P < 0.05).

Fig. 4. Effect of resveratrol on Na+K+-ATPase. Hippocampal slices were pre-treated in either the absence or presence of 100 lM resveratrol (RSV) for 30 min at 30 °C, followed by the addition of 1 mM glutamate (GLU) for 30 min/30 °C. Na+K+-ATPase activity (A) and Na+K+-ATPase expression (B) were measured as described in Section 2. Data for the activity represent the mean ± S.E.M of 4 independent experimental determinations performed in triplicate, and for immunocontent, they are expressed as percentages of the control (2 experimental determinations performed in duplicate). Data were analyzed statistically by two-way ANOVA, followed by the Tukey’s test. (a) Indicates significant differences from the control (P < 0.05) and (b) indicates significant differences from glutamate (P < 0.05).

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(Coyle and Puttfarcken, 1993; Fukui et al., 2010; Halliwell, 2006; Lalo et al., 2006). Glutamate-induced excitotoxicity via NMDA and AMPA/KA receptors is closely correlated with epilepsy. Moreover, the removal of pathological ROS during the course of a brain disorder seems to be a viable approach to neuroprotection (Waldbaum and Patel, 2010). In this sense, resveratrol, a well-established antioxidant, may be used as a neuroprotective molecule. Here, it is demonstrated that resveratrol decreases intracellular ROS production and lipid peroxidation and also prevents mitochondrial dysfunction and impairments in Na+K+-ATPase and GS in hippocampal slices after glutamate activation as well as its the putative neuroprotective mechanism.

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4.1. Oxidative stress and metabolic changes

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High glutamate concentrations induce oxidative-nitrosative stress, which can lead to lipid, protein and DNA oxidation, causing cellular damage (Coyle and Puttfarcken, 1993; Moldzio et al.,

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Fig. 5. Effect of resveratrol on GS activity. Hippocampal slices were pre-treated in either the absence or presence of 100 lM resveratrol (RSV) for 30 min at 30 °C, followed by the addition of 1 mM glutamate (GLU) for 30 min/30 °C. GS activity was measured as described in Section 2. Data represent the mean ± S.E.M of 4 independent experimental determinations performed in triplicate and analyzed statistically by two-way ANOVA followed by the Tukey’s test. (a) Indicates significant differences from the control (P < 0.05) and (b) indicates significant differences from glutamate (P < 0.05).

2013). It is important to emphasize that in this in vitro model of glutamate injury, only metabolic alterations were induced; significant cell death was not observed, but metabolic alterations could lead to future cell death. Here, under glutamate exposure, metabolic mitochondrial status (as measured by MTT) and extracellular lactate were decreased. This data could indicate the lack of an energy substrate for the CNS, because lactate can be released from astrocytes in extracellular space through monocarboxylate transporters (MCT) and be used as an energy substrate for neurons in oxidative-derived ATP production (Belanger et al., 2011; Pellerin et al., 2002). Resveratrol was able to restore lactate levels and thus can modulate cellular metabolism and energy in the brain. Many studies have shown that resveratrol possesses direct radical scavenging activity (Bobermin et al., 2012; de Almeida et al., 2007; Huang et al., 2011; Quincozes-Santos et al., 2009). Accordingly, resveratrol could protect hippocampal slices from glutamate-induced excitotoxicity, which results in increased ROS accumulation. Our group has shown that resveratrol is able to decrease intracellular ROS production in glial cells under different oxidative stimuli (Bobermin et al., 2012; Quincozes-Santos et al., 2013, 2009; Quincozes-Santos and Gottfried, 2011). Moreover, resveratrol per se also decreases intracellular ROS production and lipid peroxidation. Nardin et al. demonstrated that during the standardization of hippocampal slices for ex vivo assays, metabolic ‘‘recovery’’ is associated with cellular alterations, commonly varying from 15 to 120 min (depending on the experimental assay). Thus, in this length of time, an increase in ROS production/lipid peroxidation could have been prevented by resveratrol. HO1 is a protein by which resveratrol can induce neuroprotective effects (Bastianetto and Quirion, 2010; Quincozes-Santos et al., 2013; Sakata et al., 2010). Furthermore, a relationship exists between HO1 and NOS. The nuclear factor-erythroid-2-related factor 2 (Nrf2) regulates HO1 transcription, which acts a scavenger of NO and an inhibitor of iNOS (Wakabayashi et al., 2010). Here, resveratrol presented more effective results than L-NAME, an iNOS inhibitor, in its protection against ROS formation induced by glutamate. Furthermore, the current group recently reported that resveratrol modulates HO1 and iNOS expression in glial cells (Quincozes-Santos et al., 2013). Therefore, HO1 can also be critical to signaling the antioxidant response of resveratrol (Sakata et al., 2010). Thus, in accordance with Mazzuferi, who reported that activation of the Nrf2/HO1 system may be therapeutic for epilepsy, resveratrol has emerged as a new potential treatment for this condition (Mazzuferi et al., 2013).

Please cite this article in press as: Quincozes-Santos, A., et al. Oxidative stress mediated by NMDA, AMPA/KA channels in acute hippocampal slices: Neuroprotective effect of resveratrol. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2013.12.021

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The overstimulation of the neurotransmitter glutamate, together with other factors, can promote oxidative stress in neural cells, which is commonly associated with neurological disorders (Lau and Tymianski, 2010). Glutamate receptors activation increases the influx of Ca2+, activating proteases and stimulating NOS, which then catalyzes the synthesis of NO from L-arginine (Calabrese et al., 2007; Coyle and Puttfarcken, 1993). NO can become noxious when interacting with superoxide anions, leading to overproduction of the powerful oxidant species peroxynitrite (Bolanos and Almeida, 2006; Moncada and Bolanos, 2006). Here, we demonstrated that resveratrol potentiates the decrease in ROS production caused by NMDA and AMPA/KA inhibitors and intracellular Ca2+ chelator. Thus, these results indicate that resveratrol may partially block the overproduction of ROS in different steps of the excitotoxicity cascade induced by glutamate exposure associated with Ca2+ influx. The indirect effects of resveratrol on NMDA and AMPA receptors may indicate inhibition of glutamatemediated excitation in in vitro preparations and animal models. Moreover, resveratrol treatment significantly reduced the increased expression of KA receptor in the hippocampus of epileptic rats induced by kainic acid (Wu et al., 2009). Cellular Ca2+ signals are crucial for the control of most physiological processes and cell injuries (Parpura et al., 2012; Rosenstock et al., 2004; Smaili et al., 2003). Mitochondria, together with the endoplasmic reticulum, play pivotal roles in regulating intracellular Ca2+ content. During cellular Ca2+ overload, mitochondria take up cytosolic Ca2+, which in turn induces the opening of permeability transition pores and disrupts DWm (Lin et al., 2007; Parpura et al., 2012; Rosenstock et al., 2004). After glutamate exposure, resveratrol potentiates the effect of BAPTA-AM, decreasing intracellular ROS levels through excessive Ca2+ activation.

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Mitochondrial oxidative stress and dysfunction are contributing factors of various neuropathological conditions (Waldbaum and Patel, 2010). Here, it was demonstrated that resveratrol prevented DWm induced by glutamate exposure. Excessive ROS production induces mitochondrial dysfunction and contributes to neuronal damage (Waldbaum and Patel, 2010). Glutamate exposure and/or NMDA activation induces impaired mitochondrial function, increased intracellular Ca2+ overload, peroxynitrite generation and neuronal death, which are characteristic of neurological disorders (Bolanos and Heales, 2010; Coyle and Puttfarcken, 1993). Additionally, excessive mitochondrial ROS production can damage DNA, and resveratrol also presented genoprotective effects for glial cells (Quincozes-Santos et al., 2010, 2007). Recent evidence indicates that mitochondrial dysfunction is not merely a consequence of brain damage, but an active contributor to seizures, epileptogenesis and neurodegeneration (Kunz et al., 2000; Waldbaum and Patel, 2010). Na+K+-ATPase is an enzyme in the cytoplasmatic membrane that is responsible for the active transport of Na+ and K+ ions in the CNS, maintaining the ionic gradient necessary for neuronal excitability and regulation of neuronal cell volume, it consumes most of the ATP generated in the brain (Dobrota et al., 1999; Lingrel, 1992). It has been demonstrated that this enzyme is highly susceptible to free radical attack, and some reports have shown that Na+K+-ATPase activity is decreased in cases of epilepsy and Alzheimer’s disease (Silva et al., 2011; Zhang et al., 2013). Excessive ROS production has been reported to inhibit Na+K+-ATPase activity (Grisar et al., 1992). Resveratrol was able to prevent the decrease in Na+K+-ATPase activity in hippocampal slices, as were other antioxidants to prevent cellular damage in different models of study (Ferreira et al., 2011; Stefanello et al., 2011).

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GS is highly sensitive to oxidative and nitrosative stress and it has been hypothesized to play an important role in the pathogenesis of excitotoxicity. This enzyme is also dependent on ATP and metal ions for catalysis (Banerjee et al., 2008; Felipo and Butterworth, 2002; Hertz, 2006; Hertz and Zielke, 2004). Our group has previously demonstrated that resveratrol modulates GS activity and that NO metabolism seems to be related to GS activity failure; thus, resveratrol can act as a scavenger and may improve GS activity (Bobermin et al., 2012). Reduction of GS activity and/or expression was reported in brain models of epilepsy, ischemia and neurodegenerative diseases (Coulter and Eid, 2012; Mates et al., 2002; Walton and Dodd, 2007). Furthermore, the decrease in GS activity is coupled with the decreased capacity of glutamate metabolism and, consequently, leads to neural damage (Waldbaum and Patel, 2010).

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Resveratrol, a promising neuroprotective molecule, is able to protect against neurotoxicity and neuronal death (Fukui et al., 2010). In this study, we focused on mitochondrial bioenergetics and oxidative stress. Other antioxidants, such as vitamin C, vitamin E and N-acetyl-L-cysteine (NAC), have been suggested to exert neuroprotective effects, but the effects have been attributed to reduced lipid peroxidation and/or oxidative damage. Bobermin et al. showed that resveratrol acted more efficiently than vitamin C, L-NAME and trolox in protecting against ammonia toxicity. Here, it was demonstrated that resveratrol protected hippocampal slices against glutamate toxicity by modulating DWm, Na+K+-ATPase and GS activity. Moreover, resveratrol may activate pathways, such as HO1, that modulates excitotoxicity, mitochondrial dysfunction, oxidative stress and neuroinflammation. These results are similar to obtained in C6 glial cell suggesting that glial cells in our brain slice preparations are resveratrol targets, but further studies using neuron and astrocyte cultures will be necessary to evaluate if astrocytes are mediating the resveratrol protection against oxidative stress, particularly investigating localization and activity of HO1. Although its mechanism of action is not completely clear, this compound has a strong antioxidant/scavenger activity that prevents NMDA, AMPA/KA and intracellular Ca2+ activation and thus protects neurons against glutamate-induced neuronal damage. This study’s data suggest that resveratrol is potentially useful in neuropsychiatric disorders treatment.

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Conflict of interest

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The authors have declared that no conflict of interest to disclose.

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Acknowledgements

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This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Financiadora de Estudos e Projetos (FINEP) – IBN Net (Instituto Brasileiro de Neurociências) 01.06.0842-00, Federal University of Rio Grande do Sul (UFRGS) and Instituto Nacional de Ciência e Tecnologia para Excitotoxicidade e Neuroproteção (INCTEN/CNPq).

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tiv.2013.12.021.

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Please cite this article in press as: Quincozes-Santos, A., et al. Oxidative stress mediated by NMDA, AMPA/KA channels in acute hippocampal slices: Neuroprotective effect of resveratrol. Toxicol. in Vitro (2014), http://dx.doi.org/10.1016/j.tiv.2013.12.021