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Lithium and valproate modulate antioxidant enzymes and prevent ouabain-induced oxidative damage in an animal model of mania
Journal of Psychiatric Research 45 (2011) 162e168
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Lithium and valproate modulate antioxidant enzymes and prevent ouabain-induced oxidative damage in an animal model of mania Luciano K. Jornada a, Samira S. Valvassori a, Amanda V. Steckert a, b, Morgana Moretti a, Francielle Mina b, Camila L. Ferreira a, Camila O. Arent a, Felipe Dal-Pizzol b, João Quevedo a, * a
Laboratory of Neurosciences and National Institute for Translational Medicine (INCT-TM), Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806-000 Criciúma, SC, Brazil Laboratory of Experimental Patophysiology and National Institute for Translational Medicine (INCT-TM), Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806-000 Criciúma, SC, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 February 2010 Received in revised form 4 May 2010 Accepted 7 May 2010
In this study, we assessed the oxidative stress parameters in rats submitted to an animal model of mania induced by ouabain (OUA), which included the use of lithium (Li) and valproate (VPA). Li and VPA treatment reversed and prevented the OUA-induced damage in these structures, however, this effect varies depending on the brain region and treatment regimen. Moreover, the activity of the antioxidant enzymes, namely, superoxide dismutase (SOD) and catalase (CAT) was found to be increased and decreased, respectively, in the brain of OUA-administered rats. Li and VPA modulated SOD and CAT activities in OUA-subjected rats in both experimental models. Our results support the notion that Li and VPA exert antioxidant-like properties in the brain of rats submitted to animal model of mania induced by ouabain. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Ouabain Mania Oxidative stress Submitochondrial particles Lithium Valproate
1. Introduction Bipolar disorder (BD) is a prevalent and chronic psychiatric disorder (Belmaker, 2004). It is considered to be one of the leading causes of disability among all medical and psychiatric conditions (Murray and Lopez, 1997), and untreated BD has been associated with increased morbidity and mortality due to general medical conditions such as vascular disorders and cancer (Angst et al., 2002). Oxidative stress mechanisms have been implicated in the pathogenesis of psychiatric disorder such as bipolar disorder (Halliwell, 2006). Aerobic organisms are susceptible to oxidative stress because semi-reduced oxygen species, superoxide and hydrogen peroxide are produced by mitochondria during respiration (Chance et al., 1979). The brain is particularly vulnerable to reactive oxygen species production because it metabolizes 20% of total body oxygen and has a limited amount of antioxidant capacity (Floyd, 1999), and in fact early studies demonstrating the ease of
* Corresponding author. Fax: þ 55 48 3443 4817. E-mail address: [email protected] (J. Quevedo). 0022-3956/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2010.05.011
brain membrane peroxidation supported this notion (Zaleska and Floyd, 1985). The primary antioxidant defense system has been studied in several psychiatric disorders and involves coordinated effects induced by superoxide dismutase (SOD) and catalase (CAT) (Reddy et al., 1991; Kapczinski et al., 2008). SOD is a protective enzyme that can selectively scavenge the superoxide anion radical (O 2 ) by catalyzing its dismutation to hydrogen peroxide (H2O2) and CAT metabolizes the excess of H2O2 producing O2 þ H2O, decreasing the intracellular redox status (Andreazza et al., 2008a). Moreover, the role of Naþ/Kþ-ATPase in the pathophysiology of bipolar disorder was reported by El-Mallakh et al. (2003). The pharmacological inhibition of Naþ/Kþ-ATPase by ouabain in rats causes hyperactivity and can be used as a model of mania (El-Mallakh et al., 2003; Li et al., 1997; Decker et al., 2000). Riegel et al. (2009) found that mania-like state induced by ouabain increased superoxide generation and lipid peroxidation in submitochondrial particles of the rat brain, suggesting that the decrease in the activity of Naþ/Kþ-ATPase seen in bipolar patients may be an important link in the pathological response to oxidative stress. Mood stabilizing drugs, particularly lithium and valproate, are considered as first-line agents for both acute mania and
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maintenance treatment (Yatham et al., 2005). Several studies have suggested that the neuroprotective effects of lithium and valproate may be responsible for their therapeutical effects (Chuang et al., 2002; Li et al., 1997). Recently a study of our laboratory demonstrated that Li and VPA reversed OUA-related hyperactive behavior in the open-field test, suggesting that this model fulfills adequate face, construct and predictive validity as an animal model of mania (Jornada et al., 2009). In this context, we sought to investigate the effects of lithium and valproate on oxidative stress parameters in the brain of rats using an animal model of mania induced by ouabain. 2. Experimental methods In the present study, we have extended the investigation of the effects of Li and VPA on ouabain-induced neurochemical alterations in an animal model of mania by measuring oxidative stress parameters in prefrontal and hippocampal samples that were kept frozen at 80 C from one of our previous experiments (Jornada et al., 2009). The detailed description of the experiments has been published elsewhere (Jornada et al., 2009); therefore, we summarize here the treatment regimens and describe the subsequent steps performed for the present investigation. 2.1. Animals The subjects were adult male Wistar rats (weighing 250e350 g) obtained from our breeding colony. Animals were housed five to a cage with food and water available ad libitum and were maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.) at a temperature of 22 1 C. All experimental procedures were performed in accordance with the approval of the local Ethics Committee. All experiments were performed at the same time during the day to avoid circadian variations. 2.2. Surgical procedure Animals were intraperitoneally anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg). In a stereotaxic apparatus, the skin of the rat skull was removed and a 27-gauge 9 mm guide cannula was placed at 0.9 mm posterior to bregma, 1.5 mm right from the midline and 1.0 mm above the lateral brain ventricle. Through a 2-mm hole made at the cranial bone, a cannula was implanted 2.6 mm ventral to the superior surface of the skull and fixed with dental acrylic cement. Animals recovered from surgery within 3 days. 2.3. Treatment Reversal model: We designed our first model to reproduce the management of an acute manic episode. Animals (n ¼ 72) received a single ICV injection of 5 ml of 103 M ouabain dissolved in artificial cerebrospinal fluid (ACSF) or 5 ml of ACSF alone on the fourth day following surgery (El-Mallakh et al., 2003; Riegel et al., 2009). (Note: Hamid et al. (2009) published reports in which the ouabain dose was reported as 105 M are in error; the dose used in those studies was 103 M.) A 30-gauge cannula was placed inside the guide cannula and connected by a polyethylene tube to a microsyringe. The tip of the cannula infusion protruded 1.0 mm beyond the cannula guide aiming the right lateral brain ventricle. From the day following the injection of ouabain or ACSF, the rats were treated for 6 days with intraperitoneal (IP) injections of saline, lithium or valproate in 6 experimental groups of 12 animals per group: ACSF ICV þ saline IP (ACSF þ Sal), ACSF ICV þ lithium IP (ACSF þ Li), ACSF ICV þ valproate IP (ACSF þ VPA), ouabain ICV þ saline IP
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(OUA þ Sal), ouabain ICV þ lithium IP (OUA þ Li), ouabain ICV þ valproate IP (OUA þ VPA). Animals in the Li group received intraperitoneal injections of lithium (47.5 mg/kg) and those in the VPA group received valproate (200 mg/kg) twice a day (Jornada et al., 2009). The animals were killed 24 h after the last injection of Li, VPA or ACSF. Maintenance model: We designed the second model to mimic the maintenance phase of the treatment of BD, when the drugs still are administered even in periods of euthymia (prevention model). After recovery from surgery, animals (n ¼ 72) received IP injections of Li (47.5 mg/kg), VPA (200 mg/kg) or SAL twice a day for 12 days. In the 7 days of treatment with Sal, Li or VPA the animals received a single ICV injection of either OUA (103 M) or ACSF, totaling 6 experimental groups of 12 animals per group: Sal þ ACSF, Li þ ACSF, VPA þ ACSF, Sal þ OUA, Li þ OUA, VPA þ OUA. After the OUA or ACSF injection, the treatment with mood stabilizer was continued for 6 more days (Jornada et al., 2009). The animals were killed 24 h after the last injection of Li, VPA or ACSF. 2.4. Biochemical analysis Rats were sacrificed by decapitation and the brain transferred within 1 min to ice-cold isolation buffer (0.23 M mannitol, 0.07 M sucrose, 10 mM TriseHCl, and 1 mM EDTA, pH 7.4). The prefrontal cortex and hippocampus (n ¼ 5 animals per group) were dissected in ice-cold buffer in a Petri dish, and submitochondrial particles were prepared in parallel from the four brain regions of each animal. For biochemical analysis in total tissue, the brain structures were rapidly frozen and stored at 80 C. 2.5. Mitochondrial isolation Rat brain homogenates were centrifuged at 700 g for 10 min to discard nuclei and cell debris and the pellet was washed to enrich the supernatant that was centrifuged at 7000 g for 10 min. The obtained pellet, washed and resuspended in the same buffer, was considered to consist mainly of intact mitochondria able to carry out oxidative phosphorylation. The operations were carried out at 0e2 C. Submitochondrial particles (SMP) were obtained by freezing and thawing (three times) of isolated mitochondria. For superoxide production measurements, SMP were washed twice with 140 mM KCl, 20 mM TriseHCl (pH 7.4) and suspended in the same medium (Boveris et al., 1972). 2.6. Superoxide production in submitochondrial particles of the rat brain Superoxide production was determined in washed brain SMP using a spectrophotometric assay based on superoxide-dependent oxidation of epinephrine to adrenochrome at 37 C (l 480 nm ¼ 4.0 mM1 cm1). The reaction medium consisted of 0.23 M mannitol, 0.07 M sucrose, 20 mM TriseHCl (pH 7.4), SMP (0.3e1.0 mg protein/ml), 0.1 mM catalase, and 1 mM epinephrine. NADH (50 mM) and succinate (7 mM) were used as substrates and rotenone (1 mM) and antimycin (1 mM) were added as specific inhibitors, respectively, to assay O 2 production at the NADH dehydrogenase and at the ubiquinoneecytochrome b region. Superoxide dismutase (SOD) was used at 0.1e0.3 mM final concentration to give assay specificity (Boveris, 1984). 2.7. Thiobarbituric acid reactive species (TBARS) content in tissue and in submitochondrial particles of the rat brain As a marker of lipid peroxidation, we measured the formation of thiobarbituric acid reactive species (TBARS) during an acid-heating
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reaction, as previously described (Esterbauer and Cheeseman, 1990). Briefly, the samples were mixed with 1 ml of trichloroacetic acid 10% and 1 ml of thiobarbituric acid 0.67%, and then heated in a boiling water bath for 15 min. TBARS were determined by the absorbance at 535 nm. 2.8. Protein carbonyl content Oxidative damage to proteins was measured by the quantification of carbonyl groups based on the reaction with dinitrophenylhydrazine (DNPH), as previously described (Levine et al., 1994). Proteins were precipitated by the addition of 20% trichloroacetic acid and were redissolved in DNPH; the absorbance was read at 370 nm. 2.9. Superoxide dismutase activity This method for the assay of SOD activity is based on the capacity of pyrogallol to autoxidize, a process highly dependent on O2 2 ; a substrate for SOD (Bannister and Calabrese, 1987). The inhibition of autoxidation of this compound thus occurs when SOD is present, and the enzymatic activity can be then indirectly assayed spectrophotometrically at 420 nm, using a double-beam spectrophotometer with temperature control. A calibration curve was generated using purified SOD as the standard, in order to calculate the specific activity of SOD present in the samples. A 50% inhibition of pyrogallol autoxidation is defined as 1 unit of SOD, and the specific activity is represented as units per milligram of protein. 2.10. Catalase activity CAT activity was assayed using a double-beam spectrophotometer with temperature control. This method is based on the disappearance of H2O2 at 240 nm in a reaction medium containing 20 mM H2O2, 0.1% Triton X-100, 10 mM potassium phosphate buffer, pH 7.0, and 0.1e0.3 mg protein/ml (Aebi, 1984). One CAT unit is defined as 1 mol of hydrogen peroxide consumed per minute, and the specific activity is reported as units per milligram of protein. 2.11. Protein determination All biochemical measures were normalized to the protein content with bovine albumin as standard (Lowry et al., 1951). 2.12. Statistical analysis Biochemical data are presented as mean and standard error of the mean. Differences among the experimental groups were determined by one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test. In all comparisons, statistical significance was set at p < 0.05. 3. Results
Fig. 1. Effects of ouabain or ACSF ICV administration on superoxide levels in submitochondrial particles in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 49.213, p < 0.001; hippocampus: df ¼ 5, F ¼ 31.973, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 8.760, p < 0.001; hippocampus: df ¼ 5, F ¼ 11.029, p < 0.001). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
Fig. 1B shows that in the maintenance model a significant increase in superoxide was also detected in the Sal þ OUA group in the prefrontal and hippocampus of rats (p < 0.05), and Li and VPA prevented these values significantly in both structures (p < 0.05). 3.2. TBARS content in submitochondrial particles of the rat brain The comparison of TBARS content in submitochondrial particles among the 6 groups in the reversal model is shown in Fig. 2A. The OUA þ Sal group showed a significant increase in the amount of TBARS in submitochondrial particles in the prefrontal and hippocampus when compared to the control group (p < 0.05), and Li, but not VPA, reversed this values in both structures tested. As shown in Fig. 2B, in the maintenance model, the Sal þ OUA group showed an elevated amount of TBARS content in submitochondrial particles in prefrontal and hippocampus when compared to that of the control group (p < 0.05), and Li and VPA reversed this values in both structures tested (p < 0.05).
3.1. Superoxide production in submitochondrial particles of the rat brain
3.3. TBARS content in tissue of the rat brain
As shown in Fig. 1A, in the reversal model there was a significant increase in superoxide production in the OUA þ Sal group in the prefrontal and hippocampus when compared to the control group (p < 0.05), and Li, but not VPA, reversed this values significantly in the hippocampus of rats (p < 0.05). However, in the prefrontal neither Li nor VPA was able to reverse the OUA-induced increase of superoxide production.
Fig. 3A illustrates TBARS content of the rat brain in the reversal model. Administration of ouabain ICV (OUA þ Sal group) resulted in a marked increased amount of TBARS content in prefrontal and hippocampus (p < 0.05), and Li and VPA were able to reverse this value in the hippocampus but not in the prefrontal of rats. Likewise, administration of ouabain (Sal þ OUA group) in maintenance protocol (Fig. 3B) also resulted in a marked increased
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amount of TBARS content in prefrontal and hippocampus (p < 0.05), and once more Li and VPA were able to reverse the ouabain-induced increase of TBARS content in both structures tested. 3.4. Carbonyls protein content As shown in Fig. 4A, in the reversal model, there was a significant increase in formation of carbonyl protein in the prefrontal and hippocampus in OUA þ Sal group when compared to the control group (p < 0.05), and Li, but not VPA, reversed these values significantly in prefrontal and hippocampus of rats (p < 0.05). Fig. 4B shows protein carbonylation in the brain of rats in maintenance model. The Sal þ OUA group showed a significant increase in carbonyls protein content in hippocampus and prefrontal of rats when compared to the control group (p < 0.05), and Li and VPA were able to reverse the ouabain-induced increase of carbonyls protein content in both structures tested. 3.5. Superoxide dismutase activity
Fig. 2. Effects of ouabain or ACSF ICV administration on TBARS levels in submitochondrial particles in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 31.954, p < 0.001; hippocampus: df ¼ 5, F ¼ 29.258, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 44.923, p < 0.001; hippocampus: df ¼ 5, F ¼ 4.655, p ¼ 0.007). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
Fig. 3. Effects of ouabain or ACSF ICV administration on TBARS levels in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 40.471, p < 0.001; hippocampus: df ¼ 5, F ¼ 28.992, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 93.414, p < 0.001; hippocampus: df ¼ 5, F ¼ 118.134, p < 0.001). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
As shown in Fig. 5A, in the reversal model SOD activity was higher in the prefrontal and hippocampus homogenates from the OUA þ Sal group than in those from the control group (p < 0.05), however in the prefrontal, but not in the hippocampus, Li and VPA treatment reduced these values significantly (p < 0.05). Once more, the maintenance model (Fig. 5B) also resulted in a marked increase of SOD activity in the prefrontal and hippocampus of rats in Sal þ OUA group (p < 0.05), and Li and VPA treatment partially reduced these values in prefrontal, but not in the hippocampus (p < 0.05).
Fig. 4. Effects of ouabain or ACSF ICV administration on protein carbonyl formation in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 23.481, p < 0.001; hippocampus: df ¼ 5, F ¼ 24.541, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 49.265, p < 0.001; hippocampus: df ¼ 5, F ¼ 19.833, p < 0.001). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
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Fig. 5. Effects of ouabain or ACSF ICV administration on superoxide dismutase activity in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 65.241, p < 0.001; hippocampus: df ¼ 5, F ¼ 22.932, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 139.135, p < 0.001; hippocampus: df ¼ 5, F ¼ 70.726, p < 0.001). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
Fig. 6. Effects of ouabain or ACSF ICV administration on catalase activity in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 72.457, p < 0.001; hippocampus: df ¼ 5, F ¼ 117.860, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 40.607, p < 0.001; hippocampus: df ¼ 5, F ¼ 55.919, p < 0.001). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
3.6. Catalase activity The results of the CAT activity in the reversal model are shown in Fig. 6A. The CAT activity was significantly decreased in OUA þ Sal group (p < 0.05), and Li, but not VPA, reversed these values significantly in both structures (p < 0.05). Likewise, administration of ouabain (Sal þ OUA group) in maintenance protocol (Fig. 6B) also resulted in a marked decrease of the CAT activity in prefrontal and hippocampus (p < 0.05), and Li and VPA were able to reverse the OUA-induced decrease of the CAT activity in both structures tested. It is noteworthy that Li and VPA regimen in the control groups does not alter any of the parameters measured in any of the experimental protocols. 4. Discussion Soon after the characterization of the Naþ/Kþ-ATPase, some authors investigated its activity in mood disorders. These studies showed that Naþ pump activity is decreased in acute mania compared to recovered euthymic bipolar individuals (Reddy et al., 1992; Hesketh et al., 1977; Naylor et al., 1980). In preclinical studies, when the potent sodium pump inhibitor, ouabain, is ICV administered in rats, it induces a motor hyperactivity (El-Mallakh and Wyatt, 1995; Decker et al., 2000), which may persist for over a week after a single injection (Ruktanonchai et al., 1998). Recently, a study of our laboratory demonstrated that Li and VPA reversed OUA-related hyperactive behavior in the open-field test, suggesting that this model fulfills adequate face, construct and predictive validity as an animal model of mania (Jornada et al., 2009). Additionally, another study from our laboratory found that mania-like state induced by ouabain increased superoxide generation and lipid peroxidation in submitochondrial particles of the rat brain (Riegel et al., 2009).
In this study we demonstrated that in both experimental protocols, ouabain induces oxidative damage by increasing the amount of TBARS, protein carbonyl, and superoxide in submitochondrial particles in prefrontal and hippocampus of rats. Interestingly, the OUA-induced oxidative damage was accompanied by increased superoxide dismutase activity and decreased CAT activity. SOD is an enzyme capable of reducing the superoxide radical into hydrogen peroxide (H2O2), which acts as the substrate to CAT. When cell has increased levels of SOD without a proportional increase in peroxidases, the excess of H2O2 produced could be responsible for the oxidative damage in the cell. In addition, H2O2 can react with transitional metals and generate the radical hydroxyl, which is the most harmful radical (Halliwell and Gutteridge, 1999). Consequently, the overexpression of SOD without a compensatory increase in CAT has deleterious effects upon the cell. Interestingly, here despite Li and VPA have partially reduced the activity of SOD in rats treated with OUA, both Li and VPA decreased the amount of ouabain-induced superoxide in maintenance model and in hippocampus in the reversal model. This may be due to the fact that in the presence of Fe3þ and H2O2 in excess, hydroperoxyl radical (HO$2) and superoxide anion (O2) are also formed. Thus, Li and VPA may be decreasing SOD and H2O2, thereby decreasing O2 (Hammel et al., 2002; Liu et al., 2005). In addition, pretreatment with Li and VPA led to an increase in CAT activity in brain of OUAinjected animals, which can be preventing other reactions with H2O2. However, in this work we evaluated superoxide production in submitochondrial particles of the rat brain, which is an indirect measure of mitochondrial dysfunction. Therefore, this measure is not directly related with SOD activity. Perhaps, the main finding of this paper was that Li and VPA increased the CAT activity in prefrontal and hippocampus of rats, preventing the oxidative damage induced by OUA in maintenance
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protocol. However, in the reversal protocol, Li but not VPA increased CAT activity in both structures tested, reversing the OUA-induced increase in the amount of protein carbonyls, superoxide, TBARS in tissue and in submitochondrial particles in hippocampus. This discrepancy suggests that, when co-administered with ouabain, Li may have its antioxidant effect in an earlier time course than VPA. However, in the prefrontal cortex, Li was unable to reverse the increase of superoxide and TBARS in submitochondrial particles. This may occur due to longer treatment with Li when compared to the reversal model. In addition, Bhalla and Dhawan (2009) have demonstrated that lipid peroxidation and increase in reactive oxygen species induced by aluminum decreased following Li supplementation; besides, Li normalized activity of antioxidant enzymes that were altered by aluminum in brain of rats. In previous study with a dopaminergic animal model of mania, our laboratory found that AMPH increased rat peripheral and hippocampal DNA damage. The index of DNA damage was correlated positively with lipid peroxidation, whereas Li and VPA were able to modulate the oxidative balance and prevent recent damage to the DNA (Andreazza et al., 2008b). Machado-Vieira et al. (2007) have showed that TBARS and antioxidant enzymes’ activity (SOD and CAT)increased in unmedicated manic patients compared to controls, and acute treatment with Li showed a significant reduction in both SOD/CAT ratio and TBARS levels. Stork and Renshaw (2005) proposed a hypothesis of mitochondrial dysfunction in bipolar disorder that involves impaired oxidative phosphorylation, a resultant shift toward glycolytic energy production, a decrease in total energy production and/or substrate availability, and altered phospholipid metabolism. Impaired mitochondrial function, and as a consequence impaired cellular energy state, is an attractive hypothesis for explaining the pathophysiology of BD. The literature suggests that the decrease of ATP production leads to a dysfunction in Na pump activity which consequently may result in neuronal hyperexcitability, giving rise to manic symptoms, such as hyperactivity (El-Mallakh and Wyatt, 1995). Our results support the notion that Li and VPA exert antioxidantlike properties in the brain of rats submitted to animal model of mania induced by ouabain. Thus, the altered energy metabolism dysfunctions associated with BD may play a role in oxidative stress observed during manic episodes. Also, Li and VPA seem to exert antioxidant effects during mania. Conflict of interest None of the authors or funding sources has conflict of interest. Contributors Luciano K. Jornada designed the study and wrote the protocol. Samira S. Valvassori undertook the statistical analysis and made the first draft of the manuscript. Amanda V. Steckert and Francielle Mina made the biochemical analysis. Morgana Moretti, Camila O. Arent and Camila L. Ferreira were responsible for the surgery and pharmacological treatment. All authors contributed to and have approved the final manuscript. Role of funding source This research was supported by grants from CNPq (Felipe DalPizzol and João Quevedo), FAPESC (João Quevedo), and UNESC (Felipe Dal-Pizzol and João Quevedo). Felipe Dal-Pizzol and João Quevedo are CNPq Research Fellows. Morgana Moretti and Samira S. Valvassori are holders of CAPES studentships, Camila O. Arent, Amanda V. Steckert and Camila L. Ferreira are holders of CNPq Studentships.
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The National Council for Scientific and Technological Development (CNPq) is an agency linked to the Ministry of Science and Technology (MCT), dedicated to the promotion of scientific and technological research and to the formation of human resources for research in the country. Its history is directly linked to the scientific and technological development of Brazil. The FAPESC aims to support and promote the scientific and technological research for the advancement of all areas of knowledge for regional balance, sustainable development and improving the quality of life of people of the Santa Catarina state with respect for ethical values and based on the principles established by arts. Coordination of Improvement of Higher Education Personnel (CAPES) plays a role in the expansion and consolidation of postgraduate (masters and doctorate) in all states of Brazil. Acknowledgements We thank CNPq, FAPESC, CAPES, Instituto Cérebro e Mente and UNESC for financial support. References Aebi H. Catalase in vitro. Methods in Enzymology 1984;105:121e6. Andreazza AC, Kauer-Sant’anna M, Frey BN, Bond DJ, Kapczinski F, Young LT, et al. Oxidative stress markers in bipolar disorder: a meta-analysis. Journal of Affective Disorders 2008a;111:135e44. Andreazza AC, Kauer-Sant’Anna M, Frey BN, Stertz L, Zanotto C, Ribeiro L, et al. Effects of mood stabilizers on DNA damage in an animal model of mania. Journal of Psychiatry and Neuroscience 2008b;33:516e24. Angst F, Stassen H, Clayton PJ, Angst J. Mortality of patients with mood disorders: follow-up over 34e38 years. Journal of Affective Disorders 2002;68:167e81. Bannister JV, Calabrese L. Assays for superoxide dismutase. Methods of Biochemical Analysis 1987;32:279e312. Belmaker RH. Bipolar disorder. New England Journal of Medicine 2004;351:476e86. Bhalla P, Dhawan DK. Protective role of lithium in ameliorating the aluminiuminduced oxidative stress and histological changes in rat brain. Cellular and Molecular Neurobiology 2009;29:513e21. Boveris A. Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria. Methods in Enzymology 1984;105:429e35. Boveris A, Oshino N, Chance B. The cellular production of hydrogen peroxide. Biochemical Journal 1972;128:617e27. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiological Reviews 1979;59:527e605. Chuang DM, Chen RW, Chalecka-Franaszek E, Ren M, Hashimoto R, Senatorov V, et al. Neuroprotective effects of lithium in cultured cells and animal models of diseases. Bipolar Disorder 2002;4:129e36. Decker S, Grider G, Cobb M, Li XP, Huff MO, El-Mallakh RS, et al. Open field is more sensitive than automated activity monitor in documenting ouabain-induced hyperlocomotion in the development of an animal model for bipolar illness. Progress in Neuro-Psychopharmacology and Biological Psychiatry 2000;24:455e62. El-Mallakh RS, El-Masri MA, Uhf MO, Li XP, Decker S, Levy RS. Intracerebroventricular administration of ouabain as a model of mania in rats. Bipolar Disorder 2003;5:362e5. El-Mallakh RS, Wyatt RJ. The Na, K-ATPase hypothesis for bipolar illness. Biological Psychiatry 1995;37:235e44. Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods in Enzymology 1990;186:407e21. Floyd RA. Antioxidants, oxidative stress, and degenerative neurological disorders. Proceedings of the Society for Experimental Biology and Medicine 1999;222:236e45. Halliwell B. Oxidative stress and neurodegeneration: where are we now? Journal of Neurochemistry 2006;97:1634e58. Halliwell B, Gutteridge JM. Free Radical in Biology and Medicine. Oxford, UK: Oxford University Press; 1999. Hamid H, Gao Y, Lei Z, Hougland MT, El-Mallakh RS. Effect of ouabain on sodium pump alpha-isoform expression in an animal model of mania. Progress in Neuro-Psychopharmacology and Biological Psychiatry 2009;33:1103e6. Hammel KE, Kapich AN, Jensen Jr KA, Ryan ZC. Reactive oxygen species as agents of wood decay by fungi. Enzyme and Microbial Technology 2002;30:445e53. Hesketh JE, Glen AIM, Reading HW. Membrane ATPase activities in depressive illness. Journal of Neurochemistry 1977;28:1401e2. Jornada KL, Moretti M, Valvassori SS, Ferreira CL, Padilha PT, Arent CO, et al. Effects of mood stabilizers on hippocampus and amygdala BDNF levels in an animal model of mania induced by ouabain. Journal of Psychiatric Research; 2009. Kapczinski F, Frey BN, Andreazza AC, Kauer-Sant’Anna M, Cunha AB, Post RM. Increased oxidative stress as a mechanism for decreased BDNF levels in acute manic episodes. Revista Brasileira de Psiquiatria 2008;30:243e5.
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Levine RL, Williams JA, Stadtman ER, Shacter E. Carbonyl assays for determination of oxidatively modified proteins. Methods in Enzymology 1994;233:346e57. Li R, El-Mallakh RS, Harrison L, Changaris DG, Levy RS. Lithium prevents ouabaininduced behavioral changes. Toward an animal model for manic depression. Molecular and Chemical Neuropathology 1997;31:65e72. Liu R, Goodell B, Jellison J, Amirbahman A. Electrochemical study of 2,3-dihydroxybenzoic acid and its interaction with Cu(II) and H2O2 in aqueous solutions: implications for wood decay. Environmental Science and Technology 2005;39: 175e80. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 1951;193:265e75. Machado-Vieira R, Andreazza AC, Viale CI, Zanatto V, Cereser Jr V, da Silva Vargas R, et al. Oxidative stress parameters in unmedicated and treated bipolar subjects during initial manic episode: a possible role for lithium antioxidant effects. Neuroscience Letters 2007;421:33e6. Murray CJ, Lopez AD. Alternative projections of mortality and disability by cause 1990e2020: global burden of disease study. Lancet 1997;349:1498e504. Naylor GJ, Smith AIIW, Dick EG, Dick DA, McHarg AM, Chambers CA. Erythrocyte membrane cation carrier in manic-depressive psychosis. Psychological Medicine 1980;10:521e5.
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Lithium and valproate modulate antioxidant enzymes and prevent ouabain-induced oxidative damage in an animal model of mania Luciano K. Jornada a, Samira S. Valvassori a, Amanda V. Steckert a, b, Morgana Moretti a, Francielle Mina b, Camila L. Ferreira a, Camila O. Arent a, Felipe Dal-Pizzol b, João Quevedo a, * a
Laboratory of Neurosciences and National Institute for Translational Medicine (INCT-TM), Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806-000 Criciúma, SC, Brazil Laboratory of Experimental Patophysiology and National Institute for Translational Medicine (INCT-TM), Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806-000 Criciúma, SC, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 February 2010 Received in revised form 4 May 2010 Accepted 7 May 2010
In this study, we assessed the oxidative stress parameters in rats submitted to an animal model of mania induced by ouabain (OUA), which included the use of lithium (Li) and valproate (VPA). Li and VPA treatment reversed and prevented the OUA-induced damage in these structures, however, this effect varies depending on the brain region and treatment regimen. Moreover, the activity of the antioxidant enzymes, namely, superoxide dismutase (SOD) and catalase (CAT) was found to be increased and decreased, respectively, in the brain of OUA-administered rats. Li and VPA modulated SOD and CAT activities in OUA-subjected rats in both experimental models. Our results support the notion that Li and VPA exert antioxidant-like properties in the brain of rats submitted to animal model of mania induced by ouabain. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Ouabain Mania Oxidative stress Submitochondrial particles Lithium Valproate
1. Introduction Bipolar disorder (BD) is a prevalent and chronic psychiatric disorder (Belmaker, 2004). It is considered to be one of the leading causes of disability among all medical and psychiatric conditions (Murray and Lopez, 1997), and untreated BD has been associated with increased morbidity and mortality due to general medical conditions such as vascular disorders and cancer (Angst et al., 2002). Oxidative stress mechanisms have been implicated in the pathogenesis of psychiatric disorder such as bipolar disorder (Halliwell, 2006). Aerobic organisms are susceptible to oxidative stress because semi-reduced oxygen species, superoxide and hydrogen peroxide are produced by mitochondria during respiration (Chance et al., 1979). The brain is particularly vulnerable to reactive oxygen species production because it metabolizes 20% of total body oxygen and has a limited amount of antioxidant capacity (Floyd, 1999), and in fact early studies demonstrating the ease of
* Corresponding author. Fax: þ 55 48 3443 4817. E-mail address: [email protected] (J. Quevedo). 0022-3956/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2010.05.011
brain membrane peroxidation supported this notion (Zaleska and Floyd, 1985). The primary antioxidant defense system has been studied in several psychiatric disorders and involves coordinated effects induced by superoxide dismutase (SOD) and catalase (CAT) (Reddy et al., 1991; Kapczinski et al., 2008). SOD is a protective enzyme that can selectively scavenge the superoxide anion radical (O 2 ) by catalyzing its dismutation to hydrogen peroxide (H2O2) and CAT metabolizes the excess of H2O2 producing O2 þ H2O, decreasing the intracellular redox status (Andreazza et al., 2008a). Moreover, the role of Naþ/Kþ-ATPase in the pathophysiology of bipolar disorder was reported by El-Mallakh et al. (2003). The pharmacological inhibition of Naþ/Kþ-ATPase by ouabain in rats causes hyperactivity and can be used as a model of mania (El-Mallakh et al., 2003; Li et al., 1997; Decker et al., 2000). Riegel et al. (2009) found that mania-like state induced by ouabain increased superoxide generation and lipid peroxidation in submitochondrial particles of the rat brain, suggesting that the decrease in the activity of Naþ/Kþ-ATPase seen in bipolar patients may be an important link in the pathological response to oxidative stress. Mood stabilizing drugs, particularly lithium and valproate, are considered as first-line agents for both acute mania and
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maintenance treatment (Yatham et al., 2005). Several studies have suggested that the neuroprotective effects of lithium and valproate may be responsible for their therapeutical effects (Chuang et al., 2002; Li et al., 1997). Recently a study of our laboratory demonstrated that Li and VPA reversed OUA-related hyperactive behavior in the open-field test, suggesting that this model fulfills adequate face, construct and predictive validity as an animal model of mania (Jornada et al., 2009). In this context, we sought to investigate the effects of lithium and valproate on oxidative stress parameters in the brain of rats using an animal model of mania induced by ouabain. 2. Experimental methods In the present study, we have extended the investigation of the effects of Li and VPA on ouabain-induced neurochemical alterations in an animal model of mania by measuring oxidative stress parameters in prefrontal and hippocampal samples that were kept frozen at 80 C from one of our previous experiments (Jornada et al., 2009). The detailed description of the experiments has been published elsewhere (Jornada et al., 2009); therefore, we summarize here the treatment regimens and describe the subsequent steps performed for the present investigation. 2.1. Animals The subjects were adult male Wistar rats (weighing 250e350 g) obtained from our breeding colony. Animals were housed five to a cage with food and water available ad libitum and were maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.) at a temperature of 22 1 C. All experimental procedures were performed in accordance with the approval of the local Ethics Committee. All experiments were performed at the same time during the day to avoid circadian variations. 2.2. Surgical procedure Animals were intraperitoneally anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg). In a stereotaxic apparatus, the skin of the rat skull was removed and a 27-gauge 9 mm guide cannula was placed at 0.9 mm posterior to bregma, 1.5 mm right from the midline and 1.0 mm above the lateral brain ventricle. Through a 2-mm hole made at the cranial bone, a cannula was implanted 2.6 mm ventral to the superior surface of the skull and fixed with dental acrylic cement. Animals recovered from surgery within 3 days. 2.3. Treatment Reversal model: We designed our first model to reproduce the management of an acute manic episode. Animals (n ¼ 72) received a single ICV injection of 5 ml of 103 M ouabain dissolved in artificial cerebrospinal fluid (ACSF) or 5 ml of ACSF alone on the fourth day following surgery (El-Mallakh et al., 2003; Riegel et al., 2009). (Note: Hamid et al. (2009) published reports in which the ouabain dose was reported as 105 M are in error; the dose used in those studies was 103 M.) A 30-gauge cannula was placed inside the guide cannula and connected by a polyethylene tube to a microsyringe. The tip of the cannula infusion protruded 1.0 mm beyond the cannula guide aiming the right lateral brain ventricle. From the day following the injection of ouabain or ACSF, the rats were treated for 6 days with intraperitoneal (IP) injections of saline, lithium or valproate in 6 experimental groups of 12 animals per group: ACSF ICV þ saline IP (ACSF þ Sal), ACSF ICV þ lithium IP (ACSF þ Li), ACSF ICV þ valproate IP (ACSF þ VPA), ouabain ICV þ saline IP
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(OUA þ Sal), ouabain ICV þ lithium IP (OUA þ Li), ouabain ICV þ valproate IP (OUA þ VPA). Animals in the Li group received intraperitoneal injections of lithium (47.5 mg/kg) and those in the VPA group received valproate (200 mg/kg) twice a day (Jornada et al., 2009). The animals were killed 24 h after the last injection of Li, VPA or ACSF. Maintenance model: We designed the second model to mimic the maintenance phase of the treatment of BD, when the drugs still are administered even in periods of euthymia (prevention model). After recovery from surgery, animals (n ¼ 72) received IP injections of Li (47.5 mg/kg), VPA (200 mg/kg) or SAL twice a day for 12 days. In the 7 days of treatment with Sal, Li or VPA the animals received a single ICV injection of either OUA (103 M) or ACSF, totaling 6 experimental groups of 12 animals per group: Sal þ ACSF, Li þ ACSF, VPA þ ACSF, Sal þ OUA, Li þ OUA, VPA þ OUA. After the OUA or ACSF injection, the treatment with mood stabilizer was continued for 6 more days (Jornada et al., 2009). The animals were killed 24 h after the last injection of Li, VPA or ACSF. 2.4. Biochemical analysis Rats were sacrificed by decapitation and the brain transferred within 1 min to ice-cold isolation buffer (0.23 M mannitol, 0.07 M sucrose, 10 mM TriseHCl, and 1 mM EDTA, pH 7.4). The prefrontal cortex and hippocampus (n ¼ 5 animals per group) were dissected in ice-cold buffer in a Petri dish, and submitochondrial particles were prepared in parallel from the four brain regions of each animal. For biochemical analysis in total tissue, the brain structures were rapidly frozen and stored at 80 C. 2.5. Mitochondrial isolation Rat brain homogenates were centrifuged at 700 g for 10 min to discard nuclei and cell debris and the pellet was washed to enrich the supernatant that was centrifuged at 7000 g for 10 min. The obtained pellet, washed and resuspended in the same buffer, was considered to consist mainly of intact mitochondria able to carry out oxidative phosphorylation. The operations were carried out at 0e2 C. Submitochondrial particles (SMP) were obtained by freezing and thawing (three times) of isolated mitochondria. For superoxide production measurements, SMP were washed twice with 140 mM KCl, 20 mM TriseHCl (pH 7.4) and suspended in the same medium (Boveris et al., 1972). 2.6. Superoxide production in submitochondrial particles of the rat brain Superoxide production was determined in washed brain SMP using a spectrophotometric assay based on superoxide-dependent oxidation of epinephrine to adrenochrome at 37 C (l 480 nm ¼ 4.0 mM1 cm1). The reaction medium consisted of 0.23 M mannitol, 0.07 M sucrose, 20 mM TriseHCl (pH 7.4), SMP (0.3e1.0 mg protein/ml), 0.1 mM catalase, and 1 mM epinephrine. NADH (50 mM) and succinate (7 mM) were used as substrates and rotenone (1 mM) and antimycin (1 mM) were added as specific inhibitors, respectively, to assay O 2 production at the NADH dehydrogenase and at the ubiquinoneecytochrome b region. Superoxide dismutase (SOD) was used at 0.1e0.3 mM final concentration to give assay specificity (Boveris, 1984). 2.7. Thiobarbituric acid reactive species (TBARS) content in tissue and in submitochondrial particles of the rat brain As a marker of lipid peroxidation, we measured the formation of thiobarbituric acid reactive species (TBARS) during an acid-heating
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reaction, as previously described (Esterbauer and Cheeseman, 1990). Briefly, the samples were mixed with 1 ml of trichloroacetic acid 10% and 1 ml of thiobarbituric acid 0.67%, and then heated in a boiling water bath for 15 min. TBARS were determined by the absorbance at 535 nm. 2.8. Protein carbonyl content Oxidative damage to proteins was measured by the quantification of carbonyl groups based on the reaction with dinitrophenylhydrazine (DNPH), as previously described (Levine et al., 1994). Proteins were precipitated by the addition of 20% trichloroacetic acid and were redissolved in DNPH; the absorbance was read at 370 nm. 2.9. Superoxide dismutase activity This method for the assay of SOD activity is based on the capacity of pyrogallol to autoxidize, a process highly dependent on O2 2 ; a substrate for SOD (Bannister and Calabrese, 1987). The inhibition of autoxidation of this compound thus occurs when SOD is present, and the enzymatic activity can be then indirectly assayed spectrophotometrically at 420 nm, using a double-beam spectrophotometer with temperature control. A calibration curve was generated using purified SOD as the standard, in order to calculate the specific activity of SOD present in the samples. A 50% inhibition of pyrogallol autoxidation is defined as 1 unit of SOD, and the specific activity is represented as units per milligram of protein. 2.10. Catalase activity CAT activity was assayed using a double-beam spectrophotometer with temperature control. This method is based on the disappearance of H2O2 at 240 nm in a reaction medium containing 20 mM H2O2, 0.1% Triton X-100, 10 mM potassium phosphate buffer, pH 7.0, and 0.1e0.3 mg protein/ml (Aebi, 1984). One CAT unit is defined as 1 mol of hydrogen peroxide consumed per minute, and the specific activity is reported as units per milligram of protein. 2.11. Protein determination All biochemical measures were normalized to the protein content with bovine albumin as standard (Lowry et al., 1951). 2.12. Statistical analysis Biochemical data are presented as mean and standard error of the mean. Differences among the experimental groups were determined by one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test. In all comparisons, statistical significance was set at p < 0.05. 3. Results
Fig. 1. Effects of ouabain or ACSF ICV administration on superoxide levels in submitochondrial particles in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 49.213, p < 0.001; hippocampus: df ¼ 5, F ¼ 31.973, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 8.760, p < 0.001; hippocampus: df ¼ 5, F ¼ 11.029, p < 0.001). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
Fig. 1B shows that in the maintenance model a significant increase in superoxide was also detected in the Sal þ OUA group in the prefrontal and hippocampus of rats (p < 0.05), and Li and VPA prevented these values significantly in both structures (p < 0.05). 3.2. TBARS content in submitochondrial particles of the rat brain The comparison of TBARS content in submitochondrial particles among the 6 groups in the reversal model is shown in Fig. 2A. The OUA þ Sal group showed a significant increase in the amount of TBARS in submitochondrial particles in the prefrontal and hippocampus when compared to the control group (p < 0.05), and Li, but not VPA, reversed this values in both structures tested. As shown in Fig. 2B, in the maintenance model, the Sal þ OUA group showed an elevated amount of TBARS content in submitochondrial particles in prefrontal and hippocampus when compared to that of the control group (p < 0.05), and Li and VPA reversed this values in both structures tested (p < 0.05).
3.1. Superoxide production in submitochondrial particles of the rat brain
3.3. TBARS content in tissue of the rat brain
As shown in Fig. 1A, in the reversal model there was a significant increase in superoxide production in the OUA þ Sal group in the prefrontal and hippocampus when compared to the control group (p < 0.05), and Li, but not VPA, reversed this values significantly in the hippocampus of rats (p < 0.05). However, in the prefrontal neither Li nor VPA was able to reverse the OUA-induced increase of superoxide production.
Fig. 3A illustrates TBARS content of the rat brain in the reversal model. Administration of ouabain ICV (OUA þ Sal group) resulted in a marked increased amount of TBARS content in prefrontal and hippocampus (p < 0.05), and Li and VPA were able to reverse this value in the hippocampus but not in the prefrontal of rats. Likewise, administration of ouabain (Sal þ OUA group) in maintenance protocol (Fig. 3B) also resulted in a marked increased
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amount of TBARS content in prefrontal and hippocampus (p < 0.05), and once more Li and VPA were able to reverse the ouabain-induced increase of TBARS content in both structures tested. 3.4. Carbonyls protein content As shown in Fig. 4A, in the reversal model, there was a significant increase in formation of carbonyl protein in the prefrontal and hippocampus in OUA þ Sal group when compared to the control group (p < 0.05), and Li, but not VPA, reversed these values significantly in prefrontal and hippocampus of rats (p < 0.05). Fig. 4B shows protein carbonylation in the brain of rats in maintenance model. The Sal þ OUA group showed a significant increase in carbonyls protein content in hippocampus and prefrontal of rats when compared to the control group (p < 0.05), and Li and VPA were able to reverse the ouabain-induced increase of carbonyls protein content in both structures tested. 3.5. Superoxide dismutase activity
Fig. 2. Effects of ouabain or ACSF ICV administration on TBARS levels in submitochondrial particles in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 31.954, p < 0.001; hippocampus: df ¼ 5, F ¼ 29.258, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 44.923, p < 0.001; hippocampus: df ¼ 5, F ¼ 4.655, p ¼ 0.007). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
Fig. 3. Effects of ouabain or ACSF ICV administration on TBARS levels in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 40.471, p < 0.001; hippocampus: df ¼ 5, F ¼ 28.992, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 93.414, p < 0.001; hippocampus: df ¼ 5, F ¼ 118.134, p < 0.001). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
As shown in Fig. 5A, in the reversal model SOD activity was higher in the prefrontal and hippocampus homogenates from the OUA þ Sal group than in those from the control group (p < 0.05), however in the prefrontal, but not in the hippocampus, Li and VPA treatment reduced these values significantly (p < 0.05). Once more, the maintenance model (Fig. 5B) also resulted in a marked increase of SOD activity in the prefrontal and hippocampus of rats in Sal þ OUA group (p < 0.05), and Li and VPA treatment partially reduced these values in prefrontal, but not in the hippocampus (p < 0.05).
Fig. 4. Effects of ouabain or ACSF ICV administration on protein carbonyl formation in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 23.481, p < 0.001; hippocampus: df ¼ 5, F ¼ 24.541, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 49.265, p < 0.001; hippocampus: df ¼ 5, F ¼ 19.833, p < 0.001). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
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Fig. 5. Effects of ouabain or ACSF ICV administration on superoxide dismutase activity in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 65.241, p < 0.001; hippocampus: df ¼ 5, F ¼ 22.932, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 139.135, p < 0.001; hippocampus: df ¼ 5, F ¼ 70.726, p < 0.001). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
Fig. 6. Effects of ouabain or ACSF ICV administration on catalase activity in the prefrontal cortex and hippocampus of rats in the reversal treatment (A) (prefrontal: df ¼ 5, F ¼ 72.457, p < 0.001; hippocampus: df ¼ 5, F ¼ 117.860, p < 0.001) and in the maintenance treatment (B) (prefrontal: df ¼ 5, F ¼ 40.607, p < 0.001; hippocampus: df ¼ 5, F ¼ 55.919, p < 0.001). Bars represent means standard error of means of 5e6 animals. *p < 0.05 vs. ACSF group, according to ANOVA followed by the Tukey test. #p < 0.05 compared with OUA þ SAL group.
3.6. Catalase activity The results of the CAT activity in the reversal model are shown in Fig. 6A. The CAT activity was significantly decreased in OUA þ Sal group (p < 0.05), and Li, but not VPA, reversed these values significantly in both structures (p < 0.05). Likewise, administration of ouabain (Sal þ OUA group) in maintenance protocol (Fig. 6B) also resulted in a marked decrease of the CAT activity in prefrontal and hippocampus (p < 0.05), and Li and VPA were able to reverse the OUA-induced decrease of the CAT activity in both structures tested. It is noteworthy that Li and VPA regimen in the control groups does not alter any of the parameters measured in any of the experimental protocols. 4. Discussion Soon after the characterization of the Naþ/Kþ-ATPase, some authors investigated its activity in mood disorders. These studies showed that Naþ pump activity is decreased in acute mania compared to recovered euthymic bipolar individuals (Reddy et al., 1992; Hesketh et al., 1977; Naylor et al., 1980). In preclinical studies, when the potent sodium pump inhibitor, ouabain, is ICV administered in rats, it induces a motor hyperactivity (El-Mallakh and Wyatt, 1995; Decker et al., 2000), which may persist for over a week after a single injection (Ruktanonchai et al., 1998). Recently, a study of our laboratory demonstrated that Li and VPA reversed OUA-related hyperactive behavior in the open-field test, suggesting that this model fulfills adequate face, construct and predictive validity as an animal model of mania (Jornada et al., 2009). Additionally, another study from our laboratory found that mania-like state induced by ouabain increased superoxide generation and lipid peroxidation in submitochondrial particles of the rat brain (Riegel et al., 2009).
In this study we demonstrated that in both experimental protocols, ouabain induces oxidative damage by increasing the amount of TBARS, protein carbonyl, and superoxide in submitochondrial particles in prefrontal and hippocampus of rats. Interestingly, the OUA-induced oxidative damage was accompanied by increased superoxide dismutase activity and decreased CAT activity. SOD is an enzyme capable of reducing the superoxide radical into hydrogen peroxide (H2O2), which acts as the substrate to CAT. When cell has increased levels of SOD without a proportional increase in peroxidases, the excess of H2O2 produced could be responsible for the oxidative damage in the cell. In addition, H2O2 can react with transitional metals and generate the radical hydroxyl, which is the most harmful radical (Halliwell and Gutteridge, 1999). Consequently, the overexpression of SOD without a compensatory increase in CAT has deleterious effects upon the cell. Interestingly, here despite Li and VPA have partially reduced the activity of SOD in rats treated with OUA, both Li and VPA decreased the amount of ouabain-induced superoxide in maintenance model and in hippocampus in the reversal model. This may be due to the fact that in the presence of Fe3þ and H2O2 in excess, hydroperoxyl radical (HO$2) and superoxide anion (O2) are also formed. Thus, Li and VPA may be decreasing SOD and H2O2, thereby decreasing O2 (Hammel et al., 2002; Liu et al., 2005). In addition, pretreatment with Li and VPA led to an increase in CAT activity in brain of OUAinjected animals, which can be preventing other reactions with H2O2. However, in this work we evaluated superoxide production in submitochondrial particles of the rat brain, which is an indirect measure of mitochondrial dysfunction. Therefore, this measure is not directly related with SOD activity. Perhaps, the main finding of this paper was that Li and VPA increased the CAT activity in prefrontal and hippocampus of rats, preventing the oxidative damage induced by OUA in maintenance
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protocol. However, in the reversal protocol, Li but not VPA increased CAT activity in both structures tested, reversing the OUA-induced increase in the amount of protein carbonyls, superoxide, TBARS in tissue and in submitochondrial particles in hippocampus. This discrepancy suggests that, when co-administered with ouabain, Li may have its antioxidant effect in an earlier time course than VPA. However, in the prefrontal cortex, Li was unable to reverse the increase of superoxide and TBARS in submitochondrial particles. This may occur due to longer treatment with Li when compared to the reversal model. In addition, Bhalla and Dhawan (2009) have demonstrated that lipid peroxidation and increase in reactive oxygen species induced by aluminum decreased following Li supplementation; besides, Li normalized activity of antioxidant enzymes that were altered by aluminum in brain of rats. In previous study with a dopaminergic animal model of mania, our laboratory found that AMPH increased rat peripheral and hippocampal DNA damage. The index of DNA damage was correlated positively with lipid peroxidation, whereas Li and VPA were able to modulate the oxidative balance and prevent recent damage to the DNA (Andreazza et al., 2008b). Machado-Vieira et al. (2007) have showed that TBARS and antioxidant enzymes’ activity (SOD and CAT)increased in unmedicated manic patients compared to controls, and acute treatment with Li showed a significant reduction in both SOD/CAT ratio and TBARS levels. Stork and Renshaw (2005) proposed a hypothesis of mitochondrial dysfunction in bipolar disorder that involves impaired oxidative phosphorylation, a resultant shift toward glycolytic energy production, a decrease in total energy production and/or substrate availability, and altered phospholipid metabolism. Impaired mitochondrial function, and as a consequence impaired cellular energy state, is an attractive hypothesis for explaining the pathophysiology of BD. The literature suggests that the decrease of ATP production leads to a dysfunction in Na pump activity which consequently may result in neuronal hyperexcitability, giving rise to manic symptoms, such as hyperactivity (El-Mallakh and Wyatt, 1995). Our results support the notion that Li and VPA exert antioxidantlike properties in the brain of rats submitted to animal model of mania induced by ouabain. Thus, the altered energy metabolism dysfunctions associated with BD may play a role in oxidative stress observed during manic episodes. Also, Li and VPA seem to exert antioxidant effects during mania. Conflict of interest None of the authors or funding sources has conflict of interest. Contributors Luciano K. Jornada designed the study and wrote the protocol. Samira S. Valvassori undertook the statistical analysis and made the first draft of the manuscript. Amanda V. Steckert and Francielle Mina made the biochemical analysis. Morgana Moretti, Camila O. Arent and Camila L. Ferreira were responsible for the surgery and pharmacological treatment. All authors contributed to and have approved the final manuscript. Role of funding source This research was supported by grants from CNPq (Felipe DalPizzol and João Quevedo), FAPESC (João Quevedo), and UNESC (Felipe Dal-Pizzol and João Quevedo). Felipe Dal-Pizzol and João Quevedo are CNPq Research Fellows. Morgana Moretti and Samira S. Valvassori are holders of CAPES studentships, Camila O. Arent, Amanda V. Steckert and Camila L. Ferreira are holders of CNPq Studentships.
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The National Council for Scientific and Technological Development (CNPq) is an agency linked to the Ministry of Science and Technology (MCT), dedicated to the promotion of scientific and technological research and to the formation of human resources for research in the country. Its history is directly linked to the scientific and technological development of Brazil. The FAPESC aims to support and promote the scientific and technological research for the advancement of all areas of knowledge for regional balance, sustainable development and improving the quality of life of people of the Santa Catarina state with respect for ethical values and based on the principles established by arts. Coordination of Improvement of Higher Education Personnel (CAPES) plays a role in the expansion and consolidation of postgraduate (masters and doctorate) in all states of Brazil. Acknowledgements We thank CNPq, FAPESC, CAPES, Instituto Cérebro e Mente and UNESC for financial support. References Aebi H. Catalase in vitro. Methods in Enzymology 1984;105:121e6. Andreazza AC, Kauer-Sant’anna M, Frey BN, Bond DJ, Kapczinski F, Young LT, et al. Oxidative stress markers in bipolar disorder: a meta-analysis. Journal of Affective Disorders 2008a;111:135e44. Andreazza AC, Kauer-Sant’Anna M, Frey BN, Stertz L, Zanotto C, Ribeiro L, et al. Effects of mood stabilizers on DNA damage in an animal model of mania. Journal of Psychiatry and Neuroscience 2008b;33:516e24. Angst F, Stassen H, Clayton PJ, Angst J. Mortality of patients with mood disorders: follow-up over 34e38 years. Journal of Affective Disorders 2002;68:167e81. Bannister JV, Calabrese L. Assays for superoxide dismutase. Methods of Biochemical Analysis 1987;32:279e312. Belmaker RH. Bipolar disorder. New England Journal of Medicine 2004;351:476e86. Bhalla P, Dhawan DK. Protective role of lithium in ameliorating the aluminiuminduced oxidative stress and histological changes in rat brain. Cellular and Molecular Neurobiology 2009;29:513e21. Boveris A. Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria. Methods in Enzymology 1984;105:429e35. Boveris A, Oshino N, Chance B. The cellular production of hydrogen peroxide. Biochemical Journal 1972;128:617e27. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiological Reviews 1979;59:527e605. Chuang DM, Chen RW, Chalecka-Franaszek E, Ren M, Hashimoto R, Senatorov V, et al. Neuroprotective effects of lithium in cultured cells and animal models of diseases. Bipolar Disorder 2002;4:129e36. Decker S, Grider G, Cobb M, Li XP, Huff MO, El-Mallakh RS, et al. Open field is more sensitive than automated activity monitor in documenting ouabain-induced hyperlocomotion in the development of an animal model for bipolar illness. Progress in Neuro-Psychopharmacology and Biological Psychiatry 2000;24:455e62. El-Mallakh RS, El-Masri MA, Uhf MO, Li XP, Decker S, Levy RS. Intracerebroventricular administration of ouabain as a model of mania in rats. Bipolar Disorder 2003;5:362e5. El-Mallakh RS, Wyatt RJ. The Na, K-ATPase hypothesis for bipolar illness. Biological Psychiatry 1995;37:235e44. Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods in Enzymology 1990;186:407e21. Floyd RA. Antioxidants, oxidative stress, and degenerative neurological disorders. Proceedings of the Society for Experimental Biology and Medicine 1999;222:236e45. Halliwell B. Oxidative stress and neurodegeneration: where are we now? Journal of Neurochemistry 2006;97:1634e58. Halliwell B, Gutteridge JM. Free Radical in Biology and Medicine. Oxford, UK: Oxford University Press; 1999. Hamid H, Gao Y, Lei Z, Hougland MT, El-Mallakh RS. Effect of ouabain on sodium pump alpha-isoform expression in an animal model of mania. Progress in Neuro-Psychopharmacology and Biological Psychiatry 2009;33:1103e6. Hammel KE, Kapich AN, Jensen Jr KA, Ryan ZC. Reactive oxygen species as agents of wood decay by fungi. Enzyme and Microbial Technology 2002;30:445e53. Hesketh JE, Glen AIM, Reading HW. Membrane ATPase activities in depressive illness. Journal of Neurochemistry 1977;28:1401e2. Jornada KL, Moretti M, Valvassori SS, Ferreira CL, Padilha PT, Arent CO, et al. Effects of mood stabilizers on hippocampus and amygdala BDNF levels in an animal model of mania induced by ouabain. Journal of Psychiatric Research; 2009. Kapczinski F, Frey BN, Andreazza AC, Kauer-Sant’Anna M, Cunha AB, Post RM. Increased oxidative stress as a mechanism for decreased BDNF levels in acute manic episodes. Revista Brasileira de Psiquiatria 2008;30:243e5.
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