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Intracerebroventricular ouabain administration induces oxidative stress in the rat brain
Int. J. Devl Neuroscience 28 (2010) 233–237
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Intracerebroventricular ouabain administration induces oxidative stress in the rat brain Rafael E. Riegel a, Samira S. Valvassori a, Morgana Moretti a, Camila L. Ferreira a, Amanda V. Steckert b, Bruna de Souza b, Felipe Dal-Pizzol b, Joa˜o Quevedo a,* a
Laboratory of Neurosciences and National Institute for Translational Medicine, Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806-000 Criciu´ma, SC, Brazil Laboratory of Experimental Pathophysiology and National Institute for Translational Medicine, Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806-000 Criciu´ma, 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 15 December 2009 Received in revised form 24 January 2010 Accepted 6 February 2010
Intracerebroventricular (ICV) injection of ouabain (a potent Na+/K+-ATPase inhibitor) in rats resulted in manic-like effects. There is an emerging body of data indicating that major neuropsychiatric disorders, such as bipolar disorder and schizophrenia, are associated with increased oxidative stress. In this study, we investigated the effects of ICV ouabain injection on oxidative stress parameters in total tissue of rat brain. Our findings demonstrated that ICV injection increased thiobarbituric acid reactive species levels and protein carbonyl generation in the prefrontal cortex and hippocampus of rats. Moreover, the activity of the antioxidants enzymes catalase and superoxide dismutase was altered in several areas of the rat brain and cerebrospinal fluid of ICV ouabain-subjected rats. These results showed that Na+/K+-ATPase inhibition can lead to oxidative stress in the brain of rats. ß 2010 ISDN. Published by Elsevier Ltd. All rights reserved.
Keywords: Bipolar disorder Mania Ouabain Oxidative stress
Bipolar disorder (BD), also known as manic-depressive illness, is a brain disorder that causes unusual shifts in a person’s mood, energy, and ability to function. The defining clinical feature of the condition is the manic episodes. Mania is characterized by an elated or irritable mood, reduced need for sleep, psychomotor activation, and excessive involvement in potentially problematic behavior (El-Mallakh et al., 2003). Soon after the characterization of the Na+/K+-ATPase, some authors investigated its activity in mood disorders. These studies showed that Na+ 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). Besides, reduced enzyme activity of Na+/K+-ATPase and increased intracellular sodium and calcium in erythrocytes of bipolar patients have been reported (Looney and El-Mallakh, 1997). Nevertheless, the biochemical mechanism underling the development of the alteration caused by the Na+/K+-ATPase inhibition remains to be clarified. In preclinical studies, when the potent sodium pump inhibitor, ouabain, is intracerebroventricularly (ICV) administered in rats, it
* Corresponding author at: Laborato´rio de Neurocieˆncias, PPGCS, UNASAU, Universidade do Extremo Sul Catarinense, 88806-000 Criciu´ma, SC, Brazil. Fax: +55 48 3443 4817. E-mail address: [email protected] (J. Quevedo). 0736-5748/$36.00 ß 2010 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2010.02.002
induces a dose-dependent 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; Riegel et al., 2009). These observations suggest that ICV ouabain injection in rats evokes some Na+/K+-ATPase dysfunction, similar to that reported in bipolar patients. There is an emerging body of data indicating that major neuropsychiatric disorders, such as BD and schizophrenia, are associated with increased oxidative stress and changes in antioxidant enzymatic defense (Kuloglu et al., 2002; Ranjekar et al., 2003; Ozcan et al., 2004). It is well known that the brain is particularly prone to oxidative damage due to its relative high content of peroxidable fatty acids and limited antioxidant capacity (Floyd, 1999). Increased neuronal oxidative stress levels generate deleterious effects on signal transduction, structural plasticity and cellular resilience, mostly by inducing lipid peroxidation in membranes, proteins and genes (Mahadik et al., 2001; Scha¨fer et al., 2004). Evidence from literature have shown that individuals with bipolar disorder have increased serum thiobarbituric acid reactive substances (TBARS) in the initial manic episode (Machado-Vieira et al., 2007; Frey et al., 2006a), and in all phases of the bipolar illness (Andreazza et al., 2007). Previously we reported increased TBARS levels and superoxide generation in submitocondrial particles in the rat brain after ouabain ICV injection (Riegel et al., 2009), suggesting that oxidative stress could be causing mitochondrial damage, also related in BD. However, the
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generation of superoxide and mitochondrial membrane damage cannot extend throughout the cell, by action of antioxidant enzymes (Halliwell and Gutteridge, 1999). Oxidative and antioxidative molecules have been studied in several psychiatric disorders and many of these molecules, such as superoxide dismutase (SOD) and catalase (CAT) are involved in the pathophysiology of BD (Kunz et al., 2008). SOD is a potent protective enzyme that can selectively scavenge the superoxide anion radical (O2 ) by catalyzing its dismutation to hydrogen peroxide (H2O2) and CAT metabolizes the excess of H2O2 producing O2 + H2O, decreasing the intracellular redox status. Previous studies reported that patients with BD have significant alterations in antioxidant enzymes (Kuloglu et al., 2002; Abdalla et al., 1986). Several studies support a model of BD that involves dysfunction within subcortical (striatal-thalamic)-prefrontal networks and the associated limbic modulating region (amygdala). It seems that in BD there may be diminished prefrontal modulation of subcortical and medial temporal structures within the anterior limbic network (amygdala and anterior striatum), leading to a dysfunction of mood (see Strakowski et al., 2005). In addiction, BD postmortem studies indicate abnormal transmission in the hippocampus; also, cognitive disturbances during acute mood episodes of BD patients, suggesting that the hippocampus plays an important role in the pathophysiology of BD (see Frey et al., 2007a). Besides, impaired mitochondrial metabolism in cerebrospinal fluid (CSF) (Regenold et al., 2009) and a significant CSF increase in the adjacent outer ventricular sulci were found in BD patients (Tost et al., 2010). The present study aims to investigate the biochemical alterations induced by immediately after (to mimic an acute episode of mania) or seven days following the single ICV injection (to mimic the persistence of a manic episode) of ouabain in rats. We assessed the effects of ICV ouabain administration on lipid and protein oxidation levels (markers of oxidative stress) and SOD and CAT activity (two major antioxidant enzymes) in prefrontal, hippocampus, striatum, amygdala and CSF of rats. 1. Experimental procedures 1.1. Animal model We conducted the study using adult male Wistar rats (250–300 g) obtained from our breeding colony. The animals were housed 5 to a cage, on a 12-h light/dark cycle (lights on at 7:00 am), with free access to food and water. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behaviour (SBNeC). This study was approved by the local ethics committee (Comiteˆ de E´tica em Pesquisa da Universidade do Extremo Sul Catarinense, Protocol no 536/ 2007), and all efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. In the present study, we have extended the investigation of the effects of the administration of ouabain on oxidative stress parameters by measuring TBARS, protein carbonyl production, SOD and CAT activity in prefrontal cortex, hippocampus, striatum, amygdala and CSF that were kept frozen in 80 8C from one of our previous experiments (Riegel et al., 2009). The detailed description of the experiments has been published elsewhere (Riegel et al., 2009), therefore, here we summarize the experimental procedures and describe the subsequent steps performed for the present investigation. 1.2. Surgical procedure and treatment Animals were intraperitoneally anesthetized with ketamine (80 mg/kg) and xylasine (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 jeweler acrylic cement. Animals were tested on the third day following surgery. A 30-gauge cannula was fitted into the guide cannula and connected by a polyethylene tube to a microsyringe. The tip of the infusion cannula protruded 1.0 mm beyond the guide cannula aiming the right lateral brain ventricle. Each animal was administered 5 ml of either artificial cerebrospinal fluid (aCSF) or ouabain (10 3 M and 10 2 M; Sigma Chemical, Saint Louis, USA; dissolved in aCSF), over 30 s (El-Mallakh et al., 2003; Riegel et al., 2009) (Note: The El-Mallakh
group published reports in which the ouabain dose was reported as 10 error; the dose use in those studies was 10 3 M) (Hamid et al., 2009).
5
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1.3. Experimental design In this study were used 30 animals divided into two experimental procedures: (1) Immediately after ICV injection of ouabain at 10 3 M and 10 2 M or aCSF (n = 5 animals per group): the first model was designed in order to reproduce an acute manic episode of BD. The animals received ICV injection of ouabain or aCSF and 10 min after ICV administration were anesthetized with ketamine (30 mg/kg) and xylasine (4 mg/kg), i.p., and the CSF was drawn (80–100 ml per rat), by direct puncture of the cisterna magna with an insulin syringe (27-gauge 31/20 length). Individual samples that presented visible blood contamination were discarded. After sampling, the samples were centrifuged at 4500 g at 5 8C for 5 min, to obtain cell-free supernatants (Cruz Portela et al., 2002). CSF samples were stored at 80 8C and defrosted only when measurements were carried out. Then, the animals 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 Tris–HCl, and 1 mM EDTA, pH 7.4). The prefrontal cortex, hippocampus, striatum and amygdala were dissected in ice-cold buffer in a Petri dish. After dissection brain samples were quickly stored at 80 8C and defrosted only when measurement were carried out. (2) Seven days following the ICV injection of ouabain at 10 3 M and 10 2 M or aCSF (n = 5 animals per group): the second model was designed to mimic the persistence of manic phase of BD. The animals received a single ICV injection of ouabain or aCSF and seven days after ICV administration the CSF was draw (following the procedures mentioned above). The animals were sacrificed by decapitation and prefrontal cortex, hippocampus, striatum and amygdala were dissected (following the procedures mentioned above). Obs: The behavioral data are described in Riegel et al. (2009). 1.4. Oxidative stress parameters 1.4.1. TBARS formation To determine oxidative damage in lipid, we measured the formation of TBARS during an acid-heating reaction, as previously described (Esterbauer and Cheeseman, 1990). The samples were mixed with 1 mL of trichloroacetic acid 10% and 1 mL of thiobarbituric acid 0.67% and were then heated in a boiling water bath for 15 min. TBARS were determined spectrophotometrically by the absorbance at 535 nm. 1.4.2. Carbonyls protein formation 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. All biochemical measures were normalized to the protein content with bovine serum albumin as standard (Lowry et al., 1951). 1.4.3. Superoxide dismutase (SOD) activity This method for the assay of SOD activity is based on the capacity of pyrogallol to autoxidize, a process highly dependent on O2 ; a substrate for SOD (Marklund, 1985). 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 performed 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 U of SOD, and the specific activity is represented as units per mg of protein. 1.4.4. Catalase (CAT) 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 0.1–0.3 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 mg protein. 1.4.5. Statistical analysis All data are presented as mean SEM. Differences between experimental groups were determined by one-way ANOVA followed by Tukey post hoc test if applicable. P values less than 0.05 were considered to indicate statistical significance.
2. Results (1) The results of experiment 1 (immediately after ICV injection) are shown in Fig. 1: (1A) When compared with the control group, TBARS generation was increased in the CSF at both
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Fig. 1. Effects of the ouabain (10 3 M and 10 2 M) or saline ICV injection immediately after the administration on (A) TBARS levels, (B) protein carbonyl formation, (C) SOD and (D) CAT activity in the prefrontal cortex, hippocampus, striatum, amygdala and CSF of rats. Bars represent means standard error of means of 4–5 animals. *P < 0.05 vs. saline group, according to ANOVA followed by the Tukey test.
10 3 M and 10 2 M doses (P < 0.01). Significant changes were not detected in the TBARS generation at any dosage in the prefrontal cortex, hippocampus, striatum and amygdala. (1B) A significant increased in carbonyl group formation was detected in the prefrontal cortex at the concentrations of 10 3 M and 10 2 M (P < 0.01 for all concentrations), striatum at 10 2 M (P < 0.01), amygdala at the concentration of 10 3 M (P < 0.01) and CSF at the concentrations of 10 3 M and 10 2 M (P < 0.01).
According to Fig. 1C and D, significant changes were not detected in the SOD and CAT activity at any dosage in the prefrontal cortex, hippocampus, striatum and amygdala, however, activity of these enzymes was decreased in the CSF at both 10 3 M and 10 2 M doses. (2) The results of experiment 2 (seven days after ICV injection) are shown in Fig. 2: (2A) A significant increase in TBARS generation in the prefrontal cortex (P < 0.001), and hippocampus
Fig. 2. Effects of the ouabain (10 3 M and 10 2 M) or saline ICV injection seven days following the administration on (A) TBARS levels, (B) protein carbonyl formation, (C) SOD and (D) CAT activity in the prefrontal cortex, hippocampus, striatum, amygdala and CSF of rats. Bars represent means standard error of means of 4–5 animals. *P < 0.05 vs. saline group, according to ANOVA followed by the Tukey test.
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(P < 0.001) was detected in rats treated with ouabain at 10 3 M and in the CSF at the concentration of 10 3 M and 10 2 M (P < 0.01), compared to control group. (2B) We observed an increase in protein damage in the prefrontal (P < 0.01) and hippocampus (P < 0.01) at the concentration of 10 3M and in the CSF at the concentration of 10 3 M and 10 2 M (P < 0.01 for all concentrations). No significant changes in carbonyl group formation were detected in the striatum and amygdala of rats. Fig. 2C shows that there is a significant increase in the SOD activity in the prefrontal cortex (P < 0.01) and hippocampus (P < 0.01) and decrease in its activity in the CSF (P < 0.01) in rats treated with ouabain at 10 3 M and 10 2 M. We did not find differences in the SOD activity in the striatum and amygdale. (2D) An increase in CAT activity was detected in the prefrontal cortex (P < 0.05) and hippocampus (P < 0.05) at the concentrations of 10 2 M. In contrast, ouabain at 10 3 M decreased CAT activity in these cerebral areas (P < 0.05). In the CSF, ouabain 10 3 M and 10 2 M decreased CAT activity (P < 0.05) and there were no changes in this enzyme activity in the striatum and amygdala. 3. Discussion In this study, we demonstrated that ouabain injection induced an increased TBARS formation in the rat CSF and a long-term, but not immediate, increased TBARS generation in prefrontal and hippocampus of the rat brain. Moreover, we showed that a significant elevation of protein carbonyl formation was observed immediately after, and also seven days following the ICV administration of ouabain. Interesting enough, the increase in TBARS observed after seven days of ouabain injection coincide exactly with elevation of protein carbonyl formation, i.e. these effects are coincident in terms of ouabain dose and brain structures involved. Interestingly, in the previous study we demonstrated an increased of superoxide content and damage to lipid in submitochondrial particles in brain of rats submitted to ouabain ICV injection (Riegel et al., 2009). Together, the previous study and the present data seem to show that the ouabain-induced production of superoxide and damage to mitochondrial membrane affect the entire cell, which can lead to tissue damage. Despite the proliferation of information on oxidative stress mechanisms in the BD, the different biochemical pathways that might mediate oxidative stress in BD are not yet fully explained. With the present data we suggest that the sodium pump activity reduced in BD can be an important link in the pathological response to oxidative stress. Moreover, in the present model as in previous study (Riegel et al., 2009); we demonstrate that oxidative stresses are most evident seven days after the ICV ouabain injection. Whereas acute reactions to ouabain in animals have considerable homology to a manic episode (as reduction in the Na+/K+ATPase and consequent hyperactivity), the persistence of effect of ouabain mirrors aspects of illness progression. From these observations we suggest that ICV administration of ouabain is a good model to study the chronicity of BD.A very important finding that we can observe with the analysis of the results is that when administered ouabain at the dose of 10 3 M SOD activity increases while the activity of catalase decreases in prefrontal cortex, hippocampus and CSF (the same structures in which was observed damage to protein and lipid). SOD is an enzyme able in reducing the superoxide radical into hydrogen peroxide (H2O2), which is 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. It is important to note that seven days after ouabain ICV injection there was an increase of oxidative damage in the hippocampus and prefrontal cortex, but not in the striatum and amygdala. This may be due to the increase of SOD and decreased of CAT activity in the prefrontal cortex and hippocampus. In the striatum and amygdala the concentrations of these enzymes seem to remain constant when compared with the control group, which may be protecting these brain regions.Furthermore, when administered ouabain at the dose of 10 2 M increased both the activity of SOD and CAT, which may be protecting the tissue (note that the ouabain administration at the dose of 10 2 M does not cause damage to protein and lipid in brain tissue). In contrast, in the CSF 10 2 M ouabain administration lead to lipid and protein damage; also, increases the activity of SOD and decreases the activity of CAT. We believe that this discrepancy is by fact that the CSF is first exposed to ouabain, is the CSF that leads ouabain to all parts of the brain and perhaps by being constantly exposed cannot reverse the damage induced by ouabain. Besides, the brain essentially floats in CSF, so the CSF cushions the organ from trauma and reduces the pressure on the base of the brain, keeping its volume constant (see Siegel, 2006). After administration of ouabain may be a decrease in production of cerebrospinal fluid for the brain to maintain its volume. Moreover, CSF has low immune defense (Van-Bambeke and Tulkens, 2009), which may explain at least in part the severe oxidative damage in the CSF, both immediately after and seven days after ouabain injection. As seen in patients (Kuloglu et al., 2002; Ranjekar et al., 2003; Ozcan et al., 2004; Frey et al., 2007b), here we demonstrated that ouabain treated rats had higher SOD activity (10 2 M and 10 3 M), and lower CAT levels (10 3 M) than the control in prefrontal cortex, hippocampus and CSF, suggesting that ouabain exposure modulated SOD and CAT activity with a distinct pattern for each brain region and dosage regimen. Additionally, the amphetamine model of mania in rats also supports the view that oxidative stress is occurring during a maniclike state. In fact, chronic treatment with amphetamine increases TBARS (Frey et al., 2006a) and protein oxidation (Valvassori et al., 2008) in the hippocampus and prefrontal cortex and causes changes in SOD and CAT levels in these cerebral areas (Frey et al., 2006b). In conclusion, the results of the present study suggested that oxidative stress plays an important role in ouabain rat model of mania bipolar; i.e. an increase lipid peroxidation and protein carbonyls. This is accompanied by an imbalance between antioxidant enzymes that could result in hydrogen peroxide accumulation and potential for oxidative damage. Since that the decrease of Na+/K+-ATPase activity and oxidative stress can be observed in bipolar patients, this study may explain at least in part the underlying mechanisms of bipolar disorder. Acknowledgements This research was supported by grants from CNPq (FD-P and JQ), FAPESC (FD-P and JQ), Instituto Ce´rebro e Mente (JQ) and UNESC (FD-P and JQ). FD-P and JQ are CNPq Research Fellows. SSV and MM are holders of CAPES Studentships, AVS is holder of CNPq Studentships. References Abdalla, D.S., Monteiro, H.P., Oliveira, J.A´., Bechara, E.J., 1986. Activities of superoxide dismutase and glutathione peroxidase in schizophrenic and manic-depressive patients. Clin. Chem. 32, 805–807. Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126.
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Intracerebroventricular ouabain administration induces oxidative stress in the rat brain Rafael E. Riegel a, Samira S. Valvassori a, Morgana Moretti a, Camila L. Ferreira a, Amanda V. Steckert b, Bruna de Souza b, Felipe Dal-Pizzol b, Joa˜o Quevedo a,* a
Laboratory of Neurosciences and National Institute for Translational Medicine, Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806-000 Criciu´ma, SC, Brazil Laboratory of Experimental Pathophysiology and National Institute for Translational Medicine, Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806-000 Criciu´ma, 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 15 December 2009 Received in revised form 24 January 2010 Accepted 6 February 2010
Intracerebroventricular (ICV) injection of ouabain (a potent Na+/K+-ATPase inhibitor) in rats resulted in manic-like effects. There is an emerging body of data indicating that major neuropsychiatric disorders, such as bipolar disorder and schizophrenia, are associated with increased oxidative stress. In this study, we investigated the effects of ICV ouabain injection on oxidative stress parameters in total tissue of rat brain. Our findings demonstrated that ICV injection increased thiobarbituric acid reactive species levels and protein carbonyl generation in the prefrontal cortex and hippocampus of rats. Moreover, the activity of the antioxidants enzymes catalase and superoxide dismutase was altered in several areas of the rat brain and cerebrospinal fluid of ICV ouabain-subjected rats. These results showed that Na+/K+-ATPase inhibition can lead to oxidative stress in the brain of rats. ß 2010 ISDN. Published by Elsevier Ltd. All rights reserved.
Keywords: Bipolar disorder Mania Ouabain Oxidative stress
Bipolar disorder (BD), also known as manic-depressive illness, is a brain disorder that causes unusual shifts in a person’s mood, energy, and ability to function. The defining clinical feature of the condition is the manic episodes. Mania is characterized by an elated or irritable mood, reduced need for sleep, psychomotor activation, and excessive involvement in potentially problematic behavior (El-Mallakh et al., 2003). Soon after the characterization of the Na+/K+-ATPase, some authors investigated its activity in mood disorders. These studies showed that Na+ 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). Besides, reduced enzyme activity of Na+/K+-ATPase and increased intracellular sodium and calcium in erythrocytes of bipolar patients have been reported (Looney and El-Mallakh, 1997). Nevertheless, the biochemical mechanism underling the development of the alteration caused by the Na+/K+-ATPase inhibition remains to be clarified. In preclinical studies, when the potent sodium pump inhibitor, ouabain, is intracerebroventricularly (ICV) administered in rats, it
* Corresponding author at: Laborato´rio de Neurocieˆncias, PPGCS, UNASAU, Universidade do Extremo Sul Catarinense, 88806-000 Criciu´ma, SC, Brazil. Fax: +55 48 3443 4817. E-mail address: [email protected] (J. Quevedo). 0736-5748/$36.00 ß 2010 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2010.02.002
induces a dose-dependent 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; Riegel et al., 2009). These observations suggest that ICV ouabain injection in rats evokes some Na+/K+-ATPase dysfunction, similar to that reported in bipolar patients. There is an emerging body of data indicating that major neuropsychiatric disorders, such as BD and schizophrenia, are associated with increased oxidative stress and changes in antioxidant enzymatic defense (Kuloglu et al., 2002; Ranjekar et al., 2003; Ozcan et al., 2004). It is well known that the brain is particularly prone to oxidative damage due to its relative high content of peroxidable fatty acids and limited antioxidant capacity (Floyd, 1999). Increased neuronal oxidative stress levels generate deleterious effects on signal transduction, structural plasticity and cellular resilience, mostly by inducing lipid peroxidation in membranes, proteins and genes (Mahadik et al., 2001; Scha¨fer et al., 2004). Evidence from literature have shown that individuals with bipolar disorder have increased serum thiobarbituric acid reactive substances (TBARS) in the initial manic episode (Machado-Vieira et al., 2007; Frey et al., 2006a), and in all phases of the bipolar illness (Andreazza et al., 2007). Previously we reported increased TBARS levels and superoxide generation in submitocondrial particles in the rat brain after ouabain ICV injection (Riegel et al., 2009), suggesting that oxidative stress could be causing mitochondrial damage, also related in BD. However, the
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generation of superoxide and mitochondrial membrane damage cannot extend throughout the cell, by action of antioxidant enzymes (Halliwell and Gutteridge, 1999). Oxidative and antioxidative molecules have been studied in several psychiatric disorders and many of these molecules, such as superoxide dismutase (SOD) and catalase (CAT) are involved in the pathophysiology of BD (Kunz et al., 2008). SOD is a potent protective enzyme that can selectively scavenge the superoxide anion radical (O2 ) by catalyzing its dismutation to hydrogen peroxide (H2O2) and CAT metabolizes the excess of H2O2 producing O2 + H2O, decreasing the intracellular redox status. Previous studies reported that patients with BD have significant alterations in antioxidant enzymes (Kuloglu et al., 2002; Abdalla et al., 1986). Several studies support a model of BD that involves dysfunction within subcortical (striatal-thalamic)-prefrontal networks and the associated limbic modulating region (amygdala). It seems that in BD there may be diminished prefrontal modulation of subcortical and medial temporal structures within the anterior limbic network (amygdala and anterior striatum), leading to a dysfunction of mood (see Strakowski et al., 2005). In addiction, BD postmortem studies indicate abnormal transmission in the hippocampus; also, cognitive disturbances during acute mood episodes of BD patients, suggesting that the hippocampus plays an important role in the pathophysiology of BD (see Frey et al., 2007a). Besides, impaired mitochondrial metabolism in cerebrospinal fluid (CSF) (Regenold et al., 2009) and a significant CSF increase in the adjacent outer ventricular sulci were found in BD patients (Tost et al., 2010). The present study aims to investigate the biochemical alterations induced by immediately after (to mimic an acute episode of mania) or seven days following the single ICV injection (to mimic the persistence of a manic episode) of ouabain in rats. We assessed the effects of ICV ouabain administration on lipid and protein oxidation levels (markers of oxidative stress) and SOD and CAT activity (two major antioxidant enzymes) in prefrontal, hippocampus, striatum, amygdala and CSF of rats. 1. Experimental procedures 1.1. Animal model We conducted the study using adult male Wistar rats (250–300 g) obtained from our breeding colony. The animals were housed 5 to a cage, on a 12-h light/dark cycle (lights on at 7:00 am), with free access to food and water. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behaviour (SBNeC). This study was approved by the local ethics committee (Comiteˆ de E´tica em Pesquisa da Universidade do Extremo Sul Catarinense, Protocol no 536/ 2007), and all efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. In the present study, we have extended the investigation of the effects of the administration of ouabain on oxidative stress parameters by measuring TBARS, protein carbonyl production, SOD and CAT activity in prefrontal cortex, hippocampus, striatum, amygdala and CSF that were kept frozen in 80 8C from one of our previous experiments (Riegel et al., 2009). The detailed description of the experiments has been published elsewhere (Riegel et al., 2009), therefore, here we summarize the experimental procedures and describe the subsequent steps performed for the present investigation. 1.2. Surgical procedure and treatment Animals were intraperitoneally anesthetized with ketamine (80 mg/kg) and xylasine (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 jeweler acrylic cement. Animals were tested on the third day following surgery. A 30-gauge cannula was fitted into the guide cannula and connected by a polyethylene tube to a microsyringe. The tip of the infusion cannula protruded 1.0 mm beyond the guide cannula aiming the right lateral brain ventricle. Each animal was administered 5 ml of either artificial cerebrospinal fluid (aCSF) or ouabain (10 3 M and 10 2 M; Sigma Chemical, Saint Louis, USA; dissolved in aCSF), over 30 s (El-Mallakh et al., 2003; Riegel et al., 2009) (Note: The El-Mallakh
group published reports in which the ouabain dose was reported as 10 error; the dose use in those studies was 10 3 M) (Hamid et al., 2009).
5
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1.3. Experimental design In this study were used 30 animals divided into two experimental procedures: (1) Immediately after ICV injection of ouabain at 10 3 M and 10 2 M or aCSF (n = 5 animals per group): the first model was designed in order to reproduce an acute manic episode of BD. The animals received ICV injection of ouabain or aCSF and 10 min after ICV administration were anesthetized with ketamine (30 mg/kg) and xylasine (4 mg/kg), i.p., and the CSF was drawn (80–100 ml per rat), by direct puncture of the cisterna magna with an insulin syringe (27-gauge 31/20 length). Individual samples that presented visible blood contamination were discarded. After sampling, the samples were centrifuged at 4500 g at 5 8C for 5 min, to obtain cell-free supernatants (Cruz Portela et al., 2002). CSF samples were stored at 80 8C and defrosted only when measurements were carried out. Then, the animals 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 Tris–HCl, and 1 mM EDTA, pH 7.4). The prefrontal cortex, hippocampus, striatum and amygdala were dissected in ice-cold buffer in a Petri dish. After dissection brain samples were quickly stored at 80 8C and defrosted only when measurement were carried out. (2) Seven days following the ICV injection of ouabain at 10 3 M and 10 2 M or aCSF (n = 5 animals per group): the second model was designed to mimic the persistence of manic phase of BD. The animals received a single ICV injection of ouabain or aCSF and seven days after ICV administration the CSF was draw (following the procedures mentioned above). The animals were sacrificed by decapitation and prefrontal cortex, hippocampus, striatum and amygdala were dissected (following the procedures mentioned above). Obs: The behavioral data are described in Riegel et al. (2009). 1.4. Oxidative stress parameters 1.4.1. TBARS formation To determine oxidative damage in lipid, we measured the formation of TBARS during an acid-heating reaction, as previously described (Esterbauer and Cheeseman, 1990). The samples were mixed with 1 mL of trichloroacetic acid 10% and 1 mL of thiobarbituric acid 0.67% and were then heated in a boiling water bath for 15 min. TBARS were determined spectrophotometrically by the absorbance at 535 nm. 1.4.2. Carbonyls protein formation 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. All biochemical measures were normalized to the protein content with bovine serum albumin as standard (Lowry et al., 1951). 1.4.3. Superoxide dismutase (SOD) activity This method for the assay of SOD activity is based on the capacity of pyrogallol to autoxidize, a process highly dependent on O2 ; a substrate for SOD (Marklund, 1985). 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 performed 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 U of SOD, and the specific activity is represented as units per mg of protein. 1.4.4. Catalase (CAT) 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 0.1–0.3 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 mg protein. 1.4.5. Statistical analysis All data are presented as mean SEM. Differences between experimental groups were determined by one-way ANOVA followed by Tukey post hoc test if applicable. P values less than 0.05 were considered to indicate statistical significance.
2. Results (1) The results of experiment 1 (immediately after ICV injection) are shown in Fig. 1: (1A) When compared with the control group, TBARS generation was increased in the CSF at both
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Fig. 1. Effects of the ouabain (10 3 M and 10 2 M) or saline ICV injection immediately after the administration on (A) TBARS levels, (B) protein carbonyl formation, (C) SOD and (D) CAT activity in the prefrontal cortex, hippocampus, striatum, amygdala and CSF of rats. Bars represent means standard error of means of 4–5 animals. *P < 0.05 vs. saline group, according to ANOVA followed by the Tukey test.
10 3 M and 10 2 M doses (P < 0.01). Significant changes were not detected in the TBARS generation at any dosage in the prefrontal cortex, hippocampus, striatum and amygdala. (1B) A significant increased in carbonyl group formation was detected in the prefrontal cortex at the concentrations of 10 3 M and 10 2 M (P < 0.01 for all concentrations), striatum at 10 2 M (P < 0.01), amygdala at the concentration of 10 3 M (P < 0.01) and CSF at the concentrations of 10 3 M and 10 2 M (P < 0.01).
According to Fig. 1C and D, significant changes were not detected in the SOD and CAT activity at any dosage in the prefrontal cortex, hippocampus, striatum and amygdala, however, activity of these enzymes was decreased in the CSF at both 10 3 M and 10 2 M doses. (2) The results of experiment 2 (seven days after ICV injection) are shown in Fig. 2: (2A) A significant increase in TBARS generation in the prefrontal cortex (P < 0.001), and hippocampus
Fig. 2. Effects of the ouabain (10 3 M and 10 2 M) or saline ICV injection seven days following the administration on (A) TBARS levels, (B) protein carbonyl formation, (C) SOD and (D) CAT activity in the prefrontal cortex, hippocampus, striatum, amygdala and CSF of rats. Bars represent means standard error of means of 4–5 animals. *P < 0.05 vs. saline group, according to ANOVA followed by the Tukey test.
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(P < 0.001) was detected in rats treated with ouabain at 10 3 M and in the CSF at the concentration of 10 3 M and 10 2 M (P < 0.01), compared to control group. (2B) We observed an increase in protein damage in the prefrontal (P < 0.01) and hippocampus (P < 0.01) at the concentration of 10 3M and in the CSF at the concentration of 10 3 M and 10 2 M (P < 0.01 for all concentrations). No significant changes in carbonyl group formation were detected in the striatum and amygdala of rats. Fig. 2C shows that there is a significant increase in the SOD activity in the prefrontal cortex (P < 0.01) and hippocampus (P < 0.01) and decrease in its activity in the CSF (P < 0.01) in rats treated with ouabain at 10 3 M and 10 2 M. We did not find differences in the SOD activity in the striatum and amygdale. (2D) An increase in CAT activity was detected in the prefrontal cortex (P < 0.05) and hippocampus (P < 0.05) at the concentrations of 10 2 M. In contrast, ouabain at 10 3 M decreased CAT activity in these cerebral areas (P < 0.05). In the CSF, ouabain 10 3 M and 10 2 M decreased CAT activity (P < 0.05) and there were no changes in this enzyme activity in the striatum and amygdala. 3. Discussion In this study, we demonstrated that ouabain injection induced an increased TBARS formation in the rat CSF and a long-term, but not immediate, increased TBARS generation in prefrontal and hippocampus of the rat brain. Moreover, we showed that a significant elevation of protein carbonyl formation was observed immediately after, and also seven days following the ICV administration of ouabain. Interesting enough, the increase in TBARS observed after seven days of ouabain injection coincide exactly with elevation of protein carbonyl formation, i.e. these effects are coincident in terms of ouabain dose and brain structures involved. Interestingly, in the previous study we demonstrated an increased of superoxide content and damage to lipid in submitochondrial particles in brain of rats submitted to ouabain ICV injection (Riegel et al., 2009). Together, the previous study and the present data seem to show that the ouabain-induced production of superoxide and damage to mitochondrial membrane affect the entire cell, which can lead to tissue damage. Despite the proliferation of information on oxidative stress mechanisms in the BD, the different biochemical pathways that might mediate oxidative stress in BD are not yet fully explained. With the present data we suggest that the sodium pump activity reduced in BD can be an important link in the pathological response to oxidative stress. Moreover, in the present model as in previous study (Riegel et al., 2009); we demonstrate that oxidative stresses are most evident seven days after the ICV ouabain injection. Whereas acute reactions to ouabain in animals have considerable homology to a manic episode (as reduction in the Na+/K+ATPase and consequent hyperactivity), the persistence of effect of ouabain mirrors aspects of illness progression. From these observations we suggest that ICV administration of ouabain is a good model to study the chronicity of BD.A very important finding that we can observe with the analysis of the results is that when administered ouabain at the dose of 10 3 M SOD activity increases while the activity of catalase decreases in prefrontal cortex, hippocampus and CSF (the same structures in which was observed damage to protein and lipid). SOD is an enzyme able in reducing the superoxide radical into hydrogen peroxide (H2O2), which is 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. It is important to note that seven days after ouabain ICV injection there was an increase of oxidative damage in the hippocampus and prefrontal cortex, but not in the striatum and amygdala. This may be due to the increase of SOD and decreased of CAT activity in the prefrontal cortex and hippocampus. In the striatum and amygdala the concentrations of these enzymes seem to remain constant when compared with the control group, which may be protecting these brain regions.Furthermore, when administered ouabain at the dose of 10 2 M increased both the activity of SOD and CAT, which may be protecting the tissue (note that the ouabain administration at the dose of 10 2 M does not cause damage to protein and lipid in brain tissue). In contrast, in the CSF 10 2 M ouabain administration lead to lipid and protein damage; also, increases the activity of SOD and decreases the activity of CAT. We believe that this discrepancy is by fact that the CSF is first exposed to ouabain, is the CSF that leads ouabain to all parts of the brain and perhaps by being constantly exposed cannot reverse the damage induced by ouabain. Besides, the brain essentially floats in CSF, so the CSF cushions the organ from trauma and reduces the pressure on the base of the brain, keeping its volume constant (see Siegel, 2006). After administration of ouabain may be a decrease in production of cerebrospinal fluid for the brain to maintain its volume. Moreover, CSF has low immune defense (Van-Bambeke and Tulkens, 2009), which may explain at least in part the severe oxidative damage in the CSF, both immediately after and seven days after ouabain injection. As seen in patients (Kuloglu et al., 2002; Ranjekar et al., 2003; Ozcan et al., 2004; Frey et al., 2007b), here we demonstrated that ouabain treated rats had higher SOD activity (10 2 M and 10 3 M), and lower CAT levels (10 3 M) than the control in prefrontal cortex, hippocampus and CSF, suggesting that ouabain exposure modulated SOD and CAT activity with a distinct pattern for each brain region and dosage regimen. Additionally, the amphetamine model of mania in rats also supports the view that oxidative stress is occurring during a maniclike state. In fact, chronic treatment with amphetamine increases TBARS (Frey et al., 2006a) and protein oxidation (Valvassori et al., 2008) in the hippocampus and prefrontal cortex and causes changes in SOD and CAT levels in these cerebral areas (Frey et al., 2006b). In conclusion, the results of the present study suggested that oxidative stress plays an important role in ouabain rat model of mania bipolar; i.e. an increase lipid peroxidation and protein carbonyls. This is accompanied by an imbalance between antioxidant enzymes that could result in hydrogen peroxide accumulation and potential for oxidative damage. Since that the decrease of Na+/K+-ATPase activity and oxidative stress can be observed in bipolar patients, this study may explain at least in part the underlying mechanisms of bipolar disorder. Acknowledgements This research was supported by grants from CNPq (FD-P and JQ), FAPESC (FD-P and JQ), Instituto Ce´rebro e Mente (JQ) and UNESC (FD-P and JQ). FD-P and JQ are CNPq Research Fellows. SSV and MM are holders of CAPES Studentships, AVS is holder of CNPq Studentships. References Abdalla, D.S., Monteiro, H.P., Oliveira, J.A´., Bechara, E.J., 1986. Activities of superoxide dismutase and glutathione peroxidase in schizophrenic and manic-depressive patients. Clin. Chem. 32, 805–807. Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126.
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