Effects of mood stabilizers on oxidative stress-induced cell death signaling pathways in the brains of rats subjected to the ouabain-induced animal model of mania: Mood stabilizers exert protective effects against ouabain-induced activation of the cell death pathway

https://doi.org/10.1016/j.jpsychires.2015.04.009Get rights and content

Highlights

  • Ouabain induces hyperactivity, which is considered a manic-like behavior.

  • Ouabain decreases Bcl-2 in brains of rats.

  • Ouabain alters oxidative stress parameters, BAX and pp53 in brains of rats.

  • Lithium and Valproate protect against cellular dysfunctions ouabain-induced.

Abstract

The present study aimed to investigate the effects of mood stabilizers, specifically lithium (Li) and valproate (VPA), on mitochondrial superoxide, lipid peroxidation, and proteins involved in cell death signaling pathways in the brains of rats subjected to the ouabain-induced animal model of mania. Wistar rats received Li, VPA, or saline twice a day for 13 days. On the 7th day of treatment, the animals received a single intracerebroventricular injection of ouabain or aCSF. After the ICV injection, the treatment with mood stabilizers continued for 6 additional days. The locomotor activity of rats was measured using the open-field test. In addition, we analyzed oxidative stress parameters, specifically levels of phosphorylated p53 (pp53), BAX and Bcl-2 in the brain of rats by immunoblot. Li and VPA reversed ouabain-related hyperactivity. Ouabain decreased Bcl-2 levels and increased the oxidative stress parameters BAX and pp53 in the brains of rats. Li and VPA improved these ouabain-induced cellular dysfunctions; however, the effects of the mood stabilizers were dependent on the protein and brain region analyzed. These findings suggest that the Na+/K+-ATPase can be an important link between oxidative damage and the consequent reduction of neuronal and glial density, which are both observed in BD, and that Li and VPA exert protective effects against ouabain-induced activation of the apoptosis pathway.

Introduction

Bipolar disorder (BD) is one of the most severely debilitating medical conditions, with significant functional impairment, morbidity, and mortality. Nevertheless, little is known about the precise neurobiological underpinnings of BD (Zarate et al., 2006). Several studies have reported hypoactivation and a reduction in neuronal and glial density, primarily in limbic regions, in patients with BD (Ongür et al., 1998, Vizueta et al., 2012, Vostrikov et al., 2007).

Accumulating evidence has suggested that oxidative stress has a central role in BD (Andreazza et al., 2009, Berk et al., 2011, Gigante et al., 2010, Magalhães et al., 2011). An excessive increase in reactive oxygen species (ROS), resulting in oxidative stress and subsequent oxidative damage to lipids, proteins and nucleic acids, can lead to the activation of a cell death signaling pathway (Galluzzi et al., 2009). In fact, the protein and mRNA of BAX, a molecule known to be an apoptotic factor, have been found to be altered in BD (Kim et al., 2010, Polter et al., 2010). Moreover, studies of the brains of bipolar patients have demonstrated decreased levels of Bcl-2 and BDNF, which are anti-apoptotic factors (Kim et al., 2010, Thompson Ray et al., 2011).

Mitochondria have a central role in neuronal apoptotic signaling, primarily through ROS generation. The generation of ROS by mitochondria activates p53, which induces the transcription of pro-apoptotic proteins, such as Bax. Bax returns to mitochondria, mediating the release of intermembrane space proteins, including cytochrome c, which then leads to neuronal death (Niizuma et al., 2009). It is known that anti-apoptotic members of the Bcl-2 family, such as Bcl-2 or Bcl-xL, inhibit Bax activation. Members of the Bcl-2 protein family contain BH1 and BH2 domains, which are required for heterodimerization with Bax and the inhibition of cell apoptosis. Indeed, the ratio of Bcl-2 to Bax determines whether survival or death will occur following an apoptotic stimulus (Oltvai et al., 1993, Sedlak et al., 1995).

Animal models serve as a powerful tool for investigating the neurobiological mechanisms underlying psychiatric disorders (Valvassori et al., 2010). In recent years, many research groups have demonstrated the presence of oxidative stress factors and mitochondrial alterations in animal models of mania, similar to observations in bipolar patients. In the animal models of mania, the hyperactivity induced by stimulants is accompanied by reductions of tricarboxylic acid cycle enzymes and mitochondrial complexes I, II, I–III and IV (Lopes-Borges et al., 2015, Valvassori et al., 2010). Therefore, the study of the mitochondrial alterations in animal models of mania is very important for understanding the pathophysiology of this disorder.

Intracerebroventricular (ICV) injection of ouabain induces hyperactivity in rats by inhibiting the Na+/K+-ATPase (El-Mallakh et al., 1995), and it has been suggested as a relevant animal model of mania (Machado-Vieira et al., 2004, Young et al., 2011). Studies with this animal model have shown that the manic-like hyperactivity induced by ouabain is associated with severe brain damage due to an increased formation of lipid and protein oxidation products and a decrease of BDNF levels (Jornada et al., 2010, Jornada et al., 2011). In this context, several studies have reported Na+/K+-ATPase alterations in BD patients (Banerjee et al., 2012, El-Mallakh and Wyatt, 1995, Huff et al., 2010). Thus, the Na+/K+-ATPase may be connected to the increased apoptosis observed in BD.

Additionally, the mood stabilizers lithium (Li) and valproate (VPA) are traditionally used to treat BD. They engender reasonable protection against recurrent mood episodes and have modest antidepressant properties (Davis et al., 2005). Studies of the mechanisms of action of mood stabilizers have revealed that both Li and VPA have direct actions in neurons, including protection against cell death and the enhancement of neurogenesis (Chuang, 2005, Manji and Duman, 2001).

Because of these findings, we designed the present study to investigate mitochondrial superoxide content, lipid peroxidation and pp53, BAX and Bcl-2 levels in the brain of rats undergoing ICV administration of ouabain and to evaluate the effects of Li and VPA in this context.

Section snippets

Animals

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 Behavior (SBNeC). This study was approved by the local ethics committee, Comitê de

Results

Fig. 1 shows the effect of Li and VPA treatment on the manic-like behavior elicited by ICV ouabain administration in rats. The two-way ANOVA revealed significant effects of ouabain administration [F(1.66) = 180.94, p < 0.001] and treatment [F(2.66) = 132.56, p < 0.001] and a significant ouabain administration × treatment interaction [F(2.66) = 160.93]. Further analysis with Tukey's post hoc test showed that administration of ouabain increased locomotion in rats (crossings), and this effect was

Discussion

The present study was able to reproduce previous results from our research laboratory, in which VPA and Li prevented the hyperlocomotion induced by ouabain, a potent Na+/K+-ATPase inhibitor (Resende et al., 2013, Jornada et al., 2010). Several clinical studies have demonstrated that Na+ pump activity is decreased in acute mania compared to recovered euthymic bipolar individuals (Hesketh et al., 1977, Naylor et al., 1980, Reddy et al., 1992). Kirshenbaum and colleagues (Martin, 2011) found that

Role of funding source

CNPq, FAPESC, CAPES and UNESC.

Contributors

Samira S. Valvassori and João Quevedo designed the study, wrote the protocol and the first draft of the manuscript. Marcelo F. Vitto, Gabrielle Luz, and Claudio T. de Souza made the biochemical analysis. Samira S. Valvassori undertook the statistical analysis. Wilson R. Resende, Jéssica Lopes-Borges and Edemilson Mariot were responsible for the surgical procedures, pharmacological treatment and behavioral tests. All authors contributed to and have approved the final manuscript.

Conflict of interest

None of the authors or funding sources has conflict of interest.

Acknowledgments

We thank CNPq, FAPESC, CAPES and UNESC for financial support.

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