European Journal of Pharmacology 724 (2014) 132–139
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Neuroprotective and antioxidant effects of curcumin in a ketamine-induced model of mania in rats Marta Gazal a, Matheus R. Valente a, Bruna A. Acosta a, Fernanda N. Kaufmann a, Elizandra Braganhol b, Claiton L. Lencina b, Francieli M. Stefanello b, Gabriele Ghisleni a,n, Manuella P. Kaster a a b
Programa de Pós-Graduação em Saúde e Comportamento, Universidade Católica de Pelotas, Pelotas, Rio Grande do Sul, Brazil Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 28 September 2013 Received in revised form 12 December 2013 Accepted 18 December 2013 Available online 30 December 2013
Bipolar disorder (BD) is a chronic and debilitating illness characterized by recurrent manic and depressive episodes. Our research investigates the protective effects of curcumin, the main curcuminoid of the Indian spice turmeric, in a model of mania induced by ketamine administration in rats. Our results indicated that ketamine treatment (25 mg/kg, for 8 days) induced hyperlocomotion in the open-field test and oxidative damage in prefrontal cortex (PFC) and hippocampus (HP), evaluated by increased lipid peroxidation and decreased total thiol content. Moreover, ketamine treatment reduced the activity of the antioxidant enzymes superoxide dismutase and catalase in the HP. Pretreatment of rats with curcumin (20 and 50 mg/kg, for 14 days) or with lithium chloride (45 mg/kg, positive control) prevented behavioral and pro-oxidant effects induced by ketamine. These findings suggest that curcumin might be a good compound for preventive intervention in BD, reducing the episode relapse and the oxidative damage associated with the manic phase of this disorder. & 2013 Elsevier B.V. All rights reserved.
Keywords: Curcumin Ketamine Mania Antioxidant Neuroprotective
1. Introduction Bipolar disorder (BD) is a chronic and debilitating psychiatric condition characterized by cycling episodes of mania and depression. BD is a leading cause of disability among individuals with medical and psychiatric conditions, and it is associated with substantial morbidity and mortality. The unique hallmark of this disorder is the presence of acute mania, defined as a state of excessive energy, associated with euphoria, irritable mood, impulsivity and psychosis (Andreazza et al., 2008). Increasing evidence suggests that oxidative stress mediates neuropathological processes in neuropsychiatric disorders and is involved in the etiology and progression of BD (Andreazza et al., 2007; Machado-Vieira et al., 2007; Selek et al., 2008). Oxidative stress is a condition in which the balance between production of reactive oxygen species and levels of antioxidants is disturbed, resulting in cell damage and death. The brain is particularly susceptible to oxidative stress because it metabolizes 20% of total body oxygen and has a limited amount of antioxidant capacity
n Correspondence to: Programa de Pós-Graduação em Saúde e Comportamento, Centro de Ciências da Vida e da Saúde, Universidade Católica de Pelotas, Rua Gonçalves Chaves 373, 96015560 Pelotas, Rio Grande do Sul, Brazil. Tel.: þ 55 53 2128 8031; fax: þ 55 53 2128 8229. E-mail address: [email protected] (G. Ghisleni).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.12.028
(Halliwell and Gutteridge, 2007). Reactive oxygen species contribute to the development of neurodegeneration by targeting different substrates in the cells, causing protein, DNA and RNA oxidation, or lipid peroxidation (Gandhi and Abramov, 2012). Curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is the main curcuminoid of the popular Indian spice turmeric, obtained from rhizome of Curcuma longa Linn. The multiple beneficial effects of curcumin could be linked to its antioxidant and anti-inflammatory properties in in vivo and in vitro models (Kulkarni et al., 2008; Bhatia et al., 2011; Gupta et al., 2012; Lopresti et al., 2012; Khurana et al., 2012; Jiang et al., 2013). Curcumin is also effective against cancer, cardiovascular, liver and inflammatory diseases, neurodegenerative disorders and depression (Monroy et al., 2013). The anti-inflammatory potential of curcumin, is related to the inhibition of nuclear factor kappa B (NFkB) signaling and reduction of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α. Curcumin is also a potent inhibitor of reactive-oxygen-generating enzymes such as lipoxygenase/cyclooxygenase, xanthine dehydrogenase/oxidase, and inducible nitric oxide synthase (Lin, 2007). The efficacy of curcumin as a neuroprotective agent in several preclinical models has created considerable excitement mainly due to its low toxicity and cost, suggesting that this compound might be a worthy candidate for prophylactic intervention in mental disorders. Despite the challenge in replicating the complex symptoms associated with psychiatric disorders, animal models have been
M. Gazal et al. / European Journal of Pharmacology 724 (2014) 132–139
successfully able to mimic some of the neurochemical and physiological characteristics of conditions (Kato et al., 2007). Animal models of mania are scarce and generally based on the management of hyperlocomotion-inducing agents such as D-amphetamine, ketamine and ouabain (Frey et al., 2006; Ghedim et al., 2012). These models represent useful pharmacological tools to assess behavioral and biochemical alterations observed during the mania episode, as well as to assess the effect of possible moodstabilizing agents. In this context, the present work hypothesized that curcumin administration might prevent some of the behavioral and neurochemical modifications in a model of mania induced by ketamine in rats.
2. Materials and methods 2.1. Animals and drug treatments Female adult Wistar rats aged 11–12 weeks (250–300 g) were obtained from the Central Animal House of the Federal University of Pelotas, Pelotas, RS, Brazil. Animals were maintained under controlled environment (23 7 2 1C, 12 h-light/dark cycle, free access to food and water) and handled according to the Federation of Brazilian Societies for Experimental Biology guidelines upon approval by the Ethics Committee of the Federal University of Pelotas, Brazil. The following drugs were used: ketamine (Sigma Chemical Co., USA), dissolved in saline solution (NaCl 0.9%, w/v) and administrated by intraperitoneal route (i.p.) and curcumin (Sigma Chemical Co., USA), dissolved in peanut oil and administered by oral route (p.o.), lithium chloride was dissolved in (NaCl 0.9%, w/v) and administered by p.o. route twice a day. Appropriated vehicle groups were also assessed simultaneously. The doses of ketamine, curcumin and lithium used in the present study were chosen according to the literature (Kulkarni and Dhir, 2010; Ghedim et al., 2012; Bruning et al., 2012). 2.2. Experimental protocol of mania state This protocol was designed to mimic the prevention protocol of the mania state, as previously proposed by Ghedin et al. (2012). Rats received peanut oil, curcumin 20 mg/kg or curcumin 50 mg/ kg, once a day for 14 days. From the 8th to the 14th day the animals also received saline or ketamine (25 mg/kg), once a day, totaling six experimental groups: saline/penaut oil, ketamine/ penaut oil, saline/curcumin 20 mg/kg, ketamine/curcumin 20 mg/ kg, saline/curcumin 50 mg/kg, ketamine/curcumin 50 mg/kg. In a separate set of experiments rats received saline or lithium chloride (45 mg/kg, twice a day, used as a positive control) for 14 days. From the 8th to the 14th day the animals also received saline or ketamine (25 mg/kg), once a day, totaling four experimental groups: saline/saline, ketamine/saline, saline/lithium chloride, ketamine/lithium chloride. On the 15th day of treatment, the animals received a single injection of ketamine or saline and the locomotor activity was assessed in the open-field apparatus after 30 min (Fig. 1). 2.3. Behavioral analysis Locomotor and anxiety-related behavior was monitored using an open-field apparatus, as previously described (Kaster et al., 2004). The apparatus consisted of a wooden box measuring 40 60 50 cm3 with a frontal glass wall. The floor of the arena was divided into 12 equal squares and placed in a sound free room. Animals were placed in the rear left square and left to explore it freely for 5 min. The total number of squares crossed with all paws (crossing) was counted in order to evaluate the ambulatory
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Fig. 1. Treatment protocol.
behavior. The number of central crossings was the measure used to evaluate anxiety. The apparatus was cleaned up with a 10% alcohol solution and dried after each individual mouse session. 2.4. Biochemical assay Rats were killed by decapitation immediately after the openfield test. Prefrontal cortex (PFC) and hippocampus (HP) were manually dissected and homogenized in 10 volumes (1:10 w/v) of 20 mM sodium phosphate buffer, pH 7.4 containing 140 mM KCl. Homogenates were centrifuged at 750g for 10 min at 4 1C, the pellet was discarded and the supernatant was immediately separated and used for the stress oxidative measurements. Protein was measured using biocinchoninic acid (BCA) assay using bovine serum albumin as standard. 2.4.1. Thiobarbituric acid reactive species formation (TBARS) The measure of lipid peroxidation was determined by TBARS according to the protocol described by Esterbauer and Cheeseman (1990). Briefly, homogenates were mixed with trichloroacetic acid 10% and thiobarbituric acid 0.67% and heated in a boiling water bath for 25 min. TBARS was determined by the absorbance at 535 nm. Results were reported as nmol of TBARS per mg of protein. 2.4.2. Total sulfhydryl content This assay was performed as described by Aksenov and Markesbery (2001) which is based on the reduction of DTNB by thiols, which in turn, becomes oxidized (disulfide) generating a yellow derivative (TNB) whose absorption is measured spectrophotometrically at 412 nm. Briefly, homogenates were added to PBS buffer pH 7.4 containing EDTA. The reaction was started by the addition of 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB). Results were reported as μmol TNB per mg of protein. 2.4.3. Catalase (CAT) assay CAT activity was assayed by the method of Aebi (1984). H2O2 disappearance was continuously monitored during 90 s in a spectrophotometer adjusted at 240 nm. CAT specific activity was reported as units of enzyme per mg of protein. 2.4.4. Superoxide dismutase (SOD) assay Total SOD activity was measured by the method described by Misra and Fridovich (1972). This method is based on the inhibition of superoxide dependent adrenaline auto-oxidation in a spectrophotometer adjusted at 480 nm. The specific activity of SOD was reported as units per mg of protein. 2.5. Statistical analysis Comparisons between experimental groups were performed by one-way or two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc tests when appropriate. The values are expressed as mean 7 S.E.M. P o0.05 was considered significant.
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Fig. 2. Effect of curcumin (20 and 50 mg/kg, p.o.) on ketamine-induced hyperactivity (A); anxiety behavior (B); effect of lithium chloride (45 mg/kg, p.o., twice a day) on ketamine-induced hyperactivity (C); anxiety behavior (D) in the open-field test. The number of crossings was recorded. Data are given as the mean(s) 7 S.E.M. (n¼ 6–7 for group). nn Denotes Po 0.01 as compared to the vehicle/saline group. ♯ Denotes P o 0.05 as compared to the vehicle/ketamine group.
3. Results 3.1. Behavioral characterization focusing on ambulatory performance The treatment with ketamine was efficient to induce hyperlocomotion in rats, evaluated by the increase in the number of crossings in the open-field test, which is an indication of mania episode (Ghedim et al., 2012). Notably, as presented in Fig. 2A curcumin pretreatment (20 and 50 mg/kg) prevented the hyperlocomotion induced by ketamine in the open-field test (curcumin pretreatment: [F(2,26)¼ 4.11, Po0.05], ketamine treatment: [F(1,26)¼ 16.93, Po0.05], interaction: [F(2,26)¼7.99, Po0.05]). In addition, Fig. 2C lithium chloride pretreatment (45 mg/kg) prevented the hyperlocomotion induced by ketamine in the open-field test (lithium chloride pretreatment: [F(1,23)¼ 8.4, Po0.01], ketamine treatment: [F(1,23)¼23.08, Po0.01], interaction: [F(1,23)¼13.99, Po0.01]). As demonstrated in Fig. 2B and D, no changes in the anxiety behavior were observed for percentage of central crossings after ketamine treatment, curcumin or lithium chloride pretreatment (curcumin pretreatment: [F(2,36) ¼0.60, P ¼0.55], ketamine treatment: [F(1,36) ¼0.28, P ¼0.59], interaction: [F(2,36) ¼0.25, P ¼0.77]) and (lithium chloride pretreatment: [F(1,20)¼0.005, P ¼0.93], ketamine treatment: [F(1,20)¼0.60, P ¼0.44], interaction: [F(1,20)¼0.09, P ¼0.76]). 3.2. Measurement of oxidative stress parameters in the prefrontal cortex (PFC) In order to consolidate the notion of a neuroprotective effect of curcumin against ketamine-induced model of mania, we evaluated the effects of curcumin in oxidative stress parameters in the PFC. Fig. 3A shows that curcumin pretreatment (20 and 50 mg/kg) was
able to prevent the increase in TBARS levels in PFC induced by ketamine administration (curcumin pretreatment: [F(2,35)¼0.48, P¼ 0.62], ketamine treatment: [F(1,35)¼0.79, P¼0.38], interaction: [F(2,35)¼6.62, Po0.05]). In addition, Fig. 3B shows that curcumin (20 and 50 mg/kg) prevented the decrease in sulfhydryl content induced by ketamine in PFC (curcumin pretreatment: [F(2,36)¼4.34, Po0.05], ketamine treatment: [F(1,36)¼0.30, P¼ 0.60], interaction: [F(2,36)¼11.95, Po0.01]). We then have compared the activity of the antioxidant enzymes SOD and CAT. As shown in Fig. 3C curcumin and ketamine administration did not change the activity of SOD in the PFC (curcumin pretreatment: [F(2,28)¼0.50, P¼0.60], ketamine treatment: [F(1,35)¼ 0.71, Po0.01], interaction: [F(2,28)¼ 0.03, P¼0.97]). There was also no evident difference in the CAT activity between groups (curcumin pretreatment: [F(2,36)¼0.10, P¼ 0.94], ketamine treatment: [F (1,36)¼13.62, Po0.01], interaction: [F(2,36)¼1.61, P¼0.20]). In order to use a mood-stabilizing agent as a positive control, the effects of lithium chloride (45 mg/kg, p.o.) in oxidative stress parameters in the PFC were evaluated (Table 2). Lithium chloride was able to prevent the increase in TBARS levels induced by ketamine in the PFC (lithium chloride pretreatment: [F(1,20) ¼ 3.50, P¼ 0.07], ketamine treatment: [F(1,20)¼8.06, P o0.01], interaction: [F(1,20)¼ 14.57, Po 0.01]). In addition, lithium chloride prevented the decrease in sulfhydryl content induced by ketamine in the PFC (lithium chloride pretreatment: [F(1,19) ¼ 37.30, Po 0.01], ketamine treatment: [F(1,19) ¼ 15.50, P o0.01], interaction: [F(1,19) ¼20.42, P o0.01]). Pretreatment with lithium chloride did not change SOD activity in the PFC (lithium chloride pretreatment: [F(1,20) ¼0.69, P ¼0.42], ketamine treatment: [F (1,20) ¼0.60, P¼ 0.45], interaction: [F(1,20) ¼3.65, P ¼0.07]). In addition, no differences were found in the CAT activity (lithium chloride pretreatment: [F(1,19)¼7.93 Po0.01], ketamine treatment: [F(1,19)¼ 1.28, P¼0.27], interaction: [F(1,19)¼0.60, P¼ 0.44]).
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Fig. 3. Effects of curcumin (20 and 50 mg/kg, p.o.) on TBARS formation (A); total thiol content (B); superoxide dismutase (SOD, C); and catalase (CAT, D) activity in the prefrontal cortex of rats. Data was expressed as mean þS.E.M. (n ¼6–7 for group). nn Denotes P o 0.01 and n P o 0.05 as compared to the vehicle/saline group. ♯ Denotes Po 0.05 as compared to the vehicle/ketamine group.
3.3. Measurement of oxidative stress parameters in the hippocampus (HP) We next have evaluated the effects of curcumin in oxidative stress parameters in the HP. As depicted in Fig. 4A, curcumin pretreatment (50 mg/kg) was able to prevent increase in the TBARS levels induced by ketamine administration in rats (curumin pretreatment: [F(2,34) ¼ 2.08, P ¼0.14], ketamine treatment: [F(1,34)¼5.30, P o0.05], interaction: [F(2,34) ¼ 4.24, P o0.05]). The results in Fig. 4B demonstrated that ketamine treatment did not change the total SH content when compared to the control group. However, the treatment with curcumin alone (50 mg/kg) or (20 and 50 mg/kg) combined with ketamine increased the sulfhydryl content in the hippocampus of rats (curcumin pretreatment [F (2,33)¼7.77, P o0.01] ketamine treatment: [F(1,33) ¼0.019, P ¼0.89], interaction: [F(2,33)¼5.96, Po 0.01]). Notably, Fig. 4C shows that SOD activity was significantly decreased by ketamine and this effect was completely prevented in curcumin (20 and 50 mg/kg) pretreated groups (curcumin pretreatment: [F(2,34) ¼ 6.52, P o0.01], ketamine treatment: [F(1,34)¼0.57, P¼ 0.45], interaction: [F(2,34) ¼3.80, P o0.05]). In addition, Fig. 4D also indicated that ketamine-treated rats had a decrease in CAT activity, and the pretreatment with both doses of curcumin (20 and 50 mg/kg) was also able to prevent this effect (curcumin pretreatment: [F(2,33)¼ 11.4, P o0.01], ketamine treatment: [F(1,33) ¼0.006, P ¼0.80], interaction: [F(2,33) ¼7.99, P o0.05]). The effects of lithium chloride (45 mg/kg, p.o.) in oxidative stress parameters in the HP were also (Table 2). Lithium chloride was able to prevent increase in the TBARS levels induced by ketamine (lithium chloride pretreatment: [F(1,20) ¼5.90, P o0.05], ketamine treatment: [F(1,20)¼4.58, Po0.05], interaction: [F(1,20)¼ 6.51, Po0.05]). The results demonstrated that ketamine treatment did not change the total SH content when compared to the control group. However, the treatment with lithium chloride (45 mg/kg)
combined with ketamine increased the sulfhydryl content in the hippocampus of rats (lithium chloride pretreatment: [F(1,19) ¼ 62.23, Po 0.01], ketamine treatment: [F(1,19) ¼39.02, P o0.01], interaction: [F(1,19) ¼96.84, P o0.01]). Finally, lithium chloride was able to prevent the decrease in CAT activity induced by ketamine in the HP of rats (lithium chloride pretreatment: [F(1,20) ¼2.46, P¼ 0.13], ketamine treatment: [F(1,20)¼ 4.90, Po 0.05], interaction: [F(1,20)¼ 5.61, Po0.05]). In addition, lithium pretreatment were able to prevent the decrease in SOD activity induced by ketamine in the HP of rats, (lithium chloride pretreatment [F(1,19) ¼ 89.10, Po 0.01], ketamine treatment: [F(1,19) ¼0.23, P ¼0.64], interaction: [F(2,33)¼ 44.06, P o0.01]). The mean values and standard deviation for all the experiments and groups are represented in Tables 1 and 2.
4. Discussion The present study shows at the first time that the administration of curcumin, an active ingredient in turmeric (Curcuma longa), is able to prevent the hyperlocomotion and changes in some oxidative stress parameters induced by ketamine in rats. Additionally these data reinforce the ketamine-induced hyperlocomotion model proposed by Ghedim et al. (2012), as an important tool to study behavioral and neurochemical alterations relevant to the mania phase of BD. Animal models are consistently used to investigate intracellular mechanisms and pharmacological approaches involved in psychiatry disorders (Kato et al., 2007). However, the complexity of the biological features of BD makes difficult to mirror important aspects of the disease such as the episodic and the cyclic status. In preclinical models, hyperlocomotion is a core behavior evaluated during the manic state of BD. Indeed, several studies show that the administration of D-amphetamine or ouabain mimics the hyperlocomotion and several other behavioral and biological aspects of mania (Gould et al., 2001; Wang et al., 2013).
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Fig. 4. Effects of curcumin (20 and 50 mg/kg, p.o.) on TBARS formation (A); total thiol content (B); superoxide dismutase (SOD, C); and catalase (CAT, D) activity in the hippocampus of rats. Data was expressed as meanþ S.E.M. (n¼ 6–7 for group). nn Denotes Po 0.01 and n P o 0.05 as compared to the vehicle/saline group. ♯ Denotes Po 0.05 as compared to the vehicle/ketamine group.
Table 1 Effect of curcumin on oxidative stress parameters in the prefrontal cortex and hippocampus of rats. Groups
TBARS (nmol/mg protein)
Total SH (lmol/mg protein)
SOD (units/mg protein)
CAT (units/mg protein)
Prefrontal cortex Saline/penaut oil Ketamine/penaut oil Saline/curcumin 20 mg/kg Ketamine/curcumin 20 mg/kg Saline/curcumin 50 mg/kg Ketamine/curcumin 50 mg/kg
0.13 70.01 0.23 70.02b 0.19 70.02 0.15 70.01c 0.17 70.02 0.16 70.01c
41.767 5.14 17.667 3.20a 38.63 7 1.62 51.98 7 5.20c 30.30 7 2.49 56.36 7 8.80c
12.40 7 3.70 8.247 1.79 16.577 6.47 10.02 7 2.63 13.767 2.24 6.247 2.42
3.45 7 0.20 4.077 0.62 3.277 0.30 4.03 7 0.29 2.92 7 0.20 4.677 0.27
Hippocampus Saline/penaut oil Ketamine/penaut oil Saline/curcumin 20 mg/kg Ketamine/curcumin20 mg/kg Saline/curcumin 50 mg/kg Ketamine/curcumin 50 mg/kg
0.10 70.02 0.14 70.02b 0.09 70.02 0.10 70.01c 0.10 70.01 0.08 70.01c
54.90 7 1.13 48.307 0.80 53.137 4.50 72.217 8.98 80.93 7 10.80 62.477 2.90
5.65 7 0.78 1.677 0.60a 7.30 7 1.56 7.80 7 1.28c 6.017 1.63 7.32 7 0.71c
2.80 7 0.40 1.28 7 0.42a 2.98 7 0.71 2.90 7 0.50c 2.42 7 0.74 2.89 7 0.85c
Thiobarbituric acid reactive species formation (TBARS); total thiol content (total SH); superoxide dismutase (SOD); and catalase (CAT). Data was expressed as mean 7 S.E.M. (n¼ 6–7 animal per group). a b c
P o 0.01 as compared to the vehicle/saline group. Po 0.05 as compared to the vehicle/saline group. Po 0.05 as compared to the vehicle/ketamine group.
Recently, Ghedim et al. (2012) reported that administration of a sub-anesthetic dose of ketamine induced hyperlocomotion and modify oxidative stress parameters in the rat brain. These observations point to a good face validity of the model. In addition, the behavioral and biochemical effects were normalized by treatment with lithium and valproic acid, pointing to a predictive validity. Moreover, several clinical and pre-clinical studies demonstrated that glutamatergic system abnormalities are involved in the patophysiology of BD, pointing to a possible construct validity of this model. Our work corroborates and extends these data suggesting that this protocol fulfills adequate characteristics as an animal model of mania and further reinforces previous proposals that both glutamatergic system and oxidative stress might be
involved in this particular stage of BD (Andreazza et al., 2008; Machado-Vieira, 2012). Pre-clinical evidences show that low doses of ketamine (5–10 mg/kg) exhibit antidepressant properties (Katalinic et al., 2013). Nevertheless, in moderate doses (10–50 mg/kg) it induces hyperlocomotion and cellular dysfunction (Gould et al., 2001; Machado-Vieira et al., 2004; Ghedim et al., 2012) and higher doses possess anesthetic and dissociative effects. From the clinical point of view, infusions of low doses of ketamine induce rapid antidepressant effects (Katalinic et al., 2013). However, due to the propensity of ketamine to produce cognitive deficits, dissociative and psychotomimetic effects, its clinical use remains limited. In fact, a case report shows that continuous administration of a higher dose of ketamine is
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Table 2 Effect of lithium chloride in oxidative stress parameters in the prefrontal cortex and hippocampus of rats. Groups
TBARS (nmol/mg protein)
Total SH (lmol/mg protein)
SOD (units/mg protein)
CAT (units/mg protein)
Prefrontal cortex Saline/saline Ketamine/saline Saline/lithium 45 mg/kg Ketamine/lithium 45 mg/kg
0.12 70.01 0.2470.03a 0.16 70.01 0.14 70.01c
49.50 74.60 18.20 73.18b 55.48 71.40 57.65 74.10c
16.707 4.30 9.577 1.42 13.80 7 2.24 16.85 7 1.43
3.53 70.20 4.16 70.65 2.90 70.05 3.01 70.25
Hippocampus Saline/saline Ketamine/saline Saline/lithium 45 mg/kg Ketamine/lithium 45 mg/kg
0.08 70.02 0.15 70.02b 0.08 70.01 0.0770.01c
53.53 70.33 47.85 70.76 50.45 71.54 75.26 72.90
6.107 0.75 1.42 7 0.63a 7.94 7 0.51 11.98 7 0.64c
2.97 70.42 1.47 70.44b 2.71 70.17 2.76 70.13c
Thiobarbituric acid reactive species formation (TBARS); total thiol content (total SH); superoxide dismutase (SOD); and catalase (CAT). Data was expressed as mean 7S.E.M. (n¼ 6–7 for group). a b c
P o0.01 as compared to the vehicle/saline group. Po 0.05 as compared to the vehicle/saline group. Po 0.05 as compared to the vehicle/ketamine group.
associated with the development of manic and psychotic symptoms in humans (Ricke et al., 2011). In pre-clinical models, non-anesthetic doses of ketamin are able to induce hyperlocomotion, stereotypy, impaired cognitive function and social interaction (Lipska and Weinberger, 2000; Bubenikova-Valesova et al., 2010). In the present work we evaluated hyperlocomotion, the core symptom of mania, which was prevented by curcumin administration and by lithium chloride, used as a positive control. We also investigated anxiety behavior, but no changes were observed after ketamine, curcumin and/or lithium chloride treatment. It is important to mention that we cannot rule out the possibility that acute effects of ketamine might also be involved in the behavioral and neurochemical changes evaluated. Indeed, da Silva et al. (2010) demonstrated that a single dose of ketamine in mice induced hyperlocomotion and increased lipid peroxidation and nitrite content in the cortex of mice. Oxidative stress is an important factor involved in the pathophysiology of major neuropsychiatric disorders, including BD (Andreazza et al., 2007, 2008; Machado-Vieira et al., 2007; Selek et al., 2008). Increased reactive oxygen species levels generate deleterious effects on signal transduction, structural plasticity and cellular resilience, mostly by inducing lipid peroxidation in membranes, damage to proteins and nucleic acids (Mahadik et al., 2001). Our study also shows that ketamine induces changes in some oxidative stress parameters like increase in TBARS levels and decrease in the total thiol content in the PFC of rats. The effects of ketamine and other manic agents on lipid peroxidation were described in different brain regions (Brocardo et al., 2010; Ghedim et al., 2012). However, it is important to highlight that this is the first work reporting a decrease in the total thiol content induced by ketamine in the PFC. This finding supports the rationale that cysteine residues involved in the crucial regulation of many key proteins and peptides might be susceptible targets of oxidation in this model. Another relevant finding of our work was that ketaminetreatment reduced total SOD and CAT activity in the HP, but not in the PFC. SOD is a group of enzymes that reduces the superoxide anion (O2 ) into hydrogen peroxide (H2O2), which can react with iron to generate highly reactant hydroxyl radicals (Halliwell and Gutteridge, 2007). In contrast, CAT is the most important peroxidase responsible for detoxification of the H2O2 and prevention of hydroxyl production. The decreased activity of SOD and CAT might have deleterious effects upon the cell, generating an excess of H2O2 and oxidative damage. Differences in the vulnerability to damage between brain regions were already described, especially in models of ischemia and kainic acid-induced seizure (Zhao and Flavin, 2000; Candelario-Jalil et al., 2001; Geddes et al., 2003). The mechanisms underlying this phenomenon are far from being
understood and differences in oxidative stress susceptibility or heterogeneous distribution of glutamatergic receptor in PFC and HP might be involved. Hippocampal neurons were extensively demonstrated in the literature to be intrinsically more vulnerable to several types of insults, including mechanical insult, traumatic injury, kainic acid toxicity, stress and ischemic insults when compared to cortical neurons (Lowenstein et al., 1992; Taft et al., 1992; Zhao and Flavin, 2000; Golarai et al., 2001; Candelario-Jalil et al., 2001; Geddes et al., 2003). Further studies are needed to determine the differences in the vulnerability of these two cellular populations to ketamine-induced oxidative stress. In addition, we cannot rule out the possibly that our treatment is also changing the levels of these proteins in these areas and consequently the activity. The main finding of this study was the ability of curcumin to prevent both the hyperlocomotion and the changes in oxidative stress parameters induced by ketamine in rats. Indeed, in recent years, several studies highlighted the ability of curcumin to promote a variety of pharmacological and biological activities including anti-inflammatory and antioxidant (Kulkarni et al., 2008; Bhatia et al., 2011; Gupta et al., 2012; Lopresti et al., 2012; Khurana et al., 2012; Jiang et al., 2013). Curcumin has been recently demonstrated to provide neuroprotection by acting as a modulator of glutamergic neurotransmission, restoring the levels of glutamergic receptors, oxidative stress and imbalanced glutamate metabolism (Jayanarayanan et al., 2013). In addition, curcumin inhibit glutamate release, block GluN2B receptors (Zhang et al., 2013), and enhance neuronal survival against NMDA toxicity (Lin et al., 2011). Some of the protective effects of curcumin against glutamate-induced toxicity and cell death in vitro are comparable to those mediated by the antimanic agent lithium (Chen et al., 2003). In addition, curcumin shares with lithium several behavioral and biological activities and it was recently demonstrated to normalizes cellular antioxidant enzymes (including SOD and catalase) and decreases oxidative stress in a cellular model of Alzheimer disease by a mechanism dependent on the inhibiting of glycogen synthase kinase-3β (GSK-3β) (Huang et al., 2012). Since the inhibition of GSK-3β is one of the major known cellular target of lithium, there is a strong possibility that these two compounds might share some of the intracellular pathways to exert their antimaniac effects. As oxidative stress and inflammation are major factors involved in the etiology and progression of several disorders, curcumin administration was effective in preclinical models of numerous conditions including neurodegenerative disorders, depression and aging (Bala et al., 2006; Menon and Sudheer, 2007; Kulkarni et al., 2008; Bhatia et al., 2011; Gupta et al., 2012; Khurana et al., 2012;
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Jiang et al., 2013). Besides that, curcumin administration improves memory function, cerebral blood flow and energy metabolism (Rajasekar et al., 2013) and enhances the levels of brain derived neurothrophic factor (BDNF) and hippocampal neurogenesis (Xu et al., 2007). A recent work discussed that by acting in all those cellular targets, curcumin might be a compound of interest to treat the mood and cognitive impairments associated with BD (Brietzke et al., 2013). Here, we were able to test, for the first time, an confirm the potential of curcumin against behavioral alteration and oxidative stress in an animal model of mania.
5. Conclusion Effective treatments for preventing mood episodes in patients with BD are urgently needed, especially because BD is perhaps the psychiatric disorder with the highest mortality rate. In addition, the neurochemical alterations associated with episode-related deterioration patterns, progress with the number of episodes (Berk et al., 2011). Thus, our findings suggest that curcumin might be a good agent for preventive intervention, reducing the episode relapse and the oxidative stress associated with the manic phase of BD.
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Neuroprotective and antioxidant effects of curcumin in a ketamine-induced model of mania in rats Marta Gazal a, Matheus R. Valente a, Bruna A. Acosta a, Fernanda N. Kaufmann a, Elizandra Braganhol b, Claiton L. Lencina b, Francieli M. Stefanello b, Gabriele Ghisleni a,n, Manuella P. Kaster a a b
Programa de Pós-Graduação em Saúde e Comportamento, Universidade Católica de Pelotas, Pelotas, Rio Grande do Sul, Brazil Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 28 September 2013 Received in revised form 12 December 2013 Accepted 18 December 2013 Available online 30 December 2013
Bipolar disorder (BD) is a chronic and debilitating illness characterized by recurrent manic and depressive episodes. Our research investigates the protective effects of curcumin, the main curcuminoid of the Indian spice turmeric, in a model of mania induced by ketamine administration in rats. Our results indicated that ketamine treatment (25 mg/kg, for 8 days) induced hyperlocomotion in the open-field test and oxidative damage in prefrontal cortex (PFC) and hippocampus (HP), evaluated by increased lipid peroxidation and decreased total thiol content. Moreover, ketamine treatment reduced the activity of the antioxidant enzymes superoxide dismutase and catalase in the HP. Pretreatment of rats with curcumin (20 and 50 mg/kg, for 14 days) or with lithium chloride (45 mg/kg, positive control) prevented behavioral and pro-oxidant effects induced by ketamine. These findings suggest that curcumin might be a good compound for preventive intervention in BD, reducing the episode relapse and the oxidative damage associated with the manic phase of this disorder. & 2013 Elsevier B.V. All rights reserved.
Keywords: Curcumin Ketamine Mania Antioxidant Neuroprotective
1. Introduction Bipolar disorder (BD) is a chronic and debilitating psychiatric condition characterized by cycling episodes of mania and depression. BD is a leading cause of disability among individuals with medical and psychiatric conditions, and it is associated with substantial morbidity and mortality. The unique hallmark of this disorder is the presence of acute mania, defined as a state of excessive energy, associated with euphoria, irritable mood, impulsivity and psychosis (Andreazza et al., 2008). Increasing evidence suggests that oxidative stress mediates neuropathological processes in neuropsychiatric disorders and is involved in the etiology and progression of BD (Andreazza et al., 2007; Machado-Vieira et al., 2007; Selek et al., 2008). Oxidative stress is a condition in which the balance between production of reactive oxygen species and levels of antioxidants is disturbed, resulting in cell damage and death. The brain is particularly susceptible to oxidative stress because it metabolizes 20% of total body oxygen and has a limited amount of antioxidant capacity
n Correspondence to: Programa de Pós-Graduação em Saúde e Comportamento, Centro de Ciências da Vida e da Saúde, Universidade Católica de Pelotas, Rua Gonçalves Chaves 373, 96015560 Pelotas, Rio Grande do Sul, Brazil. Tel.: þ 55 53 2128 8031; fax: þ 55 53 2128 8229. E-mail address: [email protected] (G. Ghisleni).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.12.028
(Halliwell and Gutteridge, 2007). Reactive oxygen species contribute to the development of neurodegeneration by targeting different substrates in the cells, causing protein, DNA and RNA oxidation, or lipid peroxidation (Gandhi and Abramov, 2012). Curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is the main curcuminoid of the popular Indian spice turmeric, obtained from rhizome of Curcuma longa Linn. The multiple beneficial effects of curcumin could be linked to its antioxidant and anti-inflammatory properties in in vivo and in vitro models (Kulkarni et al., 2008; Bhatia et al., 2011; Gupta et al., 2012; Lopresti et al., 2012; Khurana et al., 2012; Jiang et al., 2013). Curcumin is also effective against cancer, cardiovascular, liver and inflammatory diseases, neurodegenerative disorders and depression (Monroy et al., 2013). The anti-inflammatory potential of curcumin, is related to the inhibition of nuclear factor kappa B (NFkB) signaling and reduction of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α. Curcumin is also a potent inhibitor of reactive-oxygen-generating enzymes such as lipoxygenase/cyclooxygenase, xanthine dehydrogenase/oxidase, and inducible nitric oxide synthase (Lin, 2007). The efficacy of curcumin as a neuroprotective agent in several preclinical models has created considerable excitement mainly due to its low toxicity and cost, suggesting that this compound might be a worthy candidate for prophylactic intervention in mental disorders. Despite the challenge in replicating the complex symptoms associated with psychiatric disorders, animal models have been
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successfully able to mimic some of the neurochemical and physiological characteristics of conditions (Kato et al., 2007). Animal models of mania are scarce and generally based on the management of hyperlocomotion-inducing agents such as D-amphetamine, ketamine and ouabain (Frey et al., 2006; Ghedim et al., 2012). These models represent useful pharmacological tools to assess behavioral and biochemical alterations observed during the mania episode, as well as to assess the effect of possible moodstabilizing agents. In this context, the present work hypothesized that curcumin administration might prevent some of the behavioral and neurochemical modifications in a model of mania induced by ketamine in rats.
2. Materials and methods 2.1. Animals and drug treatments Female adult Wistar rats aged 11–12 weeks (250–300 g) were obtained from the Central Animal House of the Federal University of Pelotas, Pelotas, RS, Brazil. Animals were maintained under controlled environment (23 7 2 1C, 12 h-light/dark cycle, free access to food and water) and handled according to the Federation of Brazilian Societies for Experimental Biology guidelines upon approval by the Ethics Committee of the Federal University of Pelotas, Brazil. The following drugs were used: ketamine (Sigma Chemical Co., USA), dissolved in saline solution (NaCl 0.9%, w/v) and administrated by intraperitoneal route (i.p.) and curcumin (Sigma Chemical Co., USA), dissolved in peanut oil and administered by oral route (p.o.), lithium chloride was dissolved in (NaCl 0.9%, w/v) and administered by p.o. route twice a day. Appropriated vehicle groups were also assessed simultaneously. The doses of ketamine, curcumin and lithium used in the present study were chosen according to the literature (Kulkarni and Dhir, 2010; Ghedim et al., 2012; Bruning et al., 2012). 2.2. Experimental protocol of mania state This protocol was designed to mimic the prevention protocol of the mania state, as previously proposed by Ghedin et al. (2012). Rats received peanut oil, curcumin 20 mg/kg or curcumin 50 mg/ kg, once a day for 14 days. From the 8th to the 14th day the animals also received saline or ketamine (25 mg/kg), once a day, totaling six experimental groups: saline/penaut oil, ketamine/ penaut oil, saline/curcumin 20 mg/kg, ketamine/curcumin 20 mg/ kg, saline/curcumin 50 mg/kg, ketamine/curcumin 50 mg/kg. In a separate set of experiments rats received saline or lithium chloride (45 mg/kg, twice a day, used as a positive control) for 14 days. From the 8th to the 14th day the animals also received saline or ketamine (25 mg/kg), once a day, totaling four experimental groups: saline/saline, ketamine/saline, saline/lithium chloride, ketamine/lithium chloride. On the 15th day of treatment, the animals received a single injection of ketamine or saline and the locomotor activity was assessed in the open-field apparatus after 30 min (Fig. 1). 2.3. Behavioral analysis Locomotor and anxiety-related behavior was monitored using an open-field apparatus, as previously described (Kaster et al., 2004). The apparatus consisted of a wooden box measuring 40 60 50 cm3 with a frontal glass wall. The floor of the arena was divided into 12 equal squares and placed in a sound free room. Animals were placed in the rear left square and left to explore it freely for 5 min. The total number of squares crossed with all paws (crossing) was counted in order to evaluate the ambulatory
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Fig. 1. Treatment protocol.
behavior. The number of central crossings was the measure used to evaluate anxiety. The apparatus was cleaned up with a 10% alcohol solution and dried after each individual mouse session. 2.4. Biochemical assay Rats were killed by decapitation immediately after the openfield test. Prefrontal cortex (PFC) and hippocampus (HP) were manually dissected and homogenized in 10 volumes (1:10 w/v) of 20 mM sodium phosphate buffer, pH 7.4 containing 140 mM KCl. Homogenates were centrifuged at 750g for 10 min at 4 1C, the pellet was discarded and the supernatant was immediately separated and used for the stress oxidative measurements. Protein was measured using biocinchoninic acid (BCA) assay using bovine serum albumin as standard. 2.4.1. Thiobarbituric acid reactive species formation (TBARS) The measure of lipid peroxidation was determined by TBARS according to the protocol described by Esterbauer and Cheeseman (1990). Briefly, homogenates were mixed with trichloroacetic acid 10% and thiobarbituric acid 0.67% and heated in a boiling water bath for 25 min. TBARS was determined by the absorbance at 535 nm. Results were reported as nmol of TBARS per mg of protein. 2.4.2. Total sulfhydryl content This assay was performed as described by Aksenov and Markesbery (2001) which is based on the reduction of DTNB by thiols, which in turn, becomes oxidized (disulfide) generating a yellow derivative (TNB) whose absorption is measured spectrophotometrically at 412 nm. Briefly, homogenates were added to PBS buffer pH 7.4 containing EDTA. The reaction was started by the addition of 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB). Results were reported as μmol TNB per mg of protein. 2.4.3. Catalase (CAT) assay CAT activity was assayed by the method of Aebi (1984). H2O2 disappearance was continuously monitored during 90 s in a spectrophotometer adjusted at 240 nm. CAT specific activity was reported as units of enzyme per mg of protein. 2.4.4. Superoxide dismutase (SOD) assay Total SOD activity was measured by the method described by Misra and Fridovich (1972). This method is based on the inhibition of superoxide dependent adrenaline auto-oxidation in a spectrophotometer adjusted at 480 nm. The specific activity of SOD was reported as units per mg of protein. 2.5. Statistical analysis Comparisons between experimental groups were performed by one-way or two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc tests when appropriate. The values are expressed as mean 7 S.E.M. P o0.05 was considered significant.
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Fig. 2. Effect of curcumin (20 and 50 mg/kg, p.o.) on ketamine-induced hyperactivity (A); anxiety behavior (B); effect of lithium chloride (45 mg/kg, p.o., twice a day) on ketamine-induced hyperactivity (C); anxiety behavior (D) in the open-field test. The number of crossings was recorded. Data are given as the mean(s) 7 S.E.M. (n¼ 6–7 for group). nn Denotes Po 0.01 as compared to the vehicle/saline group. ♯ Denotes P o 0.05 as compared to the vehicle/ketamine group.
3. Results 3.1. Behavioral characterization focusing on ambulatory performance The treatment with ketamine was efficient to induce hyperlocomotion in rats, evaluated by the increase in the number of crossings in the open-field test, which is an indication of mania episode (Ghedim et al., 2012). Notably, as presented in Fig. 2A curcumin pretreatment (20 and 50 mg/kg) prevented the hyperlocomotion induced by ketamine in the open-field test (curcumin pretreatment: [F(2,26)¼ 4.11, Po0.05], ketamine treatment: [F(1,26)¼ 16.93, Po0.05], interaction: [F(2,26)¼7.99, Po0.05]). In addition, Fig. 2C lithium chloride pretreatment (45 mg/kg) prevented the hyperlocomotion induced by ketamine in the open-field test (lithium chloride pretreatment: [F(1,23)¼ 8.4, Po0.01], ketamine treatment: [F(1,23)¼23.08, Po0.01], interaction: [F(1,23)¼13.99, Po0.01]). As demonstrated in Fig. 2B and D, no changes in the anxiety behavior were observed for percentage of central crossings after ketamine treatment, curcumin or lithium chloride pretreatment (curcumin pretreatment: [F(2,36) ¼0.60, P ¼0.55], ketamine treatment: [F(1,36) ¼0.28, P ¼0.59], interaction: [F(2,36) ¼0.25, P ¼0.77]) and (lithium chloride pretreatment: [F(1,20)¼0.005, P ¼0.93], ketamine treatment: [F(1,20)¼0.60, P ¼0.44], interaction: [F(1,20)¼0.09, P ¼0.76]). 3.2. Measurement of oxidative stress parameters in the prefrontal cortex (PFC) In order to consolidate the notion of a neuroprotective effect of curcumin against ketamine-induced model of mania, we evaluated the effects of curcumin in oxidative stress parameters in the PFC. Fig. 3A shows that curcumin pretreatment (20 and 50 mg/kg) was
able to prevent the increase in TBARS levels in PFC induced by ketamine administration (curcumin pretreatment: [F(2,35)¼0.48, P¼ 0.62], ketamine treatment: [F(1,35)¼0.79, P¼0.38], interaction: [F(2,35)¼6.62, Po0.05]). In addition, Fig. 3B shows that curcumin (20 and 50 mg/kg) prevented the decrease in sulfhydryl content induced by ketamine in PFC (curcumin pretreatment: [F(2,36)¼4.34, Po0.05], ketamine treatment: [F(1,36)¼0.30, P¼ 0.60], interaction: [F(2,36)¼11.95, Po0.01]). We then have compared the activity of the antioxidant enzymes SOD and CAT. As shown in Fig. 3C curcumin and ketamine administration did not change the activity of SOD in the PFC (curcumin pretreatment: [F(2,28)¼0.50, P¼0.60], ketamine treatment: [F(1,35)¼ 0.71, Po0.01], interaction: [F(2,28)¼ 0.03, P¼0.97]). There was also no evident difference in the CAT activity between groups (curcumin pretreatment: [F(2,36)¼0.10, P¼ 0.94], ketamine treatment: [F (1,36)¼13.62, Po0.01], interaction: [F(2,36)¼1.61, P¼0.20]). In order to use a mood-stabilizing agent as a positive control, the effects of lithium chloride (45 mg/kg, p.o.) in oxidative stress parameters in the PFC were evaluated (Table 2). Lithium chloride was able to prevent the increase in TBARS levels induced by ketamine in the PFC (lithium chloride pretreatment: [F(1,20) ¼ 3.50, P¼ 0.07], ketamine treatment: [F(1,20)¼8.06, P o0.01], interaction: [F(1,20)¼ 14.57, Po 0.01]). In addition, lithium chloride prevented the decrease in sulfhydryl content induced by ketamine in the PFC (lithium chloride pretreatment: [F(1,19) ¼ 37.30, Po 0.01], ketamine treatment: [F(1,19) ¼ 15.50, P o0.01], interaction: [F(1,19) ¼20.42, P o0.01]). Pretreatment with lithium chloride did not change SOD activity in the PFC (lithium chloride pretreatment: [F(1,20) ¼0.69, P ¼0.42], ketamine treatment: [F (1,20) ¼0.60, P¼ 0.45], interaction: [F(1,20) ¼3.65, P ¼0.07]). In addition, no differences were found in the CAT activity (lithium chloride pretreatment: [F(1,19)¼7.93 Po0.01], ketamine treatment: [F(1,19)¼ 1.28, P¼0.27], interaction: [F(1,19)¼0.60, P¼ 0.44]).
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Fig. 3. Effects of curcumin (20 and 50 mg/kg, p.o.) on TBARS formation (A); total thiol content (B); superoxide dismutase (SOD, C); and catalase (CAT, D) activity in the prefrontal cortex of rats. Data was expressed as mean þS.E.M. (n ¼6–7 for group). nn Denotes P o 0.01 and n P o 0.05 as compared to the vehicle/saline group. ♯ Denotes Po 0.05 as compared to the vehicle/ketamine group.
3.3. Measurement of oxidative stress parameters in the hippocampus (HP) We next have evaluated the effects of curcumin in oxidative stress parameters in the HP. As depicted in Fig. 4A, curcumin pretreatment (50 mg/kg) was able to prevent increase in the TBARS levels induced by ketamine administration in rats (curumin pretreatment: [F(2,34) ¼ 2.08, P ¼0.14], ketamine treatment: [F(1,34)¼5.30, P o0.05], interaction: [F(2,34) ¼ 4.24, P o0.05]). The results in Fig. 4B demonstrated that ketamine treatment did not change the total SH content when compared to the control group. However, the treatment with curcumin alone (50 mg/kg) or (20 and 50 mg/kg) combined with ketamine increased the sulfhydryl content in the hippocampus of rats (curcumin pretreatment [F (2,33)¼7.77, P o0.01] ketamine treatment: [F(1,33) ¼0.019, P ¼0.89], interaction: [F(2,33)¼5.96, Po 0.01]). Notably, Fig. 4C shows that SOD activity was significantly decreased by ketamine and this effect was completely prevented in curcumin (20 and 50 mg/kg) pretreated groups (curcumin pretreatment: [F(2,34) ¼ 6.52, P o0.01], ketamine treatment: [F(1,34)¼0.57, P¼ 0.45], interaction: [F(2,34) ¼3.80, P o0.05]). In addition, Fig. 4D also indicated that ketamine-treated rats had a decrease in CAT activity, and the pretreatment with both doses of curcumin (20 and 50 mg/kg) was also able to prevent this effect (curcumin pretreatment: [F(2,33)¼ 11.4, P o0.01], ketamine treatment: [F(1,33) ¼0.006, P ¼0.80], interaction: [F(2,33) ¼7.99, P o0.05]). The effects of lithium chloride (45 mg/kg, p.o.) in oxidative stress parameters in the HP were also (Table 2). Lithium chloride was able to prevent increase in the TBARS levels induced by ketamine (lithium chloride pretreatment: [F(1,20) ¼5.90, P o0.05], ketamine treatment: [F(1,20)¼4.58, Po0.05], interaction: [F(1,20)¼ 6.51, Po0.05]). The results demonstrated that ketamine treatment did not change the total SH content when compared to the control group. However, the treatment with lithium chloride (45 mg/kg)
combined with ketamine increased the sulfhydryl content in the hippocampus of rats (lithium chloride pretreatment: [F(1,19) ¼ 62.23, Po 0.01], ketamine treatment: [F(1,19) ¼39.02, P o0.01], interaction: [F(1,19) ¼96.84, P o0.01]). Finally, lithium chloride was able to prevent the decrease in CAT activity induced by ketamine in the HP of rats (lithium chloride pretreatment: [F(1,20) ¼2.46, P¼ 0.13], ketamine treatment: [F(1,20)¼ 4.90, Po 0.05], interaction: [F(1,20)¼ 5.61, Po0.05]). In addition, lithium pretreatment were able to prevent the decrease in SOD activity induced by ketamine in the HP of rats, (lithium chloride pretreatment [F(1,19) ¼ 89.10, Po 0.01], ketamine treatment: [F(1,19) ¼0.23, P ¼0.64], interaction: [F(2,33)¼ 44.06, P o0.01]). The mean values and standard deviation for all the experiments and groups are represented in Tables 1 and 2.
4. Discussion The present study shows at the first time that the administration of curcumin, an active ingredient in turmeric (Curcuma longa), is able to prevent the hyperlocomotion and changes in some oxidative stress parameters induced by ketamine in rats. Additionally these data reinforce the ketamine-induced hyperlocomotion model proposed by Ghedim et al. (2012), as an important tool to study behavioral and neurochemical alterations relevant to the mania phase of BD. Animal models are consistently used to investigate intracellular mechanisms and pharmacological approaches involved in psychiatry disorders (Kato et al., 2007). However, the complexity of the biological features of BD makes difficult to mirror important aspects of the disease such as the episodic and the cyclic status. In preclinical models, hyperlocomotion is a core behavior evaluated during the manic state of BD. Indeed, several studies show that the administration of D-amphetamine or ouabain mimics the hyperlocomotion and several other behavioral and biological aspects of mania (Gould et al., 2001; Wang et al., 2013).
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Fig. 4. Effects of curcumin (20 and 50 mg/kg, p.o.) on TBARS formation (A); total thiol content (B); superoxide dismutase (SOD, C); and catalase (CAT, D) activity in the hippocampus of rats. Data was expressed as meanþ S.E.M. (n¼ 6–7 for group). nn Denotes Po 0.01 and n P o 0.05 as compared to the vehicle/saline group. ♯ Denotes Po 0.05 as compared to the vehicle/ketamine group.
Table 1 Effect of curcumin on oxidative stress parameters in the prefrontal cortex and hippocampus of rats. Groups
TBARS (nmol/mg protein)
Total SH (lmol/mg protein)
SOD (units/mg protein)
CAT (units/mg protein)
Prefrontal cortex Saline/penaut oil Ketamine/penaut oil Saline/curcumin 20 mg/kg Ketamine/curcumin 20 mg/kg Saline/curcumin 50 mg/kg Ketamine/curcumin 50 mg/kg
0.13 70.01 0.23 70.02b 0.19 70.02 0.15 70.01c 0.17 70.02 0.16 70.01c
41.767 5.14 17.667 3.20a 38.63 7 1.62 51.98 7 5.20c 30.30 7 2.49 56.36 7 8.80c
12.40 7 3.70 8.247 1.79 16.577 6.47 10.02 7 2.63 13.767 2.24 6.247 2.42
3.45 7 0.20 4.077 0.62 3.277 0.30 4.03 7 0.29 2.92 7 0.20 4.677 0.27
Hippocampus Saline/penaut oil Ketamine/penaut oil Saline/curcumin 20 mg/kg Ketamine/curcumin20 mg/kg Saline/curcumin 50 mg/kg Ketamine/curcumin 50 mg/kg
0.10 70.02 0.14 70.02b 0.09 70.02 0.10 70.01c 0.10 70.01 0.08 70.01c
54.90 7 1.13 48.307 0.80 53.137 4.50 72.217 8.98 80.93 7 10.80 62.477 2.90
5.65 7 0.78 1.677 0.60a 7.30 7 1.56 7.80 7 1.28c 6.017 1.63 7.32 7 0.71c
2.80 7 0.40 1.28 7 0.42a 2.98 7 0.71 2.90 7 0.50c 2.42 7 0.74 2.89 7 0.85c
Thiobarbituric acid reactive species formation (TBARS); total thiol content (total SH); superoxide dismutase (SOD); and catalase (CAT). Data was expressed as mean 7 S.E.M. (n¼ 6–7 animal per group). a b c
P o 0.01 as compared to the vehicle/saline group. Po 0.05 as compared to the vehicle/saline group. Po 0.05 as compared to the vehicle/ketamine group.
Recently, Ghedim et al. (2012) reported that administration of a sub-anesthetic dose of ketamine induced hyperlocomotion and modify oxidative stress parameters in the rat brain. These observations point to a good face validity of the model. In addition, the behavioral and biochemical effects were normalized by treatment with lithium and valproic acid, pointing to a predictive validity. Moreover, several clinical and pre-clinical studies demonstrated that glutamatergic system abnormalities are involved in the patophysiology of BD, pointing to a possible construct validity of this model. Our work corroborates and extends these data suggesting that this protocol fulfills adequate characteristics as an animal model of mania and further reinforces previous proposals that both glutamatergic system and oxidative stress might be
involved in this particular stage of BD (Andreazza et al., 2008; Machado-Vieira, 2012). Pre-clinical evidences show that low doses of ketamine (5–10 mg/kg) exhibit antidepressant properties (Katalinic et al., 2013). Nevertheless, in moderate doses (10–50 mg/kg) it induces hyperlocomotion and cellular dysfunction (Gould et al., 2001; Machado-Vieira et al., 2004; Ghedim et al., 2012) and higher doses possess anesthetic and dissociative effects. From the clinical point of view, infusions of low doses of ketamine induce rapid antidepressant effects (Katalinic et al., 2013). However, due to the propensity of ketamine to produce cognitive deficits, dissociative and psychotomimetic effects, its clinical use remains limited. In fact, a case report shows that continuous administration of a higher dose of ketamine is
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Table 2 Effect of lithium chloride in oxidative stress parameters in the prefrontal cortex and hippocampus of rats. Groups
TBARS (nmol/mg protein)
Total SH (lmol/mg protein)
SOD (units/mg protein)
CAT (units/mg protein)
Prefrontal cortex Saline/saline Ketamine/saline Saline/lithium 45 mg/kg Ketamine/lithium 45 mg/kg
0.12 70.01 0.2470.03a 0.16 70.01 0.14 70.01c
49.50 74.60 18.20 73.18b 55.48 71.40 57.65 74.10c
16.707 4.30 9.577 1.42 13.80 7 2.24 16.85 7 1.43
3.53 70.20 4.16 70.65 2.90 70.05 3.01 70.25
Hippocampus Saline/saline Ketamine/saline Saline/lithium 45 mg/kg Ketamine/lithium 45 mg/kg
0.08 70.02 0.15 70.02b 0.08 70.01 0.0770.01c
53.53 70.33 47.85 70.76 50.45 71.54 75.26 72.90
6.107 0.75 1.42 7 0.63a 7.94 7 0.51 11.98 7 0.64c
2.97 70.42 1.47 70.44b 2.71 70.17 2.76 70.13c
Thiobarbituric acid reactive species formation (TBARS); total thiol content (total SH); superoxide dismutase (SOD); and catalase (CAT). Data was expressed as mean 7S.E.M. (n¼ 6–7 for group). a b c
P o0.01 as compared to the vehicle/saline group. Po 0.05 as compared to the vehicle/saline group. Po 0.05 as compared to the vehicle/ketamine group.
associated with the development of manic and psychotic symptoms in humans (Ricke et al., 2011). In pre-clinical models, non-anesthetic doses of ketamin are able to induce hyperlocomotion, stereotypy, impaired cognitive function and social interaction (Lipska and Weinberger, 2000; Bubenikova-Valesova et al., 2010). In the present work we evaluated hyperlocomotion, the core symptom of mania, which was prevented by curcumin administration and by lithium chloride, used as a positive control. We also investigated anxiety behavior, but no changes were observed after ketamine, curcumin and/or lithium chloride treatment. It is important to mention that we cannot rule out the possibility that acute effects of ketamine might also be involved in the behavioral and neurochemical changes evaluated. Indeed, da Silva et al. (2010) demonstrated that a single dose of ketamine in mice induced hyperlocomotion and increased lipid peroxidation and nitrite content in the cortex of mice. Oxidative stress is an important factor involved in the pathophysiology of major neuropsychiatric disorders, including BD (Andreazza et al., 2007, 2008; Machado-Vieira et al., 2007; Selek et al., 2008). Increased reactive oxygen species levels generate deleterious effects on signal transduction, structural plasticity and cellular resilience, mostly by inducing lipid peroxidation in membranes, damage to proteins and nucleic acids (Mahadik et al., 2001). Our study also shows that ketamine induces changes in some oxidative stress parameters like increase in TBARS levels and decrease in the total thiol content in the PFC of rats. The effects of ketamine and other manic agents on lipid peroxidation were described in different brain regions (Brocardo et al., 2010; Ghedim et al., 2012). However, it is important to highlight that this is the first work reporting a decrease in the total thiol content induced by ketamine in the PFC. This finding supports the rationale that cysteine residues involved in the crucial regulation of many key proteins and peptides might be susceptible targets of oxidation in this model. Another relevant finding of our work was that ketaminetreatment reduced total SOD and CAT activity in the HP, but not in the PFC. SOD is a group of enzymes that reduces the superoxide anion (O2 ) into hydrogen peroxide (H2O2), which can react with iron to generate highly reactant hydroxyl radicals (Halliwell and Gutteridge, 2007). In contrast, CAT is the most important peroxidase responsible for detoxification of the H2O2 and prevention of hydroxyl production. The decreased activity of SOD and CAT might have deleterious effects upon the cell, generating an excess of H2O2 and oxidative damage. Differences in the vulnerability to damage between brain regions were already described, especially in models of ischemia and kainic acid-induced seizure (Zhao and Flavin, 2000; Candelario-Jalil et al., 2001; Geddes et al., 2003). The mechanisms underlying this phenomenon are far from being
understood and differences in oxidative stress susceptibility or heterogeneous distribution of glutamatergic receptor in PFC and HP might be involved. Hippocampal neurons were extensively demonstrated in the literature to be intrinsically more vulnerable to several types of insults, including mechanical insult, traumatic injury, kainic acid toxicity, stress and ischemic insults when compared to cortical neurons (Lowenstein et al., 1992; Taft et al., 1992; Zhao and Flavin, 2000; Golarai et al., 2001; Candelario-Jalil et al., 2001; Geddes et al., 2003). Further studies are needed to determine the differences in the vulnerability of these two cellular populations to ketamine-induced oxidative stress. In addition, we cannot rule out the possibly that our treatment is also changing the levels of these proteins in these areas and consequently the activity. The main finding of this study was the ability of curcumin to prevent both the hyperlocomotion and the changes in oxidative stress parameters induced by ketamine in rats. Indeed, in recent years, several studies highlighted the ability of curcumin to promote a variety of pharmacological and biological activities including anti-inflammatory and antioxidant (Kulkarni et al., 2008; Bhatia et al., 2011; Gupta et al., 2012; Lopresti et al., 2012; Khurana et al., 2012; Jiang et al., 2013). Curcumin has been recently demonstrated to provide neuroprotection by acting as a modulator of glutamergic neurotransmission, restoring the levels of glutamergic receptors, oxidative stress and imbalanced glutamate metabolism (Jayanarayanan et al., 2013). In addition, curcumin inhibit glutamate release, block GluN2B receptors (Zhang et al., 2013), and enhance neuronal survival against NMDA toxicity (Lin et al., 2011). Some of the protective effects of curcumin against glutamate-induced toxicity and cell death in vitro are comparable to those mediated by the antimanic agent lithium (Chen et al., 2003). In addition, curcumin shares with lithium several behavioral and biological activities and it was recently demonstrated to normalizes cellular antioxidant enzymes (including SOD and catalase) and decreases oxidative stress in a cellular model of Alzheimer disease by a mechanism dependent on the inhibiting of glycogen synthase kinase-3β (GSK-3β) (Huang et al., 2012). Since the inhibition of GSK-3β is one of the major known cellular target of lithium, there is a strong possibility that these two compounds might share some of the intracellular pathways to exert their antimaniac effects. As oxidative stress and inflammation are major factors involved in the etiology and progression of several disorders, curcumin administration was effective in preclinical models of numerous conditions including neurodegenerative disorders, depression and aging (Bala et al., 2006; Menon and Sudheer, 2007; Kulkarni et al., 2008; Bhatia et al., 2011; Gupta et al., 2012; Khurana et al., 2012;
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Jiang et al., 2013). Besides that, curcumin administration improves memory function, cerebral blood flow and energy metabolism (Rajasekar et al., 2013) and enhances the levels of brain derived neurothrophic factor (BDNF) and hippocampal neurogenesis (Xu et al., 2007). A recent work discussed that by acting in all those cellular targets, curcumin might be a compound of interest to treat the mood and cognitive impairments associated with BD (Brietzke et al., 2013). Here, we were able to test, for the first time, an confirm the potential of curcumin against behavioral alteration and oxidative stress in an animal model of mania.
5. Conclusion Effective treatments for preventing mood episodes in patients with BD are urgently needed, especially because BD is perhaps the psychiatric disorder with the highest mortality rate. In addition, the neurochemical alterations associated with episode-related deterioration patterns, progress with the number of episodes (Berk et al., 2011). Thus, our findings suggest that curcumin might be a good agent for preventive intervention, reducing the episode relapse and the oxidative stress associated with the manic phase of BD.
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