Role of caffeine intake in lithium treated methylphenidate induced oxidative stress in an animal model of mania

 

Shanthakumar J., Tamilselvan T., Arunagiri P., Rajeshwaran K. and Balamurugan E.*

Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University,

Annamalainagar-608 002, Tamilnadu, India.

*Corresponding Author E-mail: balamurugan_au@yahoo.co.in

 

 

ABSTRACT:

In this study, we assessed the oxidative stress parameters in mice submitted to an animal model of mania induced by methylphenidate (MPH), which included the use of lithium chloride (LiCl) and caffeine (CAF).  Behavioral changes were assessed using open field tests resulted in decreased locomotor activity, frequency of crossings and rearings were observed and significantly reversed in the combined treatment (LiCl and CAF). LiCl and CAF treatment reversed and prevented the MPH induced damage in these structures; however, this effect varies depending on the brain region and treatment regimen. Moreover, the activity of the antioxidant enzymes, namely, superoxide dismutase (SOD) and catalase (CAT) was found to be increased in the brain of MPH administered mice. Combined treatment of LiCl and CAF modulated SOD and CAT activities in MPH subjected mice. Our results support the notion that LiCl and low dose CAF exert antioxidant like properties in the brain of mice induced by MPH. Further studies will be needed to discover the mechanisms responsible for these findings.

 

KEYWORDS: bipolar disorder; mania; oxidative damage; methylphenidate; lithium chloride; caffeine.

 

INTRODUCTION:

Bipolar Disorder (BD) is a mental health disorder characterized by mood swings between depression and mania. Mood swings can range from moderate to extreme and have a severe impact on relationships, career, and life in general. Bipolar disorder is a chronic, disabling and heterogeneous condition of major relevance, whose treatment needs to be considered separately through the course of the illness for manic/hypomanic, mixed and depressive episodes [1].  The use of psychostimulants in rodents can trigger a number of manic-like behaviors, such as hyperactivity, sleep disturbance, distractibility, and increased risk-taking [2].

 

Methylphenidate is a Psychostimulant structurally and pharmacologically related to amphetamine. Repeated administration of psychomotor stimulants produces an enduring and progressively enhanced behavioral response known as behavioral sensitization, which has been implicated as a model for psychiatric disorders such as mania, schizophrenia, and drug addiction [3]. The primary antioxidant defense system has been studied in several psychiatric disorders and involves coordinated effects induced by superoxide dismutase (SOD) and catalase (CAT) [4, 5]. Currently, lithium is the classic mood stabilizer and it was the first drug approved by the Food and Drug Administration (FDA) in 1974 for maintenance treatment of bipolar disorder [6]. New antidepressants and antipsychotics have been developed for the treatment of major depression and schizophrenia, all new drugs currently used for bipolar disorder were originally developed for other disorders, such as epilepsy (carbamazepine, valproate, and lamotrigine), schizophrenia (olanzapine, quetiapine, aripiprazole, and other atypical antipsychotics), or depression (selective serotonin reuptake inhibitors). Mood stabilizing drugs, particularly lithium and valproate, are considered as first-line agents for both acute mania and maintenance treatment [7]. Consumption of caffeine may be higher in psychiatric patients than in the population as a whole [8, 9]. Caffeine is regarded as the most widely consumed psychoactive substance in the world [10]. Therefore, it is likely that many depressed patients receiving treatment with TCAs could be consuming caffeine simultaneously from dietary sources, or even from over-the-counter medications. Given that caffeine can exert positive or negative mood effects depending on the dose consumed [11, 12], it is of interest to assess whether this methylxanthine could interact positively or negatively with TCA drugs. Hence, on the basis of the evidence suggesting that adenosine could be involved in the actions of TCAs, the present study was designed to assess the interaction between amitriptyline and the non-selective adenosine receptor antagonist caffeine in the forced swimming test (FST). Caffeine use has been linked with specific disorders such as anxiety disorders, sleep disorders and eating disorders, and there is a possible association with schizophrenia [12]. Surprisingly, there are no published reports linking caffeine use with mania or hypomania. The present study will be conducted to assess the role of caffeine intake in lithium treated methylphenidate induced mania in mice.

 

MATERIALS AND METHODS:

Drugs and chemicals

Methylphenidate hydrochloride (MPH, Ritalin, Novartis Pharmaceutical Inc.), Lithium chloride (LiCl), Caffeine (CAF) was purchased from Sigma-Aldrich (USA). All other chemicals used in this study were of analytical grade obtained from Merck or HIMEDIA, India.

 

Animals

Swiss albino mice (weighing 25-30 gm) will be housed in well ventilated rooms (temperature 23 ± 2°C, humidity 65-70% and 10 h light/dark cycle) at Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College, Annamalai University and feed with standard pellet diet and water ad libitum. All studies will be carried out in accordance with Indian national law of animal care and use, and committee for the purpose of control and supervision of animals of Rajah Muthiah Medical College and hospital (Reg No./160/1999/CPCSEA), Annamalai University, Annamalainagar.

 

Model of MPH-induced mania and experimental protocol

Methylphenidate (MPH) was suspended in two drops of Tween 80 and saline and administered subcutaneously (sc) at a dose of 5.0 mg/kg b w for 14 days [13]. Lithium chloride (LiCl) was administered intraperitoneally at a dose of 47.5 mg/kg b w. [14] and caffeine 10 mg/kg b w [15].  All injections were given 20 min before behavioral testing at a volume of 5-ml/kg body weight. In our experiment, a total of forty eight mice were used. The mice were divided into eight groups of six mice each. Group I: Control (saline 2mg/kg/b.w); Group II: Control + LiCl; Group III: Control + CAF; Group IV: LiCl + CAF; Group V: Mania (MPH alone); Group VI: Mania + LiCl; Group VII: Mania + CAF; Group VIII: Mania+ LiCl + CAF. LiCl and CAF will be administered for  after the significant increase in the locomotors activity, which will be monitored from the 3rd day onwards. All injections were given 20 min before behavioral testing at a volume of 5ml/kg body weight.

 

Open field test

The open field has been considered as a non-conditioned anxiety test based on the creation of a conflict between the exploratory drive of the rat and its innate fear to exposure to an open area [16]. The open field test has been employed to assess the spontaneous activity, general exploration and ambulation of the rodents. The open field consisted of a wooden box 90 cm × 90 cm × 38 cm positioned in a dimly lighted room. The walls were painted black, while the floor was painted white and was divided by 1 cm wide black lines into 25 squares of 17 cm × 17 cm (16 peripheral squares and 9 central squares). The mice were placed in the center of the open field and the number of line crossings and rearings was noted for a 10 minute period [17].

 

Biochemical assays

Animals were sacrificed by decapitation immediately after the end of the open field task and the whole brain was transferred within 1 min to ice-cold isolation buffer (0.23 M mannitol, 0.07 M sucrose, 10 mM Tris eHCl, and 1 mM EDTA, pH 7.4). The level of lipid peroxidation was determined by analyzing TBA-reactive substance according to the protocol of Niehaus and Samuelsson (1968) [18]. The pink colored chromogen formed by the reaction of 2-TBA with break-down products of lipid peroxidation was measured. Superoxide dismutase activity was assayed by the method of Kakkar et al. (1984) [19], based on the inhibition of the formation of (NADH–PMS–NBT) complex. Catalase activity was assayed by the procedure of Sinha (1972) [20] quantifying the hydrogen peroxide after reacting with dichromate in acetic acid.

 

Protein determination

All biochemical measures were normalized to the protein content with bovine albumin as standard (Lowry et al., 1951) [21].

 

Statistical analysis

All the values were expressed as mean ± S.D. of six determinations. Statistical analyses of the data were carried out by one-way ANOVA on SPSS (Statistical package for social sciences) and the group mean compared by Duncan’s Multiple Range Test (DMRT). A value of P˂0.05 was considered significant. Student’s t-test will be employed whenever necessary.

 

 

Fig. 1. Effect of Li and CAF on exploratory behavior in terms of frequency of rearings and line crossings in 10 min time interval in the open field test in MPH treated mice. Values are expressed as mean±S.E.M. with n=6 in each group; one-way ANOVA followed by Duncan’s Multiple Range Test (DMRT). Values not sharing a common marking (*, **, #, etc.) differ significantly at P<0.05 (DMRT)

 

Fig. 2. Effects of MPH administration on TBARS level in brain tissue of experimental mice. Values are expressed as mean±S.E.M. with n=6 in each group; one-way ANOVA followed by Duncan’s Multiple Range Test (DMRT). Values not sharing a common marking (*, **, #, etc.) differ significantly at P<0.05 (DMRT)

 

 

RESULTS:

As depicted in fig. 1 MPH administration greatly increased the number crossings and rearings in the open field test. Treatment with LiCl was effective in preventing the locomotor activity induced by methylphenidate. Caffeine intake did not alter the treatment compared to that of combined LiCl and CAF treated mice.  Treatment with MPH shows increased TBARS levels (fig.2) in brain tissue of mice. The levels of TBARS in animals treated with LiCl were significantly lower than those of MPH treated and control groups.  In addition the caffeine supplement along with LiCl significantly reduces the TBARS levels. As shown in fig. 3 the SOD activity was higher in MPH treated group than in those from the control group. Treatment with LiCl blocked the increase of SOD activity and combination of LiCl with caffeine also significantly reduced the SOD activity. The results of the CAT activity were shown in   fig. 4 MPH treated animals shows increased CAT activity, but LiCl was effective in blocking this effect. CAF combined with LiCl significantly decreased the CAT activity when compared to LiCl and CAF alone treatment.

 

 

Fig. 3. Effects of MPH administration on superoxide levels in brain tissue of experimental mice. Values are expressed as mean±S.E.M. with n=6 in each group; one-way ANOVA followed by Duncan’s Multiple Range Test (DMRT).  Values not sharing a common marking (*, **, #, etc.) differ significantly at P<0.05 (DMRT)

 

Fig. 4. Effects of MPH administration on catalase levels in brain tissue of experimental mice. Values are expressed as mean±S.E.M. with n=6 in each group; one-way ANOVA followed by Duncan’s Multiple Range Test (DMRT). Values not sharing a common marking (*, **, #, etc.) differ significantly at P<0.05 (DMRT)

 

 

For all the biochemical parameters studied, pretreatment with LiCl alone and CAF alone showed significant effects in all the biochemical parameters studied in MPH treated mice. But, combined treatment with LiCl and CAF normalized all the biochemical parameters studied and the effect was better than single pretreatment alone (LiCl alone or CAF alone) in MPH treated mice. Treatment with LiCl alone, CAF alone and combined treatment with LiCl and CAF to normal control mice did not show any significant effect in all the biochemical parameters studied.

 

DISCUSSION:

In this present study the CAF combined with LiCl protected against methylphenidate induced hyperlocomotion and alterations in the oxidative stress parameters in mouse brain tissue. MPH administration resulted in significant behavioral alterations (crossings and rearings) in locomotor activity. Combined treatment of LiCl and CAF blocked the MPH induced increase in locomotor activity in the open-field test in mice. The struggling and muscular exertion of the mice during the process of immobilization represent a physical dimension; while limited range of movement along with exposure in an open area represents the psychological dimensions[22]. Furthermore, lithium blocked the increased locomotor activity induced by methylphenidate [23].  According, Ellenbroek and Cools (1990) [24] the validity of animal models in psychiatric disorders should demonstrate the face, construct and predictive validities. The clinical hallmark of BD is acute mania [25], showing symptoms such as irritable mood, psychomotor activation, reduced need for sleep, and excessive involvement in potentially problematic behavior [26]. Oxidative stress has been proposed to play a significant role in the pathophysiology of major neuropsychiatric disorder such as BD and schizophrenia [27, 28]. Combined pretreatment with LiCl and CAF normalized the levels of lipid peroxidation products in MPH treated mice. Interestingly, the MPH induced oxidative damage was accompanied by increased superoxide dismutase activity and decreased CAT activity. SOD is an enzyme capable of reducing the superoxide radical into hydrogen peroxide (H2O2), which acts as the substrate to CAT. When cell has increased levels of SOD without a proportional increase in peroxidases, the excess of H2O2 produced could be responsible for the oxidative damage in the cell. In addition, H2O2 can react with transitional metals and generate the radical hydroxyl, which is the most harmful radical [29]. Consequently, the over expression of SOD without a compensatory increase in CAT has deleterious effects upon the cell. Interestingly, here despite LiCl and CAF have partially reduced the activity of SOD in mice treated with methylphenidate, both LiCl and CAF significantly decreased the amount of MPH induced SOD levels. In contrast the previous reports suggest that Li and VPA may be decreasing SOD and H2O2, thereby decreasing O2- [30,31]. Pretreatment with combined LiCl and CAF led to an decrease in CAT activity in brain of MPH treated animals, which can be preventing other reactions with H2O2.  The index of DNA damage was correlated positively with lipid peroxidation, whereas Li and VPA were able to modulate the oxidative balance and prevent recent damage to the DNA [32]. Machado-Vieira et al. (2007) have showed that TBARS and antioxidant enzymes activity (SOD and CAT) increased in unmedicated manic patients compared to controls, and acute treatment with Li showed a significant reduction in both SOD/CAT ratio and TBARS levels [33].

 

It has been demonstrated that the adenosine receptors, the main molecular target of caffeine, are involved in the regulation of ROS production, affecting the genesis and impact of free radicals in neuronal and others biological systems [34, 35, 36].  Our results support the notion that LiCl and CAF exert antioxidant like properties in the brain of mice submitted to animal model of mania induced by MPH. The altered energy metabolism dysfunctions associated with BD may play a role in oxidative stress observed during manic episodes.

 

CONCLUSION:

Thus, besides improving cognitive function, our data show that CAF consumption along with LiCl modulates the endogenous antioxidant system in the brain. Therefore, CAF ingestion, through the protection of the antioxidant system, may play an important role during mania.

 

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Received on 07.11.2013       Accepted on 01.12.2013     

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