Evaluation of sesamol and buspirone in stress induced anxiety in mice.

OBJECTIVES: The present study was designed to elucidate the effects of sesamol, buspirone and their combination in immobilization stress induced behavioral and biochemical alterations in mice.
MATERIALS AND METHODS: Male Laca mice (divided into 10 groups with 6 animals each) were pre-treated with sesamol (5 and 10 mg/kg; p.o.), buspirone (5 and 10 mg/kg; p.o.) and combination of sesamol (5 and 10 mg/kg; p.o.) with buspirone (5 mg/kg; p.o.) for consecutive five days. On the 6(th) day, animals were immobilized for 6 h and various behavioral tests such as body weight, locomotor activity, mirror chamber test and elevated plus maze were carried out. Biochemical estimations such as lipid peroxidation and nitrite concentration, glutathione and catalase levels were done. Data was analyzed using One way ANOVA followed by Tukey's test (P < 0.05) was considered statistical significant.
RESULTS: Immobilization stress significantly (P < 0.05) impaired body weight, locomotor activity, induced anxiety like behavioral and oxidative damage as compared to naοve animal. Pretreatment with sesamol (5 and 10 mg/kg; p.o.) and buspirone (5 and 10 mg/ kg; p.o.) significantly (P < 0.05) improved body weight, locomotor activity, and anxiety like behavior in mirror chamber as well as plus maze performance tasks and anti-oxidant like effect as evidenced by reduced lipid peroxidation, nitrite concentration and restoration of reduced glutathione and catalase activity as compared to control animals. Further, co- administration of sesamol (5 and 10 mg/kg) with buspirone (5 mg/kg) significantly (P < .05) potentiated the anti anxiety effects as compared to their effects alone.
CONCLUSIONS: The present study suggests that combination of sesamol and buspirone potentiated the antianxiety effects against anxiety induced by immobilization stress and oxidative damage in mice.

Introduction

Stress has been shown to affect several brain activities and promote long-term changes in multiple neural systems. The immobilization stress limits body movement resulting into psychological and physical stress.[1] Common stress symptoms include irritability, muscular tension, inability to concentrate and a variety of physical reactions such as anxiety, elevated heart rate, blood pressure, restricted mobility etc.[1] Immobilization stress causes deterioration of antioxidant enzyme activities such as catalase, reduced glutathione and increases lipid peroxidation level and nitrite concentration in brain structure of rodents.[23] This suggests the role of increased production of reactive oxygen species (ROS)in brain.[4] Studies have also reported that a short period of stress causes significant alterations in behavior and generation of free radicals (2-6 h).[5]

Role of serotonergic (5-HT) system is well documented in stress.[6] The serotonergic (5-HT) system regulates mood, emotion and sleep in mammals[7] and its dysregulation has been reported in anxiety like behavior[8] and free radical scavenging properties in vitro.[789] However, its in-vivo regulation on lipid peroxidation and antioxidant status in brain is yet to be demonstrated.[7] Buspirone is a partial 5- HT1A receptor agonist having anxiolytic properties.[10] It is devoid of side effects such as day time sedation and dependence which are generally associated with the use of benzodiazepines.[11] Hence, buspirone has been routinely used for the treatment of different types of anxiety-related disorders in humans.[12] Several studies have also reported buspirone as a potent antioxidant by promoting an increase in antioxidant enzymatic activity.[13]

Sesame lignans are reported to have physiological effects including antioxidant like effect in experimental animals.[1415] Sesamol, 3,4-methylene dioxy-phenol, obtained from Sesamum indicum is a constituent of sesame seed and scavenge hydroxyl radicals[1516] and responsible for its antioxidant properties. Thus antioxidant effect of sesamol may protect against immobilization stress in experimental animals. Further, co- administration of sesamol with buspirone could be a beneficial drug strategy against immobilization stress induced anxiety in animals.

Therefore, the aim of present study was to explore the protective effects of sesamol, buspirone and their combination against immobilization stress induced behavior alterations and oxidative damage.

Materials and Methods

Animals

Male Laca mice bred in central animal house of Punjab University, Chandigarh and weighing between 25-30 g were used. The animals were kept under standard laboratory conditions, maintained on 12 hrs light/dark cycle and had free access to food and water. The animals were acclimatized to laboratory conditions before the test and used once. All the experiments were conducted between 0900 and 1700 hrs. The experimental protocol was approved by Institutional Animal Ethics Committee and was conducted according to the Indian National Science Academy guidelines for the use and care of experimental animals.

Immobilization Stress

Animals were immobilized (IMO) for 6-h by taping all the four limbs on board by putting them on their backs using zinc oxide hospital tape.[67] The animals were released by unraveling the tape after moistening with acetone. In unstressed group, the mice were handled without any stress.

Drug and Treatment Schedule

Following drugs were used in the study-sesamol (SES) (5 mg/ kg and 10 mg/kg, p.o., Sigma Chemicals, 99% purity), buspirone (BUS) (5 mg/kg and 10 mg/kg, p.o., Ranbaxy Laboratories Ltd., Gurgaon). Buspirone was dissolved in double distilled water and sesamol was suspended in 0.5% w/v sodium carboxymethyl cellulose solution and administered by per oral (p.o.) route in constant volume of 1 ml per 100 g of body weight. Animals were divided into ten groups of six animals each. Groups 1 and 2 were treated as normal control and immobilization stress (IMO) control. Groups 3 and 4 were treated with buspirone (5 and 10 mg/kg, p.o.) and groups 5 and 6 were treated with sesamol (5 and 10 mg/kg, p.o.). Groups 7 and 8 were treated with sesamol (5 and 10 mg/kg, p.o.) in combination with lower dose of buspirone (5 mg/kg, p.o.), while groups 9 and 10 were treated as sesamol (10 mg/kg, p.o.) and buspirone (10 mg/kg, p.o.) per se.

Measurement of Body Weight

The body weights of animals were measured before the start of the experiment and thereafter prior to each behavioral quantification.

Elevated Plus Maze

It is a test for assessing anxiogenic and anxiolytic drugs effect in rodents.[17] The plus maze apparatus consist of two open (16 × 5 cm) and two closed arm (16 × 5 × 12 cm) and placed at a height of 25 cm for mice. The animals are placed individually at the centre of the elevated plus maze with their head facing toward an open arm. During the 5-min test, average time spent in the open arm of the maze was recorded.

Mirror Chamber Test

The mirror chamber consisted of a wooden chamber with a mirror enclosed. Animal was placed individually at the distal corner of mirror chamber. During the 5 min test session, the latency to enter the mirror chamber and average time spent in mirror chamber was observed. An anxiogenic response was defined as decreased average time spent in the mirror chamber.[17]

Measurement of Ambulatory Activity

The ambulatory activity was measured using actophotometer (IMCORP, Ambala, India). The animals were acclimatized by placing individually in the activity meter for 3 min. The locomotor activity was recorded for a period of 5 min and expressed in terms of total photo beam counts for 5 min per animal.[17]

Dissection and Homogenization

On day 7, after behavioral quantification, the animals were sacrificed by decapitation. The whole brains were removed and 10% (w/v) tissue homogenate was prepared in 0.1 M phosphate buffer (pH 7.4). Homogenate were centrifuged for 20 min at 15000 rpm and supernatant was used for estimation of lipid peroxidation and reduced glutathione levels. The post-nuclear fractions for catalase assay were obtained by centrifugation of the homogenate at 1000 g for 20 min, at 4°C and for other enzyme assays centrifuged at 12,000 g for 60 min at 4°C.

Lipid Peroxidation Assay

The quantitative measurement of lipid peroxidation was measured by malondialdehyde (MDA),[18] by reaction with thiobarbituric acid using Spectrophotometer (Shimadzu) at 532 nm. The values were calculated using molar extinction coefficient of choursomophore (1.56 × 105M−1cm−1) and expressed as percentage of control.

Estimation of Reduced Glutathione

Reduced glutathione in brain was estimated according to the method described by Ellman.[19] Supernatant (1 ml) was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 4°C for 1 h. The sample was centrifuged at 1200 g for 15 min at 4°C. To 1 ml of this supernatant, 2.7 ml of phosphate buffer (0.1 M, pH 8) and 0.2 ml of 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) were added. The yellow color developed was read immediately at 412 nm using spectrophotometer (Shimadzu). Results were calculated using molar extinction coefficient of choursomophore (1.36 × 104 M−1cm−1) and expressed as percentage of control.

Estimation of Catalase

Catalase activity was assayed by method of Luck[20] wherein the breakdown of H2O2 was measured at 240 nm. Briefly, the assay mixture consisted of 3 ml of H2O2 phosphate buffer (1.25 × 10−2M H2O2) and 0.05 ml of supernatant of the brain homogenate (10%), and the change in absorbance was recorded at 240 nm using the Spectrophotometer (Shimadzu). Enzyme activity was calculated using the mill molar extinction coefficient of H2O2 (0.07). The result was expressed as micromoles of H2O2 decomposed/min/mg protein.

Estimation of Nitrite

The accumulation of nitrite in the supernatant was determined with a colorimetric assay with Greiss reagent (0.1% N-(1-napthyl) ethylene diamine dihyrochloride, 1% sulfanilamide and 2.5% phosphoric acid).[21] Equal volumes of supernatant and Greiss reagent were mixed. The mixture was incubated for 10 min at room temperature. The absorbance was measured at 540 nm using spectrophotometer (Shimadzu). The concentration of nitrite in the supernatant was determined from a standard curve and expressed as percentage of control.

Protein Estimation

Protein estimation was done by biuret method using bovine serum albumin as standard.[22]

Statistical Analysis

All the values were expressed as mean ± SEM. The data was analyzed using One way analysis of variance (ANOVA) followed by Tukey's test (P < 0.05) was considered statistical significant.

Results

Effect of Sesamol, Buspirone and their Combination on the Body Weight and Locomotor Activity

Immobilization (IMO) stress significantly (P < 0.05) reduced the body weight and locomotor activity as compared to control mice. Pre-treatment with sesamol (5 and 10 mg/kg) and buspirone (5 and 10 mg/kg; p.o.) for five days significantly (P < 0.05) attenuated the decrease in body weight [Figure 1] and improved the locomotor activity [Figure 2]. Co-administration ofsesamol (5 and 10 mg/kg; p.o.) with buspirone (5 mg/kg; p.o.) further significantly (P < 0.05) attenuated the decrease in body weight and improved the locomotor activity as compared to immobilization control (IMO) animals. Further, the effect of combination was comparable to that of higher dose of buspirone (10 mg/kg; p.o.) alone.

Figure 1

Comparison of the effect of sesamol, buspirone alone and in combination on the body weight, Values are expressed as mean ± SEM,n= 6 in each group, aP<0.05 as compared to Control, bP<0.05 as compared to IMO. cP<0.05 as compared to BUS(5). dP<0.05 as compared to SES(5), eP<0.05 as compared to SES(10). (One way ANOVA followed by Tukey's test), Immobilization stress: IMO, Buspirone: BUS, Sesamol: SES

Figure 2

Comparison of the effect of sesamol, buspirone alone and in combination on the locomotor activity, Values are expressed as mean ± SEM, n= 6 in each group, aP<0.05 as compared to Control, bP<0.05 as compared to IMO, cP<0.05 as compared to BUS(5), dP<0.05 as compared to SES(5), eP<0.05 as compared to SES(10). (One way ANOVA followed by Tukey's test). Immobilization stress: IMO, Buspirone: BUS, Sesamol: SES

Effect of Sesamol, Buspirone and their Combination in Elevated Plus Maze and Mirror Chamber

Immobilization (IMO) stress for 6 h significantly (P < 0.05) increased the anxiety levels in mice by increase in first transfer latency and decrease in number of entries in mirror chamber test [Table 1]. Moreover there was also increase in number of entries and duration of time spent in closed arm and decreased number of entries as well as duration of time spent in closed armin elevated plus maze test [Table 1]. Pretreatment with sesamol (5 and 10 mg/kg; p.o.) and buspirone (5 and 10 mg/ kg; p.o.) for five days showed significant (P < .05) improvement by decrease in the first transfer latency and increase in the number of entries in mirror chamber test and decrease in number of entries and duration of time spent in closed arm and increased number of entries and duration of time spent in open arm in elevated plus maze test. Co-administration of sesamol (5 and 10 mg/kg; p.o.) with buspirone (5 mg/kg; p.o.) further significantly (P < 0.05) improved the behavior in both elevated plus maze and mirror chamber and the effect was comparable to higher dose of buspirone (10 mg/kg; p.o.) alone.

Table 1

Comparison of the effect of sesamol, buspirone alone and in combination on anxiety levels in mirror chamber and elevated plus maze

Effect of Sesamol, Buspirone and their Combination on Lipid Peroxidation, Nitrite, Reduced Glutathione and Catalase Activity

Six hour immobilization (IMO) stress significantly (P < 0.05) elevated lipid peroxidation (LPO), nitrite concentration and depleted reduced glutathione (GSH) and catalase levels [Table 2] as compared to normal control. Pretreatment with sesamol (5 and 10 mg/kg; p.o.) and buspirone (5 and 10 mg/kg; p.o.) significantly (P < 0.05)reduced lipid peroxidation (LPO), nitrite concentration and restored depleted glutathione (GSH) and catalase levels [Table 2] as compared to IMO mice. Co-administration of sesamol (5 and 10 mg/kg; p.o.) with buspirone (5 mg/kg; p.o.) further significantly (P < 0.05) improved levels of these oxidative markers.

Table 2

Comparison of the effect of sesamol, buspirone alone and in combinationon the lipid peroxidation, nitrite, reduced glutathione and catalase concentration

Discussion

In the present investigation, immobilization-induced acute stress for 6 h resulted in significant behavioral alterations including decrease in locomotor activity, body weight and caused anxiety like behavior as depicted in both mirror chamber test and elevated plus maze and induced oxidative stress. Earlier studies have also revealed that immobilization stress influences behavioral activity such as motor activity and anxiety-like effect and causes deterioration of antioxidant enzyme activities such as catalase, reduced glutathione etc.[12] Immobilization-induced stress has been the most frequently employed model of stress because stressor is of sufficient intensity to induce 2-3 fold higher rise of plasma cortisol level.[23]

The present study also showed that immobilization stress significantly impaired the antioxidant defense system as shown by increase in free radicals and impair antioxidant defenses.[4] Therefore it is possible to state that immobilization-stress-induced behavioral alterations are primarily due to the increased amount of oxidative stress in the brain animal.

Present study revealed that pretreatment with sesamol and buspirone significantly attenuated immobilization stress induced behavioral and biochemical alterations. Buspirone is a partial 5-HT1A receptor agonist and has been preferred as standard anxiolytic agent[10] over benzodiazepines because of lesser side effects such as excessive sedation and motor impairment.[11] Serotonergic (5-HT) systems are activated by stressful stimuli and dysregulation of 5-HT1A receptors have been reported to contribute towards anxiety-like behavior.[9] 5-HT activates corticotrophin- releasing factor in the paraventricular nucleus and increases the release of ACTH.[24] Recent studies suggest the antioxidant like potential of buspirone.[13] Sesamol, a constituent of sesame seed is reported for its free hydroxyl radical scavenging action.[1516] Earlier findings suggest the protective effect of sesamol in immobilization stress induced oxidative burden.[15] Also, free radical scavenging action of sesamol might be contributing towards improvement of both body weight and locomotor activity.

Further, sesamol showed enhanced anxiolytic effect when administered in combination with buspirone suggesting their therapeutic synergism against immobilization stress induced anxiety-like behavior and oxidative stress. It is known that oxidative stress plays a role in pathogenesis of motor activity.[1]

This further provides a possibility that antioxidant like effect of both sesamol and buspirone contributed towards anti-anxiety like effect of buspirone. The potentiation of anxiolytic activity in addition to antioxidant effect can be attributed to combination of sesamol with buspirone. These findings suggest that sesamol and buspirone might have acted through different pathways, thus providing beneficial actions when administered in combinations.

The present study concludes that the combination of sesamol and buspirone exerts a protective synergistic action against biochemical and behavioral alterations induced by immobilization stress in mice. Further investigations may be considered for its use in management of stress related disorders.