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Effect of combination of ketanserin and escitalopram on behavioral anomalies after olfactory bulbectomy: Prediction of quick onset of antidepressant action Abstract Objectives: Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed antidepressant drugs. The addition of low dose of 5-hydroxytryptamine type 2A enhances the therapeutic effect of SSRIs. The purpose of the present studies was to test the effects of combined treatment of a low dose of ketanserin (KET) and escitalopram (ESC) on behavioral anomalies occurring after olfactory bulbectomy (OBX). Materials and Methods: Chronic Depression was induced by OBX as shown in behavioral tests such as Open field, social interaction, and hyperemotionality tests. Acute and chronic treatment effect of KET, ESC, and combination was administered to the OBX rats. Results: Chronic (14 days) treatment with KET (1 mg/kg) or ESC (10 mg/kg) alleviated the behavioral anomalies of olfactory bulbectomized rats in modified open field exploration, social interaction, hyperemotionality. When KET treatment was combined with ESC, a short duration regimen (7 days) was sufficient to reverse the bulbectomy-induced anomalies. Conclusion: The combination therapy as a likely strategy to achieve an early-onset of antidepressant action. Introduction Antidepressants are the third most frequently prescribed therapeutic agents worldwide.[1] Selective serotonin re-uptake inhibitor (SSRI) are the most extensively used of all the antidepressants. Serotonin transporter blockade is the most common pharmacological action shared by SSRIs (SSRIs; e.g., citalopram, escitalopram (ESC), fluoxetine, sertraline), adopted for management of mood and anxiety disorders. The major shortcomings in treating depression with SSRIs are that the therapeutic response develops slowly (3-4 week), the side-efiects that often occur and that there is a significant percentage (30%) of nonresponders.[2] The mechanism behind the delay of onset could be the desensitization of receptors due to prolonged 5-HT exposure. The evidence of neuronal metabotropic 5-hydroxytryptamine type 2A (5-HT2A) receptors in the assumed correlates of depression, specifically, hippocampus, amygdala, prelimbic prefrontal cortex, striatum, and olfactory structures[34] has supported the role of these receptors in rodent's depression-like behavior and in human depression. Several antidepressants down-regulate 5-HT2A receptors after chronic treatment, supporting the role of 5-HT2A receptors in antidepressant action.[5] The paradoxical, antagonist-induced desensitization and down-regulation of 5-HT2A receptors[5] and the regulatory effects of this receptor on brain-derived neurotrophic factor pathway[6] are known to have interesting implications in the depression pathology. Interestingly, it has been shown that SSRIs increase, whereas tricyclic antidepressants decrease, binding of ligands to the 5-HT2A receptor.[7] More recently, the 5-HT2C receptor has been shown to augment the increase in extracellular 5-HT with SSRIs in microdialysis, and in 5-HT2C knockout mice, there was an augmented antidepressant effect with the administration of SSRIs as compared to their wild-type counterparts.[8] Surprisingly, despite the numerous rodents models of depression, there are few animal models currently competent of detecting a faster onset of antidepressant action over another.[9] Along with faster antidepressants, rodent's model to assess quick antidepressant action is prerequisite. Henceforth, in the current study, olfactory bulbectomy (OBX) in rats has been adopted as the animal model to evaluate the early onset of antidepressant in combination therapy[9] using ketanserin (KET) and ESC; as OBX requires chronic antidepressant treatment before a change is detected in the attenuation of hyperactivity. Materials and Methods Animals Male Wistar rats (180-200 g) were obtained from Hissar Agricultural University, Haryana, India. All animals were maintained under standard laboratory conditions (12 h light/dark cycle [lights on at 7:00 AM]; temperature 23°C ± 2°C; relative humidity; 60% ± 5%) in the Central Animal Facility. Rats were given sterilized food (standard pellet chow feed) and filtered water ad libitum. Following a quarantine period of 2 weeks, the experiment was started. The experiments on animal were performed in accordance with the protocol approved by the Institutional Animal Ethics Committee (IAEC) of Birla Institute of Technology and Science, Pilani, India (Protocol No. IAEC/RES/12/01). Drugs Escitalopram was procured from Glenmark laboratory as generous gift samples, Mumbai, India. KET tartrate (KET), a preferential 5-HT2A receptor antagonist[10] was purchased from Sigma-Aldrich chemicals private limited, New Delhi. Ketamine and Xylazine injection were obtained from Neon Lab and Indian Immunological Mumbai, India, respectively. All the drugs were dissolved in sterile distilled water and were freshly prepared for use. In OBX rat model of depression, KET (1 mg/kg)/Vehicle/ESC (10 mg/kg) was administered once a day orally for 7 or 14 days to OBX/sham rats, after post-surgical rehabilitation period (14 days). The surgery, rehabilitation, treatment, and behavioral assessment in the OBX study were done according to a schedule[11] with slight variation [Table 1], adopted for our laboratory. The animals were subjected to behavioral assessment 20 h after drug/vehicle administration. The drug treatment was continued until 23rd/30th day and to avoid the direct effect of treatment on behavior, drug administration was done after each behavioral ESC based on the treatment schedule [Table 1], The doses of standard antidepressants were selected from the pilot studies conducted in our laboratory.[12] All administrations and behavioral assessments were done between 10 and 15 h and to avoid bias, all observations were carried out by trained experimenters. Table 1 Schedule of treatments and behavioral assessments on OBX and sham-operated rats Olfactory Bulbectomy Surgery Bilateral OBX was performed as described by Kelly et al. and Ramamoorthy et al.[1314] In brief rats (220-240 g) were anesthetized using combination of xylazine and ketamine (5 and 75 mg/kg, i.p.). The animals were fixed in a stereotactic frame (Inco, India) and the skull was exposed by a midline incision and burr holes (2 mm in diameter) were drilled 8 mm anterior to bregma and 2 mm on either side of the midline at a point corresponding to the posterior margin of the orbit of the eye. The olfactory bulbs were removed by suction, the holes were then filled with hemostatic sponge in order to control excessive bleeding, and the scalp was sutured. Sham-operated rats were treated in a similar manner, including piercing of the dura mater, but their bulbs were left intact. To avoid postsurgical infection, the animals were given Sulprim injection (each ml containing 200 and 40 mg of sulphadiazine and trimethoprim respectively), intramuscularly (0.2 ml/300 g) once a day for 2 days, post-surgery. The OBX/Sham animals were housed singly in cages. Open field behavior The open field exploration was conducted as described by Kelly et al. and Ramamoorthy et al.[1314] with substantial modifications. The apparatus consisted of a circular (90-cm diameter) arena with 75-cm high aluminum walls and floor equally divided into 10 cm squares. A 60 W light bulb was positioned 90 cm above the base of the arena, which was the only source of illumination in the testing room. Each animal was individually placed in the center of the open field apparatus, and the following parameters were noted for 5 min. Ambulation scores (number of squares crossed), Thigmotaxis (movement at corner of the apparatus) and the number of rearing episodes were noted as horizontal and vertical activity, respectively. After each experiment, the apparatus was sprayed with dilute alcohol and wiped thoroughly to eliminate the residual odor. Rationale: Measurement of Hyperactivity Reflects Psychomotor Effect Social interaction test The present protocol was adapted from File and Hyde[15] and performed with slight modification. The apparatus and testing environment was similar to that of an open field test except a milder (40 W) illumination. On the day of the test, rat pairs of the same group housed in different cages were put into two different corners of the open field arena. The social interaction behavior including the running toward, probing, grooming, mounting, and crawling under the other rat were recorded for 10 min after placement of the animals into the apparatus. After each test, the apparatus was sprayed with dilute alcohol and wiped thoroughly to eliminate the residual odor. Rationale: Simulation of Human Social Behavior Hyperemotionality The procedure reported in the literature Shibata et al.[16] was adopted with slight alterations. Hyperemotionality of rats was measured by scoring the responses to the following stimuli: (1) Bite response: Bite response is scored by a rod presented 4-5 cm in front of the snout, (2) startle response: Startle response to a stream of air directed at the dorsum was scored. The air was delivered using 5-ml syringe, (3) struggle response: Struggle response was scored by handling with a gloved hand (struggle response), and (4) fight response: Fight response was scored by the tail pinching with a forceps. The rat tail is gently held from the back of a rat using blunt forceps. The responses were graded as: 0, no reaction; 1, slight; 2, moderate; 3, marked; or 4, extreme response. For each emotional response, audible vocalization were also scored and graded as follows, 0, no vocalization; 1, occasional vocalization; or 2, marked vocalization. The vocal score was added to each emotional response score. All animals in each group were observed in 1 day. The score for each animal in emotional response was given within 5 min. The observers were blind with respect to the drug treatment. Statistical Analysis All analysis was performed using Graph Pad Prism 5 for windows (GraphPad Software). The results are expressed as mean ± standard error of the mean. Statistical differences were evaluated with a Two-way analysis of variance (ANOVA) followed by the Bonferroni for multiple comparisons. The criterion for a statistically significant difference was fixed to P < 0.05. Results Effects of Escitalopram and Ketanserin on Behavior of Olfactory Bulbectomy Rats As shown in Table 2, OBX rats exhibited significant increase in ambulation (F7,40 = 30.25, P < 0.05), rearing (F7,40 = 45.23, P < 0.05), thigmotaxis (F7,40 = 14.69, P < 0.05), and defecation (F7,40 = 12.90, P < 0.05) as compared to sham treatment in modified open field paradigm. Chronic (14 days) treatment with ESC (10 mg/kg) and KET (1 mg/kg) significantly reduced the ambulation, Thigmotaxis and rearing in OBX rats as compared to vehicle treated OBX rats. OBX rats treated with ESC (10 mg/kg) and KET (1 mg/kg) exhibited decreased defecation in addition to the decrease in ambulation and number of rears. Such an effect was significant when compared to lone vehicle, ESC or KET treatments. In the sub-chronic treatment schedule (7 days), except for the ESC + KET-1, which significantly decreased the ambulation (F7,40 = 24.82, P < 0.05), rearing (F7,40 = 14.52, P < 0.05), Thigmotaxis (F7,40 = 8.05, P < 0.05and fecal pellets (F7,40 = 8.59, P < 0.05) as compared to the vehicle treated group and all other treatments fall short to show any significant reversal of abnormal behavior anomalies in OBX rats [Table 2]. Table 2 Effects of ESC, KET and the combination treatment on open field behavior of OBX and sham-operated rats In the hyper-emotional behavior paradigm assessment, it was found that OBX rats showed enhanced emotional behavior compared to sham treatment [Table 3]. Such an effect was significantly (F7,40 = 46.52, P < 0.05) reduced by chronic ESC (10 mg/kg)/KET (1 mg/kg)/ESC + KET treatments compared to vehicle treatment. However, while observing the data from 7 days treatment, it is evident that the combination of ESC + KET significantly (F7,40 = 13.72, P < 0.05) reduced the total hyperemotionality score compared to vehicle-treated OBX rats. None of the other treatments significantly changed 7th day hyperemotionality scores [Table 3]. The drug treatments did not conspicuously influence the behavior of sham animals in any of the paradigms mentioned above. Table 3 Effects of ESC, KET and the combination treatment on hyperemotionality scores of OBX and sham-operated rats Social interaction pattern revealed that OBX rats spend less time in interaction and display an increased number of crossings compared to sham treatment. Chronic treatments with ESC (10 mg/kg), KET (1 mg/kg) and ESC (10 mg/kg) + KET (1 mg/kg) significantly increased the interaction time (F7,40 = 16.59, P < 0.05) and decreased number of crossing (F7,40 = 58.53, P < 0.05) compared to vehicle treatment. Combined ESC + KET treatment was most effective in reversing the OBX induced behavior and the effects were better than the lone ESC (10 mg/kg) or KET (1 mg/kg) treatments. In the 7 days treatment schedule, the combination treatment significantly increased the interaction time (F7,40 = 14.35, P < 0.05) and decreased the crossing (F7,40 = 22.67, P < 0.05) compared to vehicle treatment [Table 4]. Table 4 Effects of ESC, KET and the combination treatment on social interaction of OBX and sham-operated rats Discussion Till date, successful attempts for early onset of action of antidepressants have been depending on pharmacological approaches based on monoaminergic systems. This encompasses the amalgamation of 5-HT reuptake blockade-which represents >80% of all antidepressant treatments on its own (e.g., SSRIs). The current study demonstrates the outcome of a combination approach on OBX induced behavior anomalies. In contrast to other animal models of depression, the OBX characterizes a model of chronic agitated hyposerotonergic depression[17] which is receptive to discover the antidepressant-like effects of agents affecting the 5-HT receptor subtypes.[18] OBX necessitates a chronic antidepressant treatment before a change is detected is the attenuation of hyperactivity. Interestingly, this model has been in front as a promising model for detecting a faster onset of antidepressant effects. Nonetheless it is believed further refinement of the model may be required.[19] The chronic mild stress model has also been proposed as a rodents model for early onset of depression.[20] In the current study, OBX rats exhibited a diverse behavioral anomalies, such as (i) ambulation (locomotion), rearing, thigmotaxis, and defecation in modified open field exploration paradigm, (ii) diminished active interaction and increased crossing in social interaction paradigm and (iii) enhanced hyperemotionality scores as reported earlier. The chronic regime of ESC significantly reversed the bulbectomy-induced behavior in all the aforementioned paradigms. This result is in line with previous reports on reversal of bulbectomy induced behavior by ESC. In addition, distinguished similarity between OBX rat and human depression is the distorted 5-HT2A receptor binding and function.[21] Mianserin and nefazodone restored the avoidance learning in bulbectomized rats, and this effect was correlated with down-regulation of 5-HT2 receptors in the frontal cortex.[22] Early onset of action is prerequisite of antidepressant drug treatment,[23] and several approaches have been predicted to achieve the same.[24] It is a notable finding that the antidepressant-like effects of the co-administration of ESC and KET were evident with a short course of treatment (7 days) in OBX rats. KET treatment can possibly lead to a blockade of serotonin type 2A receptors which eventually results in enhanced 5-HT and norepinephrine neurotransmission.[25] The synaptic serotonin levels are further increased when ESC is co-administered with KET, due to the inhibition of serotonin reuptake by ESC. Therefore, a quick-onset of antidepressant-like effect is feasible due to the involvement of both serotonergic neurotransmission systems[5] and 5-HT2A blocker.[5] Furthermore, based on clinical observations, it is noted that 5-HT2A receptor antagonism is a candidate mechanism for effectual and quick-onset antidepressant action.[34] In summary, the current study advocates that the blockade of 5-HT2A receptors may complement the antidepressant effects of ESC. Combined administration of ESC with the KET, can be considered as an approach to accomplish an early-onset of antidepressant action in humans. Further, more such studies are warranted, whether selective blockade of 5-HT2A receptors can also have an additive or synergistic action in the treatment of other diverse neuropsychiatric disorders.