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Pharma Science Monitor 6(4), Oct-Dec 2015

PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: http://www.pharmasm.com

INVESTIGATING THE POSSIBLE MECHANISM OF BENEFICIAL EFFECT OF METHYLENE BLUE AGAINST -INDUCED TARDIVE DYSKINESIA IN RATS Sandeep Goyal1*, Amandeep Kaur1, Sunil Kumar Kansal1, Uma Jyoti1, Puneet Kumar2, Arun Kaura1 1University Institute of Pharmaceutical Sciences and Research, Baba Farid University of Health Sciences, Faridkot 151 203, Punjab, India. 2ISF College of Pharmacy, Ferozepur Road, Ghal Kalan, Moga 142 001, Punjab, India.

ABSTRACT Tardive dyskinesia is a neurological iatrogenic disorder and is one of the fatal neurodegenerative movement syndromes affecting mainly orofacial region. Methylene blue, a has been reported to possess neuroprotective and antioxidant effects. The present study has been designed to investigate the possible mechanism of beneficial effect of methylene blue against haloperidol induced tardive dyskinesia in rats. Tardive dyskinesia was induced by administering haloperidol (1 mg/kg, i.p.) and concomitantly treated with methylene blue (0.5 mg/kg and 1mg/kg, i.p.), L- NAME (10mg/kg, i.p.) and L-Arginine (50 mg/kg, i.p) for 3 weeks in Wistar rats. Various behavioral parameters (vacuous chewing movements, tongue protrusions, facial jerkings, locomotor activity, motor co-ordination , gait abnormality and muscle strength) were assessed on days 1,7,14 and 21. Biochemical (oxidative stress, antioxidant defense, neuroinflammation, neurochemical estimations of & ) and histological parameters were assessed at day 22. Chronic administration of haloperidol significantly increased stereotypic behaviors, in rats, which were significantly ameliorated by administration of methylene blue. Chronic administration of haloperidol significantly enhanced oxidative stress, neuroinflammation and neuronal loss in the striatum region of the rat brain which were prevented by co-administration of methylene blue and L-NAME. Thus, the present study supports the evidence of possible beneficial effect of methylene blue in haloperidol-induced tardive dyskinesia and possible nitrosative mechanism for its antioxidant and neuroprotective effect. KEYWORDS: Methylene blue, Nitrosative mechanism, Oxidative stress, Tardive dyskinesia.

INTRODUCTION Patients of schizophrenia, schizoaffective or bipolar disorders have been prescribed with drugs for longer duration as a therapeutic approach. This causes tardive dyskinesia like symptoms in them due to long term blockade of dopamine receptors, thereby producing movement defects. Haloperidol is widely prescribed for the treatment of schizophrenia and other mental illnesses[1]. Haloperidol is bio-transformed to neurotoxic pyridinum species known as

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HPP+ that is toxic to dopaminergic neurons through inhibition of complex-I of the mitochondrial [2]. Haloperidol induced tardive dyskinesia presents a neurochemical basis of the disease, the exact mechanism is not clear. Tardive dyskinesia is a motor complication of neuroleptic therapy which involves abnormal involuntary hyperkinetic movements affecting mouth, face, tongue and sometimes limbs and trunk musculature[3,4]. TD occurs in 20-40% of patients who are on chronic neuroleptic medication[5]. Oxidative stress is implicated in the pathophysiology of tardive dyskinesia[6]. Oxidative stress is the overproduction of the free radicals such as reactive species (ROS) and reactive nitrogenous species (RNS) which may affect the various pathways leading to the various diseases. Nitric oxide (NO) is a free radical that acts as a messenger in the CNS. It is synthesized from l-arginine by enzyme nitric oxide synthase (NOS) using NADPH and molecular oxygen. Oxidative stress increases the releases of inflammatory mediators like TNF-α, IL-1β and IL-6 which are responsible for resultant neuronal cell death due to induction of the apoptosis[7]. Methylene blue is a dye[8] and also a sui generis drug because its physiological, pharmacological and clinical effects are not determined by regular drug receptor interactions[9]. It is approved by FDA for the treatment of methaemoglobinemia. The drug methylene blue has been reported to successfully complete a phase IIb for the treatment of mild to moderate Alzheimer disease showing an improvement in cognitive function after six months and slowing the progression of AD by 81% over a period of one year[10]. It has a 23 unique property of transferring electron from NADH and FADH2 to CoQ and Cyt c i.e. even on the inhibition of complex-I (NADH dehydrogenase) electron can be transferred to the Co Q and Cyt c bypassing the complex-I and II. Bypass of electron leads to decreased electron leakage, decreased ROS formation and improved energy levels. Moreover, has been reported for its beneficial effects in stroke, parkinsonism and optic neuropathy etc[8]. Thus, the present study investigated the possible mechanism of beneficial effect of methylene blue against haloperidol-induced tardive dyskinesia in rats. MATERIAL AND METHODS Animals and drug treatment Old Wistar rats (220-300g) were taken from Central Animal House of I.S.F. College of Pharmacy, Moga, Punjab and used in the present study. Animals were acclimatized to laboratory conditions at room temperature prior to experimentation. Animals were kept under standard conditions of 12h light/dark cycle with food and water ad libitum in plastic cage with soft

Sandeep et al. / Pharma Science Monitor 6(4), Oct-Dec 2015, 193-209 Impact factor: 0.3397/ICV: 4.10 195 bedding. All the behavioral assessments were carried between 9:00 and 15:00 hrs. The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC) of ISF College of Pharmacy, Moga, India and was carried out in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) for the use and care of experimental animals. Haloperidol was obtained from the “haldol”. Methylene blue was dissolve in normal and haloperidol dissolved in normal distilled water and both were administered by i.p. L-arginine and L-NAME was given for 21 days. Animals were divided into six groups, each group contain six numbers of animals. All drugs were given in constant volume of 0.5 ml per 100 g of body weight. Haloperidol or vehicle was administered daily for 21 days. Methylene blue was administered one hour prior to haloperidol treatment for 21 days. 36 rats were randomly divided into six groups. First group vehicle Control (5 ml/kg, i.p.) second group Haloperidol Control (1 mg/kg, i.p.) third group Haloperidol+ MB- low dose (0.5 mg/kg, i.p.) fourth group Haloperidol+ MB- High dose (1 mg/kg, i.p.) fifth group Haloperidol+ MB+L-NAME (10 mg/kg, i.p.) sixth group Haloperidol+MB+L-Arginine (50 mg/kg, i.p.). Measurement of body weight Animal body weight was recorded on the first and last day of experimentation[11]. Percent change in body weight was calculated. Assessment of behavioral parameters Measurements of VCM VCMs are characterized by purposeless (non-directed) mouth opening in the vertical plane with or without tongue protrusion. VCMs have been shown to develop secondary to various pharmacological (dopamine depletion, cholinomimetics) and surgical lesions. On the test day rats were placed individually in a small (30x20x30cm). Plexiglass cage for the assessment of oral dyskinesia. Animals were given 10 min to get acclimatized to the observation cage before behavioral assessments. To quantify the occurrence of oral dyskinesia, hand operated counters were employed to score VCMs, facial jerking, tongue protrusion, sniffing and grooming. If VCMs, facial jerking and tongue protrusion occurred during a period of grooming, they were not taken into account. Counting were stopped whenever the rat began grooming, and restarted when grooming stopped. Mirrors were placed under the floor and behind the back wall of the cage to permit observation of oral dyskinesia when the animal was faced away from the observer. The behavioral parameters of oral dyskinesia were measured continuously for a period of 10 min. In all the experiments, the scorer was unaware of the treatment given to the animals[7].

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Locomotor activity Locomotor activity was monitored by using an actphotometer (Medicraft, INCO, Ambala, Har- yana, India), which operates on photoelectric cells connected in a circuit with a counter. Before subjecting the animals to a cognitive task, all the animals were individually placed in the activity meter and the total activity count was measured for 10 min. The locomotor activity was expressed in terms of total photo beam counts per 10 min per animal[12]. Rotarod activity Motor co-ordination was assessed for all rats on a rota-rod. Rats were placed individually on a rotating rod with a rod of 7 cm (speed 25 rpm). Prior to any treatment, rats were trained in a single session until they attained 180 seconds on the rota-rod. Fall off time was recorded during drug treatment every week[13]. Beam crossing task Narrow beam walking is a simple test to motor coordination of the animal by allowing animals free to move on a stationary but narrow wooden beam which requires balance and equilibrium. The beam was flat (length 130 cm and width 1 cm) and was placed at a height of 100 cm from the floor to avoid the intentional falling. The mice were trained to walk in the beam from one end of the beam (Start area) to reach another end (Target area). During that, walking time to cross the beam and the number of foot errors were noted[4] Elevated plus maze The elevated plus maze consists of two opposite white open arms (16 × 5 cm), crossed with two closed walls (16 × 5 cm) with 12 cm high walls. The arms were connected with a central square of dimensions 5 × 5 cm. The entire maze was placed 25 cm high above the ground. Acquisition of memory was tested on day 20. Mice were placed individually at one end of the open arm facing away from the central square. The time taken by the animal to move from the open arm to the closed arm was recorded as the initial transfer latency (ITL). Animals were allowed to explore the maze for 10 s after recording the ITL. If the animal did not enter the enclosed arm within 90 s, it was guided to the enclosed arm and the ITL was recorded as 90 s. Retention of memory was assessed by placing the mouse in an open arm and retention latency was assessed on day 21 and day 42 of the ITL, termed as the first retention transfer latency (1st RTL) and second retention transfer latency (2nd RTL), respectively[12]. Percent (%) retention of memory was calculated by the formula: [(% retention of memory = [(TL on day 1 – TL after 24 hrs) / TL on day 1] x 100 Biochemical estimations

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Measurement of oxidative stress parameters Estimation of peroxidation The extent of lipid peroxidation in the brain was determined quantitatively by performing the method as described by Wills[14]. The amount of malondialdehyde (MDA) was measured by reaction with thiobarbituric acid at 532 nm using a Perkin Elmer Lambda 20 spectrophotometer. The values were calculated using the molar extinction co-efficient of chromophore (1.56 × 105 (mol/l)-1cm-1) Estimation of The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide, was determined by a colorimetric assay with Griess reagent (0.1 % N-(1-napththyl) ethylene diamine dihydrochloride, 1% sulphanilamide and 5% phosphoric acid). Equal volumes of the supernatant and the Griess reagent were mixed and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 540 nm using a Perkin Elmer Lambda 20 spectrophotometer. The concentration of nitrite in the supernatant was determined from standard curve[15]. Measurement of antioxidant defense Catalase estimation Catalase activity was assessed by the method of Luck, wherein the breakdown of H2O2 is measured. Briefly, the assay mixture consists of 3 ml of H2O2 buffer and 0.05 ml of the supernatant of the tissue homogenate. The change in absorbance was recorded for 2 min at 30 s intervals at 240 nm using a Perkin Elmer Lambda 20 spectrophotometer. The results were expressed as micromoles of decomposed per min per mg of protein[16]. estimation Reduced glutathione was estimated according to the method described by Ellman. Supernatant (1 ml) was precipitated with 4% sulphosalicylic acid (1 ml) and cold digested for 1 hour at 4°C. The samples were then centrifuged at 1,200 g for 15 min at 4°C. To 1 ml of the supernatant obtained, 2.7 ml of phosphate buffer (0.1 mmol/l, pH 8) and 0.2 ml of 5, 5’ dithio-bis (2- nitrobenzoic acid) (DTNB) was added. The yellow color developed was measured at 412 nm using a Perkin Elmer Lambda 20 spectrophotometer[17]. Protein estimation Proteins level was estimated in brain because they serve as catalysts that maintain metabolic processes in the cell and as signals secreted by one cell or deposited in the extracellular matrix

Sandeep et al. / Pharma Science Monitor 6(4), Oct-Dec 2015, 193-209 Impact factor: 0.3397/ICV: 4.10 198 that are recognized by other cells. The protein was measured by the Biuret method using bovine serum albumin (BSA) as a standard[18]. Measurement of neuroinflammatory estimations Estimation of Tumor Factor-alpha (TNF-α) levels TNF-α level was estimated by using rat TNF-α kit (RayBio, Rat TNF-alpha ELISA kit protocol). It is a solid phase sandwich enzyme linked immunosorbent assay (ELISA) that uses microtiter plate reader which is read at 450 nm. Concentrations of TNF-α were calculated from plotted standard curve Estimation of interleukin (IL-6) levels IL-6 level was estimated using rat IL-6 kit (RayBio, Rat IL-6 ELISA kit protocol). It is a solid phase sandwich enzyme linked immunosorbent assay (ELISA), which uses a microtitre plate reader which is read at 450 nm. From plotted standard curve concentrations of IL-6 were calculated. Neurochemical estimations All animals were sacrificed by cervical dislocation and their brains were dissected to isolate Striatum region. Biogenic amines (i.e. dopamine and norepinephrine) were estimated in different parts of the brain by HPLC (waters standard system; waters Milford, Massachusetts USA), data were recorded and analyzed with the help of Empower-3 (Orlando, Florida,USA) software. Brain samples were homogenized, containing 0.1M of perchloric acid. Samples were then centrifuged at 24000× g at 4°C for 15minutes.the supernatant was further filtered through 0.25µm nylon filters before injecting in the HPLC injection pump[19]. Histlopathological examination After completion of the above behavior tests, 3 mice in every group were sacrificed by cervical dislocation, and the brains were transcardially perfused with phosphate buffer solution (PBS, pH 7.4), followed by 4% paraformaldehyde in PBS (pH 7.4). The brains were removed and kept overnight in PBS containing 4% paraformaldehyde at 4°C (Brains should not freeze) and embedded in paraffin. Sections of 5-μm thickness were stained with hematoxylin and eosin. Hematoxylin is used to stain nuclei blue, while eosin stains cytoplasm and the extracellular connective tissue matrix pink. The lesions of brain tissues were observed with light and the images were collected by image analysis system[20]. Statistical Analysis The data was analyzed by using analysis of variance (one-way and two-way ANOVA) followed by post hoc test. All the values were expressed as means ± S.E.M in all tests, the criterion for

Sandeep et al. / Pharma Science Monitor 6(4), Oct-Dec 2015, 193-209 Impact factor: 0.3397/ICV: 4.10 199 statistical significance was p<0.05. The data was analyzed using graph pad prism 5.01 versions (San Diego, CA, USA). RESULTS AND DISCUSSION Haloperidol has a multireceptor effect in the brain. Chronic administration of haloperidol for 21 days in animals has been found to produce VCMs and related behavioral disorders. These have been hypothesized as the symptoms of orofacial dyskinetic movements, which are generally observed in patients who are on chronic treatment with typical neuroleptics[21]. In the present study chronic haloperidol treated animals showed increased frequencies of vacuous chewing movements and tongue protrusions as compared to vehicle treated control animals. In the rat striatum, with VCM induced by chronic administration of haloperidol the increase in extracellular concentration of glutamate is seen. These finding suggest that the possibility that an excessive release of glutamate is caused by the dopamine D2 receptor blocker haloperidol which further induces the development of behavioral parameters and biochemical estimations were due to its potential to increase oxidative stress, thereby producing necrosis and apoptosis in striatum are of brain to produce TD in rats. Antipsychotic drugs produce orofacial dyskinesia in humans when used chronically[22]. Clinical studies have also demonstrated decreased concentration of GABA in the cerebrospinal fluid of patients with dyskinetic problems. Orofacial dyskinesia is abnormal facial, tongue and chewing movements which are also termed as tardive dyskinesia.

FIGURE 1: Effect of pharmacological interventions on body weight against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

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FIGURE 2: Effect of pharmacological interventions on vacuous chewing movement against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

FIGURE 3: Effect of pharmacological interventions on facial jerking against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

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FIGURE 4: Effect of pharmacological interventions on tongue protrusions against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

FIGURE 5: Effect of pharmacological interventions on gait abnormality using narrow beam walk test apparatus against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

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FIGURE 6: Effect of pharmacological interventions on gait abnormality using narrow beam walk test apparatus (number of slips) against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

FIGURE 7: Effect of pharmacological interventions on locomotor activity using open field method against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

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FIGURE 8: Effect of pharmacological interventions on rearing movements using open field method against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

FIGURE 9: Effect of pharmacological interventions on motor coordination using rota rod apparatus (fall off time) against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

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FIGURE 10: Effect of pharmacological interventions on short term memory using elevated plus maze against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

FIGURE 11: Effect of pharmacological interventions on dopamine levels against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

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FIGURE 12: Effect of pharmacological interventions on norepinephrine levels against haloperidol induced tardive dyskinesia in rats. Values are expressed as Mean ± S.E.M.* vs. vehicle treated group, # vs. haloperidol group, @ vs. HPD+MB (L) (0.5 mg/kg), $ vs. HPD+ MB (H) (1 mg/kg) & HPD+MB (0.5mg/kg) +L-NAME. P<0.05 is considered statistically significant.

Haloperidol at 1 mg/kg, i.p. for 21 days produced significant decrease in body weight (Figure 1) and various behavioral alterations like increased orofacial dyskinesia (VCM, FJ, TP; Figure 2,3,4)[4] gait abnormality[23] (narrow beam walk; Figure 5,6), locomotor activity (open field; Figure 7,8), muscle grip strength (rotarod; Figure 9), spatial memory loss[24] (elevated plus maze; Figure 10), as well as biochemical alterations like increased oxidative stress (lipid peroxidation and nitrite level table 1), decreased antioxidant estimations (catalase activity and reduced glutathione estimations), increased neuroinflammatory estimations (TNF-α 213, IL-6) decreased neurochemical estimations (Post, 2014) (dopamine and norepinephrine; Figure 11,12) along with histological alterations (Plate 1) like gliosis, neuro inflammation and neuronal loss in rat brain (striatum area) as compared to vehicle treated group. The above results confirm the establishment of tardive dyskinesia after haloperidol administrated for 21 days. The present study envisages the beneficial effect of methylene blue against haloperidol induced tardive dyskinesia in rats. The neuroprotection effect of methylene blue and its mechanism has not yet been elucidating for haloperidol induced tardive dyskinesia. So, methylene blue was administrated at a dose of (0.5 mg/kg and 1 mg/kg) to haloperidol induced tardive dyskinesia in

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rats on 1st day to 21st day. For this, a statistical comparison was made to find out the level of significance. Methylene blue (0.5mg/kg and 1mg/kg) i.p. for 21 days, prevented haloperidol induced behavioral, biochemical and histological alterations in rats on comparison of both doses of methylene blue, it was found that, there is statically significant difference among groups in respect to all the parameters. In the present study, to find out the mechanism of neuroprotective effect of methylene blue. L-NAME (10 mg/kg) i.p. and L-Arginine (50 mg/kg) i.p. were administrated to haloperidol induced tardive dyskinesia in rats individually for 21 days. L- NAME treatment prevented statistical difference between behavioral, biochemical and histological alterations induced by haloperidol induced in rats. Thus, L-NAME, which is NOS inhibitor, produced beneficial effect against haloperidol induced tardive dyskinesia in rats, may be via decreasing the levels of NO It may be due to increased nitrosative stress in rat brain. Whereas administration of L-NAME along with methylene blue to haloperidol induced tardive dyskinesia have produced its neuroprotective effect by decreasing oxidative stress, neuroinflammation, neuronal loss. The combination of L- NAME with methylene blue has produced profoundly significant neuroprotective effect as compared to methylene blue alone, suggestive of its nitrosative mechanism of neuroprotection. L-Arginine does not show significant difference as compared to haloperidol treated group. Above findings suggests that L- Arginine when administering along with methylene blue to haloperidol induced tardive dyskinesia, did not shown any significant difference as compared to haloperidol induced tardive dyskinesia in rats. Table1: Effect of Pharmacological interventions on biochemical alterations on haloperidol- induced tardive dyskinesia

Group Name MDA Nitrite GSH Catalase TNF-α IL-6 (n mol/ mg (µ mol/ mg (µ mol/ mg (µmol/ mg pr) (mg/pr) (mg/pr) pr) (% of pr) pr) (% of vehicle) (% of (% of vehicle) (% of (% of vehicle) vehicle) vehicle) vehicle) NORMAL 99 ±6.34 100±5.95 99.7±6.32 100± 5.8 42.3±5.23 39.7±8.5 HPD 223.1±9.45 239.7±8.34 47.7±5.2 41.5 ±5.9 80.1±4.34 65.9±7.5 HPD+MB(L) 172.3±7.85 190.3±7.84 60.3±6.12 51.2 ±4.5 70.2±3.4 59.0±4.6 HPD+MB(H) 139.3±6.34 145.46±7.23 71.45±5.67 57.7±5.9 62.5±5.6 54.7±2.9 HPD+MB+L- 131±5.65 136.3±6.34 74.3±6.4 69.75±5.3 55.6±4.5 45.5±3.4 NAME HPD+MB+L- 223.3±9.32 238.4±10.1 46.34±5.2 40.56±5.7 78.9±7.8 63.8±6.7 Arginine

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(a) (b) (c)

(d) (e) (f)

PLATE 1: Effect of pharmacological interventions on histological alterations against haloperidol induced tardive dyskinesia in rats (a) Vehicle treated group (b) Haloperidol treated group (c) HPD + MB (L) (d) HPD+MB (H) (e) HPD+MB+L-NAME (f) HPD+MB+L-Arginine

ACKNOWLEDGEMENT Authors are thankful to ISFCP, Moga and DPSDR, Punjabi University, Patiala for providing requisite facilities to complete a part of this work. CONFLICT OF INTEREST Authors have no conflict of interest. REFERENCES 1. Barak Y, Shamir E, Zeimishlani H, Mireecki I, Toren P, Weizman R. (2002) vs. haloperidol in the treatment of elderly chronic schizophrenia patients. Prog Neuropsychopharmacol. Biol. Psychiatry 26, 1199-1202. 2. Avent K.M, Usaki E, Eyles D.W, Keeve R (1996) Haloperidol and its tetrahydropyridine derivatives are metabilosed to potentially neurotoxic pyridinum species in the baboon , life sci, 59, 1473-1482. 3. Tsai G.C. and Coyle J.T. (2002) Glutamatergic mechanisms in schizophrenia. Annual Review Pharmacology and Toxicology. 42, 165-179.

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Sandeep Kumar Goyal Email: [email protected]

Sandeep et al. / Pharma Science Monitor 6(4), Oct-Dec 2015, 193-209