Protective effect of nebivolol on reserpine-induced neurobehavioral and biochemical alterations in rats

Neurochemistry International 63 (2013) 316–321

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Protective effect of nebivolol on reserpine-induced neurobehavioral and biochemical alterations in rats V.S. Nade ⇑, N.V. Shendye, L.A. Kawale, N.R. Patil, M.L. Khatri Department of Pharmacology, M.V.P.S. College of Pharmacy, Gangapur Road, Nashik, Maharashtra 422002, India

a r t i c l e

i n f o

Article history: Received 28 February 2013 Received in revised form 29 June 2013 Accepted 7 July 2013 Available online 16 July 2013 Keywords: Nebivolol Tardive dyskinesia Vacuous chewing movements

a b s t r a c t Reserpine-induced orofacial dyskinesia is a model that shares some mechanists’ aspects with tardive dyskinesia whose pathophysiology has been related to oxidative stress. The present study was aimed to explore neuroprotective effects of nebivolol, an antihypertensive agent, on reserpine-induced neurobehavioral and biochemical alterations in rats. Reserpine (1 mg/kg, s.c.) was used to induce neurotoxicity. Administration of reserpine for 3 days every other day significantly increased the vacuous chewing movements (VCMs), tongue protrusions (TPs) and reduced the locomotor activity in rats. Pre-treatment with nebivolol (5 and 10 mg/kg, p.o. for 5 days) showed dose dependant decrease in VCMs and TP induced by reserpine. Nebivolol also showed significant improvement in locomotor activity. Reserpine significantly increased lipid peroxidation and reduced the levels of defensive antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH) in rat brain. Nebivolol reversed these effects of reserpine on oxidative stress indices; indicating amelioration of oxidative stress in rat brains. The results of the present study indicated that nebivolol has a protective role against reserpine-induced orofacial dyskinesia. Thus, the use of nebivolol as a therapeutic agent for the treatment of tardive dyskinesia may be considered. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Treatment with neuroleptics in humans can produce a serious side effect, known as tardive dyskinesia (TD) (Busanello et al., 2012; Fachinetto et al., 2007). TD has promoted substantial research into the basic mechanisms of the efficacy and adverse effects of traditional neuroleptics (Casey, 2000). It involves a serious neurodegeneration which is associated with chronic exposure to neuroleptics in experimental animals and humans (Burger et al., 2003). Neisewander et al. have suggested that reserpine-induced oral dyskinesia may provide a new model for tardive dyskinesia (Neisewander et al., 1994). Though, the neuro-pathophysiology of tardive dyskinesia is not fully understood, it has been suggested that increase in striatal dopaminergic D2 receptor expression is responsible for the onset of extrapyramidal side effects in humans and experimental rodent models of tardive dyskinesia. The dopaminergic hypersensitivity resulted as a consequence of proliferation of striatal dopamine D2 like receptors leading to the long term dopaminergic blockade and has been suggested as a potential model for antipsychotic induced tardive dyskinesia (Vital et al., 1998). ⇑ Corresponding author. Tel.: +91 253 2577250, mobile: +91 9922234934; fax: +91 253 2580250. E-mail address: [email protected] (V.S. Nade). 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.07.002

Reserpine also causes depletion of brain catecholamines leading to an akinetic state in experimental animals. It has been observed that L-DOPA administration also alleviated the reserpine induced akinetic state, indicating that behavioral recovery is dopaminedependant (Carlsson et al., 1957). The model of reserpine induced orofacial dyskinesia shows an important aspect of face validity with Parkinson’s disease (PD) (Andreatini et al., 2002). It has been reported that repeated treatment with low doses of reserpine progressively induces alterations in motor function and an increase in striatal oxidative stress, indicating its application in the study of the neuroprogressive nature of the motor signs in PD (Silva et al., 2012). The decrease in glutamate uptake was observed in the subcortical parts of brain in animals treated with reserpine, indicating that early changes in glutamate transport may be related to the development of vacuous chewing movements in rats (Burger et al., 2005). Burger et al. (2004) have reported that rats with vacuous chewing movements have significantly higher thiobarbituric acid reactive substances (TBARS) in striatum, indicating a rise in lipid peroxidation and free radical production associated with sharp reduction in activity of antioxidant enzymes like superoxide dismutase, catalase, glutathione reductase, etc. in such animals. Many researchers have tried to demonstrate that neurodegeneration involved in tardive dyskinesia is closely associated with generation of free radicals (Cadet and Lohr, 1989; Coyle and Puttfarcken,

V.S. Nade et al. / Neurochemistry International 63 (2013) 316–321

1993). The brain contains many antioxidant enzymes that prevent or suppress harmful free radical reactions (Dringen, 2005). It has been suggested that, the antioxidant treatment could be a key area in treating tardive dyskinesia in animal models (Abilio et al., 2002; Naidu et al., 2004). This particular model is advantageous because dyskinesia can be induced in short time span as compared to that induced by typical antipsychotics (Andreassen and Jorgensen, 2000). Nebivolol is a third-generation b1 adrenergic blocker with discrete pharmacological properties compared with other drugs exhibiting b blocking action (Kamp et al., 2010). One of the mechanisms of nebivolol’s antioxidant activity is due to reduction of ROS produced by a NADPH oxidase system (Cominacini et al., 2003). Nebivolol has a direct scavenging activity on oxygen radicals with peculiar antioxidant properties, which may play a key role in various diseases (Mason et al., 2006). The objective of the present study was to investigate the neuroprotective effect of nebivolol against reserpine-induced orofacial dyskinesia in rats and to unravel its mechanisms action with respect to biochemical imbalances. 2. Materials and methods 2.1. Materials Nebivolol hydrochloride (Hetero Labs, Hyderabad, India), vitamin E (Merck Ltd, Goa, India), reserpine (Research Lab, Mumbai, India), thiobarbituric acid (TBA) (Research-Lab Fine Chem Industries, Mumbai, India), nitrobluetetrazolium chloride (NBT) (Himedia Laboratories Pvt. Ltd. Mumbai, India), 5,50 - dithiobis (2-nitro benzoic acid) (DTNB) (Alfa Aesar, A Johnson Mathey Company). Bovine serum albumin (Spectrochem Pvt. Ltd., Mumbai, India). All the chemicals used were of analytical grade and purchased from standard manufacturers. 2.2. Animals Male Wistar strain rats (150–200 g) were used for the study. Animals were housed in polypropylene cages and maintained under the standard laboratory environmental conditions; temperature 25 ± 2 °C, 12: 12 h L: D cycle and 50 ± 5% RH with free access to food and water ad libitum. Animals were acclimatized to laboratory conditions before the test. Each group consisted of five (n = 5) animals. All the experiments were carried out during the light period (08:00–16:00 h). The studies were carried out in accordance with the guidelines given by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi (India). The Institution Animal Ethical Committee of M.V.P.S College of Pharmacy, Nashik approved the protocol of the study (IAEC/2013/01). 2.3. Experimental design Animals were randomly assigned into 5 groups (n = 5 for each group). Group I – Vehicle (0.2% PEG (polyethylene glycol) in distilled water p.o + 0.1% acetic acid solution s.c, vehicle for reserpine), Group II – reserpine (1 mg/kg, s.c), Group III – vitamin E (10 mg/kg, p.o) + reserpine (1 mg/kg, s.c), Group IV – nebivolol (5 mg/kg, p.o) + reserpine (1 mg/kg, s.c), Group V – nebivolol (10 mg/kg, p.o) + reserpine (1 mg/kg, s.c), Group VI – nebivolol (10 mg/kg, p.o) alone to check its own effects on vacuous chewing movements and tongue protrusions. 2.4. Induction of orofacial dyskinesia Vehicle treated group was administered with 0.2% PEG in distilled water orally for 5 days and with 0.1% acetic acid solution

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(vehicle for reserpine) subcutaneously for 3 days every other day. The first injection of acetic acid was given 24 h after the administration of PEG. Reserpine group received 1 mg/kg reserpine for 3 days every other day. Nebivolol plus reserpine groups were administered with 5 and 10 mg/kg nebivolol orally for 5 days and with 1 mg/kg reserpine s.c for 3 days every other day. The first dose of nebivolol or PEG was administered 24 h before reserpine. Nebivolol was administered 30 min before administration of reserpine. Similar procedure was carried out in vitamin E treated group of animals. Behavioral assessments were carried out on fifth day after 24 h of administration of the last dose of reserpine. 2.5. Behavioral testing To quantify the occurrence of oral dyskinesia on the test day, rats were placed individually into a small Plexiglas observation cage (30  20  20 cm) to score vacuous chewing movements (VCMs) and tongue protrusion frequencies. Animals were allowed 10 min to acclimatize to the observation cage before behavioral assessments were performed. 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 VCMs and tongue protrusion were defined as single mouth openings in the vertical plane not directed towards physical material and visible extension of the tongue outside of the mouth respectively. If VCMs or tongue protrusion occurred during a period of grooming, they were not taken into account. The behavioral parameters of oral dyskinesia were measured continuously for a period of 15 min. In all the experiments, the observer was blind to the identity of the animals (Burger et al., 2003). 2.6. Assessment of total locomotor activity by actophotometer Locomotor activity is an index of alertness of mental activity as most of the drugs acting on CNS influence locomotor activity. It is measured with the help of actophotometer which operates on photoelectric cells that are connected with circuit with counter. Interruption of light beams as a measure of movements of rats in a cage has been used by many authors. When a beam of light falling on photocell is cut-off by the animal, a count is recorded. Locomotion was measured up to 10 min for each rat (Vogel, 2002). 2.7. Biochemical estimation 2.7.1. Dissection and homogenization On the 5th day immediately after behavioral assessments the animals were killed by decapitation. The brain was removed, rinsed with isotonic saline and weighted. A 10% (w/v) tissue homogenate was prepared in 0.1 M phosphate buffer (pH 7.4). The post nuclear fraction for catalase assay was obtained by centrifugation (Remi – C - 30, Remi Industries Ltd. Mumbai, India) of the homogenate at 1000g for 20 min at 4 °C; for other enzyme assays, centrifugation was at 12,000g for 60 min at 4 °C. A Elico biospectrophotometer-BL200 was used for subsequent assays (Naidu et al., 2003). 2.7.2. Catalase activity (CAT) Catalase activity was assessed by the method of Luck (1971), where the breakdown of H2O2 was measured at 240 nm. Briefly, the assay mixture consisted of 3 ml of H2O2 phosphate buffer (0.0125 M H2O2) and 0.05 ml of supernatant of brain homogenate and the change in the absorbance was measured at 240 nm. The enzyme activity was calculated using the millimolar extension coefficient of H2O2 (0.07). The results were expressed as micro moles of H2O2 decomposed per minute per milligram of protein.

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2.7.3. Estimation of reduced glutathione (GSH) Reduced glutathione (GSH) in the brain was assayed according to the method of Ellman (1959). A 0.75 ml sample of homogenate was precipitated with 0.75 ml of 4% sulphosalicylic acid. The samples were centrifuged at 1,200g for 15 min at 4 °C. The assay mixture contained 0.5 ml of supernatant and 4.5 ml of 0.01 M DTNB [550 - dithiobis (2-nitrobenzoic acid)] in 0.1 M phosphate buffer, pH 8.0. The yellow color developed was read immediately at 412 nm. The results were expressed as micro moles of GSH per milligram of proteins.

2.7.4. Superoxide dismutase activity (SOD) Superoxide dismutase activity was assayed according to the method of Kono (1978), wherein the reduction of nitrobluetetrazolium chloride (NBT) was inhibited by the superoxide dismutase and measured at 560 nm spectrophotometrically. Briefly the reaction was initiated by the addition of hydroxylamine hydrochloride to the reaction mixture containing NBT and post nuclear fraction of brain homogenate. Results were expressed as percentage inhibition of reduction of NBT.

2.7.5. Lipid peroxidation assay (LPO) The quantitative measurement of lipid peroxidation in brain was done by the method of Wills (1966). The reaction was initiated by addition of 0.2 ml of 8% SLS, 1.5 ml of 20% acetic acid and 1.5 ml of 0.8% aqueous solution of thiobarbituric acid (TBA) to 0.1 ml of tissue homogenate. Finally, the volume was made to 4.0 ml by adding distilled water. It was then heated at 95 °C for 60 min on water bath and cooled under tap water. Then, 5 ml mixture of n-butanol: Pyridine (15:1 by volume) was added to it and further it was shaken vigorously. The amount of malondialdehyde (MDA) formed was measured by reaction with thiobarbituric acid at 532 nm. The amount of malondialdehyde (MDA) formed was measured. The results were expressed as nanomoles of MDA per milligram of protein, using the molar extension coefficient of chromophore (1.56  105 M1 cm1).

2.7.6. Protein estimation The protein content was measured according to the method of Lowry et al. (1951), using bovine serum albumin as standard and expressed as lg protein/mg of tissue.

2.8. Statistical analysis The results are expressed as mean ± SEM. Data were subjected to two-way analysis of variance (ANOVA) test.

3. Results 3.1. Assessment of orofacial dyskinesia Effect of reserpine treatment on vacuous chewing movements (VCMs) and tongue protrusions in rats were depicted in Fig 1 (a) and (b). The frequency of vacuous chewing movements and tongue protrusions were significantly (p < 0.001) increased in reserpine treated group as compared to vehicle group. Administration of nebivolol (5 mg/kg and 10 mg/kg) for a period of 5 days significantly (p < 0.001) attenuated reserpine-induced VCMs and tongue protrusions. Vitamin E also showed significant (p < 0.001) reduction in both VCMs and tongue protrusions. Nebivolol 10 mg/kg administered alone did not induce VCMs or tongue protrusions (Fig 1a, b).

3.2. Effect on total locomotor activity Total locomotor activity of rats in reserpine treated group was significantly (p < 0.001) reduced as compared to vehicle group. Administration of nebivolol showed significant (p < 0.001) rise in the locomotor activity in dose dependant manner. Results obtained by administration of Vitamin E were slightly superior to those shown by nebivolol (p < 0.001) (Fig 2). 3.3. Biochemical effects 3.3.1. Effects on SOD and CAT levels Significant (p < 0.001) decrease in levels of SOD and CAT enzymes were observed after reserpine administration as compared to vehicle group, indicating induction of neurotoxicity in rats. Pre-treatment with nebivolol 5 mg/kg and 10 mg/kg showed significant (p < 0.001) rise in levels of SOD and CAT as compared to rats treated with reserpine. These results were comparable to those obtained by administration of vitamin E (p < 0.001) (Table 1). 3.3.2. Effects on brain GSH levels The content of GSH was depleted significantly (p < 0.001) in reserpine treated group as compared to vehicle group. On the other hand, treatment with nebivolol 5 mg/kg and 10 mg/kg significantly (p < 0.001) elevated brain GSH levels which were similar to those obtained by vitamin E treatment (Table 1). 3.3.3. Effects on lipid peroxidation Levels of MDA were significantly increased in reserpine treated group, as compared to vehicle group; while administration of nebivolol at a dose of 5 mg/kg and 10 mg/kg significantly (p < 0.001) lower levels of LPO as compared to reserpine treated group. Vitamin E also significantly (p < 0.001) reduced the levels of LPO (Table 1). 4. Discussion The results of the present study indicated that nebivolol has a protective role against reserpine-induced orofacial dyskinesia. Nebivolol significantly attenuated the increase in vacuous chewing movements and tongue protrusions caused by acute exposure to reserpine. Concerning critical issues and disputes related to evaluation of animal models of tardive dyskinesia (TD), reserpine-induced VCMs and tongue protrusions seems to be a better model for tardive dyskinesia. Characteristic features of reserpine induced oral dyskinesia match very well with symptoms of TD and historically reserpine has been associated with development of TD in humans (Burger et al., 2003). Reserpine treated rats show a decrease in locomotion frequency and increased immobility accompanying as a consequence of the oral dyskinesia (Filho et al., 2002). Reserpine induced oral dyskinesia is closely associated with oxidative stress and free radicals are highly involved in development of orofacial dyskinesia in rats (Calvente et al., 2002). It has been demonstrated that antioxidant agents like monosialogangliosides significantly attenuated reserpine-induced tongue protrusions in rats, by scavenging the free radicals both in vivo and in vitro (Burger et al., 2003). In the present study, administration of reserpine (1 mg/kg) significantly increased the frequency of VCMs and tongue protrusions indicating neurotoxicity produced by reserpine. Pretreatment with nebivolol (5 and 10 mg/kg) significantly prevented an increase in the frequency of VCMs and tongue protrusions. The total locomotor activity was significantly reduced in reserpine treated group as compared to vehicle group. Administration of nebivolol showed significant improvement in locomotor activity.

319

(a) 100

I Control II Reserpine (1mg/kg, s.c.) III Vitamin E (10 mg/kg, p.o.) + Reserpine IV Nebivolol (5 mg/kg, p.o.) + Reserpine V Nebivolol (10 mg/kg, p.o.) + Reserpine VI Nebivolol (10 mg/kg, p.o.)

###

80 60 40

***

***

20

***

ns

0 I

II

III

IV

V

VI

Treatments

Tongue protrusion frequency/15 min

Vacuous chewing movements/15 min

V.S. Nade et al. / Neurochemistry International 63 (2013) 316–321

(b)

30

I II III IV V VI

###

**

20

*** 10

*** 0

I

II

Control Reserpine (1mg/kg, s.c.) Vitamin E (10 mg/kg, p.o.) + Reserpine Nebivolol (5 mg/kg, p.o.) + Reserpine Nebivolol (10 mg/kg, p.o.) + Reserpine Nebivolol (10 mg/kg, p.o.)

ns

III

IV

V

VI

Treatments

Total locomo to ractivity/10 min

Fig. 1. Protective effect of nebivolol on reserpine-induced (a) vacuous chewing movements (b) tongue protrusions in rats. Each column represents mean ± SEM. (n = 5). # Group I vs. Group II, ⁄Group II vs. Group III, ⁄Group II vs. Group IV, ⁄Group II vs. Group V, #Group I vs. Group VI. nsNon-Significant.###,⁄⁄⁄p < 0.001 (One-way ANOVA followed by Tukey’s test).

500

ns

400

***

300

**

###

200

**

I II III IV V VI

Control Reserpine (1mg/kg, s.c.) Vitamine E (10 mg/kg, p.o.) + Reserpine Nebivolol (5 mg/kg, p.o.) + Reserpine Nebivolol (10 mg/kg, p.o.) + Reserpine Nebivolol (10 mg/kg, p.o.)

100 0

I

II

III

IV

V

VI

Treatments Fig. 2. Protective effect of nebivolol on reserpine-induced reduction in total locomotor activity in rats. Each column represents mean ± SEM. (n = 5). #Group I vs. Group II, ⁄ Group II vs. Group III, ⁄Group II vs. Group IV, ⁄Group II vs. Group V, #Group I vs. Group VI. nsNon-Significant###,⁄⁄⁄, p < 0.001 (One-way ANOVA followed by Tukey’s test).

In this study, reserpine treated animals showed elevated levels of lipid peroxidation along with significant reduction in antioxidant enzymes such as SOD, CAT and GSH indicating generation of free radicals. However, administration of nebivolol significantly attenuated this reduction in enzymatic defense generated against free radicals and increase in lipid peroxidation, suggesting its possible antioxidant action. These findings supported earlier observations about attenuation of formation of reactive oxygen species secondary to ischemia/ reperfusion injury. This neuronal protection has been explained by alleviation of nitric oxide synthase (NOS) enzyme isoforms i.e. eNOS and iNOS by nebivolol (Heeba and EL-Hanafy, 2012). Vitamin E was used as standard. Vitamin E reduces production of oxygen free radicals in various organs such as brain, liver and kidney, etc. (Gurel et al., 2005). Its potential clinical usefulness in prevention of neuropathy or cardiovascular diseases in humans has been investigated (Leger, 2000; Argyriou et al., 2005). Also it has been reported that it plays a protective role in Alzheimer’s disease (Sano et al., 1997; Keltner et al., 2001). Administration of vitamin E significantly prevented

reserpine-induced VCMs and tongue protrusions; and restored defensive antioxidant enzymes with reduction in lipid peroxidation. Like nebivolol, many other adrenergic blockers have been tested previously for their antioxidant effect. Drugs like timolol, betaxolol, cartelol, nipradilol are well known beta blockers that showed antioxidant like activity and affect ROS under oxidative stress (Garbin et al., 2008; Yu et al., 2007). It has been proved that, nebivolol, but not other beta-blockers, improves endothelial function, reduces vascular superoxide production via prevention of eNOS uncoupling, reduces vascular macrophage infiltration, and inhibits NAD(P)H oxidase-dependent superoxide production (Mollnau et al., 2003). The clinical trial of nebivolol has been also done for its antioxidant activity against oxidative stress in humans (Peter et al., 2006). Thus, like other adrenergic blockers nebivolol also showed potent antioxidant property. The results obtained from the study supported the oxidative stress hypothesis of tardive dyskinesia and suggested a beneficial role of nebivolol in treatment of this disorder.

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Table 1 Protective effect of nebivolol on reserpine-induced alterations in biochemical parameters in rat brain. Treatments (Groups)

CAT (lMole of H2O2 decomposed/mg protein/min)

GSH (lMole of GSH/mg protein)

SOD (% inhibition of reduction of NBT)

LPO (nMole of MDA/mg protein)

Control (I) Reserpine (1 mg/kg, s.c) (II) Vitamin E (10 mg/kg, p.o) + Reserpine (III) Nebivolol (5 mg/kg, p.o) + Reserpine (IV) Nebivolol (10 mg/kg, p.o) + Reserpine (V) Nebivolol (10 mg/kg, p.o) alone (VI)

10.14 ± 0.59 6.53 ± 0.07### 9.08 ± 0.15⁄⁄⁄

8.13 + 0.96 1.78 ± 0.03### 6.37 ± 0.14⁄⁄⁄

84.79 ± 0.58 30.8 ± 3.43### 55.47 ± 0.80⁄⁄⁄

5.37 ± 0.38 20.99 ± 1.41### 10.96 ± 0.49⁄⁄⁄

7.87 ± 0.47⁄

5.28 ± 0.05⁄⁄⁄

44.23 ± 0.80⁄⁄⁄

12.75 ± 0.63⁄⁄⁄

8.77 ± 0.55⁄⁄⁄

5.79 ± 0.22⁄⁄⁄

52.49 ± 0.95⁄⁄⁄

12.47 ± 0.57⁄⁄⁄

8.16 ± 0.14ns

85.10 ± 0.25ns

5.4 ± 0.25ns

10.42 ± 0.33ns

All values are expressed as mean ± S.E.M. (n = 5). ns Non-Significant, ###, ⁄⁄⁄ p < 0.001 (One-way ANOVA followed by Tukey’s test), ⁄ Group II vs. Group III, Group II vs. Group IV, Group II vs. Group V, # Group I vs. Group II, Group I vs. Group VI.

5. Conclusion From the findings of the present study, we conclude that nebivolol could be used as an alternative drug for patients suffering from tardive dyskinesia accompanying oxidative stress. Moreover nebivolol seems to be a promising pharmacological agent in the reduction of tardive dyskinesia. Further, clinical trials are highly desirable to prove the efficacy of nebivolol as a therapeutic agent in the treatment of tardive dyskinesia.

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