European Journal of Pharmacology 667 (2011) 230–237

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European Journal of Pharmacology

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Behavioural Pharmacology

Protective effect of Etoricoxib against middle cerebral artery occlusion inducedtransient focal cerebral ischemia in rats

Anurag Maheshwari, Lohit Badgujar, Bonoranjan Phukan,Subhash Laxmanrao Bodhankar ⁎, Prasad ThakurdesaiDepartment of Pharmacology, Poona College of Pharmacy and Research Centre, Bharati Vidyapeeth University, Erandwane, Paud Road, Pune-411 038, India

⁎ Corresponding author at: Department of Pharmacoloand Research Centre, Bharati Vidyapeeth University, Pau038, India. Tel.: +91 20 24537237x203; fax: +91 20 2

E-mail address: sbodhind@rediffmail.com (S.L. Bodh

0014-2999/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.ejphar.2011.05.030

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 November 2010Received in revised form 21 April 2011Accepted 11 May 2011Available online 27 May 2011

Keywords:Cerebral ischemiaEtoricoxibtMCAOOxidative stressStroke

Stroke is the third leading cause of global death anddisability. Cyclooxygenase-2mRNAhas been shown tobeup-regulated after stroke and also the time window of its expression extends from 4 to 12 h. The objective of thisstudy was to elucidate the protective effect of Etoricoxib (a selective Cyclooxygenase-2 inhibitor) againsttransientmiddle cerebral artery occlusion induced behavioral, biochemical and histological alterations. Transientischemia reperfusion significantly caused behavioral (neurological deficits, decreased locomotor activity androtarod performance), biochemical (increased lipid peroxidation and nitrite concentration, while decreasedsuperoxide dismutase and catalase activity) and histological (increased infarct volume) changes. Etoricoxib(3 and 10 mg/kg, i.p.) significantly reversed the alterations caused by cerebral ischemia however, 1 mg/kg dosewas not found effective in any of the parameters. Finally, we can conclude that Etoricoxib has beneficial effectsagainst transientmiddle cerebral artery occlusion model in rats. The present study indicates that Etoricoxibmaybe considered as a potential candidate in the treatment of stroke, clinically.

gy, Poona College of Pharmacyd Road, Erandwane, Pune-4115439386.ankar).

ll rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Stroke is the third leading cause of death and disability followingcardiovascular disease and cancer. According to a projection by Elkins andJohnston, fatalities due to stroke will increase exponentially in the next30 years due to aging of the population and our inability to control riskfactors (Elkins and Johnston, 2003). Stroke can be ischemic or hem-orrhagicwith88%or12%prevalence, respectively (Rosamondetal., 2008).

Cytokines have been shown to be up-regulated in the brain after avariety of insults including stroke. They are expressed not only in cells ofthe immune system, but also in resident brain cells including glia andneurons.(Sairanen et al., 2001). Arachidonic acid released from brainphospholipids during ischemia reperfusion is converted to ProstaglandinH2 by Cyclooxygenase (COX) enzyme. There are two isoforms of COX, ofwhich COX-1 (considered to be a housekeeping gene) is constitutivelyexpressed in many cell types including microglia and leukocytes duringbrain injury (Schwab et al., 2002). COX-2, which was isolated as aninducible immediate-early gene, is believed to play a negative role inbrain injury including ischemia (Collaco-Moraes et al., 1996). Further-more COX-2 deficient mice have shown improvement following injuryafter N-methyl-D-aspartate administration (Iadecola and Alexander,2001) whereas COX-2 over expression exacerbates brain injury (Dore

et al., 2003; Iadecola and Gorelick, 2005). Interestingly, COX-2 mediatesits toxic effect through Prostaglandin E2 rather than reactive oxygenspecies even though COX-2 can generate both (Manabe et al., 2004).Inflammatory processes play a fundamental role in stroke, in both theetiology of ischemic cerebrovascular disease and the pathophysiology ofcerebral ischemia. But they are however, also considered to be atriggering factor for stroke, clinically (Lindsberg and Grau, 2003).

Nimesulide (a preferential COX-2 inhibitor) was shown to beeffective against transient (Candelario-Jalil et al., 2004) as well aspermanent middle cerebral artery occlusion in rats (Candelario-Jalilet al., 2005). Etoricoxib (5-chloro-2-[6-methyl pyridin-3-yl]-3-[4-methylsulfonylphenyl] pyridine) is ahighly selective, secondgenerationCOX-2 inhibitor. The IC50 selectivity ratio (COX1/COX2) for inhibition ofCOX-2 by Etoricoxib is 106 as compared to 7.3 for Nimesulide (Riendeauet al., 2001). Pharmacological action of Etoricoxibhasnot yet explored intransient middle cerebral artery occlusion (tMCAO) model.

The objective of this study was to evaluate the protective effect ofEtoricoxib against transient focal cerebral ischemia reperfusioninduced injury in rats. Parameters investigated included; neurobeha-vioral impairment, biochemical alterations and infarct volume.

2. Materials and methods

2.1. Animals

Male Wistar rats, weighing 270 to 300 g, were purchased fromNational Toxicology Centre, Pune, India. The rats were housed in a 12-

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h light/dark cycle at ambient temperature and had free access towater and food. All experimental procedures were approved by theInstitutional Animal Ethics Committee of Poona College of Pharmacyand were in accordance with the guidelines of the Committee forthe Purpose of Control and Supervision of Experiments on Animals(CPCSEA).

2.2. Materials

Etoricoxib was obtained as gift sample from Panacea Biotech, Indiaand 2, 3, 5-triphenyltetrazolium chloride (TTC) was purchased fromSisco Research Laboratories Pvt. Ltd. Mumbai, India.

2.3. Treatment schedule

Selected animals were randomly distributed among 5 groupsviz vehicle (n=25), SHAM (n=6), ETX (1) (n=15), ETX (3) (n=10)and ETX (10) (n=15). In the vehicle and SHAM group, 0.25% sodiumcarboxy methyl cellulose, in normal saline, was injected intraperito-neally (i.p.) immediately after tMCAO. Etoricoxib (1, 3 and 10 mg/kg i.p) was administered in three test groups designated as ETX (1), ETX(3) and ETX (10) respectively, immediately after tMCAO. The doseselection was based on the previous reports (Capone et al., 2005;Patrignani et al., 2003; Riendeau et al., 2001; Whiteside et al., 2004).

2.4. Transient middle cerebral artery occlusion (tMCAO)

Focal cerebral ischemia was induced by intraluminal suturemethod as reported previously (O'Neill and Clemens, 2001). In brief,the rats were anesthetizedwith ketamine (80 mg/kg i.p.) and xylazine(6 mg/kg i.p.). Rectal temperature was maintained at 37 °C with aheating pad for the duration of surgery and the immediatepostoperative period until the animal recovered fully from anesthesia.A longitudinal incision of 1.5 cm was made in the midline of theventral cervical skin. The left common carotid artery, left internalcarotid artery, and left external carotid artery were exposed. Thedistal portion of external carotid artery was ligated and cut. Aheparinized 3-0 Ethilon™ polyamide black monofilament (NW3328)of 40 mm length and tip rounded by heating near glowing ember(final tip diameter~0.4±0.02 mm)was inserted into the stump of leftexternal carotid artery. To occlude the blood flow to the middlecerebral artery territory, the filament was advanced 19–21 mm fromthe bifurcation of common carotid artery into the left internal carotidartery until resistance was felt. The filament was held in place bytightening the suture around the external carotid artery and placing amicrovascular clip around the artery. The wound was closed, and theanimal was returned to its cage. To allow reperfusion, the filamentwas withdrawn after 120 min of middle cerebral artery occlusion, andthen the external carotid artery was closed permanently by electrocoagulation. The SHAM operations were performed in a similarmanner except that the suture was withdrawn immediately after itwas inserted. Successful middle cerebral artery occlusionwas verified,after the animal recovered from anesthesia, using the neurologicaldeficit test which correlates with infarct volume (Garcia et al., 1995).

The dynamic changes of the micro vessel occlusion in this modelhave been characterized. General physiological parameters such asnon invasive blood pressure and electro cardio gram were recorded1 h before, 30 min after and at 24 h of tMCAO with the help of8 Channel recorder system of PowerLab (AD Instruments).

2.5. Neurological deficit scoring

Neurological deficit scores were recorded 1 h before and at 3 and24 h after tMCAO. Neurological deficit was scored on an 18 point scaleas described earlier (Garcia et al., 1995). The observational parameterswere (1) spontaneous activity (0 to 3 points); (2) symmetry in the

movement of four limbs (0 to 3 points); (3) forepaw out-stretching(0 to 3 points); (4) climbing (1 to 3 points); (5) body proprioception(1 to 3 points); and (6) response to vibrissae touch (1 to 3 points). Thescore given to each rat at the completion of evaluation at eachtime point is the summation of all six individual test scores. Theminimum neurological score is 3 and the maximum is 18. Only therats manifesting score of 9 to 11 at 3 h after tMCAO were included inthe study.

2.6. Measurement of locomotor activity (ambulation) byactophotometer

The locomotor activity (ambulatory activity) was recorded usingactophotometer (IMCORP, India). Animals were placed individually inthe activity meter for 3 min for habituation. Thereafter, locomotoractivity was recorded for a period of 5 min. Ambulatory activity wasrecorded and expressed in terms of total photo beam counts per 5 min(Bodhankar et al., 2007; Gaur et al., 2009).

2.7. Rotarod activity

All animals were evaluated for grip strength and balance using therotarod. Each rat was given a prior training session before initiation oftherapy to acclimatize them on a rotarod apparatus (Techno, Ambala,India). Animals were placed on the rotating rod with a diameter of7 cm (speed 25 rpm). Three separate trials were given to each rat at5 min interval and cut off time (180 s) was maintained throughoutthe experiment. The average results were recorded as fall of time(Gaur and Kumar, 2010c).

2.8. Dissection and homogenization

After 24 h, animals were randomized into two groups. The firstgroup of animals was used for biochemical and the second group formitochondrial complex enzyme estimation after behavioral assess-ments. In the biochemical analysis, animals were euthanized bydecapitation. Striatum was separated from each isolated brain. A 10%(w/v) tissue homogenate was prepared in 0.1 M phosphate buffer(pH 7.4). The homogenate were centrifuged at 10,000×g at 4 °C for15 min. Aliquots of supernatants were separated and used forbiochemical estimations.

2.9. Measurement of oxidative stress parameters

2.9.1. Measurement of lipid peroxidationThe quantitative measurement of lipid peroxidation (LPO) in

striatum was performed according to the method of Wills. Theamount of malondialdehyde (MDA), a measure of lipid peroxidationwas measured by reaction with thiobarbituric acid at 532 nm usingJasco V-650 spectrophotometer (Oklahoma, USA). The values werecalculated using molar extinction coefficient of chromophore(1.56×105 M−1 cm−1) and expressed as percentage of vehicletreated group (Kumar et al., 2009; Wills, 1966).

2.9.2. Estimation of nitriteThe accumulation of nitrite in the supernatant, an indicator of the

production of nitric oxide (NO), was determined with a colorimetricassay with Greiss reagent (0.1% N-(1-naphthyl) ethylenediamedihydrochloride, 1% sulfanilamide and 2.5% phosphoric acid) asdescribed by Green et al. Equal volumes of supernatant and Greissreagent were mixed and incubated for 10 min at room temperature.The absorbance of each sample was determined at 540 nm at JascoV-650 spectrophotometer (Oklahoma, USA). The concentration ofnitrite in the supernatants was determined from a sodium nitritestandard curve and expressed as percentage of vehicle treated group(Green et al., 1982; Kumar et al., 2010b).

Fig. 1. Effect of Etoricoxib on (A) mortality of rats during 24 h after tMCAO inducedischemia reperfusion injury in rats. Data analyzed by Fisher's exact test. (B) Percentinfarct volume. Values are expressed as mean±S.E.M. (n=6). Data was analyzed byone-way ANOVA followed by post hoc Tukey's test. @ Pb0.05 as compared to shamoperated animals.# Pb0.05 as compared to vehicle treated group.

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2.9.3. Superoxide dismutase activity (SOD)Superoxide dismutase activity was assayed according to the

method of Kono. In this method the reduction of nitrazobluetetrazo-lium, which is inhibited by the superoxide dismutase, is measured at560 nm using spectrophotometer. Briefly, the reaction was initiatedby the addition of the hydroxylamine hydrochloride to the mixturecontaining nitrazobluetetrazolium and sample. The results areexpressed as unit/mg protein, where one unit of enzyme is definedas the amount of enzyme inhibiting the rate of reaction by 100%(Kono, 1978).

2.9.4. Catalase estimationCatalase activity was assayed by the method of Luck, wherein the

breakdown of hydrogen peroxide (H2O2) is measured at 240 nm.Briefly, assay mixture consisted of 3 ml of H2O2 phosphate buffer and0.05 ml of supernatant of tissue homogenate (10%), and change inabsorbance was recorded at 240 nm. The results are expressed asmicromole H2O2 decomposed per milligram of protein/min (Luck,1971).

2.10. Protein estimation

Protein was assessed by biuret method using bovine serumalbumin as standard (Gornall et al., 1949; Kumar et al., 2010a).

2.11. Quantification of brain infarct volume

The quantification of infarct volume has been describedpreviously (Bederson et al., 1986). Briefly, 24 h after tMCAO, ratswere euthanized by thiopental (100 mg/kg i.p.) and intracardiacperfusion was performed with 100 ml of 1% heparinized normalsaline. The brains were dissected out immediately, transferred inice-cold saline and placed in deep freezer (~−18 °C) for 5 min. Formorphometric study, 2 mm thick coronal sections were cut usingrat brain matrix (Zivic instruments, USA). Seven coronal sectionswere collected and stained with 2% TTC solution. The infarctionappears pale white on a background of red “normal” brain. Thestained brain sections were fixed overnight in phosphate-bufferedformalin (10%) and photographed by digital camera (COOLPIX,Nikon) using validated zoom focused from predetermined height.The images were analyzed using an image processing softwareprogram (Image J, v1.34s). The total volume of each hemisphereand infarction was determined by integration of the respective areasof both the surfaces of seven coronal sections. To compensate theincrease in volume due to edema in the ipsilateral hemisphere, theinfarct volume was calculated indirectly using the followingformula: Infarct volume=[Total volume of contralateral hemi-sphere−(Volume of Non infracted zone of ipsilateral hemisphere)](Swanson et al., 1990). The infarction volume is expressed as per-centage of the volume of contralateral hemisphere using thefollowing formula

% infarction =Total indirect volume of infarction in the ipsilateral hemisphere

Total volume of the respective contralateral hemisphere× 100:

2.12. Statistical analysis

The statistical analyses were carried out using statistical software,Prism v4.0 (GraphPad software Inc) and SigmaStat 3.5 (Systat, USA).Unless otherwise stated, all the values are expressed as mean±S.E.Mand data was analyzed by One-Way Analysis of Variance (ANOVA)followed by post hoc Tukey's test. Mortality data has been analyzed byFisher's exact test. Neurological deficit scores are represented asmedian±confidence interval (C.I.). They were analyzed by Kruskal–Wallis one-way ANOVA on ranks followed by Dunn's multiple

comparison test. In all the analysis, P values less than 0.05 wereconsidered as significant.

3. Results

3.1. Effect of Etoricoxib on mortality after tMCAO in rats

Ischemia reperfusion caused a significant mortality (60%) invehicle treated group as compared to sham treated group. Adminis-tration of Etoricoxib (3 and 10 mg/kg i.p.) significantly (Pb0.01)reduced mortality in focally ischemic rats to 10% and 20% (Fisher'sexact test), respectively. However at 1 mg/kg dose a higher mortality(40%) was observed (Fig. 1A).

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3.2. Effect of Etoricoxib on brain infarct volume after tMCAO in rats

Challenging the animals with 120 min of tMCAO followed by22 h of reperfusion caused significant increase in infarct volumeafter 24 h of insertion of suture in vehicle treated animals as com-pared to sham operated animals. Etoricoxib (1, 3 and 10 mg/kg i.p.)significantly (Pb0.05) attenuated the infarct volume as compared tovehicle treated group in a dose dependant manner [F(3,20)=22.25;Pb0.05]. This reduction in the infarct volume indicates the pro-tective effect of Etoricoxib against ischemia reperfusion injury[Figs. 1B and 2].

3.3. Effect of Etoricoxib on neurological deficit score and locomotoractivity in actophotometer after tMCAO in rats

Challenging the animals with 120 min of tMCAO followed by 22 hof reperfusion caused significant neurological deficit and reductionin the locomotor activity after 3 h and 24 h of insertion of suture ascompared to sham operated animals. However, administration ofEtoricoxib (1, 3 and 10 mg/kg i.p.) significantly improved neurologicaldeficits (Pb0.05) and locomotor activity (Pb0.05) after 22 h ofreperfusion but no significant alteration is being noticed after 3 h inboth neurological deficit and locomotor activity as compared tovehicle treated group [Fig. 3A, B].

Fig. 2. Representative coronal brain sections stained with TTC showing the infarctio

3.4. Effect of Etoricoxib on fall-off time in rotarod against tMCAO in rats

The fall-off time is measured for rotarod experiment to measuremotor in coordination. A significantdecrease is beingobserved in fall-offtime in vehicle treated group as compared to sham operated animalswhich indicate motor in coordination and muscle weakness. Etoricoxibsignificantly [Pb0.05; F(4,25)=34.84] and dose dependently improvedthe fall-off latency time as compared to vehicle treated group [Fig. 3C].

3.5. Effect of Etoricoxib on oxidative stress in brain against tMCAO in rats

Oxidative stress markers (MDA and nitrite levels) were signifi-cantly enhanced by tMCAO and 22 h of reperfusion in the ipsilateralpart of the brain as compared to sham operated animals. However,single administration of Etoricoxib (3 and 10 mg/kg i.p.) significantlyattenuated the MDA [Fig. 4A] and nitrite levels [Fig. 4B] after ischemiareperfusion as compared to vehicle treated group [LPO (Pb0.05;F(4,25)=71.36), nitrite (Pb0.05; F(4,25)=19.84)]. No significant effectwas shown by Etoricoxib (1 mg/kg i.p.) dose.

3.6. Effect of Etoricoxib on antioxidant enzyme activity in brain againsttMCAO in rats

Antioxidant enzyme (SOD and catalase) activity was significantlydecreased after ischemia reperfusion injury in the ipsilateral part of

n (pale region) 24 h after tMCAO induced ischemia reperfusion injury in rats.

Fig. 3. Effect of Etoricoxib on (A) neurological deficit scores {values expressed asmedian±C.I. and data was analyzed by Kruskal–Wallis one-way ANOVA on ranksfollowed by Dunn's multiple comparison test. (B) Locomotor activity and (C) fall-offtime from rotarod in tMCAO induced ischemia reperfusion injury in rats. Values areexpressed as mean±S.E.M. (n=6). @ Pb0.05 as compared to sham operated animals.#

Pb0.05 as compared to vehicle treated group.

Fig. 4. Effect of Etoricoxib on oxidative stress parameters. (A) Lipid peroxidation(B) nitrite levels, 24 h after tMCAO induced ischemia reperfusion injury in rats. Valuesare expressed as mean±S.E.M. (n=6). Data analyzed by one-way ANOVA followed byTukey's test. @ Pb0.05 as compared to sham operated animals.# Pb0.05 as compared tovehicle treated group.

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the brain as compared to sham operated animals (Pb0.05). Further, asignificant increase in the activity of SOD [Fig. 5A] and catalase[Fig. 5B] was observed in the Etoricoxib (3 and 10 mg/kg i.p.) treatedgroups as compared to vehicle treated group. However, the lowerdose (1 mg/kg i.p.) did not show any significant improvement inantioxidant enzyme activity as compared to vehicle treated control.

Moreover no significant alteration in the activity of SOD and catalasewas observed in the contralateral region of the brain as comparedto its respective SHAM group [superoxide dismutase F(4, 25)=15.67;catalase F(4, 25)=36.51; Pb0.05].

4. Discussion

COX-2 is an inducible enzyme whose induction and expression aredynamically regulated by growth factors, mitogens, tumor growthpromoters, and physiological stresses (Dubois et al., 1998; Hwanget al., 1997). There is evidence that COX-2 participates in themechanisms of several neurodegenerative diseases (Gao et al.,2008). The up-regulation of COX-2 immunoreactivity was shown tobe confined to the area of damage. COX-2 immunoreactivity isobserved in ischemic neurons at the border of ischemic territory, aswell as in neutrophils and vascular cells. In murine animals, COX-2mRNA and protein expression up-regulate in 12–24 h after cerebral

Fig. 5. Effect of Etoricoxib on antioxidant enzyme activity. (A) Superoxidedismutase(B) Catalase, 24 h after tMCAO induced ischemia reperfusion injury in rats. Values areexpressed as mean±S.E.M. (n=6). Data analyzed by one-way ANOVA followed byTukey's test. @ Pb0.05 as compared to sham operated animals.# Pb0.05 as comparedto vehicle treated group.

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ischemia (Nogawa et al., 1998). It is observed in neurons at theperiphery of the infarct, in vascular cells, and possibly in microglia(Miettinen et al., 1997; Nogawa et al., 1997). COX-2 has also beenfound to be expressed in human brain tissue after ischemic stroke. Inpostmortem specimens of ischemic stroke patients, COX-2 is up-regulated not only in the regions of ischemic injury (Iadecola et al.,1999) but also in areas which are remote from the infarct area(Sairanen et al., 1998).

Transient MCAO model is one of the most widely and successfullyused models of ischemic stroke. Motor dysfunction is one of the mostdevastating outcomes of stroke due to tMCAO since most of the motorcortex and pyramidal tract lie within the territory supplied by middlecerebral artery. Motor dysfunction may arise from loss of corticalexcitability and/or blockade of electrical impulses at the subcorticallevel. After ischemia and recirculation, axonal conduction readily

recovers. However, a persistent failure at cortical synapses leads tomotor dysfunction (Bolay and Dalkara, 1998). Several research groupshave shown the locomotor and neurological deficits after ischemia inrats (Aggarwal et al., 2010; Gaur and Kumar, 2010c; Gupta et al.,2005). In line with these findings, our results also showed a significantdecrease in the number of counts in actophotometer and neurologicaldeficit scores. Furthermore, the fall-off time from rotarod wassignificantly decreased when compared to sham operated animalsverifying the deficit in muscle co-ordination and grip strength.Decrease in muscle co-ordination and grip strength has also beenshown by various studies in MCAO (Freret et al., 2009) as well as theBCCAO (bilateral common carotid artery occlusion) model of cerebralischemia (Gaur et al., 2009; Gaur and Kumar, 2010a). Additionally,treatment with Etoricoxib attenuated locomotor deficit and increasedneurological deficit score as well as fall-off latency time from rotarod.This supports its exhibiting protective effect against ischemiareperfusion injury. A preferential COX-2 inhibitor (Nimesulide) hasalso been shown to be effective against cerebral ischemia (Candelario-Jalil et al., 2004). Several studies reported the risk factors associatedwith the use of selective COX-2 inhibitors (Ahmad et al., 2009;Fitzgerald, 2004; Roumie et al., 2008) however, these risk factors arefound to be associated with their long term usage. In the current studywe have given the treatment as a single dose, right after the ischemicinsult.

In the present investigation, reduction in non invasive bloodpressure, heart rate, and QTc intervals was observed after induction ofischemia, which became normal after 24 h (data not shown). In thepresent study, single administration of Etoricoxib (3 and 10 mg/kg i.p.) immediately after induction of ischemia in rats, decreasedmortality and showed neuro-protection. However the mechanism ofaction of Etoricoxib still requires further investigation.

Under physiological conditions, reactive oxygen species such assuperoxide (•O2), hydrogen peroxide (H2O2), and hydroxyl radical(•OH), play important roles in signaling and metabolic pathways.Importantly reactive oxygen species levels are checked by endoge-nous antioxidants which include SOD, glutathione peroxidase,glutathione and catalase. During oxidative stress, rapid over-produc-tion of free radicals overwhelms the detoxification and scavengingcapacity of cellular antioxidant enzymes. This results in severe andimmediate damage to cellular proteins, DNA and lipids.

Oxidative stress is now well reported in the pathophysiology ofstroke. A plethora of literature indicates that ischemia reperfusioninjury causes a significant increase in oxidative stress markers suchas; reactive oxygen species, MDA and nitrite concentration. There isalso a significant decrease observed in antioxidant enzymes i.e.catalase and SOD activity, in the brain (Gaur and Kumar, 2010b,2010d; Levine, 2004; Siesjo, 2008). In the present study we havemeasured oxidative stress parameters (MDA, nitrite, SOD andcatalase) in the ipsilateral and contralateral parts of the brain. Nosignificant alteration is observed in any of the oxidative stressparameters in the contralateral region of the brain (data notshown). However, a marked increase in MDA as well as nitriteconcentration was observed. A decrease in SOD as well as catalaseactivity was also seen 24 h after tMCAO indicating oxidative stresscaused by ischemia reperfusion injury. Etoricoxib significantlyattenuated the oxidative stress and restored the antioxidant enzymeactivity. Etoricoxib has already been reported to attenuate therestrained stress induced oxidative stress in rats (Madrigal et al.,2003; Munhoz et al., 2008). In addition, it is known that COX-2mediates its toxic effect through Prostaglandin E2 rather than reactiveoxygen species (Manabe et al., 2004). Thus the ability of Etoricoxib toinhibit prostaglandin E2 synthesis (Dallob et al., 2003) as well as itspotential to attenuate oxidative stress can lead to an added protectionbeyond that conferred by antioxidants alone.

Among various selective COX-2 inhibitors, only Etoricoxib wasshown to inhibit the activation of the transcription factor, cAMP

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response element binding protein (CREB) (Niederberger et al., 2006).This led to a decreased protein expression of the pro-inflammatoryproteins, COX-2 and iNOS. CREB and NF-κB are important transcrip-tion factors that are involved in a variety of cellular responses and areresponsible for the transcription of many proteins including the pro-inflammatory proteins COX-2 and iNOS (Mayr and Montminy, 2001;Pahl, 1999). The inhibition of CREB activation by Etoricoxib togetherwith inhibition of NF-κB activation may therefore contribute to themarked decrease in COX-2 and iNOS protein expression. This furthersupports the use of Etoricoxib specifically as the treatment strategyfor stroke. Further studies however, are warranted regarding thecomparative protective profile of all selective COX-2 inhibitorsavailable to ensure the specific protective profile of Etoricoxib againststroke.

Significant damage in the neurons of ipsilateral side of the brainhas been reported to be caused by tMCAO. TTC staining only stainsviable neurons of the brain in a pink color and the region of deadneurons can be seen in white. The area of white region is thenmeasured and calculated with the help of Image J software for thesame. It has been reported by various research groups that the infarctarea increases in the ischemia reperfusion injury (Candelario-Jalilet al., 2004; Thaakur and Sravanthi, 2010). In line with the abovefindings we also observed an increased infarct volume in the vehicletreated group when compared to sham operated animals. All dosesof Etoricoxib attenuated the infarct volume dose dependently, whichfurther confirms its protective effect against ischemia reperfusioninjury.

Finally, we can conclude that Etoricoxib has manifested thebeneficial effect against ischemia reperfusion induced behavioral andbiochemical alterations. This study provides a hope that Etoricoxibmay be considered as a potential candidate in the treatment of stroke.

Acknowledgements

The authors wish to acknowledge Dr. Kolammal Nageswari, Schoolof Biosciences and Bioengineering, Indian Institute of Technology,Powai, Mumbai and Mr. Vaibhav Gaur, Department of Pharmacology,Panjab University, Chandigarh for their intellectual and technical helpin carrying out this study. We are also thankful to Panacea Biotech,Chandigarh, India for sending us a gift sample of Etoricoxib.

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