Hindwi Publishing Corportion Interntionl Journl of Alzheimer s Disese Volume 212, Article ID 97413, 8 pges doi:1.1155/212/97413 Reserch Article Neuroprotective Effects of Meloxicm nd in Scopolmine-Induced Cognitive Impirment nd Oxidtive Stress Puchchkyl Goverdhn, Akin Srvnthi, nd Thti Mmth Centre for Neurodegenertive Disese nd Aging Reserch Deprtment of Phrmcology, Vgdevi College of Phrmcy, Rmngr, Hnmkond, Wrngl 561, Indi Correspondence should be ddressed to Puchchkyl Goverdhn, gov ku@yhoo.co.in Received 29 October 211; Revised 24 December 211; Accepted 9 Jnury 212 Acdemic Editor: Frncesco Pnz Copyright 212 Puchchkyl Goverdhn et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Alzheimer s disese (AD) is progressive neurodegenertive disorder chrcterized by grdul decline in memory ssocited with shrinkge of brin tissue, with loclized loss of neurons minly in the hippocmpus nd bsl forebrin, with diminished level of centrl cholinergic neurotrnsmitter-cetylcholine nd lso reported to be ssocited with ccumultion of ubiquitinted proteins in neuronl inclusions nd lso with signs of inflmmtion. In these disorders, the bnorml protein ggregtes my themselves trigger the expression of inflmmtory meditors, such s cyclooxygense 2 (COX-2). In the present study, the effects of Meloxicm,, nd codministrtion of these drugs on scopolmine-induced lerning nd memory impirments in mice were investigted. Rectngulr mze test, Morris wter mze test, Locomotor ctivity, nd Pole climbing test were conducted to evlute the lerning nd memory prmeters. Vrious biochemicl prmeters such s cetylcholinesterse(ache), TBARS ssy, ctlse ctivity, nd DPPH ssy were lso ssessed. The present study demonstrtes tht Meloxicm,, nd co-dministrtion of these test drugs hd potentil therpeutic effects on improving the ntimnesic ctivity in mice through inhibiting lipid peroxidtion, ugmenting endogenous ntioxidnt enzymes, nd decresing cetylcholinesterse ctivity in brin. The memory enhncing cpcity of the drugs ws very significnt when compred to disese control (P <.1). 1. Introduction Alzheimer s disese (AD) is progressive neurodegenertive brin disorder tht is slow in onset but leds to dementi, unusul behvior, personlity chnges, nd ultimtely deth [1]. AD is chrcterized by the presence of excessive mounts of neuritic plques contining myloid β protein nd bnorml tu protein filments in the form of neurofibrillry tngles. Loss of cholinergic cells, prticulrly in the bsl forebrin, is ccompnied by loss of the neurotrnsmitter cetylcholine [2]. A decrese in cetyl choline in the brin of ptients with AD ppers to be criticl element in producing dementi [3]. AChE inhibitors from generl chemicl clsses such s physostigmine, tcrine, glntmine, nd heptylphysostigmine hve been tested for the symptomtic tretment of AD [4]. However, nonselectivity of these drugs, their limited efficcy, poor biovilbility, dverse cholinergic side effects in the periphery, nrrow therpeutic rnges, nd heptotoxicity re mong the severl limittions to their therpeutic success [5]. Therefore, it is worthwhile to explore the utility of other existing medicines for the tretment of vrious cognitive disorders [6]. Scopolmine, muscrinic cholinergic receptor ntgonist, hs been widely dopted to study cognitive deficits in experimentl nimls. After intrperitonel (i.p.) injection of scopolmine, the cholinergic neurotrnsmission ws blockded, leding to cholinergic dysfunction nd impired cognition in rts [7].Recently,ithsbeenreportedthtmemory impirment induced by scopolmine in rts is ssocited with ltered brin oxidtive stress sttus [8]. Therefore, rts with scopolmine-induced memory deficits were used s n niml model for screening ntidementi drugs [9]. Oxidtive stress is lso one of the ffecting fctors in AD, so severl ntioxidnts hve been studied for the reduction of oxidtive stress occurring during Alzheimer s disese [1, 11]. One of the mechnisms by which the bnorml
2 Interntionl Journl of Alzheimer s Disese Tble 1 Group-I Control Vehicle (.1% CMC). Group-II Disese control Scopolmine (1.4 mg/kg) i.p. Group-III Stndrd (5 mg/kg) orl + Scopolmine (1.4 mg/kg) i.p. Group-IV Test-I Meloxicm (5.2 mg/kg) orl + Scopolmine (1.4 mg/kg) i.p. Group-V Test-II (.49 mg/kg) p.o. + Scopolmine (1.4 mg/kg) i.p. Group-VI Test-III Meloxicm (5.2 mg/kg) orl + (.49 mg/kg) orl + Scopolmine (1.4 mg/kg) i.p. ccumultion of ubiquitinted proteins my medite neurodegenertion is by triggering n inflmmtory response. In-flmmtion is defense rection ginst diverse insults, intended to remove dmging gents nd to inhibit their detrimentl effects [12]. Those gents were found to increse neuronl levels of cyclooxygense 2 (COX-2) suggesting tht the production of such inflmmtory meditors cn be triggered by the intrcellulr ccumultion of bnorml proteins [13]. Nonsteroidl nti-inflmmtory drugs (NSAIDs) re the group of drugs which effectively interfere with the cyclooxygense pthwy which is involved in genertion of oxidtive free rdicls. In rheumtoid rthritis, NSAIDs hve showed improvement in the circulting ntioxidnt sttus on dily dosing tretment [14, 15]. For tht purpose, meloxicm (n enolic derived NSAID) hs been tken s reference drug by bsing on the possession of significnt nti-inflmmtory ctivity s well s ntioxidnt property [16]. It hs preferentil inhibitory ctivity ginst the inducible cyclooxygense-2 isoform, over the constitutive isoform cyclooxygense-1. Therefore, meloxicm nd other COX-2 selective inhibitors re promoted for their sfer profile of side effects. (L-deprenyl), n irreversible inhibitor of monomine oxidse-b (MAO-B), therpeutic gent of Prkinson s disese, is known to hve neuroprotective properties tht my involve its regultory effects on ntioxidnt enzymes. In ddition, selegiline my ct s n ntioxidnt in neurons nd protect ginst glutmte-receptor-medited toxicity. Studies of selegiline on ged mle lbortory nimls hve showed delyed cognitive impirment nd behviorl deteriortion when compred with control nimls [17]. The min purpose of the present study ws to investigte the synergistic ction of meloxicm nd selegiline in scopolmine-induced Alzheimer s disese model. 2. Mterils nd Methods 2.1. Animls. Swiss mice of mle sex weighing 2 25 g were used in the present study. They hd free ccess to food nd wter nd were mintined under stndrd lbortory conditions with lternting light nd drk cycles of 12 h ech. They were cclimtized to lbortory conditions for 2 dys before behviorl studies. All the redings were tken during the sme time of the dy, tht is, between 1 m nd 2 pm. The Institution Animls Ethics Committee (IAEC) hd pproved the experimentl protocol, nd cre of nimls ws tken s per guidelines of CPCSEA, Deprtment of Animl Welfre, nd Government of Indi [18]. 2.2. Drugs. Scopolmine (Cdil Helthcre pvt. Ltd), (INTAS phrmceuticls), nd (Alkem lbortories Ltd.) were purchsed. Meloxicm ws gifted by Dr. Reddy s Lbrotries. Scopolmine nd selegiline were diluted with distilled wter. 2.3. Experimentl Design. The nimls (n = 36) were divided into six different groups of 6 nimls per ech group. Scopolmine (1.4 mg/kg) s disese inducer ws dministered to ll groups through intrperitonel (i.p) route fter drugs dministrtion to ll the groups except norml control group. The sme procedure ws crried out for 9 dys (see Tble 1). 2.4. Behviourl Tests. All the nimls were trined for 2 dys before drugs dministrtion. 2.4.1. Rectngulr Mze Test. Assessment of lerning nd memory cn be effectively done by this method. The mze consists of completely enclosed rectngulr box with n entry nd rewrd chmber ppended t opposite ends. The box is prtitioned with wooden slts into blind pssges leving just twisting corridor leding from the entry to the rewrd chmber. Animls were trined prior to the experiment by fmilirizing with the rectngulr mze for period of 1 min for 2 h. Well-trined nimls were tken for the experiment. Trnsfer ltency (time tken to rech the rewrd chmber) ws recorded. For ech niml, four redings were tken nd the verge is tken s lerning score (trnsfer ltency) for tht niml. Lower scores of ssessment indicte efficient lerning while higher scores indicte poor lerning in nimls. The time tken by the nimls to rech the rewrd chmber from the entry chmber ws noted on dy 1, 3, 5, 7, nd 9 [19]. 2.4.2. Morris Wter Mze Test. Morris wter mze ws used to ssess lerning nd memory in experimentl mice. There re severl dvntges of Morris wter mze over other models of lerning nd memory including bsence of motivtionl stimuli such s food nd wter deprivtion, electricl stimultions, nd buzzer sounds [2, 21]. Briefly, it consists
Interntionl Journl of Alzheimer s Disese 3 of circulr wter tnk, filled with opque wter, nd one centimeter submerged pltform. First, nimls were trined to locte the pltform. During cquisition, tril escpe ltency time (ELT), time mesure to locte the hidden pltform, ws noted s n index of cquisition. Ech niml ws subjected to the four cquisition trils per dy for 4 consecutive dys. The time spent by the niml, serching for the missing pltform in trget qudrnt Q2 with respect to other qudrnt (Q1, Q3, nd Q4) on 5th dy, ws noted s n index of retrievl. For studying the effectof drug on cquisition, the drug solution ws dministered before cquisition tril [22]. 2.4.3. Locomotor Activity. Locomotor ctivity is influenced by most of the CNS drugs in both mn nd nimls. The locomotor ctivity of drug cn be studied using ctophotometer which opertes on photoelectric cells which re connected in circuit with counter when the bem of light fllingonphotocelliscutoff by the niml, then count is recorded. Animls re plced individully in the ctivity cge for 1 min nd the ctivity ws monitored. The test is done before 3 min nd fter the drug dministrtion. The photo cell count is noted nd decrese or increse in locomotor ctivity is clculted [2]. 2.4.4. Pole Climbing Test. When n electricl stimulus is given to niml, it tries to escpe from it nd move to the ner sfe plce. This equipment is designed in such wy to climb the pole when stimulus is generted. Prior to the experiment, nimls were trined. Trining nd testing is conducted in 25 25 4 cm chmber tht is enclosed in dimly light, sound ttenuted box. Scrmbled shock is delivered to the grid floor of the chmber. A smooth stinless steel pole, 2.5 cm in dimeter, is suspended by counter blnce weight through hole in the upper centre of the chmber. A micro switch is ctivted when the pole is pulled down by 3 mm. With weight greter thn 2 gm. A response is recorded when mice jumps on the pole nd ctivtes micro switch. The ctivtion of light nd speker together is used s conditioned stimulus. Ech niml ws plced six times per dy [2]. 2.5. Histopthologicl Studies. After 8-dy tretment, the brins of different groups were perfusion-fixed with 4% prformldehyde in.1 M phosphte buffer. The brins were removed nd postfixed in the sme fixtive overnight t 48 C. The brins were then routinely embedded in prffin nd stined with Hemtoxylin-Eosin. The hippocmpl lesions were ssessed microscopiclly t 4 mgnifiction [23]. 2.6. Dissection nd Homogeniztion. On dy 9, fter behviorl ssessments, nimls were scrified by cervicl disloction. The brins were removed. Ech brin ws seprtely put on ice nd rinsed with ice-cold isotonic sline. A (1% w/v) homogente ws prepred in.1 M phosphte buffer (ph 7.4). The homogente ws centrifuged t 3 rpm for 15 minutes nd liquots of superntnt were seprted nd used for biochemicl estimtion [23]. 2.7. Biochemicl Tests 2.7.1. AchE Estimtion. The cholinergic mrker, cetylcholinesterse, ws estimted in the whole brin ccording to the method of Ellmn method. Ellmn s regent is 5, 5 -dithiobis(2-nitrobenzote) nd it is lso bbrevited s DTNB. This homogente ws incubted for 5 min with 2.7 ml of phosphte buffer nd.1 ml of DTNB. Then,.1 ml of freshly prepred cetylthiocholine iodide (ph 8) ws dded nd the bsorbnce ws red t 412 nm [24, 25]. 2.7.2. Thiobrbituric Acid Rective Substnces (TBARS) Assy. This ssy is used to determine the lipid peroxidtion. Aliquots of.5 ml distilled wter were dded with1 ml of 1% trichlorocetic cid nd were dded with.5 ml of brin tissue homogente. This is centrifuged t 3 rpm for 1 min. To the.2 ml superntnt,.1 ml thiobrbituric cid (.375%) ws dded. Totl solution is plced in wter bth t 8 c for 4 min nd cooled t room temperture. Absorbnce ws red t 532 nm [26]. 2.7.3. Ctlse Activity. Ctlse ctivity ws ssessed by the method of Luck [27], wherein the brekdown of hydrogen peroxide is mesured. In this 3 ml of H 2 O 2 phosphte buffer ws dded to.5 ml of the superntnt of the tissue homogente. The bsorbnce ws recorded t 24 nm using Perkin Elmer Lmbd 2 spectrophotometer. The results were expressed s micromoles of H 2 O 2 decomposed per minute per mg protein [25]. 2.7.4. DPPH (2,2-Diphenyl-1-picrylhydrzyl) Assy. In this, mesurement is mde from the bleching of purple-coloured methnol solution of DPPH. To the 1 μl of diverse conc. of the smple, 4 ml of.4% methnolic solution of DPPH ws dded. After 3 min incubtion, bsorbnce ws red t 517 nm. Inhibition of free rdicl by DPPH in % ws clculted in the following wy: ( ) % = Ablnk A smple /A blnk 1, (1) A blnk : bsorbnce of control rection. A smple : bsorbnce of test smple. Vlues of inhibition were clculted [26]. 2.8. Sttisticl Anlysis. The sttisticl nlysis of dt ws done by the one wy nlysis of vrince (ANOVA) followed by the Dunnett s test. The probbility level less thn.5 ws considered s significnt. Results were expressed s men ± SD. 3. Results 3.1. Behviourl Tests 3.1.1. Rectngulr Mze Test. The ctivity of meloxicm nd selegiline ws evluted using rectngulr mze. The mice in ll tretment groups except scopolmine-treted group showed lower trnsfer ltency on 7th dy nd 9th dy
4 Interntionl Journl of Alzheimer s Disese Time (s) 14 12 1 8 6 4 2 Dy 1 Dy 3 Dy 5 Dy 7 Dy 9 Groups on respective dys Norml control Scopolmine c b b b b Meloxicm Meloxicm + selegiline Figure 1: Rectngulr mze test. Effect of meloxicm nd selegiline on ltency time compred to the disese control group. (Men ± SD, n = 6). Grph showing men ± SD of ltency time in seconds. P <.1, b P <.1, c P <.5 compred with corresponding vlues of disese control. Time (s) 14 12 1 8 6 4 2 b b b b Dy 1 Dy 2 Dy 3 Dy 4 Dy 5 Groups on respective dys Norml control Scopolmine c c c c b b b b Meloxicm Meloxicm + selegiline Figure 2: Morris wter mze test. Effect of meloxicm nd selegiline on ltency time compred to the disese control group. (Men ± SD, n = 6). Grph showing men ± SD of ltency time in seconds. P <.1, b P <.1, c P <.5 compred with corresponding vlues of disese control. No. of crossings in 1 min 25 2 15 1 5 c c c c Dy 1 Dy 3 Dy 5 Dy 7 Dy 9 Groups on respective dys Norml control Scopolmine Meloxicm Meloxicm + selegiline Figure 3: Locomotor ctivity. Effect of meloxicm nd selegiline on ltency time compred to the disese control group. (Men ± SD, n = 6). Grph showing men ± SD of ltency time in seconds. P <.1, b P <.1, c P <.5 compred with corresponding vlues of disese control. Time (s) 12 1 8 6 4 2 Dy 1 Dy 3 Dy 5 Dy 7 Dy 9 Norml control Scopolmine Groupsonrespectivedys Meloxicm Meloxicm + selegiline Figure 4: Pole climbing test: Effect of meloxicm nd selegiline on ltency time levels compred to the disese control group (Men ± SD, n = 6). Grph showing men ± SD of ltency time in seconds. P <.1, b P <.1, c P <.5 compred with corresponding vlues of disese control. Inhibition of enzyme (%) 4 35 3 25 2 15 1 5 Norml control Scopolmine Norml control Scopolmine Groups Meloxicm Meloxicm Meloxicm + selegiline Meloxicm + selegiline Figure 5: AchE estimtion. Effect of meloxicm nd selegiline on AchE levels compred to the disese control group. (Men ± SD, n = 6). Grph showing men ± SD of % inhibition of AchE enzyme. P<.1 compred with corresponding vlues of disese control. compred to 5th dy of the sme group s well s with the scopolmine group which ws given in Figure 1. This in dictes memory enhncing cpcity of the meloxicm nd selegiline. (5 mg/kg) treted for successive 8 dys cts s positive control, possessed significnt (P <.5) decrese in trnsfer ltency when compred to norml control nd disese control (scopolmine) using Dunnet s test.
Interntionl Journl of Alzheimer s Disese 5 MDA levels (nmoles/mg of tissue) 35 3 25 2 15 1 5 Inhibition of DPPH (%) 8 7 6 5 4 3 2 1 Norml control Scopolmine Meloxicm Meloxicm + selegiline Norml control Scopolmine Norml control Scopolmine Groups Meloxicm Meloxicm + selegiline Meloxicm Meloxicm + selegiline Figure 6: TBARS ssy. Effect of meloxicm nd selegiline on mlondildehyde levels compred to the disese control group. (Men ± SD, n = 6). Grph showing men ± SD of mlondildehyde levels. P<.1 compred with corresponding vlues of disese control. H2O2 scvenging (%) 1 9 8 7 6 5 4 3 2 1 Norml control Scopolmine Groups Meloxicm Meloxicm + selegiline Norml control Scopolmine Groups Meloxicm Meloxicm + selegiline Figure 8: DPPH ssy. Effect of meloxicm nd selegiline on inhibition of DPPH compred to the disese control group (Men ± SD, n = 6). Grph showing men ± SD of % inhibition of DPPH. P<.1 compred with corresponding vlues of disese control. 3.1.2. Morris Wter Mze Test. The ctivity of meloxicm nd selegiline was evluted using Morris wter mze. The mice tretment groups except scopolmine-treted group showed significnt trnsfer ltency on 4th dy with pltform nd on 5th dy without pltform which ws given in Figure 2. This indictes memory enhncing cpcity of the meloxicm nd selegiline. (5 mg/kg) treted for successive 8 dys cts s positive control, possessed significnt (P <.5) decrese in trnsfer ltency when compred to disese control (scopolmine) using dunnet s test. 3.1.3. Locomotor Activity. The ctivity of meloxicm nd selegiline ws evluted using photoctometer. The mice showed significnt trnsfer ltency on 7th dy compred to the 9th dy in ll tretment groups except scopolminetreted group which ws given in Figure 3. This (5mg/kg)tretedsuccessive8dysctsspositivecontrol, possessed significnt (P <.5) decrese in number of crossings which is comprble to the other tretment groups. Norml control Scopolmine Meloxicm Meloxicm + selegiline Figure 7: Ctlse ctivity. Effect of meloxicm nd selegiline on ctlse ctivity compred to the disese control group. (Men ± SD, n = 6). Grph showing men ± SD of %H 2 O 2 scvenging ctivity. P<.1 compred with corresponding vlues of disese control. 3.1.4. Pole Climbing Test. The vlues show tht there ws significnt difference tht hs been observed on dys 7 nd 9 compred to the 1, 3, nd 5. Scopolmine-treted group took more time wheres the control nd drug-treted groups showed less time to rech the pole in pole climbing pprtus. The results showed tht synergistic ction of meloxicm nd selegiline ws significnt (P <.5) nd is comprble to the stndrd drug (donepezil).
6 Interntionl Journl of Alzheimer s Disese Histopthologicl studies: () (b) (c) (d) (e) (f) Figure 9: Histopthologicl studies. These Figures (), (b), (c), (d), (e), nd (f) re norml control, scopolmine (disese control), donepezil (stndrd),meloxicm, selegiline, nd meloxicm + selegiline, respectively, representing the histologicl sections of the brin tissue showing neurologicl lesions. 3.2. Biochemicl Tests 3.2.1. AchE Estimtion. Scopolmine tretment significntly incresed the brin AchE level compred to control group (Figure 5). Stndrd drug (donepezil) nd test drugs (meloxicm, selegiline) tretment significntly inhibited the brin AchE level compred to their corresponding scopolminetreted groups. 3.2.2. TBARS Assy. Scopolmine tretment significntly incresed the brin MDA level compred to control group (Figure 6). Stndrd drug (donepezil) nd test drugs
Interntionl Journl of Alzheimer s Disese 7 (meloxicm,selegiline) tretment significntly (P <.5) decresed brin MDA level compred to their corresponding scopolmine treted groups. 3.2.3. Ctlse Activity. Ctlse levels were decresed in scopolmine-treted groups compred to the norml control group (Figure 7). Significnt (P <.5) difference hs been found in drug-treted groups. Synergistic effectwsobserved which is comprble with the stndrd group thn individul drug-treted groups. 3.2.4. DPPH Assy. Antioxidnt levels were decresed in scopolmine-treted group compred to the control group (Figure 8). Drug-treted groups showed significnt (P <.5) difference compred to the disese control group. 3.3. Histopthologicl Studies. From Figure 9, it is clerly visible tht in disese control group the degenerted cells re more compred to other groups. This will be indicted by the gps in slides. The drug-treted groups re in between the norml control nd disese control groups. The combintion group is mostly ner to the control group compred to the individul drug-treted groups. 4. Discussion The scopolmine mnesi test is widely used s primry screening test for so-clled nti-alzheimer drugs [24]. There recently hs been n incresed pprecition of the role tht inflmmtion plys in the pthogenesis of Alzheimer s disese tht hs risen principlly from epidemiologicl studies showing drmtic effect of long-term NSAID tretment on Alzheimer s disese risk. However, the molecur mechnisms by which NSAIDs intervene in the pthologicl processes tht underlie cognitive decline nd neuronl loss remin uncler [28, 29]. Recently, mny studies reported tht memory impirment in the scopolmine-induced niml model is ssocited with incresed oxidtive stress within the brin [8, 3, 31]. Oxidtive stress is the cytotoxic consequence of oxyrdicl nd oxidnt formtion nd the rection with cellulr constituents. Rective oxidtive species (ROS) re generted continuously in nervous system during norml metbolism nd neuronl ctivity. The nervous system is prticulrly vulnerble to the deleterious effects of ROS. Becuse the brin hs high consumption of oxygen, lrge mount of polyunsturted ftty cids (PUFAs), high contents of free ions, nd low levels of ntioxidnts defense were compred to other orgns [32]. Incresed MDA level s one of the ROS hs been shown to be n importnt mrker for in vivo lipid peroxidtion. From the behviorl test, tht is, rectngulr mze test nd Morris wter mze test, it is clerly seen tht there ws generl decrese in the trnsfer ltency in ll treted groups compred to the scopolmine-treted group. The memory loss effect of scopolmine is more prominent compred to the control group. In comprison with, the drugtreted groups hd lmost equl performnce which indictes synergistic effect of meloxicm nd selegiline ginst memory loss. Menwhile locomotor ctivity nd pole climbing voidnce tests re done which lso indicte the lening bility (Figure 4). The mjor ntioxidnt nd oxidtive free rdicl scvenging enzymes like glutthione, SOD, nd ctlse ply n importnt role to reduce oxidtive stress in brin. In this study, from the DPPH ssy ntioxidnt levels re estimted. These enzyme levels re decresed in the scopolmine-treted group compred to the control group. The enzyme levels re lmost equl in combintion group nd the stndrd group. Individul groups re showing less thn stndrd group. It supports the ntioxidnt ction of drugs. In the present study rts fter scopolmine tretment showed significnt increse in the brin levels of mlondildehyde, which is the mesure of lipid peroxidtion nd free rdicl genertion. In the drug-treted groups, there is significnt decrese in the levels of mlondildehyde which is nerly equl to the stndrd group. From the results, it is cler tht the nti-inflmmtory ctivity of meloxicm decreses the disese progression. 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