Brain Research Bulletin 67 (2005) 161–168

Neuroprotective effect of nicotine against 3-nitropropionic acid(3-NP)-induced experimental Huntington’s disease in rats

Mohammad Tariqa,∗, Haseeb Ahmad Khanb, Ibrahim Elfakia,Saleh Al Deeba, Khalaf Al Moutaerya

a Neuroscience Research Group, Armed Forces Hospital, P.O. Box 7897 (W-912), Riyadh 11159, Saudi Arabiab Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia

Received 23 April 2005; received in revised form 14 June 2005; accepted 16 June 2005Available online 26 July 2005

Abstract

Nicotinic acetylcholine receptors (nAChRs) are regarded as potential therapeutic targets to control various neurodegenerative diseases.Owing to the relevance of cholinergic neurotransmission in the pathogenesis of Huntington’s disease (HD) this investigation was aimed tos Systemica 0.25, 0.50a additionalg ty, inclinedp erfused withn e assessedb nd dose-d dopamine( aminergicn nicotine ine©

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tudy the effect of nicotine, a nAChR agonist, on 3-nitropropionic acid (3-NP)-induced neurodegeneration in female Wistar rats.dministration of 3-NP in rats serves as an important model of HD. The animals received subcutaneous injections of nicotine (0,nd 1.00 mg/kg) daily for 7 days. 3-NP (25 mg/kg, i.p.) was administered daily 30 min after nicotine for the same duration. Oneroup of rats served as control (vehicle only). On day 8, the animals were observed for neurobehavioral performance (motor activilane test, grip strength test, paw test and beam balance). Immediately after behavioral studies, the animals were transcardially peutral buffered formalin (10%) and brains were fixed for histological studies. Lesions in the striatal dopaminergic neurons wery immunohistochemical method using tyrosine hydroxylase (TH) immunostaining. Treatment of rats with nicotine significantly aependently attenuated 3-NP-induced behavioral deficits. Administration of 3-NP alone caused significant depletion of striatalDA) and glutathione (GSH), which was significantly and dose-dependently attenuated by nicotine. Preservation of striatal dopeurons by nicotine was also confirmed by immunohistochemical studies. These results clearly showed neuroprotective effect ofxperimental model of HD. The clinical relevance of these findings in HD patients remains unclear and warrants further studies.2005 Published by Elsevier Inc.

eywords:Nicotine; Huntington’s disease; 3-Nitropropionic acid; Neuroprotection

. Introduction

Huntington’s disease (HD) is a chronic progressive auto-omal dominant neurodegenerative disorder that is character-zed by a striatal-specific degeneration[67]. The pathologicalhanges manifest clinically in midlife as a triad of cogni-ive decline, psychiatric disturbance and impairment of motorunction. Several attempts have been made to develop experi-ental model of HD. In the most widely used animal modelsf HD, excitotoxic amino acids, such as kainic, quinolinic

∗ Corresponding author. Tel.: +966 1 477 7714x5602;ax: +966 1 470 0576.E-mail address:rkh research@yahoo.com (M. Tariq).

and ibotenic acids, are stereotactically injected into speregion of the brain. Besides being difficult, there is alwachance of error to inject the drugs into the target cells.most recent model of HD is based on systemic injectof 3-nitropropionic acid (3-NP), a mitochondrial toxin thcauses striatal neuropathy similar to that seen in clinica[4,8]. A major advantage of 3-NP model over other modeHD is that the lesions produced are bilateral, striatal speand develop spontaneously after systemic administrati3-NP. In spite of extensive research this devastating hetary disease remains incurable, warranting further studidetermine the causes and cure of HD.

The natural alkaloid nicotine present in Nicotiana tabais by far the most widely studied substance that origin

361-9230/$ – see front matter © 2005 Published by Elsevier Inc.oi:10.1016/j.brainresbull.2005.06.024

162 M. Tariq et al. / Brain Research Bulletin 67 (2005) 161–168

from tobacco smoke and exhibits widespread pharmacolog-ical effects. During the last decade, there has been a rapidexplosion of publications reporting the neuroprotective activ-ity of nicotine. Recent studies clearly suggest that nicotinicacetylcholine receptor (nAChR) in the CNS is a new potentialtherapeutic target for the management of neurodegenera-tive diseases[34]. O’Neill et al. [38] have suggested thatneuronal nAChRs agonists could provide motor improve-ment and retard the progressive course of various diseasesincluding Alzheimer’s disease and Parkinsonism. Further-more, nicotine has been shown to protect various neuronsagainst a variety of neurotoxins via neuronal nAChRs in vivo[12,27,36,40,41,50,62]as well as in vitro[23,55,56,60,61].In the present study, we demonstrate the protective effectsof nicotine (0.25–1 mg/kg) against 3-NP-induced behavioral,biochemical and histological changes in a rat model of HD.

2. Materials and methods

2.1. Animal treatment

Adult female Wistar rats (230–280 g) grown in our ani-mal breeding facility were used. The animals were housed ina temperature-controlled room (24◦C) with 12-h light/12-hdark cycle. Standard laboratory food and water were availablea ocolo andE ran-d Ther only,w ailyf with3 ico-t g/kg,r ty-f stedf sionw theb ch ofa

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the floor. Each animal was tested separately and the motoractivity was measured for a period of 2 min.

2.2.2. Balance beam testThe balance beam test was used to measure the ability

of rats to traverse a horizontal narrow beam (1 cm× 100 cm)suspended 1 m above a foam-padded cushion[20,53]. Duringtesting, the rats were given 2 min to traverse the beam. If theydid not complete the task or if they fell off the beam, thetrial was ended and the rats were placed back into their homecages. For successful performers, the latency to cross thebeam was recorded.

2.2.3. Limb withdrawal testIn this behavioral test, the animal was placed on a 20 cm

high 30 cm× 30 cm Perspex platform containing four holes,two holes of 5 cm diameter for the hind limbs and two holeswith a diameter of 4 cm for the forelimbs. The rat was placedon the platform by positioning first the hind limbs and thenthe forelimbs into the holes. The times taken by the animalto retract its first hind limb and the contralateral hind limbwere recorded. The difference between the retraction times ofboth hind limbs was determined. This is considered to be animportant parameter to measure functional abnormalities ofthe hind limbs, which are indicative for the extent of striataldegeneration[63]. The test was performed three times witha .

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d libitum throughout the study. The experimental protf this study was approved by the Institutional Researchthics Committee. The rats of matching weights wereomly divided into five groups of eight animals each.ats in group 1 served as control and received vehicleshereas rats in group 2 received 3-NP (25 mg/kg, i.p.) d

or 7 days. The animals in groups 3–5 were treated-NP similarly as in group 2, in addition they received n

ine (subcutaneously) in the doses of 0.25, 0.5 and 1 mespectively, daily 30 min before 3-NP for 7 days. Twenour hours after the last dose of 3-NP the rats were teor behavioral parameters followed by transcardiac perfuith 10% neutral buffered formalin (NBF) and fixation ofrains for immunohistochemical studies. A separate batnimals was used for biochemical studies.

.2. Behavioral studies

The animals were subjected to following behavioral ty a person unaware of the treatment protocol.

.2.1. Motor activityMotor activity was measured using Optovarimex acti

eter (Columbus Instruments, USA). The horizontal mctivity was detected by two perpendicular arrays o

nfrared beams located 2.5 cm above the floor of the terea. Each interruption of a beam on thex- ory-axis generaten electric impulse, which was presented on a digital couimilarly, the vertical motor activity was recorded using

wo additional rows of infrared sensors located 12 cm a

45 min interval and the average values were reported

.2.4. Inclined plane testInclined plane test, as described by Rivlin and Tator[49],

as used to assess motor function in rats. The inclinedpparatus consists of two rectangular boards connecach other by a hinge. A rubber mat with ridges 0.6 cmeight was fixed to the movable plane and two protractorlywood side panels with degrees (0–90) marked on

aces were fixed on the base. The maximum inclinatiohich a rat could maintain itself for 5 s with the body aerpendicular to the axis of the plane was considered a

capacity angle’ for the animals. The angle was increaseecreased by a margin of 0.5◦ gradually until the rat coulaintain its position on the inclined plane for 5 s with

alling.

.2.5. String test for grip strengthThe rat was allowed to hold with the forepaws a steel

2 mm in diameter and 35 cm in length), placed at a hef 50 cm over a cushion support. The length of time theas able to hold the wire was recorded. This latency torip loss is considered as an indirect measure of grip stre

53].

.3. Biochemical studies

After 24 h from the last dose of 3-NP the rats were saced and the brains were rapidly dissected on a pre-coetri dish. The isolated striata were quickly frozen in liq

M. Tariq et al. / Brain Research Bulletin 67 (2005) 161–168 163

nitrogen and transferred in pre-cooled vials and then storedat−80◦C until analyzed.

2.3.1. Dopamine analysisThe analysis of dopamine in striatum was carried out

according to the procedure of Patrick et al.[42]. The stri-ata from the right cerebral hemisphere were weighted andhomogenized for 10 s in 0.1 M perchloric acid containing0.05% EDTA using Teflon homogenizer. The homogenateswere immediately centrifuged at 10,000 rpm at 4◦C for10 min. The supernatants were filtered using 0.45�m porefilters and analyzed by high performance liquid chromatogra-phy (HPLC). The HPLC system consisted of an electrochemi-cal detector from Metrohm (Model 65 Herisani, Switzerland),an autoinjector (Model 712, Waters Associate Inc., Milford,MA, USA), a solvent delivery pump (Waters Model 510)and an integrator (Waters Model 745). The mobile phaseconsisted of a mixture of 0.1 M citric acid monohydrate,0.1 M sodium acetate, 7% methanol, 100�M EDTA and0.01% sodium octane sulfonic acid and the column was C-18�Bondapak (3.9 mm× 150 mm). The flow rate was main-tained at 1 ml/min and the injection volume was 20�l.

2.3.2. Glutathione analysisThe measurement of glutathione (GSH) in striatum was

carried out enzymatically according to the modified proce-d i-s 2 M)c cen-t wass ho-t i-n6o entsw Thea 2 nma ringto

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2.4.1. Tyrosine hydroxylase (TH) immunostainingThe paraffin sections of the brain were deparaffinised by

immersing the respective slides in xylene (5 min) followedby rehydration using sequential treatment with decreasingconcentrations of alcohol to water (100% alcohol, 95% alco-hol, 80% alcohol and distilled water). The rehydrated speci-mens were immersed in citrate buffer (pH 6.0) and heatedin the microwave for5 + 5 min to free the binding sitesfor TH immunoreactivity. After cooling to room tempera-ture, the brain sections were quenched with 3% H2O2 andallowed to react with specific monoclonal antibody againstrat TH (Novocastra Laboratories Ltd., UK) in 1:20 dilu-tion for 30 min at ambient temperature. After rinsing withtris-buffered saline (TBS), the sections were sequentiallyincubated with biotinyleted goat antibody, strept AB com-plex/HRP and chromogenic substrate for peroxidase, accord-ing to manufacturer’s instructions (Dako MS, Denmark). Foreach TH section, an adjacent section was stained with haema-toxylin for structure identification using light microscopy.

2.5. Statistical analysis

Data were analyzed by one-way analysis of variance(ANOVA) followed by post hoc Dunnett’s multiple com-parison tests to determine the significance level between theexperimental groups, using SPSS software (Version 10).Pv cant.

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ure of Owen[39]. The striata from the left cerebral hemphere were homogenized in ice-cold perchloric acid (0.ontaining 0.01% of EDTA. The homogenates wererifuged at 4000 rpm for 10 min. The enzymatic reactiontarted by adding 200�l of clear supernatant in a spectropometric cuvette containing 800�l of 0.3 mM reduced nicotamide adenine dinucleotide phosphate (NADPH), 100�l ofmM 5,5-dithiobis-2-nitrobenzoic acid (DTNB) and 10�lf 50 units/ml glutathione reductase (all the above reagere freshly prepared in phosphate buffer of pH 7.5).bsorbance was measured over a period of 4 min at 41t 30◦C. The glutathione level was determined by compa

he change of absorbance (�A) of test solution with the�Af standard glutathione.

.4. Histology

The histological procedure reported earlier was usedome modifications[51]. The rats were transcardially pused with 10% NBF till the outflow perfusate was absolulear and free form blood contamination. After perfusionrains were removed and immersed in the same fixatised for perfusion (10% NBF), for 3 days. The specimere then processed overnight for dehydration with incr

ng concentrations of alcohol and clearing with acetonehloroform using an automated tissue processor (Shaouthern 2L Processor Mk II, England). The specimensmbedded in paraffin blocks and coronal sections (4�m)ere made using a microtome (CUT 4050, Microtec Gmermany).

alues less than 0.05 were considered statistically signifi

. Results

.1. Effect of nicotine (NIC) on 3-NP-inducedehavioral deficits

.1.1. Motor activityAdministration of 3-NP for 7 days significantly reduc

he horizontal locomotor activity of rats (ANOVAF= 2.40,< 0.05). Concomitant treatment with nicotine doependently but insignificantly reversed the effect of 3n horizontal locomotor activity (Table 1). 3-NP also signif

cantly diminished the vertical locomotor activity (ANOV= 3.51,P< 0.05), which was significantly increased in

ats treated with the high dose of nicotine (Table 1).

.1.2. Balance beam testAll the animals in the 3-NP alone group failed to pass

eam balance test within the cut-off time of 120 s, whereaontrol rats performed this task in 7.7± 2.4 s (Table 1). Co-reatment with nicotine significantly and dose-dependemproved the performance of 3-NP treated rats in the baleam test (Table 1).

.1.3. Limb withdrawal testThe difference between the retraction times of the

ind limbs was significantly higher in 3-NP alone treaats (102.4± 34.4 s) as compared to control rats that w

164 M. Tariq et al. / Brain Research Bulletin 67 (2005) 161–168

Table 1Effect of nicotine (NIC) on 3-nitropropionic acid (3-NP)-induced behavioral deficits

Treatment group(N= 7 per group)

Motor activity (counts/2 min) Balance beamtest (s)

Limb withdrawaltest (s)

Inclined planetest (angle◦)

String test (s)

Horizontal Vertical

Control 1394± 102 273.3± 31.8 7.7± 2.4 1.0± 0.0 80.2± 1.5 78.0± 8.13-NP 288± 151# 33.3± 16.4# 120.0± 0.0## 102.4± 34.4## 60.2± 1.5## 4.3 ± 1.3##

3-NP + NIC 0.25 761± 140 71.4± 15.2 28.3± 4.9** 12.8± 3.8** 75.2± 1.5** 15.0± 4.53-NP + NIC 0.50 873± 203 168.3± 53.3 26.6± 8.7** 9.0 ± 3.2** 75.2± 1.5** 22.8± 6.8*

3-NP + NIC 1.00 882± 173 188.5± 71.6* 5.5 ± 0.5** 6.7 ± 2.9** 75.2± 1.5** 34.5± 6.1**

Values are mean± S.E.M.# P< 0.05.

## P< 0.001 vs. control group.* P< 0.05.

** P< 0.001 vs. 3-NP alone group using Dunnett’s multiple comparison test.

able to quickly retract their both hind limbs (Table 1). Theperformance of 3-NP treated rats in the limb withdrawal testwas significantly and dose-dependently improved by nicotine(ANOVA F= 7.15,P< 0.001).

3.1.4. Inclined plane testAdministration of 3-NP alone significantly impaired the

motor function of animals in the inclined plane test as indi-cated by a lower capacity angles in this group of rats (Table 1).All the three doses of nicotine appeared to be equipotent inimproving the ability of 3-NP treated rats in scoring highcapacity angles (ANOVAF= 41.06,P< 0.001).

3.1.5. String test (grip strength)The animals treated with 3-NP alone showed a signifi-

cantly lower latency to the grip loss (4.3± 1.3 s) in the stringtest as compared to control rats (78.0± 8.1 s) (Table 1). Pre-treatment with nicotine significantly and dose-dependentlyincreased the latency time of the 3-NP treated animals(ANOVA F= 24.96,P< 0.001).

3.2. Effect of nicotine on 3-NP-induced striataldopamine (DA) depletion

Administration of 3-NP (30 mg/kg) for 7 consecu-t ther ls(a lyi VAF

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lonett Hl s ofn 3-

Fig. 1. Effect of nicotine (NIC) on 3-NP-induced striatal dopamine depletionin rats. Values are mean± S.E.M.#P< 0.001 vs. control group,*P< 0.05 and** P< 0.01 vs. 3-NP alone group using Dunnett’s multiple comparison test.

Fig. 2. Effect of nicotine on 3-NP-induced striatal glutathione (GSH)depletion in rats. Values are mean± S.E.M. #P< 0.001 vs. control group,*P< 0.001 vs. 3-NP alone group using Dunnett’s multiple comparison test.

ive days significantly reduced striatal DA levels inats (5.12± 0.83�g/g) as compared to control leve12.20± 1.00�g/g) (Fig. 1). The medium (8.68± 0.64�g/g)nd high (10.16± 0.55�g/g) dose of nicotine significant

ncreased the striatal DA levels in 3-NP treated rats (ANO= 12.67,P< 0.001).

.3. Effects of nicotine on 3-NP-induced striatallutathione depletion

There was a massive depletion of GSH in 3-NP areated rats as compared to control group (Fig. 2). Althoughhe low dose of nicotine failed to modify striatal GSevels in 3-NP treated rats, medium and high doseicotine significantly protected the animals against

M. Tariq et al. / Brain Research Bulletin 67 (2005) 161–168 165

Fig. 3. Light microscopic observation of TH immunostaining of the striatal dopaminergic neurons. TH expression was markedly decreased in the striata of3-NP alone treated rats (B) as compared to intense TH immunoreactivity in control striata (A). Administration of low (C) medium (D) and high (E) doses ofnicotine dose-dependently reversed the degenerative effect of 3-NP on dopaminergic neurons.

NP-induced striatal GSH depletion (ANOVAF= 72.14,P< 0.001).

3.4. Effect of nicotine on tyrosine hydroxylaseimmunoreactivity

The striatum of control animals showed intense expressionof TH (Fig. 3A), whereas a sharp decrease in TH immunore-activity was observed in 3-NP treated rats (Fig. 3B). The lossof dopaminergic neurons due to 3-NP was dose-dependentlyreversed by nicotine showing gradually increased den-sity of TH immunostaining of the dopaminergic neu-rons (Fig. 3C–E) as compared to 3-NP alone treated rats(Fig. 3B).

4. Discussion

The treatment of rats with 3-NP produced significantmotor and behavioral abnormalities including bradykinesia,muscles weaknesses and rigidity (Table 1). These findingsare in agreement with earlier reports who also observed avariety of neurobehavioral abnormalities and motor deficitin rats following 3-NP administration[6,7,24,54,63]. Thesymptoms developed by chronic administration of 3-NP areakin to juvenile onset and late hypokinetic stages of HD[ hly

significant and dose-dependent protection of animals against3-NP-induced behavior and motor deficit (Table 1). Neuro-protective effect of nicotine has been shown against MPTP-induced straital damage[30] as well as secondary neurode-generative changes following spinal cord injury in rats[47].Several distinct mechanisms can be involved in tissue sparinginduced by nicotine in rats following exposure to neurodegen-erative agents. Involvement of nAChRs in nicotine-inducedneuroprotection against quinolinic acid[37] and MPTP[30]induced neurodegeneration has been reported earlier, sug-gesting a definite role of cholinergic neurotransmission inneuroprotective effect of nicotine[44,45].

Administration of 3-NP produced a significant depletionof striatal DA levels in rats (Fig. 1). Substantial losses inDA receptors[1] as well as DA content[3] have been sug-gested as the major contributory factors in HD. Studies onthe transgenic models of HD clearly confirmed that decline indopaminergic activity is accompanied by progressive disease[2,21]. Pretreatment of animals with medium and high dosesof nicotine significantly and dose-dependently attenuated 3-NP-induced striatal DA depletion (Fig. 1). The results ofimmunohistochemistry also demonstrated protective effect ofnicotine against 3-NP-induced reduction of tyrosine hydrox-ylase expression, which is a sensitive indicator of dopamin-ergic activity (Fig. 3). It is well established that activation ofpresynaptic nAChRs receptors results in an increased releaseo nce

9,15,28]. Treatment of rats with nicotine produced a hig f DA [29,43,46,65]. Nicotine has been shown to enha

166 M. Tariq et al. / Brain Research Bulletin 67 (2005) 161–168

DA bioavailability, which is accompanied by increased motoractivity [33,52,64]. Chronic treatment of nicotine signifi-cantly antagonized MPTP-induced depletion of DA withoutaltering the striatal levels of DA and DOPAC in control mice[18].

Furthermore, recent studies have shown that neurotrophicfactors play a critical role in neuronal survival followingexposure to neurotoxins or neurotrauma[17,68]. Nicotinestimulates the production and release of neurotrophic fac-tors, such as brain derived neurotrophic factor (BDNF),basic fibroblast growth factor (FGF-2) and nerve growthfactor (NGF) [47]. Treatment with nicotine has also beenshown to upregulate NGF receptor in a variety of neu-ronal cells[59] and to protect against apoptosis-induced byNGF deprivation[66]. Thus, nicotine may exert its neuro-protective effect at least in part by stimulating neurotrophicfactors.

There was a significant depletion of striatal GSH follow-ing exposure of animals to 3-NP (Fig. 2), clearly suggestingthe role of oxidative stress in this neurodegenerative process.Maksimovic et al.[31] also observed a significant reduc-tion in striatal GSH in quinolinic acid-induced model ofHD in rats. Nicotine significantly and dose-dependently pro-tected striatum against 3-NP-induced GSH depletion (Fig. 2).Recent studies clearly demonstrate that increased oxidativestress can be one of the major deleterious events in clin-i ase[ n topN adi-c tiono -p nerr r ofh ughb isa er-f inl na fe egen-e iveco ntt olisma no ebralb rain[

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Acknowledgements

The authors wish to thank Mr. Rajakanna Jesuraja, Mr.Khalid Abdalla Elfaki and Mrs. Biju Prasad for technicalassistance, as well as Miss Tess Jaime and Miss Audrey RoseGacutan for secretarial support and typing the manuscript.

References

[1] A. Antonini, K.L. Leenders, R. Spiegel, D. Meier, P. Vontobel, M.Weigell-Weber, R. Sanchez-Pernaute, J.G. de Yebenez, P. Boesiger,A. Weindl, R.P. Maguire, Striatal glucose metabolism and dopamineD2 receptor binding in asymptomatic gene carriers and patients withHuntington’s disease, Brain 119 (1996) 2085–2095.

[2] M.A. Ariano, N. Aronin, M. Difiglia, D.A. Tagle, D.R. Sibley, B.R.Leavitt, M.R. Hayden, M.S. Levine, Striatal neurochemical changesin transgenic models of Huntington’s disease, J. Neurosci. Res. 68(2002) 716–729.

[3] L. Backman, L. Farde, Dopamine and cognitive functioning: brain-imaging findings in Huntington’s disease and normal aging, Scand.J. Psychol. 42 (2001) 287–296.

[4] M.F. Beal, E. Brouillet, B.G. Jenkins, R.J. Ferrante, N.W. Kowall,J.M. Miller, E. Storey, R. Srivastava, B.R. Rosen, B.T. Hyman,Neurochemical and histologic characterization of striatal excitotoxiclesions produced by the mitochondrial toxin 3-nitropropionic acid,J. Neurosci. 13 (1993) 4181–4192.

[5] C.V. Borlongan, K. Kanning, S.G. Poulos, T.B. Freeman, D.W.Cahill, P.R. Sanberg, Free radical damage and oxidative stress in

.R.injec-of

.W.ioral995)

triatalort 4

toryical

[ tronion

[ ineNeu-

[ iatalsub-

[ onalto-

[ osenera-e, J.

[ ting-rsity

[ ne,and

cal [5] and experimentally induced Huntington’s dise31]. Antioxidants, on the other hand, have been showrotect nervous system against variety of toxins[35,48].icotine exerts its antioxidant effect due to its free ral chain breaking properties and/or preventing the initiaf free radical generation[19]. Nicotine can bind to comlex I of respiratory chain and inhibit the NADH-ubiquinoeductase activity and generation of superoxide (O•−) anionadical [10,11]. Nicotine can also act as a scavengeydrogen peroxide and block the fenton reaction throinding to Fe2+ [25]. Furthermore, neurotoxicity of 3-NPttributed to its ability to produce ischemic injury by int

ering with complex II of mitochondrial respiratory chaeading to depressed ATP levels[4,32]. Neurons in braire highly vulnerable to ischemia[22] and impairment onergy metabolism has been associated with neurodrative changes in brain[57]. Recently, neurodegenerathanges following exposure of rats to MPTP[14], imin-dipropionitrile [58] and harmaline[57] have been show

o accompany metabolic imbalance in brain[57,58]. Nico-ine has been shown to modulate cerebral energy metabnd conserve the energy balance[16]. Intravenous injectiof nicotine has been shown to increase regional cerlood flow (CBF) and restore energy supply in ischemic b

13,26].In conclusion, nicotine significantly and dose-depende

ttenuated 3-NP-induced striatal lesions and behaveficits in rats. The protective effect of nicotine mayttributed to its ability of restoring striatal DA levels inP intoxicated rats.

Huntington’s disease, J. FLA Med. Assoc. 83 (1996) 335–341.[6] C.V. Borlongan, T.K. Koutouzis, T.B. Freeman, D.W. Cahill, P

Sanberg, Behavioral pathology induced by repeated systemictions of 3-nitropropionic acid mimics the motoric symptomsHuntington’s disease, Brain Res. 697 (1995) 254–257.

[7] C.V. Borlongan, T.K. Koutouzis, T.S. Randall, T.B. Freeman, DCahill, P.R. Sanberg, Systemic 3-nitropropionic acid: behavdeficits and striatal damage in adult rats, Brain Res. Bull. 36 (1549–556.

[8] S.R. Bossi, J.R. Simpson, O. Isacson, Age dependence of sneuronal death caused by mitochondrial dysfunction, Neurorep(1993) 73–76.

[9] G.W. Bruyn, Huntington’s chorea: historical, clinical and laborasynopsis, in: P.J. Vinken, GW. Bruyn (Eds.), Handbook of ClinNeurology, Elsevier, Amsterdam, 1968, pp. 298–378.

10] A. Cormier, C. Morin, R. Zini, J.P. Tillement, G. Lagrue, In vieffects of nicotine on mitochondrial respiration and superoxide ageneration, Brain Res. 900 (2001) 72–79.

11] A. Cormier, C. Morin, R. Zini, J.P. Tillement, G. Lagrue, Nicotprotects rat brain mitochondria against experimental injuries,ropharmacology 44 (2003) 642–652.

12] G. Costa, J.A. Abin-Carriquiry, F. Dajas, Nicotine prevents strdopamine loss produced by 6-hydroxydopamine lesion in thestantia nigra, Brain Res. 888 (2001) 336–342.

13] G.J. Crystal, H.F. Downey, T.P. Adkins, F.A. Bashour, Regiblood flow in canine brain during nicotine infusion: effect of aunomic blocking drugs, Stroke 14 (1983) 941–947.

14] W. Duan, M.P. Mattson, Dietary restriction and 2-deoxyglucadministration improve behavioral outcome and reduce degetion of dopaminergic neurons in models of Parkinson’s diseasNeurosci. Res. 57 (1999) 195–206.

15] S.E. Folstein, The motor disorder, in: S.E. Folstein (Ed.), Hunton’s Disease: A Family of Disorder, The Johns Hopkins UnivePress, Baltimore, 1989, pp. 13–31.

16] H.M. Frankish, S. Dryden, Q. Wang, C. Bing, I.A. MacFarlaG. Williams, Nicotine administration reduces neuropeptide Y

M. Tariq et al. / Brain Research Bulletin 67 (2005) 161–168 167

neuropeptide Y mRNA concentrations in the rat hypothalamus: NPYmay mediate nicotine’s effect on energy balance, Brain Res. 694(1995) 139–146.

[17] S.J. French, T. Humby, C.H. Horner, M.V. Sofroniew, M. Rattray,Hippocampal neurotrophin and trk receptor mRNA level are alteredby local administration of nicotine, carbachol and pilocarpine, BrainRes. Mol. Brain Res. 67 (1999) 124–136.

[18] Z.G. Gao, W.Y. Cui, H.T. Zhang, C.G. Liu, Effects of nicotine on1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine-induced depression ofstriatal dopamine content and spontaneous locomotor activity in C57black mice, Pharmacol. Res. 38 (1998) 101–106.

[19] Z.Z. Guan, W.F. Yu, A. Nordberg, Dual effects of nicotine on oxida-tive stress and neuroprotection in PC12 cells, Neurochem. Int. 43(2003) 243–249.

[20] K.L. Haik, D.A. Shear, U. Schroeder, B.A. Sabel, G.L. Dunbar,Quinolinic acid released from polymeric brain implants causesbehavioral and neuroanatomical alterations in a rodent model ofHuntington’s disease, Exp. Neurol. 163 (2000) 430–439.

[21] M.A. Hickey, G.P. Reynolds, A.J. Morton, The role of dopamine inmotor symptoms in the R6/2 transgenic mouse model of Hunting-ton’s disease, J. Neurochem. 81 (2002) 46–59.

[22] F. Kagitani, S. Uchida, H. Hotta, A. Sato, Effects of nicotineon blood flow and delayed neuronal death following intermittenttransient ischemia in rat hippocampus, Jpn. J. Physiol. 50 (2000)585–595.

[23] S. Kaneko, T. Maeda, T. Kume, H. Kochiyama, A. Akaike, S. Shimo-hama, J. Kumura, Nicotine protects cultured cortical neurons againstglutamate-induced cytotoxicity via�7 neuronal receptors and neu-ronal CNS receptors, Brain Res. 765 (1997) 135–140.

[24] T.K. Koutouzis, C.V. Borlongan, T. Scorcia, I. Creese, D.W. Cahill,T.B. Freeman, P.R. Sanberg, Systemic 3-nitropropionic acid: long-

42–

[ .B.heirent60–

[ o-bral993)

[ ucedpal

[ dys-

[ pticRev.

[ ini,nico-. 104

[ .-tionthe

42.[ ate

ase106

[ oge-ced

1993)

[34] S. Nakamura, T. Takahashi, H. Yamashita, H. Kawakami, Nicotinicacetylcholine receptors and neurodegenerative disease, Alcohol 24(2001) 79–81.

[35] N. Nakao, E.M. Grasbon-Frodi, H. Widner, P. Brundin, Antioxidanttreatment protects striatal neurons against excitotoxic insults, Neu-roscience 73 (1996) 185–200.

[36] A. Nordberg, E. Hellstrom-Lindahl, M. Lee, M. Johnson, M.Mousavi, R. Hall, E. Perry, I. Bednar, J. Court, Chronic nicotinetreatment reduces beta-amyloidosis in the brain of a mouse modelof Alzheimer’s disease (APPsw), J. Neurochem. 81 (2002) 655–658.

[37] A.B. O’Neill, S.J. Morgan, J.D. Brioni, Histological and behavioralprotection by (−)-nicotine against quinolinic acid-induced neurode-generation in the hippocampus, Neurobiol. Learn Mem. 69 (1998)46–64.

[38] M.J. O’Neill, T.K. Murray, V. Lakics, N.P. Visanji, S. Duty, The roleof neuronal nicotinic acetylcholine receptors in acute and chronicneurodegeneration, Curr. Drug Targets CNS Neurol. Disord. 1 (2002)399–411.

[39] W.G. Owen, Determination of glutathione and glutathione disulfideusing glutathione reductase and 2-vinylpyridine, Anal. Biochem. 106(1980) 207–212.

[40] K. Parain, C. Hadey, E. Rousselet, V. Marchand, B. Dumery, EC.Hirsch, Cigarette smoke and nicotine protect dopaminergic neuronsagainst the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Parkinso-nian toxin, Brain Res. 984 (2003) 224–232.

[41] K. Parain, V. Marchand, B. Dumery, E. Hirsch, Nicotine, but not coti-nine, partially protects dopaminergic neurons against MPTP-induceddegeneration in mice, Brain Res. 890 (2001) 347–350.

[42] O.E. Patrick, M. Hirohisa, K. Masahira, M. Koreaki, Central nervoussystem bioaminergic responses to mechanic trauma, Surg. Neurol. 35(1991) 273–279.

[ gai,cedpus,

[ son’s

[ e to

[ ronicntral348

[ asan,ntu-251.

[ ster,ronaler’s

999)

[ nc-urg.

[ euro-atedbunit

[ ger,inerats,

[ icalon’s

[ un-d 3-

term effects on locomotor behavior, Brain Res. 646 (1994) 2246.

25] W. Linert, E. Herlinger, R.F. Jameson, E. Kienzl, K. Jellinger, MYoudim, Dopamine, 6-hydroxydopamine, iron, and dioxygen- tmutual interactions and possible implication in the developmof Parkinson’s disease, Biochim. Biophys. Acta 1316 (1996) 1168.

26] D.G. Linville, S. Williams, J.L. Raszkiewics, S.P. Arneric, Nictinic agonists modulate basal forebrain control of cortical cereblood flow in anesthetized rats, J. Pharmacol. Exp. Ther. 267 (1440–448.

27] Q. Liu, B. Zhao, Nicotine attenuates beta-amyloid peptide-indneurotoxicity, free radical and calcium accumulation in hippocamneuronal cultures, Br. J. Pharmacol. 141 (2004) 746–754.

28] E. Lugaresi, F. Cirignotta, F. Montagna, Noctural paroxysmaltonia, J. Neurol. Neurosurg. Psychiat. 49 (1986) 375–380.

29] A.B. MacDermott, L.W. Role, S.A. Siegelbaum, Presynaionotropic receptors and the control of transmitter release, Ann.Neurosci. 22 (1999) 443–485.

30] R. Maggio, M. Riva, F. Vaglini, F. Fornai, G. Racagni, G.U. CorsStriatal increase of neurotrophic factors as a mechanism oftine protection in experimental Parkinsonism, J. Neural. Trans(1997) 1113–1123.

31] I.D. Maksimovic, M.D. Jovanovic, M. Colic, R. Mihajlovic, DMicic, V. Selakovic, M. Ninkovic, Z. Malicevic, M. RusicStojiljkovic, A. Jovicic, Oxiative damage and metabolic dysfuncin experimental Huntington’s disease: selective vulnerability ofstriatum and hippocampus, Vojnosanit. Pregl. 58 (2001) 237–2

32] L. Massieu, P. Del Rio, T. Montiel, Neurotoxicity of glutamuptake inhibition in vivo: correction with succinate dehydrogenactivity and prevention by energy substrates, Neuroscience(2001) 669–677.

33] S. Nakamura, Y. Goshima, J.L. Yue, T. Miyamae, Y. Misu, Endnously released DOPA is probably relevant to nicotine-induincreases in locomotor activities of rats, Jpn. J. Pharmacol. 62 (107–110.

43] R. Pawlak, Y. Takada, H. Takahashi, T. Urano, H. Ihara, N. NaA. Takada, Differential effects of nicotine against stress-induchanges in dopaminergic system in rat striatum and hippocamEur. J. Pharmacol. 387 (2000) 171–177.

44] M. Quik, G. Jeyarasasingam, Nicotinic receptors and Parkindisease, Eur. J. Pharmacol. 393 (2000) 223–230.

45] M. Quik, J.M. Kulak, Nicotine and niconitic receptors; relevancParkinson’s disease, Neurotoxicology 23 (2002) 581–594.

46] S. Rahman, J. Zhang, W.A. Corrigal, Effects of acute and chnicotine on somatodendritic dopamine release of the rat vetegmental area: in vivo microdialysis study, Neurosci. Lett.(2003) 61–64.

47] R. Ravikumar, I. Fugaccia, S.W. Scheff, J.W. Geddes, C. SrinivM. Toborek, Nicotine attenuates morphological deficits in a cosion model of spinal cord injury, J. Neurotrauma 22 (2005) 240–

48] R.J. Reiter, J. Cabrera, R.M. Sainz, J.C. Mayo, L.C. MancheD.X. Tan, Melatonin as a pharmacological agent against neuloss in experimental models of Huntington’s disease, Alzheimdisease and Parkinsonism, Ann. N. Y. Acad. Sci. 890 (1471–485.

49] A.S. Rivlin, C.H. Tator, Objective clinical assessment of motor fution after experimental spinal cord injury in the rat, J. Neuros47 (1977) 577–581.

50] R.E. Ryan, S.A. Ross, J. Drago, R.E. Loiacono, Dose-related nprotective effects of chronic nicotine in 6-hydroxydopamine trerats, and loss of neuroprotection in alpha4 nicotinic receptor suknockout mice, Br. J. Pharmacol. 132 (2001) 1650–1656.

51] S. Sarre, H. Yuan, N. Jonkers, A. Van Hemelrijck, G. EbinY. Michotte, In vivo characterization of somatodendritic dopamrelease in the substantia nigra of 6-hydroxydopamine lesionedJ. Neurochem. 90 (2004) 29–39.

52] H. Sershen, A. Hashim, A. Lajtha, Behavioral and biochemeffects of nicotine in an MPTP-induced mouse model of Parkinsdisease, Pharmacol. Biochem. Behav. 28 (1987) 299–303.

53] D.A. Shear, J. Dong, C.D. Gundy, K.L. Haik-Creguer, G.L. Dbar, Comparison of intrastriatal injections of quinolinic acid an

168 M. Tariq et al. / Brain Research Bulletin 67 (2005) 161–168

nitropropionic acid for use in animal models of Huntington’s disease,Prog. Neuropsychopharmacol. Biol. Psychiat. 22 (1998) 1217–1240.

[54] Y. Shimano, M. Kumazaki, T. Sakurai, H. Hida, I. Fujimoto, A.Fukuda, H. Nishino, Chronically administered 3-nitropropionic acidproduces selective lesions in the striatum and reduces muscle tones,Obes. Res. 3 (Suppl. 5) (1995) 779S–784S.

[55] S. Shimohama, A. Akaike, J. Kimura, Nicotine-induced protec-tion against glutamate cytotoxicity: nicotinic cholinergic receptor-mediated inhibition of nitric oxide formation, Ann. N.Y. Acad. Sci.777 (1996) 356–361.

[56] X. Sun, Y. Liu, G. Hu, H. Wang, Protective effects of nicotine againstglutamate-induced neurotoxicity in PC12 cells, Cell. Mol. Biol. Lett.9 (2004) 409–422.

[57] M. Tariq, M. Arshaduddin, N. Biary, K. Al Moutaery, S. Al Deeb, 2-Deoxy-d-glucose attenuates harmaline induced tremors in rats, BrainRes. 945 (2002) 212–218.

[58] M. Tariq, H.A. Khan, K. Al Moutaery, S. Al Deeb, Protection by2-deoxy-d-glucose against beta,beta′-iminodiopropionitrile-inducedneurobehavioral toxicity in mice, Exp. Neurol. 158 (1999) 229–233.

[59] A.V. Terry Jr., M.S. Clarke, Nicotine stimulation of nerve growthfactor receptor expression, Life Sci. 55 (1994) PL91–PL98.

[60] Y. Tizabi, M. Al-Namaeh, K.F. Manaye, R.E. Taylor, Protectiveeffects of nicotine on ethanol-induced toxicity in cultured cerebellargranule cells, Neurotox. Res. 5 (2003) 315–321.

[61] T. Utsumi, K. Shimoke, S. Kishi, H. Sasaya, T. Ikeuchi, H.Nakayama, Protective effect of nicotine on tunicamycin-induced

apoptosis of PC12h cells, Neurosci. Lett. 370 (2004) 244–247.

[62] G. Uzum, N. Bahcekapili, A.S. Diler, Y.Z. Ziylan, Tolerance topentylentetrazol-induced convulsions and protection of cerebrovas-cular integrity by chronic nicotine, Int. J. Neurosci. 114 (2004)735–748.

[63] J.C. Vis, M.M. Verbeek, R.M.W. de Waal, H.J. Ten Donkelaar, H.P.H.Kremer, 3-Nitropropionic acid induces a spectrum of Huntington’sdisease-like neuropathology in rat striatum, Neuropathol. Appl. Neu-robiol. 25 (1999) 513–521.

[64] P. Whiteaker, H.S. Garcha, S. Wonnacott, I.P. Stolerman, Locomotoractivation and dopamine release produced by nicotine and isoare-colone in rats, Br. J. Pharmacol. 116 (1995) 2097–2105.

[65] S. Wonnacott, Presynaptic nicotinic ACh receptors, Trends Neurosci.20 (1997) 92–98.

[66] H. Yamashita, S. Nakamura, Nicotine rescues PC12 cells from deathinduced by nerve growth factor deprivation, Neurosci. Lett. 213(1996) 145–147.

[67] G.J. Yohrling 4th, G.C. Jiang, M.M. DeJohn, D.W. Miller, A.B.Young, K.E. Vrana, J.H. Cha, Analysis of cellular, transgenic andhuman models of Huntington’s disease reveals tyrosine hydroxylasealterations and substantia nigra neuropathy, Brain Res. Mol. BrainRes. 1119 (2003) 28–36.

[68] Q. Yuan, W. Wu, K.F. So, A.L. Cheung, D.M. Prevette, R.W. Oppen-heim, Effects of neurotrophic factors on motoneuron survival follow-ing axonal injury in newborn rats, Neuroreport 11 (2000) 2237–2241.