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Brain Research Bulletin 102 (2014) 57–68

Contents lists available at ScienceDirect

Brain Research Bulletin

j ourna l h o mepa ge: www.elsev ier .com/ locate /bra inresbul l

esearch report

harmacological benefit of I1-imidazoline receptors activation anduclear factor kappa-B (NF-�B) modulation in experimentaluntington’s disease

urbhi Guptaa,1, Bhupesh Sharmab,c,∗

Neuropharmacology Lab., Department of Pharmacology, School of Pharmacy, Bharat Institute of Technology, Partapur Bypass, Meerut, Uttar Pradesh, IndiaDepartment of Pharmacology, School of Pharmacy, Bharat Institute of Technology, Partapur Bypass, Meerut, Uttar Pradesh, IndiaCNS Pharmacology, Conscience Research, Pocket F-233, B, Dilshad Garden, Delhi 110095, India

r t i c l e i n f o

rticle history:eceived 28 December 2013eceived in revised form 8 February 2014ccepted 12 February 2014vailable online 28 February 2014

eywords:-Nitropropionic acidoxonidineatrium diethyl dithio carbamate

rihydrateetrabenazineitochondrial enzyme complex

a b s t r a c t

Huntington’s disease (HD), a neurodegenerative disorder, is characterized by progressive motor dysfunc-tion, emotional disturbances, dementia, weight loss and anxiety.

The tremendous amount of research work is required to identify new pharmacological agents of ther-apeutic utility to combat this condition. This study investigates the effect of selective modulator ofI1-imidazoline receptor (moxonidine) as well as nuclear factor kappa-B (NF-�B) (natrium diethyl dithiocarbamate trihydrate-NDDCT) on 3-nitropropionic acid (3-NPA) induced experimental HD condition.3-NPA was used to induce mitochondrial damage and associated HD symptoms in rats. Anxiety wasassessed using Elevated plus maze-EPM and learning-memory was assessed using EPM and Morris watermaze-MWM. Different biochemical estimations were used to assess brain striatum oxidative stress (lipidperoxide, superoxide dismutase and catalase), nitric oxide levels (nitrite/nitrate), cholinergic activity(brain striatum acetyl cholinesterase activity), and mitochondrial enzyme complex (I, II and IV) activi-ties. 3-NPA has induced anxiety, impaired learning-memory with a reduction in body weight, locomotoractivity, grip strength. It has increased brain striatum acetylcholinesterase-AChE activity, oxidative stress(lipid peroxide, nitrite/nitrate, superoxide dismutase and catalase) and impaired mitochondrial complexenzyme (I, II and IV) activities. Tetrabenazine-TBZ (monoamine storage inhibitor) was used as positive

control. Treatment with moxonidine, NDDCT and TBZ significantly attenuated 3-NPA induced reductionin body weight, locomotor activity, grip strength, anxiety as well as impaired learning and memory.Administration of these agents attenuated 3-NPA induced various biochemical impairments. Therefore,modulation of I1-imidazoline receptor as well as NF-�B may be considered as potential pharmacologicalagents for the management of 3-NPA induced HD.

© 2014 Elsevier Inc. All rights reserved.

Abbreviations: 3-NPA, 3-nitropropionic acid; 5-HT, 5-hydroxytryptamine; ACh, acetylcholine; AChE, acetylcholinesterase; BDNF, brain derived neurotrophic factor; BSA,ovine serum albumin; BW, body weight; CAG, cytosine adenine guanine; cAMP, cyclic adenosine monoamine phosphate; CMC, carboxymethyl cellulose; CPCSEA, Committeeor the Purpose of Control and Supervision of Experiments on Animals; DTNB, 5,5′-dithiobis (2-nitrobenzoic acid); EDTA, ethylene diamine tetra acetic acid; EGTA, ethylenelycol tetra acetic acid; ELT, escape latency time; EPM, elevated plus maze; GABA, gamma amino butyric acid; GSK-3, glycogen synthase kinase 3; HD, Huntington’s disease;EPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IAEC, Institutional Animal Ethics Committee; LTP, long-term potentiation; MAPK, mitogen-activated protein

inase; MWM, morris water maze; NADH, nicotinamide adenine dinucleotide; NBT, nitrazobluetetrazolium; NDDCT, Natrium diethyl dithio carbamate trihydrate; NF-�B,uclear factor kappa-B; PKA, protein kinase A; ROS, reactive oxygen species; SDH, succinate dehydrogenase; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactiveubstances; TBZ, tetrabenazine; TL, TRansfer latency; TSOA, total time spent in open arm; TSTQ, total time spent in target quadrant; VMAT2, type-2 vesicular monoamineransporter.∗ Corresponding author at: Department of Pharmacology, School of Pharmacy, Bharat Institute of Technology, Partapur Bypass, Meerut-250103, Uttar Pradesh, India;el.: +91 879 1636281/995 8219190.

E-mail addresses: sharmaslab3@gmail.com (S. Gupta), drbhupeshresearch@gmail.com, conscienceresearch@scientist.com (B. Sharma).1 Tel.: +91 992 7932746.

ttp://dx.doi.org/10.1016/j.brainresbull.2014.02.007361-9230/© 2014 Elsevier Inc. All rights reserved.

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. Introduction

Huntington’s disease (HD), a neurodegenerative disorder withn autosomal dominant expression pattern is caused by the expan-ion of a CAG (cytosine adenine guanine) repeat in the huntingtinene. Expanded polyglutamine facilitates formation of huntingtinrotein aggregates, eventually leading to deposition of cytoplasmicnd intranuclear inclusion bodies containing huntingtin protein. Its a movement disorder with a heterogeneous phenotype charac-erized by involuntary dance-like gait, motor impairment, anxiety,ognitive, psychiatric deficits and bio energetic deficits (Bhatejat al., 2012).

3-Nitropropionic acid (3-NPA) is an irreversible inhibitor ofitochondrial complex II (Brouillet et al., 1993) that inhibits the

ctivity of succinate dehydrogenase, a key enzyme of oxidativenergy production and characteristically provokes neurodegener-tion in the striatum, resembling HD symptoms. 3-NPA-inducedeurodegeneration has been widely used as an HD animal modelecause of its symptom similarity with HD (Bhateja et al., 2012).

The imidazoline receptors are available throughout the bodynd they have been reported to possess various functions. Imida-oline receptors are categorized into three main classes, I1, I2 and3-imidazoline receptor (Nechifor and Ciubotariu, 2012). I1 recep-or binding sites are located in the rostro-ventrolateral medulland also in the hypothalamus region of the brain (Raasch et al.,000). These receptors are also involved in the regulation of behav-

oral disorders such as suicidal behavior, stress, anxiety and foodntake (Nechifor and Ciubotariu, 2012) as well as in the regulationf vasomotor tone, body fat, neuroprotection (Gupta and Sharma,014), inflammation, depression and stress (Nikolic and Agbaba,012). Modulators of I1-imidazoline receptors have been reportedo exhibit a protective effect on memory (Gupta and Sharma,014) and thinking and improvement in word recall (Ostroumovat al., 2001; Wesnes et al., 1997). I1-imidazoline receptors maye involved in the brainstem control of the cholinergic outflowHaxhiu et al., 1998). I1-imidazoline receptor has been reported toeduce the cyclic adenosine monoamine phosphate (cAMP) levelsy inhibiting adenylyl cyclase. Reduced levels of cAMP and involve-ent in cholinergic outflow have been reported to show beneficial

ffects in Huntington’s disease (Haxhiu et al., 1998; Williams et al.,008). We hypothesized that I1-imidazoline receptor modulationay provide benefits in learning-memory, anxiety, cholinergic out-

ow and neuroprotection in HD. I1-imidazoline class of receptorss less explored and not much known about them in HD condition.hus, research is required to identify their utility in HD.

The nuclear factor kappa-B (NF-�B), a family of transcriptionactors, regulates the induction and resolution of inflammationAnthony et al., 2009). In neuron cell bodies, NF-�B is constitutivelyctive and involved in neuronal injury (Yakovleva et al., 2011).F-�B, a crucial regulator of dentate gyrus tissue homeostasis, sug-ests NF-�B to be a therapeutic target for treating cognitive andood disorders (Imielski et al., 2012). NF-�B is activated under

hysiological and pathological conditions including learning andemory mechanisms and neurodegenerative diseases (Napolitano

t al., 2008). In the nervous system, NF-�B is activated in neurons inesponse to excitotoxic, metabolism and oxidative stress. NF-�B isn important regulator involved in inflammatory responses, as wells in cell survival and apoptosis. It has been reported that 3-NPA-nduced activation of NF-�B in striatal treated slices (Napolitanot al., 2008). In the present study we hypothesized that inhibition ofF-�B may exert a protective effect on cognitive deficits, oxidative

tress, excitotoxicity, apoptosis and neurodegeneration. Thus, NF-

B inhibitors may deserve investigations for their potential in HD.

Tetrabenazine (TBZ), an approved drug by USFDA (Chen et al.,012), has been used for the management of various movement dis-rders including Huntington chorea (Gros and Schuldiner, 2010).

h Bulletin 102 (2014) 57–68

It is reported to offer symptomatic relief without disease mod-ifying therapy. TBZ selectively depletes central monoamines byreversibly binding to the type-2 vesicular monoamine transporter(VMAT2) (Frank, 2009), more selectively depletes dopamine thannorepinephrine. The highest binding density for TBZ is in the cau-date nucleus, putamen, and nucleus accumbens, areas known tobear the brunt of pathology in HD (Frank, 2009). We have used TBZas positive control in the present study.

In the light of above, the present study has been undertaken toinvestigate the potential of moxonidine (I1-imidazoline receptoragonist) and natrium diethyl dithio carbamate trihydrate-NDDCT(NF-�B antagonist) in 3-NPA induced experimental HD condition.

2. Material and methods

2.1. Animals

In the present study, albino Wistar rats were used which arewidely used for the induction of HD symptoms by 3-nitropropionicacid (Shivasharan et al., 2013). Adult albino Wistar rats (3–5 monthsold), of either sex, weighing 200–250 g (purchased from IndianVeterinary Research Institute, Izatnagar, India), were employedin the present study and were housed in animal house with freeaccess to water and standard laboratory pellet chow diet (KisanFeeds Ltd., Mumbai, India). The animals were exposed to naturallight (sunlight) and dark cycle. The experiments were conductedbetween 9.00 and 18.00 h in a semi-sound-proof laboratory. Theanimals were acclimatized to the laboratory condition five daysprior to behavioral study and were maintained in the laboratoryuntil the completion of the study. The protocol of the study wasduly approved by the Institutional Animal Ethics Committee (IAEC)and the care of the animals was taken as per the guidelines of theCommittee for the Purpose of Control and Supervision of Experi-ments on Animals (CPCSEA), Ministry of Environment and Forests,Government of India (Reg. No. 25/230/2011/AWD/CPCSEA).

2.2. Drugs and chemicals

Moxonidine was obtained from Unichem Laboratories, India.Tetrabenazine was obtained from Sun Pharma Pvt. Ltd., India.3-Nitropropionic acid (3-NPA), natrium diethyl dithio carbamatetrihydrate (NDDCT), Lowry’s reagent, 5,5′-dithiobis (2-nitrobenzoicacid) (DTNB), Folin-Ciocalteu reagent, bovine serum albumin(BSA) and N-naphthylethylenediamine were purchased from SigmaAldrich, USA. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES), ethylene glycol tetra acetic acid (EGTA), mannitol, glycylglycine buffer, nicotinamide adenine dinucleotide (NADH), nitra-zobluetetrazolium (NBT) were purchased from SISCO ResearchLaboratory Pvt. Ltd., Mumbai, India.

3. Experimental

3.1. 3-Nitropropionic acid (3-NPA) experimental model

3-NPA was dissolved in 0.9% saline solution and was admin-istered to rats alternatively for 28 days at a dose of 10 mg kg−1

through intraperitoneal route (Gopinath and Sudhandiran, 2012;Pandey et al., 2008). Weight, locomotor activity and grip strengthwere measured before the initiation of 3-NPA treatment (day 1)and at the end of the study (day 28).

3.2. Drugs administration

All drug solutions were freshly prepared before use. Selec-tion of doses and the dosing schedule were based on previously

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ublished reports from other labs. It has been reported that sys-emic administration of 3-NPA (10 mg kg−1 for 4 days) causedignificant body weight reduction, impaired motor function (loco-otor activity, movement pattern) and striatal lesions mimicking

ymptoms of HD (Pandey et al., 2008). After 4 doses 3-NPA exhib-ted its effect on behavioral and biochemical parameters. Hence,

e selected the administration of treatment drugs from the 10thay onwards after 4 doses of 3-NPA in rats. All the drug treatmentsere started from the 10th day onwards (from the 10th day to 28thay) daily. Saline; vehicle of 3-NPA was administered to rats fromst day to 28th day (on alternate days) and carboxymethylcellu-

ose (CMC), vehicle of treatment drugs was administered to ratsrom the 10th day to 28th day; daily till the end of the study i.e. forotal 19 days.

3-NPA and NDDCT were dissolved in saline. Moxonidine andetrabenazine were suspended in 0.5% CMC. Moxonidine (0.03 and.06 mg kg−1 orally) (Gupta and Sharma, 2014), NDDCT (5 and0 mg kg−1 intraperitoneally) (Sharma and Singh, 2012b) and tetra-enazine (3 mg kg−1 orally) (Meyer et al., 2011) were administeredo rats once a day for 19 days using oral canula. Moxonidine (0.03nd 0.06 mg kg−1), NDDCT (5 and 10 mg kg−1) and TBZ (3 mg kg−1)er se were administered orally to rats for 19 days once daily, asxact drug administration to 3-NPA treated animals was also for 19ays, i.e. starting from 10th day of 3-NPA treatment till the end ofhe study (day 28).

All animals of 3-NPA and 3-NPA + drug treatments were exposedo behavioral test on the 21st day to 28th day, whereas, vehicles andrug per se treated animals were exposed to behavioral test on the1th day to 18th day. After behavioral assessments, biochemicalstimations were also performed on the same animals.

.3. Body weight

HD patients gradually suffer from weight loss and it has beeneported that 3-NPA used for inducing HD symptoms, alters bodyeight of animals (Colle et al., 2012). Body weight was recorded onay 1 and day 28 to assess percent change in body weight.

ercent change in BW =[

BW 1st day − BW 28th dayBW 1st day

]× 100

here BW is body weight.

.4. Behavioral assessment

.4.1. Assessment of locomotor activityGait impairments or perturbed locomotion is the characteris-

ic feature of HD. It has already been reported that 3-NPA causeseduction in locomotor activity (Colle et al., 2012). ActophotometerINCO, Ambala, India) was used to assess locomotor activity. Thepparatus was placed in a darkened, sound attenuated and ven-ilated testing room during assessment. All animals were placedndividually in the activity cage for 3 min for making them habitualefore starting actual locomotor activity task for the next 5 min.ounts of basal activity of the animals were noted. Total activ-

ty including horizontal and vertical was expressed as counts per min. Counts/5 min is used as an index of locomotor activity. Allhe trials of locomotor activity were completed between 09.00 and8.00 h on 1st day and 28th day (Kumar et al., 2011).

.4.2. Rota rod testIn HD patients, involuntary choreatic movements are one of the

allmarks of motor dysfunction and it has already been reported

hat 3-NPA causes reduction in motor coordination (Bhateja et al.,012). Rota rod experiments (Rota rod, Inco, India) were used toeasure forelimb and hind limb motor coordination (Kumar et al.,

011). Animals were placed individually on the rotating rod with

h Bulletin 102 (2014) 57–68 59

a diameter of 7 cm (speed 25 rpm). Rats were trained to use theRota rod apparatus during a 2 min trial (25 rpm) on the day beforethe first day of testing. The cut off time of 180 s was fixed and eachrat performed three separate trials at 5 min interval. Each trial wasseparated by a 5 min rest period to alleviate stress and fatigue. Allthe trials were completed between 09.00 and 18.00 h on 1st dayand 28th day. Falling time latency for each rat was recorded bya trained observer blind to the experimental protocol (Yu-Taegeret al., 2012).

3.4.3. Assessment of anxiety as well as learning and memoryusing elevated plus maze (EPM)

It has been reported earlier that EPM may be utilized forassessment of learning and memory, using transfer latency (TL)as parameter for acquisition and retention of memory process onEPM for rats and mice (Haider et al., 2012; Sharma and Kulkarni,1992). The prolongation of the TL on retention testing in the EPMmethod has been considered as an indicator for impairment oflearning and memory (Itoh et al., 1991). Furthermore, Frussa-Filhoet al. (1991) have used EPM where memory was quantified bytransfer latency (the time taken by the rat to move from the openarm to the enclosed arm) and anxiety was assessed by percententries into the open arms. Briefly, the apparatus consists of twoopposing open arms (50 cm × 10 cm) perpendicular to two enclosedarms (50 cm × 10 cm × 50 cm) that extend from a central platform(10 cm × 10 cm), elevated 65 cm above the floor. The maze wasplaced in the same position throughout the test in the laboratorywhere extra maze cues were there to facilitate learning. The proce-dure and technique were same as reported earlier by Haider et al.(2012). The test comprised of three days protocol, first day was atraining session while the next two days were considered as testsessions. In the training session each rat was placed in the cen-tral square and allowed to explore the EPM for 10 min and thenreturned to the home cages. During test sessions cut off time was5 min and time spent in open arm was recorded. A significantdecrease in time spent in open arm on subsequent EPM exposurewas taken as an index of successful memory retention. This is basedon the idea that during repeated testing on EPM rat acquires infor-mation about the spatial environment and avoids the elevated andopen arms of the maze and prefers to stay in the closed arms whereit could be safe on the maze. Total time spent in the open arm mea-sured on the first day served as an index of learning and acquisitionas well as anxiety, whereas on the 2nd day it served as an index ofretention of learning task (memory) and on the 3rd day furtherserved as the index of consolidation of memory. Memory was mea-sured by the degree to which the rat remembers and avoids theelevated and unenclosed arms of the maze and prefers to stay inthe closed arms.

3.4.4. Assessment of learning and memory by Morris water maze(MWM)

HD patients and mouse models show learning and memoryimpairment even before the onset of motor symptoms. It has beensuggested that 3-NPA administration significantly impaired learn-ing and memory (Bhateja et al., 2012). MWM is one of the mostcommonly used animal models to test memory. MWM consistedof large circular pool (150 cm in diameter, 45 cm in height, filledto a depth of 30 cm with water at 28 ◦C). The water was madeopaque with white colored dye. The tank was divided into fourequal quadrants with the help of two threads, fixed at right angleto each other on the rim of the pool. A submerged platform (10 cm2),painted white was placed inside the target quadrants of this pool,

1 cm below surface of water. The position of the platform waskept unaltered throughout the training session. Each animal wassubjected to four consecutive trials on each day with a gap of5 min. The rat was gently placed in the water of the pool between

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uadrants, facing the wall of pool with the drop location changingor each trial, and allowed 120 s to locate a submerged platform.hen, it was allowed to stay on the platform for 20 s. If it failedo find the platform within 120 s, it was guided gently onto plat-orm and allowed to remain there for 20 s. Day 4 ELT to locate theidden platform in water maze was noted as an index of acquisi-ion or learning. Daily starting positions were randomized and notepeated on each day and quadrant 4 (Q4) was maintained as tar-et quadrant in all acquisition trials. On the fifth day, the platformas removed and rats were allowed to explore the pool for 120 s.

ach rat was subjected to four such trials and each trial was startedrom different quadrant. Mean time spent in all four quadrants, i.e.1, Q2, Q3 and Q4 were recorded and the time spent in the tar-et quadrant, i.e. Q4 in search of the missing platform provided anndex of retrieval. The experimenter was always standing in theame position. Care was taken that relative location of water mazeith respect to other objects in the laboratory serving, as prominent

isual clues were not disturbed during the total duration of study.ll the trials were completed during the light cycle, i.e. between9.00 and 18.00 hrs (Gupta and Sharma, 2014; Sharma and Singh,012a).

.5. Dissection and homogenization

After the estimation of behavioral parameters, the animals wereacrificed by decapitation. Brain striatum of each animal was iso-ated by putting on ice and weighed individually. A 10% (w/v) tissueomogenate was prepared in 0.1 M phosphate buffer (pH 7.4).he homogenate was centrifuged at 10,000 × g at 4 ◦C for 15 min.liquots of supernatants were separated and used for biochemicalstimations (Gupta and Sharma, 2014; Sharma and Singh, 2013).

.6. Biochemical estimations

The molecular mechanisms mediating neuronal death in HDnvolve oxidative stress and nitrosative stress as one of the impor-ant aspect. Oxidative and nitrosative stress has also been reportedn 3-NPA induced neurotoxicity (Bhateja et al., 2012).

.6.1. Assessment of striatum lipid peroxidationStriatum thiobarbituric acid reactive substances (TBARS) level

as measured spectrophotometrically (UV-1800 ENG 240V; Shi-adzu Coorporation, Japan) at 532 nm (Gupta and Sharma, 2014;

harma and Singh, 2011, 2013). The quantitative measurement ofBARS, an index of lipid peroxidation in striatum was performed..2 ml of supernatant of the homogenate was pipetted out in a testube, followed by the addition of 0.2 ml of 8.1% sodium dodecylulphate, 1.5 ml of 30% acetic acid (pH 3.5), 1.5 ml of 0.8% of thio-arbituric acid and the volume was made up to 4 ml with distilledater. The test tubes were incubated for 1 h at 95 ◦C, then cooled

nd added 1 ml of distilled water followed by the addition of 5 ml of-butanol–pyridine mixture (15:1, v/v). The tubes were centrifugedt 4000 × g for 10 min. The absorbance of developing pink color waseasured spectrophotometrically at 532 nm. A standard calibra-

ion curve was prepared using 1–10 nM of 1,1,3,3-tetra methoxyropane. The TBARS value was expressed as nanomoles per mg ofrotein.

.6.2. Assessment of striatum nitrite/nitrate levelStriatum nitrite concentration was measured spectophotomet-

ically (UV-1800 ENG 240V; Shimadzu Coorporation, Japan) at45 nm (Sharma et al., 2008; Sharma and Singh, 2012a). Briefly,

00 �l of carbonate buffer (pH 9.0) was added to 100 �l of stri-tal or standard sample followed by addition of small amounts0.15 g) of copper-cadmium alloy. The tubes were incubated atoom temperature for 1 h to reduce nitrate to nitrite. The reaction

h Bulletin 102 (2014) 57–68

was stopped by adding 100 �l of 0.35 M sodium hydroxide. Fol-lowing this, 400 �l of zinc sulphate solution (120 mM) was addedto deproteinate the samples. The samples were allowed to stand for10 min and then centrifuged at 4000 × g for 10 min. Greiss reagent(250 �l of 1.0% sulfanilamide prepared in 3 N HCl and 250 �l of 0.1%N-naphthylethylenediamine (prepared with water) was added toaliquots (500 �l) of clear supernatant and striatum nitrite wasmeasured spectophotometrically at 545 nm. The standard curve ofsodium nitrite (5–50 �M) was plotted to calculate the concentra-tion of brain striatum nitrite.

3.6.3. Assessment of striatum catalase (CAT) activityThe activity of striatum CAT was determined spectrophotome-

terically (UV-1800 ENG 240V; Shimadzu Coorporation, Japan) at240 nm by the method of Aebi (1984). Briefly, 1 ml of the brainhomogenate was taken in a test tube and 1.9 ml of phosphate buffer(50 mM, pH 7.4) was added to it. The reaction was initiated by theaddition of 1 ml of 30 mM H2O2. A mixture of 2.9 ml of phosphatebuffer and 1 ml of H2O2 without the brain homogenate served asthe blank. The decrease in absorbance due to the decomposition ofH2O2 was recorded at 240 nm against the blank. Units of CAT wereexpressed as the amount of enzyme that decomposes 1 �M of H2O2per min at 25 ◦C and the activity was expressed in terms of unitsper milligram of proteins.

3.6.4. Assessment of striatum superoxide dismutase (SOD)activity

Striatum SOD was assayed spectrophotometerically (UV-1800ENG 240V; Shimadzu Coorporation, Japan) as described byBeauchamp and Fridovich (1971). It was based on the reduc-tion of NBT to the water insoluble blue formation. The assaymixture contained 0.5 ml of brain homogenate, 1 ml of 50 mMsodium carbonate, 0.4 ml of 24 �m NBT, and 0.2 ml of 0.1 mM EDTA.The reaction was initiated by adding 0.4 ml of 1 mM hydroxy-lamine hydrochloride. The developed blue color in the reaction wasmeasured at 560 nm. Zero time absorbance was taken at 560 nm fol-lowed by recording the absorbance reading every 30 s for a periodof 5 min at 25 ◦C. The above-mentioned reaction mixtures with-out the brain homogenate served as control. The rate of increasein absorbance units (A) per minute for the control and for the testsample(s) was determined and the percentage inhibition for thetest sample(s) was calculated by the following formula:

% inhibition ={

(�A560 nM/ min)control − (�A560 nM/ min)test

(�A560 nM/ min)control

}

× 100

where (A560 nM at 5 min and 30 s – A560 nM at 30 s)/5 min = �A560nM/minute.

Units of the SOD activity were expressed as the amount ofenzyme required to inhibit the reduction of NBT by 50% and theactivity was expressed as units per mg of protein.

3.6.5. Assessment of striatum acetylcholinesterase (AChE) activityThe neuropathology of HD is characterized by progressive loss

of projection neurons in cortex and striatum; striatal choliner-gic inter neurons are relatively spared. Cholinergic projectionsplay important roles in hippocampal-dependent cognition. It hasbeen documented that cholinergic interneurons are capable ofincreased ACh release, whereas, reduced levels of extracellularstriatal ACh in HD may reflect abnormalities in the excitatory inner-

vation of cholinergic interneurons, which may have implicationsACh-dependent processes that are altered in HD, including cor-ticostriatal plasticity (Farrar et al., 2011). 3-NPA has also beenreported to cause alteration in AChE activity (Bhateja et al., 2012).

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Striatum AChE activity was measured spectrophotometericallyUV-1800 ENG 240V; Shimadzu Coorporation, Japan) at 420 nmGupta and Sharma, 2014; Sharma et al., 2008; Sharma and Singh,011). Briefly, this was measured in basis of the formation of yellowolor due to the reaction of thiocholine with dithiobisnitrobenzoateons. The rate of formation of thiocholine from acetylthiocholineodide in the presence of brain cholinesterase was measured using

spectrophotometer. 0.5 ml of clear supernatant liquid of the brainomogenate was pipetted out into 25 ml volumetric flask andilution was made with a freshly prepared DTNB [5,5′-dithiobis-2-nitro benzoic acid)] solution (10 mg DTNB in 100 ml of Sorensonhosphate buffer, pH 8.0). From the volumetric flask, two 4 ml por-ions were pipetted out into two test tubes. Into one of the testube, 2 drops of eserine solution were added. 1 ml of substrate solu-ion (75 mg of acetylcholine iodide per 50 ml of distilled water)as pipetted out into both of the test tubes. The test tube con-

aining eserine was taken as blank and the change in absorbanceer minute of the test sample was read spectrophotometerically at20 nm. AChE activity was calculated using the following formula:

= ıO.D. × volume of Assay E × mg of protein

here R = rate of enzyme activity in ‘n’ mole of acetylcholine iodideydrolyzed/minute/mg protein

O.D. = change in absorbance/minute

= extinction coefficient = 13,600 M–1/cm.

.6.6. Assessment of striatum total proteinStriatum total protein was determined spectrophotometrically

UV-1800 ENG 240V; Shimadzu Coorporation, Japan) at 750 nmccording to the method described by Sharma and Singh (2013).he brain total protein was determined by using BSA as a standard..15 ml of supernatant of tissue homogenate was diluted to 1 mlhen 5 ml of Lowry’s reagent was added. The contents were mixedhoroughly and the mixture was allowed to stand for 15 min atoom temperature. Then 0.5 ml of Folin-Ciocalteu reagent wasdded and the contents were vortexed vigorously and incu-ated at room temperature for 30 min. The standard curve waslotted using 0.2–2.4 mg/ml of BSA. The protein content was deter-ined spectrophotometrically at 750 nm. Protein concentrationas expressed as mg/ml of supernatant (Gupta and Sharma, 2014;

harma and Singh, 2013).

.7. Isolation of rat brain striatum mitochondria anditochondrial complex estimation

Mitochondrial dysfunction has been implicated in HD patho-enesis. 3-NPA has also been reported to impair the mitochondrialnzyme complex activities like NADH dehydrogenase, succinateehydrogenase and cytochrome oxidase (Colle et al., 2012).

The striatum regions were homogenized in the isolation bufferith EGTA (215 mM Mannitol, 75 mM sucrose, 0.1% BSA, 20 mMEPES, 1 mM EGTA, and pH-7.2). Homogenate was centrifuged at3,000 × g for 5 min at 4 ◦C. Pellets were resuspended in the isola-ion buffer with EGTA and spun again at 13,000 × g for 5 min. Theesulting supernatant was transferred to new tubes and topped offith isolation buffer with EGTA and again spun at 13,000 × g for

0 min. Pellets containing purified mitochondria were resuspendedn the isolation buffer without EGTA. Thus, rat brain mitochondria

ere isolated (Berman and Hastings, 1999).

.7.1. Assessment of complex I (NADH dehydrogenase) activityComplex I (NADH dehydrogenase activity) was measured spec-

rophotometrically (UV-1800 ENG 240V; Shimadzu Coorporation,

h Bulletin 102 (2014) 57–68 61

Japan). The method involves the catalytic oxidation of NADH toNAD+ with subsequent reduction of cytochrome-C. The reactionmixture contained 0.2 M glycyl glycine buffer, pH 8.5, 6 mM NADHin 2 mM glycyl glycine buffer and 10.5 mM cytochrome-C. Thereaction was initiated by the addition of a requisite amount of sol-ubilized mitochondrial sample. The absorbance change at 550 nmwas followed for 2 min (King and Howard, 1967).

3.7.2. Assessment of complex II (succinate dehydrogenase (SDH))activity

SDH was measured spectrophotometrically (UV-1800 ENG240V; Shimadzu Coorporation, Japan). The method involves theoxidation of succinate by an artificial electron acceptor, potas-sium ferricyanide. The reaction mixture contained 0.2 M phosphatebuffer pH 7.8, 1% BSA, 0.6 M succinic acid and 0.03 M potassium fer-ricyanide. The reaction was initiated by the addition of the striatummitochondrial sample, and the absorbance change at 420 nm wasfollowed for 2 min (King, 1967).

3.7.3. Assessment of complex IV (cytochrome oxidase) activityCytochrome oxidase activity was assayed in brain mitochon-

dria. The assay mixture contained 0.3 mM reduced cytochrome-C in75 mM phosphate buffer. The reaction was initiated by the additionof the solubilized striatum mitochondrial sample, and absorbancechange at 550 nm was measured spectrophotometrically (UV-1800ENG 240V; Shimadzu Coorporation, Japan) for 2 min (Sottocasaet al., 1967).

3.8. Experimental protocol

In total fourteen groups were employed in this study and eachgroup consisted of eight Albino Wistar rat.

Group I — Control group: Animals were exposed to EPM andMWM for acquisition trials and retrieval trials on MWM.

Group II and III — Vehicle control (0.9% saline and 0.5% car-boxymethylcellulose) group: Animals were administered with0.9% (w/v) saline (10 ml kg−1 intraperitoneally; once daily alter-natively for 28 days) and 0.5% (w/v) CMC (10 ml kg−1 orally; oncedaily for 19 days). All saline treated animals were exposed to EPMon 21st day to 23rd day and on MWM from the 24th day to 28thday. CMC treated animals were exposed to EPM from the 11th dayto 13th day and from 14th to 18th day on MWM.

Group IV, V, VI, VII and VIII — Moxonidine dose 1 and dose2, NDDCT dose 1 and dose 2 and TBZ per se: Animals wereadministered with moxonidine (0.03 mg kg−1 orally), NDDCT (5 and10 mg kg−1 intraperitoneally) and TBZ (3 mg kg−1 orally) once dailyfor 19 days. Animals were exposed to EPM on day 11 to day 13 andon MWM from day 14 to day 18.

Group IX — 3-Nitropropionic acid (3-NPA) treatment group:Animals were administered with 3-NPA (10 mg kg−1; intraperi-toneally) on alternate days followed by exposure to MWM on 24thday onwards. The treatment was continued during the acquisitiontrial (24th to 27th day) and retrieval trial (28th day) on MWM.

Group X, XI, XII, XIII and XIV — 3-NPA and moxonidine (dose 1and dose 2), NDDCT (dose 1 and dose 2) and tetrabenazine: Mox-onidine (0.03 and 0.06 mg kg−1 orally), NDDCT (5 and 10 mg kg−1,intraperitoneally) and tetrabenazine (3 mg kg−1; orally) once a day,were administered to the 3-NPA treated rats, starting from 10th dayof 3-NPA treatment followed by exposure to EPM on day 21 to day23 and on MWM day 24 to day 28.

3.9. Statistical analysis

Statistical analysis was done using SigmaStat v3.5. All resultswere expressed as mean ± standard deviation. Data for locomo-tor activity, grip strength, Morris water maze and elevated plus

62 S. Gupta, B. Sharma / Brain Research Bulletin 102 (2014) 57–68

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ig. 1. Effect of various agents on body weight. Results are expressed as mean ± stontrol; bp < 0.001 versus 3-NPA treated groups. C: control; S: saline; CMC: carbNDDCT); T: tetrabenazine; HD: 3-nitropropionic acid; D1: dose 1; D2: dose 2.

aze were statistically analyzed using three-way analysis of vari-nce (ANOVA) followed by Bonferroni’s post test, where, 3-NPAreatment, drug treatments and days (3-NPA/without 3-NPA × drugreatments × days) were taken as factors. On the other hand, dataor all other parameters were statistically analyzed by two-wayNOVA followed by Bonferroni’s post test, where, 3-NPA treatmentnd drug treatment (3-NPA/without 3-NPA × drug treatments)ere taken as factors. p < 0.05 was considered to be statistically

ignificant.

. Results

.1. Results of behavioral studies

3-NPA has been reported to cause a reduction in body weight,ocomotor activity, motor coordination, anxiety and impairedearning-memory (Bhateja et al., 2012; Colle et al., 2012) in the HDnimal model. Similar results are obtained in this study. Adminis-ration of moxonidine (0.03 and 0.06 mg kg−1 orally; once daily for9 days), NDDCT (5 and 10 mg kg−1 intraperitoneally; once dailyor 19 days) and tetrabenazine (3 mg kg−1 orally; once daily for 19ays) per se did not show any significant effect on the body weightFig. 1), locomotor activity (Table 1), motor coordination (Table 1),ransfer latency-TL (Table 1), total time spent in open arm-TSOAFig. 2) in elevated plus maze-EPM as well as escape latency time-LT (Table 1) and mean time spent in target quadrant-TSTQ (Fig. 3)n Morris water maze-MWM. In EPM, rise in day 1 and day 3 TLs well as TSOA is considered as an index of impairment of learn-ng and memory as well as anxiety. On MWM, rise in day 4 ELT asompared to day 1 ELT is considered as an index of impairment ofearning and memory. While decrease in day 5 TSTQ on MWM isonsidered as an index of impairment of memory. While, adminis-ration of 3-NPA (10 mg kg−1 intraperitoneally, alternatively for 28ays), significantly reduced the body weight [F(1, 84) = 2169.802;p < 0.001 versus control; F(5, 84) = 24.870; bp < 0.001 versus 3-NPA

reated groups] (Fig. 1), locomotor activity [F(1, 168) = 68,705.333;p < 0.001 versus locomotor activity of previous day within eachroup; F(1, 68) = 69,008.333; bp < 0.001 versus respective day inontrol group; F(5, 168) = 1743.533; cp < 0.001 versus respective

d deviation; two-way ANOVA followed by Bonferroni’s post test. ap < 0.001 versusethyl cellulose; M: moxonidine; N: natrium diethyl dithio carbamate trihydrate

day in 3-NPA treatment group] (Table 1), motor coordination [F(1,168) = 8237.280; ap < 0.001 versus fall of time of previous daywithin each group; F(1, 68) = 6552.013; bp < 0.001 versus respec-tive day in control group; F(5, 168) = 55.541; cp < 0.001 versusrespective day in 3-NPA treatment group] (Table 1), increase inday 1 and day 3 TL [F(2, 252) = 3133.681; ap < 0.001 versus TL ofprevious days within each group; F(2, 252) = 869.191; bp < 0.001versus TL of respective day in control group; F(10, 252) = 76.559;cp < 0.001 versus TL of respective day in 3-NPA treated group](Table 1), increase in day 1 and day 3 TSOA [F(2, 252) = 19,012.500;ap < 0.001 versus TL of previous days within each group; F(2,252) = 897.167; bp < 0.001 versus TL of respective day in controlgroup; F(10, 252) = 44.767; cp < 0.001 versus TL of respective dayin 3-NPA treated group] (Fig. 2) in EPM as well as prolongation ofday 4 ELT [F(3, 336) = 951.300; ap < 0.001 versus ELT of previousday within each group; F(3, 336) = 282.727; bp < 0.001 versus ELTof respective day in control group; F(15, 336) = 64.274; cp < 0.001versus ELT of respective day in 3-NPA treated group] (Table 1)and reduction in day 5 TSTQ [F(3, 336) = 2453.639; p < 0.001 versusTSTQ of previous day within each group; F(3, 336) = 230.288;ap < 0.001 versus TSTQ of respective day in control group; F(15,336) = 21.313; bp < 0.001 versus TSTQ of respective day in 3-NPAtreated group] (Fig. 3) on MWM, as compared to control ani-mals. Treatment with moxonidine (0.03 and 0.06 mg kg−1 orally),NDDCT (5 and 10 mg kg−1 intraperitoneally) and tetrabenazine(3 mg kg−1 orally) once daily for 19 days, significantly attenu-ated 3-NPA induced reduction in body weight [F(1, 84) = 2169.802;ap < 0.001 versus control; F(5, 84) = 24.870; bp < 0.001 versus 3-NPAtreated groups] (Fig. 1), locomotor activity [F(1, 168) = 68,705.333;ap < 0.001 versus locomotor activity of previous day within eachgroup; F(1, 68) = 69,008.333; bp < 0.001 versus respective day incontrol group; F(5, 168) = 1743.533; cp < 0.001 versus respectiveday in 3-NPA treatment group] (Table 1), motor coordination [F(1,168) = 8237.280; ap < 0.001 versus fall of time of previous daywithin each group; F(1, 68) = 6552.013; bp < 0.001 versus respec-

tive day in control group; F(5, 168) = 55.541; cp < 0.001 versusrespective day in 3-NPA treatment group] (Table 1), increase inday 1 and day 3 TL [F(2, 252) = 3133.681; ap < 0.001 versus TL ofprevious days within each group; F(2, 252) = 869.191; bp < 0.001

S. Gupta, B. Sharma / Brain Research Bulletin 102 (2014) 57–68 63

Table 1Effect of various agents on locomotor activity, grip strength, transfer latency (elevated plus maze) and escape latency time (Morris water maze) of the animals.

Group Locomotor activity (counts/5 min) Fall of time (% of control group) Transfer latency (in s) Escape latency time (s)

Day 1 Day 28 Day 1 Day 28 Day 1 Day 2 Day 3 Day 1 Day 4

C 387 ± 27.09 384 ± 23.04a 100 ± 7 100 ± 8a 150.7 ± 9.79 100.7 ± 7.04a 60.7 ± 4.06a 115 ± 7.47 42.1 ± 3.11a

S 380 ± 26.6 378 ± 22.68a 97 ± 6.79 98 ± 7.8a 152.3 ± 9.89 102.3 ± 7.16a 62.9 ± 4.21a 110 ± 7.15 45.5 ± 3.36a

CMC 375 ± 26.25 373 ± 22.38a 96 ± 6.72 98 ± 7.84a 151.6 ± 9.85 101.4 ± 7.09a 63.6 ± 4.26a 113 ± 7.34 47.8 ± 3.53a

M D1 381 ± 26.67 387 ± 23.22a 95 ± 6.65 96 ± 7.68a 150 ± 9.75 102.7 ± 7.18a 64.8 ± 4.34a 111 ± 7.21 48.9 ± 3.61a

M D2 386 ± 27.02 380 ± 22.8a 97 ± 6.79 97 ± 7.76a 153.4 ± 9.97 100.4 ± 7.02a 63.1 ± 4.22a 109 ± 7.08 44.8 ± 3.31a

N D1 379 ± 26.53 373 ± 22.38a 99 ± 6.93 98 ± 7.84a 150.9 ± 9.80 102.6 ± 7.18a 64.2 ± 4.30a 114 ± 7.41 50.7 ± 3.75a

N D2 380 ± 26.6 384 ± 23.04a 96 ± 6.72 95 ± 7.6a 152.7 ± 9.92 103.2 ± 7.22a 65.7 ± 4.40a 106 ± 6.89 47.3 ± 3.50a

T 383 ± 26.81 372 ± 22.32a 96 ± 6.72 95 ± 7.6a 150.9 ± 9.80 102.8 ± 7.19a 63.8 ± 4.27a 109 ± 7.08 49.9 ± 3.69a

HD 384 ± 26.88 129 ± 7.74b 91 ± 6.37 24.5 ± 1.96b 150.8 ± 9.80 130.4 ± 9.12b 117.6 ± 7.87b 112 ± 7.28 82.6 ± 6.11HD + M D1 374 ± 26.18 256 ± 15.36a,c 92 ± 6.44 43.9 ± 3.51a,c 152.3 ± 9.89 116.8 ± 8.17a 80.6 ± 5.40a,c 110 ± 7.15 67.8 ± 5.01a,c

HD + M D2 386 ± 27.02 271 ± 16.26a,c 93 ± 6.51 49.9 ± 3.99a,c 150.3 ± 9.76 115.1 ± 8.05a 73.2 ± 4.90a,c 113 ± 7.34 59.1 ± 4.37a,c

HD + N D1 376 ± 26.32 201 ± 12.06a,c 95 ± 6.65 48.8 ± 3.90a,c 151.7 ± 9.86 119.6 ± 8.37a 90.4 ± 6.05a,c 115 ± 7.47 65.9 ± 4.87a,c

HD + N D2 388 ± 27.16 223 ± 13.38a,c 97 ± 6.79 54.2 ± 4.33a,c 151 ± 9.81 116.2 ± 8.13a 81.7 ± 5.47a,c 108 ± 7.02 49.3 ± 3.64a,c

HD + T 389 ± 27.23 291 ± 17.46a,c 98 ± 6.86 60.3 ± 4.82a,c 151.9 ± 9.87 112.3 ± 7.86a 78.9 ± 5.28a,c 110 ± 7.15 45.2 ± 3.34a,c

Results are expressed as mean ± standard deviation; three-way ANOVA followed by Bonferroni’s post test.Locomotor activity – a p < 0.001 versus locomotor activity of previous day within each group; bp < 0.001 versus locomotor activity in respective day in control group; cp < 0.001versus locomotor activity in respective day in 3-NP treatment group.Rota rod – a p < 0.001 versus fall of time of previous day within each group; b p < 0.001 versus fall of time in respective day in control group; c p < 0.001 versus fall of time inrespective day in 3-NP treatment group.Transfer latency – a p < 0.001 versus TL of previous days within each group; b p < 0.001 versus TL of respective day in control group; c p < 0.001 versus TL of respective day in3-NP treated group.Escape latency time – ap < 0.001 versus ELT of previous day within each group; F(3, 336) = 289.419; bp < 0.001 versus ELT of respective day in control group; F(15, 336) = 61.532;c

T ethyl cT ncy ti

vc

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2giddov

Ffot

p < 0.001 versus ELT of respective day in 3-NP treated group.L: transfer latency; ELT: escape latency time; C: control; S: saline; CMC: carboxy-m: tetrabenazine; HD: 3-nitropropionic acid; TL: transfer latencies; ELT: escape late

ersus TL of respective day in control group; F(10, 252) = 76.559;p < 0.001 versus TL of respective day in 3-NPA treated group]Table 1), increase in day 1 and day 3 TSOA [F(2, 252) = 19,012.500;p < 0.001 versus TL of previous days within each group; F(2,52) = 897.167; bp < 0.001 versus TL of respective day in controlroup; F(10, 252) = 44.767; cp < 0.001 versus TL of respective dayn 3-NPA treated group] (Fig. 2) in EPM as well as prolongation of

a

ay 4 ELT [F(3, 336) = 951.300; p < 0.001 versus ELT of previousay within each group; F(3, 336) = 282.727; bp < 0.001 versus ELTf respective day in control group; F(15, 336) = 64.274; cp < 0.001ersus ELT of respective day in 3-NPA treated group] (Table 1) and

ig. 2. Effect of various agents on percent time spent in open arm (TSOA), using Elevated

ollowed by Bonferroni’s post test. ap < 0.001 versus TSOA of previous days within each grouf respective day in 3-NPA treated group. C: control; S: saline; CMC: carboxy-methyl celluetrabenazine; HD: 3-nitropropionic acid; D1: dose 1; D2: dose 2.

ellulose; M: moxonidine; N: natrium diethyl dithio carbamate trihydrate (NDDCT);me; D1: dose 1; D2: dose 2.

reduction in day 5 TSTQ [F(3, 336) = 2453.639; p < 0.001 versus TSTQof previous day within each group; F(3, 336) = 230.288; ap < 0.001versus TSTQ of respective day in control group; F(15, 336) = 21.313;bp < 0.001 versus TSTQ of respective day in 3-NPA treated group](Fig. 3) on MWM.

4.2. Results of biochemical studies

It has been reported that 3-NPA induces oxidative as well asnitrosative stress (Bhateja et al., 2012), elevates levels of brain AChE(Konagaya et al., 1992) as well as impairs the mitochondrial enzyme

plus maze. Results are expressed as mean ± standard deviation; three-way ANOVAp; bp < 0.001 versus TSOA of respective day in control group; cp < 0.001 versus TSOAlose; M: moxonidine; N: natrium diethyl dithio carbamate trihydrate (NDDCT); T:

64 S. Gupta, B. Sharma / Brain Research Bulletin 102 (2014) 57–68

Fig. 3. Effect of various agents on mean time spent in the target quadrant (TSTQ) of animals using Morris water maze. Results are expressed as mean ± standard deviation;three-way ANOVA followed by Bonferroni’s post test. ap < 0.001 versus TSTQ of previous day within each group; bp < 0.001 versus TSTQ of respective day in control group;c e; CMt ose 2.

cg2aaDodoia

Na

3dt

TE

RTc

p < 0.001 versus TSTQ of respective day in 3-NPA treated group. C: control; S: salinrihydrate (NDDCT); T: tetrabenazine; HD: 3-nitropropionic acid; D1: dose 1; D2: d

omplex activities like NADH dehydrogenase, succinate dehydro-enase and cytochrome oxidase (Bhateja et al., 2012; Colle et al.,012). Increased levels of brain striatum TBARS and nitrite/nitratere considered as indicator of oxidative stress. Increased AChEctivity is considered as an index of cholinergic dysfunction.epletion of antioxidant system is assessed by decreased levelsf brain striatum SOD and CAT. Reduced levels of mitochon-rial enzyme complexes (I, II and IV) are considered as an indexf mitochondrial dysfunction. 3-NPA produced a considerablencrease in the level of brain striatum TBARS [F(1, 84) = 42,841.500;p < 0.001 versus control; F(5, 84) = 2290.700; bp < 0.001 versus 3-PA treated groups] (Table 2), nitrite/nitrate [F(1, 84) = 11,616.000;

p < 0.001 versus control; F(5, 84) = 1136.800; bp < 0.001 versus

-NPA treated groups] (Table 2) as well as a considerableecrease in SOD [F(1, 84) = 4592.667; ap < 0.001 versus con-rol; F(5, 84) = 403.467; bp < 0.001 versus 3-NPA treated groups]

able 2ffect of various agents on brain oxidative stress, nitrite/nitrate and acetylcholinesterase

Group TBARS, nM/mg ofprotein (% ofcontrol)

Nitrite/nitrate,�mol/mg of protein (%of control)

C 100 ± 7 100 ± 6.5 1S 94 ± 6.58 94 ± 6.11

CMC 97 ± 6.79 99 ± 6.43

M D1 95 ± 6.65 93 ± 6.04

M D2 94 ± 6.58 98 ± 6.37

N D1 98 ± 6.86 92 ± 5.98

N D2 95 ± 6.65 99 ± 6.435

T 98 ± 6.86 91 ± 5.915

HD 259 ± 18.13a 201 ± 13.06a

HD + M D1 199 ± 13.93b 151 ± 9.81b

HD + M D2 146 ± 10.22b 115 ± 7.47b

HD + N D1 203 ± 14.21b 145 ± 9.42b

HD + N D2 157 ± 10.99b 120 ± 7.8b

HD + T 123 ± 8.61b 105 ± 6.82b

esults are expressed as mean ± standard deviation; two-way ANOVA followed by BonfeBARS: thiobarbituric acid reactive substances; SOD: superoxide dismutase; CAT: cataarboxy-methyl cellulose; M: moxonidine; N: natrium diethyl dithio carbamate trihydra

C: carboxy-methyl cellulose; M: moxonidine; N: natrium diethyl dithio carbamate

(Table 2), CAT [F(1, 84) = 3952.667; ap < 0.001 versus control; F(5,84) = 200.267; bp < 0.001 versus 3-NPA treated groups] (Table 2)activity as well as increase in AChE [F(1, 84) = 3634.617; ap < 0.001versus control; F(5, 84) = 111.980; bp < 0.001 versus 3-NPA treatedgroups] (Table 2), with impairment of mitochondrial enzyme com-plexes (I, II and IV) [complex I – F(1, 84) = 5704.167; ap < 0.001versus control; F(5, 84) = 188.167; bp < 0.001 versus 3-NP treatedgroups, complex II – F(1, 84) = 3082.667; ap < 0.001 versus con-trol; F(5, 84) = 385.067; bp < 0.001 versus 3-NP treated groups,complex IV – F(1, 84) = 4873.500; ap < 0.001 versus control; F(5,84) = 197.900; bp < 0.001 versus 3-NP treated groups] (Fig. 4),as compared to control animals. Administration of moxonidine,NDDCT and tetrabenazine per se did not show any significant effect

on the levels of brain striatum TBARS (Table 2), nitrite/nitrate(Table 2), SOD (Table 2) and CAT (Table 2), AChE (Table 2) andstriatal mitochondrial enzyme complexes (I, II and IV) (Fig. 4).

activity of the animals.

SOD, nU/mgprotein (% ofcontrol)

CAT, U/mg protein(% of control)

AChE, �M of AChhydrolyzed/min/mgof protein

00 ± 7.4 100 ± 8 3.4 ± 0.2697 ± 7.17 96 ± 7.68 3.5 ± 0.2799 ± 7.32 94 ± 7.52 3.3 ± 0.2697 ± 7.17 96 ± 7.68 3.4 ± 0.2695 ± 7.03 99 ± 7.92 3.5 ± 0.2797 ± 7.17 94 ± 7.52 3.6 ± 0.2896 ± 7.10 98 ± 7.84 3.4 ± 0.2698 ± 7.25 99 ± 7.92 3.6 ± 0.2841 ± 3.03a 51 ± 4.08a 6.9 ± 0.54a

59 ± 4.36b 63 ± 5.04b 5.9 ± 0.46b

85 ± 6.29b 71 ± 5.68b 5.1 ± 0.40b

61 ± 4.51b 76 ± 6.08b 6.7 ± 0.52b

81 ± 5.99b 82 ± 6.56b 5.4 ± 0.42b

90 ± 6.66b 89 ± 7.12b 4.5 ± 0.35b

rroni’s post test. ap < 0.001 versus control; bp < 0.001 versus 3-NP treated groups.lase; ACh: acetylcholine; AChE: acetylcholinesterase; C: control; S: saline; CMC:te (NDDCT); T: tetrabenazine; HD: 3-nitropropionic acid; D1: dose 1; D2: dose 2.

S. Gupta, B. Sharma / Brain Research Bulletin 102 (2014) 57–68 65

Fig. 4. Effect of various agents on striatal mitochondrial enzyme complexes I, II and IV. Results are expressed as mean ± standard deviation; two-way ANOVA followedb treaten oprop

Tdooit(vga

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otTh

y Bonferroni’s post test. ap < 0.001 versus control group; bp < 0.001 versus 3-NPA

atrium diethyl dithio carbamate trihydrate (NDDCT); T: tetrabenazine; HD: 3-nitr

reatment with moxonidine (0.03 and 0.06 mg kg−1 orally; onceaily for 19 days), NDDCT (5 and 10 mg kg−1 intraperitoneally;nce daily for 19 days) and tetrabenazine (3 mg kg−1 orally;nce daily for 19 days) significantly attenuated 3-NPA inducedncreased TBARS [F(1, 84) = 42,841.500; ap < 0.001 versus con-rol; F(5, 84) = 2290.700; bp < 0.001 versus 3-NPA treated groups]Table 2) and nitrite/nitrate [F(1, 84) = 11,616.000; ap < 0.001ersus control; F(5, 84) = 1136.800; bp < 0.001 versus 3-NPA treatedroups] (Table 2) as well as reduced SOD [F(1, 84) = 4592.667;p < 0.001 versus control; F(5, 84) = 403.467; bp < 0.001 versus-NPA treated groups] (Table 2), CAT [F(1, 84) = 3952.667;p < 0.001 versus control; F(5, 84) = 200.267; bp < 0.001 versus-NPA treated groups] (Table 2), rise in brain AChE activitiesF(1, 84) = 3634.617; ap < 0.001 versus control; F(5, 84) = 111.980;p < 0.001 versus 3-NPA treated groups] (Table 2) as well asmpairment of striatal mitochondrial enzyme complexes (I, IInd IV) [complex I – F(1, 84) = 5704.167; ap < 0.001 versus con-rol; F(5, 84) = 188.167; bp < 0.001 versus 3-NP treated groups,omplex II – F(1, 84) = 3082.667; ap < 0.001 versus control; F(5,4) = 385.067; bp < 0.001 versus 3-NP treated groups, complex IV

F(1, 84) = 4873.500; ap < 0.001 versus control; F(5, 84) = 197.900;p < 0.001 versus 3-NP treated groups] (Fig. 4).

. Discussion

In the present study, moxonidine, NDDCT and TBZ have atten-ated 3-NPA induced weight loss, reduced locomotion, impairedotor coordination, anxiety, learning and memory with reduction

f brain striatum nitroso-oxidative stress, AChE and mitochondrialysfunction.

3-NPA administration has reduced the body weight. The cause

f 3-NPA-induced body weight changes may be partially due to fac-ors outside the central nervous system (Kumar and Kumar, 2008).hough, moxonidine has been reported to reduce body weight inigher doses (Bing et al., 1999) but in our study, we have used lower

d group. C: control; S: saline; CMC: carboxy-methyl cellulose; M: moxonidine; N:ionic acid; D1: dose 1; D2: dose 2.

doses of moxonidine, which has improved 3-NPA induced reduc-tion in body weight. Inhibition of NF-�B has also been documentedto improve weight loss (Otani et al., 2012). TBZ has been reportedto regain the lost body weight (Papapetropoulos and Singer, 2006).In this study, moxonidine, NDDCT and TBZ significantly improveweight loss induced by 3-NPA, which may be or may not be due tofactors outside the central nervous system.

3-NPA treated rats developed the motor system dysfunction,which was distinguished by hypo-locomotion in Actophotome-ter, and decreased motor performance in the Rota rod task. Thebehavioral symptoms of HD are generally characterized by the lossof medium-spiny GABAergic neurons in the caudate nucleus andputamen (Han et al., 2010), which is responsible for motor dysfunc-tion in 3-NPA treated animals (Colle et al., 2012). Additionally, defi-ciencies in behavior and motor control are reminiscent of the lossof motor skills associated with increased brain protein oxidation(Forster et al., 1996). The effect of I1-imidazoline receptor agoniston locomotion or mobility has been reported due to their interac-tion with I1-imidazoline receptors (Zeidan et al., 2007). NF-�B inhi-bition has been reported to improve locomotion (Lee et al., 2012).TBZ has been reported to improve choreatic movements becauseof alteration in striatal dopamine levels (Andersson et al., 2006).

Major psychiatric symptoms of HD are cognitive dysfunctionsand mood disorders including anxiety (Hult et al., 2013). EPMis used to assess anxiety condition. In our study, on EPM 3-NPAtreated animals show anxiety like behavior. Mechanism that havebeen reported for causing anxiety like behavior in HD are increasedbrain derived neurotrophic factor (BDNF) dynamics, increaseddelivery and signaling of hippocampal BDNF (Ben et al., 2013). Spe-cific regulation of 5-HT1A receptors has been reported in mediatinganxiety like behavior in HD (Renoir et al., 2013). Glycogen synthase

kinase 3 and histone deacetylases activation have been reported forinducing anxiety in HD (Chiu et al., 2011). I1-imidazoline receptormodulators have been reported for exhibiting anti-anxiety effects(Ciubotariu and Nechifor, 2012; Taksande et al., 2010). Inhibition

6 esearc

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6 S. Gupta, B. Sharma / Brain R

f NF-�B has been reported to show anti-anxiety effects. Inhibi-ion of VMAT2 has also been reported to exhibit anti-anxiety effectNarboux-Nême et al., 2011). Moxonidine, NDDCT and TBZ reducednxiety levels in the present study, which may be because of variousffects.

3-NPA administration significantly impaired the memory asbserved in MWM paradigm. It has been reported that 3-NPAroduces lesions in hippocampal CA1 and CA3 pyramidal neurons,he area of brain that is associated with cognitive performance. Its known that the hippocampal degeneration caused by mitochon-rial dysfunction affects learning and memory, cognitive functionsPrzybyla-Zawislak et al., 2005). 3-NPA administration produces

long-term potentiation of the NMDA-mediated synaptic exci-ation (3-NPA-LTP) in striatal spiny neurons (Calabresi et al.,001), with suppression of LTD expression in the sensorimotortriatum (Dalbem et al., 2005). 3-NPA-LTP has been reported tonvolve in augmenting intracellular calcium and activation of the

itogen-activated protein kinase extracellular signal-regulatedinase. Thus, 3-NPA-LTP might play a key role in the regional andell type-specific neuronal death observed in HD (Calabresi et al.,001).

Moxonidine has been reported to improve memory and think-ng (Gupta and Sharma, 2014; Ostroumova et al., 2001). It haseen reported that moxonidine treatment improves memory tasksnd immediate word recall (Wesnes et al., 1997). Selective I1-midazoline receptor agonist has been reported to enhance thehosphorylation of mitogen-activated protein kinase (MAPK), viahe phosphatidylcholine-specific phospholipase C pathway (Zhangnd Abdel-Rahman, 2005) and phosphorylation of MAPK is impli-ated in the consolidation of memory (Luo et al., 2013). NF-�B haseen documented to play an important role in learning and mem-ry (Mattson and Meffert, 2006) by increasing AChE activity (Kuhadt al., 2009), with reduction of LTP, LTD (Kaltschmidt et al., 2006),s well as reduction of protein kinase A (PKA) activity and regula-ion of PKA/CREB signaling (Kandel, 2001). Therefore, inhibition ofF-�B may be beneficial in the improvement of cognitive deficits.oxonidine, NDDCT and TBZ have improved learning and memory

n the present study, which may be due to modulation of synapticlasticity, MAPK activation as well as GABAergic and glutamatergicodulation.Existing evidence indicates that excessive generation of free

adicals might contribute to the onset of symptoms in HD andther movement disorders (Mandavilli et al., 2005). This effectan be related, at least in part, to a reduction in specific endoge-ous antioxidant mechanisms, such as a decreased activity ofntioxidant defence enzymes, including SOD and CAT. Considerablevidence supports that the oxidative process significantly con-ributes to 3-NPA toxicity (Bhateja et al., 2012; Colle et al., 2012).-NPA diminishes oxidative phosphorylation by interfering withhe mitochondrial respiratory chain, reducing the level of availableTP, and thus causing metabolic inhibition (Bhateja et al., 2012;olle et al., 2012). Moxonidine (I1-imidazoline receptor agonist)as been reported to attenuate oxidative stress (Gupta and Sharma,014; Dorresteijn et al., 2013). It has a protective effect on theepleted antioxidant enzyme system (Gupta and Sharma, 2014;asr et al., 2010). NF-�B has been reported to regulate the cellular

esponse to oxidative stress by influencing the level of reactive oxy-en species (ROS) (Kleniewska et al., 2013). TBZ has been reportedo attenuate oxidative stress because it has an effect on oxidativetress (Milusheva et al., 2003). Moxonidine, NDDCT and TBZ haventioxidant properties due to effects on free radical generation andntioxidant enzyme system.

Mitochondrial dysfunction plays an important role in HD. Mito-hondrial dysfunction is characterized by mitochondrial swelling,embrane fluidity, rupture, release of cytochrome-C, and neu-

onal death, which may have a direct impact on membrane-based

h Bulletin 102 (2014) 57–68

processes such as fission-associated morphogenic changes, open-ing of the mitochondrial permeability transition pore or oxidativephosphorylation at the complexes of the electron transport chain,which further leads to HD (Bertoni et al., 2011). Disruption of themitochondrial enzyme complex activity is associated with ROS.In the present study, 3-NPA significantly impaired mitochondrialenzyme complex activities (complexes I, II and IV) in the stri-atum. It has been reported that I1-imidazoline receptors has aneffect on mitochondrial respiration (Raasch et al., 2000). NF-�Binhibition has been reported to induce rapid mitochondrial releaseof cytochrome-C, membrane blebbing and nuclear fragmentation(Chiarugi, 2002). It has been suggested that the massive eleva-tion of extracellular noradrenaline under conditions of oxidativestress combined with mitochondrial dysfunction may provide anadditional source of highly reactive free radicals thus initiating aself-amplifying cycle leading to neuronal degeneration. TBZ hasbeen reported to attenuate oxidative stress because it has effect onnonsynaptic release noradrenaline in response to oxidative stress(Milusheva et al., 2003). Moxonidine, NDDCT and TBZ improvedimpaired mitochondrial enzyme complexes due to the effect onmitochondrial respiration, cytochrome release and oxidative stress.

3-NPA model, though considered being a very good model, thatmimics the symptoms of HD, but as 3-NPA does not show com-plete pathophysiology of HD, thus future research with these agentsin genetic models is desirable. As this is the first study whichsuggests the utility of these agents in HD, further research in full-fledged genetic models is required to study the full potential ofthese agents as potential therapies for human subjects sufferingfrom HD. Alternatively, studies using the 3-NPA induction modelcould be designed to explore the specific signaling pathways thathave been shown to control on cognitive decline, mitochondrialdysfunction, oxidative stress and neurodegeneration.

6. Conclusions

On the basis of the results of this study and above discussion, itmay be concluded that 3-NPA has induced HD condition. Treatmentwith moxonidine, NDDCT and TBZ has recuperated 3-NPA inducedsymptoms of HD in rats. Thus, modulation of I1-imidazoline recep-tor and NF-�B may provide benefit in HD. Further, research isneeded to identify the full potential of these agents in HD condition.

Acknowledgements

Authors are thankful to Dr. Nirmal Singh, Associate Professor,Pharmacology Division, Department of Pharmaceutical Sciencesand Drug Research, Faculty of Medicine, Punjabi University, Patiala(Punjab), India, for his valuable suggestions. We are also thankful toProf. V. K. Sharma, Director, School of Pharmacy, Bharat Institute ofTechnology, Meerut, India and the Space age Research and Tech-nical Foundation Charitable Trust (SARTFCT), Bharat Institute ofTechnology, Meerut, India for providing all the necessary facilitiesand funding to conduct this research work.

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