Pharmacological investigations on potential of peroxisome proliferator-activated receptor-gamma agonists in hyperhomocysteinemia-induced vascular dementia in rats

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Neuroscience 192 (2011) 322–333

PHARMACOLOGICAL INVESTIGATIONS ON POTENTIAL OFPEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-GAMMAAGONISTS IN HYPERHOMOCYSTEINEMIA-INDUCED VASCULAR

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H. SAIN, B. SHARMA, A. S. JAGGI AND N. SINGH*

Pharmacology Division, Department of Pharmaceutical Sciences andDrug Research, Faculty of Medicine, Punjabi University, Patiala-147002, Punjab, India

Abstract—The present study has been designed to investi-gate the potential of peroxisome proliferator-activated recep-tor-gamma ([PPAR]-�) agonists, pioglitazone, and rosiglita-one in hyperhomocysteinemia-induced vascular dementiaf rats. L-methionine was administered for 8 weeks to induce

hyperhomocysteinemia and associated vascular dementia.Pioglitazone and rosiglitazone were administered to L-methi-

nine-treated rats for 4 weeks (starting from 5th to 8th weeksf methionine treatment). Donepezil served as a positive con-rol in this study. On 52nd day onward, the animals werexposed to Morris water maze (MWM) for testing learning andemory abilities. Vascular endothelial function, serum ni-

rite/nitrate levels, brain thiobarbituric acid reactive speciesTBARS), brain reduced glutathione (GSH) levels, and braincetylcholinesterase (AChE) activity were also measured. L-

methionine-treated animals have shown impairment of learn-ing, memory, endothelial function, decrease in serum nitrite/nitrate levels, and brain GSH levels along with increase inbrain TBARS levels and AChE activity. Pioglitazone, rosigli-tazone, and donepezil significantly improved hyperhomocys-teinemia-induced impairment of learning, memory, endothe-lial dysfunction, and changes in various biochemical param-eters. It is concluded that pioglitazone and rosiglitazone maybe considered as potential pharmacological agents for themanagement of hyperhomocysteinemia-induced vasculardementia. © 2011 IBRO. Published by Elsevier Ltd. All rightsreserved.

Keywords: vascular dementia, pioglitazone, rosiglitazone,oxidative stress, morris water maze.

Dementia is an organic brain disease defined as “loss ofintellectual ability of sufficient severity to interfere eitherwith occupational functioning, usual social activities or re-lationship of a person in the absence of gross clouding ofconsciousness or motor involvement” (Parle and Singh,2007; Sharma and Singh, 2010). Vascular dementia (VaD)is considered to be the second most common cause ofdementia after Alzheimer’s disease (AD) (Liu et al., 2010).

*Corresponding author. Tel: �91-981-512-9884.E-mailaddress:[email protected][email protected] (N. Singh).Abbreviations: AChE, brain acetylcholinesterase; AD, Alzheimer’s dis-ease; ELT, escape latency time; GSH, glutathione; MWM, Morriswater maze; NO, nitric oxide; PPAR-�, peroxisome proliferator-acti-

aated receptor-gamma; TBARS, thiobarbituric acid reactive species;STQ, time spent in target quadrant; VaD, vascular dementia.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All righdoi:10.1016/j.neuroscience.2011.07.002

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VaD is defined as the loss of cognitive functions to adegree that interferes with activities of daily living, resultingfrom ischemic (cerebral embolism and hypoperfusion) orhemorrhagic cerebrovascular disease (CVD) or from car-diovascular or circulatory disturbances that injure brainregions important for memory, cognition, and behavior(Schneck, 2008; Ekonomou et al., 2010). Only limited ther-apeutic interventions are available to reduce the incidenceof VaD. Patients of VaD have a highly negative predictivevalue for survival not only in comparison with populationsof similar age group, but also in comparison with patientswith AD (Schneck, 2008).

Cardiovascular disorders are considered as main riskfactors in the pathogenesis of VaD as well as AD (Wald-stein and Wendell, 2010). Hyperhomocysteinemia, or ele-vation of plasma total homocysteine, is a major risk factorfor cardiovascular disorders (Smulders and Blom, 2011),stroke (Brustolin et al., 2010), and vascular dementia(Nilsson et al., 2010). Increased levels of homocysteinehave been documented to produce changes in structureand function of cerebral blood vessels along with oxidativestress, which play a key role in cerebral vascular endothe-lial dysfunction (Rodionov et al., 2010; Rivera et al., 2010).Oxidative stress and vascular endothelial dysfunction leadto structural deformities in the cerebral blood vessels thatcan impair cerebral perfusion with subsequent neuronaldysfunction and death. These are recognized as importantcontributing factors in the pathogenesis of AD and otherdementia of vascular origin (Viswanathan et al., 2009; Dela Torre, 2010). In earlier studies from our laboratory,hyperhomocysteinemia induced by administration of L-me-thionine has been reported to produce a significant degreeof vascular dementia (Koladiya et al., 2008, 2009).

Peroxisome proliferator-activated receptors (PPARs)are ligand-activated transcription factors belonging to thenuclear receptor superfamily which are present in threeisoforms as �, �/�, and � (Arck et al., 2010). Peroxisomeroliferator-activated receptor gamma (PPAR-�) is present

on vascular cells and is known to exert protective role inthe vascular endothelial dysfunction (Beyer et al., 2008).Disruption or downregulation of these receptors have beenreported to result in vascular endothelial dysfunction(Kleinhenz et al., 2009). PPAR-� receptors are also dis-ributed broadly in central nervous system (Sarruf et al.,009), and activation of these receptors prevents neuronaleath by reduction of oxidative stress (Zhao et al., 2009)

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H. Sain et al. / Neuroscience 192 (2011) 322–333 323

PPAR-� agonists in addition to their antidiabetic activityave been shown to provide beneficial effect in variousNS disorders such as multiple sclerosis (MD) (Zhang etl., in press), amyotrophic lateral sclerosis (ALS) (Kiaei,008), stroke (Kumari et al., 2010), Parkinson’s diseasePD) (Schintu et al., 2009), neuropathic pain (Jain et al.,009), and Huntington’s disease (Chaturvedi et al., 2009).urther, it has been recently reported that PPAR-� is in-olved in improvement of memory and cognitive function inD (Hanyu and Sato, 2010). Moreover, PPAR-� agonists,

such as pioglitazone and rosiglitazone, have been demon-strated to improve experimentally induced endothelial dys-function (Kaur et al., 2010). However, the potential ofPPAR-� agonist in vascular dementia is still unexplored.Therefore, the present study has been undertaken to in-vestigate the beneficial effects of PPAR-� agonists, piogli-tazone, and rosiglitazone in L-methionine-induced vascularementia in rats.

EXPERIMENTAL PROCEDURES

Animals

Male Wistar rats weighing 200–250 g (Chaudhary Charan SinghHaryana Agricultural University, Hisar, Haryana, India) were usedin the present study. Animals were provided with standard labo-ratory food (Kisan Feeds, Ltd, Chandigarh, India) and water adlibitum and were exposed to natural cycle of light and dark. Theexperimental protocol was approved by institutional animal ethicscommittee (IAEC) and care of the animals was taken as per theguidelines of the Committee for the Purpose of Control and Su-pervision of Experiments on Animals (CPCSEA), Ministry of En-vironment and Forest, Government of India, (Reg. No. 107/1999/CPCSEA).

Drugs and chemicals

Donepezil was obtained as free sample from Wockhardt Ltd,Baddi, Himachal Pradesh, India. Pioglitazone and rosiglitazonewere obtained as free sample from Panacea Biotech Ltd. (Lalru,Punjab, India). Folin-Ciocalteu’s Phenol reagent was purchasedfrom Merck limited, Mumbai, India. L-methionine was purchasedrom SD Fine-Chemicals Limited, Mumbai, India. 5, 5, dithiobis2-nitro benzoic acid) (DTNB), reduced glutathione (GSH), bovineerum albumin (BSA), sulfanilamide, N-naphthylethylenediamineNED), and thiobarbituric acid were obtained from Loba Chem,umbai, India. 1, 1, 3, 3-tetra methoxy propane, acetylthiocholine

odide, sodium nitroprusside, phenylephrine were purchased fromigma-Aldrich, USA. Pioglitazone, rosiglitazone, and L-methionine

were suspended in 1% w/v of carboxymethyl cellulose (CMC),whereas donepezil was dissolved in saline water. All the agentswere administered orally to the animals.

L-methionine induced vascular dementia

Rats were administered L-methionine (1.7 g/kg/d, p.o.) for 8 weeksto produce hyperhomocysteinemia-induced vascular dementia(Koladiya et al., 2008, 2009). Body weight of rats was monitoredweekly. After 8 weeks, rats were subjected to Morris water maze(MWM) test for the evaluation of their learning and memory status.The L-methionine treatment was continued during acquisition trialson MWM. Serum homocysteine concentration was analyzed on1st day of the study before L-methionine treatment and 52nd day

Morris water maze test (MWM)

MWM test was employed to assess learning and memory of rats(Morris, 1984; Sharma and Singh, 2010). The MWM procedure isbased on a principle in which the animals when placed in a largepool of water have tendency to escape from the water, which isaccomplished by finding a hidden escape platform. MWM con-sisted of large circular pool (150 cm in diameter, 45 cm in height,filled to a depth of 30 cm with water at 27–29 °C). The water wasmade opaque with white colored dye. The tank was divided intofour equal quadrants with help of two threads, fixed at right angleto each other on the rim of the pool. A submerged platform (10cm2) painted in white was placed 1 cm below the surface of waterinside the target quadrant. The position of platform was keptunaltered throughout the training session.

Acquisition trial. Each animal was subjected to four consec-utive trials on each day with a gap of 5 min. The rat was gentlyplaced in the water of the pool between quadrants, facing the wallof pool with drop location changing for each trial, and allowed120 s to locate submerged platform. Then, it was allowed to stayon the platform for another 20 s. If it failed to find the platformwithin 120 s, it was guided gently onto platform and allowed toremain there for 20 s. Escape latency time (ELT) to locate thehidden platform in water maze was noted as an index of acquisi-tion or learning. Animal was subjected to four acquisition trialsdaily for four consecutive days.

Retrieval trial. On fifth day, the platform was removed, andeach rat was allowed to explore the pool for 120 s. Mean timespent in all four quadrants was noted. The mean time spent by theanimal in target quadrant searching for the hidden platform wasnoted as an index of retrieval or memory. All the trials werecompleted between 09.00 to 18.00 h in semi-soundproof labora-tory. Animals were dried with the help of a dry cloth, and theseanimals were provided with the warm air to raise their bodytemperature to normal.

Assessment of vascular endothelial dysfunction onisolated rat aortic ring preparation

The rats were decapitated, the descending thoracic aorta wasdissected, ring of thoracic aorta, approximately 3–5 mm long werecarefully excised and mounted in tissue bath chamber containingof Krebs–Henseleit solution (NaCl, 119 mM; KCl, 4.7 mM;NaHCO3, 25 mM; MgSO4, 1.0 mM; glucose, 11.1 mM; KH2PO4,1.2 mM and CaCl2, 2.5 mM) of bathing medium, pH 7.4) main-ained at 37 °C and bubbled with the carbogen (95% O2 and 5%CO2) (pH�7.4). The ring was held with the help of two oppositeparallel L-shaped stainless steel loops in opposite directions. Oneloop was connected with force–displacement transducer (Ft-2147) which was connected to Physiograph (INCO, Ambala, In-dia) and another was connected with a holder acting as an anchor,submerged in bath chamber. The preparation was allowed to bestretched with 1.5 g tension than equilibrated for 90 min withcontinuous washing with bath solution after every 10–15 min. Theisometric contractile force was measured with a force–displace-ment transducer. The aortic ring preparation incubated with Kreb-s–Henseleit solution was primed with 80 mM KCl to check itsfunctional integrity and to improve its contractility. The aortic ringpreparation stimulated with phenylephrine (3�10�6 M) until thecontractile response reached a steady tension and cumulativedose responses of acetylcholine (ACh; 10�8–10�4 M) or sodiumitroprusside (SNP; 10�8–10�4 M) was recorded with intact or

denuded endothelium at 30 min interval. The intimal layer of aorticring was rubbed gently with a moistened filter paper for 30 s toobtain endothelium-free preparation. Loss of ACh (1�10�6 M)nduced relaxation confirmed the absence of vascular endothe-

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Biochemical parameters

Collection of sample. For biochemical estimation in thebrain tissue, the animals were killed by cervical dislocation. Thebrains were removed and homogenized in phosphate buffer (pH7.4, 10% w/v) using Teflon homogenizer. The clear supernatant,obtained after centrifugation at 3000 rpm for 15 min, was used toestimate brian acetylcholinesterase (AChE) activity, thiobarbituricacid reactive species (TBARS), reduced glutathione (GSH) levels,and protein content.

Blood samples for biochemical estimation were collected justbefore killing the rats. The blood was kept at room temperature for30 min and then centrifuged at 4000 rpm for 15 min to separateserum. Serum was used to estimate homocysteine level, serumnitrite/nitrate concentration.

These biochemical estimations have been used on a regularbasis in our laboratory. To see the details of the procedures,please refer to our previous research publications (Sharma et al.,2008a,b; Koladiya et al., 2008, 2009; Sharma and Singh, 2010).

Estimation of serum homocysteine. Determination of homo-cysteine was carried out using high performance liquid chroma-tography (HPLC) (Varian Inc., CA, USA) attached with fluorescentHPLC detector according to the method of Dimitrova et al. (2002)(Koladiya et al., 2008, 2009). Rats with serum homocysteinelevels of �10 �M were considered to be hyperhomocysteinemic.

Estimation of brain AChE activity. The whole brain AChEactivity was measured by spectrophotometery (DU 640B spectro-photometer, Beckman Coulter Inc., CA, USA) using the method ofEllman et al. with slight modifications (Ellman et al., 1961; Sharmaand Singh, 2010) and excitation at 420 nm.

Estimation of brain total protein. The brain total protein wasdetermined by Lowry’s method with slight modification (Lowry etal., 1951; Sharma and Singh, 2010) using spectrophotometry andexcitation at 750 nm.

Estimation of TBARS. The quantitative measurement ofTBARS, an index of lipid peroxidation in brain was performedaccording to the method of Ohkawa et al. (1979) (Sharma andSingh, 2010) using spectrophotometry and excitation at 532 nm.

Estimation of GSH. The GSH content in tissue was esti-mated using spectrophotometery as described by Beutler et al.(1963) (Sharma and Singh, 2010) with an excitation at 412 nm.

Estimation of serum nitrite concentration. Serum nitrite con-centration was estimated spectrophotometrically using themethod of Sastry et al. (2002) (Sharma and Singh, 2010) and anexcitation at 545 nm.

Experimental protocol

Fourteen groups were employed in the present study, and eachgroup comprised of six male Wistar albino rats.

Group I: control. Normal untreated rats were subjected toacquisition trial, conducted from day 1 to day 4 and retrieval trial,conducted on day 5 using MWM.

Group II: vehicle (CMC) treated control. Rats were admin-istered 1% w/v CMC (10 ml/kg/d, p.o.) for 4 weeks and thensubjected to MWM test. The vehicle was also administered 45 minbefore acquisition trial, conducted from day 1 to day 4, and beforeretrieval trial, conducted on day 5.

Group III: vehicle (0.9% saline) treated control. Rats wereadministered 0.9% w/v saline solution (10 ml/kg/d, p.o.) for 4weeks and then subjected to MWM test. The vehicle was alsoadministered 45 min before acquisition trial, conducted from day 1

Group IV: L-methionine-treated group. In order to inducehyperhomocysteinemia, the rats were administered L-methionine1.7 g/kg/d, p.o.) for 8 weeks, and the rest of the procedure wasame as described in group II.

Group V: pioglitazone low dose per se. Rats were admin-stered pioglitazone (10 mg/kg/d, p.o.) for 4 weeks, and the rest ofhe procedure was same as described in group II.

Group VI: pioglitazone high dose per se. Rats were admin-stered pioglitazone (20 mg/kg/d, p.o.) for 4 weeks, and the rest ofhe procedure was same as described in group II.

Group VII: L-methionine�pioglitazone low–dose-treatedgroup. To the L-methionine (1.7 g/kg/d, p.o.; 8 weeks)-treatedrats, pioglitazone (10 mg/kg/d, p.o.) was coadministered for fourweeks (starting from 5th to 8th week of L-methionine administra-ion). The animals were then subjected to MWM test. The coad-inistration of pioglitazone was continued during acquisition trial,

onducted from day 1 to day 4. The animals were administeredehicle (1% w/v CMC, 10 ml/kg p.o.) 45 min before retrieval trial,onducted on day 5.

Vehicle was administered 45 min before retrieval trial just toollow the normal dosage administration schedule to prevent thehances of error in behavioral results, due to the change in thenimal’s daily handling conditions.

Group VIII: L-methionine�pioglitazone High–dose-treatedgroup. To the L-methionine (1.7 g/kg/d, p.o.; 8 weeks)-treatedrats, pioglitazone (20 mg/kg/d, p.o.) was coadministered for fourweeks (from 5th to 8th week of L-methionine administration). Theest of the procedure was same as described in group VII.

Group IX: rosiglitazone low–dose per se. Rats were admin-stered rosiglitazone (5 mg/kg/d, p.o.) for 4 weeks, and the rest ofhe procedure was same as described in group II.

Group X: rosiglitazone high dose per se. Rats were admin-stered rosiglitazone (10 mg/kg/d, p.o.) for 4 weeks and the rest ofhe procedure was same as described in group II.

Group XI: L-methionine�rosiglitazone Low–dose-treatedgroup. In this group of animals, rosiglitazone (5 mg/kg/d, p.o.)was coadministered with L-methionine, as per the protocol de-cribed in group VII.

Group XII: L-methionine�rosiglitazone High–dose-treatedroup. In this group of animals, rosiglitazone (10 mg/kg/d, p.o.)as coadministered with L-methionine, as per the protocol de-cribed in group VII.

Group XIII: donepezil per se. Rats were administered done-ezil (1 mg/kg/d, p.o.) for 4 weeks and then subjected to MWMest. The treatment was continued (administered 45 min before)uring acquisition trial, conducted from day 1 to day 4. The ani-als were administered vehicle (0.9% w/v saline solution, 10l/kg, p.o.) 45 min before retrieval trial, which was conducted onay 5.

Group XIV: L-methionine�donepezil-treated group. To theL-methionine (1.7 g/kg/d, p.o.; 8 weeks)-treated rats, donepezil (1mg/kg/d, p.o.) was coadministered for four weeks (from 5th to 8thweek of L-methionine administration). Then these animals weresubjected to MWM test. The coadministration of donepezil andL-methionine was continued during acquisition trial, conductedfrom day 1 to day 4. The animals were administered vehicle (0.9%w/v saline solution, 10 ml/kg/d, p.o.) 45 min before retrieval trial,which was conducted on day 5.

Statistical analysis

All the results of this study were statistically analyzed by software

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H. Sain et al. / Neuroscience 192 (2011) 322–333 325

deviation of mean. The data for isolated aortic ring preparationwere statistically analyzed using repeated measure analysis ofvariance (ANOVA) followed by Newman-Keul’s test. Rest of thedata obtained from various groups was statistically analyzed usingone-way ANOVA followed by Tukey’s multiple range test. TheP�0.05 was considered to be statistically significant.

RESULTS

Effect on escape latency time (ELT) and mean timespent in target quadrant (TSTQ), using MWM

Control rats showed a downward trend in their ELT valueswith a significant drop in day 2 to day 4 ELT when com-pared with day 1 ELT of these animals, hence reflectingnormal learning ability (Fig. 1, Table 1). Further on day 5,

significantly higher TSTQ was observed, when com-ared with the time spent in other quadrants, reflectingormal retrieval (Fig. 2).

Per se administration of vehicles (CMC/distilled water)r pioglitazone (low as well as high dose) or rosiglitazonelow as well as high dose) or donepezil did not show any

Fig. 1. Effect on escape latency time (ELT) of animals, using MorrisOne-way ANOVA followed by Tukey’s multiple range test. a Denotesand 4 ELT with day 1 ELT of respective group). b Denotes P�0.05 ver4 ELT of the respective group with day 1, 2, 3, and 4 ELT of coL-methionine-treated group (for the comparison of day 1, 2, 3, andrespectively). CMC, carboxymethyl cellulose; L-MET, L-methionine; P

significant effect on ELT values (Fig. 1, Table 1) and day 5TSTQ (Fig. 2).

Administration of L-methionine produced a significantincrease in day 1 to day 4 ELT, when compared to day 1 today 4 ELT of control rats, respectively, indicating impair-ment of acquisition (Fig. 1, Table 1). Further, these animalsalso showed a significant reduction in day 5 TSTQ(38�0.76) when compared with day 5 TSTQ (66�1.32) ofcontrol rats, indicating impairment of memory (Fig. 2).

Treatments of pioglitazone (low as well as high dose)/osiglitazone (low as well as high dose)/donepezil to L-me-hionine rats significantly decreased L-methionine-inducedrise in ELT values, indicating attenuation of L-methionine-nduced impairment of acquisition (Fig. 1, Table 1). Further,hese drugs also increased L-methionine-induced reduction inay 5 TSTQ indicating significant improvement of mem-ry (L-methionine�38�0.76; L-methionine�pioglitazone lowose�44�0.88; L-methionine�pioglitazone high dose�

45�0.91; L-methionine�rosiglitazone low dose�45�0.90;

aze. Values are mean�standard deviation of mean (STDEV), n�6.ersus day 1 ELT in respective group (for the comparison of day 2, 3,ctive day ELT in control group (for the comparison of day 1, 2, 3, andp, respectively). c Denotes P�0.05 versus respective day ELT inf drug-treated group with day 1, 2, 3, and 4 ELT of L-MET group,oglitazone low dose; PIO HD, pioglitazone high dose; ROS, reactive

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L-methionine�rosiglitazone high dose�45.6�0.91; L-meth-onine�donepezil�46�0.92) (Fig. 2).

ffect of various agents on changes in endothelium-ependent relaxation

dministration of L-methionine significantly attenuatedcetylcholine-induced endothelium-dependent relaxationhen compared with control rats. However it did not affectNP-induced endothelium-independent relaxation. Treat-ent of pioglitazone (low as well as high dose)/rosiglitazone

low as well as high dose)/donepezil to L-methionine-treatedrats significantly improved L-methionine-induced impairmentf endothelial-dependent relaxation (Figs 3 and 4). Adminis-

ration of vehicles (CMC/distilled water)/pioglitazone per selow as well as high dose)/rosiglitazone per se (low as well asigh dose)/donepezil per se did not show any significantffect on endothelium-dependent relaxation.

ffect on L-methionine-induced changes inbiochemical parameters

Administration of L-methionine produced a significant in-rease in serum homocysteine levels, brain AChE activity,nd brain TBARS along with decrease in brain GSH anderum nitrite/nitrate levels when compared to control rats.reatments of pioglitazone (low as well as high dose)/osiglitazone (low as well as high dose)/donepezil to L-me-hionine rats improved L-methionine-induced changes inbiochemical parameters in a significant manner. However,no significant effect was observed on body weight of ani-mals (Tables 2 and 3). Further, vehicles (CMC and distilledwater), pioglitazone per se (low as well as high dose)/rosiglitazone per se (low as well as high dose)/donepezilper se did not show any significant effect on biochemical

Table 1. Effect on escape latency time (ELT), using Morris water ma

Group Day 1 ELT

Control 99.5�3.8Vehicle (CMC) treated 105�3.7Vehicle (Saline) treated 102�3.4L-methionine treated 112�4.4b

Pioglitazone low-dose per se 100�3.6Pioglitazone high-dose per se 103.4�3.7L-methionine�pioglitazone low-dose treated 105.3�4.1b

L-methionine�pioglitazone high-dose treated 102.4�3.9c

Rosiglitazone low-dose per se 102�3.5Rosiglitazone high-dose per se 99�3.4L-methionine�rosiglitazone low-dose treated 106�4.4b

L-methionine�rosiglitazone high-dose treated 104.5�4.2b

Donepezil per se 98.7�3.5L-methionine�donepezil treated 100.2�3.8c

Values are mean�standard deviation of mean (STDEV), n�6.One-way ANOVA followed by Tukey’s multiple range test.

a Denotes P�0.05 versus day 1 ELT in respective group (for the comb Denotes P�0.05 versus respective day ELT in control group (for thend 4 ELT of control group, respectively).

c Denotes P�0.05 versus respective day ELT in L-methionine-treateday 1, 2, 3, and 4 ELT of L-methionine group, respectively).

parameters (Table 3).

DISCUSSION

Young male rats were used in the present study, as it isreported that aging and consequent variation of estrogenin blood modulate the activity of endothelial nitric oxidesynthase (eNOS), which further affects the function ofvascular endothelium and memory (Vanhoutte, 2004; Gin-gerich and Krukoff, 2005). Nitric oxide (NO) is synthesizedin the endothelium, and its levels get attenuated duringendothelial dysfunction. Endogenously formed NO ishighly unstable and gets converted to nitrate and nitrite(Sastry et al., 2002). Therefore, serum nitrite concentrationhas been employed as a specific marker of endothelialdysfunction (Abahji et al., 2007). It has already been re-ported that there is a strong correlation of cerebral vasculardisease with peripheral artery disease (Purroy et al., 2008;Poredos and Jug, 2007) and coronary artery disease(Hoshino et al., 2008; Seo et al., 2008). Very recently, ithas been reported that endothelial dysfunction is presentin patients with lacunar stroke as well as cortical (largeartery atheromatous) stroke (Stevenson et al., 2010).

MWM employed in the present study is one of the mostaccepted models to evaluate learning and memory of therodents (Morris, 1984; Parle and Singh, 2007). Controluntreated animals in our study have shown marked reduc-tion in day 4 ELT as compared to day 1 ELT duringacquisition trial, suggesting normal acquisition or learningability. Further, these animals have shown significant in-crease in day 5 mean TSTQ when compared with timespent in other quadrants, indicating normal retrieval (mem-ory) as well. These results are in line with previous studiesfrom our own laboratory (Koladiya et al., 2008; Sharmaand Singh, 2010, 2011) as well as from other laboratories

Day 2 ELT Day 3 ELT Day 4 ELT (s)

60�2.5a 38�2.4a 20�3.8a

70�2.9a 40�2.5a 21�3.9a

75�3.1a 42�2.6a 20.6�3.9a

98�4.2a,b 85�5.27a,b 62�5.8a,b

63�2.6a 41�2.5a 19�3.5a

73�3.1a 43�2.6a 20.4�3.8a

87�3.6a,b 61.6�3.8a,b,c 41.5�5.5a,b,c

82.6�3.5a,b,c 60�3.72a,b,c 39.5�4.2a,b,c

71.3�2.9a 43�2.6a 18.9�3.0a

68.3�2.9a 40�2.5a 20.6�3.9a

82.5�3.5a,b,c 57.7�3.6a,b,c 42.9�4.1a,b,c

78�3.3 a, b, c 54.2�3.3a,b,c 41.2�3.9a,b,c

66.3�2.8a 38�2.4a 19�3.1a

74.7�3.1a,b,c 48.7�3.1a,b,c 39.1�4.5a,b,c

f day 2, 3, and 4 ELT with day 1 ELT of respective group).on of day 1, 2, 3, and 4 ELT of the respective group with day 1, 2, 3,

r the comparison of day 1, 2, 3, and 4 ELT of drug-treated group with

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H. Sain et al. / Neuroscience 192 (2011) 322–333 327

L-methionine treatment for 8 weeks produced a signif-icant rise in serum homocysteine level along with impair-ment of learning (indicated by increased day 4 ELT) as wellas memory (indicated by decreased day 5 TSTQ). More-over L-methionine treatment has also shown impaired en-othelium-dependent relaxation, enhancement of AChEctivity, increase in brain oxidative stress (as reflected byise in brain TBARS and fall in GSH levels), and decreasen serum nitrite levels. L-methionine-induced hyperhomo-cysteinemia is a well-established model of experimentalendothelial dysfunction (Shah and Singh, 2006; Koladiyaet al., 2008, 2009; He et al., 2010). The result of presentstudy also supports the aforementioned contention, as asignificant decrease in serum nitrite levels and increase inoxidative stress levels (increase TBARS and decreaseGSH) were noted in L-methionine-treated rats.

It has been postulated that hyperhomocysteinemiacauses reduction in bioavailability of NO and a concomi-tant increase in vascular superoxide (O2

�) formation (Dou-pis et al., 2010). This leads to reaction of O2

� with NO toroduce peroxynitrite (ONOO�), an anion with strong oxi-

Fig. 2. Effect on mean (TSTQ) of animals using Morris water maze. Vfollowed by Tukey’s multiple range test. a Denotes P�0.01 versus dayay 5 mean time spent in the target quadrant with day 5 mean time s

the target quadrant (TSTQ) in control group (for the comparison of daytime spent in target quadrant of control group). c Denotes P�0.05 versutime spent in the target quadrant of drug-treated groups with day 5 meaD, 44�0.88; L-MET�PIO HD, 45�0.91; L-MET�ROS LD, 45�0.90;ellulose; L-MET, L-methionine; PIO LD, pioglitazone low dose; PI

rosiglitazone high dose; DON, donepezil.

dant properties, hence causing an increase in vascular

oxidative stress (Qiu et al., 2010; Sen et al., 2010). Per-oxynitrite interacts with cellular components that causeprotein nitration resulting in damage to cellular compo-nents as well as generation of nitroderivatives (Bregere etal., 2008; Szabo and Modis, 2010). These nitroderivativesinactivate regulatory receptors and enzymes such as freeradical scavengers eventually leading to further enhancedproduction of reactive oxygen species (ROS) (Szabo andModis, 2010; Zhou et al., 2010). Hyperhomocysteinemiahas been reported to produce change in structure andfunction of cerebral blood vessels because of cerebralvascular endothelial dysfunction (Rivera et al., 2010; Ro-dionov et al., 2010). It has been demonstrated that thestructural deformities in the cerebral arteries lead to im-paired cerebral perfusion, neuronal dysfunction, and death(De la Torre, 2010; Ji et al., 2010; Nishio, 2010).

Several lines of evidences have strongly advocated adirect relationship between vascular endothelial dysfunc-tion and dementia better known as vascular dementia(Atkinson, 2001; Zhu et al., 2007). It may be consideredthat because of the endothelial dysfunction in the periph-

mean�standard deviation of mean (STDEV), n�6. One-way ANOVAtime spent in other quadrants in control group (for the comparison ofther quadrants). b Denotes P�0.05 versus day 5 mean time spent inime spent in the target quadrant of respective groups with day 5 meanSTQ in L-methionine-treated group (for the comparison of day 5 meanent in target quadrant of L-MET group). L-MET, 38�0.76; L-MET�PIOROS HD, 45.6�0.91; L-MET�DON, 46�0.92. CMC-, carboxymethylioglitazone high dose; ROS LD, rosiglitazone low dose; ROS HD,

alues are5 mean

pent in o5 mean ts day 5 Tn time spL-MET�

eral blood vessels and in the cerebral vessels, there is

dil

cpaha

raaht2rc

tl

b

agd ethionine

onepezi

H. Sain et al. / Neuroscience 192 (2011) 322–333328

progressive reduction in the blood supply to the brainregions eventually leading to vascular dementia in theanimals. Earlier research work from our laboratory (includ-ing this study) has suggested that there is a direct corre-lation between endothelium-dependent relaxation andcognitive function of rats. Enhancement of brain AChEactivity and increase in brain oxidative stress (as reflectedby rise in brain TBARS and fall in GSH levels) have beenpositively correlated with memory loss in dementia, andchronic treatment of L-methionine is demonstrated to in-uce significant degree of vascular dementia along with

ncrease in brain AChE activity and oxidative stress (Ko-adiya et al., 2008, 2009; Sharma and Singh, 2010, 2011).

Experimental hyperhomocysteinemia, resulting fromhronic administration of L-methionine, has been noted toroduce cognitive dysfunction (Villa et al., 2009; Cheng etl., 2010; Hirashima et al., 2010). Hyperhomocysteinemiaas been reported to produce neurotoxicity, DNA dam-

Fig. 3. Effect on acetylcholine-induced endothelium-dependent relaxaof mean (STDEV), n�6. All the responses are expressed as percentanalysis of variance (ANOVA) followed by Newman Keul’s test. a Denoroups with % precontraction of control group). b Denotes P�0.05 vrug-treated group with % precontraction of L-MET group). L-MET, L-m

LD, rosiglitazone low dose; ROS HD, rosiglitazone high dose; DON, d

ge, apoptosis, and excitotoxicity, which are primarily p

esponsible for various deformities such as hippocampalnd white matter atrophy (Zhang et al., 2009; Firbank etl., 2010; Ho et al., 2010; Hosoi et al., 2010). Moreover,yperhomocysteinemia in recent reports has been found

o be associated with neurovascular diseases (Tyagi et al.,010) that cause dementia (Hirashima et al., 2010) and neu-odegeneration (Park et al., 2010). Therefore, hyperhomo-ysteinemia induced as a consequence to chronic L-methio-

nine treatment in our investigation has resulted in vasculardementia by virtue of its multiple effects.

In the present study, administration of pioglitazone,rosiglitazone (both PPAR-� agonists), and donepezil (anAChE inhibitor) significantly reduced the negative effect ofL-methionine on learning and memory of rats. In addition,hese drugs also improved L-methionine-induced endothe-ium dysfunction and various biochemical changes.

PPAR-� agonists, pioglitazone, and rosiglitazone areetter known as insulin sensitizers that constitute an im-

g aortic ring preparation of rat. Values are mean�standard deviationcontraction induced by 3�10�6 M phenylephrine. Repeated measure5 versus control (for the comparison of % precontraction of respectiveethionine-treated group (for the comparison of % precontraction of

; PIO LD, pioglitazone low dose; PIO HD, pioglitazone high dose; ROSl.

tion usinge of pretes P�0.0ersus L-m

ortant class of drugs, currently being used clinically in

mftP

t2P

pse

se; PIO Hr

H. Sain et al. / Neuroscience 192 (2011) 322–333 329

Type II diabetes (Bermúdez et al., 2010; Krieger-Hinck etal., 2010). These drugs act by binding to the PPAR-�, a

ember of the nuclear receptor superfamily that has a keyunction in glucose regulation, lipid metabolism, vascularone, and inflammation (Arck et al., 2010; Yu et al., 2010).PAR-� is expressed widely in CNS, where it has been

shown to regulate neuroprotection (Fatehi-Hassanabadand Tasker, 2011; Glatz et al., 2010). PPAR-� agonistshave been shown to exert neuroprotective effect in both invivo and in vitro models of global ischemia (Fatehi-Has-sanabad and Tasker, 2011). Furthermore, the deficiency ofneuronal PPAR-� has demonstrated an increased suscep-ibility to brain damage after cerebral ischemia (Zhao et al.,009; Glatz et al., 2010). Studies have indicated thatPAR-� agonists also possess excellent antioxidant activ-

ity (Li et al., in press). In recent reports, PPAR-� has beendemonstrated to play a vital role in the vasculature (Cipollaet al., 2010). Activation of PPAR-� receptors has beenshown to inhibit endothelial dysfunction (Tsuchiya et al.,

Fig. 4. Effect on sodium nitroprusside-induced endothelium independResponses are expressed as percentage of precontraction inducedNewman-Keul’s test. L-MET, L-methionine; PIO LD, pioglitazone low doosiglitazone high dose; DON, donepezil.

2009), and pioglitazone is noted to improve acetylcholine-

dependent endothelial function and evoked cerebral bloodflow in AD mice (Nicolakakis et al., 2008). Further, PPAR-�is being considered as novel target to manage cognitivedecline in AD patients (Landreth, 2007; Landreth et al.,2008). PPAR-� agonists have the potential to modulatevarious signaling molecules/pathways, including matrixmetalloproteinase-9, mitogen-activated protein kinases,signal transducer and activator of transcription, mitochon-drial uncoupling protein 2, mitoNEET expression, amyloidprecursor protein degradation, beta-site amyloid precursorprotein cleaving enzyme 1, and Wnt signaling (Kaundaland Sharma, 2010). Administration of PPAR-� agonistioglitazone to APP transgenic mice reduced the expres-ion of BACE-1 (beta-amyloid precursor protein-convertingnzyme, i.e., �-secretase) and hence beta-amyloid load

(Heneka and Landreth, 2007). However, in another study,pioglitazone had no reducing effect of amyloid pathology ormemory deficit in aged APP-transgenic mice (Nicolakakiset al., 2008). In several animal studies, pioglitazone has

ation. Values are mean�standard deviation of mean (STDEV), n�6.0�6 M phenylephrine. Repeated measures of ANOVA followed byD, pioglitazone high dose; ROS LD, rosiglitazone low dose; ROS HD,

ent relaxby 3�1

been reported to exhibit anti-inflammatory effects (Diab et

n(f

SSP

h

PP

RR

D

C

V

P

R

H. Sain et al. / Neuroscience 192 (2011) 322–333330

al., 2002; Thomas et al., 2004), excellent antioxidant ac-tivity (Ishida et al., 2004; Jayesh et al., 2005; Pathan et al.,2006, 2008; Nicolakakis et al., 2008), and anticholinester-ase activity (Pathan et al., 2006; Kaur et al., 2009).PPAR-� agonists exhibit anti-inflammatory properties be-cause of negative regulation of the expression of proin-flammatory molecules, such as interleukin-1b (IL-1b), IL-6,and tumor necrosis factor-� (TNF-�) (Halvorsen et al.,2010; Zhang et al., in press).

Although some reports imply that PPAR-� agonists doot provide significant beneficial effects in memory deficitsGeldmacher et al., 2011; Gold et al., 2010), others haveound beneficial effects in memory-related tasks (Hanyu and

Table 2. Effect on serum homocysteine level and body weight of anim

Groups Homocysteine le

Basal value

Control 6.2�0.24Vehicle (CMC) treated 6.4�0.31Vehicle (distilled water) treated 6.23�0.29L-methionine treated 6.74�0.32

ioglitazone low-dose per se 6.12�0.29ioglitazone high-dose per se 6.2�0.31

L-methionine�pioglitazone low dose 6.75�0.24L-methionine�pioglitazone high dose 6.32�0.28

osiglitazone low-dose per se 5.92�0.34osiglitazone high-dose per se 6.3�0.28

L-methionine�rosiglitazone low dose 6.35�0.31L-methionine�rosiglitazone high dose 5.65�0.29

onepezil per se 6.8�0.33L-methionine�donepezil treated 6.3�0.32

Values are mean�standard deviation of mean (STDEV), n�6.One-way ANOVA followed by Tukey’s multiple range test.

a Denotes P�0.05 versus final values in control.b Denotes P�0.05 versus final values in L-methionine-treated group.

Table 3. Effect on serum nitrite/nitrate, brain thiobarbituric acid reactiv(AChE) activity

Groups Brain AChE (�M of AChhydrolyzed/minute/mg prot

ontrol 3.1�0.38Vehicle (CMC) treated 3.22�0.39

ehicle (distilled water) treated 3.1�0.38L-methionine treated 8.93�0.76a

Pioglitazone low-dose per se 3.0�0.37ioglitazone high-dose per se 3.2�0.39

L-methionine�pioglitazone low dose 4.83�0.62b

L-methionine�pioglitazone high dose 4.6�0.65b

Rosiglitazone low-dose per se 3.12�0.38osiglitazone high-dose per se 3.11�0.39

L-methionine�rosiglitazone low dose 4.93�0.64b

L-methionine�rosiglitazone high dose 4.64�0.65b

Donepezil per se 2.9�0.38L-methionine�donepezil treated 4.20�0.62b

Values are mean�standard deviation of mean (STDEV), n�6.One-way ANOVA followed by Tukey’s multiple range test.

a Denotes P�0.05 versus control.

ato, 2010; Rodriguez-Rivera et al., 2011; Kaundal andharma, 2010; Yao et al., 2009). In some clinical studies,PAR-� agonists have exhibited cognitive and functional im-

provements, and stabilization of the disease in diabetic pa-tients with AD (Sato et al., in press; Hanyu and Sato, 2010).In earlier report, pioglitazone has shown to improve memorydeficits induced by intracerebroventricular streptozotocin (Pa-than et al., 2006) and in our recent study, we have demon-strated that pioglitazone mediated beneficial effect in intrace-rebroventricular streptozotocin-induced dementia that mayinvolve nitric oxide-dependent pathway (Kaur et al., 2009).PPAR-� agonists such as pioglitazone and rosiglitazoneave also been reported to improve endothelial function via

easured on day 1 of Morris water maze exposure

Body weight (g)

Final value Basal value Final value

6.2�1.10 221�12.60 223�10.806.16�1.04 213�11.45 219�10.106.27�1.20 212�10.14 214�9.8221.2�3.20a 223�14.56 225 �12.906.84�1.12 211�9.15 214�9.456.12�1.30 202�9.45 204�10.8512.9�2.12b 209�12.30 218�11.1011.6�2.17b 221�9.74 222�10.255.26�0.98 210�11.32 213�11.506.32�1.60 223�14.76 224�12.1012.4�2.20b 215�12.52 216�12.50

11.82�2.19b 203�11.60 206�11.426.59�1.2 211�9.75 216�10.6013.8�2.34b 222�13.90 222�12.60

(TBARS), brain reduced GSH levels, and brain acetylcholinesterase

Serum nitrite/nitrate(�M/L)

Brain TBARS (nM/mg of protein)

Brain GSH (�M/mgof protein)

13.8�1.12 3.32�0.22 16.4�0.9613.30�1.20 3.31�0.30 16.38�0.9513.38�1.80 3.35�0.24 16.32�0.85

4.9�0.90a 5.2�0.0.38a 6.8�1.10a

13.71�1.02 3.29�0.19 16.48�0.8013.9�1.20 3.26�0.23 16.45�0.939.85�0.98b 4.15�0.23b 11.6�1.13b

10.3�0.98b 4.12�0.26b 10.56�0.98b

13.63�1.15 3.31�0.18 16.32�0.8013.7�1.18 3.38�0.26 16.37�0.8710.1�.97b 4.19�0.32b 11.8�0.83b

10.21�1.05b 4.17�0.29b 11.68�1.1b

12.4�1.24 3.1�0.20 16.4�0.0.9811.0�1.1b 4.2�0.17b 10.8�1.20b

als as m

vel (�M)

e species

ein)

e

dv2dflseo

bc

dot(cSm

blil

D

D

D

E

E

F

F

G

G

H. Sain et al. / Neuroscience 192 (2011) 322–333 331

activation of endothelial PPAR-� (Duan et al., 2008; Pascerit al., 2000). PPAR-� activation has been shown to decrease

the expression of adhesion molecules that induce endothelialinflammation by adherence of monocytes to the endothelium(Pasceri et al., 2000, Jackson et al., 1999; Verrier et al.,2004). Furthermore, PPAR-� agonists have been shown toirectly enhance NO production in cultured endothelial cellsia PPAR-� dependent mechanism (Polikandriotis et al.,005). These findings suggest that PPAR-� agonists couldirectly improve endothelial function by decreasing local in-ammation and increasing NO production. Therefore, withupport from literature and data in hand it appears quitevident that pioglitazone and rosiglitazone mediated reversalf L-methionine-induced vascular dementia involve coordi-

nated activity of their multiple actions such as antioxidative,anti-AChE activity, anti-inflammatory, and neuroprotectiveactions. Although this may be the first report documentingpotential of PPAR-� agonists in vascular dementia, similarenefits have been reported with pioglitazone on cerebrovas-ular deficits observed in APP mice (Nicolakakis et al., 2008).

Acetylcholinesterase inhibitors are the main class ofrugs that are frequently used for the management of mem-ry deficits. In our previous reports, we have demonstratedhat donepezil in addition to its usefulness in dementia of ADSharma et al., 2008a,b) also exerts beneficial effect in vas-ular dementia (Koladiya et al., 2008, 2009; Sharma andingh, 2010, 2011). Donepezil is already in clinical use for theanagement of dementia of AD.

CONCLUSIONS

In lieu of the above discussion, it may be concluded thatPPAR-� agonists, pioglitazone, and rosiglitazone provideeneficial effects by improving learning, memory, endothe-

ial function, brain cholinergic activity, and oxidative stressn L-methionine-induced vascular dementia in rats. Modu-ation of PPAR-� may be considered as important target forvascular dementia. Nevertheless, further studies areneeded to substantiate these findings.

Acknowledgement—Authors are thankful to Department of Phar-maceutical Sciences and Drug Research, Punjabi University Pa-tiala, Punjab, India for providing necessary facilities and fundingfor the research work. We are also thankful to All India Council forTechnical Education (AICTE), India. Further, we thank toWokhardt, Ltd, Baddi, Himachal Pradesh, India and PanaceaBiotech, Ltd, Lalru, India, for providing the free sample of drugs.

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(Accepted 4 July 2011)(Available online 14 July 2011)