Pharmacology, Biochemistry and Behavior 102 (2012) 101–108

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Pharmacology, Biochemistry and Behavior

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Experimental hypertension induced vascular dementia: Pharmacological,biochemical and behavioral recuperation by angiotensin receptor blocker andacetylcholinesterase inhibitor

Bhupesh Sharma 1, Nirmal Singh ⁎CNS and CVS lab., Pharmacology division, Department of Pharmaceutical Sciences and Drug Research, Faculty of Medicine, Punjabi university, Patiala-147002, Punjab, India

⁎ Corresponding author. Tel.: +91 981 5129884.E-mail addresses: bhupeshresearch@gmail.com (B. S

nirmalresearch@gmail.com (N. Singh).1 Tel.: +91 964 6523233.

0091-3057/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.pbb.2012.03.029

a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 November 2011Received in revised form 25 March 2012Accepted 31 March 2012Available online 6 April 2012

Keywords:TelmisartanDonepezilDeoxycorticosterone acetateEndothelial dysfunctionMorris water mazeOxidative stress

Involvement of vascular pathology has been suggested in hypertension as well as vascular dementia (VaD),which also have a very high degree of co-occurrence in ageing population. We have recently reported thatexperimental diabetes as well as hyperhomocystenemia induces VaD. In the present research work, for thefirst time we are reporting the genesis of VaD by deoxycorticosterone acetate (DOCA)-salt induced experi-mental hypertension. Furthermore, we have also investigated the beneficial effect of telmisartan, an angio-tensin II type 1 receptor blocker (ARB) and donepezil, an acetylcholinesterase inhibitor (AChEI), on DOCA-salt hypertension induced VaD in rats. DOCA-salt hypertensive rats performed poorly on Morris watermaze, reflecting impairment in their learning and memory. Furthermore, DOCA-salt treatment has shown asignificant impairment of vascular endothelial function (DOCA attenuated acetylcholine induced endotheli-um dependent relaxation), with a significant reduction in serum nitrite/nitrate levels, along with increasedaortic, serum and brain oxidative stress levels (aortic superoxide anion, serum and brain thiobarbituricacid reactive species, brain glutathione) and brain acetylcholinesterase activity. Treatments of telmisartanas well as donepezil significantly attenuated DOCA-salt hypertension induced learning and memory deficits,endothelial dysfunction, and changes in various biochemical parameters. It may be concluded that DOCA-salthypertension induces VaD in rats. ARBs and AChEIs may be considered as potential pharmacological agentsfor the management of hypertension induced VaD.

© 2012 Elsevier Inc. All rights reserved.

1. Introduction

Cardiovascular risk factors, including hypertension, have beenlinked to subsequent increased incidence, onset and progressionrate of dementia of vascular origin and other etiologies (Monsuez et al.,2011; Moretti et al., 2011). Vascular dementia (VaD) represents the sec-ond most common cause of dementia after Alzheimer's disease (AD) inthe elderly, and is referred as the “silent epidemic of the twenty-first cen-tury” (Battistin and Cagnin, 2010). Optimal treatment of cardiovascularrisk factors has been observed to prevent and slow down age-relatedcognitive disorders (Monsuez et al., 2011; Wehling and Groth, 2011).

Both endothelial dysfunction and VaD are reported to have highprobability of occurrence with hypertension (Muiesan et al., 2011;Yang et al., 2011; Zhang et al., 2011). Aging-related structural and func-tional disturbances in the macro- or microcirculation of the brain makeit vulnerable to cognitive dysfunction, leading to dementing illness(Kalaria, 2010). In previous reports from our lab, we have shown that

harma),

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endothelial dysfunction occurred due to different metabolic disordersresults in VaD. Furthermore, we have observed impairment of learningand memory as the major behavioral alteration in VaD. We have alsoreported that in VaD, there is an enhancement of central & peripheraloxidative stress, brain acetylcholinesterase activity with reduction ofserum nitrite levels (Koladiya et al., 2008, 2009; Sain et al., 2011;Sharma and Singh, 2010, 2011a, 2011b).

Since chronic hypertension is associated with an increased risk ofboth VaD and AD, the role of anti-hypertensive therapy for the pre-vention and delay of cognitive decline and dementia is of central im-portance. Most longitudinal studies have shown a significant inverseassociation between anti-hypertensive therapies and dementia inci-dence (Duron and Hanon, 2010). But the effect of experimental hy-pertension, in the genesis of VaD, is yet to be investigated.

The local renin–angiotensin system (RAS) in the brain is a multi-tasking system. Aside from its vasoactive actions, brain angiotensinII (AT-II) has also been implicated in the pathogenesis of cognitive de-cline, and beneficial effects of angiotensin receptor blockers (ARBs) inAD are suggested (Danielyan et al., 2010). AT II type 1 receptor blockers(ARBs) have been demonstrated to reduce the onset of stroke, strokeseverity, the incidence and progression of dementia, as ARBs provideprotection against ischemic brain damage and associated cognitive

102 B. Sharma, N. Singh / Pharmacology, Biochemistry and Behavior 102 (2012) 101–108

decline owing to an increase in cerebral blood flow and reduction inoxidative stress (Horiuchi and Mogi, 2011). However, the effect ofARB in hypertension associated VaD remain to be studied.

Acetyl cholinesterase inhibitors (AChEIs) including donepezilcomprises one of the prominent class of drugs currently being usedclinically for memory deficits of patients with dementia of AD(Riordan et al., 2011). Several studies have also documented benefi-cial effect of donepezil in dementia of other etiologies (Delrieu etal., 2011; Riordan et al., 2011; Rosenbloom et al., 2011). Howeverbeneficial effect of donepezil in hypertension associated VaD remainsto be explored.

In light of the above the present study has been undertaken to in-vestigate the genesis of VaD by experimental hypertension in rats.Furthermore, the potential of telmisartan, an angiotensin II type 1 re-ceptor blocker (ARB) as well as donepezil, an acetylcholinesterase in-hibitor (AChEI) in DOCA hypertension induced VaD in rats has alsobeen studied.

2. Material and methods

2.1. Animals

Adult albino Wistar rats (male), weighing 200–250 g wereemployed in the present study and were housed in animal housewith free access to water and standard laboratory pellet chow diet(Kisan Feeds Ltd, Mumbai, India). The animals were exposed to12 hlight and 12 h dark cycle. The experiments were conducted between9.00 and 18.00 h in a semi sound-proof laboratory. The animalswere acclimatized to the laboratory condition five days prior to be-havioral study and were maintained in the laboratory until the com-pletion of the study. The protocol of the study was duly approvedby Institutional Animal Ethics Committee (IAEC) and care of the ani-mals was taken as per the guidelines of Committee for the Purposeof Control and Supervision of Experiments on Animals (CPCSEA),Ministry of Environment and Forests, Government of India (Reg. No.107/1999/CPCSEA).

2.2. Drugs and reagents

Telmisartan was obtained as ex-gratia from Cipla, India. Deoxycor-ticosterone acetate (DOCA) and 1,1,3,3 tetra methoxy propane werepurchased from Sigma Aldrich (St. Louis, MO, USA). 5,5′-dithiobis(2-nitro benzoic acid) [DTNB], Bovine serum albumin (BSA), Glutathi-one reduced (GSH) standard and Nitrobluetetrazolium (NBT) werepurchased from Sisco Research Laboratories Pvt Ltd., India. Thiobarbi-turic acid was purchased from Loba Chemie, India. Donepezil was dis-solved in saline and administered intraperitoneally. Telmisartan wassuspended in 1% w/v of sodium carboxy methyl cellulose (CMC).DOCA was dissolved in arachis oil and administered subcutaneously.DOCA treated animals were administered with 0.9% NaCl and 0.2%KCl solution instead of drinking water, whereas all other groupswere maintained on normal drinking water. Telmisartan and CMCwere administered orally with the help of an oral tube (canulla). Allthe drug solutions were freshly prepared before use.

Hypertension and subsequent VaD in rats was induced by ad-ministering DOCA and salt solution for 90 days (85 days+5 daysduring Morris water maze exposure). Our pilot study suggests theinduction of a stable hypertension in 50 days (2 months approxi-mately). To study the effects of the therapeutics on hypertension in-duced VaD, both the drugs were administered for last 40 days viz.day 51 to day 90 (35 days+5 days during MWM exposure) inDOCA treated animals. Thus, the vehicle (Saline & CMC) and drugper se (telmisartan and donepezil) control groups were treatedwith the respective agent alone, for total 40 days (35 days+5 daysduring MWM exposure).

2.3. Deoxycorticosterone acetate (DOCA) hypertension induced vasculardementia

Rats, were administered DOCA (20 mg/kg, s.c. twice weekly) for90 days and the drinking water was replaced with solution of 1% so-dium chloride (NaCl) and 0.2% potassium chloride (KCl), to producehypertension (Bockman et al., 1992). The animals were used on86th day for the behavioral and other assessment. Mean arterialblood pressure (MABP) was measured by BIOPAC MP100, using Acq-Knowledge 3.8.2. analysis system.

2.4. Telmisartan and donepezil administration

Rats, were administered telmisartan (5 mg kg−1 p.o., daily and10mg kg−1 p.o., daily) by suspending it in CMC (1% w/v) for 40 daysin telmisartan per se (low dose and high dose) group. Furthermore,telmisartan (5 mg kg−1 p.o., daily and 10 mg kg−1 p.o., daily) was ad-ministered for last 40 days viz. day 51 to day 90 (35 days+5 days dur-ing Morris water maze exposure) in DOCA treated animals.

Similarly, rats, were administered donepezil (0.5 mg kg−1 i.p.,daily) by dissolving it in 0.9% saline, for 40 days in donepezil per segroup. Furthermore, donepezil (0.5 mg kg−1 i.p., daily) was adminis-tered for last 40 days viz. day 51 to day 90 (35 days+5 days duringMorris water maze exposure) in DOCA treated animals.

Doses of the drugs were selected on the bases of on our pilot stud-ies and previously published research reports (Hamed and Malek,2007; Mogi et al., 2008; Sharma and Singh, 2010, 2011a, 2011b).

2.5. Experimental protocol

In total 88 male Wistar rats were employed which were divided into eleven groups and each group was consisted of eight animals.

2.5.1. Group I—Control groupAnimals were exposed to Morris water maze for acquisition trial

from Day 1 to Day 4 and retrieval trial on Day 5. These animalswere not administered with any vehicle or chemical agent.

2.5.2. Group II—Vehicle control group (0.9% saline)Animals were administered saline (10 ml kg−1 i.p., daily) for

35 days followed by exposure to Morris water maze. The treatmentwas continued during acquisition (from 36th to 39th day) and re-trieval trials (on 40th day) on Morris water maze.

2.5.3. Group III—Vehicle control group (CMC)Animals were administered CMC (10 ml kg−1 i.p., daily), for

35 days rest of the procedure was same as described in group II.

2.5.4. Group IV—Vehicle control group (Arachis oil)Animals were administered with arachis oil (maximum

2.5 ml kg−1 s.c., twice weekly), for 85 days followed by exposure toMorris water maze. The treatment was continued during acquisition(from 86th to 89th day) and retrieval trials (on 90th day) on Morriswater maze.

2.5.5. Group V—DOCA treatment groupAnimals were administered DOCA (20 mg kg-1 s.c., twice weekly),

for 85 days followed by exposure to Morris water maze. The treat-ment was continued during acquisition (from 86th to 89th day) andretrieval trials (on 90th day) on Morris water maze. Drinking waterof these animals was replaced with the solution of 1% NaCl and 0.2%KCl.

2.5.6. Group VI—Telmisartan low dose per seAnimals were administered telmisartan (5 mg kg−1 p.o., daily),

for 35 days rest of the procedure was same as described in group II.

103B. Sharma, N. Singh / Pharmacology, Biochemistry and Behavior 102 (2012) 101–108

2.5.7. Group VII—Telmisartan high dose per seAnimals were administered telmisartan (10 mg kg−1 p.o., daily),

for 35 days rest of the procedure was same as described in group II.

2.5.8. Group VIII—DOCA and Telmisartan low doseTelmisartan (5 mg kg−1 p.o., daily) was administered to the DOCA

treated rats, starting from 51st day of DOCA treatment followed byexposure to Morris water maze on 86th day of DOCA administration.The treatment was continued during acquisition (from 86th to 89thday) and retrieval trials (on 90th day) on Morris water maze. Drink-ing water of these animals was replaced with the solution of 1%NaCl and 0.2% KCl.

2.5.9. Group IX—DOCA and Telmisartan high doseTelmisartan (10 mg kg−1 p.o., daily) was administered to the

DOCA treated rats rest of the procedure was same as described ingroup VIII. Drinking water of these animals was replaced with the so-lution of 1% NaCl and 0.2% KCl.

2.5.10. Group X—Donepezil per seAnimals were administered donepezil (0.5 mg kg−1 i.p., daily), for

35 days rest of the procedure was same as described in group II.

2.5.11. Group XI—DOCA and DonepezilDonepezil (0.5 mg kg−1 i.p., daily) was administered to the DOCA

(20 mg kg−1 s.c., twice weekly), treated rats, starting from 51st dayof DOCA treatment followed by exposure to Morris water maze on86th day of DOCA administration. The treatment was continued dur-ing acquisition (from 86th to 89th day) and retrieval trials (on 90thday) on Morris water maze. Drinking water of these animals wasreplaced with the solution of 1% NaCl and 0.2% KCl.

2.6. Assessment of learning and memory by Morris water maze

Morris water maze (Morris, 1984; Parle and Singh, 2004; Sharmaand Singh, 2010, 2011a, 2011b) is one of the most commonly usedanimal models to test memory. MWM consisted of large circularpool (150 cm in diameter, 45 cm in height, filled to a depth of30 cm with water at 28 °C). The water was made opaque with whitecolored dye. The tank was divided into four equal quadrants withhelp of two threads, fixed at right angle to each other on the rim ofthe pool. A submerged platform (10 cm²), painted white was placedinside the target quadrants of this pool, 1 cm below surface ofwater. The position of platform was kept unaltered throughout thetraining session. Each animal was subjected to four consecutive trialson each day with gap of 5 min. The rat was gently placed in the waterof the pool between quadrants, facing the wall of pool with drop loca-tion changing for each trial, and allowed 120 s to locate submergedplatform. Then, it was allowed to stay on the platform for 20 s. If itfailed to find the platform within 120 s, it was guided gently ontoplatform and allowed to remain there for 20 s. Day 4 escape latencytime (ELT) to locate the hidden platform in water maze was notedas index of acquisition or learning. Daily starting positions were ran-domized and not repeated on each day and quadrant 4 (Q4) wasmaintained as target quadrant in all acquisition trials. On fifth day,platform was removed and rats were allowed to explore in the poolfor 120 s. Each rat was subjected to four such trials and each trialwas started from different quadrant. Mean time spent in all fourquadrants i.e. Q1, Q2, Q3 and Q4 were recorded and the time spent inthe target quadrant i.e. Q4 in search of missing platform provided anindex of retrieval. The experimenter was always stood at the same po-sition. Care was taken that relative location of water maze with respectto other objects in the laboratory serving, as prominent visual clueswere not disturbed during the total duration of study. All the trialswere completed during the light cycle i.e. between 09.00 and 18.00 hrs.

2.7. Biochemical parameters

2.7.1. Collection of sampleBlood samples for biochemical estimation were collected by retro-

orbital bleeding. The blood was kept at room temperature for 30 minand then centrifuged at 4000 rpm for 15 min to separate serumwhichwas then used for biochemical estimation.

After retro-orbital bleeding, animals were sacrificed by cervicaldislocation; thoracic aorta and brain tissue were carefully removed.Thoracic aorta was used for endothelium dependent and independentrelaxation (as per the procedure defined below in Section 2.8), as wellas for the estimation of superoxide anion, whereas brains were sub-jected to various biochemical estimations (brain TBARS, GSH, AChEand proteins). The removed brains were homogenized in phosphatebuffer (pH 7.4, 10% w/v) using Teflon homogenizer and centrifugedat 3000 rpm for 15 min to obtain the clear supernatant. This clear su-pernatant (TBARS, GSH, AChE and proteins) containing was removedcarefully from the centrifugation tube and it was then used for differ-ent biochemical estimations.

2.7.2. Estimation of serum nitrite concentrationSerum nitrite concentration was measured spectophotometrically

(DU 640B Spectrophotometer, Beckman Coulter Inc., CA, USA) at545 nm, using method of Sastry and colleagues (Sastry et al., 2002;Sharma and Singh, 2010).

2.7.3. Estimation of aortic production of super oxide anionThe superoxide anion was determined spectrophotometrically

(DU 640B Spectrophotometer, Beckman Coulter, Inc.) at 540 nmusing method of Wang and colleagues (Sharma and Singh, 2010;Wang et al., 1998).

2.7.4. Estimation of brain Acetyl Cholinesterase (AChE) ActivityThe whole brain AChE activity was measured spectrophotometeri-

cally (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at420 nm by the method of Ellman and colleagues (Ellman et al., 1961;Sharma and Singh, 2010; Voss and Sachsse, 1970).

2.7.5. Estimation of thiobarbituric acid reactive substances (TBARS)The brain/serum TBARS was measured spectrophotometrically

(DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at532 nm using method of Ohkawa and colleagues (Ohokawa et al.,1979; Sharma and Singh, 2010).

2.7.6. Estimation of reduced glutathione (GSH)The reduced glutathione (GSH) content in brain was estimated

spectrophotometrically (DU 640B spectrophotometer, Beckman Coul-ter Inc., CA, USA) at 412 nm using method of Beutler and colleagues(Beutler et al., 1963; Sharma and Singh, 2010).

2.7.7. Estimation of brain total proteinThe brain total protein was determined spectrophotometrically

(DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at750 nm using method of Lowry and colleagues (Lowry et al., 1951;Sharma and Singh, 2010).

2.8. Assessment of vascular endothelial function using isolated rat aorticring preparation

Thoracic aorta was removed (as per the procedure defined abovein Section 2.7.1), cut into a ring of 4 to 5 mm width, and mountedin organ bath containing Krebs–Henseleit bubbled with carbonatedoxygen (95% O2:5% CO2), and maintained at 37.8 C. The preparationwas allowed to equilibrate for 90 min under 1.5 g tension. The iso-metric contractions were recorded (Pieper, 1997) with a force-displacement transducer (Ft-2147) connected to Physiograph (INCO,

Table 1Effect of various agents, on mean arterial blood pressure (MABP) and day 4 escape la-tency time (ELT) of animals, using Morris water maze (MWM).

Group Name of thegroup

Mean arterial bloodpressure (mm Hg)

Escape latency time (in s)

Basal Final(Day 1 MWM)

Day 1 Day 4

I Control 88.1±2.3 92.2±2.9 99.2±2.3 42.2±2.6a

II Vehicle control(0.9% NaCl)

90.3±3.2 91.1±2.7 101.5±3.2 48.2±4.3a

III Vehicle control(CMC)

89.5±2.8 90.6±2.5 103.4±2.7 49.6±3.6a

IV Vehicle control(Arachis oil)

87.7±3.3 89.1±3.1 97.4±3.5 44.2±2.8a

V DOCA 90.7±2.4 165.6±4.2⁎ 106.3±2.8 82.2±3.6b

VI Telmisartan lowdose per se

88.3±3.9 91.1±2.8 103.1±3.8 45.2±4.1a

VII Telmisartan highdose per se

89.6±2.8 90.8±2.4 101.2±3.7 49.4±3.5a

VIII DOCA andTelmisartan lowdose

89.2±4.1 128.1±2.1⁎, # 103.5±2.2 64.2±1.7a, c

IX DOCA andTelmisartan highdose

90.1±3.6 109.4±3.3⁎, # 104.2±3.4 57.7±2.5a, c

X Donepezil per se 88.4±3.3 92.2±2.9 103.5±2.7 44.2±3.6a

XI DOCA andDonepezil

91.5±3.4 161.5±3.3⁎ 112.1±3.2 53.1±4.4a, c

n=8, Results are mean±standard error of means; one way ANOVA followed byTukey's multiple range test.NaCl—sodium chloride; CMC—sodium carboxymethylcellulose;DOCA—deoxycorticosterone acetate.⁎ pb0.05 versus MABP of control group.# pb0.05 versus MABP of DOCA treated group.a pb0.05 versus day 1 ELT in respective group.b pb0.05 versus day 4 ELT of control group.c pb0.05 versus day 4 ELT of DOCA treated group.

104 B. Sharma, N. Singh / Pharmacology, Biochemistry and Behavior 102 (2012) 101–108

Ambala, India). The preparation was primed with 80 mmol L–1− KCl tocheck its functional integrity and to improve its contractility. The cumula-tive dose responses of acetylcholine (ACh; 10–8 to 10–4 mol L−1) orsodium nitroprusside (SNP; 10–8 to 10–4 mol L−1) were recorded inphenylephrine (3×10–6 mol L−1) precontracted preparations (Koladiyaet al., 2008, 2009; Sharma and Singh, 2010, 2011a, 2011b). The intimallayer of aortic ring was rubbed gently with a moistened filter paperfor 30 s to obtain endothelium-free preparations. Loss of ACh(1×10–6 mol L−1) induced relaxation confirmed the absence of vas-cular endothelium (Koladiya et al., 2008, 2009; Sharma and Singh,2010, 2011a, 2011b).

2.9. Statistical analysis

Statistical analyses were done using GraphPad Prism v5.01. All re-sults were expressed as mean±S.E.M. Data for isolated aortic ringpreparation were statistically analyzed using repeated measures ofanalysis of variance (ANOVA) followed by Newman Keul's test. Allother results were analyzed using one-way ANOVA followed byTukey's multiple range test. Pb0.05 was considered to be statisticallysignificant.

3. Results

3.1. Effect on escape latency time (ELT) and time spent in target quadrant(TSTQ), using Morris water maze (MWM)

Before subjecting the animals to MWM test, their motor coordina-tion scores were measured by employing Rota rod test. However, nosignificant difference was noted between scores of hypertensionand control animals (data not shown). Control rats showed a down-ward trend in their ELT. There was a significant fall in day 4 ELT,when compared to day 1 ELT of these rats (Table 1), reflecting normallearning ability.

Further on day 5 a significant rise in TSTQ was observed, whencompared to time spent in other quadrants (Fig. 1), reflecting normalretrieval as well. Administration of 0.9% saline water (10 ml kg−1 i.p.,40 days), CMC (10 ml kg−1 p.o., 40 days) and arachis oil (maximum

Fig. 1. Effect on time spent in target quadrant of animals using Morris water-maze. n=8, Resrange test. a pb0.05 versus mean time spent in other quadrants in control; b pb0.05 versus mtarget quadrant (TSTQ) in DOCA treated group. NaCl—sodium chloride; CMC—sodium carbo

2.5 ml kg−1 s.c., twice weekly for 90 days) did not show any signifi-cant effect on ELT and TSTQ as compared to control rats. Administra-tion of telmisartan (5 mg kg−1 p.o. and 10 mg kg−1 p.o., 40 days) aswell as donepezil (0.5 mg kg−1 i.p., 40 days) did not show any

ults are mean±standard error of means; one way ANOVA followed by Tukey's multipleean time spent in target quadrant in control group; c pb0.05 versus mean time spent inxymethylcellulose; DOCA—deoxycorticosterone acetate.

Fig. 2. Effect on acetylcholine induced endothelium dependent relaxation using aorticring preparation. n=8, Responses are expressed as percentage of precontraction in-duced by 3×10−6 M phenylephrine. Results are mean±standard error of means; Re-peated measure ANOVA followed by Newman Keul's test. a pb0.05 versus control;b pb0.05 versus DOCA treated group. Ach—acetylcholine; DOCA—deoxycorticosteroneacetate.

105B. Sharma, N. Singh / Pharmacology, Biochemistry and Behavior 102 (2012) 101–108

significant per se effect on ELT and TSTQ (Table 1 and Fig. 1). HoweverDOCA (20 mg kg−1 s.c., twice weekly for 90 days) treated rats ?>showed a significant increase in day 4 ELT (89th day of DOCA treat-ment), when compared to day 4 ELT of control animals (Table 1) in-dicating impairment of acquisition. Further DOCA administrationalso produced a significant decrease in day 5 TSTQ (90th day of DOCAtreatment), when compared day 5 TSTQ of control animals (Fig. 1), in-dicating impairment of memory as well.

Administration of telmisartan (low and high dose) as well asdonepezil, significantly prevented DOCA induced rise in day 4 ELT, indi-cating reversal of DOCA induced impairment of acquisition (Table 1).Further treatment of these drugs also attenuated DOCA induced de-crease in day 5 TSTQ in a significant manner, indicating reversal ofDOCA induced impairment of memory (Fig. 1).

3.2. Effect on endothelium dependent and independent relaxation

Acetylcholine (ACh) and sodium nitroprusside (SNP) in a dose de-pendent manner produced endothelium dependent and independentrelaxation in phenylephrine (3×10−6 M) precontracted isolated rataortic ring preparation. DOCA administration significantly attenuatedacetylcholine induced endothelium dependent relaxation (Fig. 2),

however it did not affect SNP induced endothelium independent re-laxation (data not shown).

Treatment of telmisartan (low and high dose) as well as donepezil,significantly abolished the effect of DOCA on endothelial dependentrelaxation. Further telmisartan (low and high dose) as well as done-pezil, did not show any per se effect on endothelium dependent relax-ation. There was no significant effect observed in vehicle (0.9% NaCl,1% CMC and arachis oil) as well as per se (telmisartan and donepezil)treated groups (for better representation of the significant effects ofDOCA and drug treated DOCA groups, vehicle and per se groups arenot included in Fig. 2).

3.3. Effect on mean arterial blood pressure (MABP)

Administration of DOCA produced a significant increase in MABP ascompared to control rats. Treatment with donepezil, did not show anysignificant change in DOCA induced increase in MABP (Table 1). Telmi-sartan (low and high dose) has significantly reduced the DOCA inducedincrease in MABP. Further, telmisartan (low and high dose) as well asdonepezil, did not show any significant per se effect on MABP of the an-imals (Table 1).

3.4. Effect on serum nitrite level

Administration of DOCA produced a significant decrease in serumnitrite, when compared to control rats. Treatment with telmisartan(low and high dose) as well as donepezil, prevented DOCA induceddecrease in serum nitrite level in a significant manner (Table 2). Fur-ther, telmisartan (low and high dose) as well as donepezil, did notshow any significant per se effect on serum nitrite level (Table 2).

3.5. Effect on brain acetyl cholinesterase (AChE) activity

Administration of DOCA produced a significant, increase in brainAChE activity, when compared to control rats. Treatmentwith telmisar-tan (low and high dose) as well as donepezil, significantly preventedDOCA induced rise in brain AChE activity. Further, telmisartan (lowand high dose) as well as donepezil, did not show any significant perse effect on brain AChE activity (Table 2).

3.6. Effect on oxidative stress levels

Administration of DOCA produced a significant increase, in aorticsuperoxide anion level, brain and serum thiobarbituric acid reactivespecies (TBARS) (Table 2) and a significant decrease, in the brainlevels of reduced form of glutathione (GSH) (Table 2), when com-pared to control rats; hence reflecting induction of oxidative stress.Treatment with telmisartan (low and high dose) as well as donepezil,significantly prevented DOCA induced oxidative stress. Further, telmi-sartan (low and high dose) as well as donepezil, did not show any sig-nificant per se effect on oxidative stress level (Table 2).

4. Discussion

In our study chronic administration of DOCA (20 mg kg−1 s.c., twiceweekly for 90 days) has resulted in marked hypertension. DOCA hyper-tensive rat is a widely used model for the assessment of hypertensivecondition and its secondary complications including vascular endothelialdysfunction (Bockman et al., 1992; Kandlikar and Fink, 2011; Ocaranzaet al., 2011; Uddin et al., 2011).

DOCA hypertensive rats performed poorly on MWM test, indicatingimpairment in their learning abilities and memory capacities. Further-more, a significant rise in brain AChE activity, brain and serum TBARS,aortic superoxide anion along with a fall in brain GSH and serumnitrite/nitrate levels was also noted. Morris water maze employed inthe present study is one of the most widely used and accepted model

Table 2Effect of various agents on oxidative stress (superoxide anion, TBARS, GSH) and brain acetyl cholinesterase (AChE) activity and serum nitrite/nitrate levels of animals.

Group Name of the group Serum nitrite/nitrate (μM/L)

Serum TBARS(μM/L)

Aortic superoxide anion(Reduced NBT—pM/min/mg)

Brain AchE activity (μM of AChhydrolyzed/min/mg protein)

Brain TBARS(nM/mg protein)

Brain GSH(μM/mg of protein)

I Control 12.8±2.2 3.8±0.6 3.7±0.4 3.1±0.5 4.2±0.7 18.2±2.1II Vehicle control (0.9%

NaCl)11.2±3.1 3.7±0.4 3.6±0.5 3.3±0.7 4.1±0.6 17.3±2.4

III Vehicle control (CMC) 12.2±2.2 3.8±0.4 3.5±0.4 3.0±0.4 3.9±0.9 18.4±2.9IV Vehicle control

(Arachis oil)11.6±1.8 3.6±0.3 3.3±0.7 2.9±0.9 4.5±0.5 17.5±2.6

V DOCA 6.5±2.5a 7.3±1.1a 6.9±0.7a 11.6±1.7a 11.2±1.2a 7.2±3.1a

VI Telmisartan low doseper se

12.6±2.5 3.4±0.7 3.3±0.4 3.4±0.5 4.4±1.5 18.4±2.1

VII Telmisartan high doseper se

13.1±2.4 3.2±0.4 3.6±0.6 3.2±0.8 4.9±0.9 17.6±3.5

VIII DOCA and Telmisartanlow dose

9.6±2.1b 5.7±1.8b 5.2±0.6b 7.8±1.7b 6.9±1.4b 11.5±1.3b

IX DOCA and Telmisartanhigh dose

9.9±1.4b 4.4±2.2b 4.4±0.5b 6.9±2.1b 6.1±2.2b 12.8±2.1b

X Donepezil per se 12.1±3.2 3.7±0.4 3.9±0.7 3.4±0.6 4.2±0.7 17.5±3.3XI DOCA and Donepezil 9.9±1.2b 6.1±0.7b 6.0±0.3b 4.5±0.9b 7.6±0.8b 13.7±3.2b

n=8, Results are mean±standard error of means; one way ANOVA followed by Tukey's multiple range test.TBARS—thiobarbituric acid reactive species; GSH—reduced form of glutathione; NaCl—sodium chloride; CMC—sodium carboxymethylcellulose; DOCA—deoxycorticosteroneacetate.

a pb0.05 versus control group.b pb0.05 versus DOCA treated group.

106 B. Sharma, N. Singh / Pharmacology, Biochemistry and Behavior 102 (2012) 101–108

to test learning and memory of rodents (Koladiya et al., 2008, 2009;Morris, 1984; Sain et al., 2011; Sharma and Singh, 2010, 2011a,2011b). Chronic administration of DOCA, in our study produced signif-icant degree of vascular endothelial dysfunction reflected by impair-ment of acetylcholine induced endothelial dependent relaxation ofaortic strips, reduction in serum nitrite/ nitrate levels and increase oxi-dative stress (increase in aortic superoxide anion in thoracic aorta andserum TBARS) was also noted. these findings are in line with previousfindings (Borde et al., 2011; Szasz and Watts, 2010; Sahan-Firat et al.,2010; Szasz and Watts, 2010). Moreover in our earlier studies wehave demonstrated that vascular endothelial dysfunction in additionto impairment of memory and oxidative stress produces rise in brainAChE activity (Koladiya et al., 2008, 2009; Sharma and Singh, 2010,2011a, 2011b). Previous studies from other labs also suggest thatAChE activity is enhanced in oxidative stress (Giridharan et al., 2011),memory impairment (Giridharan et al., 2011; Agrawal et al., 2009)and in vascular dysfunction (Valsecchi et al., 2011). To claim that risein brain AChE activity is due to vascular dysfunctionwill needmore rig-orous and specifically planned research work on this aspect. Therefore,the observed DOCA induced vascular dementia may be due to increasein oxidative stress both in peripherally as well as centrally, impairmentof endothelial function and increase in brain acetylcholinesterase activ-ity. Further this is the first study of its kind which is reporting the gen-esis of VaD by DOCA-salt induced experimental hypertension.

In this investigation treatments of telmisartan as well as donepezilto DOCA hypertensive rats attenuated endothelial dysfunction, learn-ing and memory deficits as well as altered biochemical parameters. Inaddition, telmisartan also produced a significant reduction in MABP ofhypertensive rats.

Pharmacotherapy targeting theRAS is one of themost effectivemeansof reducing hypertension and cardiovascularmorbidity (Braunwald et al.,2004; Matchar et al., 2008). Both, T1 and AT2 receptors have been foundto bewidely distributed throughout the brain and vasculature (Gallinat etal., 1998). Activation of AT1 receptors leads to vasoconstriction, cellularproliferation, and inflammation, which results into vascular malfunction,remodeling and atherosclerosis; eventually causing cerebrovascular in-sufficiency and neuronal dysfunction (Lu et al., 2005; Takeda et al.,2009. An increasing number of studies have shown a relation betweenARB and preservation of cognitive function (Hanon et al., 2008; Fogariet al., 2003; Poon, 2008; Raghavendra et al., 1998; Tedesco et al., 1999).It has also been reported that ARBs improved neurovascular dysfunction

and decreased ROS production in dementia (Girouard and Iadecola,2006; Saavedra, 2005; Ozacmak et al., 2007. Further ARBs have beenreported to restore long term potentiation in the hippocampus of ADmice and ameliorated the impairment of cerebrovascular regulation.Brain activity critically depends on a continuous blood supply, and afocal increase in cerebral blood flow in a functionally activated areasupports normal cognitive function (Girouard and Iadecola, 2006).This depends on the brain microcirculation (Haydon and Carmignoto,2006) and ARBs have been suggested to improve the microcirculationor evenmay act directly on neurons in the brain to exert a neuroprotec-tive effect (Takeda et al., 2009). Telmisartan and other ARBs have beenreported to improve the endothelial dysfunction in periphery (Satoh etal., 2010; Takiguchi et al., 2011) and even in cerebral cortex (Oyama etal., 2010). Therefore, the beneficial effect of telmisartan in DOCA hyper-tension induced vascular dementia is due to its multiple peripheral andcentral effects viz. antihypertensive, enhancement of endothelial func-tion, antioxidative, antiacetylcholinesterase activity, neuroprotectiveetc. It is important to note here that this research work for the firsttime is reporting that telmisartan, an ARB recuperate the experimentalhypertension induced VaD.

It has been reported that VaD patients present cholinergic deficitsin the brain and cerebrospinal fluid that may be closely related tothe pathophysiology of cognitive impairment. Moreover, cholinergictherapies have shown promising effects on cognitive improvementin VaD patients (Wang et al., 2009). Acetylcholinesterase inhibitors(AChEIs) are the main class of drugs which are frequently used forthemanagement of memory deficits. AChEIs has been reported to pro-vide protection to the endothelial function (Borroni et al., 2005;Koladiya et al., 2008, 2009; Sharma and Singh, 2010, 2011a, 2011b),that leads in enhanced cerebral perfusion (Maltz et al., 2004) and im-proved dynamic cerebrovascular regulation (Rosengarten et al., 2006).The precise mechanisms of these cholinergic agents are currently notfully understood; however, accumulating evidence indicates that thesedrugs may act through the cholinergic anti-inflammatory pathway, inwhich the efferent vagus nerve signals suppress pro-inflammatory cyto-kine release and inhibit inflammation, although regulation of oxidativestress and energy metabolism, alleviation of apoptosis may also be in-volved (Wang et al., 2009). Various reports from our lab have shownthe well defined anti-oxidative stress activity of donepezil along withanti-acetylcholinesterase activity. Donepezil induced reversal of learningand memory is in line with our previous research work (Koladiya et al.,

107B. Sharma, N. Singh / Pharmacology, Biochemistry and Behavior 102 (2012) 101–108

2008, 2009; Sain et al., 2011; Sharma et al., 2008a, 2008b; Sharma andSingh, 2010, 2011a, 2011b). But for the first time we are reporting thatdonepezil, an AChEI, recuperate the experimental hypertension inducedVaD.

Thus, it is concluded that DOCA-salt induced experimental hyper-tension is responsible for genesis of endothelial dysfunction and sub-sequent vascular dementia in rats. Telmisartan (an ARB) as well asdonepezil (an AChEI), have recuperated DOCA- salt hypertension in-duced endothelial dysfunction as well as vascular dementia in ratsand thus may be considered as potential pharmacological agents forthe management of hypertension associated vascular dementia. Nev-ertheless, this is the first report of its kind which shows induction ofvascular dementia by experimental hypertension, further studies areneeded to explore the impact of experimental hypertension in induc-tion of vascular dementia.

Conflict of interest

None.

Role of the funding source

None.

Acknowledgments

Authors are thankful to Department of Pharmaceutical Sciencesand Drug Research, Faculty of Medicine, Punjabi University, Patiala,Punjab, India for providing all the necessary facilities to conduct thisresearch. We are also thankful to Mr. A.S. Jaggi, Assistant Prof. Phar-macology for his valuable suggestions.

References

Agrawal R, Tyagi E, Shukla R, Nath C. A study of brain insulin receptors, AChE activityand oxidative stress in rat model of ICV STZ induced dementia. Neuropharmacolo-gy 2009;56(4):779–87.

Battistin L, Cagnin A. Vascular cognitive disorder: a biological and clinical overview.Neurochem Res 2010;35(12):1933–8.

Beutler E, Duron O, Kelly B. Reduced glutathione estimation. J Lab Clin Med 1963;61:82.Bockman CS, Jeffries WB, Pettinger WA, Abel PW. Reduced contractile sensitivity and

vasopressin receptor affinity in DOCA-salt hypertension. Am J Physiol 1992;262:1752–8.

Borde P, Mohan M, Kasture S. Effect of myricetin on deoxycorticosterone acetate(DOCA)-salt-hypertensive rats. Nat Prod Res 2011;25(16):1549–59.

Borroni B, Agosti C, Martini G, Volpi R, Brambilla C, Caimi L, et al. Cholinesterase inhib-itors exert a protective effect on endothelial damage in Alzheimer disease patients.J Neurol Sci 2005;229–230:211–3.

Braunwald E, Domanski MJ, Fowler SE, Geller NL, Gersh BJ, Hsia J, et al. PEACE TrialInvestigators: angiotensin-converting-enzyme inhibition in stable coronary arterydisease. N Engl J Med 2004;351:2058–68.

Danielyan L, Klein R, Hanson LR, Buadze M, Schwab M, Gleiter CH, et al. Protectiveeffects of intranasal losartan in the APP/PS1 transgenic mouse model of Alzheimerdisease. Rejuvenation Res 2010;13(2–3):195–201.

Delrieu J, Piau A, Caillaud C, Voisin T, Vellas B. Managing cognitive dysfunction throughthe continuum of Alzheimer's disease: role of pharmacotherapy. CNS Drugs2011;25(3):213–26.

Duron E, Hanon O. Antihypertensive treatments, cognitive decline, and dementia.J Alzheimers Dis 2010;20(3):903–14.

Ellman GL, Courtney DK, Andres V, Feathstone RM. A new and rapid colorimeteric de-termination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88–95.

Fogari R, Mugellini A, Zoppi A, Derosa G, Pasotti C, Fogari E, et al. Influence of losartanand atenolol onmemory function in very elderly hypertensive patients. J HumHypertens 2003;17:781–5.

Gallinat S, Yu M, Dorst A, Unger T, Herdegen T. Sciatic nerve transection evokes lastingup-regulation of angiotensin AT2 and AT1 receptor mRNA in adult rat dorsal rootganglia and sciatic nerves. Brain Res Mol Brain Res 1998;57:111–22.

Giridharan VV, Thandavarayan RA, Sato S, Ko KM, Konishi T. Prevention ofscopolamine-induced memory deficits by schisandrin B, an antioxidant lignanfrom Schisandra chinensis in mice. Free Radic Res 2011;45(8):950–8.

Girouard H, Iadecola C. Neurovascular coupling in the normal brain and in hyperten-sion, stroke, and Alzheimer disease. J Appl Physiol 2006;100:328–35.

Hamed AA, Malek HA. Effect of telmisartan in experimentally induced diabetes mellitusin rats. Int J Health Sci (Qassim) 2007;1(2):249–56.

Hanon O, Berrou JP, Negre-Pages L, Goch JH, Nádházi Z, Petrella R, et al. Effects ofhypertension therapy based on eprosartan on systolic arterial blood pressure and

cognitive function: primary results of the Observational Study on Cognitive Functionand Systolic Blood Pressure Reduction Open-label Study. J Hypertens 2008;26:1642–50.

Haydon PG, Carmignoto G. Astrocyte control of synaptic transmission and neurovascularcoupling. Physiol Rev 2006;86:1009–31.

Horiuchi M, Mogi M. Role of angiotensin II receptor subtype activation in cognitivefunction and ischaemic brain damage. Br J Pharmacol 2011;163(6):1122–30.

Kalaria RN. Vascular basis for brain degeneration: faltering controls and risk factors fordementia. Nutr Rev 2010;68(2):S74–87.

Kandlikar SS, Fink GD. Mild DOCA-salt hypertension: sympathetic system and role ofrenal nerves. Am J Physiol Heart Circ Physiol 2011;300(5):H1781-7.

Koladiya RU, Jaggi AS, Singh N, Sharma BK. Ameliorative role of atorvastatin and pita-vastatin in L-methionine induced vascular dementia in rats. BMC Pharmacol2008;8:14.

Koladiya RU, Jaggi AS, Singh N, Sharma BK. Beneficial effects of Donepezil on vascularendothelial dysfunction-associated dementia induced by L-Methionine in rats. JHealth Sci 2009;55(2):215–25.

Lowry OH, Rosebrough NJ, Far AL, Randall RJ. Protein measurement with folin–phenolreagent. J Biol Chem 1951;193:265–75.

Lu Q, Zhu YZ, Wong PT. Neuroprotective effects of candesartan against cerebral ische-mia in spontaneously hypertensive rats. Neuroreport 2005;16:1963–7.

Maltz JS, Eberling JL, Jagust WJ, Budinger TF. Enhanced cutaneous vascular response inAD subjects under donepezil therapy. Neurobiol Aging 2004;25(4):475–81.

Matchar DB, McCrory DC, Orlando LA, Patel MR, Patel UD, Patwardhan MB, et al. Sys-tematic review: comparative effectiveness of angiotensin-converting enzyme in-hibitors and angiotensin II receptor blockers for treating essential hypertension.Ann Intern Med 2008;148:16–29.

Mogi M, Li JM, Tsukuda K, Iwanami J, Min LJ, Sakata A, et al. Telmisartan prevented cog-nitive decline partly due to PPAR-gamma activation. Biochem Biophys Res Com-mun 2008;375(3):446–9.

Monsuez JJ, Gesquière-Dando A, Rivera S. Cardiovascular prevention of cognitive de-cline. Cardiol Res Pract 2011;2011:250970.

Moretti A, Gorini A, Villa RF. Pharmacotherapy and prevention of vascular dementia.CNS Neurol Disord Drug Targets 2011;10(3):370–90.

Morris RGM. Developments of a water maze producer for studying spatial learning inthe rats. J Neurosci Methods 1984;11:47–60.

Muiesan ML, Salvetti M, Belotti E, Paini A, Rosei CA, Aggiusti C, et al. Effects of barnidipinein comparison with hydrochlorothiazide on endothelial function, as assessed by flowmediated vasodilatation in hypertensive patients. Blood Press 2011;20(4):244–51.

OcaranzaMP, Rivera P, NovoaU, PintoM,González L, ChiongM, et al. Rho kinase inhibitionactivates the homologous angiotensin-converting enzyme-angiotensin-(1–9) axis inexperimental hypertension. J Hypertens 2011;29(4):706–15.

Ohokawa H, Ohishi N, Yagi K. Assay for lipid peroxided in animal tissues by thiobarbi-turic acid reaction. Anal Biochem 1979;95:351–8.

Oyama N, Yagita Y, Sasaki T, Omura-Matsuoka E, Terasaki Y, Sugiyama Y, et al. An an-giotensin II type 1 receptor blocker can preserve endothelial function and attenu-ate brain ischemic damage in spontaneously hypertensive rats. J Neurosci Res2010;88(13):2889–98.

Ozacmak VH, Sayan H, Cetin A, Akyildiz-Igdem A. AT1 receptor blocker candesartan-induced attenuation of brain injury of rats subjected to chronic cerebral hypoper-fusion. Neurochem Res 2007;32(8):1314–21.

Parle M, Singh N. Animalmodels for testingmemory. Asia Pac J Pharmacol 2004;16:101–20.Pieper GM. Diabetic induced endothelial dysfunction in rat aorta: role of hydroxyl rad-

icals. Cardiovasc Res 1997;34:145–56.Poon IO. Effects of antihypertensive drug treatment on the risk of dementia and cogni-

tive impairment. Pharmacotherapy 2008;28:366–75.Raghavendra V, Chopra K, Kulkarni SK. Involvement of cholinergic system in losartan-

induced facilitation of spatial and short-term working memory. Neuropeptides1998;32:417–21.

Riordan KC, Hoffman Snyder CR, Wellik KE, Caselli RJ, Wingerchuk DM, DemaerschalkBM. Effectiveness of adding memantine to an Alzheimer dementia treatment reg-imen which already includes stable donepezil therapy: a critically appraisedtopic. Neurologist 2011;17(2):121–3.

Rosenbloom MH, Brown J, Berlowitz DR, Budson AE. Safety and tolerability of galanta-mine SA in dementia patients transitioned from donepezil. Consult Pharm2011;26(2):108–10.

Rosengarten B, Paulsen S, Molnar S, Kaschel R, Gallhofer B, Kaps M. Acetylcholine ester-ase inhibitor donepezil improves dynamic cerebrovascular regulation in Alzheimerpatients. J Neurol 2006;253(1):58–64.

Saavedra JM. Brain Angiotensin II: New developments, unanswered questions andtherapeutic opportunities. Cell Mol Neurobiol 2005;25:485–512.

Sahan-Firat S, Jennings BL, Yaghini FA, Song CY, Estes AM, Fang XR, et al. 2,3′,4,5′-Tetra-methoxystilbene prevents deoxycorticosterone-salt-induced hypertension: contribu-tion of cytochrome P-450 1B1. Am J Physiol Heart Circ Physiol 2010;299(6):H1891–901.

Sain H, Sharma B, Jaggi AS, Singh N. Pharmacological investigations on potential of perox-isome proliferator-activated receptor-gamma agonists in hyperhomocysteinemia-induced vascular dementia in rats. Neuroscience 2011;192:322–33.

Sastry KV, Moudgal RP, Mohan J, Tyagi JS, Rao GS. Spectrophotometric determination ofserum nitrite and nitrate by copper–cadmium alloy. Anal Biochem 2002;306(1):79–82.

Satoh M, Haruna Y, Fujimoto S, Sasaki T, Kashihara N. Telmisartan improves endothelialdysfunction and renal autoregulation in Dahl salt-sensitive rats. Hypertens Res2010;33(2):135–42.

Sharma B, Singh N. Pitavastatin and 4′-hydroxy-3′-methoxyacetophenone (HMAP) re-duce cognitive dysfunction in vascular dementia during experimental diabetes.Curr Neurovasc Res 2010;7(3):180–91.

108 B. Sharma, N. Singh / Pharmacology, Biochemistry and Behavior 102 (2012) 101–108

Sharma B, Singh N. Attenuation of vascular dementia by sodium butyrate in streptozo-tocin diabetic rats. Psychopharmacology 2011a;215(4):677–87.

Sharma B, Singh N. Behavioral and biochemical investigations to explore pharmacolog-ical potential of PPAR-gamma agonists in vascular dementia of diabetic rats. Phar-macol Biochem Behav 2011b;100(2):320–9.

Sharma B, Singh N, Singh M. Modulation of celecoxib- and streptozotocin-induced ex-perimental dementia of Alzheimer's disease by Pitavastatin and Donepezil. J Psy-chopharmacol 2008a;22(2):162–71.

Sharma B, Singh N, Singh M, Jaggi AS. Exploitation of HIV protease inhibitor Indinavir asa memory restorative agent in experimental dementia. Pharmacol Biochem Behav2008b;89(4):535–45.

Szasz T, Watts SW. Uric acid does not affect the acetylcholine-induced relaxation ofaorta from normotensive and deoxycorticosterone acetate-salt hypertensive rats.J Pharmacol Exp Ther 2010;333(3):758–63.

Takeda S, Sato N, Takeuchi D, Kurinami H, Shinohara M, Niisato K, et al. Angiotensin re-ceptor blocker prevented beta-amyloid-induced cognitive impairment associatedwith recovery of neurovascular coupling. Hypertension 2009;54(6):1345–52.

Takiguchi S, Ayaori M, Uto-Kondo H, Iizuka M, Sasaki M, Komatsu T, et al. Olmesartanimproves endothelial function in hypertensive patients: link with extracellular su-peroxide dismutase. Hypertens Res. 2011;34(6):686–92. (Jun).

Tedesco MA, Ratti G, Mennella S, Manzo G, Grieco M, Rainone AC, et al. Comparison oflosartan and hydrochlorothiazide on cognitive function and quality of life in hyper-tensive patients. Am J Hypertens 1999;12:1130–4.

Uddin MN, Horvat D, Demorrows S, Agunanne E, Puschett JB. Marinobufagenin is anupstreammodulator of Gadd45a stress signaling in preeclampsia. Biochim BiophysActa 2011;1812(1):49–58.

Valsecchi AE, Franchi S, Panerai AE, Rossi A, Sacerdote P, ColleoniM. The soy isoflavone genis-tein reverses oxidative and inflammatory state, neuropathic pain, neurotrophic and vas-culature deficits in diabetes mouse model. Eur J Pharmacol 2011;650(2–3):694–702.

Voss G, Sachsse K. Red cell and plasma cholinesterase activities in microsamples ofhuman and animal blood determined simultaneously by a modified acetylthiocho-line/DTNB Procedure. Toxicol Appl Pharmacol 1970;16:764–72.

WangHD, Pagano PJ, Du Y, Cayatte AJ, QuinnMT, Brecher P, et al. Superoxide anion from theadventitia of the rat thoracic aorta inactivates nitric oxide. Circ Res 1998;82:810–8.

Wang J, ZhangHY, Tang XC. Cholinergic deficiency involved in vascular dementia: possiblemechanism and strategy of treatment. Acta Pharmacol Sin 2009;30(7):879–88.

Wehling M, Groth H. Challenges of longevity in developed countries: vascular preven-tion of dementia as an immediate clue to tackle an upcoming medical, social andeconomic stretch. Neurodegener Dis 2011;8(5):275–82.

Yang Q, Xue HM, Wong WT, Tian XY, Huang Y, Tsui SK, Underwood MJ, He GW, et al.AVE3085, an enhancer of endothelial nitric oxide synthase, restores endothelialfunction and reduces blood pressure in spontaneously hypertensive rats. Br J Phar-macol 2011;163(5):1078–85.

Zhang LN, Vincelette J, Chen D, Gless RD, Anandan SK, Rubanyi GM, et al. Inhibition ofsoluble epoxide hydrolase attenuates endothelial dysfunction in animal models ofdiabetes, obesity and hypertension. Eur J Pharmacol 2011;654(1):68–74.