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Behavioural Brain Research 226 (2012) 317– 330

Contents lists available at ScienceDirect

Behavioural Brain Research

j ourna l ho mepage: www.elsev ier .com/ locate /bbr

esearch report

entral angiotensin converting enzyme facilitates memory impairment inntracerebroventricular streptozotocin treated rats

antoshkumar Totaa, Pradeep Kumar Kamata, Gunjan Saxenaa, Kashif Hanifa,bul Kalam Najmic, Chandishwar Nathb,∗

Division of Pharmacology, Central Drug Research Institute, Lucknow U.P.IndiaDivision of Toxicology, Central Drug Research Institute, Lucknow U.P., IndiaDepartment of Pharmacology, Faculty of Pharmacy, Jamia Hamdard University, New Delhi, India

r t i c l e i n f o

rticle history:eceived 19 June 2011eceived in revised form 18 July 2011ccepted 25 July 2011vailable online 16 August 2011

eywords:ngiotensin converting enzymeemory

erindopriltreptozotocinerebral blood flowrain energy metabolismeurodegeneration

a b s t r a c t

Preclinical and clinical studies indicated involvement of renin angiotensin system (RAS) in memory func-tions. However, exact role of RAS in cognition is still ambiguous. Our aim was to explore how angiotensinconverting enzyme (ACE) modulates memory in experimental model of memory impairment. Memorydeficit was induced by intracerebroventricular administration of streptozotocin (STZ, 3 mg/kg) in rats.Perindopril, an ACE inhibitor, was given for 21 days and memory function was evaluated by Morris watermaze test. Cerebral blood flow (CBF) was measured by laser doppler flowmetry. The biochemical andexpression studies were done in cortex and hippocampus of rat brain after the completion of behavioralstudies. STZ caused impairment in memory along with significant reduction in CBF, ATP level and elevatedoxidative and nitrosative stress. The activity and mRNA expression of acetylcholinesterase (AChE) andACE were also increased in rat brain regions following STZ administration. However, serum ACE activityremained unaffected. Treatment with perindopril dose dependently improved memory by increasingenergy metabolism and CBF. Perindopril also decreased oxidative and nitrosative stress, activity and

mRNA expression of AChE and ACE in STZ treated rat. Further, ACE inhibition mitigated STZ induced neu-rodegeneration as observed in histopathological studies. Moreover, perindopril per se improved memoryand CBF, decreased oxidative stress with no effect on AChE activity and expression. However, perindoprilper se significantly reduced ACE activity but increased mRNA expression of ACE in rat brain. These resultssuggest that ACE occupies a pivotal role in STZ induced memory deficit thus implicating central RAS incognition.

. Introduction

Presence of the renin angiotensin system (RAS) has been estab-

ished in central nervous system where it is involved in theegulation of several homeostatic/physiological and behavioralrocesses such as thirst, temperature, sexual behavior and cog-itive functions [1,2]. Although the RAS plays a detrimental role

n neuronal damage during pathological conditions like cerebral

Abbreviations: ACE, angiotensin converting enzyme; AChE, acetyl-holinesterase; aCSF, artificial cerebrospinal fluid; AD, Alzheimer’s disease;�, amyloid beta; BSA, bovine serum albumin; DCF-DA, 2′ ,7′-dichlorofluorescein-iacetate; DTNB, 5,5′-dithiobis (2-nitrobenzoic acid); GSH, glutathione; ICV,

ntracerebroventricular; IP, intraperitoneal; MDA, malondialdehyde; RAS, reninngiotensin system; ROS, reactive oxygen species; SHR, spontaneously hyperten-ive rats; STZ, streptozotocin; TBA, 2-thiobabituric acid; TCA, trichloroacetic acid;EP, 1,1,3,3-tetraethoxypropane.

Communication No. 8120.∗ Corresponding author. Tel.: +91 522 2612411 18x4259; fax: +91 522 2623405.

E-mail address: cnathcdri@rediffmail.com (C. Nath).

166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2011.07.047

© 2011 Elsevier B.V. All rights reserved.

ischemia [3], its involvement in neurodegenerative diseases is stillbeing explored. The reports show that continuous exaggeration ofRAS is involved in the impaired cognitive functions [4] and evenour previous study had shown involvement of AT1 receptors inmodel of memory deficit induced by intracerebral (IC) strepto-zotocin (STZ) [5]. An increase in ACE activity has been reportedin the AD brain [6] but a recent study found no change in ACElevel with age [7]. Recent clinical evidences suggested that ACE isinvolved in cognitive dysfunction/dementia in AD patients becauseACE inhibitors delayed onset of dementia [8–11] and significantlydecreased the ACE activity in cerebrospinal fluid (CSF) [12]. Further,studies showed that ACE inhibitors positively influenced cognitivefunction independent of their blood pressure lowering effects, withpatients displaying better results than those on diuretics and beta-blockers [13]. Moreover, an in vitro study shows that ACE is involved

in A� cleavage as captopril promoted accumulation of cell-derivedA� in the media of beta-amyloid precursor-protein expressing [14].Conversely, it had also been shown that ACE did not have any effecton the A� metabolism [15]. Further, Miners et al. [7] showed that

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CE level correlated directly with insoluble and inversely with sol-ble A� in controls but not AD brain. However, Danielyan et al. [16]howed that treatment with losartan decreased amyloid beta leveln APP/PS1 transgenic mouse model of AD and one recent study byhu et al. [17] also reported facilitatory effect of central Ang II onmyloid beta production.

Further, a number of experimental studies tried to clear ambigu-us role of ACE in memory. It was reported that ACE inhibitionmproved memory function in aged rats [18]. Streptozotocin-iabetic rats, which had impaired memory in the water mazeask, demonstrated improved performance after inhibition of ACEy enalapril [19]. Further, memory impairment in spontaneouslyypertensive rats, Wistar-Kyoto rats and in scopolamine inducedemory deficit rodents was reversed by ACE inhibition by capto-

ril [20,21]. In another study, rats made hypertensive by Goldblattethod had a poor acquisition and retrieval of the learned behavior

ut this state was reversed by ACE inhibition [22]. Recently, experi-ental studies also revealed the beneficial effects of ACE inhibition

n cerebral hypoperfusion [23] and A� [24] induced models ofemory impairment in rodents.Evidences suggest that AD and other types of dementia are

ssociated with reduced cerebral blood flow (CBF), impaired brainnergy metabolism, cholinergic dysfunction and oxidative stress25–28]. Central injection of streptozotocin, in a sub diabetogenicose, in rodents had been found to cause impairment in learningnd memory. The ICV STZ induced memory impairment is reportedo be due to the increased oxidative stress, impairment in glucosetilization and energy metabolism, insulin receptor dysfunctionnd reduced acetylcholine synthesis because of decreased cholinecetyltransferase activity in brain [5,29–32]. Though the role ofCE in memory functions has been reported earlier to some extent

18–24], but its involvement in oxidative stress, cerebral blood flow,holinergic dysfunction and impaired brain energy metabolismssociated with memory dysfunction and neurodegeneration hasot been explored so far. Therefore, in this study, the role of cen-ral ACE in memory function was evaluated by using perindopril, alinically used ACE inhibitor, as an experimental pharmacologicalool, at the doses having no effect on blood pressure.

. Materials and methods

.1. Animals

The experiments were carried out with adult male Sprague Dawly (SD) rat225–250 g) procured from the Laboratory Animal Services Division of Central Drugesearch Institute, Lucknow, India. Experiments were performed according to inter-ationally followed ethical standards and approved by Institutional Animal Ethicsommittee (IAEC). They were kept in a polyacrylic cages (22.5 × 37.5 cm) with oneat per cage and maintained under standard housing conditions (room temperature4–27 ◦C and humidity 60–65%) with a 12 h light and dark cycle. Food and waterere available ad libitum but were withdrawn 1 h prior to administration of drugs

ill completion of the trial.

.2. Materials

Streptozotocin, chloral hydrate, sodium chloride (NaCl), 2-thiobarbituric acidTBA), 1,1,3,3 tetraethoxypropane (TEP), potassium chloride (KCl), magnesiumhloride (MgCl2), calcium chloride (CaCl2), bovine serum albumin (BSA), HEPES,cetylthiocholine iodide (AChI), N-(1-naphthyl) ethylenediamine dihydrochloride,ulfanilamide, Phosphoric acid, 2′ ,7′-dichlorofluorescein diacetate (DCF-DA), 5,5′-ithiobis (2-nitrobenzoic acid) (DTNB), hematoxylin and eosin (HE) were purchasedrom Sigma–Aldrich, USA. ATP colorimetric/fluorometric assay kit was purchasedrom Biovision, USA. Perindopril was purchased from Glenmark, India (Perigardablets).

.3. Drug administration

.3.1. Intracerebroventricular (ICV) injection of streptozotocin (STZ)Rats were anaesthetized with chloral hydrate (300 mg/kg, IP). Streptozotocin

STZ, 3 mg/kg) was dissolved in artificial CSF (aCSF) and slowly injected in a volume of0 �l into each lateral cerebral ventricle (ICV) on day 1 and 3 using the coordinates:.8 mm posterior to bregma, 1.5 mm lateral to sagittal suture, 3.6 mm ventral by

search 226 (2012) 317– 330

Hamilton microsyringe. In the aCSF group, artificial CSF (147 mM NaCl, 2.9 mM KCl,1.6 mM MgCl2, 1.7 mM CaCl2 and 2.2 mM dextrose) was injected in the same volumeas STZ on day 1 and 3 [31,32].

2.3.2. Experimental protocol and administration of perindoprilThe study was done with six rats per group. To study preventive effect in Morris

water maze test, different doses of perindopril (0.05 and 0.1 mg/kg) were adminis-tered orally for 21 days starting from first dose of STZ. Perindopril was administeredorally 1 h before STZ administration on day 1 and 3. The control, aCSF and STZgroup received saline for 21 days orally. Another group of animals was treated withperindopril (0.1 mg/kg, p.o) for 21 days to study per se (Rat + Perindopril − STZ) effect.

2.4. Assessment of learning and memory by Morris water maze test

The Morris water maze consisted of a large circular black pool of 120 cm diam-eter, 50 cm height, filled to a depth of 30 cm with water at 26 ± 2 ◦C. A black coloredround platform of 8 cm diameter was placed 1 cm below the surface of water ina constant position. The water was colored with non-toxic black dye to hide thelocation of the submerged platform. The pool was divided into four hypotheticalquadrants. Data were acquired through a video camera connected to the comput-erized tracking system (Columbus Instruments, USA) fixed above the center of thepool.

On the 14th day from 1st STZ injection, spatial learning and memory of animalswere tested in Morris water maze [33]. The rat could climb on the platform to escapefrom the necessity of swimming. Trials were given on 14th, 15th and 21st day after1st STZ injection in preventive study. The rat were given a maximum time of 120 s(cut-off time) to find the hidden platform and were allowed to stay on it for 30 s. Ratthat failed to locate the platform were put on it manually during first session only(acquisition trial). The animals were given a daily session of 5 trials per day. Latencytime to reach the platform was recorded in each trial. First trial of session 1 wasconsidered as acquisition trial and last trial of each session was taken as retentiontrials.

In order to assess the possibility of drug interference with animal sensory andmotor coordination or the animal motivation, the capability of rats to escape to avisible platform was tested in this study. For the test, platform was placed on a newlocation inside the pool 1 cm above the water line. Rats were allowed to swim for60 s. Time to reach the platform was recorded as escape latency [34].

A probe trial was performed 1 h after the last water maze session (on day 21) toaccess the extent of memory consolidation. The time spent in the target quadrantindicates the degree of memory consolidation that has taken place after learning.The individual rat was placed into the pool as in the training trial, except that thehidden platform was removed from the pool [34]. The time spent in target quad-rant was measured for 60 s. In probe trial, each rat was placed at a start positiondirectly opposite to platform quadrant. Further, the path length in target quadrantand number of times crossing over the platform site of each rat was also measuredand calculated.

2.5. Spontaneous locomotor activity

Spontaneous locomotor activity (SLA) was assessed Optovarimex activity meter(Columbus Inc, USA) prior to trial on water maze test to check any change in SLA,which might affect the memory tests. Each animal was observed for 5 min after aperiod of 10 min for acclimatization.

2.6. Measurement of blood pressure

Blood pressure (BP) of rats was measured by tail cuff method using non inva-sive blood pressure measurement technique (Columbus-8, USA). After having ratsacclimatized in restrainer for a week, basal BP of all the rats was measured beforeadministration of STZ (ICV) and/or perindopril. BP was again measured on day 21after the completion of treatment.

2.7. Measurement of cerebral blood flow

Cerebral blood flow was measured by laser doppler flowmetry (LDF 100, BIOPAC,USA). LDF qualitatively measures CBF in arbitrary blood perfusion units (BPU) [35].The rats were anesthetized with chloral hydrate (300 mg/kg, IP) and a 0.5 mm diam-eter micro-fiber laser doppler probe was fixed on the skull (6 mm lateral and 1 mmposterior of bregma) and CBF was monitored within cortical region [36]. CBF wasmeasured continuously at above-mentioned co-ordinates for a period of 10 minrecording values after each 30 s and then average values of blood flow were cal-culated [29,30].

2.8. Estimation of biochemical parameters

For biochemical assays, rats were sacrificed with over dose of ether anesthe-sia after completion of behavioral studies. Brain was removed quickly from eachrat, kept on ice-cold plate and then dissected into cerebral cortex and hippocam-pus according to the method of Glowinski and Iversen [37] and homogenized in

S. Tota et al. / Behavioural Brain Research 226 (2012) 317– 330 319

Table 1Primer sequences and specific conditions for RT-PCR study of AChE, ACE and actin.

Primer Sequence Annealing temperature (◦C) Product size (bp)

AChE Forward: 5′-GATCCCTCGCTGAACTACACC-3′ 60 331Reverse: 5′-GGTTCTTCCAGTGCACCATGTAGGAG-3′

ACE Forward: 5′-CGCTACAACTTCGACTGGTGG-3′ 62 881Reverse: 5′-TATTTCCGGGATGTGGCCAT-3′

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odium phosphate buffer (0.03 M, pH 7.4, 10% w/v) by using an Ultra-Turrax T25USA) homogenizer at a speed of 9500 rpm.

.8.1. Measurement of lipid peroxidationMDA, a measure of lipid peroxidation was measured spectrophotometrically by

sing 2-thiobarbituric acid as described previously [4,29,30,39].

.8.2. Measurement of glutathioneGlutathione (GSH), a natural antioxidant, level was determined by its reaction

ith 5,5′-dithiobis 2-nitrobenzoic acid as described in our previous research articles4,29,30,39].

.8.3. Measurement of nitrite levelNitrite level, a measure of nitric oxide production, was estimated by Griess

eagent according to our previous reports [4,29,30].

.8.4. Intracellular ROS estimationHomogenous suspension of rat brain regions (cortex and hippocampus) from

ach group was prepared in HEPES–Tyrode solution (145 mM NaCl, 5 mM KCl, 2 mMaCl2, 1 mM MgCl2, 5 mM glucose, 5 mM HEPES, pH 7.4) by treatment of the finelyhopped brain tissue with collagenase (750 unit/ml) for 45 min at 37 ◦C [38,39].ntracellular ROS was estimated by fluorimetric method using the oxidation sen-itive fluorescent probe 2′ ,7′-dichlorofluorescein (DCF) diacetate. After collagenasereatment the dissociated neurons were incubated with DCF (5 �M) for 15 min at7 ◦C and then washed with HBSS and analyzed by fluorimetry.

.8.5. Acetylcholinesterase (AChE) assayThe brain homogenate was mixed with 1% Triton X-100 and samples were cen-

rifuged at 100,000 × g at 4 ◦C in a Beckman Ultracentrifuge (LE 80, USA) for 60 min.upernatant was collected and acetylcholinesterase (AChE) activity was estimateds described previously [4,29,30].

.8.6. Estimation of angiotensin converting enzyme (ACE) activityAngiotensin converting enzyme (ACE) activity was measured by a spectropho-

ometric mono-reagent assay. This assay involved the enzymatic hydrolysis of theynthetic substrate, N-[3-(2-furyl) acryloyl]-l-phenylalanyl-glycylglycine (FAPGG)o N-[3-(2-furyl) acryloyl]-l-phenylalanine (FAP) and glycylglycine (GG). The reac-ion was monitored at 340 nm [40,41].

To estimate ACE activity in serum, the reaction mixture containing 10 �l oferum and 200 �l of the substrate solution were kept at 37 ◦C in ELISA plate readerBIOTEK, USA) and reaction was monitored at 340 nm. Reading was taken 0 min and0 min. Serum ACE activity was expressed as unit/liter.

The brain homogenate in volume of 500 �l was mixed with 1% Triton X-1001% w/v in 0.03 M sodium phosphate buffer, pH 7) and samples were centrifuged at00,000 × g at 4 ◦C in a Beckman Ultracentrifuge (LE 80, USA), using a fixed angleotor (80 Ti) for 60 min. Supernatant was collected and stored at 4 ◦C for estima-ion of ACE activity. To estimate ACE activity 10 �l of this supernatant was mixedith 200 �l of substrate solution and reaction was monitored at 340 nm at 37 ◦C for

0 min in ELISA plate reader. Brain ACE activity was expressed in units/mg protein.

.8.7. ATP assayATP was estimated in cortex and hippocampus of rat brain using ATP colorimet-

ic/fluorometric assay kit [29,42].

.9. Protein estimation

Protein was measured in all brain samples for nitrite, GSH and MDA by theethod of Lowry et al. [43] and protein for acetylcholine esterase and ACE activ-

ty was estimated by the method of Wang and Smith [44] by using bovine serumlbumin as standard.

.10. Angiotensin converting enzyme and acetylcholinesterase mRNA expressiony reverse transcription polymerase chain reaction

ACE and AChE mRNA expression were studied in cortex and hippocampus of ratrain by reverse transcription polymerase chain reaction. RNA was isolated from

55.7 352

brain using TRIzol reagent (Sigma) as directed by the manufacturer. Concentra-tion and purity of RNA were determined spectrophotometrically using GeneQuant.Approximately 2 �g of total RNA was reverse transcribed using reverse transcrip-tase (RT) in a 20-�l mixture containing oligo-(dT)-primer, RNase Inhibitor, dNTPmix and 5X reaction buffer (Omniscript RT kit).

The resultant cDNA was amplified separately with specific primer for ACE, AChEand actin using Taq PCR core kit (Qiagen, USA). Briefly, cDNA (2 �l) was amplified in a20 �l reaction volume containing 1 U Taq polymerase, 200 �M (each) dNTP mix and2 �l 10X Taq buffer with specific primers. The polymerase chain reaction mixturewas amplified in a DNA thermal cycler (Bioer XP cycler) through 35 cycles at thespecifications described in Table 1.

The PCR products were detected by electrophoresis on a 1% agarose gelcontaining ethidium bromide. Band intensities were quantified by computerizeddensitometry (Alpha Imager gel documentation system) and normalized withrespect to actin mRNA.

2.11. Histopathological studies

The histopathological studies in brain tissue were conducted according to Li et al.[45]. Rats were anesthetized under ether anesthesia and perfused intracardially withphosphate-buffered saline (0.02 M, pH 7.4) followed by 4% paraformaldehyde in0.1 M phosphate-buffered saline, pH 7.4 for pre-fixation of the tissue. Then the braintissue was dissected out carefully and was kept in 4% paraformaldehyde overnightfor post-fixation. After post-fixation the tissue was dehydrated and embedded inparaffin for 4 h in infiltration unit. Block was prepared in block preparation unit(Shandon Histocenter-2) and coronal sections (10 �m) were cut with the help ofa microtome (Leica RM 2255, Lab India) and picked up on poly-l-lysine coatedslides. Sections from the rostral to the caudal portion of the brain were stained withhematoxylin and eosin. Stained sections were captured (light microscopy) at ×200magnifications by Leica Application suite V3.1.0 software. Three identical micro-scopic fields of each section were selected for dead cell count in per mm2 area byLeica QWin V3 software [31].

2.12. Statistical analysis

The results were expressed as mean ± S.E.M. Statistical analysis of Morris watermaze, histological, molecular and biochemical experiments was performed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. InMorris water maze test, mean latency time and mean path length of different trial(acquisition and retention trials) were compared within and between the groups. Inhistological, molecular and biochemical experiments, the mean values of STZ groupwere compared with control and aCSF group. The data of perindopril + STZ (0.05and 0.01 mg/kg) groups was compared with STZ group whereas the mean values ofperindopril per se group were compared with control and aCSF groups.

3. Results

3.1. Effects of perindopril on STZ induced memory deficit in theMorris water maze test

3.1.1. Analysis of latency timeThe role of central ACE in the STZ induced memory deficit was

studied by using perindopril as an experimental pharmacologicaltool. One way ANOVA followed by Tukey’s multiple comparisontest revealed no significant difference in the mean escape latencytime of acquisition trial in Morris water maze test [F(5, 30) = 0.53,P > 0.05]. But the control [F(3, 20) = 65.66, P < 0.01] and aCSF [F(3,20) = 34.16, P < 0.01] groups showed learning during the reten-tion trials as revealed by significant decrease in latency time as

compared to acquisition trial. However, no significant decreasein latency time [F(3, 20) = 0.55, P > 0.05] was observed through-out all the sessions in the STZ treated animals (Fig. 1a). Theinter-group analysis of latency time suggest that the STZ treated

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nimals showed significantly higher latency time as compared tohe control and aCSF groups [F(11, 60) = 10.91, P < 0.001]. The treat-

ent with the perindopril significantly decreased [0.05 mg/kg:(3, 20) = 17.31, P < 0.01 and 0.1 mg/kg: F(3, 20) = 40.34, P < 0.001]

ig. 1. Effect of perindopril pretreatment on STZ (ICV) induced memory impairment in ras) ± S.E.M. (n = 6). (*) Significant decrease in latency time (*P < 0.01 and **P < 0.001) vs. acrial of control and aCSF group (one way ANOVA followed by Tukey’s multiple comparison

cm) ± S.E.M. (n = 6). (*) Significant decrease in path length (*P < 0.01 and **P < 0.001) vs. arial of control and aCSF group (one way ANOVA followed by Tukey’s multiple comparisoCSF, STZ, Peri 0.05 mg/kg + STZ, Peri 0.1 mg/kg + STZ and Peri 0.1 mg/kg per se treated anersion of the Morris water maze test.

search 226 (2012) 317– 330

latency time in the STZ treated animals suggesting spatial mem-ory improvement (Fig. 1a). The inter-group analysis of latency timesuggest that perindopril treated animals took significantly less timeto reach hidden platform in comparison to the STZ injected rats

ts. (a) Comparison of latency time. Data values are expressed as mean latency timequisition trial and ($) significant decrease in latency time ($P < 0.01) vs. respectivetest). (b) Comparison of path length. Data values are expressed as mean path lengthcquisition trial and ($) significant decrease in path length ($P < 0.01) vs. respectiven test). (c) Figure shows the representative Morris water maze tracking of control,imals during 3rd retention trial. (d) Mean latency time (s) ± S.E.M. during a visible

S. Tota et al. / Behavioural Brain Research 226 (2012) 317– 330 321

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one way ANOVA followed by Tukey’s multiple comparison test,cquisition: [F(2, 15) = 0.12, P > 0.05), 1st Retention: F(2, 15) = 9.45,

< 0.01), 2nd Retention: F(2, 15) = 10.97, P < 0.01), 3rd Retention:(2, 15) = 8.35, P < 0.01].

Further, perindopril (0.1 mg/kg) per se treated animals showedignificant decrease [0.05 mg/kg: F(3, 20) = 10.09, P < 0.01] inatency time during retention trials as compared to acquisitionrial. Moreover, comparison of latency time of control, vehicle ander se group by one way ANOVA revealed a significant decrease ofatency time in 1st retention trial [F(2, 15) = 6.46, P < 0.01] of per seroup in comparison to respective trials of control and aCSF groups.owever, no significant change was observed in Acquisition: [F(2,5) = 1.37, P > 0.05], 2nd Retention: [F(2, 15) = 0.86, P > 0.05] and 3rdetention: [F(2, 15) = 0.04, P > 0.05] trials of control, aCSF and per seroup (Fig. 1a).

.1.2. Analysis of path lengthInvestigation of path length revealed that STZ treated animals

ad significantly longer path lengths as compared to control andCSF group [one way ANOVA followed by Tukey’s multiple compar-son test, F(11, 60) = 10.55, P < 0.001]. Further, intra-group analysisanimals × trials) showed a significant reduction of path lengthn control [F(3, 20) = 34.16, P < 0.01] and aCSF [F(3, 20) = 32.72,

< 0.01] group during retention trials as compared to acquisitionrial. Perindopril treated group had significantly [F(11, 60) = 9.65,

< 0.001] reduced path length than STZ injected rats (Fig. 1b).Further, statistical analysis showed a significant correlation

Pearson r = 0.995; R2 = 0.989 and P < 0.01) between mean latencyime and mean path length of all the groups in all the trials. Theepresentative swim pattern of different groups of animals duringhird retention trial was shown in Fig. 1c.

.1.3. Visible platform testAll the groups showed equivalent efficacy in locating a visible

latform [F(5, 30) = 0.25, P > 0.05] implying absence of motor-ensory differences between the experimental groups (Fig. 1d).

.1.4. Probe trialThe probe trial data is depicted in Fig. 2a–d, which provides

our representations of selective performance in the retention test.ig. 2a is a standard measure and compares time spent in the targetuadrant against the average time spent in other three quadrants.he time spent in target quadrant was significantly high in controlnd vehicle groups [one way ANOVA followed by Tukey’s multi-le comparison test, F(2, 15) = 8.37, P < 0.01] in comparison to theTZ group. It was further observed that the target quadrant pref-

rence was completely lost in STZ injected animals (P > 0.05). Thereatment with perindopril at all the doses prevented the memorympairment as indicated by the significant (P < 0.01) increase in theime spent in target quadrant in comparison to average time spent

inued).

in other three quadrants. Further, inter group analysis showed asignificant increase [F(2, 15) = 5.12, P < 0.05] in the time spent intarget quadrant of perindopril treated animals as compared to STZgroup. However there was no significant [F(2, 15) = 0.79, P > 0.05]difference in the time spent in target quadrant of control, vehicleand perindopril per se groups.

The total path length in target quadrant and average path lengthin other three quadrants is shown in Fig. 2b. Except the STZ injectedgroup, all other animals showed a significantly high (P < 0.05) pathlength in target quadrant in comparison to average path length inother three quadrants. Further, in the STZ group, the path length intarget quadrant was significantly less [F(2, 15) = 17.28, P < 0.05] ascompared to control and aCSF groups. Perindopril group exhibitedsignificantly [F(2, 15) = 7.28, P < 0.05] higher path length in targetquadrant in comparison to that of STZ group. However there wasno significant [F(2, 15) = 0.26, P > 0.05] difference in path length intarget quadrant of control, vehicle and perindopril per se groups.

Probe trial study also revealed that STZ treated animals showedsignificantly less [F(2, 15) = 13.47, P < 0.05] platform crossings whencompared with control and aCSF groups, indicating their inferiorsearch accuracy for the hidden platform. Perindopril administra-tion in STZ injected rats improved search accuracy as indicated bysignificantly higher [F(2, 15) = 10.68, P < 0.05] platform crossings incomparison to STZ group (Fig. 2c). The representative swim pat-tern of different groups of animals during probe trial was shown inFig. 2d.

3.2. Effects of perindopril on locomotor activity

The spontaneous locomotor activity did not differ significantlyamong different groups [Total: F(5, 30) = 0.25, P > 0.05, Ambulatory:F(5, 30) = 1.65, P > 0.05, and Vertical: F(5, 30) = 2.98, P > 0.05].

3.3. Effect of perindopril on mean arterial blood pressure (MAP)

The treatment of rats with both doses (0.05 and 0.1 mg/kg) ofperindopril did not affect the blood pressure significantly (P > 0.05)(Table 2).

3.4. Effect of perindopril on cerebral blood flow

The CBF was measured by LDF and expressed in blood perfusionunits. STZ administration significantly reduced [F(2, 15) = 16.95,P < 0.01] CBF in comparison to control and aCSF groups. One wayANOVA followed by Tukey’s test showed that perindopril (0.05 and

0.1 mg/kg) dose dependently restored CBF in STZ injected rats [F(2,15) = 133.4, P < 0.001]. CBF was significantly higher [F(2, 15) = 10.29,P < 0.01] (P < 0.01) in perindopril per se group in comparison to con-trol and aCSF groups (Fig. 3).

322 S. Tota et al. / Behavioural Brain Research 226 (2012) 317– 330

Table 2Effect of perindopril on mean blood pressure (mmHg) as measured by non invasive blood pressure instrument in rats.

Groups BP (mmHg) on day 1 (before treatment)(Mean ± S.E.M.)

BP (mmHg) on day 21 (at the end of treatment)(Mean ± S.E.M.)

P value vs. day 1

Control 104.9 ± 9.5 106.8 ± 1.46 P > 0.05STZ 105.3 ± 3.84 104.3 ± 2.33 P > 0.05

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Perindopril 0.05 mg/kg + STZ 103.8 ± 1.43

Perindopril 0.1 mg/kg + STZ 103.3 ± 2.09

Perindopril 0.1 mg/kg per se 103.5 ± 1.55

.5. Estimation of biochemical parameters

.5.1. Estimation of activity of angiotensin converting enzyme

ACE activity was measured by using synthetic substrate FAPGG.

CV administration of STZ did not affect serum ACE activity.erindopril caused a significant reduction in serum ACE activityn comparison to STZ group [F(2, 15) = 36.38, P < 0.01]. Perindopril

ig. 2. Effect of perindopril on probe trial performance in Morris water maze. (a) Probe trn average time spent in all three non-target quadrants. Data values are expressed as mes. average time spent in other three non-target quadrants of respective group, (#) Signroup and ($) significant difference in time spent in target quadrant ($P < 0.05) vs. STZ grouadrant with an average path length in all three non-target quadrants. Data values are e

ength (*P < 0.01) vs. average path length in other three non-target quadrants of respectiontrol and aCSF group and ($) significant difference in path length in target quadrant ($Probe trial. (#) Significant decrease (#P < 0.01) vs control and aCSF group and (*) Significanaze tracking of control, aCSF, STZ, Peri 0.05 mg/kg + STZ, Peri 0.1 mg/kg + STZ and Peri 0.13 – quadrant 3 and TQ – target quadrant. In probe trial, each rat was placed at a start po

103.0 ± 1.55 P > 0.05100.2 ± 1.72 P > 0.05100.8 ± 0.62 P > 0.05

per se also caused a significant [F(2, 15) = 64.30, P < 0.01] reductionin serum ACE activity in comparison to control and aCSF groups(Fig. 4a). ICV STZ treatment significantly elevated ACE activity in

cortex [F(2, 15) = 22.52, P < 0.01] and hippocampus [F(2, 15) = 98.47,P < 0.01] as compared to the control and aCSF groups. Treatmentwith perindopril significantly decreased the ACE activity in cortex[F(2, 15) = 8.19, P < 0.01] and hippocampus [F(2, 15) = 46.2, P < 0.001]

ial performance as measured by comparing time spent in the target quadrant withan time spent (s) ± S.E.M. (n = 6). (*) Significant difference in time spent (*P < 0.01)ificant difference in time spent in target quadrant (#P < 0.05) vs. control and aCSFup. (b) Probe trial performance as measured by comparing path length in the targetxpressed as mean path length (cm) ± S.E.M. (n = 6). (*) Significant difference in pathve group, (#) significant difference in path length in target quadrant (#P < 0.05) vs.

< 0.05) vs. STZ group. (c) Number of crossings of training site (±S.E.M.) during thet increase (*P < 0.01) vs. STZ group. (d) Figure shows the representative Morris water

mg/kg per se treated animals during probe trial. Q1 – quadrant 1, Q2 – quadrant 2,sition in the quadrant 2 and allowed to swim for 60 s.

S. Tota et al. / Behavioural Brain Research 226 (2012) 317– 330 323

(Cont

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Fig. 2.

f STZ injected rat brain (Fig. 4b). Further, perindopril per se alsoignificantly decreased ACE activity in brain regions [Cortex: F(2,5) = 22.52, P < 0.01, Hippocampus: F(2, 15) = 98.47, P < 0.001].

.5.2. ATP assayEnergy metabolism was studied by estimating ATP content in

ortex and hippocampus of rat brain. The ATP levels in differentroups were analyzed by one way ANOVA followed by Tukey’s test

ig. 3. Effect of perindopril pretreatment on cerebral blood flow (CBF) in STZ (ICV)nduced memory deficit rats. Data values are expressed as mean CBF in arbitrarylood perfusion units (BPU) ± S.E.M. (n = 6). (#) Significant decrease (#P < 0.01) inomparison to control group, (*) significant increase (*P < 0.01) in comparison to STZICV) group and ($) significant increase ($P < 0.01) in comparison to control groupone way ANOVA followed by Tukey’s multiple comparison test).

inued).

for multiple comparisons. As shown in Fig. 5, the STZ administra-tion significantly decreased ATP content in cortex [F(2, 15) = 19.99,P < 0.01] and hippocampus [F(2, 15) = 24.26, P < 0.01] of rat brain ascompared to control and aCSF groups. Pretreatment with perindo-pril dose dependently attenuated STZ induced reduction in ATPlevel in both the brain regions [Cortex: F(2, 15) = 47.39, P < 0.01,Hippocampus: F(2, 15) = 24.78, P < 0.01]. However, perindopril perse had no significant effect on ATP level as compared to controland aCSF groups [Cortex: F(2, 15) = 3.01, P > 0.05, Hippocampus: F(2,15) = 3.08, P > 0.05].

3.5.3. Malondialdehyde (MDA) levelThe MDA level (nmol/mg protein) in the rat brain regions

was measured after the completion of behavioral experiments. Incomparison to control and aCSF groups, the level of MDA rosesignificantly in cortex [F(2, 15) = 188, P < 0.01] and hippocampus[F(2, 15) = 37.9, P < 0.01] of STZ treated rats. Preventive treatmentwith perindopril significantly [Cortex: F(2, 15) = 104.6, P < 0.01 andHippocampus: [F(2, 15) = 50.7, P < 0.01] decreased MDA level inboth the brain regions. However, per se treatment of perindo-pril decreased MDA in hippocampus only [Cortex: F(2, 15) = 2.67,P > 0.05 and Hippocampus: [F(2, 15) = 10.91, P < 0.01] (Fig. 6a).

3.5.4. Glutathione (GSH) levelAs shown in Fig. 6b administration of the aCSF (ICV) had no sig-

nificant (P > 0.05) effect on GSH (�g/mg protein) level in any brainregion as compared to control. However a significant fall in theGSH level, as compared to control and aCSF groups, was observed incortex [F(2, 15) = 14.55, P < 0.01] and hippocampus [F(2, 15) = 11.47,

324 S. Tota et al. / Behavioural Brain Research 226 (2012) 317– 330

Fig. 4. (a) Effect of perindopril pretreatment on serum ACE activity in STZ(ICV) induced memory deficit rats. Serum ACE activity was expressed as meanUnits/liter ± S.E.M. (n = 6). (*) Significant decrease (*P < 0.01) in comparison to STZ(ICV) group, ($) significant decrease ($P < 0.01) in comparison to control group. (b)Effect of perindopril pretreatment on brain ACE activity in STZ (ICV) induced mem-ory deficit rats. Brain ACE activity was expressed as mean Units/mg protein ± S.E.M.(nd

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Fig. 6. (a) Effect of perindopril pretreatment on MDA level in STZ (ICV)induced memory deficit rats. Data values are expressed as mean MDA level(nmol/mg protein) ± S.E.M. (n = 6). (#) Significant increase (#P < 0.01 and ##P < 0.001)in comparison to control group, (*) significant decrease (**P < 0.001) in comparisonto STZ (ICV) group and ($) significant decrease ($P < 0.05) in comparison to respec-tive region of control group; (b) Effect of perindopril pretreatment on GSH level inSTZ (ICV) induced memory deficit rats. Data values are expressed as mean GSH level

Pretreatment with 0.1 mg/kg perindopril significantly reducedamount of ROS in both brain parts whereas 0.05 mg/kg perindopril

n = 6). (#) Significant increase (#P < 0.05) in comparison to control group, (*) sig-ificant decrease (*P < 0.05) in comparison to STZ (ICV) group and ($) significantecrease ($P < 0.05) in comparison to control group.

< 0.01] of STZ group. This reduction in GSH level was amelioratedy preventive treatment with perindopril [Cortex: F(2, 15) = 41.68,

< 0.05 and Hippocampus: [F(2, 15) = 12.78, P < 0.01]. Perindopriler se significantly increased GSH levels in hippocampus as com-ared to control and aCSF group [Cortex: F(2, 15) = 3.44, P > 0.05 andippocampus: F(2, 15) = 8.27, P < 0.05].

.5.5. Nitrite levelNitrite level was measured by Griess method. As shown in Fig. 7,

significant increase in nitrite level was observed in cortex [F(2,

5) = 61.27, P < 0.001] and hippocampus [F(2, 15) = 17.79, P < 0.01]f STZ treated animals. Perindopril caused a significant decrease initrite level in cortex [F(2, 15) = 41.68, P < 0.001] and hippocampusF(2, 15) = 7.37, P < 0.01]. However, perindopril per se had no effect

ig. 5. Effect of perindopril on brain energy metabolism in rat brain regions. Dataalues are expressed as mean ATP level (nmol/mg protein) ± S.E.M (n = 6). (#) Sig-ificant decrease (#P < 0.01) vs. respective region of control and aCSF group and (*)ignificant increase (*P < 0.05 and **P < 0.01) vs. respective region of STZ group.

(�g/mg protein) ± S.E.M. (n = 6). (#) Significant decrease (#P < 0.01) in comparison tocontrol group, (*) significant increase (*P < 0.05 and **P < 0.01) in comparison to STZ(ICV) group and ($) significant increase ($P < 0.01) in comparison to control group.

on nitrite level in rat brain regions [Cortex: F(2, 15) = 0.35, P > 0.05and Hippocampus: [F(2, 15) = 1.43, P > 0.05].

3.5.6. Measurement of reactive oxygen species (ROS)Production of ROS was measured relative to control. Treat-

ment with STZ increased ROS generation in cortex [F(2, 15) = 40.15,P < 0.01] and hippocampus [F(2, 15) = 30.26, P < 0.01] of rat brain.

reduced ROS only in hippocampus [Cortex: F(2, 15) = 8.0, P < 0.05and Hippocampus: [F(2, 15) = 33.53, P < 0.01]. In comparison to

Fig. 7. Effect of perindopril pretreatment on nitrite level in STZ (ICV)induced memory deficit rats. Data values are expressed as mean nitrite level(�g/mg protein) ± S.E.M. (#) Significant increase (#P < 0.05 and ##P < 0.001) in com-parison to control group, (*) significant decrease (*P < 0.05 and **P < 0.001) incomparison to STZ (ICV) group.

S. Tota et al. / Behavioural Brain Re

Fig. 8. Effect of perindopril pretreatment on ROS level in STZ (ICV) induced mem-ory deficit rats. Data values are expressed as % of control values (fluorescenceisd

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ntensity). (#) Significant increase (#P < 0.01) in comparison to control group, (*)ignificant decrease (*P < 0.01) in comparison to STZ (ICV) group. ($) Significantecrease ($P < 0.01) in comparison to control group.

ontrol and aCSF groups there was a significant decrease inOS level in hippocampus [F(2, 15) = 25.2, P < 0.01] of perindopriler se group (Fig. 8). However, in cortex, per se treatment didot show any significant effect [F(2, 15) = 0.37, P > 0.05] in ROS

evel.

.5.7. Acetylcholine esterase assayAChE activity was significantly higher in cortex [F(2, 15) = 74.21,

< 0.01] and hippocampus [F(2, 15) = 35.18, P < 0.01] of STZ treatedats in comparison to control and aCSF groups. As shown inig. 9, preventive treatment of perindopril significantly restored theltered AChE activity in STZ treated rat brain regions [Cortex: F(2,5) = 38.01, P < 0.01 and Hippocampus: [F(2, 15) = 59.34, P < 0.001].owever, perindopril per se had no effect on AChE activity in cortex

F(2, 15) = 0.31, P > 0.05] and hippocampus [F(2, 15) = 1.41, P > 0.05].

.6. Effect of perindopril on AChE and ACE mRNA expression inTZ induced memory deficit rat brain.

As shown in Fig. 10a and b, the ACE mRNA expression was signif-cantly increased in cortex and hippocampus of STZ treated animals

s compared to that of control group [Cortex: F(2, 15) = 10.35,

< 0.01; Hippocampus: F(2, 15) = 9.33, P < 0.01]. Preventive treat-ent with perindopril 0.05 mg/kg in STZ treated rats significantly

P < 0.05) decreased ACE mRNA levels in cortex and hippocampus

ig. 9. Effect of perindopril pretreatment on AChE activity in STZ (ICV)nduced memory deficit rats. Data values are expressed as mean AChE activity�mol/min/mg protein) ± S.E.M. (#) Significant increase (#P < 0.01) in comparisono control group, (*) significant decrease (*P < 0.05 and **P < 0.01) in comparison toTZ (ICV) group.

search 226 (2012) 317– 330 325

but higher dose of perindopril had significant (P < 0.05) effect inhippocampus only. However, per se treatment of perindopril sig-nificantly increased ACE mRNA expression in cortex [F(2, 15) = 2.75,P < 0.05] and hippocampus [F(2, 15) = 1.68, P < 0.05].

Further, we found a significant increase in AChE mRNA expres-sion in cortex and hippocampus of STZ treated rats [Cortex: F(2,15) = 5.9, P < 0.05; Hippocampus: F(2, 15) = 7.7, P < 0.05] in com-parison to control and aCSF groups (Fig. 10a and c). Preventivetreatment with perindopril significantly [Cortex: F(2, 15) = 4.5,P < 0.05; Hippocampus: F(2, 15) = 7.76, P < 0.05] decreased AChEmRNA levels in STZ treated rats but per se perindopril had no sig-nificant effect [Cortex: F(2, 15) = 1.36, P > 0.05; Hippocampus: F(2,15) = 0.11, P > 0.05].

3.7. Histological changes

As shown in Fig. 11, ICV administration of STZ caused manyhistological changes in hippocampus and cortex of rat brain. HEstained sections revealed shrinkage (red arrows) and decrease indensity of neurons in hippocampal area. Quantitative analysis ofcell counts in hippocampal region showed a significant reductionin the number of cells in the STZ treated rats compared to con-trol [F(4, 20) = 61.47, P < 0.01]. We also found a significant decreasein number of neurons in periventricular region of the STZ-treatedgroup in comparison to control and CSF. Treatment with perindoprilattenuated STZ induced neurodegenerative changes in rats. Therewas a significant increase (P < 0.01) in cell number in hippocampusof perindopril treated rat as compared to STZ group.

In deep seated cortical region, control and CSF did not revealany remarkable difference while STZ showed degeneration of neu-rons (red arrows). The changes were less in perindopril treated rats[F(4, 20) = 231.99, P < 0.001] (Fig. 12). The quantitative comparisonin terms of dead cell counts per mm2 of different brain regions isdepicted in Fig. 13. Perindopril per se group did not show any signif-icant change in the morphology of cells in cortex and hippocampus.

4. Discussion

The present study demonstrated that angiotensin convertingenzyme lays a pivotal role in memory deficit induced by intracere-broventricular (ICV) streptozotocin (STZ) in rat because treatmentwith perindopril, an ACE inhibitor, prevented the impairmentof learning and memory, improved cerebral blood flow, cholin-ergic function, brain energy metabolism and reduced oxidativestress.

We first examined the involvement of central ACE in STZinduced memory deficit using perindopril, a brain penetratingACE inhibitor [46], as an experimental pharmacological tool. STZ(ICV) caused impairment in memory as shown by no significantchange in latency time and path length in water maze test. Fur-ther, probe trial study also confirmed the memory impairment asSTZ injected rats lost the target quadrant preference and showedreduced number of platform crossings. STZ injected animals weretreated with perindopril and their performance in the water mazetest was compared to age-matched, vehicle-treated controls. ACEinhibition by perindopril improved memory in STZ injected rats asevidenced by significant decrease in latency time and path length.Perindopril treated animals also exhibited improved target quad-rant preference as shown by time spent in platform zone andnumber of platform crossings. Further, per se treatment of perindo-pril improved memory function as shown by significantly reduced

retention latencies as compared to control animals. Although thecognitive effects of ACE inhibitors have previously been inves-tigated with doses affecting blood pressure in rodents [18–24],this study importantly used a dosage regime with no significant

326 S. Tota et al. / Behavioural Brain Research 226 (2012) 317– 330

Fig. 10. (a) mRNA expression of AChE, ACE and �-actin. 1 – Control, 2 – aCSF, 3 – STZ, 4 – Peri 0.05 mg/kg + STZ, 5 – Peri 0.1 mg/kg + STZ and 6 – Peri 0.1 mg/kg per se. (b) ACEmRNA levels (normalized to level of �-actin). (#) Significant increase (#P < 0.05) in comparison to respective region of control and aCSF groups and (*) significant decrease(*P < 0.05) in comparison to STZ. (c) AChE mRNA levels (normalized to level of �-actin). (#) Significant increase (#P < 0.05) in comparison to respective region of control andaCSF groups and (*) significant decrease (*P < 0.05) in comparison to STZ.

S. Tota et al. / Behavioural Brain Research 226 (2012) 317– 330 327

Fig. 11. Micrograph showing effect of STZ on neurons of the hippocampus. (A) Control, (B) aCSF and (E) Peri 0.1 mg/kg per se did not reveal difference in cell numbers (blackarrow showing intact cells) in the neurons. (C) Note: shrinkage of neurons in STZ-treated rat brain (red arrow in C) revealed degeneration in the hippocampus of STZ-treatedr campur

eipnem

Fclt

ats. (D) Less degeneration is seen in Peri 0.1 mg/kg + STZ treated rats in the hippoeferred to the web version of the article.)

ffect on blood pressure. Therefore, it can be said that memorynfluencing effect of ACE observed in the present study is inde-

endent of its blood pressure regulating effect. Further, there waso significant effect on blood glucose level and locomotor activityxcluding the possibility that theses parameters may have alteredemory function in STZ treated rats. Moreover, all the animals

ig. 12. Micrograph showing effect of STZ on deep seated cortical neurons (entorhinal coortical cells (black arrow showing intact cells) while (C) STZ showed degeneration in deeive cells). (D) Peri 0.1 mg/kg + STZ treated rats showed significantly less cortical neuronalhis figure legend, the reader is referred to the web version of the article.)

s. (For interpretation of the references to color in this figure legend, the reader is

showed similar preference for searching visible platform indicatingabsence of motor-sensory differences between the experimental

groups.

Elevation in ACE activity has been reported in various brainregions of Alzheimer’s disease (AD) patients [6,7]. We also gotsimilar findings as in this study STZ significantly increased ACE

rtex). (A) Control, (B) aCSF and (E) Peri 0.1 mg/kg per se did not reveal difference inp seated cortical neurons (red arrows showing dead cells and black arrows showing

degeneration than STZ treated rats. (For interpretation of the references to color in

328 S. Tota et al. / Behavioural Brain Research 226 (2012) 317– 330

Fig. 13. Dead cell counts in the brain of control, aCSF, STZ, Peri 0.1 mg/kg + STZ and Peri 0.1 mg/kg per se treated group. (*) Significant difference (P < 0.001) in dead cells fromc

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ontrol, CSF, Peri 0.1 mg/kg + STZ and Peri 0.1 mg/kg per se treated group.

ctivity in rat brain without affecting serum ACE activity indicatingnvolvement of central ACE in memory deficit. Further we studiedCE mRNA expression in cortex and hippocampus and found signif-

cant increase in ACE mRNA following STZ administration. Previouseports showed that diabetes caused by STZ (IP) is associated withncreased ACE activity and mRNA expression [47]. Therefore, thencrease in ACE activity and expression in brain by STZ (ICV) maye due to hyperglycemia like condition [48,49] in the brain fol-

owing STZ administration. However, administration of perindoprilecreased ACE activity and mRNA expression in brain regions ofTZ treated rat. Further perindopril per se treatment significantlyncreased ACE expression while decreasing its activity in brain. Thisnding is in agreement with previous report which showed thatreatment with quinapril, an ACE inhibitor, increases ACE mRNAxpression and decreased ACE activity indicating a feedback regu-ation of RAS [50].

In our study we found aggravated nitrosative (increased nitriteevel) and oxidative stress (decreased GSH and, increased MDA andOS). This enhancement in oxidative stress markers is likely due to

ncreased formation of Ang II, due to increased ACE activity, whichtimulates NADPH oxidase that plays a pivotal role in the devel-pment of oxidative stress by producing superoxides [51,52]. ACEnhibition exhibits anti oxidative action as evidenced in this study

hen perindopril reduced lipid peroxidation product MDA andlevated antioxidant i.e. glutathione. Further, perindopril pretreat-ent reduced the superoxide formation as shown by fluorimetry.CE inhibition, by decreasing Ang II, limits the stimulation of vas-ular NADPH oxidase, thereby preventing the increased superoxideux associated with activation of the RAS [53]. Pretreatment witherindopril reduced nitrite level also.

In this study, we found a significant reduction in cerebral bloodow in STZ treated rats as measured by laser doppler flowme-ry. The decrease in CBF was corresponds with the impairmentn memory functions following STZ administration. Many clinicaltudies also showed similar alteration in cerebral microcircula-ion in patients with AD [27,28]. Though the exact mechanismor this microcirculation impairment is not known, the proba-le reasons include oxidative stress and endothelial dysfunctionausing restriction of blood flow to the brain [54]. Further,ecently Inaba et al. [4] showed that continuous activation of reninngiotensin system reduced the CBF and increased oxidative stressn renin/angiotensinogen transgenic mice. Thus, increased ACE

ctivity, which will elevate level of Ang II, may lead to reductionn cerebral blood flow [4] either due to vasoconstriction (by Ang II)r due to oxidative stress (due to activation of NADPH oxidase byng II) induced endothelial dysfunction.

Modulatory role of ACE in decreasing CBF was confirmed byusing perindopril which ameliorated decrease in CBF in STZ treatedrats. Further we found a significant increase in CBF in perindo-pril per se group, the values being higher than that of control andvehicle groups. This increase in CBF may be responsible for thememory enhancing effect of perindopril observed in this study. Thisobservation is backed by another study where ACE inhibition hasbeen shown to increase hippocampal blood flow and improve hip-pocampal long-term potentiation in diabetic rats compared withuntreated diabetics [19]. Improvement in CBF is due to reducedoxidative stress as our previous studies have shown that antioxi-dants like curcumin and quercetin improved CBF in intracerebralSTZ injected mice [29,30].

ICV-STZ model of memory deficit shows a disturbed glucosemetabolism [48] may be due to increased oxidative stress and cere-bral hypoperfusion [29,30]. Glucose, its metabolites and products ofenergy metabolism (like ATP) are the brain’s main source of energyand therefore normal glucose metabolism is critical for proper brainfunctions like protein synthesis and basic cellular and molecularprocesses [55]. In neurodegenerative disorders like AD, the brainglucose and energy metabolism are disturbed impairing learningand memory [56]. In our study, the results showed a significantdecrease in ATP level in STZ injected rat brain regions suggestinga disturbed energy metabolism, an observation supported by ourprevious findings also [29,42]. Perindopril improved brain energymetabolism in STZ injected rats as shown by increased ATP levelsin cortex and hippocampus. A recent study also corroborates thisobservation when it was shown that Ang II enhances ROS produc-tion by activating NADPH oxidase which depresses mitochondrialenergy metabolism [57]. This suggests that Ang II contributes to theageing process by prompting mitochondrial dysfunction and block-ade of RAS will reduce ROS and enhances mitochondrial contentand function.

Cholinergic system in the hippocampus plays an important rolein memory formation and retrieval [25,26]. As reported in our pre-vious studies [4,29,30,42], in this study also we found impairedcholinergic system as shown by increased acetylcholinesteraseactivity in STZ injected rat brain regions. RT-PCR study also revealedan increase in AChE mRNA expression in cortex and hippocampusof STZ treated rats. Better performance of perindopril treated ratsin behavioral tests can also be attributed to improved cholinergicsystem due to decreased activity of AChE resulting in increased

acetylcholine (ACh). Ang II inhibits release of ACh from the humantemporal cortex associated with cognitive performance and thatthis effect is reversed by Ang II receptor antagonists [58]. There-fore, it can be said that in this study also ACE inhibitor, perindopril,

S. Tota et al. / Behavioural Brain Research 226 (2012) 317– 330 329

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Fig. 14. Figure shows various pathways involved in STZ induced

mproved cholinergic system and learning and memory in treatedats by decreasing Ang II.

Further, we found that brain sections of STZ-treated rats stainedy HE staining showed increased vacuoles in cortical area, dam-ged periventricular cells, and disorganization of hippocampus.his finding is in agreement with our previous report which showedhat STZ induced memory impairment is associated with neuronalegeneration in hippocampal and cortical areas of rat brain [31].his neuronal damage is due to increased oxidative stress, reducederebral blood flow and cholinergic dysfunction as discussed above.nhibition of ACE by perindopril reduced extent of neuronal dam-ge in hippocampus, entorhinal cortex and periventricular corticalegion of STZ injected rat brain by improving cerebral blood, oxida-ive stress and cholinergic function. These observations reveal thenvolvement of the RAS in neurodegeneration.

Based on these findings we can say that ACE plays a pivotal rolen memory deficit by causing oxidative stress, cholinergic dysfunc-ion, reduced cerebral circulation and impaired energy utilizationFig. 14). The results of this study are in agreement with somelinical reports which showed involvement of central ACE in neu-odegenerative conditions. Miners et al. [6] showed elevated ACEctivity in AD which was correlated with Braak stage. Recently, A�2 (ICV) induced memory impairment in rats was also associatedith increased ACE activity in hippocampus and perindopril treat-ent improved learning and memory [24]. In the present studye found a significant impairment in brain cholinergic function

long with increased oxidative stress, the changes also implicatedn A�-induced memory impairment [59].

. Conclusion

This study supports many clinical observations like PROGRESSrial where ACE inhibition shows positive effects on memory. Iteems that mechanism behind memory enhancement involves

ntioxidative action and improving effect on cerebral blood flownd cholinergic system. Therefore, due to this neuroprotectiveffect, ACE inhibitors can be a better option for hypertensiveatients with memory impairment.

[

ory deficit along with possible role of renin angiotensin system.

Acknowledgments

Financial support to Santoshkumar Tota and Pradeep KumarKamat from Council of Scientific and Industrial Research (CSIR) NewDelhi, India, is gratefully acknowledged.

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