Progress in Neuro-Psychopharmacology & Biological Psychiatry 43 (2013) 79–88

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Progress in Neuro-Psychopharmacology & BiologicalPsychiatry

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Inhibition of central angiotensin II enhances memory function and reduces oxidativestress status in rat hippocampus

Walther Bild a,c, Lucian Hritcu b, Cristinel Stefanescu a, Alin Ciobica b,c,⁎a Gr. T. Popa University of Medicine and Pharmacy, 16 Universitatii Street, 700115, Iasi, Romaniab Alexandru Ioan Cuza University, 11 Carol I Blvd., 700506, Iasi, Romaniac Center of Biomedical Research of the Romanian Academy, Iasi Branch, Romania

Abbreviations: Abeta, amyloid beta; ACE, angioteAlzheimer's disease; aMCI, amnestic mild cognitive imANOVA, analysis of variance; GPX, glutathione peroxidoxidized glutathione; i.c.v., intracerebroventricularly; Imolecule-1; LTP, long term potentiation; MDA, malondiaotinamide adenine dinucleotide phosphate-oxidase; RAROS, reactive oxygen species; SEM, standard error odismutase; TOP, 3-thienylalanine-ornithine-proline; WS⁎ Corresponding author at: Alexandru Ioan Cuza Univ

Carol I, 11, 700506, Iasi, Romania. Tel.: +40 751218264E-mail address: alin.ciobica@uaic.ro (A. Ciobica).

0278-5846/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.pnpbp.2012.12.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 July 2012Received in revised form 11 December 2012Accepted 11 December 2012Available online 20 December 2012

Keywords:Angiotensin IIMemoryOxidative stress

While it is now well established that the independent brain renin–angiotensin system (RAS) has some im-portant central functions besides the vascular ones, the relevance of its main bioactive peptide angiotensinII (Ang II) on the memory processes, as well as on oxidative stress status is not completely understood.The purpose of the present workwas to evaluate the effects of central Ang II administration, as well as the effects ofAng II inhibition with either AT1 and AT 2 receptor specific blockers (losartan and PD-123177, respectively) or anangiotensin-converting enzyme (ACE) inhibitor (captopril). These effects were studied on the short-termmemory(assessed through Y-maze) or long-termmemory (as determined in passive avoidance) and on the oxidative stressstatus of the hippocampus.Our results demonstrate memory deficits induced by the administration of Ang II, as showed by the signifi-cant decrease of the spontaneous alternation in Y-maze (p=0.015) and latency-time in passive avoidancetask (p=0.001) when compared to saline. On the other side, the administration of all the aforementioned AngII blockers significantly improved the spontaneous alternation in Y-maze task, while losartan also increasedthe latency time as compared to saline in step-through passive avoidance (p=0.042).Also, increased oxidative stress statuswas induced in the hippocampus by the administration of Ang II, as demon-strated by increased levels of lipid peroxidationmarkers (malondialdehyde-MDA concentration) (pb0.0001) anda decrease in both antioxidant enzymes determined: superoxide dismutase-SOD (pb0.0001) and glutathioneperoxidase-GPX (p=0.01), as compared to saline. Additionally, the administration of captopril resulted in anincrease of both antioxidant enzymes and decreased levels of lipid peroxidation (p=0.001), while PD-123177significantly decreased MDA concentration (p>0.0001) vs. saline.Moreover, significant correlations were found between all of the memory related behavioral parameters andthe main oxidative stress markers from the hippocampus, which is known for its implication in the processesof memory and also where RAS components are well expressed.This could be relevant for the complex interactions between Ang II, behavioral processes and neuronal oxidativestress, and could generate important therapeutic approaches.

© 2012 Elsevier Inc. All rights reserved.

1. Introduction

It is now well known that the brain has its own intrinsic renin–angiotensin system (RAS) and this could serve as amodel for the actionof peptides on neuronal function in general (Haulica et al., 2005; von

nsin-converting enzyme; AD,pairment; Ang, angiotensin;ase; GSH, glutathione; GSSG,CAM-1, intercellular adhesionldehyde; NADPH oxidase, nic-S, renin–angiotensin system;

f the mean; SOD, superoxideT, water soluble tetrazolium.ersity, Dept. of Biology, B dul; fax: +40 232201472.

rights reserved.

Bohlen und Halbach and Albrecht, 2006). Additionally, it is now knownthat brainRAS is implicatednot only in themechanismsof bloodpressure,but also in the modulation of complex functions in the brain, includingemotional responses and memory (Braszko et al., 2003b; Ciobica et al.,2009; Gard, 2002; Gard and Rusted, 2004; McKinley et al., 2003;Saavedra, 2005). We have previously demonstrated the implications ofbrain RAS in anxiety-related processes (Bild and Ciobica, 2012; Ciobicaet al., 2011). Also, our group was among the first to demonstrate the in-volvement of the brain RAS in pain perception (Haulica et al., 1986).

The brain RAS is represented by a number of bioactive angiotensin(Ang) peptides, which could have variable and sometimes opposite neu-robiological activities (Llorens-Cortes and Mendelsohn, 2002; Santos etal., 2000; von Bohlen und Halbach, 2003; von Bohlen und Halbach andAlbrecht, 2006). These include Ang II, Ang IV and Ang-(1–7). However,the most important angiotensin peptide is Ang II, which acts through

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two different highly-specific receptors called AT1 and AT2 (Culman etal., 2001, 2002).

Although it is accepted that this peptide has interesting cognitiveproperties (Ciobica et al., 2009; Haulica et al., 2005; McKinley et al.,2003), behavioral data regarding Ang II have been difficult to interpret,considering that there are reports showing beneficial (Braszko, 2002,2005; Braszko et al., 1988a,b, 2006), negative (Bonini et al., 2006;Inaba et al., 2009; Kerr et al., 2005; Lee et al., 1995; Maul et al., 2008)or no significant effect at all (Shepherd et al., 1996; Walther et al.,1999) for Ang II on cognitive processes.

Thus has been stated that central administration of Ang II induces afacilitated aversive memory in rodents (Braszko, 2002), while the use ofsimilar behavioral tests demonstrated impaired or no changes onmemo-ry retention, following Ang II administration (Bonini et al., 2006; Kerr etal., 2005). Controversial results were reported too, concerning the Ang IIreceptor antagonists, losartan and PD-123177 (selective for the AT1 andAT2 receptor, respectively), since Shepherd et al. reported no effects ofeither compound in two different models of working memory in rats(Shepherd et al., 1996), while other studies have shown that low dosesof losartan and PD123177 improve scopolamine-impaired performancein a light/dark box habituation task (Chalas and Conway, 1996).

Several authors (Kumaran et al., 2008;Manschot et al., 2003) allowedfor limited beneficial effects on memory functions for the angiotensin-converting enzyme (ACE) inhibitors, commonly used as antihypertensivedrugs, have been demonstrated, but there are also very recent reportsstating that the role of ACE inmemory function is still ambiguous or insuf-ficiently explored (Tota et al., 2012a,b).

Additionally, the effects of Ang II on the oxidative status are contro-versial, with reports stating both pro-oxidant actions, exerted by an in-crease of reactive oxygen species (ROS) generation, mainly through thestimulation of NAD(P)H oxidase, which thenmeditates the activation ofsuperoxide (Basso et al., 2007; Inaba et al., 2009; Miller et al., 2007;Wang et al., 2006), aswell as authors stating no changes of the oxidativestress status as a result of Ang II administration in terms of all oxidativestress markers determined, as in the work of Gonzales group, whichshowed no significant change of SOD, GPX and catalase specific activity,as well as no changes in MDA levels, as an index of lipid peroxidationprocesses (Gonzales et al., 2002).

In this context, the aim of the presentworkwas to evaluate the effectsof central Ang II inhibition using either AT1 and AT 2 receptor specificblockers (losartan and PD-123177, respectively) or an ACE inhibitor(captopril) on short-term memory (assessed through Y-maze) orlong-term memory (as determined in passive avoidance), and on theoxidative stress status from the hippocampus, which is known for itsimplication on memory processes (Eichenbaum and Cohen, 1993) andalso where RAS components are very well expressed (von Bohlen undHalbach and Albrecht, 2006).Moreover, wewere interested in studyingif there is a correlation between the behavioral parameters we deter-mined in Ymaze or passive avoidance tasks and the levels of the oxidativestress markers (two antioxidant enzymes: superoxide dismutase-SODand glutathione peroxidase-GPX, as well as a lipid peroxidation marker:malondialdehyde-MDA) within the hippocampus.

2. Material and methods

2.1. Animals

AdultmaleWistar (n=25) rats,weighing200–250 g at the beginningof the experiment, were housed in groups of five animals per cage andkept in a room with controlled temperature (22 °C) and a 12:12-hlight/dark cycle (starting at 08:00 h), with food and water ad libitum.

The animals were treated in accordancewith the guidelines of animalbioethics from the Act on Animal Experimentation and Animal Healthand Welfare Act from Romania and all procedures were in compliancewith the European Communities Council Directive of 24 November1986 (86/609/EEC). This study was approved by the local Ethics

Committee and also efforts were made to minimize animal sufferingand to reduce the number of animals used.

2.2. Materials

Ang II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), Captopril (N-[(S)-3-Mercapto-2-methylpropionyl]-L-proline), Losartan (2-Butyl-4-chloro-1-{[2′-(1H-tetrazol-5-yl)(1,1′-biphenyl)-4-yl]methyl}-1H-imidazole-5-methanol monopotassium) and PD 123177 ((S)-1-[(4-Amino-3-methylphenyl)methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-Imidazo[4,5-c]pyridine-6-carboxylic acid trifluoroacetate salt hydrate)were obtainedfrom Sigma-Aldrich.

SOD Assay Kit was obtained from Fluka (product number: 19160),while GPX cellular activity assay kit CGP-1 was also purchased fromSigma Chemicals.

2.3. Experimental design

2.3.1. NeurosurgeryAll surgical procedures were conducted in aseptic conditions, anes-

thesia with sodium pentobarbital (45 mg/kg b.w., i.p., Sigma). Ratswere mounted in the stereotaxic apparatus with the nose oriented 11°below the horizontal zero plane. A plastic cannula (Portex, 0.44 innerdiameter, 0.9 mm outer diameter) was stereotaxically implanted inthe left lateral ventricle at the following coordinates: 0.5 mm posteriorto bregma; 1.3 mm lateral to themidline; 4.3 mmventral to the surfaceof the cortex (Paxinos and Watson, 2006). The cannula was positionedwith acrylic dental cement and secured with one stainless steel screw.

2.3.2. Pharmacological treatmentThe animalswere randomlydivided intofive groups of 5 animals each.

All the drug solutions were freshly prepared before use. Angiotensin II,losartan, PD-123177 and captopril were dissolved in saline (0.9% NaCl)a few minutes before the injection and administered intracerebro-ventricularly (i.c.v.) in doses of 0.1 mg/kg/b.w. for 7 consecutive days.The control rats were also injected with saline. The i.c.v. injections weremade manually with a 10 μl Hamilton syringe. The procedure was non-traumatic for the rat, which was gently held in the hand of the experi-menter. The injection volume was always 2 μl introduced over approxi-mately 5 s.

The treatment began 3 days after the neurosurgery and lasted for7 days. Memory functions were tested through Y-maze and passiveavoidance tasks, performed during the last 3 days of treatment (Y mazeon the 5th day and passive avoidance in the last 2 days). Thus, only oneset of animals was used for both behavioral tasks.

In the testing days, the aforementioned drugs were given 15 minbefore performing the behavioral task.

The aforementioned dosage and the duration of treatment wereselected using our pilot studies and previously published reports re-garding RAS behavioral effects (Bild and Ciobica, 2012; Braszko, 2002;Braszko et al., 1988a,b; Ciobica et al., 2010; Tota et al., 2012b;Winnicka et al., 1998).

2.4. Evaluation of memory function

2.4.1. Y-maze taskShort-termmemorywas assessed by spontaneous alternation behav-

ior in the Y-maze task. TheY-maze used in the present study consisted ofthree arms (35 cm long, 25 cmhigh, and 10 cmwide) and an equilateraltriangular central area. The rat was placed at the end of one arm andallowed to move freely through the maze for 8 min. An arm entry wascounted when the hind paws of the rat were completely within thearm. Also, themazewas cleanedwith alcohol-free disinfectantwipes be-tween each trial. Spontaneous alternation behavior was defined as theentry into all three arms on consecutive choices. The number of maxi-mum spontaneous alternation behaviors was calculated as the total

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number of arms entered minus 2 and percent spontaneous alternationwas calculated as (actual alternations/maximum alternations)×100.Spontaneous alternation behavior is considered to reflect spatial work-ingmemory, which is a form of short-termmemory (Hritcu et al., 2007).

2.4.2. Step-through passive avoidance taskIn brief, a step-through type passive avoidance apparatus (Coulbourn

Instruments) consisting of two compartments (25×15×15 cm high),one illuminated and one dark, both equipped with a grid floor wasused. The two compartments were separated by a guillotine door. Inthe acquisition trial, each ratwas placed in the illuminated compartment;when the animal entered the dark compartment, the doorwas closed andan inescapable foot shock (0.3 mA, 5 s) was delivered through the gridfloor. The rat was removed after receiving the foot shock and was placedback into the light compartment. The doorwas again opened 30 s later tostart the next trial. The training continued until the rat stayed in the lightcompartment for a 120-s period on a single trial. The rats were given 3–5trials and trained to avoid punishment (remain on shock-free zone).After 24 h, each rat was placed in the light compartment and thestep-through latency was recorded until 300 s had elapsed (retentiontrial). The step-through latency in the retention trial was used as theindex of retention of the training experience. Longer retention latencieswere interpreted as indicating better retention of the training experi-ence (Hefco et al., 2003; Hritcu et al., 2007).

2.4.3. Histological controlAfter the behavioral test, all rats were anesthetized, rapidly decapi-

tated and thewhole brainwas removed. The location of the i.c.v. cannu-las was verified by injecting a dye (Trypan Blue, Sigma) through eachcannula at the endof the experiment. In thisway, after the endof exper-iments the brains were removed and the cannula placement was veri-fied under light microscopy. All cannulas were found to be in the rightposition.

2.4.4. Hippocampus dissectionAlso, considering the importance of hippocampus in memory pro-

cesses (EichenbaumandCohen, 1993) and also the fact that RAS compo-nents are very well expressed here (von Bohlen und Halbach andAlbrecht, 2006), the hippocampi were then collected. Each of the sam-pleswasweighed andhomogenizedwith a Potter Homogenizer coupledwith Cole-Parmer ServodyneMixer in bidistilledwater (1 g tissue/10 mlbidistilled water). Samples were centrifuged for 15 min at 3000 rpm.Following centrifugation, the supernatant was separated and pipettedinto tubes.

2.5. Biochemical estimations

2.5.1. Determination of superoxide dismutaseSuperoxide dismutase (SOD) activity was measured by the percent-

age of reaction inhibition rate of enzyme with WST-1 substrate (a watersoluble tetrazolium dye) and xanthine oxidase using a SOD Assay Kit(Fluka, product number: 19160) according to themanufacturer's instruc-tions. Each endpoint assay was monitored by absorbance at 450 nm (theabsorbance wavelength for the colored product of WST-1 reaction withsuperoxide) after 20 min of reaction time at 37 °C. The percent inhibitionwas normalized by mg protein and presented as SOD activity units.

2.5.2. Determination of glutathione peroxidaseGlutathione peroxidase (GPX) activity was measured using the GPX

cellular activity assay kit CGP-1 (Sigma Chemicals). This kit uses an indi-rectmethod, based on the oxidation of glutathione (GSH) to oxidized glu-tathione (GSSG) catalyzed by GPX, which is then coupled with recyclingGSSG back to GSH utilizing glutathione reductase (GR) and NADPH. Thedecrease in NADPH at 340 nm during oxidation of NADPH to NADP is in-dicative of GPX activity.

2.5.3. Determination of malondialdehydeMalondialdehyde (MDA) levels were determined by thiobarbituric

acid reactive substances (TBARs) assay. 200 μl of supernatant wasadded and briefly mixed with 1 ml of trichloroacetic acid at 50%,0.9 ml of Tris–HCl (pH 7.4) and 1 ml of thiobarbituric acid 0.73%. Aftervortex mixing, samples were maintained at 100 °C for 20 min. After-wards, samples were centrifuged at 3000 rpm for 10 min and superna-tant read at 532 nm. The signal was read against an MDA standardcurve and the results were expressed as nmol/mg protein (Ciobica etal., 2012).

2.5.4. Data analysisThe animal's behavior in Ymaze (as expressed through spontane-

ous alternation and number of arm entries) and passive avoidance(as expressed through the step-through latency time) and the levelsof oxidative stress markers (SOD, GPX and MDA) were statisticallyanalyzed by using one-way analysis of variance (ANOVA). All resultsare expressed as mean±SEM. Post hoc analysis were performedusing Tukey's honestly significant difference test in order to comparegroups. F values for which pb0.05 were regarded as statistically signif-icant. Pearson's correlation coefficient was used to investigate possiblecorrelations between the behavioral parameters in Y maze and passiveavoidance tasks and hippocampal oxidative stress markers.

3. Results

Regarding the behavioral performance of rats in Y-maze task, we ob-served a significant group difference in terms of spontaneous alternation(F(4,20)=25, pb0.0001) (Fig. 1), suggesting significant effects on short-term spatial memory.

Post hoc comparisons showed a significant decrease of spontaneousalternation in Ang II treated rats (p=0.015) vs. saline group and also anincrease in captopril (p=0.01), losartan (p=0.001) or PD-123177 (p=0.007) groups when compared to saline treated rats. A significant in-crease of the spontaneous alternation was also found for losartan(pb0.0001), PD-123177 (pb0.0001) and captopril (pb0.0001) treatedrats, when each group was compared to Ang II group (Fig. 1).

However, the comparison between the Ang II blockers and the ACEblocker showed no significant differences regarding the spontaneousalternation: losartan vs captopril (p=0.07), PD-123177 vs captopril(p=0.6) and losartan vs. PD-123177 (p=0.1).

Moreover, this effect could not be attributed to the motor activity,since the number of armentries in the Y-maze taskwas not significantlychanged in the groups (F(4,20)=0.8, p=0.5), as showed by Fig. 2.

As for the multi-trial passive avoidance task, the behavior of rats wasassessed through the step-through-latency. In this way, we also observeda significant overall effect of the treatment on the latency time (F(4,20)=26, pb0.0001), suggesting significant effects on long-termmemory. Addi-tionally, post hoc analysis revealed a significant decrease of the latencytime in Ang II group when compared to saline-treated rats (p=0.001)and a significant increase of this time in the case of losartan when com-pared to saline group (p=0.042). Still, no significant differenceswere no-ticed in the case of saline vs. PD-123177 (p=0.6) and saline vs. captopril(p=0.4) (Fig. 3).

Also, a significant increase of latency time was seen in losartan(pb0.0001), PD-123177 (p=0.001) and captopril (pb0.0001) groups,when they were compared individually with the Ang II-treated rats.

Regarding the group differences between the Ang II blockers, we no-ticed a significant increase in the latency time for the losartan groupwhen compared to PD-123177 (p=0.008) and no significant differencesbetween losartan vs. captopril (p=0.077) and PD-123177 vs. captopril(p=0.067) (Fig. 3).

In what concerns the oxidative stress markers, when we analyzedthe overall effect of treatment on SOD specific activity we also foundsignificant differences (F(4,20)=30, pb0.0001). Post hoc comparisonsalso showed a significant decreased of SOD activity in Ang II group

Fig. 1. The effects of angiotensin II, captopril, losartan and PD-123177 administration on the spontaneous alternation percentage in the Y-maze task. The values are mean±S.E.M.(n=5 per group). *p=0.01 vs. saline, **p=0.007 vs. saline and ***p=0.001 vs. saline.

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(pb0.0001) when compared with saline, as well as increase of SOD incaptopril (p=0.005) group, as compared to saline-treated rats. Still,no significant differences were noted between saline and losartangroups (p=0.1), while in the case of PD-123177 group we even saw asignificant decrease (p=0.049) of SOD, as compared to saline (Fig. 4).

Additionally, we observed a significant increase of SOD specificactivity in losartan (pb0.0001), PD-123177 (p=0.007) and captopril(pb0.0001) groups when every one of them was compared to AngII-treated rats.

When we compared the Ang II receptor blockers with ACEI-blockers,we observed a significant increase of SOD specific activity in captoprilgroup, as compared to losartan (pb0.0001) and PD-123177 (pb0.0001)groups, while no significant difference was seen between losartan vs.PD-123177 (p=0.49) treated rats (Fig. 4).

Also we observed significant modifications of GPX specific activity inour experimental groups (F(4,20)=25, pb0.0001). Additionally, posthoc comparisons revealed a significant decrease of GPX in Ang II group

Fig. 2. The effects of angiotensin II, captopril, losartan and PD-123177 administration on the n

(p=0.01) when compared to saline treated rats, as well as an increasein captopril group (p=0.002), as compared to saline group. Still, no sig-nificant modifications were reported in the case of losartan (p=0.8)and PD-123177 (p=0.3), as compared to saline (Fig. 5).

Additionally, we also observed a significant increase of GPX specificactivity in losartan (pb0.0001), PD-123177 (p=0.017) and captopril(pb0.0001) groups, when which one of them was compared to Ang IItreated rats.

Also, we observed a significant increase of GPX specific activity incaptopril group, as compared to losartan (pb0.0001) and PD-123177(pb0.0001) groups, while no significant differences were noticed be-tween losartan and PD-123177 (p=0.09) groups (Fig. 5).

Regarding the levels of MDA from the hippocampus we also foundsignificant differences between our treatment groups (F(4,20)=51,pb0.0001).Moreover,whenwe performed the post hoc analysis,we ob-served a significant increase for the MDA levels in the Ang-II group(p>0.0001), as compared to saline rats, and a significant decrease of

umber of arm entries in the Y-maze task. The values are mean±S.E.M. (n=5 per group).

Fig. 3. The effects of angiotensin II, captopril, losartan and PD-123177 on the step-through-latency (s) in the multi-trial passive avoidance task. The values are mean±S.E.M. (n=5per group). *p=0.04 vs. saline and ***p=0.001 vs. saline.

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MDA in captopril (p=0.001) and PD-123177 (p>0.0001) groupswhencompared to saline. However, no significant modifications are reportedfor the losartan-treated group (p=0.6) vs. saline (Fig. 6).

Also a very significant decrease of MDA levels was observed in thecase of captopril (p>0.0001), losartan (p>0.0001) and PD-123177(p>0.0001) groups when each one of them was compared to AngII-treated rats. Additionally, we noticed a significant decrease of MDAin captopril (p=0.005) and PD-123177 (p>0.0001) groupswhen com-pared to losartan. Also, the MDA concentration was significantly de-creased in PD-123177 (p=0.003) group, as compared to captopril(Fig. 6).

Interestingly, when we performed the Pearson correlations betweenthe behavioral parameters which we determined in here (spontaneousalternation percentage in Y-maze/step-through-latency in passive avoid-ance task) and the main oxidative stress markers from the hippocampus,we obtained significant correlations in all six cases: spontaneous alterna-tion vs. GPX (n=25, r=0.478, p=0.016), spontaneous alternation vs.SOD (n=25, r=0.385, p=0.047), spontaneous alternation vs. MDA(n=24, r=−0.593, p=0.002), and also for step-through-latency vs.SOD (n=25, r=0.610, p=0.001), step-through-latency vs. GPX

Fig. 4. The effects of angiotensin II, captopril, losartan and PD-123177 on SOD specific activi**p=0.005 vs. saline and ***p=0.0001 vs. saline.

(n=25, r=0.6, p=0.002) or step-through-latency vs. MDA (n=25,r=−0.476, p=0.016).

4. Discussion

The present study investigated the effects of central Ang II inhibitionwith either AT1 and AT 2 receptor specific blockers or an ACE inhibitoron short-term and long-termmemory and on the oxidative stress statusof the hippocampus, known for its implication in memory processes(Eichenbaum and Cohen, 1993) and also where RAS components arevery well expressed (von Bohlen und Halbach and Albrecht, 2006).Our results provide additional evidence regarding the memory alter-ation and increased oxidative stress induced by the administration ofAng II, while its blocking through the aforementionedmethods resultedin opposite effects.Moreover, we found here significant correlations be-tween thememory related behavioral parameters from the Y-maze andpassive avoidance tasks and the main three markers of the oxidativestress status from the hippocampus.

As previouslymentioned, a complete brain RAS exists aside from theperipheral one and has all necessary precursors and enzymes required

ty in hippocampus. The values are mean±S.E.M. (n=5 per group). *p=0.04 vs. saline,

Fig. 5. The effects of angiotensin II, captopril, losartan and PD-123177 on GPX specific activity in hippocampus. The values are mean±S.E.M. (n=5 per group). *p=0.01 vs. salineand **p=0.002 vs. saline.

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for the formation and metabolism of the biologically active forms ofangiotensin (von Bohlen und Halbach & Albrecht, 2000; von Bohlenund Halbach and Albrecht, 2006; Llorens-Cortes and Mendelsohn,2002; McKinley et al., 2003).

However, aswepreviouslymentioned,when it comes to the cognitiveeffects of Ang II the results are controversial. Thus, when angiotensin II isadministered in the dorsal neostriatum, the retention in the step-downshock avoidance is significantly decreased (Morgan and Routtenberg,1977), while retrieval in the passive avoidance task was significantlyincreased after intracerebroventricular administration of the sameangiotensin II (Braszko, 2002; Braszko et al., 1988a,b; von Bohlenund Halbach and Albrecht, 2006).

It was also demonstrated that Ang II administered to the hippocam-pus impaired retention of the single trial step through shock avoidanceresponse by the activation of AT1 receptors (Lee et al., 1995). Otherstudies have provided evidence that Ang II applied to the hippocampalarea CA1 blocked memory formation through a mechanism involving

Fig. 6. The effects of angiotensin II, captopril, losartan and PD-123177 on MDA concentrationand ***pb0.0001 vs. saline.

the activation of AT2 receptors (Kerr et al., 2005). Additionally, it wasalso stated that there is a possible role of hippocampal angiotensin II re-ceptors in voluntary exercise-induced enhancement of learning andmemory in rat (Akhavan et al., 2008). Still, it has been shown that an-giotensin II-deficient mice present normal retention of spatial memory(Walther et al., 1999).

Also, Maul et al. (2008) demonstrated that mice deficient for theAT2 receptor gene have poor performances in spatial memory tasksand one-way active avoidance. The same authors stated that thesemice have abnormal dendritic spine morphology and length, featureswhich are associated with mental retardation (Mavroudis et al.,2011).

It has been also reported that Ang II receptor blockers could facilitatelong term potentiation (LTP), in the hippocampus of ADmice (Manschotet al., 2003), while it was previously demonstrated that Ang II blockslong-term potentiation in both hippocampus (Armstrong et al., 1996)and amygdala (von Bohlen und Halbach and Albrecht, 1998). Still, the

in hippocampus. The values are mean±S.E.M. (n=5 per group). **p=0.001 vs. saline

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effects of losartan vs PD 123319 on this kind of specific forms of synapticplasticity could be extremely different, as showed by Tchekalarova andAlbrecht, 2007.

Additionally, it was demonstrated that the administration oftelmisartan, an AT1 receptor blocker, significantly decreased somehypertension-induced learning and memory deficits in the water mazetask (Sharma and Singh, 2012). Also, Inaba group demonstrated thatthe administration of olmesartan (another AT1 blocker) diminishedthe cognitive alterations observed in shuttle-box avoidance task forthe human renin and human angiotensinogen gene chimeric transgenicmice, together with an increase of the cerebral blood flow (Inaba et al.,2009). On the other side, it was very recently reported that the adminis-tration of Compound 21, an AT2 receptor agonist, can in fact amelioratethe cognitive decline observed in a specific type 2 diabetes mellitusmodel in mice (Mogi et al., 2012).

The abovementioned conflicting results can be explained by thefact that the cognitive effects of Ang II are very sensible to the variousmethodological approaches (e.g. time and type of administration),differences in dosage, duration of treatment, number and frequencyof training sessions, animal strains or type of memory task evaluated.

This is why authors stated that data suggesting the facilitatory effectof angiotensin II on the memory should be interpreted with care, sinceAng II is also a precursor for neuroactive angiotensin fragments such asAng IV, which is known for its enhancing effects on the cognitive pro-cesses (Gard, 2008; von Bohlen und Halbach, 2003). Thus, different re-sults might be obtained also depending on the time interval betweenthe injection of Ang II and the performing of the behavioral paradigm(Braszko et al., 2006).

ACE inhibitors were reported to enhance conditioned avoidance andalso effects of other enzymes involved in the degradation of Ang II to var-ious other bioactive fragments and habituation memory (Braszko et al.,2003a; Nikolova et al., 2000). Therefore, when administrated prior totraining, captopril facilitated learning in the second trial of the activeavoidance task in mice (Raghavendra et al., 2001), while ACE inhibitionimproved the impaired performance in different models of animal learn-ing (Manschot et al., 2003; Wyss et al., 2003). This could be perhapsexplained by the fact that treatment with ACE inhibitors, such as capto-pril, improves cerebral blood flow and protects against damage inducedby cerebral ischemia in normal (Kumaran et al., 2008) and hypertensive(Braszko et al., 2003a) rats. Also it was very recently demonstrated thatseven days of oral administration of perindopril (a long-acting ACE inhib-itor), resulted in significant improvements in scopolamine-induced defi-ciencies on transfer latency time, path length and platform crossings, asstudied in the water-maze task (Tota et al., 2012b). Interestingly enough,it was reported that perindopril per se had no significant effects on thesaid behavioral parameters, suggesting that besides an improvement ofthe cerebral blood flow (Tota et al., 2011), the beneficial effects of ACE in-hibitors could be also connected with the cholinergic neurotransmission(Tota et al., 2012b).

It was also demonstrated that perindopril could improve thememorydeficits generated in Water maze task, as a result of streptozotocin ad-ministration. Additionally, this improvementwas followed by an increaseof the brain energy metabolism and of the cerebral blood flow, as well asdiminished streptozotocin-induced neuronal damage in hippocampus,entorhinal cortex and periventricular cortical region (Tota et al., 2012a).

Similar aspects were also demonstrated for another ACE inhibitor,enalapril, which partially prevents some deficits in water maze task,by facilitating the long-term potentiation in the hippocampus, as wellas the blood flow of this area in diabetic rats, when compared withuntreated diabetics (Manschot et al., 2003).

Regarding our results, we report here an increase of the spontaneousalternation percentage in Y-maze task, which is known to be an indexfor the short-term spatial memory, in the groups treated with AT1and AT2 receptor blockers (losartan and PD-123177, respectively) orwith an ACE inhibitor (captopril). Additionally, Ang II administrationresulted in decreased alternation percentage. Similar aspects were

also observed in the multi-trial passive avoidance task, where we founda decrease in the step-through-latency, a parameter believed to be rele-vant for the long-termmemory, in the Ang II-treated group,while the ad-ministration of losartan resulted in increased values of this latency.

This confirms that some of these antihypertensive drugs, particularlyACE inhibitors (such as captopril) and some angiotensin receptor blockers(losartan andPD-123177)maybe associatedwith a lower rate ofmemorydecline.

Regarding the effects of Ang II on the oxidative stress, previous reportsalso showed varying results. Thence, it has been reported that the admin-istration of this peptide increases oxidative stress levels (Basso et al.,2007; Wang et al., 2006) by stimulating the activation of the NAD(P)Hoxidase (Chabrashvili et al., 2003; Miller et al., 2007; Rajagopalan et al.,1996), which then triggers the formation of free radicals such as superox-ide anion (Griendling et al., 2000; Inaba et al., 2009). These aspects areconfirmed by the hypertensive bouts induced by the activation of RASin experimental animals, later demonstrated to be associated with an in-creased production of vascular superoxide (Laursen et al., 1997; Munzeland Keaney, 2001).

However, on the other side it was also reported that Ang II admin-istration results in no significant changes of the lipid peroxidationlevels, as expressed through the MDA concentration or in SOD, GPXand catalase enzymatic activity (Gonzales et al., 2002).

In what concerns the Ang II blockers, it was shown that the adminis-tration of losartan or PD-123319 resulted in a decreased oxidative stressstatus in the cerebral blood or glial cells (Antelava et al., 2007; Yaoet al., 2007), while in other experiments losartan significantly de-creased angiotensin II-induced oxidative stress, but PD-123319 did not(Yanagitani et al., 1999).

Additionally, it has been previously demonstrated that the adminis-tration of two different doses of the AT1 blocker telmisartan resulted inthe significant improvement of an experimental hypertension-inducedoxidative stress shown by a significant increase in the aortic superoxideanion levels, brain and serum thiobarbituric acid reactive species, aswell as a significant decrease in the brain levels of the reduced form ofglutathione (Sharma and Singh 2012).

Advanced genetic studies also demonstrated that blocking the AT1receptors with olmesartan results in decreased oxidative stress levelsin transgenic mice for renin and angiotensinogen (Inaba et al., 2009).

Very recent reports also showed that the administration of perindoprilresulted in a significant decrease of a scopolamine-induced oxidativestress status in mice, both in the cortex and the hippocampus, as showedby the decreased levels of MDA and increased levels of glutathione (Totaet al., 2012b). Additionally, the increased oxidative and nitrosative stressstatus, as a result of STZ-induced diabetes in rats, was ameliorated byperindopril within the hippocampus and cortex. This improvement wasmainly shown by the increased levels of GSH, as well as the decreased in-tracellular reactive oxygen species, MDA and nitrite concentrations in theaforementioned central areas (Tota et al., 2012a).

It was also reported that the use of sulfur-containing ACE inhibitors,such as 3-thienylalanine-ornithine-proline (TOP), was associated withreduced oxidative stress in Spontaneously Hypertensive Rats, as showedby the increased levels of antioxidants and decreased levels of lipid per-oxidation markers (Hanif et al., 2009). Additionally, the same authorsreported an increased scavenging activity of TOP, as compared to capto-pril (Hanif et al., 2009).

As for our results, we observed a significant decrease of both SOD andGPX specific activities in theAng II-treated rats, togetherwith an increaseof MDA concentrations from the hippocampus, suggesting pro-oxidanteffects. Also, blocking Ang II with the ACE inhibitor captopril, resultedin decreased levels of MDA and increased specific activities for bothSOD and GPX. Still, no significant modifications of the oxidative stressmarkers were observed in the case of AT 1 blocker losartan. Moreover,we observed a significant decrease of SOD specific activity in thePD-123177 treated group, as compared to saline. This could be perhapsexplained by the fact that SOD is the first line of defense against ROS,

86 W. Bild et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 43 (2013) 79–88

catalyzing the conversion of superoxide radicals to hydrogen peroxide,which is then converted into water by GPX and catalase (Sies, 1997),and in this way this could be a compensatory mechanism, especiallysince the levels of MDA were significantly decreased in PD-123177group, as compared to saline-treated rats. Also, the decrease of SOD inPD-123177 treated ratswasminimally significantwith a p value of 0.049.

The abovementioned results can be linked to the fact that Ang II maycoordinate some important events in the brain inflammatory processesthat togetherwithROS generation could explain someof the neurodegen-erative effects of this peptide (Benigni et al., 2010). Vargas et al. (2012)reported the increased expression of Ang II, monocyte/macrophage(ED-1 positive cells), CD8, the intercellular adhesion molecule-1(ICAM-1) and the lymphocyte function-associated antigen-1 (LFA-1)were reported in an experimental model of diabetes in rats. More impor-tantly, the expression of these molecules was reduced by the administra-tion of losartan and enalapril. Still, the administration of Ang II receptorblockers had no effect on some oxidative and nitrosative stress markers,such as superoxide anion and catalase expression, nitrite content orlipid peroxidation markers (Vargas et al., 2012).

Other reports also stated that the administration of AT1 blockerscould ameliorate the levels of inflammatory stress in rats (Saavedra,2012). Moreover, a significant correlation was demonstrated betweenAng II expression and the levels of some pro-inflammatory moleculesin diabetic rats' cerebellum (Vargas et al., 2012). Also, Zhang groupdemonstrated that Ang II induces cerebral microvascular inflamma-tion via oxidative stress (Zhang et al., 2010).

The role of Ang II-induced oxidative stress was also demonstrated indepression, another very well known inflammatory disorder (Maes etal., 2012; Stefanescu and Ciobica, 2012), since rats performing the forcedswimming test displayed increased concentrations of both superoxideanion and angiotensin II in the cerebrum and cerebellum (Pedreanezet al., 2006). Our group also previously demonstrated that the adminis-tration of Ang-(1–7), which is known for its opposite effects to Ang II(Haulica et al., 2003;Machado et al., 2000), resulted in anxiolytic actionsexerted in the elevated plus maze task, as well as decreased levels ofoxidative stress in the amygdala (Bild and Ciobica, 2012).

The results we report in the present work could be also connectedwith the theories stating that inhibition of the RAS, with these pharma-cological agents (ACE inhibitor or AT1 and AT2 blockers) could play animportant role in the protection of both the nervous and cardiovascularsystems during aging (Akhavan et al., 2008; Culman et al., 2002;Saavedra, 2005; Thone-Reineke et al., 2004). Also this could be relevantfor the implications of oxidative stress in aging, as well as in differentneuropsychiatric disorders and especially in dementia (Padurariu etal., 2010a,b). On the other hand, it has been shown that these types ofcompounds substantially prolonged life span (Yao et al., 2007). Addi-tionally, the analysis of the oxidative stress markers suggests that thisprotective effect is related to an antioxidant action of the RAS inhibitors(as we demonstrated here mainly in the case of captopril) and also to areduced formation of reactive oxygen species.

Increasing amounts of evidence are also showing that RAS is involvedin several neurodegenerative diseases, such as AD and PD (Becker et al.,2008; Danielyan et al., 2010; Davies et al., 2011; Li et al., 2011;Lopez-Real et al., 2005; Ohrui et al., 2004; Patrick and Gordon, 2007; Ro-senberg et al., 2008;Wright and Harding, 2011; Yasar et al., 2008). In thisway, a significant negative correlation was recently reported betweenanti-hypertensive therapies and the incidence of dementia (Duron andHanon, 2010). Additionally, increased ACE concentrations were demon-strated in the various brain regions involved in themodulation of learningand memory processes (Wright and Harding, 2011), while it also seemsthat ACE inhibitors could delay the onset of dementia (Li et al., 2011).

A clear association was demonstrated by several authors betweenthe ACE gene and AD, as well as between the gene for ACE and the atro-phy of hippocampus and amygdala (Sleegers et al., 2005). Advanced ge-netic studies have shown that there is a significant correlation betweenthe angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D)

polymorphism and the incidence of amnestic mild cognitive impairment(aMCI), an intermediate state between normal aging and dementiawhich mainly refers to episodic memory impairment and decline in theability to learn new information (Zhang et al., 2012). Additionally, thesame authors reported that ACE activity in aMCI group was negativelycorrelatedwith the scores of AuditoryVerbal Learning Test-delayed recall(Zhang et al., 2012).

Barnes et al. (1990) stated that Ang II receptor antagonists could re-verse the Ang II-induced inhibitory effects on the acetylcholine releasefrom the temporal cortex (Barnes et al., 1990). In addition, immunohisto-chemical data demonstrated an increased activity of ACE and angiotensinII in the parietal cortex of AD patients (Savaskan et al., 2001), as well as inthe brain of AD rat models (Hou et al., 2008).

There are also reports showing that treatment with Ang II blockersresulted in delayed AD pathogenesis (Hou et al., 2008), as well as largestudies (e.g. PROGRESS) describing a protective effect of ACE inhibitorson the cognitive impairment from the vascular dementia (Tzourio etal., 2003). Additionally, it was demonstrated that ACE blockers could di-minished the hypoperfusion-induced hippocampal neurodegeneration(Kumaran et al., 2008).

On the other side, it was showed that the administration of both ACEand AT2 blockers was not associated in any way with better cognitivefunction in the GEMS study, which was however mainly designed tostudy the effects of Gingko biloba on memory function (Yasar et al.,2012). Similar studies regarding the lack of efficacy for Ang II blockerswere reported in patients with recent ischemic stroke (Diener et al.,2008) or diabetes (Anderson et al., 2011).

In fact, many results in this area of research are contradictory, withreports stating a strong correlations between ACE activity in AD andBraak stage (Miners et al., 2009), while other studies showed no corre-lations at all between ACE activity and age, aswell as between insolubleand soluble Amyloid beta (Abeta) and ACE levels of AD patients (Minerset al., 2010).When it comes to the relation between Ang II blockers andAbeta the results are also conflicting, with human genetic studies dem-onstrating that ACE ismodulating the susceptibility and the progressionof AD via the degradation of Abeta (Hemming and Selkoe, 2005), whilestudies on mice showed that ACE deficiency does not alter in any waythe concentrations of beta-amyloid (Eckman et al., 2006).

Further studies are warranted in order to determine the role of thevarious angiotensin peptides and angiotensin receptors in some neuro-pathological states. Our group has also undergo studies regarding thepossible co-administration of AT1 and AT2 receptor antagonists andtheir relevance onmemory functions and oxidative stress status, consid-ering that a functional interaction between AT1 and AT2 receptorsseems to exist (Gelband et al., 1997), with reports stating that the simul-taneous administration of AT1 and AT2 blockers could generate summa-tive effects for example on the acquisition processes in a conditionedavoidance response task, as compared to their individual administration(Braszko, 2002).

However, based on the increased memory performance and de-creased oxidative stress mediated by Ang II inhibitors, which werealso demonstrated in the present work, the manipulation of the centralRAS could be considered as a promising therapeutic target in the treat-ment of cognitive dysfunctions. For this purpose, a better understandingof the complex interactions between Ang II, behavioral processes andneuronal oxidative stress is necessary and could lead to new importanttherapeutic aspects.

5. Conclusions

This study demonstrates that the inhibition of central Ang II witheither an ACE inhibitor (captopril) or AT1 and AT 2 blockers (losartanand PD-123177, respectively) resulted in a significant enhancementof both short term and long term memory as showed in Y-maze andpassive avoidance task, as well as a significant decrease of the oxida-tive stress status in the hippocampus. Moreover, we demonstrated

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here a significant correlation between thesememory related behavioralparameters and the specific markers of the oxidative stress status.

Acknowledgments

Ciobica Alin is supported by a POSDRU grant /89/1.5/S/49944,Alexandru Ioan Cuza University, Iasi.

References

Akhavan M, Emami-Abarghoie M, Sadighi-Moghaddam B, Safari M, Yousefi Y,Rashidy-Pour A. Hippocampal angiotensin II receptors play an important role inmediating the effect of voluntary exercise on learning and memory in rat. BrainRes 2008;1232:132–8.

Armstrong DL, Garcia EA, Ma T, Quinones B, Wayner MJ. Angiotensin II blockade oflong-term potentiation at the perforant path–granule cell synapse in vitro. Pep-tides 1996;17(4):689–93.

Anderson C, Teo K, Gao P, Arima H, Dans A, Unger T. Renin angiotensin system blockadeand cognitive function in patients at high risk of cardiovascular disease: analysis ofdata from the ONTARGET and TRANSCEND studies. Lancet Neurol 2011;10:43–53.

Antelava N, Gongadze N, Gogolauri M, Kezeli T, Pachkoriia K. Comparative characteris-tic of angiotensin-converting enzyme inhibitor – captopril and the angiotensin IIreceptor blockers – losartan action on the oxidative metabolism in experimentalhyperlipidemia in rabbits. Georgian Med News 2007;150:57–60.

Barnes J, Barnes N, Costall B, Horovitz Z, Ironside J, Naylor R. Angiotensin II inhibits ace-tylcholine release from human temporal cortex: implications for cognition. BrainRes 1990;507:341–3.

Basso N, Cini R, Pietrelli A, Ferder L, Terragno N. Protective effect of long-term angio-tensin II inhibition. Am J Physiol Heart Circ Physiol 2007;293:1351–8.

Becker C, Jick S, Meier C. Use of antihypertensives and the risk of Parkinson disease.Neurology 2008;70:1438–44.

Benigni A, Cassis P, Remuzzi1 G. Angiotensin II revisited: new roles in inflammation,immunology and aging. EMBO Mol Med 2010;2:247–57.

Bild W, Ciobica A. Angiotensin-(1–7) central administration induces anxiolytic-like ef-fects in elevated plus maze and decreased oxidative stress in the amygdala. J AffectDisord 2012. http://dx.doi.org/10.1016/j.jad.2012.07.024. [Epub ahead of print][PubMed PMID:22868060].

Bonini J, Bevilaqua L, Zinn C, Kerr D, Medina J, Izquierdo I, et al. Angiotensin II disruptsinhibitory avoidance memory retrieval. Horm Behav 2006;50:308–13.

Braszko J. AT(2) but not AT(1) receptor antagonism abolishes angiotensin II increase ofthe acquisition of conditioned avoidance responses in rats. Behav Brain Res2002;131:79–86.

Braszko J. Valsartan abolishes most of the memory-improving effects of intracerebro-ventricular angiotensin II in rats. Clin Exp Hypertens 2005;27:635–49.

Braszko J, Kupryszewski G, Witczuk B, Wiśniewski K. Angiotensin II-(3–8)-hexapeptideaffects motor activity, performance of passive avoidance and a conditioned avoid-ance response in rats. Neuroscience 1988a;27:777–83.

Braszko J, Kupryszewski G, Witczuk B, Wisniewski K. Angiotensin II(3–8) hexapeptideaffects motor activity, performance of passive avoidance and a conditioned avoid-ance response in rats. Neuroscience 1988b;27:777–83.

Braszko J, Karwowska-Polecka W, Halicka D, Gard P. Captopril and enalapril improvecognition and depressed mood in hypertensive patients. J Basic Clin PhysiolPharmacol 2003a;14:323–43.

Braszko J, Kulakowska A, Winnicka M. Effects of angiotensin II and its receptor antag-onists on motor activity and anxiety in rats. J Physiol Pharmacol 2003b;54:271–81.

Braszko J, Walesiuk A, Wielgat P. Cognitive effects attributed to angiotensin II may re-sult from its conversion to angiotensin IV. J Renin Angiotensin Aldosterone Syst2006;7:168–74.

Chabrashvili T, Kitiyakara C, Blau J, Karber A, Aslam S, Welch W, et al. Effects of ANG IItype 1 and 2 receptors on oxidative stress, renal NADPH oxidase, and SOD expres-sion. Am J Physiol Regul Integr Comp Physiol 2003;285:117–24.

Chalas A, Conway E. No evidence for involvement of angiotensin II in spatial learning inwater maze in rats. Behav Brain Res 1996;81:199–205.

Ciobica A, Bild W, Hritcu L, Haulica I. Brain renin–angiotensin system in cognitivefunction: pre-clinical findings and implications for prevention and treatment ofdementia. Acta Neurol Belg 2009;109:171–80.

Ciobica A, Hritcu L, Nastasa V, Padurariu M, Bild W. Inhibition of central angiotensinconverting enzyme exerts anxiolytic effects by decreasing brain oxidative stress.J Med Biochem 2010;30(2):109–14.

Ciobica A, Hritcu L, Bild V, Padurariu M, Bild W. The effects of angiotensin II and its re-ceptor blockers on anxiety status and central oxidative stress in rat. J Neurol2011;258:69–70.

Ciobica A, Olteanu Z, Padurariu M, Hritcu L. The effects of pergolide on memory and ox-idative stress in a rat model of Parkinson's disease. J Physiol Biochem 2012;68:59–69.

Culman J, Baulmann J, Blume A, Unger T. The renin–angiotensin system in the brain: anupdate. J Renin Angiotensin Aldosterone Syst 2001;2:96-102.

Culman J, Blume A, Gohlke P, Unger T. The renin–angiotensin system in the brain: pos-sible therapeutic implications for AT(1)-receptor blockers. J Hum Hypertens2002;16:64–70.

Danielyan L, Klein R, Hanson LR, Buadze M, Schwab M, Gleiter C. Protective effects ofintranasal losartan in the APP/PS1 transgenic mouse model of Alzheimer disease.Rejuvenation Res 2010;13:195–201.

Davies N, Kehoe P, Ben-Shlomo Y, Martin R. Associations of antihypertensive treatmentswith Alzheimer's disease, vascular dementia, and other dementias. J Alzheimers Dis2011;26:699–708.

Diener H, Sacco R, Yusuf S, Cotton D, Ounpuu S, Lawton W, et al. Effects of aspirin plusextended-release dipyridamole versus clopidogrel and telmisartan on disabilityand cognitive function after recurrent stroke in patients with ischaemic stroke inthe prevention regimen for effectively avoiding second strokes (PRoFESS) trial: adouble-blind, active and placebo-controlled study. Lancet Neurol 2008;7:875–84.

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

Eckman E, Adams S, Troendle F, Stodola B, Kahn M, Fauq A. Regulation of steady-statebeta-amyloid levels in the brain by neprilysin and endothelin-converting enzymebut not angiotensin-converting enzyme. J Biol Chem 2006;281:30471–8.

Eichenbaum H, Cohen N. Memory, amnesia, and the hippocampal system. MIT Press;1993.

Gard P. The role of angiotensin II in cognition and behaviour. Eur J Pharmacol 2002;438:1-14.

Gard P. Cognitive-enhancing effects of angiotensin IV. BMC Neurosci 2008;9:S15.Gard PR, Rusted JM. Angiotensin and Alzheimer's disease: therapeutic prospects. Ex-

pert Rev Neurother 2004;4(1):87–96.Gelband C, ZhuM, Lu D, Reagan L, Fluharty S, Posner P. Functional interactions between

neuronal AT1 and AT2 receptors. Endocrinology 1997;138:2195–8.Gonzales S, Noriega G, Tomaro M, Peña C. Angiotensin-(1–7) stimulates oxidative

stress in rat kidney. Regul Pept 2002;106:67–70.Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular bi-

ology and disease. Circ Res 2000;86:494–501.Hanif K, Snehlata Pavar C, Arif E, Biswas P, Fahim M, Pasha M, et al. Effect of

3-thienylalanine-ornithine-proline, new sulfur-containing angiotensin-convertingenzyme inhibitor on blood pressure and oxidative stress in spontaneously hyperten-sive rats. J Cardiovasc Pharmacol 2009;53:145–50.

Haulica I, Neamtu C, Stratone A, Petrescu G, Branisteanu D, Rosca V, et al. Evidence forthe involvement of cerebral renin–angiotensin system (RAS) in stress analgesia.Pain 1986;27:237–45.

Haulica I, Bild W, Mihaila CN, Ionita T, Boisteanu CP, Neagu B. Biphasic effects of angio-tensin (1–7) and its interactions with angiotensin II in rat aorta. J Renin Angioten-sin Aldosterone Syst 2003;4:124–8.

Haulica I, Bild W, Serban N. Angiotensin peptides and their pleiotropic actions. J ReninAngiotensin Aldosterone Syst 2005;6:121–31.

Hefco V, Yamada K, Hefco A, Hritcu L, Tiron A, Nabeshima T. Role of themesotelencephalicdopamine system in learning and memory processes in the rat. Eur J Pharmacol2003;475:55–60.

HemmingM, Selkoe D. Amyloid beta-protein is degraded by cellular angiotensin-convertingenzyme (ACE) and elevated by an ACE inhibitor. J Biol Chem 2005;280:37644–50.

Hou Y, Wang L, Zhou K, Chen Y, Tian Z, Song J, et al. Altered angiotensin-converting enzymeand its effects on the brain in a rat model of Alzheimer disease. Chin Med J (Engl)2008;121:2320–3.

Hritcu L, Clicinschi M, Nabeshima T. Brain serotonin depletion impairs short-termmemory, but not long-term memory in rats. Physiol Behav 2007;91:652–7.

Inaba S, Iwai M, Furuno M, Tomono Y, Kanno H, Senba I, et al. Continuous activation ofrenin–angiotensin system impairs cognitive function in renin/angiotensinogentransgenic mice. Hypertension 2009;53:356–62.

Kerr D, Bevilaqua L, Bonini J, Rossato JI, Kohler C, Medina J, et al. Angiotensin II blocksmemory consolidation through an AT2 receptor-dependent mechanism. Psycho-pharmacology (Berl) 2005;179:529–35.

Kumaran D, Udayabanu M, Kumar M, Aneja R, Katyal A. Involvement of angiotensinconverting enzyme in cerebral hypoperfusion induced anterograde memory im-pairment and cholinergic dysfunction in rats. Neuroscience 2008;155:626–39.

Laursen J, Rajagopalan S, Galis Z, Tarpey M, Freeman B, Harrison D. Role of superoxidein angiotensin II-induced but not catecholamine-induced hypertension. Circulation1997;95:588–93.

Lee E, Ma Y,Wayner M, Armstrong D. Impaired retention by angiotensin-II mediated bythe AT(1) receptor. Peptides 1995;16:1069–71.

Li N, Lee A, Whitmer R, Kivipelto M, Lawler E, Kazis L. Use of angiotensin receptorblockers and risk of dementia in a predominantly male population: prospective co-hort analysis. BMJ 2011;340:b5465.

Llorens-Cortes C, Mendelsohn F. Organisation and functional role of the brain angioten-sin system. J Renin Angiotensin Aldosterone Syst 2002;1:39–48.

Lopez-Real A, Rey P, Soto-Otero R, Mendez-Alvarez E, Labandeira-Garcia JL.Angiotensin-reduces oxidative stress and protects dopaminergic neurons in a6-hydroxydopamine rat model of Parkinsonism. J Neurosci Res 2005;81:865–73.

Machado R, Santos R, Andrade S. Opposing actions of angiotensins on angiogenesis. LifeSci 2000;66:67–76.

Maes M, Fišar Z, Medina M, Scapagnini G, Nowak G, Berk M. New drug targets indepression: inflammatory, cell-mediated immune, oxidative and nitrosative stress, mi-tochondrial, antioxidant, and neuroprogressive pathways. And new drug candidates—Nrf2 activators and GSK-3 inhibitors. Inflammopharmacology 2012;20:127–50.

Manschot S, Biessels G, Cameron N, Cotter M, Kamal A, Kappelle L, et al. Angiotensinconverting enzyme inhibition partially prevents deficits in water maze performance,hippocampal synaptic plasticity and cerebral blood flow in streptozotocin-diabeticrats. Brain Res 2003;966:274–82.

Maul B, Von Bohlen Und Halbach O, Becker A, Sterner-Kock A, Voigt JP. Impaired spatialmemory and altered dendritic spine morphology in angiotensin II type 2 receptor-deficient mice. J Mol Med 2008;86:563–71.

Mavroudis I, Fotiou D, Manani M, Njaou S, Frangou D, Costa V, et al. Dendritic pathologyand spinal loss in the visual cortex in Alzheimer's disease: a Golgi study in pathol-ogy. Int J Neurosci 2011;121:347–54.

88 W. Bild et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 43 (2013) 79–88

McKinleyM, AlbistonA, AllenA,MathaiM,MayC,McAllenR, et al. The brain renin–angioten-sin system: location and physiological roles. Int J Biochem Cell Biol 2003;35:901–18.

Miller S, Norton L, Murphy M, Dalsing M, Unthank J. The role of the renin–angiotensinsystem and oxidative stress in spontaneously hypertensive rat mesentericcollateral growth impairment. Am J Physiol Heart Circ Physiol 2007;292:2523–31.

Miners S, Ashby E, Baig S, Harrison R, Tayler H, Speedy E. Angiotensinconverting enzymelevels and activity in Alzheimer's disease: differences in brain and CSF ACE and asso-ciation with ACE1 genotypes. Am J Transl Res 2009;1:163–77.

Miners J, van Helmond Z, Kehoe P, Love S. Changes with age in the activities ofbeta-secretase and the Abeta-degrading enzymes neprilysin, insulin-degrading en-zyme and angiotensin-converting enzyme. Brain Pathol 2010;20:794–802.

Mogi M, Iwanami Jun, Jing Fei, Tsukuda Kana, Horiuchi Masatsugu. Co-treatment withmemantine and compound 21, an angiotensin II type 2 receptor agonist, preventscognitive decline in type 2 diabetic mice. Alzheimers Dement 2012;8:P707.

Morgan J, Routtenberg A. Angiotensin injected into the neostriatum after learning dis-rupts retention performance. Science 1977;196:87–9.

Munzel T, Keaney Jr JF. Are ACE inhibitors a “magic bullet” against oxidative stress? Cir-culation 2001;104:1571–4.

Nikolova J, Getova D, Nikolov F. Effects of ACE inhibitors on learning and memory pro-cesses in rats. Folia Med (Plovdiv) 2000;42:47–51.

Ohrui T, Matsui M, Yamaya H, Arai S, Ebihara M, Maruyama H. Angiotensin-convertingenzyme inhibitors and incidence of Alzheimer's disease in Japan. J Am Geriatr Soc2004;52:649–50.

Padurariu M, Ciobica A, Dobrin I, Stefanescu C. Evaluation of antioxidant enzymes ac-tivities and lipid peroxidation in schizophrenic patients treated with typical andatypical antipsychotics. Neurosci Lett 2010a;479:317–20.

Padurariu M, Ciobica A, Hritcu L, Stoica B, Bild W, Stefanescu C. Changes of some oxidativestressmarkers in the serum of patients withmild cognitive impairment and Alzheimer'sdisease. Neurosci Lett 2010b;469:6-10.

Patrick G, Gordon KW. Is inhibition of the renin–angiotensin system a new treatmentoption for Alzheimer's disease? Lancet Neurol 2007;6:373–8.

Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 6th ed. San Diego: Aca-demic Press Elsevier; 2006.

Pedreanez A, Arcaya JL, Carrizo E, Mosquera J. Forced swimming test increases superoxideanion positive cells and angiotensin II positive cells in the cerebrum and cerebellumof the rat. Brain Res Bull 2006;71:18–22.

Raghavendra V, Chopra K, Kulkarni S. Comparative studies on the memory-enhancingactions of captopril and losartan in mice using inhibitory shock avoidance para-digm. Neuropeptides 2001;35:65–9.

Rajagopalan S, Kurz S, Münzel T, Tarpey M, Freeman B, Griendling K, et al. AngiotensinII-mediated hypertension in the rat increases vascular superoxide production viamembrane NADH/NADPH oxidase activation. J Clin Invest 1996;97:1916–23.

Rosenberg P, Mielke M, Tschanz J, Cook L, Corcoran C, Hayden K, et al. Effects of cardio-vascular medications on rate of functional decline in Alzheimer disease. Am JGeriatr Psychiatry 2008;16:883–92.

Saavedra J. Brain angiotensin II: new developments, unanswered questions and thera-peutic opportunities. Cell Mol Neurobiol 2005;25:485–512.

Saavedra J. Angiotensin II AT(1) receptor blockers ameliorate inflammatory stress: a benefi-cial effect for the treatment of brain disorders. Cell Mol Neurobiol 2012;32:667–81.

Santos RAS, Campagnole-Santos MJ, Andrade SP. Angiotensin-(1–7): an update. RegulPept 2000;91:45–62.

Savaskan KC, Hock G, Olivieri S, Bruttel C, Rosenberg C, Hulette F, et al. Cortical alter-ations of angiotensin converting enzyme, angiotensin II and AT1 receptor inAlzheimer's dementia. Neurobiol Aging 2001;22:541–6.

Sharma B, Singh N. Experimental hypertension induced vascular dementia: pharmaco-logical, biochemical and behavioral recuperation by angiotensin receptor blockerand acetylcholinesterase inhibitor. Pharmacol Biochem Behav 2012;102:101–8.

Shepherd J, Bill D, Dourish C, Grewal S, Mclenachan A, Stanhope K. Effects of the selec-tive angiotensin II receptor antagonists losartan and PD123177 in animal modelsof anxiety and memory. Psychopharmacology (Berl) 1996;126:206–18.

Sies H. Oxidative stress: oxidants and antioxidants. Exp Physiol 1997;82:291–5.Sleegers KT, Heijer EJ, van Dijk A, Hofman AM, Bertoli-Avella PJ, Koudstaal MM, et al.

ACE gene is associated with Alzheimer's disease and atrophy of hippocampusand amygdala. Neurobiol Aging 2005;26:1153–9.

Stefanescu C, Ciobica A. The relevance of oxidative stress status in first episode and re-current depression. J Affect Disord 2012;143(1–3):34–8.

Tchekalarova J, Albrecht D. Angiotensin II suppresses long-term potentiation in the lat-eral amygdala of mice via L-type calcium channels. Neurosci Lett 2007;415:68–72.

Thone-Reineke C, Zimmermann M, Neumann C, Krikov M, Li J, Gerova N, et al. Are an-giotensin receptor blockers neuroprotective? Curr Hypertens Rep 2004;6:257–66.

Tota S, Kamat P, Saxena G, Hanif K, Najmi A, Nath C. Central angiotensin converting en-zyme facilitates memory impairment in intracerebroventricular streptozotocintreated rats. Behav Brain Res 2011;226:317–30.

Tota S, Kamat P, Saxena G, Hanif K, Najmi A, Nath C. Central angiotensin converting en-zyme facilitates memory impairment in intracerebroventricular streptozotocintreated rats. Behav Brain Res 2012a;226:317–30.

Tota S, Nath C, Najmi AK, Shukla R, Hanif K. Inhibition of central angiotensin convertingenzyme ameliorates scopolamine induced memory impairment in mice: role ofcholinergic neurotransmission, cerebral blood flow and brain energy metabolism.Behav Brain Res 2012b;232:66–76.

Tzourio C, Anderson N, Chapman M, Woodward B, Neal S, MacMahon J, et al. Effects ofblood pressure lowering with perindopril and indapamide therapy on dementiaand cognitive decline in patients with cerebrovascular disease. Arch Intern Med2003;163:1069–75.

Vargas R, Rincón J, Pedreañez A, Viera N, Hernández-Fonseca JP, Peña C, et al. Role ofangiotensin II in the brain inflammatory events during experimental diabetes inrats. Brain Res 2012;1453:64–76.

von Bohlen und Halbach O, Albrecht D. Angiotensin II inhibits long-term potentiationwithin the lateral nucleus of the amygdala through AT1 receptors. Peptides1998;19(6):1031–6.

von Bohlen und Halbach O, Albrecht D. Identification of angiotensin IV binding sites inthe mouse brain by a fluorescent binding study. Neuroendocrinology 2000;72(4):218–23.

von Bohlen und Halbach O. Angiotensin IV in the central nervous system. Cell TissueRes 2003;311:1–9.

von Bohlen und Halbach O, Albrecht D. The CNS renin–angiotensin system. Cell TissueRes 2006;326:599–616.

Walther T, Voigt J, Fukamizu A, Fink H, Bader M. Learning and anxiety in angiotensin-deficient mice. Behav Brain Res 1999;100:1–4.

Wang D, Chabrashvili T, Borrego L, Aslam S, Umans J. Angiotensin II infusion alters vas-cular function in mouse resistance vessels: roles of O and endothelium. J Vasc Res2006;43:109–19.

Winnicka M, Braszko J, Wiśniewski K. 6-OHDA lesions to amygdala and hippocampusattenuate memory-enhancing effect of the 3–7 fragment of angiotensin II. GenPharmacol 1998;30:801–5.

Wright J, Harding J. The brain RAS and Alzheimer's disease. Exp Neurol 2011;223:326–33.

Wyss J, Kadish I, van Groen T. Age-related decline in spatial learning and memory:attenuation by captopril. Clin Exp Hypertens 2003;25:455–74.

Yanagitani Y, Rakugi H, Okamura A,Moriguchi K, Takiuchi S, OhishiM. Angiotensin II type1 receptor-mediated peroxide production in human macrophages. Hypertension1999;33:335–9.

Yao E, Fukuda N, Matsumoto T, Kobayashi N, Katakawa M, Yamamoto C, et al. Losartanimproves the impaired function of endothelial progenitor cells in hypertension viaan antioxidant effect. Hypertens Res 2007;30:1119–28.

Yasar S, Zhou J, Varadhan R, Carlson C. The use of angiotensin converting enzyme inhib-itors and diuretics is associated with a reduced incidence of impairment on cognitionin elderly women. Clin Pharmacol Ther 2008;84:119–26.

Yasar S, Lin F, Fried L, Kawas C, Sink K, DeKosky S, et al, Ginkgo Evaluation of Memory(GEM) Study Investigators. Diuretic use is associated with better learning andmemory in older adults in the Ginkgo Evaluation of Memory Study. AlzheimersDement 2012;8:188–95.

Zhang M, Mao Y, Ramirez SH, Tuma RF, Chabrashvili T. Angiotensin II induced cerebralmicrovascular inflammation and increased blood–brain barrier permeability viaoxidative stress. Neuroscience 2010;171:852–8.

Zhang Z, Deng L, Yu H, Shi Y, Bai F, Xie C, Yuan Y, Jia J, Zhang Z. Association ofangiotensin-converting enzyme functional gene I/D polymorphism with amnesticmild cognitive impairment. Neurosci Lett 2012;514(1):131–5.