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Contents lists available at SciVerse ScienceDirect

Neurochemistry International

journal homepage: www.elsevier .com/locate /nci

The levels of renin–angiotensin related components are modifiedin the hippocampus of rats submitted to pilocarpine model of epilepsy

Telma Luciana Furtado Gouveia a, Maria Isabel Berzaghi Frangiotti a, Joíse Marques Vieira de Brito a,Eduardo Ferreira de Castro Neto a, Maisa Mayume Sakata c, Andreia Cristina Febba c, Dulce Elena Casarini c,Débora Amado a, Esper Abrão Cavalheiro a, Sandro Soares Almeida e, Martha Trindade Manchini d,Ronaldo Carvalho Araújo e, José Antonio Silva Jr. d, Maria da Graça Naffah-Mazzacoratti a,b,⇑a Neurology and Neurosurgery Department, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazilb Biochemistry Department, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazilc Medicine Department, Nephrology Laboratory, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazild Rehabilitation Department, Universidade Nove de Julho, São Paulo, Brazile Biophysical Department, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil

a r t i c l e i n f o

303132333435363738394041

Article history:Received 14 September 2011Received in revised form 9 April 2012Accepted 11 April 2012Available online xxxx

Keywords:PilocarpineAngiotensin IAngiotensin IITemporal lobe epilepsyAngiotensin converting enzyme (ACE)

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0197-0186/$ - see front matter � 2012 Published byhttp://dx.doi.org/10.1016/j.neuint.2012.04.012

⇑ Corresponding author at: Biochemistry DepartmSão Paulo (UNIFESP), São Paulo, Brazil. Tel.: +5555494743.

E-mail address: naffahmazz.nexp@epm.br (M.d.G.

Please cite this article in press as: Gouveia, T.L.Fmitted to pilocarpine model of epilepsy. Neuro

a b s t r a c t

We previously showed patients with temporal lobe epilepsy (TLE) present an increased expression ofangiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulationof renin–angiotensin system (RAS) in this disease.

This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels ofthe peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1–7 Ang(1–7), were detected byHPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin convertingenzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampusof Wistar rats by real time PCR, during acute (n = 10), silent (n = 10) and chronic (n = 10) phases of pilo-carpine-induced epilepsy.

We observed an increased peptide level of Ang1–7 into acute and silent phases, decreasing importantly(p 6 0.05) in the chronic phase, suggesting that AngI may be converted into Ang(1–7) by NEP, which ispresent in high levels in these periods.

Our results also showed increased peptide level of AngII in the chronic phase of this model. In contra-position, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngImay be cleaved to AngII by tonin, which showed increased expression in all phases. We found changesin AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1–7 could act at Mas receptor duringthe silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides,their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-inducedepilepsy.

� 2012 Published by Elsevier Ltd.

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1. Introduction

Renin–angiotensin system (RAS) is originally known as a bloodpressure controller and its role in inflammation has been only re-cently studied. This system is characterized by a precursor mole-cule angiotensinogen, and through the renin enzyme is cleavedto angiotensin I (AngI), which is a decapeptide with little or nonebiological action. AngI can circulate in blood and tissues beingcleaved by a key enzyme called angiotensin converting enzyme

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., et al. The levels of renin–angchem. Int. (2012), http://dx.doi

(ACE), which removes the dipeptide His-Leu from C-terminal sideof the molecule, transforming AngI into AngII, which has an impor-tant biological action (review in de Gasparo et al. (2000)).

Although widely distributed in peripheral systems, all RAS com-ponents have also been found in the brain where they activelymodulate several functions, including exploratory behavior, stress,anxiety, learning and memory acquisition (Chen et al., 1993; Meff-ert et al., 1996; Saavedra et al., 2005; Wright and Harding, 2004;Yamada et al., 1996).

AngII has been considered to be the major RAS active peptide,acting in the periphery as paracrine and endocrine hormone. Thispolypeptide could also be considered a neurotransmitter/neuro-modulator through its action on AT1 and AT2 receptors presentin several brain regions (Wright et al., 2002).

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It is already known that AngII promotes inflammatory responseinducing oxidative stress, NADPH oxidase and NF-jb activationand consequently production of several cytokines and adhesionmolecules (Benicky et al., 2011). In addition, many experimentalstudies suggest that NF-jb and cytokines participate in the neuro-nal excitability and/or in glial scar formation in epilepsy, showing arelationship between this chronic disease and inflammatoryresponse (Ferder et al., 2003, 2006). In addition, evidences haveemerged indicating RAS participation in some neurodegenerativedisorders such as Alzheimer (Barnes et al., 1991; Tian et al.,2004), Parkinson (Grammatopoulos et al., 2005) and Huntington(Bird, 1980) diseases.

In epilepsy, clinical and experimental findings showed impor-tant role of inflammatory processes linked to this disease (Argana-raz et al., 2004a,b; Vezzani and Granata, 2005), particularly in themechanisms underlying generation of seizures (ictogenesis) andtransformation of a normal neuronal network into a seizure-gener-ating one (Pitkanen and Sutula, 2002). Recently, we observed an in-creased expression of several inflammatory markers such as kininreceptors, cytokines and nitric oxide in limbic areas of subjectswith TLE (Perosa et al., 2007; Silva et al., 2008; Gouveia et al.,2011; Varella et al., 2011). Our group also showed an up-regulationof AT1 and AT2 receptors in the cortex and in the hippocampal for-mation of patients with TLE with mesial temporal sclerosis (MS)(Arganaraz et al., 2008), supporting RAS0 involvement on thispathology.

AngII may be generated in the brain via the classical pathway,through renin and ACE action or can be directly released from angi-otensinogen by cathepsin G or tonin actions (Lippoldt et al., 1995).According to Araujo et al. (2002), tonin may be an alternative path-way to AngII generation in the brain and could participate in thispeptide’s effects.

Recently it is become evident that another peptide namedAngiotensin (1–7) Ang(1–7) is active in several organs includingthe brain (Alenina et al., 2008), and several endopeptidases suchas neprilysin, thimet and neutral endopeptidase (NEP) may metab-olize the AngII in the brain generating Ang(1–7) (Karamyan andSpeth, 2007).

Mas is the main receptor for Ang(1–7) peptide being a physio-logical antagonist of AT1 receptor (Santos et al., 2003; Kosteniset al., 2005), which is also related to neuronal regulation. Mas-deficient mice exhibit increased anxiety; despite of an enhancedlong-term potentiation in the hippocampus. Furthermore, briefseizure episodes led to a significant and transient increase in MasmRNA in rat hippocampus (Martin and Hockfield, 1993).

Several animal models have been used to study TLE, such as thoseinduced by kainic acid (Ben-Ari et al., 1981), kindling (McNamara,1994) and pilocarpine (Turski et al., 1983) as they show similarityto human phenomena. Pilocarpine administered acutely to rats/ormice produces electrographic and behavioral limbic seizures andstatus epilepticus that last up to 12 h (acute phase). The statusepilepticus is accompanied by widespread brain damage that ismainly observed in limbic areas. This acute period is followed bysilent (seizure-free) period and finally by a chronic period character-ized by spontaneous recurrent seizures (Cavalheiro et al., 1991).

Then, this study was designed to contribute for better under-standing of the relationship between the RAS and TLE during epi-leptogenesis process, without anti-epileptic drugs interference.

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2. Methods

2.1. Animals and SE induction

The experiments were performed under the approval of UNI-FESP Ethical Committee Institutional ethical approval of protocol

Please cite this article in press as: Gouveia, T.L.F., et al. The levels of renin–angmitted to pilocarpine model of epilepsy. Neurochem. Int. (2012), http://dx.doi

and all efforts were made to minimize animal suffering. Wistaradult male rats, weighing 250 g, were housed in groups of threeor four per cage and maintained in controlled room temperature,humidity and light–dark cycle (12:12 h) with chow pellets andtap water available ad libitum.

The rats received a single dose of pilocarpine [350 mg/kg,intraperitoneal (i.p.)]. To prevent peripheral cholinergic effects,scopolamine methylnitrate was injected subcutaneously at adose of 1 mg/kg, 30 min before pilocarpine administration. Onlyrats that displayed status epilepticus (SE) were included in thisstudy.

The SE was monitored and terminated with an injection of diaz-epam (10 mg/kg, ip; Roche, Brazil) 5 h after SE onset. The silent andchronic periods were followed by video-recordings of the animals24 h/day during 60 days. The animals were killed by decapitation;the brain was quickly removed and stored at �80 �C until assay.

Four groups of rats were analyzed: Control group (saline); Acutegroup (rats killed 5 h after SE onset); Silent group (animals killed5 days after pilocarpine injection) and Chronic group (rats killedduring the period of spontaneous seizures, 60 days after pilocar-pine injection). A group of animals, which received pilocarpinebut did not develop SE was initially used as a second control. Asthey did not present statistical difference when compared to con-trol group, they were excluded in this study. There were 10 ani-mals in each group.

2.2. Quantification of angiotensin-related peptides in the hippocampus

Quantification of AngI, AngII and Ang(1–7) were done accordingto assay previously described by Ronchi et al. (2007). Briefly,dissected hippocampi were weighed and homogenized with100 mM phosphate buffer, pH 7.2, containing 340 nM,sucrose, 300 mM NaCl and a mix of proteases inhibitors ethylene-diaminetetraacetic (EFTA) tripotassium salt dehydrate (25 mM),o-phenanthroline (0.44 mM), pepstatin A (0.12 mM) and 4-chloro-mercuribenzoic acid (1 mM). Twenty milliliters of solvent wasadded to 1 g of tissue, followed by centrifugation at 15,000g for20 min. The samples were concentrated in C18 Sep-Pak columnsactivated with methanol (5 mL), tetrahydrofuran (5 mL), hexane(5 mL), methanol (5 mL) and water (10 mL). Peptides were elutedwith ethanol, acetic acid and water (90:4:6).

Samples were then lyophilized and resuspended in 500 lL ofmobile phase A: 5% acetonitrile (50 mL) in 0.1% orthophosphoricacid (1 mL). Peptides were separated in a reverse-phase columnAquapore ODS 300 (250 � 4.6 mm), 7l, using the gradient 5–35%of mobile phase B: 95% acetonitrile in H3PO4 0.1% in the flow1.5 mL/min for 40 min in the Milton Roy System, constituted oftwo constaMetric 3000 pumps, a UV detector spectroMonitor3100, a programmer GM 4000 and a mixer. Synthetic standardswere used and peptide detection was carried out at 214 nm. Theresults were expressed in ng/mL and corrected by the weight ofthe tissue.

2.3. Quantitative real-time PCR for RAS molecules

Dissected hippocampi were frozen in liquid nitrogen and storedat �80 �C. Thawed tissue was homogenized in 1 mL TRIzol reagent(Invitrogen, Carlsbad, CA, USA) and total RNA was isolated accord-ing to the manufacturer’s instructions. RNA was subjected toDNase I digestion, followed by reverse transcription to cDNA andPCR was performed in a 7000 Sequence Detection System (ABIPrism, Applied Biosystems, Foster City, CA) using the SYBRGreencore reaction kit (Applied Biosystems).

Primers used for mRNA quantification were:

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Pm

AT1 (

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GenBank™ Accession No. NM_031009.2)forward primer: 5́-ATGCCAGTGTGTTTCTGCTC-3́reverse primer: 5́-AACTCAACACTCCCCATTGG-3́

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AT2 (

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GenBank™ Accession No. NM012494.3)forward primer: 5́-CAGTGGTCTGCTGGGATTGC-3́reverse primer: 5́-CCATCCAGGTCAGAGCATCC-3́

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Mas (

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GenBank™ Accession No. NM012757.2)forward primer: 5́-ACTGCCGGGCGGTCATCATC-3́reverse primer: 5́-GGTGGAGAAAAGCAAGGAGA-3́

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NEP (

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GenBank Accession No. NM 18272.1)forward primer: 5́-GGATCTTGTAAGCAGCCTCAGC-3́

reverse primer: 5́-AGTTGGCACACCGTCTCCAG-3́

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Tonin (

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GenBank Accession No. NM 012677.1)forward primer: 5́-CCCTAGTCCTGTCCGTGGGACGAAT-3́reverse primer: 5́-CTTACTACCATCTCAGAGGGGTTG-30

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ACE (

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GenBank Accession No. NM 001012006.1)forward primer: 5́-GCAGTACAAAGACTTGCCTG-3́reverse primer: 5́-TGGCAGAGGCTGACATGTTA-30

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GAPDH ( 279

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GenBank Accession No. NM 017008.3)forward primer: 50-GGGCAGCCCAGAACATCAT-30

reverse primer: 50-CCGTTCAGCTCTGGGATGAC-30

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Quantitative values for AT1, AT2, Mas, NEP, Tonin and ACEmRNA transcription were obtained from the threshold cycle num-ber, where the increased signal associated with an exponentialgrowth of PCR products begins to be detected. Melting curves weregenerated at the end of every run to ensure product uniformity.The relative target gene expression level was normalized on thebasis of GAPDH expression as endogenous RNA control.

DCt values of the samples were determined by subtracting theaverage Ct value mRNA from the average Ct value of the internalcontrol GAPDH. As it is uncommon to use DCt as a relative datadue to this logarithmic characteristic, the 2�DCt parameter was usedto express the relative expression data.

2.4. Statistical analysis

Concentration of peptides and mRNA expression levels was pre-sented as an n-fold difference relative to the levels expressed andthe different values were compared using the one-way ANOVA fol-lowed by Tukey–Kramer t-test. The data are presented asmean ± SD and p 6 0.05 was considered to be statisticallysignificant.

3. Results

3.1. Animal behavior

Pilocarpine administration induced the following behavioralchanges: akinesia, facial automatisms and limbic seizures consist-ing of forelimb clonus with rearing, salivation, and masticatory jawmovements and falling.

This type of behavior built up progressively into motor limbicseizures that recurred repeatedly evolving to long-lasting SE. Afteracute phase, the rats presented normal behavior, which character-ized the seizure-free period (silent phase), which was recorded andno seizure was detected, at least 24 h before sacrifice. Chronicphase only started with first spontaneous seizure occurrence.

3.2. Quantification of peptides in hippocampus

Decreased levels of hippocampal AngI was observed duringacute, silent and chronic phase (Fig. 1A). The concentration of AngII

this article in press as: Gouveia, T.L.F., et al. The levels of renin–angilocarpine model of epilepsy. Neurochem. Int. (2012), http://dx.doi

was very low in control, acute and silent periods, however, it in-creased dramatically into the chronic phase (p 6 0.001) (Fig. 1B).In addition, the levels of Ang(1–7) increased into acute and silentphases, decreasing importantly into the chronic phase (p 6 0.001)(Fig. 1C).

The ratio between AngII/AngI and Ang(1–7)/AngII were alsoanalyzed to verify the conversion rate between these peptides.The data showed increased ratio of AngII/AngI (Fig. 2A) into thechronic phase (p 6 0.05). In contrast, the conversion of AngII toAng(1–7) was decreased during acute (p 6 0.05), silent (p 6 0.05)and even lower at chronic phase (p 6 0.01) (Fig. 2B), in the hippo-campus of rats submitted to pilocarpine-induced model of epi-lepsy. The ratio between peptides amount was used to visualizethe main peptide that remain in the hippocampus during all phasesof this model.

3.3. Expression analysis of RAS -related molecules

The mRNA expression levels of AngII AT1 receptors showed adecreased expression in acute (p 6 0.01) and silent (p 6 0.001)groups (Fig. 3A). In chronic group, an increased expression of AngIIAT1 was observed when compared with control group (p 6 0.001).

AngII AT2 receptors mRNA expression was increased in acute(p 6 0.001) and silent (p 6 0.001) groups, showing no alterationinto chronic group (Fig. 3B). Mas mRNA expression was signifi-cantly increased only into the silent period (p 6 0.001) (Fig. 3C).

ACE mRNA levels were decreased in acute (p 6 0.001), silent(p 6 0.001) and chronic (p 6 0.001) phases as shown on Fig. 4A,while NEP mRNA was increased only into acute (p 6 0.05) and si-lent period (p 6 0.05) (Fig. 4B). Tonin mRNA levels was increasedin all periods (Fig. 4C). The Fig. 5 summarizes the main foundresults of comparison between pilocarpine injected group (acute,silent and chronic groups) with control group in several steps ofAngII formation and their related molecules. mRNA expression lev-els of endogenous RNA control (GAPDH) did not change in allgroups during all phases in pilocarpine model of epilepsy.

4. Discussion

Our results showed an increased concentration of AngII in thehippocampus of rats in the chronic phase of pilocarpine epilepsymodel. Usually, angiotensin II (AngII) is the major point of interestof investigations about RAS role on the central nervous system(CNS). This peptide is related to synaptic plasticity since it blockslong-term potentiation (LTP) in the hippocampus (Denny et al.,1991; Armstrong et al., 1996). Moreover, AngII reduces NMDAreceptors signaling through AT2 (angiotensin II receptor type 2)receptors-mediated mechanisms (Schelman et al., 1997, 2004; Jinget al., 2004).

Several other angiotensin-related peptides are recognized asbeing biologically active in brain structures and, among them,Ang(1–7), which is thought to counterbalance most of the AngII ef-fects (Daemen et al., 1991; Strawn et al., 1999; Tallant et al., 2005).In fact, in acute phase, we observed a low quantity of AngII, withlow expression of ACE but an increased amount of Ang(1–7),accompanied by high expression levels of NEP. In chronic phase,the ratio between AngII/AngI was increased, showing a predomi-nant form of AngII. Differently, the ratio between Ang(1–7)/AngIIwas decreased into the chronic phase, demonstrating that the mainpeptide in the hippocampus really is Ang(1–7) at this time.

Previous report suggested an anti-inflammatory action ofAng(1–7) in cardiovascular diseases (Ferrario, 2006). This peptidesignificantly attenuates the inflammatory action of AngII, such asinduced reactive oxygen species generation, c-SRC and ERK1/2activation and other molecules related to the death of endothelial

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Fig. 1. Hippocampal peptides concentration: (A) AngI concentration was decreased during acute, silent and chronic phase of this epilepsy model. (B) AngII concentration wasvery low in control, acute and silent periods, increasing dramatically into the chronic phase. (C) Ang1–7 concentration was increased during acute and silent phases,decreasing importantly in chronic phase.

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cells (Sampaio et al., 2005). Previously reports have shown aninvolvement of inflammatory mediators in epilepsy (Naffah-Mazz-acoratti et al., 1995; Arganaraz et al., 2004a,b; Perosa, 2008; Gou-

Please cite this article in press as: Gouveia, T.L.F., et al. The levels of renin–angmitted to pilocarpine model of epilepsy. Neurochem. Int. (2012), http://dx.doi

veia et al., 2011). Thus, the release of Ang1–7 in tissues could belinked to neuroprotection as well as its main receptor (Mas) thatpresented increased expression. Mas receptor was initially

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Fig. 2. Ratio between peptides concentration. (A) AngII/AngI was increased in chronic phase. (B) Ang1–7/AngII was decreased during acute, silent and was very low in chronicphase. The difference between groups was evaluated using one-way ANOVA followed by Tukey–Kramer t-test. ⁄p 6 0.05, ⁄⁄p 6 0.01 and ⁄⁄⁄p 6 0001.

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described as a protooncogene and it is expressed in high concen-tration in the hippocampus, mainly in the dentate gyrus, hilusand pyramidal cell of CA3 region (Alenina et al., 2007). Moreover,Ang(1–7)/Mas axis integrity seems to be essential for normalobject recognition memory (Lazaroni et al., 2012). In peripheralsystem, the activation of Mas receptor decreases neutrophil influxand cytokine production in experimental arthritis showing an anti-inflammatory effect (Silveira et al., 2010).

According to Martin and Hockfield (1993), brief seizures epi-sodes led to a transient increase in Mas mRNA in the hippocampus.Thus, our finding of an increased expression of this receptor couldalso be related to hippocampal plasticity occurring during thesilent period of pilocarpine model of epilepsy, in response to initialinjury induced by status epilepticus as postulated by Cavalheiroet al. (1991). In our study, in acute and silent periods, AngII levelswere very low as well as AngII AT1 expression. In contrast, thechronic phase was characterized by an increased level of AngIIand by its AT1 receptor.

Previous results of our group reported that humans with tem-poral lobe epilepsy showed an increased expression of both recep-tors at the hippocampus supporting the idea of an up-regulation ofrenin–angiotensin system in temporal lobe epilepsy (Arganaraz et

Please cite this article in press as: Gouveia, T.L.F., et al. The levels of renin–angmitted to pilocarpine model of epilepsy. Neurochem. Int. (2012), http://dx.doi

al., 2008). Another study suggests that enalapril (ACE inhibitor)and losartan (AngII AT1 receptors blocker) were able to decreaseseizures (Pereira et al., 2010). AngII AT1 receptor, when blockedby losartan, also has a protective function in mouse model of Alz-heimer disease, showing that this receptor has deleterious functionin nervous system, when overexpressed (Danielyan et al., 2010).

In contraposition, our data showed that ACE expression was re-duced in all periods. This data suggests that angiotensinogen maybe cleaved to AngII by tonin, which showed increased expression inall phases. Angiotensinogen concentration in cerebrospinal fluidwas 1000-fold higher than of AngI levels and astrocytes was shownto be the major site of angiotensinogen synthesis in the brain(Deschepper et al., 1986). According to Araujo et al. (2002), toninis also expressed by astrocytes in rat brain. Taken together, thesedata suggest that AngII may be generated due to tonin action,directly from angiotensinogen (Borges et al., 2003).

In this context, this study shows for the first time, changes inthe profile of angiotensin-related polypeptides and their receptorsand converting enzymes in the hippocampus of rats submitted topilocarpine model of temporal lobe epilepsy. This informationmay allow the development of innovative strategies to block theactivation of RAS signaling in epilepsy.

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Fig. 3. Quantification of mRNA expression of receptors by real-time PCR. (A) AngII AT1 receptor showed a decreased expression in acute and silent groups and an increasedexpression in chronic group. (B) AngII AT2 receptor mRNA expression was increased in acute and silent groups showing no alteration into chronic group. (C) Mas mRNAexpression was increased only in silent period. The difference between groups was evaluated using one-way ANOVA followed by Tukey–Kramer t-test. ⁄p 6 0.05, ⁄⁄p 6 0.01and ⁄⁄⁄p 6 0001.

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Please cite this article in press as: Gouveia, T.L.F., et al. The levels of renin–angiotensin related components are modified in the hippocampus of rats sub-mitted to pilocarpine model of epilepsy. Neurochem. Int. (2012), http://dx.doi.org/10.1016/j.neuint.2012.04.012

Fig. 4. Quantification of mRNA expression of enzymes by real-time PCR. (A) ACE mRNA level was decreased in acute, silent and chronic phases. (B) NEP mRNA was increasedin acute and silent period. (C) Tonin mRNA levels was increased in chronic phase. The difference between groups was evaluated using one-way ANOVA followed by Tukey–Kramer t-test. ⁄p 6 0.05, ⁄⁄p 6 0.01 and ⁄⁄⁄p 6 0001.

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Please cite this article in press as: Gouveia, T.L.F., et al. The levels of renin–angiotensin related components are modified in the hippocampus of rats sub-mitted to pilocarpine model of epilepsy. Neurochem. Int. (2012), http://dx.doi.org/10.1016/j.neuint.2012.04.012

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Fig. 5. Summary of main found results in all periods of pilocarpine model of epilepsy. " or ; ⁄p 6 0.05, "" or ;;, ⁄⁄p 6 0.01 and """ or ;;; ⁄⁄⁄p 6 0001.

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5. Uncited references

De Mello and Frohlich (2011) and Silva et al. (2003).

Acknowledgements

This study was supported by research Grants from Coordenaçãode Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conse-lho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP),Cooperação Interinstitucional de Apoio à Pesquisa sobre o Cérebro(ClnAPCe) and Instituto Nacional de Neurociência Translacional(INNT) (Brazil).

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