ORIGINAL RESEARCH

Strain-Dependent Effects of Sub-chronically Infused LosartanAgainst Kainic Acid-Induced Seizures, Oxidative Stress, and HeatShock Protein 72 Expression

Jane Tchekalarova • Natasha Ivanova •

Daniela Pechlivanova • Kalina Ilieva •

Milena Atanasova

Received: 28 August 2013 / Accepted: 26 September 2013 / Published online: 22 October 2013

� Springer Science+Business Media New York 2013

Abstract We studied the involvement of angiotensin

(Ang) II AT1 receptors in the pathophysiology of kainate

(KA)-induced neurotoxicity, focusing on the regulation of

the oxidative stress state and expression of HSP 72 in the

frontal cortex and hippocampus in two strains, spontane-

ously hypertensive rats (SHRs) and normotensive Wistar

rats. The KA injection was executed after the rats were

infused subcutaneously via osmotic mini-pumps with lo-

sartan (10 mg/kg day) for 14 days. Losartan delayed the

onset of KA-induced seizures in SHRs but not in Wistar

rats without affecting the seizure intensity score. This

selective AT1 receptor antagonist decreased the lipid per-

oxidation only in naive SHRs. However, it attenuated the

KA-induced increase in lipid peroxidation in both SHRs

and Wistar rats. The adaptive enhancement of cytosolic

superoxide dismutase (SOD) activity in KA-treated SHRs

was recovered to control level after sub-chronic losartan

infusion while no change in mitochondrial SOD activity

was detected in the two strains. Both losartan and KA

produced a higher expression of HSP 72 in the hippo-

campus of the two strains compared to naive rats infused

with vehicle. Taken together, our findings demonstrate that

the efficacy of a sub-chronic systemic losartan infusion in

preventing the KA-induced seizure activity and neurotox-

icity is more pronounced in SHRs, considered as a model

of essential hypertension, than in normotenisve Wistar rats.

The results suggest that the blockade of AT1 receptors,

commonly used as a strategy for prevention of high blood

pressure, may be useful as an adjunctive treatment in status

epilepticus to reduce oxidative stress and neurotoxicity.

Keywords Kainic acid � Losartan �Oxidative stress �Heat shock protein 72 � Spontaneously hypertensive

rats �Wistar rats

Introduction

The brain renin–angiotensin system (RAS) is involved in

the regulation of classic physiology and behaviors includ-

ing blood pressure, sodium and body water balance, pitu-

itary gland hormones, and reproductive hormones, as well

as thirst-related and sexual behaviors. Most of these func-

tions are mediated through the AT1 receptor subtype acti-

vation (Wright et al. 2008). The non-peptide and selective

AT1 receptor antagonist losartan is routinely used for

studying the functions of the biologically active brain

neuropeptide angiotensin (Ang) II and the role of AT1

receptor subtype, in particular. There is growing literature

data suggesting that Ang peptides mediate brain excit-

ability, including seizure susceptibility. Thus, the Ang II-

induced inhibition of hippocampal and gyrus dentatus

long-term potentiation (LTP) is prevented by co-injection

with losartan suggesting that this effect was mediated via

activation of the AT1 receptor subtype (Armstrong et al.

1996a, b; Denny et al. 1991; Wayner et al. 1993, 1995).

The Ang II suppressed the NMDA- and/or kainate (KA)-

evoked increase in the discharge rate of dorsal lateral

geniculate nucleus probably mediated by AT1 receptors

(Albrecht et al. 1997). The above-mentioned Ang II effects

on some electrophysiological parameters are in accordance

with our previous pharmacological results on animal

J. Tchekalarova (&) � N. Ivanova � D. Pechlivanova

Institute of Neurobiology, Bulgarian Academy of Sciences,

Acad. G. Bonchev Str., Bl. 23, Sofia 1113, Bulgaria

e-mail: janetchekalarova@gmail.com

K. Ilieva � M. Atanasova

Department of Biology, Medical University of Pleven,

1 Kliment Ohridski Str., Pleven 5800, Bulgaria

123

Cell Mol Neurobiol (2014) 34:133–142

DOI 10.1007/s10571-013-9994-8

seizure tests (Tchekalarova and Georgiev 2005). In this

line, we have found that the anticonvulsant activity of Ang

II in acute seizure models and pentylenetetrazol (PTZ)

kindling model is mediated via activation of AT1 receptor

subtype (Tchekalarova and Georgiev 2005). In addition,

recently, we have found that losartan treatment at a dose of

10 mg/kg for 10 days exhibit an anticonvulsant effect in

acute PTZ seizure model in normotensive Wistar rats

(Pechlivanova et al. 2011).

In SHRs, brain Ang II receptors are mostly distributed in

structures associated with cardiovascular regulation and

endogenous Ang II has been considered an active neuro-

peptide responsible for pathogenesis of arterial blood

pressure (Saavedra 1992). The SHR strain is characterized

by an increased pressor sensitivity to the intracerebrovas-

cular (icv) infusion of Ang II and Ang III as compared with

normotensive Wistar–Kyoto and Sprague–Dawly rats

(reviewed in Wright and Harding 1992). Together with the

well-known effects of the selective AT1 receptor antagonist

losartan on the hypertension in SHRs (Kawano et al. 1994),

our recent data have shown that losartan is able to alleviate

stress-induced behavioral changes in SHRs (Pechlivanova

et al. 2011). Experimental data, including ours, support the

suggestion that SHRs could be considered as a tool for

studying the link between hypertension and epilepsy

(Greenwood et al. 1989; Scorza et al. 2005; Tchekalarova

2010, 2011).

In the recent years, the crucial role of oxidative stress in

the occurrence and the development of hypertension have

received an increased attention. The brain is particularly

sensitive to attacks of reactive oxygen species (ROS).

Currently, a number of protective approaches have been

applied with potential antioxidants to improve hyperten-

sion. Ang II exerts many of its detrimental effects through

its interaction with the AT1 receptor (Manrique et al.

2009). The activation of AT1 receptors in non-adrenal tis-

sues results in a myriad of intracellular events including

production of ROS, which contribute to endothelial dys-

function, with secondary increases in arterial blood pres-

sure (Mehta et al. 2007). Losartan has been shown to

attenuate oxidative stress damage in kidney, liver, and the

whole brain of SHRs without affecting the antioxidant

defense system in normotensive rats (Polizio and Pena

2005). It has been suggested that differences in types,

severity, and complications of diseases as well as strains

may influence the responses to blood pressure and oxida-

tive stress (Erejuwa et al. 2011).

Oxidative stress has been also implicated in a variety of

acute and chronic neurologic disturbances, including status

epilepticus (SE). The expression of heat shock protein 72

(HSP 72) in the brain was considered as marker of KA-

induced neurotoxicity and literature data suggest that the

expression of stress proteins can be directly attributed to

oxidative stress (Ambrosio et al. 1995; Gupta and Briyal

2006). In addition, increased expression of HSP 72 was

seen after KA-induced SE in rat brain (Gupta and Briyal

2006).

Agents with antioxidant properties might be considered

as a potential therapeutic for adjunctive antiepileptic ther-

apy. Both the KA and pilocarpine-induced SE models are

useful to investigate the development of excitotoxic neu-

ronal injury, ROS and HSP 72 production in rodents

(Bonan et al. 2000; Gupta and Briyal 2006). The brain,

including the frontal cortex and the hippocampus, which

are connected with each other through different neuro-

transmitter systems, is particularly vulnerable to attacks of

ROS and to KA-induced SE (Chen and Buckmaster 2005).

Based on this evidence, our goal was to compare the effi-

cacy of chronic systemic pretreatment with the AT1

receptor antagonist losartan on seizure activity and SE-

induced production of oxidative damage and HSP 72 in the

frontal cortex and the hippocampus of two strains, SHRs,

as a model of essential hypertension, and normotensive

Wistar rats.

Materials and Methods

Animals

The experiments were performed on adult male normo-

tensive Wistar rats obtained from the animal facility of the

Bulgarian Academy of Sciences and spontaneously

hypertensive rats (SHRs) from the local breeding house

(Medical University, Pleven). The rats weighing

200–250 g were adapted for a week under standardized

laboratory conditions (12 h/12 h light/dark cycle, temper-

ature 22 ± 2 �C, 50 % relative humidity) in groups of

three in plastic cages with soft bedding. Food and water

were available ad libitum throughout the study except

during the tests. The experimental protocol was in com-

pliance with the European Communities Council Directive

of 24th November 1986 (86/609/EEC) and the experi-

mental design was approved by the local Institutional

Ethics Committees of the Institute of Neurobiology, Bul-

garian Academy of Sciences and Medical University

(Pleven).

Experimental Design

The animals were divided into eight experimental groups

(n = 10) as follows: Group I: Control and sham normo-

tensive Wistar rats treated with vehicle (Wis-C-veh);

Group II: control and sham Wistar rats treated with losar-

tan ? vehicle (Wis-C-los); Group III: Wistar rats treated

with vehicle ? KA (Wis-KA-veh); Group IV: Wistar rats

134 Cell Mol Neurobiol (2014) 34:133–142

123

treated with losartan ? KA (Wis-KA-los); Group V:

Control and sham SHRs treated with vehicle (SHRs-C-

veh); Group VI: Control and sham SHR treated with lo-

sartan ? vehicle (SHRs-C-los); Group VII: SHRs treated

with vehicle ? KA (SHRs-KA-veh); Group VIII: SHRs

treated with losartan ? KA (SHRs-KA-los).

Implantation of Osmotic Mini-Pumps

For implantation of the osmotic mini-pumps, animals were

anesthetized with sodium pentobarbital (50 mg/kg). Lo-

sartan (kindly gifted by MERCK&CO., INC, New Jersey,

USA) was infused chronically for a period of 2 weeks via

osmotic mini-pumps at a dose of 10 mg/kg day (concen-

tration of losartan in the fluid inside the osmotic mini-

pumps: 700 mg/ml). This dose was chosen on the basis of

previous data showing that it is appropriate for blockade of

the dipsogenic response to Ang II (Dourish et al. 1992).

Alzet osmotic mini-pumps were filled with drug dissolved

or vehicle (0.9 % NaCl, pumping rate 0.5 ll/h, information

provided by manufacture). No differences between control

and sham-operated rats were detected. The method of lo-

sartan infusion via s.c. osmotic mini-pumps provided

constant steady-state hormonal concentrations.

Arterial Blood Pressure Measurements

The systolic arterial blood pressure (ABP) was measured

non-invasively in conscious unrestrained SHRs by a tail

cuff method (Ugo Basile Blood Pressure Recorder 5800)

before the start of experimental procedures to confirm the

hypertension. The ABP value for each rat was calculated as

a mean of three measurements.

Kainic Acid-Induced Status Epilepticus

On the 14th day of vehicle/losartan s.c. infusion the ani-

mals from groups III, IV, VII, and VIII received i.p.

injection of KA (Ascent Scientific, UK) at a dose of 12

mg/kg dissolved in sterile saline (0.9 % NaCl) or saline

(groups I, II, V, VI) in a volume of 1 ml/kg of body weight.

The protocol used to elicit KA-induced SE was based on

previous studies (Lopez-Meraz et al. 2005; Morales-Garcia

et al. 2009). After the KA injection, the animals were put in

individual plexiglas cages and observed for a period of 4 h

to evaluate the appearance of seizures and their intensity.

Seizures were assessed according to the Racine’s scale

(1972) consisting of six stages (0–5), which correspond to

the successive developmental stages of motor seizures: (0)

normal non-epileptic activity; (1) facial automatism,

sniffing, scratching, wet dog shakes; (2) head nodding,

staring, tremor; (3) forelimb clonus with lordotic posture;

(4) rearing and continued forelimb clonus, salivation; (5)

forelimb clonus and loss of posture. Latency for the onset

of the first seizure of stage 4 was also evaluated.

Biochemical Experiments

Biochemical tests were conducted 4 h after KA injection.

The animals were sacrificed by decapitation under a light

anesthesia (CO2). Brains were quickly dissected on ice and

the frontal cortex and hippocampi were bilaterally

removed. The tissue samples were frozen in liquid nitro-

gen, and stored at -70 �C before analysis.

Measurement of Lipid Peroxidation

The extent of lipid peroxidation was determined quantita-

tively by direct measurement of hydroperoxides in redox

reactions with ferrous ions. Therefore, the tissue samples

were homogenized in cold 20 mM HEPES buffer (pH 7.2)

and extracted with chloroform. The extracted lipid perox-

ides were assayed with LPO assay kit (Cayman Chemical

Company, USA) according the instructions provided. The

resulting ferric ions were detected using thiocyanate ion as

the chromogen and by reading the absorbance at 500 nm.

The extent of lipid peroxidation was expressed in nmol.

Measurement of Cytosolic and Mitochondrial Superoxide

Dismutase

The tissue samples were homogenized in cold 20 mM

HEPES buffer (pH 7.2) and centrifuged at 1,5009g for

5 min, at 4 �C. To separate cytosolic and mitochondrial

SOD, the 1,5009g supernatant was again centrifuged at

10,0009g for 15 min, at 4�C. The resulting supernatant

was tested for cytosolic SOD and the pellet—for mito-

chondrial SOD with SOD assay kit (Cayman Chemical

Company, USA). The results were expressed in U/ml.

Western Blotting

Tissues were washed once in ice-cold PBS and homoge-

nized in 5 ml of cold 20 mM HEPES buffer, pH 7.2,

containing 1 mM EGTA, 210 mM mannitol, and 70 mM

sucrose. Protein concentration was determined by spec-

trophotometric measuring of the homogenates at 280 nm.

Equal amounts (20 mg/lane) of protein samples were run

on 12 % SDS polyacrylamide gel. The proteins were

transferred onto nitrocellulose membrane and blocked with

3 % bovine serum albumin in TBS-0.05 % Tween. The

membrane was incubated with the primary mouse anti-

Hsp72 antibody l chain (invitrogen) 1:500, for 2 h at room

temperature or overnight at 4 �C. The membrane was

washed 3 times with TBS-Tween and further incubated in

the secondary antibody anti-mouse l chain, raised in goat

Cell Mol Neurobiol (2014) 34:133–142 135

123

and conjugated with alkaline phosphatase (Vector Labs,

USA) 1:250. After 3 times washing in TBS-Tween the

membrane was incubated in 10 ml ABC-AmP reagent

(Vector Labs, USA) for 10 min at room temperature and

washed again. The membrane was equilibrated in TBS for

substrate (pH 9.5) and incubated in substrate solution

BCIP/NBT (Vector Labs, USA) at room temperature for

about 30 min. After developing appropriate density color

bands, the membrane was rinsed in PBS and air-dried. For

appropriate load control is used standard sample obtained

by mixing aliquots of several of the homogenates in order

to make a large quantity of mixed homogenate for appro-

priate load control. A standard volume of this homogenate

was loaded onto a single lane of each gel. All other bands

on each gel were expressed relative to this standard as

relative area (RA) as ratio between the standard and sam-

ple. Blots were scanned and analyzed with the ImageJ

software (V 1.42q).

Statistical Analysis

All results were expressed as mean ± S.E. The data with

seizure severity scores were analyzed by Student’s t test

(two groups comparison) and the biochemical parameters

by means of three-way ANOVA (factors: Strain, KA-

treatment and Drug) (SigmaStat� SPSS). The incidence of

seizures was evaluated by Fisher’s exact test. The level of

statistical significance was set at 5 %.

Results

Effect of Losartan Treatment on KA-Induced Seizures

The SHRs were characterized with a higher ABP

(178 ± 1.6 mmHg, p \ 0.05) compared to the normoten-

sive Wistar rats (137 ± 1.5 mmHg) before the start of

experiments. The behavior observed after a systemic i.p.

injection of a single excitotoxic dose of KA (12 mg/kg)

consisted of initial wet-dog shakes, facial automatisms, and

head nodding (partial seizures). Further, this motor activity

progressed to forelimb clonus with lordotic posture (class

III) followed by rearing (class IV) and occasionally fore-

limb clonus and loss of posture (class V) (secondary gen-

eralized seizures). In agreement with our previous work

(Atanasova et al. 2013), the SHR-KA-veh group showed a

significant decrease in the latency for KA-induced seizures

(stage 4) compared to Wis-KA-veh group (p = 0.01)

(Table 1). However, while the sub-chronic losartan infu-

sion failed to modify the latency for the onset of the first

seizure in Wistar rats, it significantly elongated the latency

for the appearance of the first seizure in SHRs

(*p = 0.016). Neither incidence nor seizure intensity was

significantly changed in Wis-KA-los and SHR-KA-los

groups, respectively.

Effects of Losartan Treatment on Level of Lipid

Peroxidation

The effects of a sub-chronic s.c. infusion of losartan on lipid

peroxidation during the KA-induced SE are presented in

Fig. 1a, b. The pretreatment with losartan caused a significant

decrease in the level of lipid peroxidation both in the frontal

cortex and the hippocampus in control SHRs (p = 0.020 vs.

SHR-veh). Furthermore, sub-chronic blockade of AT1 recep-

tors significantly attenuated the KA-induced increase of the

lipid peroxidation in the hippocampus of Wistar rats and SHRs

(*p = 0.02 vs. SHR-C-veh; p = 0.014 vs. SHR-KA-veh),

respectively.

Effects of Losartan on Cytosolic Superoxide Dismutase

(SOD) Cu/Zn Activity

Figure 2a, b presents the effect of a sub-chronic losartan

pretreatment on SOD Cu/Zn activity in the frontal cortex

and hippocampus of Wistar rats and SHRs, respectively.

Post hoc test showed that losartan significantly increased

the activity of the cytosolic antioxidant enzyme SOD Cu/

Zn in the frontal cortex of control SHRs (*p = 0.015).

However, the long-term blockade of AT1 receptors pre-

vented the adaptive increase of the enzyme activity in the

frontal cortex of the two strains (p \ 0.01 vs. Wis-KA-veh;

*p \ 0.001 vs. SHR-C-veh and p = 0.01 vs. SHR-KA-veh,

Table 1 Effect of pretreatment with losartan on KA-induced seizures

in Wistar and spontaneously hypertensive rats (SHRs)

Group Seizures (n/N)a Latency (min)b Seizure

intensityc

Wis-C-veh 0/10 ns 0

Wis-C-los 0/10 ns 0

Wis-KA-veh 9/10 86.50 ± 4.41 4.2 ± 0.3

Wis-KA-los 5/10 86.0 ± 9.6 2.9 ± 0.5

SHRs-C-veh 0/10 ns 0

SHRs-C-los 0/10 ns 0

SHRs-KA-veh 9/10 64.1 ± 2.1* 3.7 ± 0.47

SHRs-KA-los 7/10 89 ± 13.2** 3.6 ± 0.4

Rats were pretreated with losartan (10 mg/kg day) or saline via Alzet

osmotic mini-pumps (pumping rate 0.5 ll/h). On the 14th day of the

perfusion they received KA (12 mg/kg, i.p.)a Number of animals that showed seizure episodes (4th stage)/num-

ber of animals per groupb Time to the onset of the first seizure episode (min)c Seizure intensity according to scale of five points of severity

(Racine’s scale)

* p = 0.01 versus Wistar–KA-veh group; ** p = 0.016 versus SHR-

KA-veh group (Kruskal–Wallis test)

136 Cell Mol Neurobiol (2014) 34:133–142

123

respectively) and in the hippocampus of SHRs,

respectively.

Effects of Losartan on Mitochondrial Superoxide

Dismutase (SOD) Mn Activity

The sub-chronic losartan exposure decreased mitochondrial

SOD activity in the frontal cortex of KA-treated Wistar rats

(p \ 0.01 vs. Wis-KA-veh) (Fig. 3a). However, the selective

AT1 receptor antagonist failed to exert any changes in SOD-

Mn activity in the hippocampus of Wistar rats and in the frontal

cortex and hippocampus of SHRs, respectively (Fig. 3a, b).

Effects of Losartan on Heat Shock Protein 72

Post hoc test revealed that the sub-chronic losartan pre-

treatment exerted a significant increase in the expression of

the HSP 72 in the frontal cortex of Wis-C-los group

(*p = 0.006 vs. Wis-C-veh) and in the hippocampus of

Wis-C-los (*p = 0.015 vs. Wis-C-veh), Wis-KA-los

(*p \ 0.001 vs. Wis-C-veh), SHR-C-los (*p = 0.001 vs.

SHR-C-veh), and SHR-KA-los group (*p \ 0.001 vs.

SHR-C-veh; p \ 0.001 vs. SHR-KA-veh), respectively,

similar to the increased adaptive expression of this chap-

erone in the hippocampus of the KA-treated Wistar rats

Fig. 1 Lipid peroxidation in the frontal cortex (a) and the hippo-

campus (b) of controls (c) and KA-treated Wistar and SHRs infused

with vehicle (veh) or losartan (los) (details in the text to Table 1).

Data are presented as mean ± SEM (n = 10). Analysis of data by

three-way ANOVA indicated a main Strain effect [F1,59 = 32.844,

p \ 0.001] in (a), a main Strain effect [F1,68 = 11.889, p = 0.001], a

main KA effect [F1,68 = 14.395, p \ 0.001], and a main Drug effect

[F1,68 = 13.397, p \ 0.001], and strain 9 drug interaction

[F1,68 = 4.510, p = 0.038] in (b). *p \ 0.05 versus C-veh group;

p \ 0.05 vs KA-veh group, #p \ 0.05 versus Wistar rats

Fig. 2 Cytosolic superoxide dismutase (SOD-Cu/Zn) activity in the

frontal cortex (a) and the hippocampus (b) of controls (c) and KA-

treated Wistar and SHRs pretreated with either vehicle (veh) or

losartan (los) (details in Table 1). Data are presented as mean ± SEM

(n = 10). Analysis of data by three-way ANOVA indicated a main

KA-treatment effect [F1,75 = 16.975, p \ 0.001] and KA-treatment

9 Drug interaction [F1,75 = 14.531, p \ 0.001] in (a); a main KA-

treatment effect [F1,72 = 5.462, p = 0.023] in (b). *p \ 0.05 versus

C-veh group; p \ 0.05 versus KA-veh group

Cell Mol Neurobiol (2014) 34:133–142 137

123

(*p = 0.001 vs. Wis-C-veh) and SHRs (*p = 0.05 vs

SHR-C-veh), respectively (Fig. 4a, b).

Discussion

In the present study, the sub-chronic losartan pretreatment

showed a mild anticonvulsant effect in the SHRs by

increasing the latency for onset of the KA-induced limbic

seizures while this AT1 receptor antagonist failed to affect

the seizure activity in normotensive Wistar rats. Previ-

ously, we have found that the sub-chronic treatment with

losartan at the same dose had an antihypertensive activity

in SHRs while it failed to affect the arterial pressure in

normotensive Wistar rats (Pechlivanova et al. 2010).

Although the link between the anticonvulsant and antihy-

pertensive efficacy of losartan might be speculative, there

are literature data in support of the close relationship

between hypertension and epilepsy (Devinsky et al. 2004;

Hilz et al. 2002; Tomson et al. 1998). Furthermore, our

recent results have demonstrated that although long-term

treatment with losartan after SE is unable to prevent epi-

leptogenesis and the development of chronic epileptic state

in KA model of TLE both in Wistar rats and SHRs, this

AT1 receptor antagonist elongates the latency to the first

spontaneous seizure in the KA-treated SHRs without

affecting the latency in normotensive Wistar rats

Fig. 3 Mitochondrial superoxide dismutase (SOD-Mn) activity in the

frontal cortex (a) and the hippocampus (b) of controls (c) and KA-

treated Wistar and SHRs pretreated with either vehicle (veh) or

losartan (los) (details in Table 1). Data are mean ± SEM (n = 10).

Analysis of data by three-way ANOVA indicated a main drug effect

[F1,64 = 10.706, p \ 0.002] in (a). p \ 0.05 versus KA-veh group

Fig. 4 Heat shock protein (HSP) 72 in the frontal cortex (a) and the

hippocampus (b) of controls (c) and KA-treated Wistar and SHRs

pretreated with either vehicle (veh) or losartan (los) (details in

Table 1). Data are mean ± SEM (n = 10). Analysis of data by three-

way ANOVA indicated a main strain effect [F1,69 = 5.323,

p = 0.024], a main drug effect [F1,69 = 41.304, p \ 0.001], and

KA-treatment 9 Drug interaction [F1,69 = 10.799, p = 0.002].

*p \ 0.05 versus C-veh group; #p \ 0.05 versus Wistar rats

138 Cell Mol Neurobiol (2014) 34:133–142

123

(unpublished data). The present results agree with the

above-mentioned findings showing a losartan efficacy only

on the latency for seizure appearance in SHRs without

changing the incidence and intensity of KA-induced sei-

zures in the two strains. Although accumulating experi-

mental and clinical data support the presumption that the

selective AT1 antagonists can exert an anticonvulsant

effect (Arganaraz et al. 2008; Lukawski et al. 2010; Pereira

et al. 2010), our data suggest that a blockade of AT1

receptor might be efficient for add-on therapy in epileptic

patients with hypertension. The AT1 receptors are mostly

localized in brain structures associated with regulation of

arterial blood pressure and heart functions such as the

anterior pituitary, hypothalamus, and circumventricular

organs (CVOs) but they are also detected in areas involved

in seizure susceptibility, including the piriform cortex,

hippocampus, lateral geniculate, caudate putamen, amyg-

dala, and septum (Wright et al. 2008). We were the first to

demonstrate that angiotensin peptides are involved in sei-

zure susceptibility (Tchekalarova and Georgiev 2005). In

addition, recently, we have found that losartan treatment at

a dose of 10 mg/kg for 10 days exhibits an anticonvulsant

effect in acute PTZ seizure model in normotensive Wistar

rats (Pechlivanova et al. 2011).

In agreement with our previous data and that of others

control SHRs were characterized with a disturbed oxidative

defense system compared to normotensive rats in physio-

logical conditions (Atanasova et al. 2013; Polizio and Pena

2005). Previously, Haugen et al. (2000) reported that

hyperactivity of RAS triggers oxidative stress in SHRs

considered as a model of essential hypertension. In this

regard, several reports support the suggestion that Ang II-

dependent generation of superoxide and oxygen species

present a key mechanism of cardiac and renal impairment,

secondary to diverse pathologies (Kazama et al. 2004;

Zhang et al. 1999; Zimmerman et al. 2004). Moreover,

some of the beneficial effects associated with RAS inhi-

bition can be ascribed to the prevention of oxidant-medi-

ated damage (de Cavanagh et al. 2004). The observed

significant elevation of lipid peroxidation in the hippo-

campus as sequence of KA-induced neurotoxicity is in

agreement with our previous report and those of others

(Atanasova et al. 2013; Dal-Pizzol et al. 2000; Tan et al.

1998; Tejada et al. 2006). In the present study, we have

found that the sub-chronic losartan infusion can attenuate

the level of lipid peroxidation of control hypertensive rats

both in the frontal cortex and the hippocampus but failed to

affect the oxidative defense system of control normoten-

sive Wistar rats. A large body of in vivo and in vitro evi-

dence revealed that the antagonists of AT1 receptors

function as a free radical scavenging antioxidant in SHRs.

Thus, losartan treatment at the same dose of 10 mg/kg day

in the drinking water for a period of 14 days increased the

SOD activity and glutathione peroxidase to protect brain,

kidney, and liver in SHR but did not change these

parameters in normotensive Wistar–Kyoto rats (Polizio and

Pena 2005).

The neurotoxin KA increased ROS production and the

consequent mitochondrial dysfunction in a number of brain

areas, particularly in the hippocampus (Liang et al. 2000).

The increase of protein oxidation and lipid peroxidation in

the hippocampus, which is evident during the acute seizure

phase after KA injection in our previous study and others

(Atanasova et al. 2013) can induce an adaptive increase in

the activity of some of the antioxidant enzymes. One of

these defense enzyme is SOD, which is responsible for

degradation of superoxide. The balance between these

antioxidant systems and ROS is crucial for cell homeo-

stasis and neuronal function, in particular. The activity of

antioxidant enzymes are vulnerable to the level of oxida-

tive stress and the direction of changes (increase or

decrease) depends on the mechanism underlying different

pathologies, in which an increase of ROS can cause an

adaptive enhancement of enzyme activity or not as a

consequence of a disturbed balance in a defense enzyme

system.

In our study, in line with our previous report (Atanasova

et al. 2013), the KA-induced neurotoxicity significantly

enhanced the activity of defensive cytosolic antioxidant

enzymes SOD in a region-specific manner in the two

strains. Specifically, in Wistar rats, this differential adap-

tive response of oxidative markers in the selected brain

regions may arise from the differences in the antioxidant

buffering capacities. Interestingly, no changes in mito-

chondrial SOD activity were detected as a result of KA-

induced limbic seizures, which suggests that the cytosolic

defense system is more sensitive in the acute KA seizure

model than the mitochondrial antioxidant enzyme system

in the two studied strains. Literature data support the pre-

sumption that seizures provoke different changes in the

oxidative defense system, which are influenced by previous

level of oxidative stress, brain area, strain used and time

points detected for the direction of changes. Thus, Can-

delario-Jalil (2001) revealed that the systemic administra-

tion of an excitotoxic dose of KA decreased the

hippocampal SOD activity with respect to basal levels

detected 24 h after KA application. The finding that SOD

activity was increased as a consequence of the KA-induced

neurotoxicity in an area-specific manner in the two strains,

and that losartan prevented this enhancement to control

level suggests that SOD activity does not contribute

directly to the protective effects of AT1 receptor blockade

on oxidative stress. Probably losartan affected the SOD

activity indirectly by suppressing the ROS production in

the brain areas critical for limbic seizures and thereby

preventing the respective adaptive response connected with

Cell Mol Neurobiol (2014) 34:133–142 139

123

an enhancement of the defense enzyme system. In this

regard, the high cytosolic SOD activity most probably

represents a functional mechanism of adaptation to dis-

turbed balance between the antioxidant defense system and

the production of ROS in the KA model of neurotoxicity.

In accordance with our recent study and that of other

authors, an increased expression of HSP 72 have been

detected specifically in the hippocampus both in the nor-

motensive Wistar rats and SHRs during the acute phase of

the KA-induced neurotoxicity. These data agree with other

reports demonstrating time-dependent changes in the

expression of the stress proteins with maximal increased

expression at 3–24 h after KA (Akbar et al. 2001; Arm-

strong et al. 1996a, b). In addition, the HSPs expression in

the hippocampus positively correlates with the severity of

KA-elicited limbic seizures (Zhang et al. 1997). The sub-

chronic losartan infusion over 14 days via s.c. implanted

osmotic mini-pump not only did not prevent the KA-

induced expression of HSP 72 but per se provoked a pro-

tein expression in the hippocampus in the two strains. The

chaperones HSPs, which form a highly conserved system,

are considered important modulators of neuronal function,

responsible for the preservation, and repair of the correct

protein conformation (Calabrese et al. 2010; Witt 2010).

Recent studies have shown that the HSPs contribute to

cytoprotection in a number of human diseases including

inflammation, cancer, aging, and neurodegenerative disor-

ders. The results of several laboratories support the

hypothesis that HSPs are crucial cellular mechanism par-

ticipating in neuroprotection from excitotoxic overactiva-

tion of glutamate receptors. Thus, Ekimova et al. (2010)

reported that exogenous HSPs are able to penetrate into the

brain and exert an anticonvulsant activity against chemi-

cally induced seizures (NMDA and PTZ). Overexpression

of HSP 70, which reduce neuronal injury after seizures, are

suggested to attenuate apoptotic cell death (Tsuchiya et al.

2003) and produce a protection against hippocampal neu-

rodegeneration induced by endogenous glutamate in vivo

(Ayala and Tapia 2008). Our finding that losartan can cause

a profound increase in HSP 72 expression specifically in

the hippocampus of the two strains supports the suggestion

that the blockade of AT1 receptors represents a neuropro-

tective mechanism against KA-induced neurotoxicity. Our

results are also in agreement with another finding that the

antiinflammatory response of losartan in the early stage of

an obstruction includes a suppression of oxidative stress

and an increase in HSP 72 expression independent from

changes in blood pressure (Manucha et al. 2005).

Taken together, our findings demonstrated that the

efficacy of a sub-chronic systemic losartan infusion in

preventing the KA-induced seizure activity and neurotox-

icity was more pronounced in the SHRs, considered as a

model of essential hypertension, than in the normotensive

Wistar rats. Our results suggest that the blockade of the

AT1 receptors, commonly used as a strategy for prevention

of high blood pressure, may be useful as an adjunctive

treatment in status epilepticus to reduce oxidative stress

and neurotoxicity.

Acknowledgments This work was supported by the Medical Sci-

ence Council, Medical University of Pleven contract No. 3/2011 and

National Science Fund (research Grant # DTK 02/56 2009-1012).

Conflicts of Interest None.

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