ORIGINAL PAPER

Effect of Anacyclus pyrethrum on Pentylenetetrazole-InducedKindling, Spatial Memory, Oxidative Stress and Rho-Kinase IIExpression in Mice

Monika Pahuja • Jogender Mehla • K. H. Reeta •

Manjari Tripathi • Yogendra Kumar Gupta

Received: 18 May 2012 / Revised: 29 November 2012 / Accepted: 1 December 2012 / Published online: 15 December 2012

� Springer Science+Business Media New York 2012

Abstract Anacyclus pyrethrum (A. pyrethrum) has been

reported to exhibit anticonvulsant activity. In the present

study, the effect of hydro-alcoholic extract of A. pyrethrum

root (HEAP) on pentylenetetrazole (PTZ) induced kind-

ling, spatial memory, oxidative stress and rho kinase

(ROCK II) was assessed. Male albino mice (25–30 g) were

used in the study. PTZ (35 mg/kg, i.p. on alternate days)

was injected to induce kindling and PTZ (70 mg/kg, i.p)

challenge was given 7 days post-kindling. HEAP was

administered orally daily in the doses of 100, 250 and

500 mg/kg along with PTZ injections during the kindling

process and continued till PTZ challenge post kindling.

Spatial memory was assessed using Morris water maze test.

Oxidative stress parameters [malondialdehyde (MDA) and

reduced glutathione (GSH)] and ROCK II expression were

estimated in whole brain at the end of the study. Pre-

treatment with HEAP (250 and 500 mg/kg) showed sig-

nificant increase in the myoclonic jerk latency and delay in

the development of kindling. A significant decrease in

mortality was observed at higher doses of HEAP (250 and

500 mg/kg). Pre-treatment with HEAP significantly

increased the number of platform crossings and decreased

the escape latency, as opposed to the PTZ group, thus

showing protection against memory deficit. HEAP pre-

treatment also attenuated the oxidative stress induced by

PTZ kindling. PTZ induced kindling increased the ROCK

II expression whereas, HEAP pre-treatment attenuated the

increase in ROCK II expression. To conclude, HEAP pre-

treatment showed antiepileptic effect and also showed

protection against cognitive impairment by decreasing

oxidative stress and ROCK II expression in PTZ kindled

mice.

Keywords Epilepsy � Anacyclus pyrethrum � PTZ

kindling � Spatial memory � Oxidative stress � Rho kinase II

Introduction

Epilepsy is a common neurological disorder affecting 1 %

of population worldwide [1]. Despite a large number of

antiepileptic drugs in the market, 30 % of the patients do

not become seizure free. Chronically used antiepileptic

drugs (AEDs) are often associated with adverse drug

reactions which further require therapeutic drug monitor-

ing. This leads to an unmet demand for an effective and

safe therapy for epilepsy patients throughout the globe.

Sodium valproate is the most commonly used drug for the

treatment of epilepsy because of its broad spectrum action.

It is also used for treatment of bipolar disorder. Both the

underlying disease and AEDs therapy have untoward

effects on cognition in epileptic patients, resulting in

memory deficits, learning disabilities and behavioural

problems [2, 3]. There is increasing evidence suggesting

that oxidative stress plays an important role either in

development or progression of the seizures, leading to

membrane lipid peroxidation and depletion of antioxidant

enzymes [4, 5]. Therefore, the potential therapeutic value

of antioxidants is worthy of further investigation.

Pentylenetetrazole (PTZ) kindling is most commonly

used as an experimental model for induction of seizures

and evaluating the antiepileptic effect of drugs [6].

M. Pahuja � J. Mehla � K. H. Reeta � Y. K. Gupta (&)

Department of Pharmacology, All India Institute of Medical

Sciences, New Delhi 110029, India

e-mail: ykykgupta@gmail.com; yk.ykgupta@gmail.com

M. Tripathi

Department of Neurology, All India Institute of Medical

Sciences, New Delhi 110029, India

123

Neurochem Res (2013) 38:547–556

DOI 10.1007/s11064-012-0947-2

Kindling process initially does not induce seizures but

decrease the seizure threshold which ultimately leads to the

occurrence of seizures [6]. Generalized tonic–clonic sei-

zures in kindling process have been associated with cog-

nitive deficit [7–9]. Moreover, drug screening in kindling

models have been shown to capitulate results which are

more foretelling of clinical efficacy and adverse effects

[10]. The neuronal damage in the hippocampus due to

kindling may be responsible for cognitive dysfunctions

[11]. Kindling also increased the expression of rho kinase

(ROCK II) which is associated with glutamate activation

[12]. An increased activity of the glutamatergic transmis-

sion has also been reported to play a crucial role in neu-

ronal cell death of the PTZ kindled rats due to free radicals

generation [13, 14].

Anacyclus pyrethrum has been used as a brain tonic in

complementary and alternative medicine [15]. Pyrethrins,

N-isobutyldienediynamide and polysaccharides are the

principle constituents of A. pyrethrum root [16, 17]. In our

previous study, hydro-alcoholic extract of A. pyrethrum

(HEAP) root showed the dose dependant anticonvulsant

effect in PTZ and maximal electroshock (MES) models of

seizure in rats. HAEP also ameliorated the cognitive

impairment induced by PTZ and MES induced seizures

[18]. It has also been reported to possess anti-inflammatory,

antioxidant, immunostimulating and anti-mutagenic activ-

ity [17, 19–21]. In the present study, hydro-alcoholic root

extract of A. pyrethrum (HEAP) has been evaluated for its

antiepileptic potential in PTZ induced kindling model of

epilepsy. Its effect on cognition, ROCK II and oxidative

stress were also evaluated.

Materials and Methods

Plant Extract

Anacyclus pyrethrum root were purchased from local

market in Delhi and authenticated at the Department of

Pharmacognosy, Hamdard University, Delhi. A voucher

specimen was deposited at the Department of Pharma-

cognosy, Hamdard University, Delhi (Voucher No.-PRL/

JH/08/21). Plant extraction was performed as described

earlier [18]. The hydoalcoholic extract of A. pyrethrum was

fully soluble in distilled water.

Animals

Male Swiss albino mice (25–30 g; aged 6–7 weeks) were

used for the present study. Animals were obtained from the

Central Animal Facility of All India Institute of Medical

Sciences, New Delhi, India. Animals were group housed in

polyacrylic cages with not more than 5 animals per cage.

Animals were maintained under standard laboratory con-

ditions with natural dark and light cycle, standard dry

rodent pellet diet and tap water was provided ad libitum.

All experiments were performed between 9:00 a.m. to

3:00 p.m. The study protocol was approved by the Insti-

tutional Animal Ethics Committee (503/IAEC/2011). All

efforts were made to minimize animal suffering and to

reduce the number of animals used.

Drugs and Treatments

Pentylenetetrazole and HEAP were freshly prepared

throughout the study. PTZ was dissolved in normal saline

and injected intraperitoneally. Commercial saline was

obtained from Bexter India Ltd. HEAP was dissolved in

distilled water and administered orally daily by gavage in

the doses of 100, 250 and 500 mg/kg along with PTZ

injections during the kindling process and continued till

PTZ challenge post kindling. Sodium valproate (VAL) was

used as positive control. The animals were randomly

divided into seven groups of 10 animals per group. Group I

(vehicle control) received normal saline only. Group II

received normal saline and PTZ (35 mg/kg, i.p). Group III

received valproate 100 mg/kg, p.o daily and PTZ. Groups

IV, V and VI were administered HEAP at the doses of 100,

250 and 500 mg/kg, p.o daily and PTZ, respectively. Group

VII was administered HEAP at the dose of 500 mg/kg, p.o

daily and served as per se group. The extract or drugs were

given in a volume not exceeding 10 ml/kg. Thirty minutes

before the administration of PTZ, vehicle, valproate and

HEAP (100, 250 and 500 mg/kg) were administered to

their respective groups.

Pentylenetetrazole Induced Kindling

Pentylenetetrazole was administered at a sub-convulsive

dose of 35 mg/kg, three times a week (Monday, Wednes-

day and Friday). After each injection, the mice were placed

singly in transparent plexiglass cages and were observed

for 30 min. The intensity of convulsions was rated

according to 6-point scale [8].

Stage 0: No response

Stage 1: Ear and facial twitching

Satge 2: Head nodding, head clonus and myoclonic jerks

Stage 3: Unilateral forelimb clonus

Stage 4: Rearing with bilateral forelimb clonus

Stage 5: Generalized tonic–clonic seizure (GTCS) with

loss of righting reflex

The latency, number of myoclonic jerks and latency of

GTCS were recorded. The animals considered kindled if they

exhibited stage 4 or (and) 5 of seizures on two consecutive

trials. The animals were given PTZ challenge (70 mg/kg, i.p)

548 Neurochem Res (2013) 38:547–556

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7 days after the development of kindling. In the meantime,

acquisition trials were given to the animals in Morris water

maze. The retention of memory in animals was checked 24 h

after PTZ challenge. HEAP was administered during the

acquisition trials in Morris water maze.

Morris Water Maze

Morris water maze was used to test the spatial memory of the

animals. Training in the maze took place during the light

phase of the cycle between 800 and 1400 hours. Morris water

maze consisted of large circular pool (1.22 m in diameter,

0.61 m in height, filled with water at 28 ± 1 �C). Trans-

parent clear plexiglass platform (10 9 10 cm, 0.29 m high)

was used. To render it invisible to the mice, platform was

submerged 1 cm below the surface of the water. The testing

procedure was used as described earlier by Szyndler et al.

[22]. Briefly, the water tank was divided into four equal

quadrants (Q1, Q2, Q3 and Q4) and the platform was kept in

Q4 quadrant. The animals received four trials during four

daily acquisition sessions. A trial was started by placing mice

into the pool, facing the wall of tank. Each of the four starting

points was used once in a series of trials. The position of

platform was fixed for each trial. The trial was terminated

automatically as soon as the animal reached the platform or

when 120 s had elapsed. The animal was allowed to stay on

the platform for 5 s. Then it was taken out of the platform and

new trial was started. Mice which did not find the platform

within 120 s were put on the platform gently and allowed to

stay there for 5 s. After each trial, the animals were gently

dried with a towel and returned to home cage. On fifth day, a

spatial probe trial (60 s) was tested to detect spatial memory

of the animal. During the probe trial, the same protocol as

described above was followed; however, the platform had

been removed from the tank. The path of each mouse was

analyzed by using the Any-maze video tracking system

(Catterpillar Instrumentation Pvt). Latency to reach the

platform in the acquisition trials and latency to reach the

platform, number of platform crossings and the time spent in

the target quadrant during the probe trial were noted.

Biochemical Estimations

After Morris water maze test, animals were quickly

decapitated under ether anaesthesia and the brains were

removed, cleaned with ice cold saline and stored at -80 �C

till further analysis.

Tissue Preparation

Brain tissue samples were thawed and each brain sample

was divided into two parts. One part was used to assess

lipid peroxidation product and reduced glutathione by

preparing 10 % (w/v) homogenate with ice-cold 0.1 M

phosphate buffer (pH 7.4) and another part was used to

study the expression of ROCK II by western blot.

Measurement of Brain Lipid Peroxidation

Malondialdehyde (MDA), an indicator of lipid peroxida-

tion was estimated as described by Ohkawa et al. [23].

1.5 ml of 20 % (v/v) acetic acid pH-3.5, 1.5 ml of 0.8 %

(w/v) of thiobarbituric acid and 0.2 ml of 8.1 % (w/v) of

sodium dodecyl sulphate were added to 0.1 ml of brain

homogenate, and then heated at 95 �C for 60 min. 5 ml of

n-butanol/pyridine (15:1) was added to the mixture after

cooling. The organic layer was separated by centrifugation

at 4,000 rpm for 10 min and absorbance was measured at

532 nm using spectrophotometer. 1,1,3,3-Tetra-ethoxy

propane was used as a standard. The concentration of MDA

is expressed in nmol/g wet-tissue.

Measurement of Brain Reduced Glutathione (GSH)

Glutathione was measured according to the method of

Ellman [24]. The homogenate was mixed with equal

quantity of 10 % trichloroacetic acid and centrifuged to

separate the proteins. 2 ml of 0.3 M phosphate buffer (pH

8.4), 0.5 ml of DTNB and 0.4 ml of double distilled water

were added to 100 ll supernatant thus obtained. A parallel

standard GSH was run to determine the concentration of

GSH in test samples. The absorbance was read in a spec-

trophotometer at 412 nm within 15 min. The concentration

of reduced glutathione is expressed as lg/g wet-tissue.

Western Blot

Brain homogenate was prepared in lysis buffer (50 mM

Tris–HCl, pH 7.4), 400 mM NaCl, 3 mM EDTA, 1 mM

dithiothreitol, 1 mM phenylmethylsulphonyl fluoride and

2 % SDS). The homogenate was centrifuged at 17,000 rpm

for 30 min at 4 �C and supernatant was removed. Equal

amounts of proteins were loaded in wells and separated by

electrophoresis on 8 % polyacrylamide–sodium dodecyl

sulphate gels. Then, protein was transferred to a nitrocel-

lulose membrane. The membrane was blocked with the

blocking agent for 2 h. It was then incubated with a pri-

mary antibody raised against rho kinase (ROCK II)

(monoclonal IgG; Merck Millipore, USA) at 1:1,000

dilution (overnight) followed by horseradish peroxidase-

conjugated secondary antibody (goat anti-rabbit, 1:2,000

for 2 h; Merck Millipore, USA). The optical density of

bands was analyzed using Alpha Imager gel documentation

system. Rho kinase (ROCK II) expression was normalized

to the optical density of the b-tubulin band to minimize

variations in sample loading.

Neurochem Res (2013) 38:547–556 549

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Statistical Analysis

Results are expressed as mean ± SEM. Statistical analysis

was performed using one way analysis of variance

(ANOVA) followed by Bonferroni’s post hoc test.

P \ 0.05 was considered as significant. Analysis of vari-

ance (ANOVA) for repeated measures was used to assess

differences in the escape latencies in Morris water maze

test among the groups over a period of 5 days. LSD post

hoc test was performed to identify the origin of any sig-

nificant differences. Student’s t test was used to analyze the

latencies to GTCS data pre- and post-PTZ challenge. All

statistical analyses were performed using SPSS statistical

software package version 16.0.

Results

Effect of HEAP on Seizures

Effect of HEAP on Development of Kindling

Repeated administration of sub-convulsive dose of PTZ on

alternate days caused development of stage 5 of seizures on

19.7 ± 0.6 days in the control mice. One way ANOVA

showed significant difference [F(4,45) = 69.138, P \ 0.001]

in the mean duration for development of kindling among the

groups. Post hoc analysis by Bonferroni’s test showed sig-

nificant delay in kindling in HEAP pre-treated groups at the

doses of 250 and 500 mg/kg in comparison to PTZ group

(Table 1). No significant delay in development of kindling in

valproate (100 mg/kg) and HEAP (100 mg/kg) group was

observed.

Effect of HEAP on Seizures in PTZ Kindled Mice

HEAP caused dose dependent increase in mean myoclonic

jerk latency [F(4,45) = 21.097, P \ 0.001] and decrease in

mean number of myoclonic jerks [F(4,45) = 7.431,

P \ 0.001] as compared to the PTZ group (Fig. 1a, b). Post

hoc analysis showed significant (P \ 0.001) increase in

mean myoclonic jerk latency in HEAP treated groups at the

doses of 100, 250 and 500 mg/kg as compared to PTZ

group. A significant decrease in mean number of myo-

clonic jerks were observed in 100 (P \ 0.01), 250

(P \ 0.001) and 500 mg/kg (P \ 0.001) of HEAP treated

groups as compared to PTZ group (Fig. 1b).

Paired ‘t’ test showed the significant difference

(P \ 0.001) in the GTCS latency pre and post PTZ chal-

lenge amongst the experimental groups (Fig. 2a). There

was a significant difference in mean GTCS duration

[F(4,45) = 7.581, P \ 0.001] amongst the different

groups. A significant (P \ 0.001) decrease in mean GTCS

duration was observed in HEAP treated groups at 100, 250

and 500 mg/kg as compared to PTZ group (Fig. 2b).

Effect of HEAP on PTZ Challenge

On PTZ challenge, kindled mice showed a prototype of

GTCS, starting with myoclonic jerks, clonus and then gen-

eralized tonic–clonic seizures with loss of righting response.

The mortality was found to be decreased from 6/10 in PTZ

treated group to 5/10, 2/10 and 1/10 in HEAP treated group at

100, 250 and 500 mg/kg, respectively. Valproate (100 mg/

kg) group showed a mortality of 4 out of 10 mice (Table 1).

Chi square test revealed no significant difference in per-

centage of mortality in between the groups.

Effect of HEAP on Spatial Learning in Morris Water

Maze Test

In the baseline of Morris water maze test (before initiation of

kindling process), no significant change in escape latency,

mean number of platform crossings and mean time spent in

target quadrant amongst the groups was observed. Post

kindling, analysis of variance for repeated measures for

Morris water maze test indicated a significant change in the

escape latency among the groups. Post hoc analysis revealed

a significant decrease in escape latency within the groups 1

and 3–7, during the acquisition trial over the period of 4 days

{day 1[F(6,63) = 0.379, P \ 0.890], day 2 [F(6,63)

=8.319, P \ 0.001], day 3 [F(6,63) = 43.300, P \ 0.001],

day 4 [F(6,63) = 406.738, P \ 0.001]} (Fig. 3a). A signif-

icant decrease in escape latency was observed in group 1 and

groups 3–7 on days 2, 3 and 4 as compared to day1 whereas

no significant change in escape latency in acquisition trials

was observed in PTZ group (group 2). A significant decrease

in escape latency was observed on days 2, 3 and 4 in vehicle

control group as compared to PTZ group, thus showing the

impairment of learning ability of the animals in PTZ group.

On days 2, 3 and 4 there was a significant decrease in escape

latency in HEAP (250 and 500 mg/kg) treated groups as

compared to PTZ group.

Table 1 Effect of HEAP on development of kindling by PTZ

Groups Development of kindling Mortality

PTZ (35 mg/kg) 19.7 ± 0.6 6/10 (60 %)

Valproate (100 mg/kg) 22.2 ± 0.5 4/10 (40 %)

HEAP (100 mg/kg) 21.1 ± 0.6 5/10 (50 %)

HEAP (250 mg/kg) 28.9 ± 1.0*** 2/10 (20 %)

HEAP (500 mg/kg) 34.0 ± 0.5*** 1/10 (10 %)

Data represent mean ± SEM (n = 10), *** P \ 0.001, as compared

to PTZ group, parenthesis showed percentage mortality

550 Neurochem Res (2013) 38:547–556

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There was a significant difference in mean escape latency

[F(6,45) = 22.686, P \ 0.001] (Fig. 3b), mean number of

platform crossings [F(6,45) = 6.436, P \ 0.001] and mean

time spent in target quadrant [F(6,45) = 27.681, P \ 0.001]

in probe trial after PTZ challenge among the groups. A

significant increase in escape latency was observed in PTZ

Fig. 1 Effect of HEAP on

a myoclonic jerk latency,

b number of myoclonic jerks in

PTZ induced kindling in mice.

Data represent mean ± SEM,

**P \ 0.01; ***P \ 0.001,

asterisks as compared to PTZ

group

Fig. 2 Effect of HEAP on

a GTCS latency, b GTCS

duration in PTZ induced

kindling in mice. Data represent

mean ± SEM, ###,

***P \ 0.001, hash symbols as

compared to pre PTZ challenge,

asterisks as compared to PTZ

group

Neurochem Res (2013) 38:547–556 551

123

group as compared to vehicle control group, indicating

impairment of memory. Pre-treatment with HEAP at 250

(P \ 0.05) and 500 mg/kg (P \ 0.001) showed significant

decrease in mean escape latency in comparison to PTZ

group, but the decrease in mean escape latency was not

found to be returned to the vehicle control levels, thus

showing a significant difference (P \ 0.01) in comparison

to vehicle control group. Although the treatment with VAL

and HEAP 100 showed decrease in mean escape latency in

comparison to PTZ group, but it was not found to be sta-

tistically significant and thus the difference in mean escape

latency in VAL and HEAP 100 as compared to vehicle

control group was found to be significant, indicating no

improvement in cognitive function. Post hoc analysis

showed a significant (P \ 0.01) decrease in mean number of

platform crossings during the probe trial in PTZ group as

compared to vehicle control group (Fig. 4a). Significant

increase in mean number of platform crossings were

observed in groups pre-treated with HEAP at 250 (P \ 0.05)

and 500 mg/kg (P \ 0.01), respectively in comparison to

PTZ group (Fig. 4a). Mean time spent in target quadrant was

significantly (P \ 0.001) decreased in PTZ group as com-

pared to vehicle control group. A significant increase in the

mean time spent in target quadrant was observed in groups

pre-treated with HEAP at 250 (P \ 0.05) and 500 mg/kg

(P \ 0.001), respectively in comparison to PTZ group

(Fig. 4b). HEAP per se showed no significant difference

(P [ 0.05) in the mean escape latency, mean number of

platform crossings and mean time spent in target quadrant in

the probe trial as compared to the vehicle control group

(Figs. 3, 4). No significant difference was found in swim

speed and thigmotaxis between all experimental groups in

water maze test during the acquisition and probe trials.

Effect of HEAP on Oxidative Stress Parameters

Effect of HEAP on Brain MDA Levels

A significant difference was observed in the mean MDA

level of whole brain of mice among various groups

[F(6,63) = 107.968, P \ 0.001]. On Post hoc analysis

mean MDA level was found to be increased significantly

(P \ 0.001) in PTZ kindled group as compared to vehicle

control group. A significant decrease in mean MDA level

was observed in HEAP 100 (P \ 0.05), 250 (P \ 0.001)

and 500 mg/kg (P \ 0.001), respectively as compared to

PTZ group. HEAP per se showed no significant difference

(P [ 0.05) in the mean MDA level as compared to the

vehicle control group (Fig. 5a).

Effect of HEAP on Brain GSH Levels

The whole brain mean GSH content was found to be

significantly different among various groups [F(6,63) =

30.666, P \ 0.001]. Post hoc analysis revealed a significant

decrease (P \ 0.001) in the mean GSH level in PTZ group

Fig. 3 Effect of HEAP on

a escape latency in acquisition

trial, b escape latency in probe

trial. Data represent

mean ± SEM, *P \ 0.05;

**p \ 0.01; ***P \ 0.001;###P \ 0.001, asterisks as

compared to day 1 in each

group; hash symbols as

compared to vehicle control

group; aas compared to vehicle

control; bas compared to PTZ

group

552 Neurochem Res (2013) 38:547–556

123

Fig. 4 Effect of HEAP on

a number of platform crossings

in probe trial, b time spent in

target quadrant in probe trial.

Data represent mean ± SEM,

*P \ 0.05; **P \ 0.01;

***P \ 0.001; aas compared to

vehicle control group; bas

compared to PTZ group

Fig. 5 Effect of HEAP on

a MDA levels, b GSH levels in

PTZ induced kindling in mice.

Data represents mean ± SEM,

*P \ 0.05; ***P \ 0.001; aas

compared to vehicle control

group; bas compared to PTZ

group

Neurochem Res (2013) 38:547–556 553

123

in comparison to vehicle control group. Mean GSH level

was found to be increased significantly in HEAP 100

(P \ 0.05), 250 (P \ 0.001) and 500 mg/kg (P \ 0.001),

respectively as compared to PTZ group. No significant

difference (P [ 0.05) was observed in mean GSH level in

HEAP per se group as compared to vehicle control group

(Fig. 5b).

Effect of HEAP on Rho Kinase (ROCK II)

Chronic PTZ administration (35 mg/kg) significantly

up-regulated the ROCK II expression in comparison to

vehicle control as demonstrated by western blotting (Fig. 6).

A significant decrease (P \ 0.001) in the expression of

ROCK II (Fig. 6) was observed in HEAP (500 mg/kg)

group. HEAP per se did not cause any significant change in

ROCK II expression in comparison to vehicle control group.

Discussion

Kindling model is a widely accepted tool to study seizure

mechanisms, epileptogenesis and neuronal plasticity [25].

Sub-convulsive doses of PTZ administered on alternate

days induced kindling in the mice. The present study

demonstrated the potent anticonvulsant property of HEAP

against the development of kindling in PTZ-kindled mice.

The most interesting finding of the study was the delay in

kindling development in mice pretreated with HEAP. The

possibility of HEAP showing the protection against GTCS

because of altered kinetics of PTZ is negligible as PTZ was

injected intra-peritoneally after 30 min of HEAP oral

administration. Further, there is no published evidence of

PTZ kinetics alteration by any oral drug administration.

Pre-treatment with HEAP significantly increases the latent

period of occurrence of myoclonic jerks, clonus and further

the development of kindling, dose dependently. The num-

ber of myoclonic jerks were also decreased in a dose

dependent manner in HEAP pre-treated groups. HEAP at

the dose of 500 mg/kg showed maximum protection

against PTZ-induced kindling. The significant decrease in

number of myoclonic jerks showed persistent insensitivity

to chemical stimuli. The results of the present study are in

concordance with our earlier reports of anticonvulsant

activity of HEAP against single administration of PTZ and

maximal electroshock induced seizures in rats [18]. Fur-

ther, in addition to suppression of development of kindling

HEAP pre-treatment decreased seizure severity even when

the disease state i.e. kindling is fully developed, which is

evident from the fact that there is significant decrease in

mortality in HEAP pre-treated mice.

Numerous studies have shown that convulsions have both

short- and long-term effects on animal behavior. Neuronal

loss in various regions of brain including dentate gyrus of the

hippocampus following kindled seizures is well reported

[11]. Hippocampus has been recognized for its participation

in memory processes, thus damage to this area may result in

cognitive impairment including spatial and contextual

memory processes [26, 27]. In agreement with the previous

studies [8, 9, 28], the present study demonstrates the spatial

memory deficits in PTZ kindled mice. The escape latency

showed a significant change in the acquisition trials in

Fig. 6 Effect of HEAP on rho

kinase II expression in PTZ

kindled mice. Data represents

mean ± SEM. *P \ 0.05;

***P \ 0.001; aas compared to

vehicle control group; bas

compared to PTZ group

554 Neurochem Res (2013) 38:547–556

123

kindled mice, indicating a deficit in learning process. How-

ever, pre-treatment with HEAP causes significant decrease

in the time to reach the platform, during the training sessions,

which shows the protective effect of HEAP against kindling

induced learning deficits. Post PTZ challenge, the PTZ kin-

dled animals spent less time in the target quadrant (where

platform was placed) in comparison to HEAP pre-treated and

valproate group. Therefore, the results of this study dem-

onstrate that the PTZ kindling model produced spatial

learning deficits and pre-treatment with HEAP significantly

decreased the learning impairment in kindled mice. More-

over, no significant difference was found in swim speed and

thigmotaxis between all experimental groups in water maze

test during the acquisition and probe trials, which may rule

out the possibility of involvement of these factors in learning

and memory abilities.

Experimental models of epilepsy have been associated

with increased oxidative stress in the central nervous

system (CNS) [14]. PTZ induced kindling may also elicit

various other biochemical processes including the acti-

vation of membrane phospholipases, proteases, and nuc-

leases. Increased membrane phospholipids’ metabolism

results in generation of free radicals and lipid peroxides.

Both the hemisphere of the brain is equally affected by

the seizures. Different buffer systems are required for the

estimation of oxidative stress parameters and ROCK II

expression. Therefore, in the present study one hemi-

sphere was homogenized in phosphate buffer for estima-

tion of oxidative parameters and another hemisphere was

homogenized in lysis buffer for studying ROCK II

expression by western blot analysis. The increase in MDA

level and decrease in GSH level in PTZ control group in

the present study supports the previous reports of

increased oxidative stress in PTZ induced seizures. The

elevated level of MDA, a marker of lipid peroxidation,

indicates increased free radical generation in the PTZ

kindled animals [29]. GSH is an endogenous antioxidant

and it reacts with the free radicals and prevents the

generation of free radicals and the significant decrease in

GSH levels were observed in the PTZ kindled animals

[30]. However, HEAP administration prevented the rise in

MDA levels in dose dependent manner, which indicates

the attenuation of lipid peroxidation. Moreover, the sig-

nificant increase in GSH levels were also observed with

HEAP pre-treatment as compared to the PTZ kindled

mice. The results in the present study are in concordance

with the recent report of potential antioxidant and free

radical scavenging activity of 50 % methanolic extract of

A. pyrethrum [20]. As an imbalance in oxidant and anti-

oxidant system also contributes to the impairment of

cognition, the increased oxidative stress observed in PTZ

kindled animals may be at least in part is responsible for

the spatial learning deficit.

Recently, the activation of rhoA has been reported in

vulnerable brain regions following traumatic and epileptic

insults to the CNS [31]. Borisoff et al. [32] has shown that

the suppression of rho kinase activity may enhance axonal

regeneration. In the present study, the expression of ROCK

II has been increased by PTZ kindling. Schroder et al. [13,

33] have reported that PTZ kindling enhanced glutamate

receptor density, which at least in part mediating its epi-

leptogenic action. Glutamate is also found to activates rho

kinase in neuronal cells and responsible for brain damage

and epileptogenesis [34]. In concordance with the previous

findings, which showed the protective effect of two rho

kinase inhibitors in PTZ kindling [12], the results of the

present study showed that pre-treatment with HEAP sig-

nificantly decrease the expression of ROCK II, which is

comparable to that of the standard drug valproate. There-

fore, drugs with rho kinase inhibitory activity could be

potentially novel anti-epileptic agents.

Thus from the present findings it can be concluded that

pre-treatment with HEAP protects against the development

of kindling and the spatial memory deficits. Pre-treatment

with HEAP also decreased the marked increase in oxida-

tive stress, which at least in part is responsible for its

anticonvulsant activity. Further, the present study also

showed that rho/rho kinase signaling may be involved in

epileptogenesis and HEAP pre-treatment decreased the

expression of ROCK II, which in addition to its antioxidant

activity may contribute to its anti-epileptic potential.

Acknowledgments The authors are thankful to Indian Council of

Medical Research (ICMR), Ministry of Science, Government of

India, New Delhi, India, for providing financial assistance to Monika

Pahuja under the guidance of Dr. Y. K. Gupta, for carrying out this

research work.

References

1. Prilipko L, De Boer MH, Dua T, Bertolote J (2006) Epilepsy

care—the WHO/ILAE/IBE global campaign against epilepsy.

U.S. Neurological Disease, pp 39–40

2. Jambaque I, Dellatolas G, Dulac D, Ponsot G, Signoret JL (1993)

Verbal and visual memory impairment in children with epilepsy.

Neuropsychologia 31:1321–1337

3. Thompson PJ, Trimble MR (1983) Anticonvulsant serum levels:

relationship to impairments of cognitive functioning. J Neurol

Neurosurg Psychiatry 46:227–233

4. Erakovic V, Zupan G, Varljen J, Laginja J, Simonic A (2001)

Altered activities of rat brain metabolic enzymes caused by

pentylenetetrazol kindling and pentylenetetrazol-induced sei-

zures. Epilepsy Res 43:165–173

5. Bruce AJ, Baudry M (1995) Oxygen free radicals in rat limbic

structures after kainate-induced seizures. Free Rad Biol Med

18:993–1002

6. Morimoto K, Fahnestock M, Racine RJ (2004) Kindling and

status epilepticus models of epilepsy: rewiring the brain. Prog

Neurobiol 73(1):1–60

Neurochem Res (2013) 38:547–556 555

123

7. Becker A, Grecksch G (1992) dTyr-D-Phe3 (Pro-D-Phe-Pro-Gly)

interacts specifically with amygdaloid-kindled seizures and is

capable of preventing the learning deficit occurring after kindling.

Peptides 13(1):73–76

8. Gupta YK, Veerendra Kumar MH, Srivastava AK (2003) Effect

of Centella asiatica on pentylenetetrazole-induced kindling,

cognition and oxidative stress in rats. Pharmacol Biochem Behav

74(3):579–585

9. Mehla J, Reeta KH, Gupta P, Gupta YK (2010) Protective effect

of curcumin against seizures and cognitive impairment in a

pentylenetetrazole-kindled epileptic rat model. Life Sci

87(19–22):596–603

10. Loscher W (2011) Critical review of current animal models of

seizures and epilepsy used in the discovery and development of

new antiepileptic drugs. Seizure 20:359–368

11. Cavazos JE, Sutula TP (1990) Progressive neuronal loss induced

by kindling: a possible mechanism for mossy fiber synaptic

reorganization and hippocampal sclerosis. Brain Res 527:1–6

12. Inan SY, Buyukafsar K (2008) Antiepileptic effects of two Rho-

kinase inhibitors, Y-27632 and fasudil, in mice. Br J Pharmacol

155:44–51

13. Schroder H, Becker A, Lossner B (1993) Glutamate binding to

brain membranes is increased in pentylenetetrazole-kindled rats.

J Neurochem 60:1007–1011

14. Rauca C, Zerbe R, Jantze H (1999) Formation of free hydroxyl

radicals after pentylenetetrazol-induced seizure and kindling.

Brain Res 847:347–351

15. Prajapati ND, Purohit SS, Sharma AK, Kumar T (2003) A

handbook of medicinal plants: a complete source book. Agrobios,

India, pp 43–44

16. Crombie L (1954) Isolation and structure of an N-isobutyld-

ienediynamide from pellitory (Anacyclus pyrethrum DC.). Nature

174:832–833

17. Bendjeddou D, Lalaoui K, Satta D (2003) Immunostimulating

activity of the hot water-soluble polysaccharide extracts of

Anacyclus pyrethrum, Alpinia galanga and Citrullus colocynthis.

J Ethnopharmacol 88(2–3):155–160

18. Pahuja M, Mehla J, Reeta KH, Joshi S, Gupta YK (2012) Root

extract of Anacyclus pyrethrum ameliorates seizures, seizure-

induced oxidative stress and cognitive impairment in experi-

mental animals. Epilepsy Res 98(2–3):157–165

19. Rimbau V, Cerdan C, Vila R, Iglesia J (1999) Antiinflammatory

activity of some extracts from plants used in the traditional medi-

cine of North-African countries. Phytother Res 10(5):421–423

20. Kalim MD, Bhattacharyya D, Banerjee A, Chattopadhyay S

(2010) Oxidative DNA damage preventive activity and antioxi-

dant potential of plants used in Unani system of medicine. BMC

Complement Altern Med 10:77–87

21. Sukumaran K, Kuttan R (1995) Inhibition of tobacco-induced

mutagenesis by eugenol and plant extracts. Mutat Res 343(1):

25–30

22. Szyndler J, Piechal A, Blecharz-Klin K, Skorzewska A, Maciejak

P, Walkowiak J et al (2006) Effect of kindled seizures on rat

behavior in water Morris maze test and amino acid concentrations

in brain structures. Pharmacol Rep 58:75–82

23. Ohkawa H, Ohishi N, Yagi K (1979) Assay of lipid peroxides in

animal tissue by thiobarbituric acid reaction. Anal Biochem

95:351–358

24. Ellman GL (1959) Tissue sulphydryl groups. Arch Biochem

Biophys 82:70–73

25. Genkova-Papazova MG, Petkova B, Shishkova N, Lazarova-

Bakarova M (2001) Effect of calcium channel blockers nifedipine

and diltiazem on pentylenetetrazole kindling-provoked amnesia

in rats. Eur Neuropsychopharmacol 11:91–96

26. Broadbent NJ, Squire LR, Clark RE (2004) Spatial memory,

recognition memory, and the hippocampus. Proc Natl Acad Sci

USA 101:14515–14520

27. Ramos JM (2000) Long-term spatial memory in rats with hip-

pocampal lesions. Eur J Neurosci 12:3375–3384

28. Hamm RJ, Pike BR, Temple MD, Dell DM, Lyeth BG (1995)

The effect of postinjury kindled seizures on cognitive perfor-

mance of traumatically brain-injured rats. Exp Neurol 136:

143–148

29. Ilhan A, Gurel A, Armutcu F, Kamisli S, Iraz M (2005) Anti-

epileptogenic and antioxidant effects of Nigella sativa oil against

pentylenetetrazol-induced kindling in mice. Neuropharmacology

49:456–464

30. Ono H, Sakamoto A, Sakura N (2000) Plasma total glutathione

concentrations in epileptic patients taking anti-convulsants. Clin

Chim Acta 298:135–143

31. Dubreuil CI, Marklund N, Deschamps K, McIntosh TK,

McKerracher L (2006) Activation of Rho after traumatic brain

injury and seizure in rats. Exp Neurol 198(2):361–369

32. Borisoff JF, Chan CC, Hiebert GW, Oschipok L, Robertson GS,

Zamboni R et al (2003) Suppression of Rho-kinase activity pro-

motes axonal growth on inhibitory CNS substrates. Mol Cell

Neurosci 22:405–416

33. Schroder H, Schlichthaar R, Krug M (1994) Specific [3H]L-

glutamate binding and [3H]D-aspartate release in the hippo-

campus of rat after pentylenetetrazol kindling and long-term

potentiation. Neuroscience 60:337–342

34. Jeon S, Kim S, Park JB, Suh PG, Kim YS, Bae CD et al (2002)

RhoA and Rho kinase-dependent phosphorylation of moesin at

Thr-558 in hippocampal neuronal cells by glutamate. J Biol

Chem 277:16576–16584

556 Neurochem Res (2013) 38:547–556

123