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Original ArticleJournal of Epilepsy Research

pISSN 2233-6249 / eISSN 2233-6257

Anti-kindling Effect of Bezafibrate, a Peroxisome Proliferator-activated Receptors Alpha Agonist, in Pentylenetetrazole Induced Kindling Seizure ModelLekha Saha1, Swati Bhandari1, Alka Bhatia2, Dibyajyoti Banerjee2, Amitava Chakrabarti1

1Department of Pharmacology, Postgraduate Institute of Medical Education & Research, Sector 12, Chandigarh;2Department of Experimental Medicine and Biotechnology, Postgraduate Institute Of Medical Education &

Research, Sector 12, Chandigarh, India

Received September 26, 2014Accepted November 17, 2014

Corresponding author: Lekha SahaDepartment of Pharmacology, Postgraduate Institute of Medical Education & Research, Sector 12, Chandigarh, IndiaTel. +91-01722755253Fax. +91-01722744401E-mail; [email protected]

Background and Purpose: Studies in the animals suggested that Peroxisome proliferators activated

receptors (PPARs) may be involved in seizure control and selective agonists of PPAR α or PPAR γ raise

seizure thresholds. The present study was contemplated with the aim of evaluating the anti kindling

effects and the mechanism of bezafibrate, a Peroxisome proliferator-activated receptors α (PPAR-α)

agonist in pentylenetetrazole (PTZ) induced kindling model of seizures in rats.

Methods: In a PTZ kindled Wistar rat model, different doses of bezafibrate (100 mg/kg, 200 mg/kg and

300 mg/kg) were administered intraperitoneally 30 minutes before the PTZ injection. The PTZ injection

was given on alternate day till the animal became fully kindled or till 10 weeks. The parameters

measured were the latency to develop kindling and incidence of kindling, histopathological study of

hippocampus, hippocampal lipid peroxidation studies, serum neuron specific enolase, and hippocampal

DNA fragmentation study.

Results: In this study, bezafibrate significantly reduced the incidence of kindling in PTZ treated rats and

exhibited a marked prolongation in the latencies to seizures. In the present study bezafibrate decreased

the thiobarbituric acid-reactive substance i.e. Malondialdehyde levels, increased the reduced gluta-

thione levels, catalase and superoxide dismutase activity in the brain. This added to its additional neuro-

protective effects. Bezafibrate also reduced the neuronal damage and apoptosis in hippocampal area of

the brain. Therefore bezafibrate exerted anticonvulsant properties in PTZ induced kindling model in rats.

Conclusions: These findings may provide insights into the understanding of the mechanism of

bezafibrate as an anti kindling agent and could offer a useful support to the basic antiepileptic therapy in

preventing the development of PTZ induced seizures, suggesting its potential for therapeutic

applications in temporal lobe epilepsy. (2014;4:45-54)

Key words: Bezafibrate, Pentylenetetrazole, Kindling, Mechanism, Antiepileptic drug

Introduction

Epilepsy affects 1-2% of children and 0.5-1% of the population

worldwide. Approximately 30% of patients with epilepsy have re-

fractory epilepsy, that is, seizures persist despite accurate diagnosis

and carefully monitored treatment with antiepileptic drugs.1,2

Kindling is a model of epilepsy, with the advantage of being both

an epileptogenic and a spontaneous seizure model. Kindled seizures

are widely accepted as an animal model of human temporal lobe epi-

lepsy (TLE), wherein repeated sub threshold brain stimulation

(electrical or chemical) leads to behavioral signs of seizures (tonic

and clonic)3,4 and such procedure results in a state mimicking many

signs of human TLE: hippocampal atrophy, loss of neurons in the lim-

bic areas: Cal, CA3, and dentate gyrus of the hippocampus, glass

and growth of new neuronal connections.5-7

Studies have focused on the role of oxidative stress in seizures.

Free radicals have been implicated in the development of seizures.8,9

Animal data also suggest that even a single seizure may be damag-

ing and can cause DNA fragmentation which is the characteristic fea-

ture of apoptosis. Also Gamma-enolase, a neuron-specific enzyme

46 Journal of Epilepsy Research Vol. 4, No. 2, 2014

Copyright ⓒ 2014 Korean Epilepsy Society

critical in energy metabolism, is elevated following anoxia, stroke, or

status epilepticus.10

Peroxisome proliferator-activated receptors (PPARs) have been

considered as potential drug targets for seizure control.11 Both PPAR

α and PPARγ are activated by the polyunsaturated fatty acids (PUFA)

and their eicosanoid derivatives, as well as by synthetic legands such

as the fibrates.12 Activation of peroxisome proliferator- activated re-

ceptors stimulates the transcription of the genes involved in fatty

acid oxidation via the formation of heterodimeric transcription-factor

complexes with the retinoid-X-receptor (RXR).13

Based on animal studies that have shown that selective agonists

of PPAR α or PPAR γ raise seizure thresholds, it is suggested that

PPARs may be involved in seizure control.14 Fenofibrate, for instance,

an agonist of PPAR α, has been reported to raise the seizure thresh-

old in the acute seizure models like tail-infusion pentylenetetrazole

(PTZ) and Pilocarpine seizure tests in rats.15 The involvement of of

PPARα in seizure control has been documented by Puligheddu M et

al in nicotine induced seizures in mice and rats.16 The role of PPARα in a genetic model of absence epilepsy has also been demonstrated

in a study by Citraro R et al in rat.17 However, the role of PPAR-α in

the kindling seizure models have not been reported in the published

literatures.

The purpose of the present study was to evaluate the anti kindling

effects and the mechanism of bezafibrate, a PPARα agonist in PTZ in-

duced kindling model of seizures in rats. These findings may provide

insights into the understanding of the mechanism of bezafibrate as

an anti kindling agent and could offer a useful support to the basic

antiepileptic therapy in preventing the development of PTZ induced

seizures, suggesting its potential for therapeutic applications in tem-

poral lobe epilepsy.

Methods

Animals

Young male wistar rats (200 to 250 gm) were housed 2 per cage

and maintained at 23±20 C with a relative humidity of 65±5% in 12

hours light/dark cycle. Animals were acclimatized to laboratory con-

ditions for at least 7 days prior to experimentation. All procedures in-

volving animals and their care were performed with the agreement of

the local ethics committee for animal experimentation and in com-

pliance with our institutional guidelines, which in turn comply with

current national and international laws and recommendations.

Drug preparation and dosing schedule

PTZ was dissolved in 0.9% saline in strength of 0.31% and in-

jected intraperitoneally (i.p.) in a volume not exceeding 1.0 mL/kg, at

a sub convulsive dose of 30 mg/kg every alternate day until develop-

ment of kindling or up to 10 weeks. Bezafibrate (100, 200, 300

mg/kg) was dissolved in 5% dimethyl sulfoxide (DMSO) and Sodium

Valporate (200 mg/kg) in 0.9% saline and were given intra-

peritoneally, 30 minutes before the PTZ injection till the animal de-

velops kindling or up to 10 weeks. All chemicals were procured from

Sigma Pharmaceutical Industry Co.

Diets and treatments

Animals had free access to standard pellet chow diet and tap wa-

ter ad libitum. Rats were weighed and divided into 7 groups: Group I:

Vehicle Control (DMSO 5%+PTZ 30 mg/kg) (n=8); Group II: Saline

Control (Normal Saline+PTZ 30 mg/kg) (n=8); Group III: sodium val-

porate (VPA) 200 group (Sodium Valproate 200 mg/kg+PTZ 30

mg/kg) (n=8); Group IV: bezafibrate (BEZF) 100 group (Bezafibrate

100 mg/kg+PTZ 30 mg/kg) (n=8); Group V: BEZF 200 group

(Bezafibrate 200 mg/kg+PTZ 30 mg/kg) (n=8); Group VI: BEZF 300

group (Bezafibrate 300 mg/kg+PTZ 30 mg/kg) (n=8), Group VII: un-

anaesthetized group. To see the impact of pentobarbital on apop-

totic biomarkers and oxidative stressed parameters, an un-

anaesthetized group was included where animals were sacrificed by

stunning.

Pentylenetetrazole induced kindling in rats

PTZ was injected i.p. as mentioned above. After each injection of

PTZ, the rats were placed singly in isolated transparent Plexiglas cag-

es and were observed for 2hrs/day. The intensity of convulsions was

rated according to modified Racine scale as follows:18 0-No re-

sponse; 1 - Ear and facial twitching; 2 - Myoclonic jerks without rear-

ing; 3 - Myoclonic jerks with rearing; 4 - Turn over into side position,

clonic - tonic seizures; 5 - Turn over into back position, generalized

tonic - clonic convulsions. An animal was considered kindled when it

exhibits, stage 4 or 5 of seizure score on three consecutive trials.

Collection of samples

When the animal became fully kindled (exhibits, stage 4 or 5 of

seizure score on three consecutive trials), on the next day, it was sac-

rificed by decapitation under the overdose of intraperitoneal pento-

barbitone (50 mg/kg) anesthesia. The hippocampus was carefully

dissected out of the brain. 2 mL of blood was also collected from

Lekha Saha, et al. Understanding Mechanism of Bezafibrate in Kindling 47

www.kes.or.kr

each anesthetized animal by cardiac puncture prior to decapitation.

Studies with hippocampus

Histopathology of the hippocampus

One half of each brain was fixed in 10% formalin and stored for

histopathological studies using hemotoxylin and eosin (H&E) stain.

Thereafter, tissue was sliced, routinely processed, and embedded in

paraffin wax. 5 μm coronal paraffin sections were cut, mounted and

stained by haematoxylin and eosin. The acidophilus neuron, identi-

fied by intense cytoplasmic eosinophilia accompanied by chromatin

dispersion with loss of nuclear membrane integrity, was perceived as

the marker for irreversible neuronal damage at the cellular level. The

numbers of acidophilus neurons in different regions of the hippo-

campus were estimated on a 0-3 grading scale, 0=none, 0.5=slight

(<10%), 1.0=mild (10-25%), 1.5=mild-to-moderate (26-45%), 2.0

=moderate (46-54%), 2.5=moderate-to-severe (55-75%), and 3.0

=severe (>75%), as previously published.19 The surviving cells were

defined as round-shaped, cytoplasmic membrane-intact cells,with-

out any nuclear condensation or distorted aspect. The surviving pyr-

amidal cells in the hippocampal CA3 region were seen at high magni

fication (400×). Sections were examined by a blinded investigator

without knowledge of any other data on that animal.

Hippocampal DNA fragmentation study

DNA was isolated from hippocampal brain specimens using DNA

isolation kits and was subjected to agarose gel electrophoresis.

Hippocampal lipid peroxidation studies

Estimations of thiobarbituric acid-reactive substance

The extent of lipid peroxidation was estimated according to the

method of Ohokawa et al20 in tissue homogenate. Tissue homoge-

nate was prepared in a ratio of 1g of wet tissue to 9 mL of phosphate

buffer (pH 7.2) using a homogenizer. To 0.1 mL of the homogenate,

0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid

solution, 1.5 mL of a 0.8% aqueous solution of thiobarbituric acid

were added. The mixture was finally made up to 4.0 mL with distilled

water, and heated at 95ºC for 60 min. After cooling with tap water,

1.0 mL of distilled water and 5.0 mL of the mixture of n-butanol and

pyridine (15:1, v/v) were added, and the mixture was shaken

vigorously. After centrifugation at 4,000 rpm for 10 min, the absorb-

ance of the organic layer (upper layer) was measured at 532 nm in a

spectrophotometer against a blank containing all the reagents ex-

cept the homogenate. The malondialdehyde (MDA) equivalents of

the samples were calculated using the extinction coefficient 1.56×

105 M-1cm-1.

Determination of catalase activity

The activity of catalase was measured by the method of Luck.21 A

10% w/v homogenate of the hippocampus was prepared in phos-

phate buffer. The homogenate was centrifuged and the supernatant

was used for the enzyme assay. In short, the reaction mixture con-

tained Tris (50 mM) - EDTA (5 mM) buffer, pH 7.0, 10 mM H2O2 (in

0.1 M KH2PO4 buffer, pH 7.0) in test cuvette. The reference cuvette

contained Tris-EDTA solution and distilled water only. The contents

of both the cuvettes were incubated at 37ºC for 10 min. The reaction

was started by the addition of tissue homogenate to both reference

as well as test cuvettes. The rate of elimination of H2O2 by catalase

was measured by recording the rate of change of absorbance per min

at 240 nm for 4 min. Catalase activity was expressed as μmol of H2O2

consumed /min/mg protein using a molar extinction coefficient of

43.6 mM-1cm-1.

Reduced glutathione estimations

Assay of glutathione (GSH) was performed in tissue homogenates

by the method of Moron et al.22 To 500 μL homogenate 100 μL of

25% trichloroacetic acid (TCA) was added. The precipitated proteins

were separated by centrifugation at 2,000×g for 15 min. Superna-

tants were diluted with 0.2 M sodium phosphate buffer, pH 8.0. To

this, 2.0 mL of 0.6 mM 5,5 Dithiobis (2-Nitrobenzoic acid) was

added. The colored complex formed by DTNB and GSH was meas-

ured spectrophotometricaly at 412 nm against a reference cuvette

containing 0.1 or 0.2 mL of 5% TCA. A standard curve of GSH was

plotted with every set of assays. All the assays were done in du-

plicates. The levels of GSH were expressed as μg of GSH/mg protein.

Measurement of serum neuron specific enolase

2 mL of blood was collected from each anesthetized animal by car-

diac puncture prior to decapitation. Blood samples were allowed to

clot for 2 hours at room temperature or overnight at 4ºC before cen-

trifugation for 15 minutes at 1,000×g. Serum was removed and sam-

ples were stored at -20ºC or -80º. Repeated freeze-thaw cycles were

avoided. The sample was subjected to the estimation of Neuron-

Specific Enolase (NSE) by ELISA kit (CUSABIO BIOTECH CO., LTD.,

China) according to the manufacturer’s instructions.

Statistical analysis

Data are expressed as the mean±SD. One-way analysis of var-



Table 1. Effect of bezafibrate on the average seizure score of three readings for every week in PTZ kindled rats

Time (week)

Vehicle control group(No of animals

seized/no of animals

tested)

Saline control group(No of animals


tested)

VPA 200 group (No of animals


tested)

BEZF 100 group (No of animals


tested)



tested)



tested)

1 0.79±0.26(8/8)

0.88±0.31(8/8)

0±0*†‡§

(0/8)0.83±0.31

(5/8) 0.67±0.36

(3/8) 0.75±0.35

(4/8)

2 0.95±0.57 (8/8)

1.50±0.18 (8/8)

0.12±0.17*†‡§

(2/8)1.50±0.31

(5/8) 1.45±0.31

(4/8) 1.67±0.31

(6/8)

3 1.54±0.24 (7/7)

1.91±0.15 (6/6)

0.17±0.25*†‡§

(3/8)2±0.47

(4/7) 1.92±0.15

(5/8)2.21±0.35

(7/8)

4 2.21±0.15 (7/7)

2.33±0.25 (5/5)

0.13±0.17*†‡§

(3/8)2.54±0.47

(4/5) 2.21±0.36§

(6/7)2.75±0.24

(8/8)

5 3.73±0.27 (5/5)

2.91±0.30 (4/4)

0±0*†‡§

(0/8)3.21±0.62

(4/4) 2.33±0.25*†§

(7/7)2.96±0.22

(7/7)

6 3.99±0.17 (4/4)

3.29±0.12 (4/4)

0±0*†‡§

(0/8)3.38±0.33

(4/4) 2.25±0.15*†§

(6/7)2.83±0.31*†

(7/7)

7 3.95±0.13 (3/3)

3.42±0.24 (3/3)

0.08±0.15*†‡§

(2/8)3.13±0.31

(4/4) 1.92±0.1*†§

(4/7)2.58±0.30*†

(7/7)

8 4.21±0.23 (3/3)

3.50±0.18 (2/2)

0.42±0.12*†‡§

(4/8)3.13±0.31*

(3/3)1.96±0.15*†§

(4/7)2.58±0.30*†

(7/7)

9 4.10±0.22(2/2)

3.71±0.12(2/2)

0.42±0.12*†‡§

(4/8)3.13±0.31*

(3/3)1.96±0.15*†§

(4/7)2.58±0.30*†

(7/7)

10 4.16±0.12 (1/1)

3.96±0.12 (1/1)

0.42±0.12*†‡§

(4/8)3.17±0.25*

(3/3)1.96±0.15*†§

(4/7)2.58±0.30*†

(7/7)

Data are expressed as Mean±SD. n=8. One way ANOVA with repeated measures followed by Bonferroni post hoc analysis. *p<0.05 compared to vehicle control and saline control groups. †p<0.05 compared to BEZF 100 group; ‡p<0.05 compared to BEZF 200 group; §p<0.05 compared to BEZF 300 group.PTZ, pentylenetetrazole; VPA, sodium valporate; BEZF, bezafibrate; SD, standard deviation; ANOVA, analysis of variance.

Figure 1. Effect of various treatments on the percent of animals develops

kindling at various time points. Data expressed as % of animals with SD.

N=8. Fischer’s exact test was done for comparison. *p<0.05 compared to

vehicle and Saline control groups; †p<0.05 compared to VPA 200 group.

PTZ, pentylenetetrazole; VPA, sodium valporate; BEZF, bezafibrate; SD,

standard deviation.

iance (ANOVA) using bonferroni post hoc analyses were performed

using SPSS (20.0 version; LEAD Technologies, Chicago, IL, USA).

Statistical significance was considered at p<0.05.

Results

Effect of bezafibrate in PTZ- induced kindling model

In vehicle control and saline control groups, the rats showed a

gradual increase in the seizure score throughout the 10 weeks of the

study period and 7 animals out of 8 (87.5%) became kindled at the

end of 10 weeks in both the groups (Table 1, Fig. 1).

Sodium valproate (200 mg/kg, ip) pre-treatment significantly re-

duced the seizure score in the PTZ treated animals throughout the 10

weeks of study period (Table 1). In the VPA 2,000 group, none of the

animal developed kindling at any point of time. The data for sodium

valproate treated group were significantly less as compared to that of

vehicle control and saline control group at all time points (p<0.05,

Fig. 1).

Bezafibrate showed dose-dependent protection against PTZ in-

duced kindling in rats. At 200 mg/kg, the effect of bezafibrate was


www.kes.or.kr

Table 2. Effect of bezafibrate on hippocampal reduced glutathione levels, malondialdehyde levels, catalase activity and serum neuron specific enolase levels in PTZ treated rats

GroupsGSH levels

(nmol/mg protein)MDA levels

(nmol/mg protein)Catalase activity

(nmol/mg protein)s-NSE levels

(nmol/mg protein)

Vehicle Control 0.0079±0.0012† 253.45±1.05† 6.16±0.05† 15.03±0.27†

Saline Control group 0.0086±0.0005† 241.23±0.88† 6.96±0.18† 17.46±1.06†

VPA 200 group 0.0197±0.0006*‡§∥ 209.28±1.81*‡§∥ 8.43±0.11*‡§ 6.12±0.28*‡∥

BEZF 100 group 0.0145±0.0012* 233.16±1.17* 7.38±0.29* 13.37±1.10*

BEZF 200 group 0.0166±0.0008*‡∥ 215.72±0.50*‡∥ 7.69±0.24* 6.85±0.21*‡∥

BEZF 300 group 0.0138±0.0007* 222.67±0.37*‡ 8.21±0.08*‡∥ 8.94±0.73*‡

Unanaesthetized group 0.0221±0.0013 205.24±1.04 9.25±0.08 7.21±0.54

Data are expressed as Mean±SD. n=8. One way ANOVA followed by Bonferroni post hoc analysis.*p<0.05 compared to vehicle control and saline control groups; †p<0.05 compared to unanaesthetized group, ‡p<0.05 compared to BEZF 100 group; §p<0.05 compared to BEZF 200 group; ∥p<0.05 compared to BEZF 300 group.GSH, glutathione; MDA, malondialdehyde; sNSE, serum neuron specific enolase; PTZ, pentylenetetrazole.

significantly better when compared to that of 100 mg/kg of

bezafibrate. However, the beneficial anti kindling effect of bezafi-

brate reached a ceiling at 200 mg/kg dose, since the maximum seiz-

ure score was 1.96±0.15 in 200 mg/kg group as compared to the

maximum seizure score of 2.58±0.30 in 300 mg/kg group which was

statistically significant. The anti kindling effect (particularly the seiz-

ure score) of bezafibrate was significantly less as compared to that of

sodium valproate. At 200 mg/kg, bezafibrate showed an anti kin-

dling effect which increased over the period of study and was sig-

nificantly better as compared to that of vehicle control and saline

control groups from the 5th week onwards till the end of the study

period i.e. 10th week both in terms of reduction in seizure score and

the number of animals developing PTZ induced kindling behavior. In

the bezafibrate groups, the seizure score increased gradually up to

week 5 and then gradually decreased because chronic admin-

istration of bezafibrate takes some time to produce its effect on the

seizure scores and also due to animals being withdrawn because

they have met the full-kindled criteria. Whereas in the control

groups, the seizure scores increase consistently, which indicate that

in the control group individual animal shows high seizure scores.

Sodium valproate on the other hand, showed a significant anti kin-

dling effect from 1st week onwards till the end of the study (Table 1).

Lipid peroxidation parameters

Effect of bezafibrate on reduced glutathione levels in the

rat brain

Treatment with PTZ in vehicle control and saline control groups

significantly reduced the GSH levels in rat brain when compared with

unanaesthetized normal control rat. Pre-treatment with sodium val-

proate (200 mg/kg) and three different doses of bezafibrate (100

mg/kg, 200 mg/kg, 300 mg/kg, i.p.) significantly increased the level

of GSH in brain towards the normal. Among all the three doses of be-

zafibrate 200 mg/kg was more effective in increasing the GSH level

in the brain as compared to that with 100 and 300 mg/kg doses

(Table 2).

The effect of bezafibrate on malondialdehyde levels in rat

brain

Pre-treatment with sodium valproate (200 mg/kg) and three dif-

ferent doses of bezafibrate (100 mg/kg, 200 mg/kg, 300 mg/kg, i.p)

also significantly reduced the whole brain MDA level in PTZ treated

animals and it was statistically significant when compared to vehicle

and saline treated groups. The reduction in the brain MDA level was

significantly more with bezafibrate 200 mg/kg pre treated group

when compared to bezafibrate 100 and 300 mg/kg pre treated

group (Table 2).

The effect of bezafibrate on catalase activity in rat brain

The catalase activity was significantly decreased in vehicle and sal-

ine treated rats when compared to the unanaesthetized control

animals. In the sodium valproate and bezafibrate pre treated groups,

the catalase activity in rat brain was comparable with that of the con-

trol group but significantly higher as compared to that of the vehicle

and saline treated groups (Table 2).

Effect of bezafibrate on serum neuron specific enolase

levels in rats

The level of NSE in serum was significantly elevated in vehicle and

saline treated group as compared to the unanaesthetized control



Table 3. Effect of various treatments on damage scores in the hippocampus of the rats assessed

GroupsBrain region

CA1 CA3 H DG

Vehicle control 0.15±0.11 0.17±0.11 0.17±0.11 0.17±0.11

Saline control 0.17±0.11 0.09±0.08 0.08±0.08 0.17±0.11

VPA 200 0.00±0.00 0.08±0.08 0.08±0.08 0.00±0.00

BEZF 100 0.16±0.11 0.17±0.11 0.00±0.00 0.25±0.11

BEZF 200 0.15±0.11 0.08±0.08 0.08±0.08 0.17±0.11

BEZF 300 0.17±0.04 0.12±0.04 0.10±0.04 0.13±0.04

Unanaesthetized 0.16±0.12 0.14±0.12 0.10±0.06 0.16±0.07

The data represent means±SEM of neuronal damage scores assessed by numbers of acidophilic neurons by H&E stain. H, hilus; DG, dentate gyrus; SEM, standard error of mean.

A B C

D E F

G

H

I

Figure 2. The morphology of hippocampal neurons in different experimental groups. H&E stain clearly shows gliosis in hilus (B) and area CA1 (E) in the PTZ treated rats’ hippocampus as compared to hilus (A) and area CA1 (D) in control rats. C, F are magnifications of B and E respectively. Arrow head point to gliosis. Arrows point to glial cells. Scale bars=50 μm (A, B, D, E), 100 μm (C, F). G-I show neurons in hippocampal CA1 regions (×400) in the VPA 200 treated rat (G), BEZF 200 treated rat (H) and BEZF 300 treated rats (I) stained with H&E. PTZ, pentylenetetrazole; VPA, sodium valporate; BEZF, bezafibrate.

group. In the sodium valproate pre treated group (6.12±0.28 nmol/

mg protein), there was significant reduction in serum neuron specific

enolase (sNSE) level as compared to that observed with vehicle and

saline treated groups. All the three doses of bezafibrate also showed

significant reduction in sNSE level as compared to vehicle and saline

treated groups. Bezafibrate at 200 mg/kg and 300 mg/kg showed

significant reduction in sNSE levels as compared to that with bezafi-

brate 100 mg/kg, while the effect observed with the dosage of 200

mg/kg was most effective and was statistically significant as com-

pared to that with 300 mg/kg dose of bezafibrate (Table 2).

Histopathological changes in the hippocampus

We rarely observed acidophilic neurons in different sectors of the

PTZ-treated rats’ hippocampus. Despite lacking of evidence for

PTZ-induced cell necrosis, our study found gliosis in area CA1 and

the hilus of the hippocampus in PTZ treated rats (Table 3, Fig. 2),

suggesting the existence of neuronal loss. In VPA 200 group, there

was less or no gliosis. Pre-treatment of BEZF 200 and 300, there was

also less gliosis as compared to PTZ group. H&E staining showed that

dead neurons in hippocampal CA3 region with pyknotic nuclei are

clearly distinguishable from surviving cells that show round and pale-


www.kes.or.kr

A

B

Figure 3. High power (40) photomicrographs showing H&E staining with hematoxylin and eosin of the hippocampal CA3 pyramidal neurons in control, PTZ and BEZF 200 mg treated groups. (A) The changes of the hippocampal CA3 pyramidal neurons in control, PTZ and BEZF 200mg treated groups. Bar=20 mm. (B) The number of surviving cells in different groups. Compared with control rats, the surviving cells of rats of PTZ group showed decrease significantly. BEZF 200 mg could keep from cells damage. Bars indicate mean±SD. *p<0.05 vs. control. †p<0.05 vs. PTZ. PTZ, pentylenetetrazole; VPA, sodium valporate; BEZF, bezafibrate; SD, standard deviation.

Figure 4. Effect of variuos treatments on DNA fragmentation in PTZ treated

rats in different experimental groups. DL, DNA Ladder; 1-2, control group;

3-4, VPA 200 group; 5-6, PTZ group; 7-8, BEZF 100 group; 9-10, BEZF 200

group; 11-12, BEZF 300 group. PTZ, pentylenetetrazole; VPA, sodium

valporate; BEZF, bezafibrate.

ly stained nuclei. As shown in Fig 3A and 3B, the surviving neuron

numbers in PTZ alone treated group were significantly decreased

compared with that of control group (p<0.05). Moreover, BEZF 200,

significantly attenuated the neuron loss induced by seizures (p<

0.05) (Fig. 3A and 3B).

DNA fragmentation studies in the hippocampus of PTZ

induced kindled rats

All the brain samples from different groups were subjected to DNA

fragmentation. The PTZ and BEZF 100 groups showed laddering pat-

tern with the formation of a large number of fragments ranging from

1,517 base pairs to 100 base pair fragments indicating DNA

fragmentation. In contrast, VPA 200, BEZF 200 and BEZF 300 groups

did not show formation of small fragments and hence no prominent

laddering pattern (Fig. 4).

Discussion

In the present study, bezafibrate exhibited a marked non dose de-

pendent anti kindling activity, suggesting its beneficial role in re-

ducing seizure severity. Bezafibrate pre-treated groups showed sig-

nificant reduction in the percent of animals (12.5%) developed

kindling. PTZ thresholds were increased and the onset of seizure was

delayed in the PTZ-induced kindling model.

As per a previous study, a PPAR-γ ligand (FMOC-Lieucine), ex-

erted anticonvulsant properties against audiogenic seizures induced

in adult magnesium-deficient mice.23



However, in the same model, rosiglitazone, other PPAR-γ agonist,

did not protect mice. The seizure protection induced by FMOC-L-

Leucine was also reduced by a PPAR-γ antagonist. Study on PPAR-α agonist, fenofibrate has reported the anticonvulsant property of the

drug comparable to kitogenic diet in adult rats using PTZ induced

acute seizure model and lithium -Pilocarpine induced status epi-

lepticus (SE) model .15

PPAR-α is likely to regulate the expression of many genes that en-

code enzymes of fatty acid oxidation, ketogenesis, amino acids, and

neurotransmitters in the brain and/or liver.24-27 One principal mecha-

nism of action proposed for antiepileptic drugs is their ability to cor-

rect disturbances in neurotransmitter concentrations.28,29

Recent years have focused on the role of oxidative stress in

seizures. Free radicals have been implicated in the development of

seizures, causing cellular dysfunction by attacking at the poly-

unsaturated sites of the biological membranes leading to lipid

peroxidation.8,9 Frantseva et al,8 using mice with amygdala con-

vulsion, showed increased oxygen radical production and lipid perox-

idation following a seizure, which was inhibited using antioxidants.

A study by Gupta YK, et al,9 also demonstrated increased oxidative

stress in PTZ induced kindling model in rats. Some antioxidants have

been shown to be effective in reducing the oxidative stress in the

models of epilepsy.30-32

In our study PTZ also produced oxidative stress and there was a

significant increase in the brain MDA levels (an indicator of the lipid

peroxidation due to free radical generation), reduction in GSH

(endogenous antioxidant) levels and decreased catalase activity as

compared to control. Both sodium valproate and bezafibrate re-

versed this increase in brain MDA levels and reduced glutathione lev-

els in addition to elevating brain catalase activity which was found to

be at par with the previous studies where bezafibrate showed anti-

oxidative properties.33,34 Possibly, the antioxidant property and free

radical scavenging potential of bezafibrate which preserved the anti-

oxidant enzyme catalase activity, consequently lead to reductive ef-

fect on seizure intensity and duration.

Studies by Cavazos JE, et al35 and Pitkanen et al36 have shown that

seizure activity is associated with neuronal damage and both ne-

crotic and apoptotic forms of cell death contribute to brain damage

in the PTZ-induced epilepsy model. Also, as per previous studies,

chronic treatment with sodium valproate after kainate-induced SE

prevented spontaneous recurrent seizures and reduced histological

lesions in the hippocampus.37,38 Bezafibrate also proves to be a

promising agent for the treatment of neurodegenerative diseases.39

In the present study, sodium valproate and bezafibrate main-

tained near normal morphology of the neurons while PTZ kindled

rats showed diffuse neuronal injury with most of the neurons demon-

strating nuclear chromatin clumping, hypereosinophilia, condensa-

tion of the cytoplasm and fragmentation of the cells.

Neuron specific enolase, a sensitive marker of neuronal damage

increases in animal models of traumatic and ischemic brain injury,

cerebral hypoxia, and epileptic seizures.40-45 Study by Wang et al,46

has demonstrated increased level of sNSE in PTZ kindled rats and pre

treatment with topiramate and folic acid decreased sNSE levels.46

Our data also showed that there is an increase in sNSE in vehicle and

saline treated groups and decrease in sodium valproate and bezafi-

brate pre treated groups, accompanied by histological evidence of

neuronal damage in the hippocampus. Thus we postulate that the in-

creased sNSE in vehicle and saline control groups may be a con-

sequence of neuronal injuries in the hippocampus and bezafibrate

decreased the sNSE by reduced the oxidative stress induced neuronal

injuries in the hippocampus. In order to investigate the extent of neu-

ronal cell damage caused by PTZ induced epileptic seizures, we car-

ried out nuclear DNA fragmentation studies. In a study, Zhand et al

demonstrated that in amygdala kindled seizures with increasing seiz-

ure severity, DNA fragmentation increases.47 In the present study all

the brain samples of the PTZ treated group showed laddering pattern

indicating DNA fragmentation. While the sodium valproate and be-

zafibrate groups did not show any major fragmentations.

These findings suggest, seizures cause an early production of oxi-

dative damage to DNA bases before significant DNA strand breaks

appear, indicating that reactive oxygen species may be a contributory

factor in the mechanism by which seizures cause cell death and beza-

fibrate by anti-oxidative activity prevent such DNA damage.

In many brain regions, there are expression of PPAR-α on the

neurons.48,49 PPAR-α is activated by both endogenous ligands

[N-acylethanolamines oleoylethanolamide (OEA) and palmitoyletha-

nolamide (PEA)]50 and synthatic ligands such as hypolipidemic

fibrates.51 In vitro and in vivo experiments have demonstrated that

nicotine-evoked excitation of dopamine neurons as well as nicotine

addictive properties in rats and monkeys, are suppressed by ligands

to the PPARα.51-54 Evidences reveal the non - transcriptional inter-

action between PPARα and nAChR, via phosphorylation52,53 and this

mechanism might account for the PPAR-α ligands induced blockade

of neuronal and behavioral responses to nicotine.51,52,54 Study by

Piligheddu M et al, demonstrated the antiepileptic effects of PPAR-α agonists in their study.16 In their study both the acute administration


www.kes.or.kr

of PPAR-α agonist WY 14643 (WY 80 mg/kg, i.p.) or chronic admin-

istration of fenofibrate in diet (0.2%) for 14 days significantly re-

duced or abolished behavioral and EEG expressions of nicotine-in-

duced seizures. Acute WY effects were reverted by the PPARα antag-

onist MK886 (3 mg/kg, i.p.). Another study by Citraro R et al, also

documented the antiepileptic action of PEA through CB1 and PPAR-

α activation in genetic model of absence epilepsy in rat.17 In this

study, the authors administered PEA, anandamide (AEA), a PPAR-α antagonist (GW6471) and a synthetic CB1 receptor antagonist/in-

verse agonist (SR141716) to WAG/Rij rats in order to evaluate the ef-

fects on epileptic spike-wave discharges (SWDs) on EEG recordings.

GW6471 antagonized PEA's effects whereas it did not modify AEA's

effects. Furthermore, the authors have also measured PEA, AEA and

2-AG (2-arachidonoylglycerol) brain levels identifying significant dif-

ferences between epileptic and control rats such as decreased PEA

levels in both thalamus and cortex that might contribute to absence

epilepsy. Therefore, these data demonstrate that PEA has anti-ab-

sence properties in the WAG/Rij rat model and that such properties

depend on PPAR-α and indirect activation of CB1 receptors.17

The underlying mechanisms of anticonvulsive effects of Bezafi-

brate such as PPAR-α activation, which in turn is responsible for acti-

vation of several pathways and other mechanisms, should be

investigated. Further studies are needed to explore its roles. If our

data can be confirmed by clinical trials, the management of dyslipide-

mia may be modified, in particular for epileptic patients.

To conclude these findings may provide insights into the under-

standing of the mechanism of bezafibrate as an anti kindling agent

and could offer a useful support to the basic antiepileptic therapy in

preventing the development of PTZ induced seizures, suggesting its

potential for therapeutic applications in temporal lobe epilepsy.

Conflict of Interest

None of the authors have any conflict of interest to disclose.

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