research article the effects of quinacrine, proglumide...

Research ArticleThe Effects of Quinacrine Proglumide and Pentoxifyllineon Seizure Activity Cognitive Deficit and Oxidative Stress inRat Lithium-Pilocarpine Model of Status Epilepticus

Mohammad Ahmad1 Gasem M Abu-Taweel2

Ahmad E Aboshaiqah3 and Jamaan S Ajarem4

1 Department of Medical Surgical Nursing College of Nursing King Saud University PO Box 642 Riyadh 11421 Saudi Arabia2Department of Basic Sciences College of Education Dammam University PO Box 2375 Dammam 31451 Saudi Arabia3 Department of Nursing Administration and Education College of Nursing King Saud University PO Box 642Riyadh 11421 Saudi Arabia

4Department of Zoology College of Science King Saud University PO Box 2455 Riyadh 11451 Saudi Arabia

Correspondence should be addressed to Mohammad Ahmad mbadshahksuedusa

Received 26 July 2014 Revised 5 October 2014 Accepted 19 October 2014 Published 16 November 2014

Academic Editor Vladimir Jakovljevic

Copyright copy 2014 Mohammad Ahmad et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present data indicate that status epilepticus (SE) induced in adult rats is associated with cognitive dysfunctions and cerebraloxidative stress (OS)This has been demonstrated using lithium-pilocarpine (Li-Pc)model of SEOS occurring in hippocampus andstriatum of mature brain following SE is apparently due to both the increased free radicals production and the limited antioxidantdefense Pronounced alterations were noticed in the enzymatic glutathione-S transferase (GST) catalase (CAT) and superoxidedismutase (SOD) as well as in the nonenzymatic thiobarbituric acid (TBARS) and reduced glutathione (GST) indices of OS in thehippocampus and striatum of SE induced animals Quinacrine (Qcn) proglumide (Pgm) and pentoxifylline (Ptx) administered toanimals before inducing SE were significantly effective in ameliorating the seizure activities cognitive dysfunctions and cerebralOS The findings suggest that all the drugs were effective in the order of Ptx lt Pgm lt Qcn indicating that these drugs arepotentially antiepileptic as well as antioxidant however further studies are needed to establish this fact It can be assumed thatthese antiepileptic substances with antioxidant properties combined with conventional therapies might provide a beneficial effectin treatment of epilepsy through ameliorating the cerebral OS

1 Introduction

Status epilepticus (SE) is an emergency neurological con-dition where recurrent generalized seizures last for morethan 30 minutes and if not controlled neuronal injury occurs[1 2] Besides neurobehavioral deficits SE is preferentiallyassociated with a wide range of neurochemical imbalance insome areas of the brain [3ndash6] Such neuronal hyperactivityandor excitotoxicity have been associated with excessivegeneration of free radicals [7 8] particularly in the brainwhich contains large quantities of oxidizable lipids andmetals and moreover the brain has fewer mechanisms of

antioxidation than other tissues [9] Oxidative stress which isgenerally defined as the overproduction of free radicals candramatically alter neuronal function and has been linked tothe pathogenesis of epilepsy [8 10ndash12] There exist variousendogenous antioxidant defense mechanisms both enzy-matic and nonenzymatic which within certain limits cancounteract increased ROS production

Several neurochemical studies in animal models haverevealed that oxidative stress-related seizures producechanges in antioxidant enzymatic activity and receptorbinding [8 13 14] The pathological process and underlyingmechanisms involved in the oxidative stress during SE are still

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 630509 11 pageshttpdxdoiorg1011552014630509

2 Oxidative Medicine and Cellular Longevity

far from clear There is ample evidence to suggest that braintissues are highly vulnerable to the oxidative stress [15] Reac-tive oxygen species (ROS) can dramatically affect the struc-ture and function of neurons and recently Freitas et al [15]suggested an evidence based role of ROS in the pathophys-iology of SE Importantly epilepsy induced by pilocarpine inrodent models can provide information regarding oxidativestress-related epileptic activity [3 4 16ndash18] Besides epilepticseizure activities oxidative stress has also been related withcognitive impairment [19ndash22] Furthermore in such animalmodels pharmacological approaches designed to reduce theoxidative stress afford significant neuroprotection attestingto the critical role that oxidative stress plays in a diversearray of neurological diseases and disorders Inhibition of theneurochemical pathways responsible for neuronal excitabilityand behavioral changes may lead to the development of newtherapeutic agents for the treatment of SE

The anticonvulsant effect of several agents having antiox-idant property such as curcumin vineatrol transresveratrolmelatonin adenosine alpha lipoic acid pentoxifylline bus-pirone aloe vera and metformin has been demonstrated invarious studies [2 6 23ndash28]

Proglumide (Pgm) is a known cholecystokinin (CCK)antagonist [29] and changes CCK level and receptor popu-lation of CCK has been associated with SE [30] Quinacrine(Qcn) is a potent phospholipase A2 (PLA2) inhibitor [31] andhas been shown to exert neuroprotective activity in ischemicbrain [32] PLA2 are a family of ubiquitous enzymes thatdegrade membrane phospholipids and produce a variety oflipid mediators to regulate neuronal functions [32] PLA2has also been shown to have a direct role in regulation ofneurotransmitters and electrolytes in normal and reducedOS-mediated cellular toxicity Pentoxifylline (Ptx) has alsobeen shown to have neuroprotective effects against a host ofneurobehavioral disorders including ischemic brain injuryneurotrauma dementia stroke improved cognitive effectfollowing hippocampal lesions and ameliorated effects onlithium-pilocarpine induced SE in young rats [6 33ndash35]

The pilocarpine- (Pc-) induced seizure in rodents isgenerally considered as one of themost suitable experimentalmodels that has been frequently used to study the patho-physiology and management strategies of SE [3 8 36] Dueto high mortality of animals in pilocarpine (alone) model[37] a combination of lithium chloride and pilocarpine (Li-Pc) model is often preferred Lithium (Li) potentiates theepileptogenic action of Pc but at the same time significantlyreduces the mortality of animals [38 39] The Li-Pc model ofSE reproducesmost clinical temporal andneuropathologicalfeatures of SE [3 6 40 41]

The present study was designed to study the comparativeeffects of Pgm Qcn and Ptx on epileptic seizure activitiescognitive tests and oxidative stress in the brain regions of ratsin which SE was induced by lithium-pilocarpine Our primeobjective was to assess if the oxidative stress observations inthe SE animals can be of any help in identifying effectiveantiepileptic drugs

2 Materials and Methods

21 Experimental Animals The animals used in the presentstudy were adult male Sprague Dawley rats (weighing 200ndash250 g 2 months old) and were housed under controlledconditions with 12 hours light-dark diurnal cycle at 22 plusmn1∘C with humidity at 50ndash60 and with free access tofood and water except during experimental handlings Allstudy protocols and animal handling procedures were inaccordance with and approved by the Research and EthicsCommittee of King Saud University Riyadh Saudi ArabiaThe prior permission was obtained from this committee forexecuting the experiments

22 Induction of SE Animals were randomly assigned intothirteen groups The animals in groups 1 2 and 3 servedas controls and received saline Li (3mEqmLkg ip) andPc alone (20mgmLkg sc) respectively SE was induced ingroups 4 to 13 by administering an aqueous (saline) solutionof Li (BDH Laboratory Supplies Poole England in a doseas in control) followed by (20 h later) Pc (Sigma ChemicalCo St Louis MO USA in the dose as used for control)Group 4 served as the experimental control of SE group andgroups 5 to 13 served as the drug test groups Qcn Pgmand Ptx (Sigma USA) were dissolved in saline and wereadministered at doses of 5 15 and 30mgmLkg ip 250500 and 750mgmLkg ip and 20 40 and 60mgmLkg ip(one hour before Pc injection) to groups 5 to 13 respectivelyGroups receiving Qcn Pgm and Ptx alone in the highestdose (30 750 and 60mgkg resp) served as the controls forthe drugs used in this study However the results of thesedrug alone controls are not included in the results of thisstudy since these drugs administered alone in the animals didnot show any abnormal behavioral activities and resembledthe untreated controls Furthermore even all investigatedparameters in this study for the drug alone groups werealmost similar as the naıve control groups Thus none ofthe results for the drug alone treated groups are includedin the results of this study in order to avoid unnecessaryconfusion and crowding in the numbers of bars shown in theresult figures After Pc injections the animals (119899 = 10 pergroup)were observed for a sequence of behavioral alterationsincluding peripheral cholinergic signs (PCS) stereotypedmovements (STM) clonic movements of forelimbs headbobbing tremors and seizures which developed progres-sively within 1-2 h into SE [42] For comparative observationson the drug effects all seizure activities were presented aslatency to develop seizure and SE The seizure behaviorconsists of head bobbing with intermittent forelimb and hindlimb clonus hyperextension of tails loss of posture fallingback and myoclonic jerks whereas SE is a condition wherethese recurrent generalized seizures last for more than 30minutes in the animalsMortality (if any) within 24 hwas alsorecorded

221 Morris Water-Maze Test After induction of SE forseizure activities analyses the animals (119899 = 6 to 8 per group)were subjected to cognitive test Animals were allowed to

Oxidative Medicine and Cellular Longevity 3

acclimatize to the testing room for 2 h before testing All testswere performed between 1000 and 1500 hours of the lightedphase

The test has been extensively used to assess cognitivefunctions in a variety of epilepsy models [43 44] The ratswere tested for visual-spatial memory using a water-maze[45]The water-maze consisted of a galvanized white circularwater tank (117 cm diameter 55 cm height) filled with cleartap water (26 plusmn 1∘C) to a depth of 30 cm A 10 cm diameterstainless steel white escape platform was placed 1 cm belowthe water level and the water was made opaque by addition of1 liter of milk which prevented visualization of the platformFour points on the rim of the tank were designated north (N)south (S) east (E) and west (W) thus dividing the pool intofour quadrants (NW NE SE and SW)

On the first day each rat was allowed to swim freely inthe pool for 60 sec without the platform present in the poolThis free swim enabled the rat to become habituated to thetraining environment On days 2ndash5 rats were trained for 24trials (six trials a day with an intertrial interval of 30 sec) tolocate and escape onto the submerged platform At the startof each trial the rat was held facing the perimeter of thewater tank and dropped into the pool to ensure immersionThe latency from immersion into the pool to escape ontothe hidden platform (maximum trial duration 120 sec) wasrecorded On mounting the platform each rat was givena 30 sec intertrial interval for rest and for learning andmemorizing the spatial cues to reach the platform for escapeThe testing procedures used during the four days of locatingthe hidden platform provide a measure of hippocampal-dependent spatial reference memory [46]

23 Biochemical Studies Based on our pilot studies andliterature survey [5 47 48] the biochemical studies wereundertaken 1 h after Li-Pc treatment and immediately afterkilling the rest of the animals by decapitation their brainswere dissected on ice and cerebral areas (hippocampus andstriatum) were removed and frozen in liquid nitrogen andstored at minus70∘C for determination of some nonenzymatic andenzymatic oxidative stress indices

24 Determination of Nonenzymatic OS Indices

241 Lipid Peroxides Lipid peroxides (LP) in striatum andhippocampus were determined spectrophotometrically asthiobarbituric acid-reactive substances (TBARS) accordingto the method of Ohkawa et al [49] Tissue lipid peroxidelevels were quantified using extinction coefficient of 156 times105mminus1 cmminus1 and expressed as nanomoles of TBARS formed

per g tissue weight The results are expressed as nmolg wetweight

242 Glutathione Reduced glutathione (GSH) level in stria-tum and hippocampus was measured enzymatically in thebrain tissues by a slightly modified method [50] The slopeof the change in absorbance was used to quantitate total GSHby comparing the slope of the samples with a standard curve

prepared with pure glutathione (Sigma) The specific activityis expressed into umolg tissue weight

25 Determination of Enzymatic OS Indices

251 Glutathione-S-Transferase Glutathione S-transferase(GST) was estimated by the method of Habig et al [51]by using 1-chloro-24-dinitrochlorobenzene (CDNB) as sub-strate at 340 nm The GST activity is expressed as Ug tissueweight

252 Catalase Catalase (CAT) activity was measured bythe method of Aebi [52] by tracking the decomposition ofhydrogen peroxide by measuring decrease in extinction ofH2O2

at 240 nm The activity of CAT is expressed as rateconstant of first order reaction K per gram tissue weight

253 Superoxide Dismutase Superoxide dismutase (SOD)activity was estimated by the method of Misra and Fridovich[53] Activity is expressed as the amount of enzyme thatinhibits the oxidation of epinephrine by 50 which is equalto U per gram tissue weight

26 Statistical Analysis The data were analyzed by Bartlettrsquostest for equal variance and by Gaussian-shaped distributionfor normality using the Kolmogorov-Smirnov goodness-of-fit test As the data passed the normality test (119875 gt 010) groupmeans were compared with one-way ANOVA with post hoctesting using Tukey-Kramer Multiple Comparisons Test orStudent-Newman-Keuls Multiple Comparisons Tests Differ-ences in seizure and SE incidences and mortality were testedby Student-Newman-Keuls Multiple Comparisons Tests Allresults were expressed as means plusmn SEM and the significancewas defined as 119875 lt 005 for all tests

3 Results

31 Features of Li-Pc Induced SE Within 5min after injectionof Pc all animals started developing a gradual and significantchange in behavior including PCS (miosis piloerectiondiarrohea mild tremors scratching and salivation) and STM(sniffing paw licking and rearing) followed by seizures in100 of the animals with a mean latency of 962 plusmn 12minto develop seizure (Table 1)The convulsive episode consistedof head bobbing with intermittent forelimb and hind limbclonus hyperextension of tails loss of posture falling backandmyoclonic jerks building up to SE in 100of animalsThemean latency to onset of SE was 2386 plusmn 154min (Table 1)and on average the SE lasted for more than one hour Atotal of 10 mortality were observed over a period of 24 hfollowing Pc injections (Table 1)

32 Effect of Drugs Pretreatment on Li-Pc Induced SE PgmQcn and Ptx dose-dependently and significantly increasedthe latencies to seizure and SE and decreased the percentagesof seizures and SE (Table 1) and also reduced the intensityand frequency of seizure PCS and STM episodes (not shown


Table1Dose-depend

entantiepilepticactiv

ityof

pentoxify

lline

(Ptx)proglumide(Pg

m)andqu

inacrin

e(Qcn)a

gainstLi-Pcind

uced

statuse

pilepticus

(SE)

inadultrats

Behavioralparameterso

bserved

Con

trol

Ptx(m

gkg)

Pgm

(mgkg)

Qcn

(mgkg)

2040

60250

500

750

515

30Latencyto

seizures

(min)

962plusmn

120

1459n

splusmn14

22462aplusmn244

3815

aplusmn18

01246n

splusmn117

2247aplusmn15

93533

aplusmn16

41021n

splusmn14

21938aplusmn261

3172aplusmn15

3Seizures

()

100

850

550

a100

a852

613

a213

a874

665

a298

a

Latencyto

SE(m

in)

2386plusmn

154

3461aplusmn086

4870aplusmn096

NoSE

3252

aplusmn10

744

61aplusmn12

25611

aplusmn263

2962aplusmn16

140

33aplusmn257

588

aplusmn19

1SE

()

100

500

a200

a0a

614

a368

a235

a683

a392

a254

a

Mortality(

)with

in24

hours

100

00

00

00

00

Animalsw

ereo

bservedform

orethan1h

after

Li-Pc(lithium

-pilo

carpine)

injections

forind

ucingSE

inallgroup

sandallanimalsw

ereo

bservedform

orethan24

hoursfor

mortality

nsStatisticallyno

nsignificant

a 119875lt0001as

comparedto

control(0m

gkg)b

yANOVA

follo

wed

byStud

ent-N

ewman-K

eulsMultip

leCom

paris

onsT

est


1 20

50

100

150

Test days

Mea

n lat

ency

to re

ach

the p

latfo

rm

lowastlowastlowastlowastlowastlowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

ControlLiPiloLi-Pilo (SE)

Ptx-1Ptx-2Ptx-3Pgm-1

Pgm-2Pgm-3Qcn-1Qcn-2Qcn-3

(a)

Test days3 4

0

50

100

150

Mea

n lat

ency

to re

ach

the p

latfo

rm

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




(b)

Figure 1 Performance in water-maze of animals that experienced SE along with their controls as well as the SE groups that were pretreatedwith Ptx Qcn and PgmMean latency to reach the hidden platform in secondsplusmn SEM (119910-axis) on each testing day (119909-axis) shows that animalssubjected to SE were slower in finding the platform (cognitive effect) than the controls on all four testing days Treatment with Ptx Qcn andPgm in varying doses was effective in improving the cognitive function in the order Ptx lt Pgm lt Qcn and dose-dependently Li = lithiumchloride Pilo = pilocarpine SE = status epilepticus doses of drugs in mgkg body weight Ptx-1 Ptx-2 and Ptx-3 = 20 40 and 60 Pgm-1Pgm2 and Pgm3 = 250 500 and 750 and Qcn-1 Qcn-2 and Qcn-3 = 5 15 and 30 respectively represents significance as compared tocontrol (119875 lt 0001) whereas lowast lowastlowast and lowast lowast lowast show 119875 lt 001 119875 lt 005 and 119875 lt 0001 respectively as compared to SE group by one-wayANOVA

in Table 1) The severity of SE was significantly and dose-dependently reduced and latency to attain SE was increasedin the drug treated animals The drugs were effective in theorder Ptx gt Pgm gt Qcn The highest dose (60mgkg) of Ptxcompletely abolished SE (Table 1) Furthermore nomortalitywas observed in the rats pretreated with all the three drugsas compared to 10 mortality in the Li-Pc treated group(Table 1) The control groups that received Li or the drugsalone did not show any signs of seizure or SE

33 Morris Water-Maze Test Rats with Li-Pc treatmentexhibited longer escape latencies to reach the platform ascompared with control group (119875 lt 001 Figure 1) howeverall groups displayed gradual improvement in performanceover the 4 days of testing (training) period The number ofsuccessful animals to reach the platform was significantlyhigher in the drug pretreated groups as compared to Li-Pc(SE) group on all the four testing days but in the order Ptx gtPgm gt Qcn (119875 lt 0001 Figure 1)

34 Biochemical Studies

341 Nonenzymatic OS Indices

TBARS in Hippocampus and Striatum The lipid peroxida-tion level (TBARS) in the hippocampus and striatum wasmarkedly (119875 lt 0001) increased after 1 h of Li-PC (SE)treatment as compared to the control group (Figure 2(a))

Pretreatment with drugs significantly (119875 lt 0001) and dose-dependently attenuated Li-Pc induced increase in TBARS inthe hippocampus and striatum in the order Ptx gt Pgm gtQcn(Figure 2(a)) as compared to Li-Pc (SE) group

GSH in Hippocampus and Striatum A highly significant(119875 lt 0001) depletion of hippocampal and striatal GSHwas observed in Li-Pc (SE) group (Figure 2(b)) Pretreatmentwith drugs significantly and dose-dependently attenuatedthis depletion of GSH in the hippocampus and striatum inthe order Ptx gt Pgm gt Qcn (Figure 2(b)) as compared to Li-Pc (SE) group

342 Enzymatic OS Indices

GST in Hippocampus and Striatum A highly significant (119875 lt0001) depletion of hippocampal (Figure 3(a)) and striatal(Figure 3(b)) GST was also observed in Li-Pc (SE) groupPretreatment with drugs significantly and dose-dependentlyattenuated this depletion of GST in the hippocampus andstriatum in the order PtxgtPgmgtQcn (Figures 3(a) and 3(b))as compared to Li-Pc (SE) group

CAT in Hippocampus and Striatum The CAT level in thehippocampus (Figure 3(c)) and striatum (Figure 3(e)) wasmarkedly (119875 lt 0001) increased after 1 h of Li-PC (SE)treatment as compared to the control group (Figures 3(c) and3(d)) Pretreatment with drugs significantly (119875 lt 0001) and


0

5

10

15

0

50

100

150

lowastlowastlowast lowastlowastlowast lowastlowast


Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

(a)

0

100

200

300

400

500

0

250

500

750

1000



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




GSH

leve

l as n

mol

gtis

sue w

tplusmnSE

MG

SH le

vel a

s nm

olg

tissu

e wtplusmn

SEM

(b)

Figure 2 Comparative effect of Ptx Pgm and Qcn on the nonenzymatic oxidative stress indices like (a) lipid peroxidation content (TBARS)and (b) total glutathione level (GSH) in hippocampus and striatum of rats after Li-Pc induced SE The comparative effects are in the orderPtx lt Pgm lt Qcn and dose-dependent Abbreviations and statistical significance are the same as in Figure 1

dose-dependently attenuated Li-Pc induced increase in CATin the hippocampus and striatum in the order Ptx gt Pgm gtQcn (Figures 3(c) and 3(d)) as compared to Li-Pc (SE) group

SOD in Hippocampus and Striatum The SOD level in thehippocampus (Figure 3(e)) and striatum (Figure 3(f)) wassignificantly (119875 lt 0001) decreased after 1 h of Li-PC (SE)treatment as compared to the control group Pretreatmentwith drugs significantly (119875 lt 0001) and dose-dependentlyattenuated Li-Pc induced decrease in SOD in the hippocam-pus and striatum in the order Ptx gt Pgm gtQcn (Figures 3(e)and 3(f)) as compared to Li-Pc (SE) group

4 Discussion

The present findings indicate that Pc administration to ratspretreated with Li initiated cholinergic symptoms includingmiosis piloerection diarrhea and mild tremors followed byseizures SE developed between 20 to 30 minutes after Pc

administration which consisted of head bobbing intermit-tent forelimb and hind limb clonus and hyperextension oftail and hind limb along with loss of posture No mortalitywas found in any of the drug treated groups as compared to10 mortality in the Li-Pc (SE) group The present Morriswater-maze results showed that rats with SE took longertime to reach escape platform spent lesser time in the targetquadrant or completely failed to reach the platform clearlysuggesting impaired visual-spatial memory and cognitivedeficit Cognitive dysfunctionswere also evident from the factthat the animals swam along with the wall of the test tank andrarely tried to find the escape platform in the target quadrantImpairment of learning andmemory in rats with SE has beenreported by several investigators [6 54 55]The specific causeof motor and cognitive deterioration following SE is far fromclear However according to recent reports neurochemicalimbalance and alteration of neuronal structure following SEmight be responsible for neurobehavioral changes [39 47]Cha et al [56] reported that hippocampus receives dense


0

25

50

75

100 G

ST (U

g ti

ssue

wt)

Hippocampus1 2 3 1 2 3 1 2 3


(a)

0

3

6

9

12

GST

(Ug

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(b)

0

25

50

75

CAT

(kg

wet

tiss

ue w

t)

Hippocampus1 2 3 1 2 3 1 2 3


(c)

0

10

20

30

40

CAT

(kg

wet

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3

lowastlowastlowast lowastlowastlowastlowast

(d)

0

20

40

60

80

Hippocampus1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(e)

0

15

30

45

60

Striatum1 2 3 1 2 3 1 2 3

lowastlowastlowastlowastlowastlowast lowastlowast

SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(f)

Figure 3 Comparative effect of Ptx Pgm and Qcn on the enzymatic oxidative stress indices like glutathione-S-transferase level (GST) inhippocampus (a) and striatum (b) catalase (CAT) activity in hippocampus (c) and striatum (d) and superoxide dismutase (SOD) activity inhippocampus (e) and striatum (f) of rats after Li-Pc induced SE The comparative and dose-dependent effects are in the order Ptx lt Pgm ltQcn Abbreviations and statistical significance are the same as in Figure 1


cholinergic projections and overactivation of these afferentsby Pc may directly produce neuronal hyperexcitation andtrigger seizure activity accompanied by alterations in neu-ronal plasticity within the hippocampal circuitry causingimpairment of learning and memory in rats [44 57 58]

The results of the present study clearly showed theantiepileptic activity of all three drugs (Pgm Qcn andPtx) against Li-Pc induced seizure as revealed by highlysignificant decrease in frequency of epileptic episodes andincrease in the latency to SE The mortality was absent in allthe three drug treated groups and the comparative efficacyof the tested drugs was in the order Ptx gt Pgm gt Qcn(Table 1) Furthermore our study consistently demonstratedthat pharmacological intervention using Pgm Ptx and Qcnsignificantly and dose-dependently attenuated SE inducedimpaired memory and the comparative efficacy of the testeddrugs was in the same order Ptx gt Pgm gt Qcn throughout

The present biochemical studies indicated a significantand dose-dependent increase in TBARS and CAT (Figures2(a) and 2(b)) and decrease in GSH GST and SOD levels(Figures 3(a)ndash3(f)) in the hippocampus and striatum of therats treated with Li-Pc clearly suggesting a high level ofenzymatic (GST SOD and CAT) and nonenzymatic (TBARSand GSH) oxidative stress in these brain regions It is widelyaccepted that tissue injury is dependent not only on thenature of offending pathogen but also on the quality of hostdefense system including the type and levels of antioxidantsOxidative stress is particularly facilitated in brain since theneuronal cells contain large quantities of oxidizable lipidsand metals and the antioxidant defense mechanism in brainis relatively weaker as compared to other organs [8] Thedrugs used in the present study significantly and dose-dependently attenuated Li-Pc induced OS in hippocampusand striatum but in the order Ptx gt Pgm gt Qcn (Figures 2and 3) Earlier it has been reported [6] that Ptx may exertits pharmacological effects by several mechanisms includinginhibition of phosphodiesterase enzymes (PDEs) leadingto increases in cyclic adenosine monophosphate (cAMP)blockade of adenosine receptors translocation of extracellu-lar calcium an inhibitory effect on inflammatorymechanismand free radical scavenging activity The role of cAMP in theetiopathology of epileptic seizures has been widely studied[59 60] Besides its definite association with seizure activitycAMP also plays a key role in biochemical regulation ofthe cognitive process of memory consolidation [61] Earlierstudies have also revealed that OS-related seizures producechanges in antioxidant enzyme activities [6 8 13 14] andstill other studies have related Pc-induced epileptic activitywith the disturbance in OS activities [3 4 16] Neuronalhyperexcitability and excessive production of free radicalshave been implicated in the pathogenesis of a considerablerange of neurological disorders including epilepsy [62ndash64]Lipid peroxidation in a tissue is an index of irreversiblebiological damage of the cell membrane phospholipid whichin turn leads to inhibition ofmost of the sulphydryl and somenonsulphydryl enzymes [26 65] Lipid peroxidation can beinduced by many chemicals like kainic acid Pc and tissueinjuries and it has been suggested for a possible mechanismfor the neurotoxic effects observed during epileptic activity

[5 26]The present findings clearly indicated that lipid perox-idation levels (TBARS) in the striatum and hippocampus ofrats were increased and the reduced glutathione (GSH) con-centrationwas decreased after seizure activities of SE inducedby Li-Pc In normal conditions there is a steady state balancebetween the ROS production (TBARS) and their scavengingby the cellular antioxidant system (GSH) Presence of Pc maybe associated with marked alterations of enzymatic (SODCAT and GST) and nonenzymatic components (TBARS andGSH) of antioxidant defense system (AOS) The comparativestudy of the three drugs (Qcn Pgm and Ptx) demonstratesthat these drugs act as effective antioxidants and effectivelyprotect against Pc-induced lipid peroxidation and amelioratethe negative effect of Pc on antioxidant status by protectingthe effects from oxidative stress and have an amelioratingeffect on the AOS Thus it is likely that this pathomechanismmay contribute at least in part to the pathophysiology ofthe seizure activity Antioxidant therapies have been of greatinterest in the treatment of neurodegenerative disorders[66] Among the three drugs studied herein Ptx is foundto have a 100 survival rate in the SE animals withoutany mortality and has the highest neuroprotection from SEin the cognitive behavioral and biochemical parameters asobserved herein and as reported in an earlier study [6] Ptxhas been reported as a potent free radical scavenger [67]and the neuroprotective effect of Ptx has been attributedto its antioxidant activity by earlier investigators [34 35]The present study clearly suggests that Ptx Qcn and Pgmare potent free radical scavengers and have antiepileptic aswell as antioxidant activity in the order Ptx gt Pgm gt QcnAntioxidants and free radical scavengers have been shown toreduce neuropathology associated with SE [6]

5 Conclusion

In conclusion the present study suggests that all the drugsused in the present study have potential for being antiepilep-tic as well as antioxidant in an effective order of Ptx lt Pgmlt Qcn However further studies can confirm these effectsand also can indicate whether OS plays a definite role inthe pathophysiology of installation andor propagation ofepileptic seizures It is however assumed that Ptx Pgm andQcn combined with conventional therapies might providea beneficial effect in the treatment of epilepsy throughameliorating the cerebral OS

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud UniversityRiyadh SaudiArabia for funding of this research through theResearch Group Project no RGP-VPP-240


References

[1] S Chen S Fujita N Koshikawa and M Kobayashi ldquoPilo-carpine-induced status epilepticus causes acute interneuronloss and hyper-excitatory propagation in rat insular cortexrdquoNeuroscience vol 166 no 1 pp 341ndash353 2010

[2] M Ahmad ldquoProtective effects of curcumin against lithium-pilocarpine induced status epilepticus cognitive dysfunctionand oxidative stress in young ratsrdquo Saudi Journal of BiologicalSciences vol 20 no 2 pp 155ndash162 2013

[3] E A Cavalheiro M J Fernandes L Turski and M G Naffah-Mazzacoratti ldquoSpontaneous recurrent seizures in rats aminoacid and monoamine determination in the hippocampusrdquoEpilepsia vol 35 no 1 pp 1ndash11 1994

[4] R M de Freitas F C F de Sousa S M M Vasconcelos G SB Viana and M M D F Fonteles ldquoAcute alterations of neu-rotransmitters levels in striatum of young rat after pilocarpine-induced status epilepticusrdquo Arquivos de Neuro-Psiquiatria vol61 no 2 pp 430ndash433 2003

[5] RM Freitas SMMVasconcelos F C F Souza G S B Vianaand M M F Fonteles ldquoMonoamine levels after pilocarpine-induced status epilepticus in hippocampus and frontal cortex ofWistar ratsrdquoNeuroscience Letters vol 370 no 2-3 pp 196ndash2002004

[6] M Tariq M Ahmad K A Moutaery and S A DeebldquoPentoxifylline ameliorates lithium-pilocarpine induced statusepilepticus in young ratsrdquo Epilepsy and Behavior vol 12 no 3pp 354ndash365 2008

[7] A J Bruce and M Baudry ldquoOxygen free radicals in rat limbicstructures after kainate-induced seizuresrdquo Free Radical Biologyand Medicine vol 18 no 6 pp 993ndash1002 1995

[8] RM Freitas SMMVasconcelos F C F Souza G S B Vianaand M M F Fonteles ldquoOxidative stress in the hippocampusafter pilocarpine-induced status epilepticus in Wistar ratsrdquoFEBS Journal vol 272 no 6 pp 1307ndash1312 2005

[9] M I Bellissimo D Amado D S P Abdalla E C Ferreira EA Cavalheiro and M Da Graca Naffah-Mazzacoratti ldquoSuper-oxide dismutase glutathione peroxidase activities and thehydroperoxide concentration are modified in the hippocampusof epileptic ratsrdquo Epilepsy Research vol 46 no 2 pp 121ndash1282001

[10] J Liu A Wang L Li Y Huang P Xue and A Hao ldquoOxidativestress mediates hippocampal neuron death in rats after lithium-pilocarpine-induced status epilepticusrdquo Seizure vol 19 no 3pp 165ndash172 2010

[11] C Ikonomidou and A M Kaindl ldquoNeuronal death and oxida-tive stress in the developing brainrdquo Antioxidants and RedoxSignaling vol 14 no 8 pp 1535ndash1550 2011

[12] J Folbergrova and W S Kunz ldquoMitochondrial dysfunction inepilepsyrdquoMitochondrion vol 12 no 1 pp 35ndash40 2012

[13] S M Xavier C O Barbosa D O Barros R F Silva A AOliveira and R M Freitas ldquoVitamin C antioxidant effectsin hippocampus of adult Wistar rats after seizures and statusepilepticus induced by pilocarpinerdquo Neuroscience Letters vol420 no 1 pp 76ndash79 2007

[14] D O Barros S M L Xavier C O Barbosa et al ldquoEffects ofthe vitamin E in catalase activities in hippocampus after statusepilepticus induced by pilocarpine inWistar ratsrdquoNeuroscienceLetters vol 416 no 3 pp 227ndash230 2007

[15] R M Freitas ldquoInvestigation of oxidative stress involvementin hippocampus in epilepsy model induced by pilocarpinerdquoNeuroscience Letters vol 462 no 3 pp 225ndash229 2009

[16] H Shibley and B N Smith ldquoPilocarpine-induced status epilep-ticus results in mossy fiber sprouting and spontaneous seizuresin C57BL6 and CD-1micerdquo Epilepsy Research vol 49 no 2 pp109ndash120 2002

[17] H S Aboul Ezz Y A Khadrawy and N A Noor ldquoTheneuroprotective effect of curcumin and nigella sativa oil againstoxidative stress in the pilocarpine model of epilepsy a compar-ison with valproaterdquoNeurochemical Research vol 36 no 11 pp2195ndash2204 2011

[18] P DU H-Y Tang X Li et al ldquoAnticonvulsive and antioxidanteffects of curcumin on pilocarpine-induced seizures in ratsrdquoChinese Medical Journal vol 125 no 11 pp 1975ndash1979 2012

[19] K H Reeta J Mehla and Y K Gupta ldquoCurcumin is protectiveagainst phenytoin-induced cognitive impairment and oxidativestress in ratsrdquo Brain Research vol 1301 pp 52ndash60 2009

[20] K H Reeta J Mehla and Y K Gupta ldquoCurcumin amelioratescognitive dysfunction and oxidative damage in phenobarbitoneand carbamazepine administered ratsrdquo European Journal ofPharmacology vol 644 no 1ndash3 pp 106ndash112 2010

[21] K H Reeta J Mehla M Pahuja and Y K Gupta ldquoPhar-macokinetic and pharmacodynamic interactions of valproatephenytoin phenobarbitone and carbamazepine with curcuminin experimental models of epilepsy in ratsrdquo PharmacologyBiochemistry and Behavior vol 99 no 3 pp 399ndash407 2011

[22] A Ataie M Sabetkasaei A Haghparast A H Moghaddamand B Kazeminejad ldquoNeuroprotective effects of the polypheno-lic antioxidant agent curcumin against homocysteine-inducedcognitive impairment and oxidative stress in the ratrdquo Pharma-cology Biochemistry and Behavior vol 96 no 4 pp 378ndash3852010

[23] Y K Gupta and S Briyal ldquoProtective effect of vineatrolagainst kainic acid induced seizures oxidative stress and on theexpression of heat shock proteins in ratsrdquo European Neuropsy-chopharmacology vol 16 no 2 pp 85ndash91 2006

[24] Y K Gupta S Briyal and M Sharma ldquoProtective effect ofcurcumin against kainic acid induced seizures and oxidativestress in ratsrdquo Indian Journal of Physiology and Pharmacologyvol 53 no 1 pp 39ndash46 2009

[25] R L M de Freitas I M Santos G F de Souza A D RTome G B Saldanha and R M de Freitas ldquoOxidative stressin rat hippocampus caused by pilocarpine-induced seizures isreversed by buspironerdquo Brain Research Bulletin vol 81 no 4-5pp 505ndash509 2010

[26] J C Gilbert and A H Sawas ldquoATPase activities and lipidperoxidation in rat cerebral cortex synaptosomesrdquo ArchivesInternationales de Pharmacodynamie et deTherapie vol 263 no2 pp 189ndash196 1983

[27] N Rathor T Arora SManocha AN Patil P KMediratta andK K Sharma ldquoAnticonvulsant activity of Aloe vera leaf extractin acute and chronic models of epilepsy in micerdquo Journal ofPharmacy and Pharmacology vol 66 no 3 pp 477ndash485 2014

[28] R-R Zhao X-C Xu F Xu et al ldquoMetformin protects againstseizures learning and memory impairments and oxidativedamage induced by pentylenetetrazole-induced kindling inmicerdquo Biochemical and Biophysical Research Communicationsvol 448 no 4 pp 414ndash417 2014

[29] B S Bunney L A Chiodo and A S Freeman ldquoFurther studieson the specificity of proglumide as a selective cholecystokininantagonist in the central nervous systemrdquo Annals of the NewYork Academy of Sciences vol 448 pp 345ndash351 1985

[30] C Schwarzer J M Williamson E W Lothman A Vezzaniand G Sperk ldquoSomatostatin neuropeptide Y neurokinin B and


cholecystokinin immunoreactivity in two chronic models oftemporal lobe epilepsyrdquoNeuroscience vol 69 no 3 pp 831ndash8451995

[31] C Vigo G P Lewis and P J Piper ldquoMechanisms of inhibitionof phospholipase A2rdquo Biochemical Pharmacology vol 29 no 4pp 623ndash627 1980

[32] J W Phillis ldquoCerebroprotective action of the phospholipaseinhibitor quinacrine in the ischemiareperfused gerbil hip-pocampusrdquo Life Sciences vol 58 no 6 pp PL97ndashPL101 1996

[33] G M A Cunha P J P Bezerra M D D Saldanha M CCavalcante M S D de Brun and G S B Viana ldquoPentoxifyllineimproves learning and memory in glutamate-lesioned ratsrdquoPharmacology Biochemistry and Behavior vol 6 pp 687ndash6942000

[34] S Savas N Delibas C Savas R Sutcu and A Cindas ldquoPentox-ifylline reduces biochemical markers of ischemia-reperfusioninduced spinal cord injury in rabbitsrdquo Spinal Cord vol 40 no5 pp 224ndash229 2002

[35] C Banfi L Sironi G de Simoni et al ldquoPentoxifylline preventsspontaneous brain ischemia in stroke-prone ratsrdquo Journal ofPharmacology and ExperimentalTherapeutics vol 310 no 3 pp890ndash895 2004

[36] F Dal-Pizzol F Klamt M M R Vianna et al ldquoLipid perox-idation in hippocampus early and late after status epilepticusinduced by pilocarpine or kainic acid in Wistar ratsrdquo Neuro-science Letters vol 291 no 3 pp 179ndash182 2000

[37] W A Turski ldquoPilocarpine-induced seizures in rodentsmdash17years onrdquo Polish Journal of Pharmacology vol 52 no 1 pp 63ndash65 2000

[38] R Druga H Kubova L Suchomelova and R HaugvicovaldquoLithiumpilocarpine status epilepticus-induced neuropathol-ogy of piriform cortex and adjoining structures in rats is age-dependentrdquo Physiological Research vol 52 no 2 pp 251ndash2642003

[39] H Kubova P Mares L Suchomelova G Brozek R Drugaand A Pitkanen ldquoStatus epilepticus in immature rats leadsto behavioural and cognitive impairment and epileptogenesisrdquoEuropean Journal of Neuroscience vol 19 no 12 pp 3255ndash32652004

[40] L Turski C Ikonomidou W A Turski Z A Bortolottoand E A Cavalheiro ldquoReview cholinergic mechanisms andepileptogenesis The seizures induced by pilocarpine a novelexperimental model of intractable epilepsyrdquo Synapse vol 3 no2 pp 154ndash171 1989

[41] C Dube S Boyet C Marescaux and A Nehlig ldquoRelationshipbetweenneuronal loss and interictal glucosemetabolismduringthe chronic phase of the lithium-pilocarpine model of epilepsyin the immature and adult ratrdquoExperimental Neurology vol 167no 2 pp 227ndash241 2001

[42] M A Persinger Y R J Bureau M Kostakos O Perederyand H Falter ldquoBehaviors of rats with insidious multifocalbrain damage induced by seizures following single peripheralinjections of lithium and pilocarpinerdquo Physiology and Behaviorvol 53 no 5 pp 849ndash866 1993

[43] S Faverjon D C Silveira D D Fu et al ldquoBeneficial effects ofenriched environment following status epilepticus in immatureratsrdquo Neurology vol 59 no 9 pp 1356ndash1364 2002

[44] A RuttenM VanAlbada D C Silveira et al ldquoMemory impair-ment following status epilepticus in immature rats time-courseand environmental effectsrdquo European Journal of Neurosciencevol 16 no 3 pp 501ndash513 2002

[45] R Morris ldquoDevelopments of a water-maze procedure forstudying spatial learning in the ratrdquo Journal of NeuroscienceMethods vol 11 no 1 pp 47ndash60 1984

[46] H J Spiers N Burgess T Hartley F Vargha-Khadem and JOrsquoKeefe ldquoBilateral hippocampal pathology impairs topograph-ical and episodic memory but not visual pattern matchingrdquoHippocampus vol 11 no 6 pp 715ndash725 2001

[47] V S Nascimento A A Oliveira R M Freitas et alldquoPilocarpine-induced status epilepticus monoamine levelmuscarinic and dopaminergic receptors alterations in striatumof young ratsrdquoNeuroscience Letters vol 383 no 1-2 pp 165ndash1702005

[48] R M Freitas A D A Oliveira S M M Vasconcelos F C FSousa G S B Viana and M M F Fonteles ldquoExpression ofmuscarinic and dopaminergic receptors and monoamine levelsfrontal cortex of epileptic ratsrdquo Pharmacology Biochemistry andBehavior vol 83 no 2 pp 302ndash306 2006

[49] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[50] M J Mangino M K Murphy G G Grabau and C BAnderson ldquoProtective effects of glycine during hypothermicrenal ischemia-reperfusion injuryrdquo The American Journal ofPhysiologymdashRenal Fluid and Electrolyte Physiology vol 261 no5 pp F841ndashF848 1991

[51] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS-transferases The first enzymatic step in mercapturic acidformationrdquoThe Journal of Biological Chemistry vol 249 no 22pp 7130ndash7139 1974

[52] H Aebi ldquoCatalaserdquo in Methods of Enzymatic Analysis H UBergmeter Ed vol 2 Academic Press New York NY USA1972

[53] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquoThe Journal of Biological Chemistry vol 247 no 10pp 3170ndash3175 1972

[54] Z Liu C E Stafstrom M Sarkisian et al ldquoAge-dependenteffects of glutamate toxicity in the hippocampusrdquo Developmen-tal Brain Research vol 97 no 2 pp 178ndash184 1996

[55] C-L Wu L-T Huang C-W Liou et al ldquoLithium-pilocarpine-induced status epilepticus in immature rats result in long-term deficits in spatial learning and hippocampal cell lossrdquoNeuroscience Letters vol 312 no 2 pp 113ndash117 2001

[56] B H Cha D C Silveira X Liu Y Hu andG L Holmes ldquoEffectof topiramate following recurrent and prolonged seizures dur-ing early developmentrdquo Epilepsy Research vol 51 no 3 pp 217ndash232 2002

[57] P L Hung M C Lai S N Yang et al ldquoAminophyllineexacerbates status epilepticus-induced neuronal damages inimmature rats a morphological motor and behavioral studyrdquoEpilepsy Research vol 49 no 3 pp 218ndash225 2002

[58] R G M Morris E I Moser G Riedel et al ldquoElementsof a neurobiological theory of the hippocampus the role ofactivity-dependent synaptic plasticity inmemoryrdquoPhilosophicalTransactions of the Royal Society B Biological Sciences vol 358no 1432 pp 773ndash786 2003

[59] M Higashima K Ohno H Kinoshita and Y KoshinoldquoInvolvement of GABA(A) and GABA(B) receptors in afterdis-charge generation in rat hippocampal slicesrdquo Brain Researchvol 865 no 2 pp 186ndash193 2000

[60] J E Walker E Lewin J R Sheppard and R Cromwell ldquoEnzy-matic regulation of adenosine 3101584051015840 monophosphate (cyclic


AMP) in the freezing epileptogenic lesion of rat brain and inhomologous contralateral cortexrdquo Journal of Neurochemistryvol 21 no 1 pp 79ndash85 1973

[61] G M Rose A Hopper M de Vivo and A Tehim ldquoPhos-phodiesterase inhibitors for cognitive enhancementrdquo CurrentPharmaceutical Design vol 11 no 26 pp 3329ndash3334 2005

[62] M M F Marinho F C F de Sousa V M S de Bruin M RVale and G S B Viana ldquoEffects of lithium alone or associatedwith pilocarpine on muscarinic and dopaminergic receptorsand on phosphoinositide metabolism in rat hippocampus andstriatumrdquo Neurochemistry International vol 33 no 4 pp 299ndash306 1998

[63] M Patel ldquoMitochondrial dysfunction and oxidative stresscause and consequence of epileptic seizuresrdquo Free RadicalBiology and Medicine vol 37 no 12 pp 1951ndash1962 2004

[64] M T Lin and M F Beal ldquoMitochondrial dysfunction andoxidative stress in neurodegenerative diseasesrdquoNature vol 443no 7113 pp 787ndash795 2006

[65] M Hayashi ldquoOxidative stress in developmental brain disor-dersrdquo Neuropathology vol 29 no 1 pp 1ndash8 2009

[66] T R Golden andM Patel ldquoCatalytic antioxidants and neurode-generationrdquo Antioxidants and Redox Signaling vol 11 no 3 pp555ndash569 2009

[67] V B Bhat and K M Madyastha ldquoAntioxidant and radicalscavenging properties of 8-oxo derivatives of xanthine drugspentoxifylline and lisofyllinerdquo Biochemical and BiophysicalResearch Communications vol 288 no 5 pp 1212ndash1217 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


far from clear There is ample evidence to suggest that braintissues are highly vulnerable to the oxidative stress [15] Reac-tive oxygen species (ROS) can dramatically affect the struc-ture and function of neurons and recently Freitas et al [15]suggested an evidence based role of ROS in the pathophys-iology of SE Importantly epilepsy induced by pilocarpine inrodent models can provide information regarding oxidativestress-related epileptic activity [3 4 16ndash18] Besides epilepticseizure activities oxidative stress has also been related withcognitive impairment [19ndash22] Furthermore in such animalmodels pharmacological approaches designed to reduce theoxidative stress afford significant neuroprotection attestingto the critical role that oxidative stress plays in a diversearray of neurological diseases and disorders Inhibition of theneurochemical pathways responsible for neuronal excitabilityand behavioral changes may lead to the development of newtherapeutic agents for the treatment of SE

The anticonvulsant effect of several agents having antiox-idant property such as curcumin vineatrol transresveratrolmelatonin adenosine alpha lipoic acid pentoxifylline bus-pirone aloe vera and metformin has been demonstrated invarious studies [2 6 23ndash28]

Proglumide (Pgm) is a known cholecystokinin (CCK)antagonist [29] and changes CCK level and receptor popu-lation of CCK has been associated with SE [30] Quinacrine(Qcn) is a potent phospholipase A2 (PLA2) inhibitor [31] andhas been shown to exert neuroprotective activity in ischemicbrain [32] PLA2 are a family of ubiquitous enzymes thatdegrade membrane phospholipids and produce a variety oflipid mediators to regulate neuronal functions [32] PLA2has also been shown to have a direct role in regulation ofneurotransmitters and electrolytes in normal and reducedOS-mediated cellular toxicity Pentoxifylline (Ptx) has alsobeen shown to have neuroprotective effects against a host ofneurobehavioral disorders including ischemic brain injuryneurotrauma dementia stroke improved cognitive effectfollowing hippocampal lesions and ameliorated effects onlithium-pilocarpine induced SE in young rats [6 33ndash35]

The pilocarpine- (Pc-) induced seizure in rodents isgenerally considered as one of themost suitable experimentalmodels that has been frequently used to study the patho-physiology and management strategies of SE [3 8 36] Dueto high mortality of animals in pilocarpine (alone) model[37] a combination of lithium chloride and pilocarpine (Li-Pc) model is often preferred Lithium (Li) potentiates theepileptogenic action of Pc but at the same time significantlyreduces the mortality of animals [38 39] The Li-Pc model ofSE reproducesmost clinical temporal andneuropathologicalfeatures of SE [3 6 40 41]

The present study was designed to study the comparativeeffects of Pgm Qcn and Ptx on epileptic seizure activitiescognitive tests and oxidative stress in the brain regions of ratsin which SE was induced by lithium-pilocarpine Our primeobjective was to assess if the oxidative stress observations inthe SE animals can be of any help in identifying effectiveantiepileptic drugs

2 Materials and Methods

21 Experimental Animals The animals used in the presentstudy were adult male Sprague Dawley rats (weighing 200ndash250 g 2 months old) and were housed under controlledconditions with 12 hours light-dark diurnal cycle at 22 plusmn1∘C with humidity at 50ndash60 and with free access tofood and water except during experimental handlings Allstudy protocols and animal handling procedures were inaccordance with and approved by the Research and EthicsCommittee of King Saud University Riyadh Saudi ArabiaThe prior permission was obtained from this committee forexecuting the experiments

22 Induction of SE Animals were randomly assigned intothirteen groups The animals in groups 1 2 and 3 servedas controls and received saline Li (3mEqmLkg ip) andPc alone (20mgmLkg sc) respectively SE was induced ingroups 4 to 13 by administering an aqueous (saline) solutionof Li (BDH Laboratory Supplies Poole England in a doseas in control) followed by (20 h later) Pc (Sigma ChemicalCo St Louis MO USA in the dose as used for control)Group 4 served as the experimental control of SE group andgroups 5 to 13 served as the drug test groups Qcn Pgmand Ptx (Sigma USA) were dissolved in saline and wereadministered at doses of 5 15 and 30mgmLkg ip 250500 and 750mgmLkg ip and 20 40 and 60mgmLkg ip(one hour before Pc injection) to groups 5 to 13 respectivelyGroups receiving Qcn Pgm and Ptx alone in the highestdose (30 750 and 60mgkg resp) served as the controls forthe drugs used in this study However the results of thesedrug alone controls are not included in the results of thisstudy since these drugs administered alone in the animals didnot show any abnormal behavioral activities and resembledthe untreated controls Furthermore even all investigatedparameters in this study for the drug alone groups werealmost similar as the naıve control groups Thus none ofthe results for the drug alone treated groups are includedin the results of this study in order to avoid unnecessaryconfusion and crowding in the numbers of bars shown in theresult figures After Pc injections the animals (119899 = 10 pergroup)were observed for a sequence of behavioral alterationsincluding peripheral cholinergic signs (PCS) stereotypedmovements (STM) clonic movements of forelimbs headbobbing tremors and seizures which developed progres-sively within 1-2 h into SE [42] For comparative observationson the drug effects all seizure activities were presented aslatency to develop seizure and SE The seizure behaviorconsists of head bobbing with intermittent forelimb and hindlimb clonus hyperextension of tails loss of posture fallingback and myoclonic jerks whereas SE is a condition wherethese recurrent generalized seizures last for more than 30minutes in the animalsMortality (if any) within 24 hwas alsorecorded

221 Morris Water-Maze Test After induction of SE forseizure activities analyses the animals (119899 = 6 to 8 per group)were subjected to cognitive test Animals were allowed to

















3 Results




Table1Dose-depend


ityof

pentoxify

lline

(Ptx)proglumide(Pg

m)andqu

inacrin

e(Qcn)a

gainstLi-Pcind

uced

statuse

pilepticus

(SE)

inadultrats


bserved

Con

trol

Ptx(m

gkg)

Pgm

(mgkg)

Qcn

(mgkg)

2040

60250

500

750

515

30Latencyto

seizures

(min)

962plusmn

120

1459n

splusmn14

22462aplusmn244

3815

aplusmn18

01246n

splusmn117

2247aplusmn15

93533

aplusmn16

41021n

splusmn14

21938aplusmn261

3172aplusmn15

3Seizures

()

100

850

550

a100

a852

613

a213

a874

665

a298

a

Latencyto

SE(m

in)

2386plusmn

154

3461aplusmn086

4870aplusmn096

NoSE

3252

aplusmn10

744

61aplusmn12

25611

aplusmn263

2962aplusmn16

140

33aplusmn257

588

aplusmn19

1SE

()

100

500

a200

a0a

614

a368

a235

a683

a392

a254

a

Mortality(

)with

in24

hours

100

00

00

00

00

Animalsw

ereo

bservedform

orethan1h

after

Li-Pc(lithium

-pilo

carpine)

injections

forind

ucingSE

inallgroup

sandallanimalsw

ereo

bservedform

orethan24

hoursfor

mortality

nsStatisticallyno

nsignificant

a 119875lt0001as

comparedto

control(0m

gkg)b

yANOVA

follo

wed

byStud

ent-N

ewman-K

eulsMultip

leCom

paris

onsT

est


1 20

50

100

150

Test days

Mea

n lat

ency

to re

ach

the p

latfo

rm


lowastlowast


lowast




(a)

Test days3 4

0

50

100

150

Mea

n lat

ency

to re

ach

the p

latfo

rm

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




(b)













0

5

10

15

0

50

100

150



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

(a)

0

100

200

300

400

500

0

250

500

750

1000



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




GSH

leve

l as n

mol

gtis

sue w

tplusmnSE

MG

SH le

vel a

s nm

olg

tissu

e wtplusmn

SEM

(b)




4 Discussion




0

25

50

75

100 G

ST (U

g ti

ssue

wt)

Hippocampus1 2 3 1 2 3 1 2 3


(a)

0

3

6

9

12

GST

(Ug

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(b)

0

25

50

75

CAT

(kg

wet

tiss

ue w

t)

Hippocampus1 2 3 1 2 3 1 2 3


(c)

0

10

20

30

40

CAT

(kg

wet

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(d)

0

20

40

60

80

Hippocampus1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(e)

0

15

30

45

60

Striatum1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(f)







5 Conclusion




Acknowledgment



References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































3 Results




Table1Dose-depend


ityof

pentoxify

lline

(Ptx)proglumide(Pg

m)andqu

inacrin

e(Qcn)a

gainstLi-Pcind

uced

statuse

pilepticus

(SE)

inadultrats


bserved

Con

trol

Ptx(m

gkg)

Pgm

(mgkg)

Qcn

(mgkg)

2040

60250

500

750

515

30Latencyto

seizures

(min)

962plusmn

120

1459n

splusmn14

22462aplusmn244

3815

aplusmn18

01246n

splusmn117

2247aplusmn15

93533

aplusmn16

41021n

splusmn14

21938aplusmn261

3172aplusmn15

3Seizures

()

100

850

550

a100

a852

613

a213

a874

665

a298

a

Latencyto

SE(m

in)

2386plusmn

154

3461aplusmn086

4870aplusmn096

NoSE

3252

aplusmn10

744

61aplusmn12

25611

aplusmn263

2962aplusmn16

140

33aplusmn257

588

aplusmn19

1SE

()

100

500

a200

a0a

614

a368

a235

a683

a392

a254

a

Mortality(

)with

in24

hours

100

00

00

00

00

Animalsw

ereo

bservedform

orethan1h

after

Li-Pc(lithium

-pilo

carpine)

injections

forind

ucingSE

inallgroup

sandallanimalsw

ereo

bservedform

orethan24

hoursfor

mortality

nsStatisticallyno

nsignificant

a 119875lt0001as

comparedto

control(0m

gkg)b

yANOVA

follo

wed

byStud

ent-N

ewman-K

eulsMultip

leCom

paris

onsT

est


1 20

50

100

150

Test days

Mea

n lat

ency

to re

ach

the p

latfo

rm


lowastlowast


lowast




(a)

Test days3 4

0

50

100

150

Mea

n lat

ency

to re

ach

the p

latfo

rm

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




(b)













0

5

10

15

0

50

100

150



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

(a)

0

100

200

300

400

500

0

250

500

750

1000



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




GSH

leve

l as n

mol

gtis

sue w

tplusmnSE

MG

SH le

vel a

s nm

olg

tissu

e wtplusmn

SEM

(b)




4 Discussion




0

25

50

75

100 G

ST (U

g ti

ssue

wt)

Hippocampus1 2 3 1 2 3 1 2 3


(a)

0

3

6

9

12

GST

(Ug

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(b)

0

25

50

75

CAT

(kg

wet

tiss

ue w

t)

Hippocampus1 2 3 1 2 3 1 2 3


(c)

0

10

20

30

40

CAT

(kg

wet

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(d)

0

20

40

60

80

Hippocampus1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(e)

0

15

30

45

60

Striatum1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(f)







5 Conclusion




Acknowledgment



References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Dose-depend


ityof

pentoxify

lline

(Ptx)proglumide(Pg

m)andqu

inacrin

e(Qcn)a

gainstLi-Pcind

uced

statuse

pilepticus

(SE)

inadultrats


bserved

Con

trol

Ptx(m

gkg)

Pgm

(mgkg)

Qcn

(mgkg)

2040

60250

500

750

515

30Latencyto

seizures

(min)

962plusmn

120

1459n

splusmn14

22462aplusmn244

3815

aplusmn18

01246n

splusmn117

2247aplusmn15

93533

aplusmn16

41021n

splusmn14

21938aplusmn261

3172aplusmn15

3Seizures

()

100

850

550

a100

a852

613

a213

a874

665

a298

a

Latencyto

SE(m

in)

2386plusmn

154

3461aplusmn086

4870aplusmn096

NoSE

3252

aplusmn10

744

61aplusmn12

25611

aplusmn263

2962aplusmn16

140

33aplusmn257

588

aplusmn19

1SE

()

100

500

a200

a0a

614

a368

a235

a683

a392

a254

a

Mortality(

)with

in24

hours

100

00

00

00

00

Animalsw

ereo

bservedform

orethan1h

after

Li-Pc(lithium

-pilo

carpine)

injections

forind

ucingSE

inallgroup

sandallanimalsw

ereo

bservedform

orethan24

hoursfor

mortality

nsStatisticallyno

nsignificant

a 119875lt0001as

comparedto

control(0m

gkg)b

yANOVA

follo

wed

byStud

ent-N

ewman-K

eulsMultip

leCom

paris

onsT

est


1 20

50

100

150

Test days

Mea

n lat

ency

to re

ach

the p

latfo

rm


lowastlowast


lowast




(a)

Test days3 4

0

50

100

150

Mea

n lat

ency

to re

ach

the p

latfo

rm

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




(b)













0

5

10

15

0

50

100

150



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

(a)

0

100

200

300

400

500

0

250

500

750

1000



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




GSH

leve

l as n

mol

gtis

sue w

tplusmnSE

MG

SH le

vel a

s nm

olg

tissu

e wtplusmn

SEM

(b)




4 Discussion




0

25

50

75

100 G

ST (U

g ti

ssue

wt)

Hippocampus1 2 3 1 2 3 1 2 3


(a)

0

3

6

9

12

GST

(Ug

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(b)

0

25

50

75

CAT

(kg

wet

tiss

ue w

t)

Hippocampus1 2 3 1 2 3 1 2 3


(c)

0

10

20

30

40

CAT

(kg

wet

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(d)

0

20

40

60

80

Hippocampus1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(e)

0

15

30

45

60

Striatum1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(f)







5 Conclusion




Acknowledgment



References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 20

50

100

150

Test days

Mea

n lat

ency

to re

ach

the p

latfo

rm


lowastlowast


lowast




(a)

Test days3 4

0

50

100

150

Mea

n lat

ency

to re

ach

the p

latfo

rm

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




(b)













0

5

10

15

0

50

100

150



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

(a)

0

100

200

300

400

500

0

250

500

750

1000



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




GSH

leve

l as n

mol

gtis

sue w

tplusmnSE

MG

SH le

vel a

s nm

olg

tissu

e wtplusmn

SEM

(b)




4 Discussion




0

25

50

75

100 G

ST (U

g ti

ssue

wt)

Hippocampus1 2 3 1 2 3 1 2 3


(a)

0

3

6

9

12

GST

(Ug

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(b)

0

25

50

75

CAT

(kg

wet

tiss

ue w

t)

Hippocampus1 2 3 1 2 3 1 2 3


(c)

0

10

20

30

40

CAT

(kg

wet

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(d)

0

20

40

60

80

Hippocampus1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(e)

0

15

30

45

60

Striatum1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(f)







5 Conclusion




Acknowledgment



References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

0

50

100

150



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

tissu

e wtplusmn

SEM

TBRA

S co

nten

ts as

nm

olg

(a)

0

100

200

300

400

500

0

250

500

750

1000



Striatum

Hippocampus

1 2 3 1 2 3 1 2 3

1 2 3 1 2 3 1 2 3




GSH

leve

l as n

mol

gtis

sue w

tplusmnSE

MG

SH le

vel a

s nm

olg

tissu

e wtplusmn

SEM

(b)




4 Discussion




0

25

50

75

100 G

ST (U

g ti

ssue

wt)

Hippocampus1 2 3 1 2 3 1 2 3


(a)

0

3

6

9

12

GST

(Ug

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(b)

0

25

50

75

CAT

(kg

wet

tiss

ue w

t)

Hippocampus1 2 3 1 2 3 1 2 3


(c)

0

10

20

30

40

CAT

(kg

wet

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(d)

0

20

40

60

80

Hippocampus1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(e)

0

15

30

45

60

Striatum1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(f)







5 Conclusion




Acknowledgment



References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

25

50

75

100 G

ST (U

g ti

ssue

wt)

Hippocampus1 2 3 1 2 3 1 2 3


(a)

0

3

6

9

12

GST

(Ug

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(b)

0

25

50

75

CAT

(kg

wet

tiss

ue w

t)

Hippocampus1 2 3 1 2 3 1 2 3


(c)

0

10

20

30

40

CAT

(kg

wet

tiss

ue w

t)

Striatum1 2 3 1 2 3 1 2 3


(d)

0

20

40

60

80

Hippocampus1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(e)

0

15

30

45

60

Striatum1 2 3 1 2 3 1 2 3


SOD(times10minus1)

(Ug

wet

tiss

ue w

t)




(f)







5 Conclusion




Acknowledgment



References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusion




Acknowledgment



References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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