Pharmacology, Biochemistry and Behavior 125 (2014) 55–64

Contents lists available at ScienceDirect

Pharmacology, Biochemistry and Behavior

j ourna l homepage: www.e lsev ie r .com/ locate /pharmbiochembeh

Curcumin supplementation improves mitochondrial and behavioraldeficits in experimental model of chronic epilepsy

Harpreet Kaur a, Amanjit Bal b, Rajat Sandhir a,⁎a Department of Biochemistry, Panjab University, Chandigarh 160014, Indiab Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

⁎ Corresponding author at: Department of BiochemistryPanjab University, Sector-14, Chandigarh, 160014, India. Te172 2541022.

E-mail address: sandhir@pu.ac.in (R. Sandhir).

http://dx.doi.org/10.1016/j.pbb.2014.08.0010091-3057/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 June 2014Received in revised form 28 July 2014Accepted 2 August 2014Available online 10 August 2014

Keywords:CurcuminChronic epilepsyMitochondriaCognitionOxidative stress

The present studywas aimed to investigate the potential beneficial effect of curcumin, a polyphenolwith pleiotropicproperties, onmitochondrial dysfunctions, oxidative stress and cognitive deficits in a kindledmodel of epilepsy. Kin-dled epilepsy was induced in rats by administering a sub-convulsive dose of pentylenetetrazole (PTZ, 40 mg/kgbody weight) every alternate day for 30 days. PTZ administered rats exhibited marked cognitive deficits assessedusing active and passive avoidance tasks. This was accompanied by a significant decrease in NADH:cytochrome-creductase (complex I) and cytochrome-c oxidase (complex IV) activities alongwith an increase in ROS, lipid perox-idation and protein carbonyls. The levels of glutathione also decreased in the cortex and hippocampus. Electronmicrographs revealed disruption ofmitochondrialmembrane integritywith distorted cristae in PTZ treated animals.Histopathological examination showed pyknotic nuclei and cell loss in the hippocampus as well as in the cortex ofPTZ treated animals. Curcumin administration at a dose of 100 mg/kg, p.o. throughout the treatment paradigmwasable to ameliorate cognitive deficits with no significant effect on seizure score. Curcumin was able to restore theactivity of mitochondrial complexes. In addition, significant reduction in ROS generation, lipid peroxidation andprotein carbonyls was observed in PTZ animals supplemented with curcumin. Moreover, glutathione levels werealso restored in PTZ treated rats supplemented with curcumin. Curcumin protected mitochondria from seizureinduced structural alterations. Further, the curcumin supplemented PTZ rats had normal cell morphology andreduced cell loss. These results suggest that curcumin supplementation has potential to prevent mitochondrialdysfunctions and oxidative stress with improved cognitive functions in a chronic model of epilepsy.

© 2014 Elsevier Inc. All rights reserved.

1. Introduction

Epilepsy is a brain disorder characterized by recurrent unprovokedseizures with prolonged epileptic discharge (Panayiotopoulos, 2005).The type of epileptic seizure depends on the brain region affected andthe cause of epilepsy. Complex partial seizures arising from the limbicsystem(hippocampus) led to a condition knownas temporal lobe epilep-sy (TLE). TLE is themost commonly studied type of epilepsy because of itshigh prevalence and severity (Engel et al., 2003). The burst-firing of neu-rons in an epileptic condition leads to a cascade of events that include in-crease in oxidative stress, inflammation and activation of cell deathpathways (Fujikawa, 2005; Macdonald and Kapur, 1999). Increased oxi-dative stress is considered as a contributing factor to the pathogenesis ofepilepsy (Sudha et al., 2001).Mitochondria are the primary organelles in-volved in several important metabolic activities and play a central role inenergy transduction within the cell (Szewczyk and Wojtczak, 2002).

, Basic Medical Science Building,l.: +91 172 2534131; fax: +91

Defects inmitochondrial dynamics alongwith change in energymetabo-lismgenerally contribute to generation of superoxide, hydrogenperoxideand hydroxyl radicals which damage cellular components like DNA,lipids and proteins (Yan et al., 2013). Mitochondrial dysfunctions havebeen observed to play a cardinal role in the pathogenesis of various neu-rodegenerative diseases such as Alzheimer's disease (Eckert et al., 2012),Huntington's disease (Sandhir et al., 2014), and Parkinson's disease(Navarro and Boveris, 2010).

Seizures have also been observed in animals treated with potassiumcyanide inhibitor of cytochrome-c oxidase, i.e. complex IV (Yamamotoand Tang, 1996), and 3-nitroproprionic acid, inhibitor of succinate dehy-drogenase, i.e. complex II (Urbanska et al., 1998) suggesting that inhibi-tion of mitochondrial respiratory chain components lead to epilepsy.Studies suggest that defects inmitochondrial functionmight be underly-ing the pathogenesis of seizures in temporal lobe epilepsy (Azakli et al.,2013). The currently available antiepileptic drugs for treatment of seizuredisorder have numerous adverse effects including some degree of cogni-tive, behavioral or psychiatric reactions (Walia et al., 2004). Therefore, anobvious way to intervene seizure induced mitochondrial damage in epi-lepsy is through the use of natural antioxidant compoundswithwide ap-plicability and therapeutic efficacy with no adverse effects.

Fig. 1. The schematic diagram indicating the treatment paradigm.

56 H. Kaur et al. / Pharmacology, Biochemistry and Behavior 125 (2014) 55–64

Curcumin (diferuloylmethane), a yellow dietary pigment in the rhi-zome Curcuma longa, belongs to the family Zingiberaceae which hasbeen used as Ayurvedic medicine for decades to treat various diseases(He et al., 2010). Numerous studies have shown curcumin as an antiox-idant and anti-inflammatory compoundwith neuroprotective propertiesin various neurological conditions such as Parkinson's disease, multiplesclerosis, and Alzheimer's disease (Hishikawa et al., 2012). Many clinicaltrials have been completed and some are being undertaken to evaluatethe efficacy of curcumin against a wide range of human diseases includ-ing neurological disorders. A studywas designed to generate tolerability,pharmacokinetics, and preliminary clinical and biomarker efficacy dataon curcumin in Alzheimer's patients which was unable to demonstrateclinical and biochemical evidences of efficacy against Alzheimer's diseasedue to a small sample size (30 patients) and short duration of study(Ringman et al., 2012). Moreover, curcumin in the form of capsules,nanoparticles, powder and solution has been in clinical trials to improvecognitive functions and neuropathy in patients (Gupta et al., 2013).Curcumin has been shown to provide protection against amyloid peptideinduced mitochondrial dysfunction in Alzheimer's disease (Huang et al.,2012). Curcumin administration to aluminum-treated animals has beenobserved to decrease brain mitochondrial damage by normalizing theactivities of mitochondrial electron transport complexes affected follow-ing aluminum treatment (Sood et al., 2011). A recent study has shownthe neuroprotective effect of CNB-001, a pyrazole derivative of curcuminin rotenone induced toxicity (Jayaraj et al., 2013). To the best of ourknowledge, this is the first study designed with an aim to evaluate theneuroprotective potential of curcumin in preventing seizure inducedmitochondrial dysfunctions and cognitive deficits in a chronic model ofepilepsy.

2. Material and methods

2.1. Chemicals

Pentylenetetrazole and curcumin were purchased from Sigma Chem-ical Co. (St. Louis, MO, USA) and Hi-Media Laboratories (Mumbai, India)respectively. Other chemicals used in the present studywere of analyticalgrade and were procured from either Sigma Chemical Co. (St. Louis, MO,USA) or Sisco Research Laboratories (Mumbai, India).

2.2. Animals and treatment schedule

Adult male Wistar rats weighing 200–220 g were procured from theCentral Animal House facility of Panjab University. All the animals werehoused under standard housing and acclimatized for a week. They hadfree access to rat pellet diet (Ashirwad Industries, Ropar, India) andwater. The procedures followed were approved by the InstitutionalAnimal Ethics Committee (IAEC) of the university and were in accor-dancewith the guidelines for humaneuse and care of laboratory animals.Animalswere randomly segregated into the following four groupswith 8animals in each group. The schematic representation of the treatmentparadigm is provided in Fig. 1 and is based on a report in literature(Rajabzadeh et al., 2012).

Control Animals were administered with vehicle daily for the durationof the treatment.

Curcumin Animals were administered with curcumin (suspended in 1%carboxymethyl cellulose in distilled water) daily at a dose of100 mg/kg body weight, orally for 40 days.

PTZ Animals were administered PTZ intraperitoneally (dissolvedin saline) at a dose of 40 mg/kg on alternate days for a periodof 30 days and a single dose of PTZ on the 40th day(challengedose) to ensure kindling success.

PTZ + curcumin Animals were administered with curcumin daily30 min before PTZ injection at a dose of 100 mg/kg orally for40 days while PTZ was administered for 30 days only.

Adose of 100 mg/kg curcuminwas selected on the basis of literatureindicating the average intake of turmeric of 2–2.5 g per day correspond-ing to 100 mg of curcumin with no adverse effect (Shah et al., 1999).Curcumin at a similar dose has been found to be neuroprotective invarious neurological conditions including epilepsy (Du et al., 2009; Maet al., 2013; Thiyagarajan and Sharma, 2004; Yadav et al., 2009).

2.3. Kindling procedure

The kindlingmodel is an accepted experimental model for TLE and isgenerally used for screening of potential antiepileptic drugs (Morimotoet al., 2004). Pentylenetetrazole (PTZ), a GABAA antagonist commonlyused as a convulsant agent, which when administered (repeatedly)in a sub-convulsive dose for a specific period of time, slowly resultsin the development of a kindled model of epilepsy, also referred to aschronic epilepsy (Hansen et al., 2004). A sub-convulsive dose of PTZ40 mg/kg in the volume of 1 ml/kg was administered to rats alterna-tively for 30 days resulting in a permanent change with generalizedtonic–clonic convulsions. After every PTZ injection, each animal wasplaced separately in a cage and the occurrence and intensity of seizureswere observed for over 25–30 min. The intensity of seizures was calcu-lated according to Racine's scale depicted in Table 1 (Racine, 1972). Thesuccess of kindling was assessed by giving a challenge dose of PTZ(40 mg/kg) 10 days after the last PTZ injection (on day 40) and seizureswere scored. Many investigators have used a similar model (Kumaret al., 2013). The PTZ + curcumin group was administered PTZ for30 days followed by a single dose of PTZ to access the success of kindlingas done in the PTZ treated group. The cumulative kindling scorewas cal-culated for each group. Rats that fulfilled the kindling criteria and hadtonic–clonic seizures following the challenge dose were included inthe study.

2.4. Isolation of mitochondria

Animals were sacrificed by decapitation undermild ether anesthesia;the hippocampus and cerebral cortexwere dissected from the brain. Eachregion was weighed and homogenized separately in ice cold buffer con-taining 10 mM Tris HCl (pH 7.4), 0.44 M sucrose, 10 mM EDTA and 0.1%BSA and centrifuged at 2100 g for 15min at 4 °C. The pelletwas discardedand the supernatant obtained was further centrifuged at 14,000 g for15 min at 4 °C. The mitochondrial pellet obtained was washed with thesame buffer and again spun at 7000 g for 15 min at 4 °C. The final mito-chondrial pellet was re-suspended in a buffer containing 10mM Tris HCl(pH 7.4) and 0.44 M sucrose.

2.5. Activity of mitochondrial complexes

2.5.1. NADH:cytochrome c reductase (EC1.6.99.3, complex I)The activity of NADH:cytochrome c reductase was measured as

described by King and Howard (1967). The method involves catalyt-ic oxidation of NADH to NAD+ with subsequent reduction of cyto-chrome c on addition of mitochondrial preparation. The increase inabsorbance was read at 550 nm for 3 min. Results were expressed as

57H. Kaur et al. / Pharmacology, Biochemistry and Behavior 125 (2014) 55–64

nmol NADH oxidized/min/mg of protein using the molar extinction co-efficient of reduced cytochrome c at 550 nm (1.96 mM−1 cm−1).

2.5.2. Succinate dehydrogenase (EC 1.3.5.1, complex II)The activity of succinate dehydrogenasewas assayed according to the

method of King et al. (1976). Succinate dehydrogenase catalyzes the ox-idation of succinate to fumarate on addition of potassium ferricyanidethat acts as an artificial electron acceptor. The reaction was initiated onaddition of a requisite amount of mitochondrial preparation and thechange in absorbance was read at 420 nm for 3 min. Results wereexpressed as nmol of succinate oxidized/min/mg of protein using themolar extinction coefficient of potassium ferricyanide (1000M−1 cm−1).

2.5.3. Cytochrome-c oxidase (EC 1.9.3.1, complex IV)The activity of cytochrome oxidase was assayed according to the

method of Sottocasa et al. (1967). This assay measures the oxidationof reduced cytochrome c by the enzyme. The cytochrome c was firstreduced by adding a few crystals of sodium borohydride and then theoxidation of reduced cytochrome cwasmeasured by a decrease in absor-bance at 550 nm after the addition of an appropriate amount of mito-chondrial suspension. Results were expressed as nmol of cytochromec oxidized/min/mg of protein using the molar extinction coefficient ofcytochrome c (1.96 mM−1 cm−1).

2.5.4. F1–F0 ATP synthase (EC 3.6.3.14, complex V)The activity of mitochondrial F1–Fo ATP synthase was assayed using

the method of Griffiths and Houghton (1974). Appropriate amount ofmitochondrial sample was added to the ATPase buffer and incubated at37 °C for 10 min. The reaction was stopped by addition of 10% (w/v)trichloroacetic acid (TCA) and contents were centrifuged at 3000 g for20min. The phosphate releasedwas analyzed on the basis of the reactionof the inorganic phosphate with ammonium molybdate as described byFiske and Subbarow (1927). Results were expressed as nmol of ATPhydrolyzed/min/mg protein.

2.6. Mitochondrial membrane permeability

Mitochondria transition pore opening leads to swelling ofmitochon-dria. Mitochondrial swelling and contraction were used as a functionaltest for the mitochondrial membrane integrity which was measured at520 nm as described by Tedeschi and Harris (1958). Mitochondrialswelling was assessed by adding an appropriate amount of sample tothe buffer containing 0.12 M KCl in 0.02 M Tris HCl (pH 7.4) and theabsorbancewasmeasured for 6min at 520 nm. The contraction ofmito-chondriawas initiated by addingMg+2-ATP and absorbancewas read at520 nm.

2.7. Mitochondrial ROS generation

Intracellular generation of ROS was assessed using the methoddescribed by Wang and Joseph (1999). Oxidation of intracellular 2′,7′-dichlorofluorescein-diacetate (DCFH-DA, nonflorescence) to highly fluo-rescent 2′,7′ dichlorofluorescein (DCF) wasmeasured using a spectroflu-orometer with an excitation at 488 nm and an emission at 530 nm after

Table 1The behavioral stages and the characterization of seizure score according to Racine's scale.

Stages of seizure Characterization

Stage 0 No responseStage 1 Facial movements, ear and whisker twitchingStage 2 Myoclonic convulsions without rearingStage 3 Myoclonic convulsions with rearingStage 4 Clonic–tonic convulsionStage 5 Generalized clonic–tonic seizures with loss of postural control

addition of the mitochondrial sample. Results were expressed as pmolDCF/min/mg protein.

2.8. Protein carbonyls

The protein carbonyls were determined using 2,4-dinitrophenyl-hydrazine (DNPH) assay as described by Levine et al. (1990). The mito-chondrial sample was dissolved in 20 mM DNPH prepared in 2 N HCland kept at room temperature for 1 h in the dark. Respective negativecontrol was prepared in which mitochondria were dissolved in 2 N HClonly. Following 1 h incubation, 20% (w/v) TCAwas added for protein pre-cipitation and the mixture was centrifuged at 10,000 g for 10 min.Supernatant was discarded and the pellet obtained was washed threetimes with an ethyl acetate:ethanol mixture (1:1) to remove excessDNPH. The final pellet was later dissolved in 6 M guanidine HCl solutionat 50 °C and the yellow colored complex obtained was read at 370 nmagainst their negative controls. Negative controls were read at 280 nmto determine protein concentration. The results were expressed asnmol carbonyls/mg protein using the extinction coefficient of DNPH(22 mM−1 cm−1).

2.9. Lipid peroxidation

Malondialdehyde (MDA), a measure of lipid peroxidation, was esti-mated according to themethod of Ohkawa et al. (1979). The amount ofMDA formed was measured by the reaction with thiobarbituric acid at532 nm using a spectrophotometer. The results were expressed asnmol MDA/mg protein using the molar extinction coefficient of MDA-thiobarbituric acid chromophore (1.56 × 105 M−1 cm−1).

2.10. Glutathione (GSH)

GSH levels were estimated by the method of Roberts and Francetic(1993). DTNB added during estimation was reduced by free\SH ofGSH to form 5-mercapto-2-nitrobenzoate which was read at 412 nm.Results were expressed as μmol GSH/mg protein by using the molarextinction coefficient (13,100 M−1 cm−1).

2.11. Estimation of protein

The protein content was estimated according to the method ofLowry et al. (1951) using bovine serum albumin (BSA) as standard.

2.12. Electron microscopy

Transmission electron microscopy was done as described by Gaoet al. (2013). The hippocampus and cerebral cortex were removed afterdecapitation undermild anesthesia (ether) and fixed in 3% (w/v) glutar-aldehyde (diluted in 0.2 M Sorenson's buffer) for 24 h at 4 °C. Brain sec-tions were diced into small cubes of 1mm size and post fixed in osmiumtetroxide, dehydrated in a graded ethanol series, cleared in propyleneoxide at room temperature and finally embedded in an EPON mixturecontaining Taab/812. Thick sections of 0.5 μmsizewere cut and toluidineblue staining was done to confirm the presence of neurons. 60 nm ultra-thin sections were cut, mounted on Nickel grids and were double con-trast stained using uranyl acetate and lead citrate. Sections wereexamined and photographed using Hitachi H-7500 transmission elec-tron microscope (Hitachi, Tokyo, Japan).

2.13. Cresyl violet staining

The brain sections were stained with cresyl violet (0.1% w/v) by themethod described byOgita et al. (2003). Briefly, ratswere deep anesthe-tized and perfused transcardially with saline followed by 0.4% (w/v)paraformaldehyde prepared in phosphate buffered saline. Brains wereremoved and processed for routine paraffin embedding. Coronal sections

Fig. 2. Effect of curcumin supplementation on PTZ induced kindling. Values are expressed asmean ± SEM; n = 8/group.

58 H. Kaur et al. / Pharmacology, Biochemistry and Behavior 125 (2014) 55–64

were prepared and mounted on poly-L-lysine coated slides. Paraffinsections were de-waxed in xylene solution twice for 20 min, followedby rehydration in 100% ethanol for 10 min. Then the slides were im-mersed once in 90% (v/v) ethanol, and once in 70% (v/v) ethanol follow-ed by immersion in 50% (v/v) ethanol. The slides were dipped in 30% (v/v) ethanol for 10 min, rinsed two times in distilled water and finally im-mersed in cresyl violet staining solution (0.1% w/v) for 5 min. A finalrinse in water was given to all slides and later air dried. Two changes ofn-butanol were given at a final step followed by cleaning in xylene solu-tion for 5min. Slides weremounted in DPX and dried overnight. Imageswere acquired using a Leica DM2500 microscope (Leica microsystems,Wetzlar, Germany) attached with a Leica DFC295 CCD camera.

2.14. Neurobehavioral studies

The animals in treatment groups were assessed for cognitive deficitsusing active and passive avoidance tests.

2.14.1. Active avoidance testActive avoidance task was performed according to the method

described by Kamboj and Sandhir (2007). The behavior apparatusconsisted of a two way shuttle box equipped with a stainless steel gridfloor and divided into two equal sized chambers (dark and lit compart-ments). The chambers were interconnectedwith an opening enough forthe animal to pass through. Acoustic and visual stimulators in the formof buzzer and lightwere supplied. Each animal was kept inside the shut-tle box for 15min for habituation one day before starting the task duringwhich the animal can explore freely. During the learning session, the an-imalwas placed in the lit compartmentwhere it learned to enter the safecompartment after experiencing light and sound stimulus (conditionedstimulus) for 10 s each and avoiding the electric shock (un-conditionedstimulus). Electric shock (10 s)was given to animal that did not respondto the conditioned stimulus. The total of 10 trials were given to each an-imal in a day. The number of trials in which the animal responded onlyto un-conditioned stimulus were recorded as escape trials.

2.14.2. Passive avoidance testPassive avoidance task was performed to evaluate the learning and

long-term memory in animals as described by Uzum et al. (2010). Theapparatus consisted of two chambers (a dark and an illuminated cham-ber), with a stainless steel grid floor to deliver foot-shocks. The rat wasplaced in the illuminated compartment and had free access to all partsof the apparatus for 300 s for habituation. One day after the last PTZ in-jection, the animal was placed in the illuminated chamber and allowedto explore for 30 s. After 30 s, the door between the two chambers waslifted up and thereafter its latency to enter the dark compartment wasrecorded. The interval between the placement of rat in the light cham-ber and its entry into the dark chamber was measured as entrance la-tency. Immediately, the door was closed once the animal enters thedark compartment and a single electric foot shock (65 V AC, 50 Hz)for 5 s was delivered. Consolidation and long-term memory wereassessed. One day after this learning session, the animal was placed inthe illuminated chamber and the latency to re-enter the dark chamberwas measured. This was known as consolidation memory. The sameprocedure was used for the long-term memory test which was per-formed three days after the learning/training session. Foot shockswere not administered during the retention tests. Rats that did notenter the dark compartment during the given 5-min period wasremoved from the apparatus and the latencywas considered to be 300 s.

2.15. Statistical analysis

Values are expressed as mean ± standard error mean (SEM). Thestatistical significance was analyzed by one-way analysis of variancefollowed by Newman–Keuls test using SPSS 16 software. Values withp b 0.05 were considered as statistically significant.

3. Results

3.1. PTZ kindling score

Fig. 2 shows the effect of curcumin supplementation on seizurescore. 30 days of PTZ treatment in animals resulted in generalizedtonic–clonic seizures. It was observed that curcumin administered at adose of 100 mg/kg had no significant effect on the severity and progres-sion of seizures in PTZ treated animals.

3.2. Mitochondrial respiratory chain enzymes

The activities of mitochondrial electron transport chain componentsin the cortex and hippocampus are presented in Table 2. NADH:cyto-chrome c reductase activity was inhibited by 40% in the cortex and42% in the hippocampus of PTZ treated animals as compared to controls.However, the activity was found to be increased by 68% in the cortexand by 76% in the hippocampus on curcumin supplementation to thePTZ treated animals. The activity of cytochrome c oxidase was foundto be significantly lowered by 62% in the cortex and by 47% in the hippo-campus of PTZ treated animals as compared to controls. Supplementa-tion with curcumin increased cytochrome c oxidase activity by 85% inthe cortex and 88% in the hippocampus in comparison to PTZ treatedanimals. The activity of succinate dehydrogenase and F1–Fo ATP syn-thase was found to be unaffected in both the regions of brain on PTZtreatment as compared to controls. Administration of curcumin alsohad no effect on these electron transport chain complexes.

3.3. Mitochondrial swelling

Mitochondrion swelling was observed to be increased by 8 fold inthe hippocampus and by 3 fold in the cortex of PTZ treated animalswhen compared to controls. However, mitochondrial swelling wasobserved to be decreased by 2 fold in the hippocampus and cortex of an-imals co-administeredwith curcuminwhen compared to PTZ treated an-imals (Fig. 3).

3.4. Reactive oxygen species, lipid peroxidation and protein carbonyls

ROS productionwas found to be significantly increased in the cortex(90%) and hippocampus (94%) of PTZ treated animals in comparison tocontrols. However, curcumin administration to PTZ animals decreasedthe levels by 36% in the cortex and by 46% in the hippocampus whencompared to the PTZ treated group (Table 3). Malondialdehyde, abyproduct of lipid peroxidation, was observed to be increased

Table 2Effect of curcumin administration on the activity of mitochondrial complexes in cortex and hippocampus of PTZ treated rats.

Groups NADH:cytochrome creductase (nmol NADHoxidized/min/mg protein)

Succinate dehydrogenase(nmol succinate oxidized/min/mg protein)

Cytochrome oxidase(nmol cytochrome coxidized/min/mg protein)

F1F0 synthase(nmol ATP hydrolyzed/min/mgprotein)

Cortex Hippocampus Cortex Hippocampus Cortex Hippocampus Cortex Hippocampus

Control 42.55 ± 4.41 25.62 ± 3.01 43.24 ± 10.53 28.97 ± 2.79 3.16 ± 0.65 5.63 ± 1.08 161.39 ± 6.56 231.36 ± 25.07Curcumin 29.71 ± 3.96 29.87 ± 6.64 25.96 ± 3.03 30.72 ± 2.47 4.08 ± 0.94 3.23 ± 0.29 153.37 ± 15.99 288.94 ± 42.94PTZ 25.39 ± 4.04⁎ 14.76 ± 1.77⁎ 41.68 ± 6.52 23.21 ± 3.06 1.21 ± 0.34⁎ 2.99 ± 0.38⁎ 155.65 ± 11.09 312.31 ± 40.66PTZ + Curcumin 42.56 ± 5.38# 25.97 ± 3.37# 32.59 ± 9.04 24.46 ± 8.29 2.24 ± 0.78 5.64 ± 0.79# 175.95 ± 17.15 225.46 ± 27.22

Values are expressed as mean ± SEM; n = 8/group.⁎ Significantly different from control group (p b 0.05).# Significantly different from PTZ treated group (p b 0.05).

59H. Kaur et al. / Pharmacology, Biochemistry and Behavior 125 (2014) 55–64

remarkably in the cortex (40%) and hippocampus (24%) of PTZ treatedanimals as compared to controls. Curcumin supplementation signifi-cantly lowered PTZ induced lipid peroxidation in the cortex (22%) andhippocampus (25%) when compared to PTZ treated animals (Table 3).Protein carbonyl levels were also found to be increased by 87% in thecortex and by 188% in the hippocampus while curcumin supplementa-tion to PTZ treated animals decreased the levels by 91% in the hippocam-pus and 54% in the cortex when compared to the PTZ treated group(Table 3).

3.5. Glutathione (GSH)

GSH, a first line of defense, was observed to significantly decreasedafter PTZ treatment in both the hippocampus (13%) and cortex (16%)when compared to controls. However, curcumin supplementation re-sulted in restoration of GSH levels in both regions of the brain.

3.6. Electron microscopy

The electron micrographs of the cortex and hippocampus wereexamined for ultrastructure changes in mitochondria (Figs. 4 and 5).PTZ treatment to animals resulted in severe damage to mitochondrialstructure characterized by disruption of mitochondrial membraneintegrity and distorted cristae with clearing of matrix density in bothregions. However, normal morphology of mitochondria was observedin saline and curcumin treated animals. Curcumin supplementation toPTZ animals was able to reverse the detected structural abnormalities.

Fig. 3. Effect of curcumin supplementation on the mitochondrial swelling in cortex andhippocampus of animals with PTZ induced chronic epilepsy. Values are expressed asmean ± SEM; n = 8/group. *Significantly different from the control group (p b 0.05)and #significantly different from the PTZ treated group (p b 0.05).

3.7. Cresyl violet staining

PTZ treated animals showed shrunken nucleus (pyknotic) and cyto-plasm in both the cortex and hippocampus when compared to controlanimals (Fig. 6). However, in the PTZ animals supplemented withcurcumin, a decrease in the number of pyknotic neurons was observed.Curcumin supplementation preserved the normal morphology and in-tegrity of cells in both the regions (increase in viable cells). The curcumintreated animals also exhibited normal morphology with well-roundedcells devoid of pyknosis in the cortex and hippocampus similar to thatof the control animals.

3.8. Active avoidance test

Cognitive behavior was assessed by the number of times the animalsavoid foot shock after training (Fig. 7a). Animals avoid foot shock andprefer to go to the shock free chamber with light and buzzer stimuli.In the present study, the PTZ treated animals showed a significantincrease in the number of escapes as compared to control animals. How-ever, curcumin co-administration with PTZ proved to be beneficial as inthis group the animals entered the safe compartment on conditionedstimulus (buzzer).

3.9. Passive avoidance test

This test is generally used tomeasure cognitive alterations after drugadministration which is based on the animal's ability to remember theprevious shock experience and to avoid the re-entry into the darkchamber. In the acquisition/learning phase, the latency to enter intothe dark compartmentwas observed to be the same for all groups. How-ever, a significant impairment in both consolidation as well as long-term memory occurred after PTZ treatment as a significant decrease inthe entrance latency was observed. The animals treated with PTZ tookless time to enter the dark compartment than the controls. On theother hand, the PTZ treated animals supplemented with curcuminshowed prolonged latency to enter the dark compartment when com-pared to animals in the PTZ treated group which depicts that curcuminattenuate PTZ induced decrease in entrance latency. Control andcurcumin treated animals avoided entering the dark compartmentand performed better in consolidated as well as long term memory as-sessment (Fig. 7b).

4. Discussion

In the present study, repeated administration of a sub-convulsivedose of PTZ on alternative days resulted in the development of kindlingin animals and these results are in accordance with the earlier studieswhere repetitive administration of a sub-convulsive dose of PTZ resultsin the appearance and progressive increase in convulsant activityresulting in generalized seizures (Corda et al., 1990). On the otherhand, curcumin administration with PTZ had no significant effect on

Table 3Effect of curcumin administration on lipid peroxidation, reactive oxygen species, protein carbonyls and glutathione levels in cortex and hippocampus of PTZ treated animals.

Groups ROS (pmol/min/mg/protein) LPO (nmolMDA/mg protein)

Protein carbonyls(nmol/mg protein)

GSH (μmol/mg protein)

Cortex Hippocampus Cortex Hippocampus Cortex Hippocampus Cortex Hippocampus

Control 34.22 ± 7.79 40.88 ± 3.55 1.62 ± 0.21 3.04 ± 0.11 0.25 ± 0.09 0.23 ± 0.08 8.96 ± 0.44 9.04 ± 0.29Curcumin 46.88 ± 4.82 50.66 ± 6.11 1.77 ± 0.18 2.98 ± 0.21 0.11 ± 0.01 0.12 ± 0.04 8.77 ± 0.61 9.03 ± 0.14PTZ 64.88 ± 9.61⁎ 79.28 ± 14.24⁎ 2.26 ± 0.09⁎ 3.77 ± 0.24⁎ 0.47 ± 0.07⁎ 0.65 ± 0.01⁎ 7.53 ± 0.11⁎ 7.85 ± 0.17⁎

PTZ + Curcumin 41.77 ± 9.58# 42.66 ± 11.99# 1.77 ± 0.15# 2.80 ± 0.18# 0.22 ± 0.03# 0.05 ± 0.01# 8.73 ± 0.28# 8.51 ± 0.18#

Values are expressed as mean ± SEM; n = 8/group.⁎ Significantly different from control group (p b 0.05).# Significantly different from PTZ treated group (p b 0.05).

60 H. Kaur et al. / Pharmacology, Biochemistry and Behavior 125 (2014) 55–64

the seizure score suggesting that curcumin at a dose of 100 mg/kg couldnot prevent epileptic seizure. This is in accordance with the study per-formed by Sumanont et al., (2007) wherein curcumin failed to delaythe onset of kainic acid induced seizure and had no effect on seizurescores.

A significant decrease in NADH:cytochrome c reductase and cyto-chrome c oxidase activity has been observed in PTZ animals which isin accordance with the study by Kudin et al. (2002). Inhibition of mito-chondrial complexes could be due to ROS generation following PTZtreatment (Celikyurt et al., 2011). A recent report shows seizuresinduced dysfunction of mitochondrial complex IV in animals (Gaoet al., 2014). Furthermore, disturbance in glycolytic rates and lactate/pyruvate ratios and loss of mitochondrial N-acetylaspartate have alsobeen observed in human epileptic tissue which confirms the involve-ment of mitochondria in TLE (Rowley and Patel, 2010). Inhibition ofmitochondrial electron transport components contributes to incom-plete electron transport and decrease in intracellular ATP productionthat disturb the normal calciumhomeostasis thereby affecting neuronalexcitability and synaptic transmission (Folbergrova and Kunz, 2012).Curcumin supplementation to PTZ animals restored the activities ofthese complexes in both the regions possibly because curcumin scav-enges ROS because of its antioxidant properties (Barzegar andMoosavi-Movahedi, 2011). Thesefindings are in agreementwith the re-cent report which showed that curcumin reduces hepatotoxicity byprotecting against mitochondrial alterations: oxidative phosphoryla-tion disruption, decrease in the cellular ATP levels, mitochondrial

Fig. 4. Transmission electron micrographs of cortex showing mitochondrial ultrastructure channification. Lower panel shows magnified view of individual mitochondria.

permeability transition, calcium homeostasis disruption and oxidativestress (Garcia-Nino and Pedraza-Chaverri, 2014). Furthermore,curcumin encapsulated solid lipid nanoparticles have been shown toameliorate mitochondrial impairments induced in 3-NP inducedHuntington's disease through activation of the NRF2 pathway (Sandhiret al., 2014).

The activities of succinate dehydrogenase and ATP synthase wereunaffected in both brain regions of PTZ treated animals. The results arein line with previous studies on animal models of epilepsy where nosignificant change was observed in the activity of succinate dehy-drogenase and ATP synthase (Gao et al., 2013; Rahman, 2012). Clin-ical profiles of patients with intractable childhood epilepsy haveshown major changes in NADH:cytochrome c reductase and cyto-chrome c oxidase activity whereas the activity of other complexesremained almost unchanged (Kang et al., 2007). Therefore, our re-sults provide supporting evidence for previous observations that mi-tochondrial complexes NADH:cytochrome c reductase and cytochrome coxidase are major contributors in impaired mitochondrial function inchronic epilepsy (Kunz et al., 2000).

Impairment in mitochondrial respiration can accentuate oxidativestress (Ankarcrona et al., 1995). In this study, protein carbonyl, ROS andLPO levels were found to be elevated in the hippocampus and cortexof PTZ treated animals suggesting an increase in oxidative stress.Increased ROS levels might be due to decreasedmitochondrial complexI activity which is considered to be the main source of ROS production(Kudin et al., 2004). It has been found that partial inhibition of complex

ges in PTZ treated rats. Upper panel shows mitochondrial ultrastructure at 50000× mag-

Fig. 5. Transmission electron micrographs of hippocampus showing mitochondrial ultrastructure changes in PTZ rats. Upper panel showsmitochondrial ultrastructure at 50000×magni-fication. Lower panel shows magnified view of individual mitochondria.

61H. Kaur et al. / Pharmacology, Biochemistry and Behavior 125 (2014) 55–64

I contributes to increased ROS production which causes cell death(Folbergrova et al., 2010). Excess ROS formed canmodify the proteinleading to increase in protein carbonyl content. Previous studies alsoreported increased protein carbonyls in rat cortex and hippocampusafter status epilepticus (Chuang et al., 2012). Curcumin administration

Fig. 6. Effect of curcumin supplementation on neuronal cell death assessed after cresyl vio(a) magnification at 20× and (b) magnification at 40×.

to PTZ animals resulted in a decrease in ROS, protein carbonyl and LPOlevels which demonstrates the role of curcumin as anti-oxidant in epi-lepsy (Du et al., 2012). The presence of hydroxyl moiety and carbonylgroups in curcumin is considered to be a major contributor for its directfree radical scavenging ability (Anand et al., 2008).

let staining of cortex and hippocampus of animals with PTZ induced chronic epilepsy.

Fig. 7. Effect of curcumin supplementation on cognitive behavior as assessed using active avoidance task (a) and passive avoidance task (b). Values are expressed asmean± SEM; n= 8.*Significantly different from the control group (*p b 0.05), #significantly different from the PTZ treated group (p b 0.05).

62 H. Kaur et al. / Pharmacology, Biochemistry and Behavior 125 (2014) 55–64

Glutathione is the most abundant non-enzymatic antioxidant whichprovides the first line of defense against free radicals. The data from thepresent study showed a significant decrease in GSH levels in both regionsof the brain following PTZ treatment; however, curcumin supplementa-tion restored GSH levels in both brain regions indicating curcumin inincreasing endogenous antioxidant defense of brain. These results are inagreement with the previous findings where curcumin has been shownto provide neuroprotection both in vivo and in vitro by restoring the de-pleted GSH levels and protecting the neuronal cells against protein oxida-tion and mitochondrial dysfunctions (Jagatha et al., 2008).

Increased oxidative stress can alter the mitochondrial membranepotential that can contribute to mitochondrial swelling (Kroemeret al., 2007). Mitochondrial swelling was observed in the cortex aswell as hippocampus of PTZ treated animals. Previous reports suggestseizure associated changes in neurons which were characterized byswollen and often disrupted mitochondria (Kudin et al., 2009). Severedegenerative damage including mitochondrial swelling had been seenin animals administered with PTZ (Asadi-Shekaari et al., 2012). The mi-tochondria swelling might be an effect of increased calcium concentra-tion due to PTZ administration (Papp et al., 1987). It has beendocumented that excessive intracellular calcium results in an increasein free radicals which causemitochondria pore to open leading to mito-chondria swelling (Brustovetsky et al., 2003). However, we found thatcurcumin supplementation to PTZ animals prevented mitochondriaswelling and restored othermitochondrial functions whichmight be at-tributed to the presence of phenolic as well as β-diketone functionalgroups in curcuminwhich scavenge the free radicals and ameliorate ox-idative stress (Reddy and Lokesh, 1994). Curcumin has been observed toreduce mitochondrial swelling (decreased permeability transition poreopening) in potassium dichromate treated rats and prevented mito-chondrial dysfunctions (Garcia-Nino et al., 2013).

Ultrastructural analysis indicated severe damage to mitochondriaas seen by the disruption of mitochondrial membrane integrity withdistorted cristae and cleared matrix density in the hippocampus as wellas cortex of PTZ treated animals. Our results are in agreement with theprevious findings where seizures are known to be associated with mito-chondrial swelling and mitochondrial dysfunctions (Zhang et al., inpress). Functional defects in mitochondria along with abnormalities inmitochondrial ultrastructure have also been seen in the patients of TLE(Folbergrova and Kunz, 2012). These results suggest that abnormities inbrainmitochondrial activity are associatedwith seizure induced changes.Furthermore, curcumin supplementation to PTZ animals protected thebrainmitochondria fromdamage suggesting that curcumin can attenuateoxidative stress and restoremitochondrial functions in chronic models ofepilepsy. These changes could be directly correlated with impairment inmitochondrial enzyme activities as it has been suggested that respiratory

enzymes andmitochondrialmembranes are synthesized in a coordinatedmanner, any changes in respiratory component can affect its membranestructure (King et al., 1972). A recent study has shown that curcumincan prevent ultrastructural changes in brainmitochondria induced by ar-senic bymodulating the expression of pro- and anti-apoptotic mitochon-drial proteins in the brain (Srivastava et al., in press).

Neuronal cell loss is one of the hallmarks of human TLE. Oxidativestress and mitochondrial dysfunctions have been found to be involvedin neuronal cell death in epilepsy (Cavazos et al., 1994). In the presentstudy, increased neuronal cell death was observed in both regions ofPTZ treated animals and these results are in agreement with previousstudies where neuronal death was found in brain regions followingprolonged seizure activity (Meldrum, 1993). Increased free radical pro-duction has been implicated in neuronal damage in mouse brain withkainate induced seizures (Baik et al., 1999). It has been documentedthat seizures result in excessive increase in excitatory amino acid (glu-tamate) levels, which leads to neuronal cell death via the activation ofNMDA receptors (Rothman and Olney, 1986). Morphological changesin mitochondria including swelling and disruption of mitochondrialmembrane may also contribute to cell damage as observed in a studyconducted in epileptic patients (Chen et al., 2010). Curcumin supple-mentation attenuated PTZ-induced neuronal cell death in both regionswhich could be due to an increase in GSH levels and decrease in oxida-tive stress parameters such as LPO, ROS and protein carbonyls oncurcumin administration.

An impaired memory performance is considered to be a hallmark ofTLE (Dietl et al., 2004). In the present study, cognitive impairment wasobserved in PTZ treated animals when compared to controls as assessedusing passive avoidance task. PTZ administration resulted in disturbanceof both consolidation and long termmemory. These results are in accor-dance with the previous studies where memory deficits were observedon PTZ treatment (Genkova-Papazova and Lazarova-Bakarova, 1995).However, curcumin supplementation ameliorated memory functions asseen by the increase in latency of animals to enter into the dark compart-ment. It has been reported that curcuminhas potential to reverse alumin-ium-induced cognitive dysfunctions and oxidative damage in rats(Kumar et al., 2009). Reports are also available where curcumin signifi-cantly enhanced learning and memory in mouse models of Alzheimer'sdisease (Pan et al., 2008). Curcumin is found to improve cognition by reg-ulating the expression of neurotransmitter-related genes such as synap-totagmin (Syt IV and Syt I) and complexins (Cplxs) which are known tobe associated with spatial memory in rats (Dong et al., 2012).

Various reports have indicated that prolonged or recurrent seizuresdisrupt cognitive behavior in kindled models of epilepsy (Rossler et al.,2000; Sutula et al., 1994; Zhang et al., 2013). Our study further providesevidence that animals treated with PTZ exhibit significant cognitive

63H. Kaur et al. / Pharmacology, Biochemistry and Behavior 125 (2014) 55–64

impairments. This alteration in memory function could be because PTZadministration inhibited GABAergic activity which leads to saturation ofendogenous long term potentiation (Moser et al., 1998). However,curcumin supplementation to PTZ animals, resulted in improvement inlearning and memory. This finding is in line with the previousstudies reporting the neuroprotective effect of curcumin whereits administration ameliorates cognitive impairment in PTZ induced kin-dled rats (Mehla et al., 2010). Similar reports showing the role ofcurcumin in protecting rats against lead inducedmemory deficits and at-tenuating amyloid-beta induced cognitive deficits in animals are alsoavailable (Dairam et al., 2007; Frautschy et al., 2001).

In conclusion, our results demonstrates that PTZ induced kindlingresults in impairment of mitochondrial functions contributing to eleva-tion of oxidative stress that leads tomitochondria ultrastructure changesaccompanied by cognitive impairment in animals. This study showsthat curcumin supplementation has a potential to preventmitochondrialdysfunction, oxidative stress and cognitive impairment in chronicmodels of epilepsy which might involve multiple mechanisms. There-fore, curcumin could be used as an adjuvant therapy in preventingmito-chondrial deficits and cognitive impairment in chronic epilepsy.

Acknowledgments

The authors acknowledge the financial assistance received from theUniversity Grants Commission, New Delhi [No. 36-196/2009 (SR)].

References

Anand P, Thomas SG, Kunnumakkara AB, Sundaram C, Harikumar KB, Sung B, et al.Biological activities of curcumin and its analogues (congeners) made by man andmother nature. Biochem Pharmacol 2008;76:1590–611.

Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA, et al.Glutamate-induced neuronal death: a succession of necrosis or apoptosis dependingon mitochondrial function. Neuron 1995;15:961–73.

Asadi-Shekaari M, Kalantaripour TP, Nejad FA, Namazian E, Eslami A. The anticonvulsantand neuroprotective effects of walnuts on the neurons of rat brain cortex. Avicenna JMed Biotechnol 2012;4:155–8.

Azakli H, Gurses C, Arikan M, Aydoseli A, Aras Y, Sencer A, et al. Whole mitochondrial DNAvariations in hippocampal surgical specimens and blood samples with high-throughputsequencing: a case of mesial temporal lobe epilepsy with hippocampal sclerosis. Gene2013;529:190–4.

Baik EJ, Kim EJ, Lee SH, Moon C. Cyclooxygenase-2 selective inhibitors aggravate kainic acidinduced seizure and neuronal cell death in the hippocampus. Brain Res 1999;843:118–29.

Barzegar A, Moosavi-Movahedi AA. Intracellular ROS protection efficiency and freeradical-scavenging activity of curcumin. PLoS One 2011;6:e26012.

Brustovetsky N, Brustovetsky T, Purl KJ, Capano M, Crompton M, Dubinsky JM.Increased susceptibility of striatal mitochondria to calcium-induced permeabilitytransition. J Neurosci 2003;23:4858–67.

Cavazos JE, Das I, Sutula TP. Neuronal loss induced in limbic pathways by kindling: evidencefor induction of hippocampal sclerosis by repeated brief seizures. J Neurosci 1994;14:3106–21.

Celikyurt IK, Mutlu O, Ulak G, Akar FY, Erden F. Gabapentin, a GABA analogue, enhancescognitive performance in mice. Neurosci Lett 2011;492:124–8.

Chen SD, Chang AY, Chuang YC. The potential role of mitochondrial dysfunction in seizure-associated cell death in the hippocampus and epileptogenesis. J Bioenerg Biomembr2010;42:461–5.

Chuang YC, Lin TK, Huang HY, Chang WN, Liou CW, Chen SD, et al. Peroxisome proliferator-activated receptors gamma/mitochondrial uncoupling protein 2 signaling protectsagainst seizure-induced neuronal cell death in the hippocampus following experimentalstatus epilepticus. J Neuroinflammation 2012;9:184.

Corda MG, Giorgi O, Longoni B, Orlandi M, Biggio G. Decrease in the function of the gamma-aminobutyric acid-coupled chloride channel produced by the repeated administrationof pentylenetetrazol to rats. J Neurochem 1990;55:1216–21.

Dairam A, Limson JL, Watkins GM, Antunes E, Daya S. Curcuminoids, curcumin, and deme-thoxycurcumin reduce lead-induced memory deficits in male Wistar rats. J Agric FoodChem 2007;55:1039–44.

Dietl T, Urbach H, Helmstaedter C, Staedtgen M, Szentkuti A, Grunwald T, et al. Persistentsevere amnesia due to seizure recurrence after unilateral temporal lobectomy. EpilepsyBehav 2004;5:394–400.

Dong S, Zeng Q, Mitchell ES, Xiu J, Duan Y, Li C, et al. Curcumin enhances neurogenesisand cognition in aged rats: implications for transcriptional interactions related togrowth and synaptic plasticity. PLoS One 2012;7:e31211.

Du P, Li X, Lin HJ, Peng WF, Liu JY, Ma Y, et al. Curcumin inhibits amygdaloid kindledseizures in rats. Chin Med J (Engl) 2009;122:1435–8.

Du P, Tang HY, Li X, Lin HJ, PengWF, Ma Y, et al. Anticonvulsive and antioxidant effects ofcurcumin on pilocarpine-induced seizures in rats. Chin Med J (Engl) 2012;125:1975–9.

Eckert GP, Renner K, Eckert SH, Eckmann J, Hagl S, Abdel-Kader RM, et al. Mitochondrialdysfunction—a pharmacological target in Alzheimer's disease. Mol Neurobiol 2012;46:136–50.

Engel Jr J, Wilson C, Bragin A. Advances in understanding the process of epileptogenesisbased on patient material: what can the patient tell us? Epilepsia 2003;44(Suppl.12):60–71.

Fiske CH, Subbarow Y. The nature of the “inorganic phosphate” in voluntary muscle.Science 1927;65:401–3.

Folbergrova J, Kunz WS. Mitochondrial dysfunction in epilepsy. Mitochondrion 2012;12:35–40.

Folbergrova J, Jesina P, Haugvicova R, Lisy V, Houstek J. Sustained deficiency ofmitochondri-al complex I activity during long periods of survival after seizures induced in immaturerats by homocysteic acid. Neurochem Int 2010;56:394–403.

Frautschy SA, Hu W, Kim P, Miller SA, Chu T, Harris-White ME, et al. Phenolic anti-inflammatory antioxidant reversal of Abeta-induced cognitive deficits and neuropa-thology. Neurobiol Aging 2001;22:993–1005.

Fujikawa DG. Prolonged seizures and cellular injury: understanding the connection.Epilepsy Behav 2005;7(Suppl. 3):S3–S11.

Gao J, Yao H, Pan XD, Xie AM, Zhang L, Song JH, et al. Alteration of mitochondrial functionand ultrastructure in the hippocampus of pilocarpine-treated rat. Epilepsy Res 2013;108:162–70.

Gao J, Yao H, Pan XD, Xie AM, Zhang L, Song JH, et al. Alteration of mitochondrial functionand ultrastructure in the hippocampus of pilocarpine-treated rat. Epilepsy Res 2014;108:162–70.

Garcia-Nino WR, Pedraza-Chaverri J. Protective effect of curcumin against heavy metals-induced liver damage. Food Chem Toxicol 2014;69C:182–201.

Garcia-NinoWR, Tapia E, Zazueta C, Zatarain-Barron ZL, Hernandez-Pando R, Vega-GarciaCC, et al. Curcumin pretreatment prevents potassium dichromate-induced hepato-toxicity, oxidative stress, decreased respiratory complex I activity, and membranepermeability transition pore opening. Evid Based Complement Alternat Med 2013;2013:424692.

Genkova-Papazova MG, Lazarova-Bakarova MB. Pentylenetetrazole kindling impairslong-term memory in rats. Eur Neuropsychopharmacol 1995;5:53–6.

Griffiths DE, Houghton RL. Studies on energy-linked reactions: modified mitochondrialATPase of oligomycin-resistant mutants of Saccharomyces cerevisiae. Eur J Biochem1974;46:157–67.

Gupta SC, Patchva S, Aggarwal BB. Therapeutic roles of curcumin: lessons learned fromclinical trials. AAPS J 2013;15:195–218.

Hansen SL, Sperling BB, Sanchez C. Anticonvulsant and antiepileptogenic effects of GABAAreceptor ligands in pentylenetetrazole-kindled mice. Prog NeuropsychopharmacolBiol Psychiatry 2004;28:105–13.

He LF, Chen HJ, Qian LH, Chen GY, Buzby JS. Curcumin protects pre-oligodendrocytes fromactivated microglia in vitro and in vivo. Brain Res 2010;1339:60–9.

HishikawaN, Takahashi Y, Amakusa Y, Tanno Y, Tuji Y, Niwa H, et al. Effects of turmeric onAlzheimer's disease with behavioral and psychological symptoms of dementia. Ayu2012;33:499–504.

Huang HC, Xu K, Jiang ZF. Curcumin-mediated neuroprotection against amyloid-beta-inducedmitochondrial dysfunction involves the inhibition of GSK-3beta. J AlzheimersDis 2012;32:981–96.

Jagatha B, Mythri RB, Vali S, Bharath MM. Curcumin treatment alleviates the effects ofglutathione depletion in vitro and in vivo: therapeutic implications for Parkinson'sdisease explained via in silico studies. Free Radic Biol Med 2008;44:907–17.

Jayaraj RL, Tamilselvam K, Manivasagam T, Elangovan N. Neuroprotective effect of CNB-001, a novel pyrazole derivative of curcumin on biochemical and apoptotic markersagainst rotenone-induced SK–N–SH cellular model of Parkinson's disease. J MolNeurosci 2013;51:863–70.

Kamboj A, Sandhir R. Perturbed synaptosomal calcium homeostasis and behavioral deficitsfollowing carbofuran exposure: neuroprotection by N-acetylcysteine. Neurochem Res2007;32:507–16.

Kang HC, Lee YM, Kim HD, Lee JS, Slama A. Safe and effective use of the ketogenic diet inchildren with epilepsy and mitochondrial respiratory chain complex defects. Epilepsia2007;48:82–8.

King TE, Howard RL. Preparation and properties of soluble NADH dehydrogenase fromcardiac muscle. Methods in enzymology. New York: Academic Press; 1967. p. 275–6.

King ME, Godman GC, King DW. Respiratory enzymes and mitochondrial morphology ofHeLa and L cells treated with chloramphenicol and ethidium bromide. J Cell Biol1972;53:127–42.

King TE, Ohnishi T, Winter DB, Wu JT. Biochemical and EPR probes for structure–functionstudies of iron sulfur centers of succinate dehydrogenase. Adv ExpMed Biol 1976;74:182–227.

Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in celldeath. Physiol Rev 2007;87:99–163.

Kudin AP, Kudina TA, Seyfried J, Vielhaber S, Beck H, Elger CE, et al. Seizure-dependentmodulation of mitochondrial oxidative phosphorylation in rat hippocampus. Eur JNeurosci 2002;15:1105–14.

Kudin AP, Bimpong-Buta NY, Vielhaber S, Elger CE, Kunz WS. Characterization ofsuperoxide-producing sites in isolated brain mitochondria. J Biol Chem 2004;279:4127–35.

Kudin AP, Zsurka G, Elger CE, Kunz WS. Mitochondrial involvement in temporal lobeepilepsy. Exp Neurol 2009;218:326–32.

Kumar A, Dogra S, Prakash A. Protective effect of curcumin (Curcuma longa), against alu-minium toxicity: possible behavioral and biochemical alterations in rats. Behav BrainRes 2009;205:384–90.

64 H. Kaur et al. / Pharmacology, Biochemistry and Behavior 125 (2014) 55–64

Kumar A, Lalitha S, Mishra J. Possible nitric oxide mechanism in the protective effectof hesperidin against pentylenetetrazole (PTZ)-induced kindling and associatedcognitive dysfunction in mice. Epilepsy Behav 2013;29:103–11.

KunzWS, Kudin AP, Vielhaber S, Blumcke I, ZuschratterW, Schramm J, et al. Mitochondrialcomplex I deficiency in the epileptic focus of patients with temporal lobe epilepsy.Ann Neurol 2000;48:766–73.

Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, et al. Determination ofcarbonyl content in oxidatively modified proteins. Methods Enzymol 1990;186:464–78.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Proteinmeasurement with the Folin phenolreagent. J Biol Chem 1951;193:265–75.

Ma J, Liu J, Yu H, Wang Q, Chen Y, Xiang L. Curcumin promotes nerve regeneration andfunctional recovery in rat model of nerve crush injury. Neurosci Lett 2013;547:26–31.

Macdonald RL, Kapur J. Acute cellular alterations in the hippocampus after status epilep-ticus. Epilepsia 1999;40(Suppl. 1):S9–S20. [discussion S21–22].

Mehla J, Reeta KH, Gupta P, Gupta YK. Protective effect of curcumin against seizures andcognitive impairment in a pentylenetetrazole-kindled epileptic rat model. Life Sci2010;87:596–603.

Meldrum BS. Excitotoxicity and selective neuronal loss in epilepsy. Brain Pathol 1993;3:405–12.

Morimoto K, Fahnestock M, Racine RJ. Kindling and status epilepticus models of epilepsy:rewiring the brain. Prog Neurobiol 2004;73:1–60.

Moser EI, Krobert KA, Moser MB, Morris RG. Impaired spatial learning after saturation oflong-term potentiation. Science 1998;281:2038–42.

Navarro A, Boveris A. Brain mitochondrial dysfunction in aging, neurodegeneration, andParkinson's disease. Front Aging Neurosci 2010;2:1–11.

Ogita K, Okuda H, Yamamoto Y, Nishiyama N, Yoneda Y. In vivo neuroprotective roleof NMDA receptors against kainate-induced excitotoxicity in murine hippocampalpyramidal neurons. J Neurochem 2003;85:1336–46.

Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituricacid reaction. Anal Biochem 1979;95:351–8.

Pan R, Qiu S, Lu DX, Dong J. Curcumin improves learning and memory ability and its neu-roprotective mechanism in mice. Chin Med J (Engl) 2008;121:832–9.

Panayiotopoulos CP. Clinical aspects of the diagnosis of epileptic seizures and epilepticsyndromes. In: Panayiotopoulos CP, editor. The epilepsies: seizures, syndromes andmanagement. Oxford: Bladon Medical Publishing; 2005. p. 1–47.

Papp A, Feher O, Erdelyi L. The ionic mechanism of the pentylenetetrazol convulsions.Acta Biol Hung 1987;38:349–61.

Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure.Electroencephalogr Clin Neurophysiol 1972;32:281–94.

Rahman S. Mitochondrial disease and epilepsy. Dev Med Child Neurol 2012;54:397–406.Rajabzadeh A, Bideskan AE, Fazel A, Sankian M, Rafatpanah H, Haghir H. The effect of PTZ-

induced epileptic seizures on hippocampal expression of PSA-NCAM in offspringborn to kindled rats. J Biomed Sci 2012;19:56.

Reddy AC, Lokesh BR. Studies on the inhibitory effects of curcumin and eugenol on theformation of reactive oxygen species and the oxidation of ferrous iron. Mol CellBiochem 1994;137:1–8.

Ringman JM, Frautschy SA, Teng E, Begum AN, Bardens J, Beigi M, et al. Oral curcumin forAlzheimer's disease: tolerability and efficacy in a 24-week randomized, double blind,placebo-controlled study. Alzheimers Res Ther 2012;4:43.

Roberts JC, Francetic DJ. The importance of sample preparation and storage in glutathioneanalysis. Anal Biochem 1993;211:183–7.

Rossler AS, Schroder H, Dodd RH, Chapouthier G, Grecksch G. Benzodiazepine receptorinverse agonist-induced kindling of rats alters learning and glutamate binding.Pharmacol Biochem Behav 2000;67:169–75.

Rothman SM, Olney JW. Glutamate and the pathophysiology of hypoxic–ischemic braindamage. Ann Neurol 1986;19:105–11.

Rowley S, Patel M. Mitochondrial involvement and oxidative stress in temporal lobeepilepsy. Free Radic Biol Med 2010;62:121–31.

Sandhir R, Yadav A, Mehrotra A, Sunkaria A, Singh A, Sharma S. Curcumin nanoparticlesattenuate neurochemical and neurobehavioral deficits in experimental model ofHuntington's disease. Neuromolecular Med 2014;16:106–18.

Shah BH, Nawaz Z, Pertani SA, Roomi A, Mahmood H, Saeed SA, et al. Inhibitory effect ofcurcumin, a food spice from turmeric, on platelet-activating factor- and arachidonicacid-mediated platelet aggregation through inhibition of thromboxane formationand Ca2+ signaling. Biochem Pharmacol 1999;58:1167–72.

Sood PK, Nahar U, Nehru B. Curcumin attenuates aluminum-induced oxidative stress andmitochondrial dysfunction in rat brain. Neurotox Res 2011;20:351–61.

Sottocasa GL, Kuylenstierna B, Ernster L, Bergstrand A. An electron-transport systemassociatedwith the outermembrane of livermitochondria. A biochemical andmorpho-logical study. J Cell Biol 1967;32:415–38.

Srivastava P, Yadav RS, Chadravanshi LP, Shukla RK, Dhuriya YK, Chauhan LK, et al.Unraveling themechanism of neuroprotection of curcumin in arsenic induced cholin-ergic dysfunctions in rats. Toxicol Appl Pharmacol 2014. (in press).

Sudha K, Rao AV, Rao A. Oxidative stress and antioxidants in epilepsy. Clin Chim Acta2001;303:19–24.

Sumanont Y, Murakami Y, Tohda M, Vajragupta O, Watanabe H, Matsumoto K. Effects ofmanganese complexes of curcumin and diacetylcurcumin on kainic acid-inducedneurotoxic responses in the rat hippocampus. Biol Pharm Bull 2007;30:1732–9.

Sutula TP, Cavazos JE, Woodard AR. Long-term structural and functional alterationsinduced in the hippocampus by kindling: implications for memory dysfunction andthe development of epilepsy. Hippocampus 1994;4:254–8.

Szewczyk A, Wojtczak L. Mitochondria as a pharmacological target. Pharmacol Rev 2002;54:101–27.

Tedeschi H, Harris DL. Some observations on the photometric estimation of mitochondrialvolume. Biochim Biophys Acta 1958;28:392–402.

Thiyagarajan M, Sharma SS. Neuroprotective effect of curcumin in middle cerebral arteryocclusion induced focal cerebral ischemia in rats. Life Sci 2004;74:969–85.

Urbanska EM, Blaszczak P, Saran T, Kleinrok Z, Turski WA. Mitochondrial toxin 3-nitropropionic acid evokes seizures in mice. Eur J Pharmacol 1998;359:55–8.

Uzum G, Akgun-Dar K, Aksu U. The effects of atorvastatin on memory deficit and seizuresusceptibility in pentylentetrazole-kindled rats. Epilepsy Behav 2010;19:284–9.

Walia KS, Khan EA, Ko DH, Raza SS, Khan YN. Side effects of antiepileptics—a review. PainPract 2004;4:194–203.

Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assayusing microplate reader. Free Radic Biol Med 1999;27:612–6.

Yadav RS, Sankhwar ML, Shukla RK, Chandra R, Pant AB, Islam F, et al. Attenuation ofarsenic neurotoxicity by curcumin in rats. Toxicol Appl Pharmacol 2009;240:367–76.

Yamamoto H, Tang HW. Preventive effect of melatonin against cyanide-induced seizuresand lipid peroxidation in mice. Neurosci Lett 1996;207:89–92.

Yan MH, Wang X, Zhu X. Mitochondrial defects and oxidative stress in Alzheimer diseaseand Parkinson disease. Free Radic Biol Med 2013;62:90–101.

Zhang LS, Chen JF, Chen GF, Hu XY, Ding MP. Effects of thioperamide on seizure develop-ment and memory impairment induced by pentylenetetrazole-kindling epilepsy inrats. Chin Med J (Engl) 2013;126:95–100.

Zhang X, Chen G, Lu Y, Liu J, Fang M, Luo J, et al. Association of mitochondrial Letm1 withepileptic seizures. Cereb Cortex 2013. (in press).