Understanding the anti-kindling role and its mechanism of Resveratrol in Pentylenetetrazole induced-kindling in a rat model

Pharmacology, Biochemistry and Behavior 120 (2014) 57–64

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Understanding the anti-kindling role and its mechanism of Resveratrol in Pentylenetetrazole induced-kindling in a rat model Lekha Saha ⁎, Amitava Chakrabarti Department of Pharmacology, Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh, India

a r t i c l e

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Article history: Received 8 November 2013 Received in revised form 18 January 2014 Accepted 22 January 2014 Available online 30 January 2014 Keywords: Pentylenetetrazole Resveratrol Epileptogenesis Kindling Rat Antioxidant

a b s t r a c t Background: Resveratrol is a polyphone chemical found in a number of plant species, including peanuts and grapes, but with significant amounts in red wine. In normal plant physiology, Resveratrol is produced as a defensive response to injury or parasitic attacks. Resveratrol has diverse biological properties and actions with potential clinical applications, including anti-inflammatory, antioxidant, anti proliferative, and neuroprotective effects. Aim: The aim of the present study was to explore the effect and mechanism of Resveratrol in Pentylenetetrazole (PTZ) induced kindling in rats. Materials and methods: In a PTZ kindled Wistar rat model, different doses of Resveratrol (25 mg/kg, 50 mg/kg and 75 mg/kg) were administered orally 30 min before the PTZ injection. The PTZ injection was given on alternate day till the animal became fully kindled or till 10 weeks. The following parameters were compared between control and various experimental groups: the course of kindling, stages of seizures, histopathological scoring of hippocampus, antioxidant parameters, DNA fragmentation and caspase-3 expression in the hippocampus, and neuron-specific enolase in the blood. One way ANOVA followed by Bonferroni post hoc analysis and Fischer's Exact test were used for statistical analyses. The results: In the present study, Resveratrol showed dose-dependent anti-seizure effect. Resveratrol (75 mg/kg) significantly increased the latency to myoclonic jerks, clinic seizures as well as generalized tonic-clinic seizures, improved the seizure score and decreased the number of myoclonic jerks. PTZ induced kindling caused a significant neuronal injury, oxidative stress and apoptosis which were reversed by pretreatment with Resveratrol in a dose-dependent manner. Conclusion: Our study suggests that Resveratrol has a potential antiepileptogenic effect on PTZ-induced kindling in rats. The possible underlying mechanisms of Resveratrol as an antiepileptic agent may be due to its antioxidative property and neuroprotective effect. © 2014 Elsevier Inc. All rights reserved.

1. Introduction About a dozen of new drugs have become available for epilepsy over the last 15–20 years. Current medications primarily act to symptomatically suppress seizure; however, there is minimal clinical evidence that they correct the underlying brain abnormalities causing epilepsy (epileptogenesis) or alter its natural history and long-term prognosis (Temkin, 2001). Thus, there is a need to identify drugs (antiepileptogenic drugs) which modify the disease as well as can inhibit the progression of epilepsy or completely prevent its development. However, at this point, no proven antiepileptogenic therapies have been developed for clinical use. Many antiepileptic medications were identified through screening assays that assessed efficacy against acutely provoked seizures in nonepileptic animals. As a result, they inhibit seizures through mechanisms that directly decrease neuronal excitability, such as by modulating neurotransmitter receptors and ion channels. A ⁎ Corresponding author at: Deptt of Pharmacology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh 160012, India. Tel.: +91 1722755253. E-mail address: [email protected] (L. Saha). 0091-3057/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pbb.2014.01.010

better strategy for developing antiepileptogenic therapies might be to interrupt the initial mechanistic events that trigger downstream cellular and molecular changes in the brain that lead to seizures. This approach is particularly plausible and clinically relevant for acquiring epilepsies that are caused by a remote brain injury (e.g., head trauma, stroke), with seizures starting after a prolonged period, from months to years later. During the latent period of epileptogenesis, histopathological and molecular changes (e.g., neuronal death, synaptic reorganization) that promote epileptogenesis occur and could be targeted for correction by an antiepileptogenic therapy. Epileptogenesis can be studied in numerous rodent models of symptomatic epilepsy, including kindling, post-status epilepticus models of Temporal Lobe Epilepsy, Traumatic Brain Injury, and stroke models, and models of febrile seizures (Walker et al., 2002; Stables et al., 2003; Pitkanen et al., 2007). There are two types of experimental protocol to evaluate drug effects on kindling acquisition: 1) drug is administered before each stimulation and the effects on kindling acquisition are determined relative to vehicle controls; 2) anticonvulsant drug effects study in fully kindled rats (Loscher and Brandt, 2010). In the present study we have followed the first protocol and demonstrated the effect on kindling acquisition.

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After the realization of reduced cardiac risk by red wine popularly referred as French paradox, much interest has emerged in Resveratrol, which is the active constituent of red wine. Resveratrol (RESV; 3, 5, 4′-tri-hydroxy stilbene) is a type of polyphenol and an antimicrobial substance synthesized de novo by plants (a phytoalexin). RESV is found in the skin of red grapes and is a component of red wine (Fremont, 2000; Orallo, 2008). The other sources of RESV include raspberries, mulberries, plums, peanuts, bilberries, blueberries, cranberries, Scots pine, and Japanese knotweed. RESV is synthesized instinctively by the above plants as a protection to counter the bacterial and fungal infections, stress and injury (Balestrazzi et al., 2009; Maddox et al., 2010). Studies in animal models imply a number of other beneficial health effects of RESV, which comprise anti-ischemic, antiviral, antioxidant and anti-inflammatory properties (Belguendouz et al., 1997; Jang et al., 1999; Manna et al., 2000; Sato et al., 2000; Kraft et al., 2009; Campagna and Rivas, 2010; Robich et al., 2010; Sun et al., 2010). Pertaining to the central nervous system, multiple cell culture investigations and in vivo studies in animal models of neurodegenerative diseases/brain injury point out that RESV is a potent neuroprotective compound (Sun et al., 1997; Bastianetto et al., 2000; Jang and Surh, 2003; Han et al., 2004; Sinha et al., 2002; Wang et al., 2002; Baur and Sinclair, 2006; Sakata et al., 2010; Liu et al., 2010; Shindler et al., 2010; Singleton et al., 2010). Previous studies by various researchers demonstrated that RESV protects against neuronal death and acute seizures induced by the ionotropic glutamate receptor agonist, kainate and FeCl3 (Gupta et al., 2001, 2002; Virgili and Contestabile, 2000). Studies have also explored the potential mechanism of RESV mediated neuroprotection against acute seizures (Shetty, 2011; Wang et al., 2004; Gao and Hu, 2005; Li et al., 2005). Study by Wu et al. demonstrated the effect of RESV on SE-induced epileptogenesis and chronic epilepsy induced by kainite (Wu et al., 2009). With this background the present study was planned to explore the effect of RESV on the kindling acquisition by Pentylenetetrazole (PTZ) in rats and its mechanism. 2. Materials and methods 2.1. Experimental animals 15–20 weeks old male Wistar rats weighing between 200 to 250 g were used for the present study. The animals were obtained from the maintained inbred colony of the institute central animal house and maintained at 23 ± 2 °C with a relative humidity of 65 ± 5% in 12 h light/dark cycle. Animals had free access to standard pellet chow diet and tap water ad libitum. Animals were acclimatized to laboratory conditions for at least 7 days prior to experimentation. Institutional Animal Ethics Committee (IAEC) approval (No.55/IAEC/267 dated 27.07.2011) was obtained before the start of the study and the study was carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals guidelines. 2.2. Treatment scheduled A total of 83 rats were divided into six groups: Group I: Control (physiological saline) (n = 8); Group II: PTZ (physiological saline + PTZ 30 mg/kg) (n = 15); Group III: VPA 200 (Sodium Valproate 200 mg/kg + PTZ 30 mg/kg) (n = 15); Group IV: RESV 25 (Resveratrol 25 mg/kg + PTZ 30 mg/kg) (n = 15); Group V: RESV 50 (Resveratrol 50 mg/kg + PTZ 30 mg/kg) (n = 15); Group VI: RESV 75 (Resveratrol 75 mg/kg + PTZ 30 mg/kg) (n = 15). 2.3. Drug preparation and dosing schedule PTZ was dissolved in 0.9% physiological saline and injected intraperitoneally (i.p.) in a volume not exceeding 10 ml/kg, at a sub convulsive dose of 30 mg/kg every alternate day until the animal developed

kindling or up to 10 weeks. Resveratrol (25, 50, 75 mg/kg) was given orally 30 mins before PTZ injection till the animal developed kindling or up to 10 weeks. Sodium Valporate (200 mg/kg) was dissolved in 0.9% physiological saline and administered by intraperitoneal injection 30 min before PTZ injection till the animal develops kindling or up to 10 weeks. Resveratrol, sodium valproate and Pentylenetetrazole was purchased from Sigma-Aldrich, USA. 2.4. Pentylenetetrazole (PTZ) induced kindling in rats PTZ was injected i.p. as mentioned above. After each injection of PTZ, the rats were placed singly in isolated transparent Plexiglas cages and were observed for 2 hrs. The intensity of convulsions was rated according to the Racine scale (Racine, 1972) as follows: 0 — no response; 1 — ear and facial twitching; 2 — myoclonic jerks without rearing; 3 — myoclonic jerks with rearing; 4 — turn over into side position, clonic–tonic seizures; and 5 — turn over into back position, generalized tonic–clonic convulsions. an animal was considered fully kindled when it exhibits stage 4 or 5 of seizure score on three consecutive trials. there was no mortality or morbidity in any of the animals in any group. 2.5. Studies with Hippocampus When the animal became fully kindled (exhibits stage 4 or 5 of seizure score on three consecutive trials), on the next day, it was sacrificed by decapitation under the overdose of intraperitoneal pentobarbitone anesthesia. The hippocampus was carefully dissected out of the brain and the following parameters were evaluated: 2.5.1. Histopathology of the hippocampus One half of each brain was fixed in 10% formalin and stored for histopathological studies using hemotoxylin and eosin (H&E) stain. Thereafter, tissue was sliced, routinely processed, and embedded in paraffin wax. 5 μm coronal paraffin sections were cut, mounted and stained by haematoxylin and eosin. The acidophilic neuron, identified by intense cytoplasmic eosinophilia accompanied by chromatin dispersion with loss of nuclear membrane integrity (Fujikawa et al., 2000), was perceived as the marker for irreversible neuronal damage at the cellular level. The numbers of acidophilic neurons in different regions of the hippocampus were estimated on a 0–3 grading scale, 0 = none, 0.5 = slight (b 10%), 1.0 = mild (10–25%), 1.5 = mild-to-moderate (26–45%), 2.0 = moderate (46–54%), 2.5 = moderate-to-severe (55–75%), and 3.0 = severe (N75%), as previously published (Fujikawa et al., 2000). The boundaries of each region were shown in Fig. 1. Sections were examined by a blinded investigator without knowledge of any other data on that animal. 2.5.2. Hippocampal DNA fragmentation study DNA was isolated from hippocampal brain specimens using DNA isolation kits and stored for Agarose gel electrophoresis. 2.5.3. Hippocampal oxidative stress studies Once the animal became fully kindled, on the next day, it was sacrificed. Brain was perfused with ice cold 0.9% physiological saline through cardiac puncture before decapitation. The hippocampus was promptly excised after decapitation, weighed and chilled in ice cold 0.9% physiological saline and the oxidative stress parameters were measured immediately. 2.5.3.1. Estimations of thiobarbituric acid-reactive substance (TBARS). The extent of lipid peroxidation was estimated according to the method of Ohokawa et al. in tissue homogenate (Ohokawa et al., 1979). Tissue homogenate was prepared in a ratio of 1 g of wet tissue to 9 ml of phosphate buffer (pH 7.2) using a homogenizer. To 0.1 ml of the homogenate, 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid solution, 1.5 ml of a 0.8 % aqueous solution of thiobarbituric acid were

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Fig. 1. Example of a hippocampal region where the extent of hippocampal injury was evaluated. CA3 region begins in the region bisected by an imaginary line connecting the two edges of the dentate granule cells of the CA2 region. CA1 extended from the CA2 region to an imaginary line drawn perpendicular to the crest of the dentate gyrus (DG; arrow). The hilus (H) was defined as the inner border of the granule cell layer (GCL) together with the area formed by two imaginary straight lines connecting the two tips of the GCL with the proximal end of the CA3c area. Scale bar = 100 μm.

added. The mixture was finally made up to 4.0 ml with distilled water, and heated at 95 ºC for 60 min. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of the mixture of n-butanol and pyridine (15:1, v/v) were added, and the mixture was shaken vigorously. After centrifugation at 4000 rpm for 10 min, the absorbance of the organic layer (upper layer) was measured at 532 nm in a spectrophotometer against a blank containing all the reagents except the homogenate. The MDA equivalents of the samples were calculated using the extinction coefficient 1.56 × 105 M−1 cm−1. 2.5.3.2. Determination of catalase activity (CAT). The activity of catalase was measured by the method of Luck (Luck, 1963). A 10% w/v homogenate of the hippocampus was prepared in phosphate buffer. The homogenate was centrifuged and the supernatant was used for the enzyme assay. In short, the reaction mixture contained Tris (50 mM)EDTA (5 mM) buffer, pH 7.0, 10 mM H2O2 (in 0.1 M KH2PO4 buffer, pH 7.0) in test cuvette. The reference cuvette contained Tris-EDTA solution and distilled water only. The contents of both the cuvettes were incubated at 37 °C for 10 mins. The reaction was started by the addition of tissue homogenate to both reference as well as test cuvettes. The rate of elimination of H2O2 by catalase was measured by recording the rate of change of absorbance per min at 240 nm for 4 min. Catalase activity was expressed as μmol of H2O2 consumed/min/mg protein using a molar extinction coefficient of 43.6 mM−1 cm−1. 2.5.3.3. Reduced glutathione (GSH) estimations. Assay of GSH was performed in tissue homogenates by the method of Moron et al. (Moron et al., 1979). To 500 μl homogenate 100 μl of 25% trichloroacetic acid (TCA) was added. The precipitated proteins were separated by centrifugation at 2000 ×g for 15 mins. Supernatants were diluted with 0.2 M sodium phosphate buffer, pH 8.0. To this, 2.0 ml of 0.6 mM DTNB was added. The colored complex formed by DTNB and GSH was measured spectrophotometricaly at 412 nm against a reference cuvette containing 0.1 or 0.2 ml of 5% TCA. A standard curve of GSH was plotted with every

set of assays. All the assays were done in duplicates. The levels of GSH were expressed as μg of GSH/mg protein.

2.5.4. Protein expression study of caspase 3 in hippocampus by western blot analysis The frozen brain tissue was homogenized in lysis buffer. Insoluble materials were removed by centrifugation. Approximately 50 μg of total protein extracts was subjected to SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) followed by electrophoretic transfer on nitrocellulose membrane (Towbin et al., 1979). Bovine serum albumin in TBS20 mM(pH7.2)/Tween-20 was used to block the membranes. Specific primary antibody against active caspase 3, and β-actin was added to the membrane separately followed by washing and addition of respective HRP-conjugated secondary antibody. The blots were developed using enhanced chemilumenescence plus detection reagents A and B (Amersham Biosciences, UK Limited). The change in band intensity in the case of each protein was quantified by scanning densitometry using scion image 4.3 software.

2.6. Estimation of neuron-specific enolase (NSE) in the blood 2 ml of blood was collected from each anesthetized animal by cardiac puncture prior to decapitation. The blood sample was subjected to the estimation of Neuron-Specific Enolase (NSE) by ELISA kit (CUSABIO BIOTECH CO., LTD., China) according to the manufacturer's instructions.

2.7. Statistical analysis Data are expressed as the mean ± SD. One-way analysis of variance (ANOVA) with bonferroni/fisher's exact post hoc analyses were performed using SPSS (20.0 version; LEAD Technologies, Chicago, IL, U.S.A.). Statistical significance was considered at P b 0.05.

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Table 1 The seizure score in PTZ treated rats in various treatment groups at different time point. PTZ was administered on alternate day. Data are expressed as Mean ± SD. Data were compared by one way ANOVA followed by Bonferroni post hoc analysis. * P b 0.05 compared to PTZ group; # p b 0.05 compared to RESV 25 group; $ P b 0.05 compared to RESV 50 group; + p b 0.05 compared to RESV 75 group. Time Control (week)

PTZ

10 20 30 40 50 60 70 80 90 10 0

0.80 1.45 1.94 2.35 2.93 3.39 4.42 4.50 4.71 4.96

± ± ± ± ± ± ± ± ± ±

0.31 0.18 0.15 0.25 0.30 0.12 0.24 0.18 0.12 0.12

VPA 200

RESV25

0 0.62 0.67 0.63 0 0 0.08 0.42 0.43 0.42

0.83 1.50 2 2.54 3.21 3.38 3.13 3.13 3.13 3.17

± ± ± ± ± ± ± ± ± ±

0 *#$+ 0.17*#$+ 0.25 *#$+ 0.17 *#$+ 0 *#$+ 0*#$+ 0.15*#$+ 0.12*#$+ 0.12 *#$+ 0.12*#$+

± ± ± ± ± ± ± ± ± ±

0.31 0.31 0.47 0.47 0.62 0.33 0.31 0.31* 0.31* 0.25*

RESV 50

RESV 75

0.67 1.45 1.92 2.21 2.33 2.25 1.92 1.96 1.96 1.96

0.75 1.67 2.21 2.75 2.96 2.83 2.58 2.58 2.58 2.58

± ± ± ± ± ± ± ± ± ±

0.36 0.31 0.15 0.36 + 0.25* # + 0.15 * #+ 0.15* # + 0.15* # + 0.15* # + 0.15* # +

± ± ± ± ± ± ± ± ± ±

0.35 0.31 0.35 0.24 0.22 0.31*# 0.30*# 0.30*# 0.30* # 0.30* #

3. Results

3.3. The effect of various treatments on DNA fragmentation in hippocampus

3.1. Effect of Resveratrol on PTZ induced kindling model

All the brain samples from different treatment groups were subjected to DNA fragmentation. The PTZ group showed laddering pattern with the formation of a large number of fragments ranging from 1517 base pairs to 100 base pair fragments indicating DNA fragmentation. In contrast, VPA 200, RESV 50 and RESV 75 group did not show formation of small fragments and hence no prominent laddering pattern (Fig. 3).

Rats in the control group showed a seizure score of zero (0) throughout the 10 weeks of the study period. In PTZ treated group, the rats showed a gradual increase in the seizure score of 0.80 ± 0.30 at 1st week to 4.96 ± 0.11 at the end of 10 weeks of the study period (Table 1). Further, in the PTZ treated group, the number of animals developed kindling showed progressive increase in a time dependent manner from 0/8 (0%) at 1st week to 4/8 (50%) at 4th week to 7/8 (87.5%) at the 10th week (Table 2). In VPA 200 treatment group, none of the animal developed kindling at any point of time (Table 2). RESV showed dose-dependent protection against PTZ induced kindling in rats. All the three doses of RESV showed decrease in seizure scoring and significantly less scoring was seen with both 50 mg and 75 mg/kg doses when compared with a PTZ group (Table 1). Regarding the number of animals developed kindling, significantly less number of animals developed kindling with all three doses of RESV. In RESV 50 and RESV 75 groups' only one animal in each group showed kindling at the end of 10 weeks (Table 2).

3.2. Histopathological changes in the hippocampus We rarely observed acidophilic neurons in different sectors of the PTZ-treated rats' hippocampus. Despite lacking of evidence for PTZinduced cell necrosis, our study found gliosis in area CA1 and the hilus of the hippocampus in PTZ treated rats (Table 3, Fig. 2), suggesting the existence of neuronal loss. In VPA 200 group, there was less or no gliosis. Pretreatment of RESV 50 and 75, there was also less gliosis as compared to PTZ group.

3.4. Lipid peroxidation parameters 3.4.1. Effect of Resveratrol on malondialdehyde (MDA) level in rat brain Pretreatment with VPA 200 and three different doses of RESV (25 mg/kg, 50 mg/kg, 75 mg/kg, i.p.) significantly reduced the whole brain MDA level in the animals and it was statistically significant when compared to PTZ treated groups (Table 4).

3.4.2. Effect of Resveratrol on reduced glutathione (GSH) levels in the rat brain Treatment with PTZ alone significantly reduced the GSH levels in rat brain when compared with normal control rat. Pre-treatment with VPA 200 and three different doses of RESV (25 mg/kg, 50 mg/kg, 75 mg/kg, i.p.) significantly increased the level of GSH in the brain as compared to that of PTZ alone treated group (Table 4).

3.4.3. Effect of Resveratrol on catalase activity in rat brain The catalase activity was significantly decreased in PTZ treated rats when compared to the control animals and it was significantly higher when pretreated with VPA 200 and RESV (Table 4).

Table 2 The number of animals (%) developed kindling in various experimental groups at different time point. PTZ was administered on alternate day. Data are expressed as number (%) of animals. Data were analyzed by Fisher's exact test. @ P b 0.05 compared to control; * P b 0.05 compared to PTZ; $ P b 0.05 compared to VPA 200; # P b 0.05 within group comparison between 1st and 10th week. Time (week)

1 2 3 4 5 6 7 8 9 10

Control

PTZ

VPA 200

RESV 25

RESV 50

No. of animals (%)

No. of animals (%)

No. of animals (%)

No. of animals (%)

No. of animals (%)

RESV 75 No. of animals (%)

0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

0 (0) 2 (25) 3 (37.5) 4 (50) 4 (50) 5 (62.5) 6 (75) 6 (75) 7 (87.5) 7 (87.5) @ # $

0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) *

0 (0) 1 (12.5) 3 (37.5) 3 (37.5) 4 (50) 4 (50) 4 (50) 5 (62.5) 5 (62.5) 5 (62.5) @ # $

0 (0) 0 (0) 1 (12.5) 1 (12.5) 1 (12.5) 1 (12.5) 1 (12.5) 1 (12.5) 1 (12.5) 1 (12.5) *

0 (0) 0 (0) 0 (0) 1 (12.5) 1 (12.5) 1 (12.5) 1 (12.5) 1 (12.5) 1 (12.5) 1 (12.5) *

L. Saha, A. Chakrabarti / Pharmacology, Biochemistry and Behavior 120 (2014) 57–64 Table 3 Effect of various treatments on Damage scores in the hippocampus of the rats assessed. The data represent means ± SEM of neuronal damage scores assessed by numbers of acidophilic neurons by H&E stain. Brain region CA1 Control PTZ VPA 200 RESV 25 RESV 50 RESV 75

0.15 0.17 0.00 0.16 0.15 0.17

CA3 ± ± ± ± ± ±

0.11 0.11 0.00 0.11 0.11 0.04

0.17 0.09 0.08 0.17 0.08 0.12

H ± ± ± ± ± ±

0.11 0.08 0.08 0.11 0.08 0.04

0.17 0.08 0.08 0.00 0.08 0.10

DG ± ± ± ± ± ±

0.11 0.08 0.08 0.00 0.08 0.04

0.17 0.17 0.00 0.25 0.17 0.13

± ± ± ± ± ±

0.11 0.11 0.00 0.11 0.11 0.04

3.5. Effect of Resveratrol on serum neuron specific enolase (sNSE) level in rats The level of sNSE in serum was significantly elevated in PTZ (17.46 ± 1.06 ng/ml) treated group as compared to the control animals (5.03 ± 0.28 ng/ml). In the sodium valproate pre-treated group (VPA 200), there was significant reduction (6.12 ± 0.28 ng/ml) in s-NSE level as compared to PTZ treated group. All the three doses of RESV also showed significant reduction in s-NSE level. As compared to 25 mg/kg, RESV 75 mg/kg and 50 mg/kg showed significant reduction in s-NSE levels (Table 4).

3.6. Protein expression study of caspase 3 in hippocampus by western blot analysis Fig. 4 shows the protein expression of β actin (Fig. 4a) and Caspase 3 (Fig. 4b) in hippocampus of the rats in different groups. Induction of kindling with PTZ caused a significant increase in expression of caspase 3 in the hippocampus as compared to control group. Pre treatment with RESV (25–75 mg/kg) caused appreciable decreased in the expression of caspase 3 in the hippocampus and the expression was negligible with RESV 75 mg/kg dose (Fig. 4). In the VPA 200 group, the expression of caspase 3 was also negligible.

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3.7. Discussion The present study demonstrated the protective effect of RESV in PTZ induced kindling in a rat model. The antiepileptogenic effect of RESV demonstrated in our study is in consistent with the previous studies. In the present study we have also tried to demonstrate the underlying antiepileptogenic mechanism of RESV by exploring the oxidative neuronal injury and apoptosis pathways. A study by Virgili and Contestabile (2000) was the first to suggest a neuroprotective property for RESV against excitotoxic brain injury (Virgili and Contestabile, 2000). They compared the effect of systemic administration of an excitotoxin kainic acid (KA) between young adult rats that were chronically treated with RESV and control young adult rats. They found that chronic RESV treatment prior to the KA administration considerably reduced the damage caused by KA in the olfactory cortex and the hippocampus. Following this, a study demonstrated the protective effect of RESV pretreatment on KA induced seizures and oxidative stress (Gupta et al., 2002). They found that a single dose of RESV (at 40 mg/kg i.p.) five-minutes prior to KA treatment (10 mg/kg i.p.) increased the latency to convulsions but was unable to completely inhibit the convulsions. However, with multiple doses of RESV treatment (i.e. at 5 min prior to KA injection and at 30 and 90 min post-KA injection), the incidence of convulsions was significantly reduced. The above RESV treatment regimen also inhibited the KA-injury related increases in the level of MDA, suggesting that antioxidant function is one of the mechanisms by which RESV mediates neuroprotection against excitotoxic injury and acute seizures. Furthermore, a study has shown neuroprotection when RESV was administered prior to intracortical placement of FeCl3 (Gupta et al., 2001). In the absence of RESV treatment, FeCl3 treated animals exhibited significant epileptiform EEG discharges and increased levels of the oxidative stress marker MDA in the brain tissue. However, in animals that received RESV (20 or 40 mg/kg i.p.) 30 min prior to FeCl3 treatment, the onset of the epileptiform EEG discharges was delayed and MDA levels were reduced. A subsequent study investigated this issue and showed that prior RESV treatment protects against neurotoxicity induced by KA (Wang et al., 2004). Collectively, the above studies suggest the promise of a RESV treatment (commencing either prior to

Fig. 2. The morphology of hippocampal neurons in different experimental groups. A-D show neurons in area CA1, CA3, DG (dentate gyrus), hilus in the control group from left to right, while EH shows neurons in corresponding areas in the PTZ group. No necrotic neurons (eosinophilic cytoplasm and large, round, basophilic chromatin clumps) are found in these areas. H&E stain clearly shows gliosis in hilus (I) and area CA1 (K) in the PTZ treated rats' hippocampus. J, L are magnifications of I and K respectively. Arrow head point to gliosis. Arrows point to glial cells. Scale bars = 50 μm (A–H, I, K), 100 μm (J, L). M–O show neurons in hippocampal CA1 regions (×400) in the VPA 200 treated rat (M), RESV 50 treated rat (N) and RESV 75 treated rat (O) stained with H&E.

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1517 bp 1000 bp 500 bp 300 bp 100 bp DL 12

11

10

9

8

7

6

5

4

3

2

1

Fig. 3. Effect of various treatments on the DNA fragmentation in the hippocampus of the rats. DL: DNA Ladder; 1–2: Control group; 3–4: VPA 200 group; 5–6: PTZ group; 7–8: RESV 25group; 9–10: RESV 50 group; and 11–12: RESV 75 group.

or at the time of the excitotoxic injury) for minimizing the excitotoxininduced seizures, oxidative stress and hippocampal neurodegeneration. Oxidative stress resulting from excessive free-radical release is likely implicated in the initiation and progression of epilepsy (Shin et al., 2011). PTZ is a selective blocker of the GABAA receptor chloride ionophore complex. It has convulsant effects after repeated or single administration and it affects several neurotransmitter systems, such as GABAergic, adenosinergic, and glutamatergic systems (Bell-Horner et al., 2001; Shin et al., 2011). After PTZ-induced seizures, significant decreases in GSH, GSSG, cysteine, and protein thiols as well as increases in the protein carbonyl and protein disulfides levels were observed in the mouse cerebral cortex (Patsoukis et al., 2004). PTZ-induced reductions in total SOD activity and lipid antioxidant (a-tocopherol) content were observed in rat brain homogenates (Rauca et al., 2004). Study by Wu et al. analyzed the effects of intragastric administration of RESV (15 mg/kg) for 10 days following an intrahippocampal injection of 1.0 μg of KA into anesthetized rats (Wu et al., 2009). Analyses of behavioral spontaneous seizures at an early time-point (8 weeks) after KA injection suggested that only a smaller percentage of animals exhibit spontaneous seizures among rats receiving both KA and RESV, in comparison to rats receiving KA alone. Furthermore, EEG recordings for a brief period of 2 hr suggested that epileptiform-like waves were reduced in rats receiving both KA and RESV, in comparison to rats receiving KA alone. Histological analyses revealed considerable neuroprotection in the CA1 cell layer and CA3a sub region in the KA plus RESV treated rats, in comparison to a widespread neurodegeneration observed in rats receiving KA alone. However, the above regimen of RESV treatment was unable to protect neurons in the CA3b and CA3c sub regions. The synaptic reorganization of dentate mossy fibers (in the form of aberrant mossy fiber sprouting into the inner molecular layer of the dentate gyrus) was also reduced when examined at ~8 weeks after KA injection. The KA plus RESV treated animals also exhibited reduced expression of kainate receptors than rats treated with KA alone. The above

observations support the promise of RESV treatment commencing after the hippocampal injury or acute seizures for reducing the incidence/intensity of injury or acute seizure induced chronic TLE. In the present study the chronic administration of RESV showed neuroprotection against the PTZ induced kindling in rats and also showed antioxidant effects by decreasing the MDA level, increasing GSH level and catalase activity in the rat brain. Free radicals are normal products of cellular aerobic metabolism involved in the development of seizures (Rauca et al., 1999; Patel, 2002). However, when the production of free radicals increases or defense mechanism of the body decreases, they cause cellular dysfunction by attacking at the polyunsaturated sites of the biological membranes causing lipid peroxidation. The increase in levels of malondialdehyde (MDA) is a marker of lipid peroxidation. In the present study we have measured oxidative stress parameters viz. malondialdehyde (MDA), reduced glutathione and catalase activity in kindled brain tissues to ascertain the involvement of oxidative stress in epileptogenesis and its modulation by an antioxidant, RESV. Repeated PTZ administration has significantly increased the free radical generation as indicated by increased MDA in the PTZ-kindled rats. RESV dose dependently decreased the MDA levels in the brain tissue of PTZ treated rats. In the present study the decrease level of glutathione was also observed in the PTZ treated rats and the level was increased when pretreated with RESV. These results indicate that during kindling there was excessive oxidative stress pertaining as a consequence glutathione levels were depleted while combating oxidative stress. However, RESV treatment has restored the reduced glutathione level in the brain tissues of PTZ treated rat. In the present study, the decrease levels of catalase activity were also observed in the PTZ treated rats and RESV administration at all doses demonstrated increase in the level of catalase activity in kindled rat brain tissue. Serum neuron specific enolase (s-NSE) is a sensitive marker of neuronal damage in several central nervous system (CNS) diseases including epilepsy (Rodriquez-Nunez et al., 2000; DeGiorgio et al., 1996). Studies have also demonstrated the increased level of sNSE in various animal models of epilepsy and also in patients with epilepsy (Rodriquez-Nunez et al., 2000; DeGiorgio et al., 1996; Steinhoff et al., 1999; Schreiber et al., 1999; Lima et al., 2004). Elevation of sNSE after status eplipticus can be correlated with overall histologic evidence for damage (Schreiber et al., 1999). Our data also showed increases in sNSE in PTZ treated group and decrease in VPA 200 and RESV pretreated groups, accompanied by histological evidence of neuronal damage in hippocampus suggests that such elevations of sNSE appear to be roughly proportional to the extent of histological damage. Thus we postulate that the increased sNSE here is a consequence of death in distinct neuronal populations and in different brain structures and Resveratrol has been successful in preventing this excitotoxicity induced neuronal damage in PTZ treated rats. To investigate the extent of neuronal cell damage caused by PTZinduced epileptic seizures, we carried out nuclear DNA fragmentation studies and expression study of proapoptotic caspase 3 in hippocampus. Animal data also suggest that even a single seizure may be damaging and can cause DNA fragmentation which is the characteristic feature of apoptosis (Zhang LX et al.,1998). Thus with the increasing severity

Table 4 Effect of various treatments on the levels of reduced glutathione (GSH), malondialdehyde (MDA) and catalase in the hippocampus and neuron specific enolase (sNSE) in the serum of PTZ treated rats. Data are expressed as Mean ± SD. Data were analyzed by one way ANOVA followed by Bonferroni post hoc analysis. *P b 0.05 compared to PTZ group; # P b 0.05 compared to RESV 25 group; $ P b 0.05 compared to RESV 50 group; +P b 0.05 compared to RESV 75 group. Groups

GSH levels (μg of GSH/mg protein)

MDA levels (nmol of MDA/mg protein)

Catalase activity (μmol of H2O2decomposed/min/mg protein)

s-NSE levels (ng/ml)

Control PTZ VPA 200 RESV 25 RESV 50 RESV 75

0.0219 0.0086 0.0197 0.0145 0.0166 0.0138

201.45 241.23 209.28 233.16 215.72 222.67

9.16 6.96 8.43 7.38 7.69 8.21

5.03 17.46 6.12 13.37 6.85 8.94

± ± ± ± ± ±

0.0012 0.0005⁎ 0.0006 * # $ + 0.0012* 0.0008 * # + 0.0007 *

± ± ± ± ± ±

1.05 0.88⁎ 1.81*#$+ 1.17* 0.50 *#+ 0.37*#

± ± ± ± ± ±

0.05 0.18⁎ 0.11* # $ 0.29* 0.24 * 0.08 * # +

± ± ± ± ± ±

0.27 1.06⁎ 0.28* # + 1.10* 0.21 * # + 0.73 * #

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a) β actin expression

Marker

1

2

3

63

b) Caspase 3 expression

4

5

Marker 1

2

3

4

5

6

Fig. 4. Effect of various treatments on the expression of Caspase 3 in the hippocampus of the rats. 1: control group, 2: PTZ group, 3: RESV 25 group; 4: RESV 50 group; 5: RESV 75 group; and 6: VPA 200 group.

of seizure there was an increase in DNA fragmentation. In the present study all the brain samples of the PTZ treated group showed laddering pattern with the formation of a large number of small fragments indicating DNA fragmentation and there were increased expression of caspase 3. While in the VPA 200 and RESV groups the brain samples did not show any major fragmentations and the caspase 3 expression was also less. These findings suggest seizures cause an early production of oxidative damage to DNA bases before significant DNA strand breaks appear, indicating that reactive oxygen species may be a contributory factor in the mechanism by which seizures cause cell death. To conclude, the present data support that RESV offers protection against PTZ kindling in rats and it could be a promising candidate to control both developments of seizure and oxidative stress induced neuronal injury and apoptosis during epilepsy. However, further biochemical, molecular and clinical studies are required to ascertain its effectiveness and mechanism of action during epilepsy. Conflict of interest None declared. Acknowledgements The research work was supported by the Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh – 160012, India by providing funding. References Balestrazzi A, Bonadei M, Calvio C, Mattivi F, Carbonera D. Leaf-associated bacteria from transgenic white poplar producing Resveratrol-like compounds: isolation, molecular characterization, and evaluation of oxidative stress tolerance. Can J Microbiol 2009;55:829–40. Bastianetto S, Zheng WH, Quirion R. Neuroprotective abilities of Resveratrol and other red wine constituents against nitric oxide-related toxicity in cultured hippocampal neurons. Br J Pharmacol 2000;131:711–20. Baur JA, Sinclair DA. Therapeutic potential of Resveratrol: the in vivo evidence. Nat Rev Drug Discov 2006;5:493–506. Belguendouz L, Fremont L, Linard A. Resveratrol inhibits metal ion-dependent and independent peroxidation of porcine low-density lipoproteins. Biochem Pharmacol 1997;53:1347–55. Bell-Horner CL, Dibas MI, Covey DF, Drewe JA, Dillon GH. Pentylenetetrazole-induced inhibition of recombinant gamma-aminobutyric acid type A GABA (A) receptors: mechanism and site of action. J Pharmacol Exp Ther 2001;298:986–95. Campagna M, Rivas C. Antiviral activity of Resveratrol. Biochem Soc Trans 2010;38:50–3. DeGiorgio CM, Gott PS, Rabinowicz AL, Heck CN, Smith TD, Correale JD. Neuron-specific enolase, a marker of acute neuronal injury, is increased in complex partial status epilepticus. Epilepsia 1996;37:606–9. Fremont L. Biological effects of Resveratrol. Life Sci 2000;66:663–73. Fujikawa DG, Shinmei SS, Cai B. Kainic acid-induced seizures produce necrotic, not apoptotic, neurons with internucleosomal DNA cleavage: implications for programmed cell death mechanisms. Neuroscience 2000;98:41–53.

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