Anticonvulsant potential of the peroxisome proliferator-activated receptor gamma agonist pioglitazone in pentylenetetrazole-induced acute seizures and kindling in mice

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Anticonvulsant potential of the peroxisome proliferator-activated receptor gamma agonist pioglitazone in pentylenetetrazole-induced acute seizures and kindling in mice Dalaal M. Abdallah⁎ Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Kasr El-Aini Str., 11562 Cairo, Egypt

A R T I C LE I N FO

AB S T R A C T

Article history:

Pioglitazone, a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, is used in

Accepted 14 June 2010

inflammatory brain diseases, and it was shown to protect against seizures in genetically

Available online 21 June 2010

epileptic mice. The present study, therefore, verified its potential antiepileptic effect in pentylenetetrazole (PTZ)-induced acute seizures and kindling in mice. Kindling was induced

Keywords:

in male Swiss albino mice using a subconvulsive dose of PTZ (40 mg/kg, i.p., on alternate

Pioglitazone

days) for 17 days, while acute epileptic animals received a single dose of PTZ (60 mg/kg, i.p.).

Valproate

Animals were pretreated with either pioglitazone (10 mg/kg, p.o.) or the standard

Pentylenetetrazole

antiepileptic drug valproate (50 mg/kg, p.o.). Kindled mice showed elevated cortical levels

Acute seizures

of TNF-α, IL-10, PGE2, and caspase-3, while acute PTZ increased only the cytokines. However,

Kindling

inducible nitric oxide synthase (iNOS) was not expressed in the hippocampi of both acutely

iNOS

convulsed and kindled animals. In acute PTZ convulsion, as well as kindled mice,

Inflammation

pioglitazone and valproate protected against PTZ-induced seizures and delayed seizure

Apoptosis

latency onset. Pioglitazone normalized all altered parameters except for PGE2 in PTZ-kindled animals and, unpredictably, further elevated TNF-α in the acute model. Valproate showed also the same pattern but reinstated IL-10 partially in kindled mice. The present results revealed that both models increase pro- and anti-inflammatory cytokines, while only kindling elevates PGE2 and caspase-3; nonetheless, neither model affects the expression of iNOS. The anticonvulsive effect of either pioglitazone or valproate is presumably associated with attenuating inflammation and preventing apoptosis. © 2010 Elsevier B.V. All rights reserved.

⁎ Fax: +20 2 24073411. E-mail address: [email protected]. Abbreviations: HO, hemeoxygenase; IL, interleukin; NO, nitric oxide; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; PIO, pioglitazone; PG, prostaglandin; PPAR, peroxisome proliferator-activated receptor; PTZ, pentylenetetrazole; PTZ-a, pentylenetetrazole acute; PTZ-k, pentylenetetrazole kindled; SOD, superoxide dismutase; TZD, thiazolidinedione; TNF, tumor necrosis factor; VPA, valproate 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.06.034

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1.

Introduction

The therapeutic merit of the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists, thiazolidinediones (TZDs), reaches far beyond their insulin sensitizing action. Recently, these drugs were found to confer neuroprotection in several animal models, including acute cerebral ischemia, as well as Parkinson's and Alzheimer's diseases (Dehmer et al., 2004; Kiaei et al., 2005; Shimazu et al., 2005; Roses et al., 2007). Effects of TZDs extend to protect against epileptic disorders, where few attempts traced the anticonvulsant efficacy of PPAR-γ agonists (Okada et al., 2006; Maurois et al., 2008; Sun et al., 2008; Yu et al., 2008). Increased inflammatory mediators are produced secondary to the epileptogenic insult and are important in the development and maintenance of seizure responses (Vezzani and Granata, 2005), possibly by modulating glutamate homeostasis (Vezzani et al., 2008). Elevated levels of proinflammatory cytokines, such as IL-1β and TNF-α, mRNA were documented in status epilepticus and electrical convulsive rats (Okada et al., 2002; Du et al., 2007). Moreover, prostaglandin E2 (PGE2) possesses a proconvulsive effect, which is attributed to increased glutamate release-mediated neuronal injury (ColeEdwards and Bazan, 2005). Nitric oxide (NO) is known to play a role in epilepsy, where its anti- or proconvulsive effects have been revealed (Paul and Subramanian, 2002; El-Abhar and El Gawad, 2003; Itoh et al., 2004). Excessive NO production is linked to the activation of neuronal nitric oxide synthase (nNOS) in both acute (Bikjdaouene et al., 2003) and kindled (Itoh et al., 2004) PTZ models, while De Luca et al. (2006) showed that inducible nitric oxide synthase (iNOS) is associated with kindling responses. Beside the well-recognized anti-inflammatory effect of TZDs (Dehmer et al., 2004; Kim et al., 2005; Michalik et al., 2006; Krag et al., 2009), their antioxidant properties may add to their antiepileptic efficacy (Okada et al., 2006; Sun et al., 2008; Yu et al., 2008). PPAR-γ agonists enhance the antioxidant enzymes superoxide dismutase-1 and -2 (SOD-1 and -2), as well as catalase gene expression (Girnun et al., 2002; Hwang et al., 2005; Ding et al., 2007). Although Maurois et al. (2008) documented that rosiglitazone was ineffective against audiogenic and ibotenate-induced epilepsy, Yu et al. (2008) con-

Fig. 1 – Effect of pioglitazone (PIO-k, 10 mg/kg, p.o.) and valproate (VPA-k, 50 mg/kg, p.o.) on seizure stage in PTZ kindled (PTZ-k; 40 mg/kg, i.p., nine injections on alternate days) mice. Values are median of 11–13 mice; as compared to PTZ-k group (§); Kruskal– Wallis test (nonparametric ANOVA) followed by Dunn's multiple comparisons test.

veyed that the anticonvulsive effect endowed by this PPAR-γ agonist was linked to its antioxidant properties in lithium– pilocarpine-induced status epilepticus model. These authors concluded that the suppression of hemeoxygenase-1 (HO-1) expression, a stress-related protein, and superoxide anion generation are important entities in its antiepileptic action. Since the available data on PPAR-γ agonists in experimental seizure models are contentious, ranging from inhibition to ineffectiveness (Maurois et al., 2008; Yu et al., 2008), and the study of Okada et al. (2006) is the first to document pioglitazone's antiepileptic efficacy, the current investigation aimed to evaluate further its effect in PTZ-induced acute seizures and kindling mice models. In this context, the influence of pioglitazone, in comparison with the antiepileptic drug, valproate, on inflammation, apoptosis, and iNOS was assessed.

Table 1 – Effect of pioglitazone (PIO; 10 mg/kg, p.o.) and valproate (VPA; 50 mg/kg, p.o.) on PTZ-induced acute seizures (PTZ-a; 60 mg/kg, i.p.) and kindling (PTZ-k; 40 mg/kg, nine injections on alternate days) in mice. Groups PTZ-a PTZ-k VPA –a VPA-k PIO-a PIO-k

Median seizure stage 4 4 0 3 3 3

Stage 4/5 seizure latency (min) a

9:3 ± 0.02 4:2 ± 1.03b 16:6 ± 3.40b 16:1 ± 1.91a 18:3 ± 1.70b 19:1 ± 1.33a

Stage 4/5 seizure incidence (%) 100 100 20b 9a 20b 31a

In the seizure stage, values are median of 11–13 mice; statistical comparisons were carried out using Kruskal–Wallis test (nonparametric ANOVA) followed by Dunn's multiple comparisons test. In the parametric analysis (stage 4/5 seizure latency), values are means of 11–13 animals ± SEM; statistical comparisons were carried out using one-way ANOVA followed by Student–Newman–Keuls multiple comparisons test. Stage 4/5 seizure incidence (11–13 animals) was compared using Fisher's exact probability test. As compared with PTZ-k (a) and PTZ-a (b) groups, P < 0.05. VPA-a, valproate-acute; VPA-k, valproate-kindled; PIO-a, pioglitazone-acute; PIO-k, pioglitazone-kindled.

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2.

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Results

During the course of kindling, mice exhibited seizures starting from the 4th dose of PTZ, but reached stage 4 or 5 at the 7th injection, and lasted thereafter (Fig. 1). On the last day, animals showed convulsions after 4 min of observation, while an acute dose of PTZ invoked clonic–tonic convulsion, with a latency of 9 min, a delay that was significant from kindled mice (Table 1). On the other hand, the protective effects of valproate and pioglitazone started from day 13 in the kindled group (Fig. 1). Pioglitazone and valproate caused 69% and 91% protection and prolonged convulsion latency to 16 and 19 min, respectively, on the last day of the experiment (Table 1). Similarly, acute doses of both drugs protected 80% of animals and delayed seizure latency to 18 min (pioglitazone) and 17 min (valproate) (Table 1). Compared to normal animals, PTZ-kindling (PTZ-k) increased cortical caspase-3 activity by 4-folds, while PTZ-a (PTZ-acute) produced no alteration (Fig. 2). Pretreatment with pioglitazone or valproate leveled off caspase-3 by 3.3-folds and 2.6-folds, respectively, in PTZ kindled animals (Fig. 2). Similarly, cortical PGE2 was increased in kindled mice (123%) but not in the acute PTZ group; however, neither pioglitazone nor valproate opposed it (Fig. 3). Moreover, both PTZ dose regimens elevated cortical levels of TNF-α by 74% (PTZ-k) and 90% (PTZ-a) (Fig. 4), as well as IL-10 by 567% (PTZ-k) and 311% (PTZ-a) (Fig. 5). In kindled mice, levels of both cytokines were decreased by pioglitazone and valproate to different extents. Though IL-10 was suppressed by both treatments in the PTZ-a

Fig. 2 – Effect of pioglitazone (PIO; 10 mg/kg, p.o.) and valproate ( VPA; 50 mg/kg, p.o.) on cortical caspase-3 activity in PTZacutely convulsed (PTZ-a; 60 mg/kg, i.p.) and PTZ-kindled (PTZk; 40 mg/kg, i.p., nine injections on alternate days) mice. Values are means of seven to nine mice±SEM; as compared to normal control (*), PTZ-a ( @), and PTZ-k (§) groups, using one-way ANOVA followed by Student–Newman–Keuls multiple comparisons test, P<0.05. VPA-a, valproate-acute; VPA-k, valproatekindled; PIO-a, pioglitazone-acute; PIO-k, pioglitazone-kindled.

Fig. 3 – Effect of pioglitazone (PIO; 10 mg/kg, p.o.) and valproate (VPA; 50 mg/kg, p.o.) on cortical PGE2 content in PTZ-acutely convulsed (PTZ-a; 60 mg/kg, i.p.) and PTZ-kindled (PTZ-k.; 40 mg/kg, i.p., nine injections on alternate days) mice. Values are means of seven to nine mice ± SEM; as compared to normal control (*) group, using one-way ANOVA followed by Student–Newman–Keuls multiple comparisons test, P<0.05. VPA-a, valproate-acute; VPA-k, valproate-kindled; PIO-a, pioglitazone-acute; PIO-k, pioglitazone-kindled.

Fig. 4 – Effect of pioglitazone (PIO; 10 mg/kg, p.o.) and valproate (VPA; 50 mg/kg, p.o.) on cortical TNF-α content in PTZ-acutely convulsed (PTZ-a; 60 mg/kg, i.p.) and PTZ-kindled (PTZ-k; 40 mg/kg, i.p., nine injections on alternate days) mice. Values are means of seven to nine mice ± SEM; as compared to normal control (*), PTZ-a ( @), PTZ-k (§), VPA-a (†), and PIO-a (●) groups, using one-way ANOVA followed by Student–Newman–Keuls multiple comparisons test, P < 0.05. VPA-a, valproate-acute; VPA-k, valproate-kindled; PIO-a, pioglitazone-acute; PIO-k, pioglitazone-kindled.

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3.

Fig. 5 – Effect of pioglitazone (PIO; 10 mg/kg, p.o.) and valproate (VPA; 50 mg/kg, p.o.) on cortical IL-10 content in PTZ-acutely convulsed (PTZ-a; 60 mg/kg) and PTZ-kindled (PTZ-k; 40 mg/ kg, i.p., nine injections on alternate days) mice. Values are means of seven to nine mice ± SEM; as compared to normal control (*), PTZ-a (@), PTZ-k (§), VPA-a (†), and VPA-k (#) groups, using one-way ANOVA followed by Student–Newman–Keuls multiple comparisons test, P < 0.05. VPA-a, valproate-acute; VPA-k, valproate-kindled; PIO-a, pioglitazone-acute; PIO-k, pioglitazone-kindled.

model, TNF-α was unexpectedly further elevated (Figs. 4 and 5). Regarding iNOS, immunohistochemical study revealed no expression in the hippocampi of both PTZ-a and PTZ-k mice (Figs. 6).

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Discussion

In the present work, a single PTZ administration elevated cortical TNF-α and IL-10, effects that also persisted in the kindled animals. The proinflammatory cytokine, TNF-α, is known to participate in the induction and maintenance of epilepsy as proven in other epileptic models (Okada et al., 2006; Sun et al., 2008; Vezzani et al., 2008), where the former authors demonstrated that the overexpression of TNF-α, among other inflammatory cytokines in EL mice brains, is linked to sporadic spontaneous seizures pointing out to its pivotal role in epilepsy, regardless of the animal model. Hence, elevated levels of brain TNF-α in PTZ-kindled and acutely convulsed mice, documented in this study, may signify a proepileptogenic potential and may lower the threshold for seizure induction, thus setting the basis for the onset of epilepsy (Vezzani et al., 2008). As compensation, it is believed that these proinflammatory molecules mediate the synthesis of anti-inflammatory cytokines, including IL-10 (Vezzani and Granata, 2005). Such cytokines were found to impose a crucial role in major brain diseases, such as epilepsy, to promote survival of neurons and glia, via antagonizing the devastating effects of their proinflammatory counter partners (Strle et al., 2001; Vezzani and Granata, 2005). Thus, this may explain the elevated level of cortical IL-10, in PTZ-induced epilepsy models. Vezzani and Granata (2005) stated that TNF-α stimulates the production of arachidonic acid and triggers the transcription of various inflammatory genes, thus increasing the expression of COX-2 enzyme. Such effect was verified in the current study, where PTZ-kindled mice showed elevated cortical PGE2, an outcome that was not observed in acutely convulsed animals. As a support to the current results, COX-2 inhibitors protected against PTZ-kindling (Dhir et al., 2007)

Fig. 6 – Effect of PTZ-acute (PTZ-a; 60 mg/kg, i.p.) and PTZ-kindled (PTZ-k; 40 mg/kg, i.p., nine injections on alternate days) on hippocampal iNOS expression in mice. Original magnification x200 (panel A) and x400 (panel B).

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and were ineffective in PTZ acute convulsions (Akarsu et al., 2006). These findings implicate that PGE2 synthesis might be time-dependent or might possess a role in increased seizure suitability. PGE2 triggers neuroinflammation and activates the intrinsic apoptotic pathway in neurons (Mirjany et al., 2002; Takadera and Ohyashiki, 2006), while TNF-α stimulates the external death pathway (Kraft et al., 2009), where both pathways can activate the apoptosis executer caspase-3. The current investigation showed that PTZ kindling, rather than acute convulsions, is associated with caspase-3 activation. These data support a previous study by Pavlova et al. (2004) who reported that PTZ-induced kindling elevated caspase-3, an effect that was not reached by its acute administration (Kamińska et al., 1994). Apoptosis may influence seizure susceptibility during epileptogenesis (Henshall, 2007), thus possibly decreasing the seizure threshold of PTZ subconvulsive dose that induced kindling in this study. Itoh et al. (2004) showed that NO is produced by the activation of nNOS in PTZ kindling model; however, De Luca et al. (2006) revealed that iNOS knock-out mice reached kindling more slowly when administered PTZ. The data of the present investigation confirm the findings of Rauca et al. (2004), who documented that iNOS is not expressed in the kindling model. Albeit pioglitazone protected against PTZ-induced acute convulsions, it surprisingly elevated TNF-α level further above that induced by PTZ, an effect that mimics that of valproate. Vezzani et al. (2008) stated that the TNF-α modulatory effect on seizures depends on its brain level and on the receptor subtypes predominantly activated by this cytokine. In addition, Balosso et al. (2005) revealed that increased brain level of TNF-α inhibits seizures in mice, an action that is mediated by neuronal p75 receptors. In the present work, the spike in TNFα was associated by a delay in PTZ-induced convulsions and normal level of the neurotrophic molecule IL-10, thus pointing to the potential modulating effect of TNF-α on seizure susceptibility, in the PTZ-acute model. In the kindled model, however, the antiepileptic effect of pioglitazone was accompanied by suppression in the TNF-α level, thus opposing the acute pattern, but with no effect on PGE2. These findings may be linked with the pioglitazone inhibitory action on TNF-α mRNA but not that of COX-2, as documented by Okada et al. (2006) in brains of genetically epileptic mice. These authors concluded that transcriptional factors involved in the inhibition of TNF-α did not necessarily suppress COX-2, stating that other factors, not affected by PPAR-γ agonist, were responsible for the negative influence on COX-2 expression. This report offers an explanation for the unaltered level of PGE2 shown in the current study. Rosiglitazone, another TZD, protected neurons in status epilepticus rats by impeding TNF-α expression via hampering CD40 expression and microglia activation (Sun et al., 2008). Previously, TZDs were shown to suppress transcription of proinflammatory genes and to confer an anti-inflammatory effect possibly by antagonizing NF-κB or STAT signaling pathways and/or by inhibiting induction of TNF-α (Okada et al., 2006; Tureyen et al., 2007; Sun et al., 2008). The effect of pioglitazone on IL-10 is controversial, where, in different animal models, it was reported to exert no effect (Krag et

al., 2009) or to further elevate IL-10 level (Kim et al., 2005). These findings counteract the current results, where pioglitazone lowered the PTZ-induced IL-10 elevation in both acutely convulsed and kindled animals; a plausible explanation could be related to normalization of TNF-α. The antiapoptotic effect endowed by the PPAR-γ agonist, represented by the decreased activity of caspase-3 in the current work, goes in line with that of Li et al. (2008), who reported the antiapoptotic effect of pioglitazone in ischemia/ reperfused heart model. Such an effect is likely attributed to the drug anti-inflammatory properties depicted by the suppression of TNF-α in kindled animals. A decreased blood glucose level (Velísek et al., 2008), as well as a ketogenic diet (Fenoglio-Simeone et al., 2009), have been previously shown to modulate seizures and hence could affect seizures/kindling evoked by PTZ herein. Several studies (Plulutzky, 2003; Shimazu et al., 2005; Sundararajan et al., 2005), as well as the present one (data not shown), displayed that TZDs do not affect serum glucose in nondiabetic human or rodents. Hence, the anticonvulsive effect of pioglitazone is not correlated to its antidiabetic effect. Valproate showed the same pattern as did pioglitazone, where it protected against PTZ-kindling, hampered cortical TNF-α, and did not affect PGE2. Ichiyama et al. (2000) referred the valproate-suppressing effect on TNF-α to the inhibition of NF-κB, an effect that may offer an explanation to the partially restored cortical IL-10 in kindled mice by valproate herein. The drug also reinstated cortical caspase-3 activity, which is in accordance with the in vitro work of Lasseck et al. (2010), which could again be linked to the reduction in TNF-α level provoked by kindling. Taken together, the results of the present study emphasize the role of cytokines in the process of kindling, as well as in acute seizures, and favor the modulatory action of PPAR-γ agonists including pioglitazone against the epilepsy-associated neuroinflammatory processes.

4.

Experimental procedures

4.1.

Animals

Adult male Swiss albino mice 8 weeks old, weighing 20–25 g (National Research Center Laboratory, Cairo, Egypt), were kept under controlled environmental conditions, at a constant temperature (23 ± 2 °C), humidity (60 ± 10%), and a light/ dark (12 h:12 h) cycle. Mice were allowed standard pellet diet and tap water ad libitum before, during 1 week of acclimatization period, and throughout experimentation. Methods for investigating PTZ-induced acute seizures and kindling were approved by the Animal Care and Use Committee of the Faculty of Pharmacy Cairo University (Cairo, Egypt) before the commencement of experiments. Experiments were done between 9:00 AM and 12:00 PM to minimize circadian influences on seizure susceptibility.

4.2.

PTZ-induced acute seizures, kindling, and treatments

Mice were allocated into seven groups and divided into two subsets, one (n = 7–9 per group) for biochemical estimations

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and another (n = 4 per group) for the immunohistochemical study. Animals received nine subconvulsive doses of PTZ (40 mg/kg, i.p.; Sigma-Aldrich, MO, USA; Ammon-Treiber et al., 2007) dissolved in saline on alternate days for 17 days to serve as the positive control group (PTZ-k). In PTZ-acute (PTZ-a) group, animals were injected with a single convulsive dose of PTZ (60 mg/kg, i.p.; Uma Devi et al., 2006). For each acute and kindled experiments, other two groups were pretreated orally, 30 min before PTZ injection and animals received either sodium valproate (Sanofi-Aventis, Paris, France) equivalent to 50 mg/kg valproic acid (Nagatomo et al., 2000) dissolved in saline or pioglitazone hydrochloride (10 mg/ kg; Takeda, Osaka, Japan; Kubota et al., 2006), suspended in 0.5% carboxymethylcellulose (CMC). The last group was given CMC and was injected intraperitoneally with saline, to serve as the normal control group. After acute or after each PTZ injection, animals were observed for 20 min in Plexiglas cages, and seizure stages were recorded on a 6-point scale according to AmmonTreiber et al. (2007): stage 0—no response; stage 1—ear and facial twitching; stage 2—convulsive waves through the body, without rearing; stage 3—myoclonic jerks, upright position; stage 4—clonic–tonic convulsions, turn over into side position; and stage 5—generalized clonic–tonic convulsions, loss of postural control. The maximum response reached was recorded in each animal, and mice that experienced convulsions during the observation period of the first three injections as well as those died of convulsions during the experimental period were excluded from this study. Kindling was achieved when mice had a seizure stage of 4 on three consecutive administrations. During the experimental course, the median seizure stage was calculated for each group, as well as the latency onset of first stage 4/5 seizure, and the incidence of convulsing animals were recorded.

4.3.

Biochemical assays

Mice were euthanized by decapitation 24 h after the acute or last PTZ administration, and brains were quickly removed on ice plates. The cerebral cortex was carefully excised and homogenized either in ice-cold saline for assessment of TNF-α, IL-10, and caspase-3 or in 0.1 M phosphate buffer (pH 7.4), containing 1 mM EDTA and 0.1 μM indomethacin for PGE2 measurement and frozen at −80 °C. Cortical TNF-α, IL-10, and PGE2 were assessed using ELISA kits purchased from Invitrogen (CA, USA), Bender MedSystems (Vienna, Austria), and Cayman Chemical (MI, USA), respectively, and results were expressed as picograms per gram (pg/g) of tissue. The activity of cortical caspase-3 was determined using colorimetric assay kit (Biosource International, CA, USA) according to the manufacturer's protocol. Briefly, the levels of the chromophore p-nitroanilide (pNA) released by caspase-3 activity in the tissue lysates were quantified spectrophotometrically at 405 nm. The increase in caspase-3 activity was determined by comparing the absorbance of pNA from samples of a different group with that of normal control. Protein concentrations in samples were estimated according to Lowry et al. (1951), and values were expressed as OD per milligram (mg) of protein.

4.4.

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Immunohistochemistry of hippocampal iNOS

Another set of animals was anesthetized with sodium pentobarbital (50 mg/kg, i.p.) 24 h after last PTZ or vehicle treatment, and the procedure reported for immunohistochemistry for light microscopy was followed with modification. Briefly, mice were transcardially perfused with 4% paraformaldehyde (PFA) in Tris-buffered saline (TBS), and their brains were removed, postfixed for 2 h in 4% PFA/TBS, and placed in 30% sucrose until they sank. Brains were embedded in paraffin blocks, and sagittal sections through the entire hippocampus were cut at 4 μm. Subsequently, sections were deparaffinized, dehydrated, and incubated in 3% hydrogen peroxide for 15 min to block endogenous peroxidase. Nonspecific protein binding was blocked for 10 min with normal serum using Universal Quick Kit (Novocastra, Newcastle, UK). For immunohistochemical detection of iNOS, slides were incubated with primary mouse monoclonal antibody (anti-iNOS 1:3500; Novocastra) for 1 h at room temperature in a humidified chamber. After rinsing twice with TBS, sections were treated with a labeled streptavidin– biotin kit (Novocastra). The sections were then incubated in 3,3′-diaminobenzidine (Novocastra) for 5 min and counterstained with Mayer hematoxylin. The sections were examined under a microscope for the appearance of brown staining.

4.5.

Statistical analysis

Parametric data are expressed as mean of seven to nine experiments ± SEM, and statistical comparisons were carried out using one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls multiple comparisons test. In nonparametric data, values are median and statistical comparisons were analyzed using Kruskal–Wallis test (nonparametric ANOVA) followed by Dunn's multiple comparisons test. For stage 4/5 seizure incidence, the Fisher's exact probability test was used. The minimal level of significance was identified at P < 0.05.

Acknowledgment The author would like to thank Prof Dr. Soheir Assaad, Histology Department, Faculty of Medicine, Cairo University, Cairo, Egypt, for her assistance with the immunohistochemical testing.

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