Brain Research Bulletin 113 (2015) 1–7

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Antiepileptogenic effects of the selective COX-2 inhibitor etoricoxib, on the development of spontaneous absence seizures in WAG/Rij rats Rita Citraro a , Antonio Leo a , Rosario Marra b , Giovambattista De Sarro a , Emilio Russo a,∗ a b

University of Catanzaro, School of Medicine, Department of Science of Health, Catanzaro, Italy National Council of Research (CNR), Institute of Neurological Science, Catanzaro, Italy

a r t i c l e

i n f o

Article history: Received 6 December 2014 Received in revised form 9 February 2015 Accepted 13 February 2015 Available online 19 February 2015 Keywords: Etoricoxib Cyclooxygenase (COX) Neuroinflammation Absence epileptogenesis Spike-wave discharges (SWDs) WAG/Rij rats

a b s t r a c t Different data suggest the involvement of specific inflammatory pathways in the pathogenesis of epilepsy. Cyclooxygenase (COX), which catalyses the production of pro-inflammatory prostaglandins, may play a significant role in seizure-induced neuroinflammation and neuronal hyperexcitability. COX-2 is constitutively expressed in the brain and also increased during/after seizures. COX-2 inhibitors may thus attenuate inflammation associated with brain disorders. We studied whether early long-term treatment (17 consecutive weeks starting from 45 days postnatal age) with the non-steroidal anti-inflammatory drug etoricoxib (10 mg/kg/day per os), a selective COX-2 inhibitor, was able to prevent/reduce the development of absence seizures in WAG/Rij rats, a recognized animal model of absence epilepsy and epileptogenesis. Drug effects on the incidence, duration and properties of absence seizure spike-wave discharges (SWDs) were measured both 1 and 5 months after treatment withdrawal; furthermore, the acute effects of etoricoxib on SWDs in 6-month-old WAG/Rij rats were measured. Early long-term treatment (ELTT) with etoricoxib led to an ∼40% long-lasting (5 months) reduction in the development of spontaneous absence seizures in adult WAG/Rij rats thus exhibiting antiepileptogenic effects. Acutely administered etoricoxib (10 and 20 mg/kg i.p.) also had anti-absence properties, significantly reducing the number and duration of SWDs by ∼50%. These results confirm the antiepileptogenic effects of COX-2 inhibitors and suggest the possible role of COX-2, prostaglandin synthesis and consequent neuroinflammation in the epileptogenic process underlying the development of absence seizures in WAG/Rij rats. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Epilepsy is a chronic brain disorder affecting ∼1% of the human population worldwide; the underlying mechanisms of seizure generation and epileptogenesis however, still remain unclear (Vezzani et al., 2013a). Immune responses are implicated in seizure induction and the development of epilepsy (Marchi et al., 2014). Indeed, clinical and experimental evidence have implicated a positive feedback cycle between brain neuroinflammation and epileptogenesis (Xu et al., 2013). Brain inflammation may also be a common substrate contributing to seizures in drug-resistant epilepsies of different aetiologies, and recurrent seizures can per se be a major cause of long-term inflammation (Liu et al., 2012; Vezzani, 2014). In the brain, inflammation involves both resident cells, such as glia and neurones, as well as cells of the peripheral innate immune system

∗ Corresponding author at: Department of Science of Health, School of Medicine, University of Catanzaro, Via T. Campanella, 115, 88100 Catanzaro, Italy. Tel.: +39 0961 3694191; fax: +39 0961 3694192. E-mail address: [email protected] (E. Russo). http://dx.doi.org/10.1016/j.brainresbull.2015.02.004 0361-9230/© 2015 Elsevier Inc. All rights reserved.

such as granulocytes and macrophages (Wilcox and Vezzani, 2014). Recently brain inflammation has gained recognition as a crucial factor to the etiopathogenesis of epilepsy development (Choi and Koh, 2008; Vezzani et al., 2013b). Neuroinflammation is characterized by glial activation, oedema, and synthesis of inflammatory mediators such as cytokines and prostaglandins (PGs) such as PGE2 (Takemiya et al., 2006; Vezzani et al., 2008). These proinflammatory changes predominantly occur in activated microglia and astrocytes and increase the risk of epilepsy development. Ongoing brain inflammation has the potential to lower seizure threshold, which in turn may promote neuronal excitability through modifications of neuronal ion channels, alterations of neurotransmitter uptake or release, and regulation of blood–brain barrier (BBB) permeability (Vezzani, 2014; Xu et al., 2013). Pharmacological studies have therefore been designed to interfere with specific proinflammatory pathways during epileptogenesis (Vezzani, 2014). Cyclooxygenase (COX) catalyses the first committed step in the synthesis of prostanoids, a large family of arachidonic acid metabolites comprising PGs, prostacyclin and thromboxane. Two isoforms of COX enzymes have been identified: the constitutively expressed COX-1 and the inducible highly regulated COX-2 (Aid and Bosetti, 2011).

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COX has been found to be constitutively expressed in different brain areas, and inhibitors of COX enzyme(s), particularly the COX2 inhibitors, may attenuate inflammation associated with brain disorders (Kulkarni and Dhir, 2009; Minghetti, 2004). COX-2 is expressed in discrete populations of neurones and it is induced in the brain after various insults, thus contributing to brain inflammatory processes (Strauss, 2008; Vezzani et al., 2013b). It has been shown that COX-2 is also up-regulated by seizures (Shimada et al., 2014; Tu and Bazan, 2003) and COX-2-selective inhibitors have been found to either facilitate or inhibit seizures depending on the experimental model used (Baik et al., 1999; Shafiq et al., 2003). It has been reported that COX-2 induction can have an important role during epileptogenesis (Jung et al., 2006; Tu and Bazan, 2003). The increase in COX-2 activity contributes to neurodegeneration by glutamate excitotoxicity, oxidative stress, or the neurotoxic actions of PGs (Bezzi et al., 1998). Selective COX-2 inhibitors may thus constitute a novel approach for anti-epileptogenesis or diseasemodifying treatments. In this context, different COX-2 inhibitors were previously tested in post-status epilepticus models. For example, celecoxib shows antiepileptogenic and neuroprotective effects in the lithium–pilocarpine model both in mature and developing rats (Jung et al., 2006; Zhang et al., 2008). At odds, other studies have shown that parecoxib, another COX-2 inhibitor, was neuroprotective but not antiepileptogenic in the pilocarpine model of temporal lobe epilepsy (Polascheck et al., 2010). Conflicting results with COX-2 inhibitors have been obtained towards kainic acid-induced seizures, with both pro-convulsant and anticonvulsant effects being reported (Baik et al., 1999; Kim et al., 2008). Notably, rofecoxib was found to reduce seizure frequencies and hippocampal cell death in the kainate-induced epilepsy model (Kunz and Oliw, 2001). Indomethacin, which inhibits both COX1 and 2 (Perrone et al., 2010), was also previously found to reduce seizures in absence epilepsy animal models (Kovacs et al., 2014) and to slow the course of electrical kindling in mice (Tanaka et al., 2009). Based on this background, we decided to investigate the possible antiepileptogenic effects of etoricoxib, a selective COX-2 inhibitor, in a genetic animal model of absence epilepsy and epileptogenesis, the WAG/Rij (Wistar Albino Glaxo rats from Rijswijk) rat. More specifically, WAG/Rij rats, as well as GAERS (genetically absence epileptic rats from Strasbourg), are genetically prone to develop spontaneous absence seizures during their lifespan with only few early immature SWDs appearing on the EEG (WAG/Rij rats after P50 and GAERS likely earlier). SWDs increase in number and duration with ageing and their electroencephalographic morphology become fully matured in all rats of the WAG/Rij strain only after 2–3 months of age (Dezsi et al., 2013; van Luijtelaar et al., 2011, 2014). Keeping with this, both strains of rats can be considered models of epileptogenesis and it is known that an early pharmacological intervention can modify the underlying process and the development of spontaneous absence seizures in adulthood (over 5 months) (Blumenfeld et al., 2008; Giblin and Blumenfeld, 2010; Pitkanen and Engel, 2014; White and Loscher, 2014). Furthermore, in WAG/Rij rats, the role of neuroinflammation still remains to be clarified; however, several previous reports already indicated a substantial contribution of inflammation on the underlying disease (Gyorffy et al., 2014; Kovacs et al., 2011, 2014; Russo et al., 2013, 2014a). On the other hand, several previous studies have already identified alterations in some neurotransmitter systems and ion channels function contributing to absence seizures in this rat strain (Sitnikova, 2010; van Luijtelaar and Sitnikova, 2006; van Luijtelaar and Zobeiri, 2014). The resulting absence seizures represent the final picture of a large amount of dysfunctions, on the role of (mainly thalamic) GABA (Liu et al., 2007; Pisu et al., 2008; van Luijtelaar and Zobeiri, 2014) and glutamate receptors (Citraro et al., 2006b; Coenen et al., 1992; Ngomba et al., 2008; Peeters et al.,

1994; Russo et al., 2008), various peptide and steroid (ovarian and corticosteroids) hormones (Citraro et al., 2006a; Schridde and van Luijtelaar, 2004) and others (Citraro et al., 2013b; Ehling et al., 2012; Kanyshkova et al., 2012; Kole et al., 2007; Russo et al., 2014b; Takacs et al., 2010; van Luijtelaar et al., 2012), all contributing to the generation and maintenance of epileptic SWDs. It is generally accepted that the SWDs, typical for absence epilepsy, have their origin and are maintained in the cortico-thalamo-cortical network, although the exact initiating site is a matter of discussion and might be different for different species, models and between subjects of the same species (Meeren et al., 2005; van Luijtelaar and Zobeiri, 2014). Our results indicate that etoricoxib can exert significant absence antiseizure and antiepileptogenic activity in the WAG/Rij rats. 2. Materials and methods 2.1. Animals Male WAG/Rij rats were used in all experiments. Rat progenitors were purchased from Charles River Laboratories s.r.l. (Calco, Lecco, Italia) at a body weight of ∼60 g (3–4 weeks old). Following arrival, animals were housed three/four per cage and kept under controlled environmental conditions (60 ± 5% humidity; 22 ± 2 ◦ C; 12/12 h reversed light/dark cycle; lights on at 8 PM). Female rats at 10 weeks of age were placed with same-age group males for mating for 16 h in the ratio 1:1 and examined the next morning for the presence of a vaginal plug, a sign of successful copulation. Dams of all strains were housed 2 per cage, whereas, all offspring after weaning (P21) were housed three/four per cage. Animals were allowed free access to standard laboratory chow and water until the time of experiments. Procedures involving animals and their care were conducted in conformity with the international and national law and policies (EU Directive 2010/63/EU for animal experiments, ARRIVE guidelines and the Basel declaration including the 3R concept). The experimental protocols and procedures described in this manuscript were approved by the local ethical committee of the University of Catanzaro. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.2. Experimental protocol in WAG/Rij rats The present work aimed at the evaluation of (1) the effects of etoricoxib (Arcoxia, Merck Sharp & Dohme, Rome, Italy) on the epileptogenic process underlying the development of absence seizures in WAG/Rij rats (early long-term treatment); (2) effects of eterocoxib on established absence seizures (acute treatment) in the same model. For the early long-term treatment (ELTT), etoricoxib was administered orally at a dose of 10 mg/kg/day dissolving 30 mg of etoricoxib in 360 ml of drinking water. Doses used were based on previously published articles (Jayaraman et al., 2010; Tanaka et al., 2009). The final dilution was calculated on the evidence that rats drink on average 12 ml/100 g/day (Citraro et al., 2014); this was further confirmed by checking the volume drunk by rats. ELTT treatment was started in rats at 45 days of age (n = 10 per group; before seizure onset, which occurs around P50–60) and continued for a further 17 weeks up to the age of ∼5 months, in agreement with our previously published protocol (Russo et al., 2011, 2013). Agematched control rats (n = 10) were kept under the same housing conditions over the same period of time with water. Furthermore, particular attention was paid to the possible appearance of any obvious drug-induced side-effects. At the age of ∼6 months, after surgery (see Section 2.3), all WAG/Rij rats from each group (treated and untreated) underwent three recording periods, for 3 consecutive days, within 30 and 36 days after treatment discontinuation (Citraro et al., 2014; Russo et al., 2011). Every recording session

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lasted 3 h without administration of any drug. The same recording schedule was performed at the age of 10 months in the same animals in order to evaluate the duration of drugs’ effects. For acute drug treatment, adult WAG/Rij rats (6 months old, 280 g; n = 24) were intraperitoneally (i.p.) administered with different doses of etoricoxib (5, 10 and 20 mg/kg; n = 6 for each dose) in order to evaluate drug effects on SWDs parameters. Every EEG recording session lasted 5:1 h baseline without injection, and 4 h after the i.p. administration of etoricoxib or vehicle (Citraro et al., 2013b). 2.3. Animal surgery and EEG recording All WAG/Rij rats around the age of 6 months were chronically implanted, under anaesthesia obtained by administration of a mixture of tiletamine/zolazepam (1:1; Zoletil 100® ; 50 mg/kg i.p.; VIRBAC Srl, Milan, Italy), using a Kopf stereotaxic instrument, with five cortical electrodes for EEG recordings (Citraro et al., 2014; Russo et al., 2013). Stainless-steel screw electrodes were implanted on the dura mater over the cortex: two in the frontal region (AP = −1 mm; L = ±2.5 mm) and two in the parietal region (AP = −5 mm; L = ±2.5 mm); the ground electrode was placed over the cerebellum (Citraro et al., 2013a). All animals were allowed at least 1 week of recovery and handled twice a day. In order to habituate the animals to the recording conditions, rats were connected to the recording cables, for at least 3 days before the experiments. Every EEG recording was always performed starting at 9.00 am in order to avoid circadian alterations within groups. The animals were attached to a multichannel amplifier (Pinnacle Technology’s 8400–9000 video/EEG system with Sirenia Software, Kansas, USA) by a flexible recording cable and an electric swivel, fixed above the cages, permitting free movements for the animals. All EEG signals were amplified and conditioned by analogue filters (filtering: below 1 Hz and above 30 Hz at 6 dB/octave) and subjected to an analogueto-digital conversion with a sampling rate of 250 Hz. The blinded quantification of absence seizures was based on the number and the duration of EEG SWDs, as previously described (Citraro et al., 2013b, 2014; Russo et al., 2011). 2.4. Statistical analysis All statistical procedures were performed using SPSS 15.0.0 software (SPSS Inc., Chicago, IL, USA). EEG recordings were subdivided into 30 min epochs, and the duration and number of SWDs were treated separately for every epoch. Such values were averaged and data obtained were expressed as mean ± SEM for every dose of compound. Early long-term treated animals were analyzed and compared by repeated measures one-way ANOVA followed by Tukey’s post hoc test. Data obtained from acutely treated animals were compared statistically to their respective control group using one-way ANOVA since treatment was the only variable and followed by a post hoc, two-sided Bonferroni test. All tests used were two-sided and P ≤ 0.05 was considered significant. 3. Results EEG recordings were performed at the age of 6 and 10 months in drug-free conditions (withdrawal at 5 months of age) in order to measure antiepileptogenic effects besides antiseizure effects (Citraro et al., 2014; Russo et al., 2013). Control (untreated) WAG/Rij rats, at 6 months of age, had a mean number of SWDs (nSWDs) of 6.92 with a mean total duration (dSWDs) of 36.93 s and a mean single duration (sSWD) of 5.35 s for a 30 min epoch. SWDs incidence and duration at 10 months of age were increased but this was not significant.

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ELTT with etoricoxib (10 mg/kg/day), for 17 consecutive weeks up to the age of 5 months was associated with a significant (P < 0.05) reduction in the development of spontaneous SWDs (both number and total duration, but not single seizure duration) recorded at 6 months of age; additionally, this effect was maintained for 4 further months up to the age of 10 months (Fig. 1). More specifically, compared to control, ELTT with etoricoxib attained an ∼45% reduction in seizure development, which was persistent for 5 months after treatment withdrawal; no gross behavioural effects were observed during this period and animal growth was not modified (data not shown). No significant difference was observed when comparing etoricoxib effects between 5 and 10 months of age. Accordingly, statistical comparison between etoricoxib-treated rats and control groups either at 6 or 10 months of age was always significant for both nSWDs and dSWDs. Considering the previously reported acute effects of indomethacin in this animal model (Kovacs et al., 2014; Rimoli et al., 2009), we performed a dose–response study for acutely administered etoricoxib (5, 10 and 20 mg/kg, i.p.). At the higher tested doses (10 and 20 mg/kg), etoricoxib had significant antiseizure effects (P < 0.01) on both the number and total duration of SWDs with a peak average reduction (∼50%) 1 h after administration (Fig. 2). These effects were already evident 30 min after injection and lasted for 180 min. In contrast, the lower dose of etoricoxib (5 mg/kg) had no significant effects on absence seizures (Fig. 2). No obvious statistical dose–response relationship was therefore observed for etoricoxib within this dose range tested.

4. Discussion Different studies suggest a key role of brain inflammation in the development of epilepsy (epileptogenesis) and the initiation of seizures (Choi and Koh, 2008; Vezzani et al., 2013b). CNS inflammation affects the integrity of the BBB, enhances neuronal excitability, decreases seizure threshold and may exacerbate seizure-induced brain injury (Kim et al., 2012; Wilcox and Vezzani, 2014). In the present work, we showed that an early long-term treatment (ELTT) with etoricoxib, a selective COX-2 inhibitor, started prior to seizure onset, displayed antiepileptogenic properties, reducing the subsequent development of absence seizures in adult (greater than 6 months) WAG/Rij rats. In agreement with our results, other findings reported antiepileptogenic properties of COX-2 inhibitors in some other animal models of epilepsy (Kulkarni and Dhir, 2009; Rojas et al., 2014; Tanaka et al., 2009). In particular, it was shown that prolonged administration of celecoxib, another selective COX-2 inhibitor, after a pilocarpineinduced status epilepticus, prevented neuronal damage in the hippocampus and epileptogenesis (Jung et al., 2006; Zhang et al., 2008). In contrast, a similar agent parecoxib exerted a significant neuroprotective effect on hippocampal neurones but no antiepileptogenic effects in the same model (Polascheck et al., 2010). COX-2 participates in the inflammatory response, neuronal death, neuronal hyperexcitability and astrocyte activation (Shimada et al., 2014; Voutsinos-Porche et al., 2004). COX-2 expression, which catalyses the production of pro-inflammatory PGs, is induced in the brain after various insults, thus contributing to brain inflammatory processes involved in epileptogenesis (Kawaguchi et al., 2005; Okada et al., 2001). PGs (such as PGF2␣ ) in the mammalian brain are both directly and indirectly involved with neuronal activity (Kim et al., 2001; Tandon et al., 2003). Activation of the PGE2 receptor exacerbates the rapid up-regulation of interleukin (IL)-6 and IL-1␤ in activated microglia (Quan et al., 2013). In agreement, the concentrations of PGs are increased in the cerebrospinal fluid of epileptic patients (Desjardins et al., 2003). Considering that PGE2 has an excitatory

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Fig. 1. Effects of etoricoxib on the development of absence seizures in WAG/Rij rats. Effects of early long-term treatment (ELLT) with etoricoxib on SWD parameters recorded in WAG/Rij rats at both 6 (6 m) and 10 (10 m) months of age. Data are expressed as percentage change relative to 6-month-old control WAG/Rij rats (dotted line). Data values are means ± SEM (n = 10 per group). *Significantly different (P < 0.05) from age-matched untreated control WAG/Rij rats. CTRL = control untreated rats; nSWDs = mean number of SWDs for every 30-min epoch; dSWDs = mean cumulative duration of SWDs for every 30-min epoch expressed in seconds(s); sSWD = mean duration of a single SWD expressed in (s). Note that significant reductions in nSWDs and dSWDs (but not sSWDs) were seen relative to control at 6 and 10 months of age, after ELTT with etoricoxib.

effect on the brain, COX enzyme inhibition has been indicated as a viable therapeutic target for neuroprotective and antiepileptogenic treatment in humans (Vezzani et al., 2013b). It has previously been shown that celecoxib markedly reduces the COX-dependent increase in PGE2 levels in the brain of kainatetreated rats independently from any peripheral action (Ciceri et al., 2002). At odds, other authors have indicated no protective and no antiepileptogenic effects or even a seizure aggravating effect by COX-2 inhibitors (Holtman et al., 2009; Polascheck et al., 2010). Indeed, in the CNS, COX-2 is mainly expressed in glutamatergic neurones particularly within the hippocampus and the cerebral cortex, areas highly involved in seizure onset (Choi and Koh, 2008); furthermore, COX-2 induction in cortical neurons contributes to seizures, and it has been suggested that COX-2 might be an indicator for neuronal seizure activity during epileptogenesis (Serrano et al., 2011). COX-2 is responsible for glial cell activation by prostanoids leading to neuroinflammation maintained by cytokine release. As a consequence, pro-inflammatory cytokine levels (e.g. IL-1␤ and tumour necrosis factor-␣ (TNF-␣)) synthesized by glial cells increase susceptibility to seizures (Vezzani, 2014; Vezzani et al., 2008). Besides these chronic protective effects, acute inhibition of COX-2 also appears to be beneficial in most seizure models (Dhir et al., 2006a); acute treatment with COX-2 inhibitors significantly decreases the incidence of PTZ-induced convulsions, increases seizure threshold and inhibits the development of PTZinduced kindling (Akula et al., 2008; Dhir et al., 2006b, 2007). It also protects from rapid electrical stimulation in rats or mice (Takemiya et al., 2003; Tu and Bazan, 2003) and electroshockinduced convulsions (Shafiq et al., 2003). Nevertheless, the level of neuronal COX-2 expression inside the CNS appears to be coupled to excitatory neuronal activity as evident from the up-regulation of COX-2 protein expression in the brain after seizures (Rojas et al., 2014). This seizure-dependent up-regulation of COX-2 expression is dependent on N-methyl-d-aspartate (NMDA)-type glutamate receptor activity (Adams et al., 1996). Both basal expression and seizure-induced over-expression of COX-2 are reduced by blocking NMDA receptors (Toscano et al., 2008).

Overall, COX-2 appears to have a major role in brain excitability; it is involved in seizure generation and the epileptogenic process including neurodegeneration. Suggestive data also support the antiseizure and antiepileptogenic effects of COX-2 inhibitors even if contrasting results have also been reported, suggesting a possible different role for this enzyme in different types of seizures and epilepsy. Finally, other indirect mechanisms might be involved in the antiepileptogenic/antiseizure effects of COX-2 inhibitors; e.g. the decrease in PG synthesis is directly linked to the accumulation of arachidonic acid, which has previously been demonstrated to inhibit T-type calcium channels (Talavera et al., 2004). This latter effect might indeed contribute to the acute etoricoxib antiseizure effects observed in our model, considering the relevant role of this type of calcium channel in absence epilepsy (Broicher et al., 2008; van Luijtelaar et al., 2000). However, other mechanisms cannot be excluded as in the case of indomethacin (which is also able to block T-type calcium channels) (Rimoli et al., 2009); clearly, a modulation of inflammatory processes through a reduction in brain prostaglandin levels must be considered. Different research groups have reported a strong link between absence seizures and inflammation in animal models and particularly in WAG/Rij rats (Akin et al., 2011; Kovacs et al., 2014; Russo et al., 2014a; van Luijtelaar and Zobeiri, 2014). We have previously suggested that a neuroinflammatory process might also been involved in the epileptogenic process underlying the development of spontaneous absence seizures in WAG/Rij rats; however, consistent data and an exhaustive description of the epileptogenic process in this strain of rats are still lacking (Giblin and Blumenfeld, 2010; Russo et al., 2013; van Luijtelaar and Zobeiri, 2014). While basal cytokine levels are not substantially different between epileptic WAG/Rij and non-epileptic control ACI rats (van Luijtelaar et al., 2012), the bacterial endotoxin lipopolysaccharide (LPS) enhances absence epileptic activity in WAG/Rij rats (Kovacs et al., 2011; Russo et al., 2014a). LPS effects are known to be mediated by increased levels of pro-inflammatory cytokines (i.e. IL-1␤, TNF-␣) and COX-2/PGE2 in the CNS that can enhance the excitation in the cortico-thalamic network, aggravating epileptic seizure activity in

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Fig. 2. Effects of i.p. administration of various doses of etoricoxib on the number (A) and duration (B) of SWDs in WAG/Rij rats. At the higher tested doses (10 and 20 mg/kg), etoricoxib had significant antiseizure effects (P < 0.01; relative to vehicle control) on both the number and total duration of SWDs between 30 and 180 min after administration. These effects were indistinguishable in peak and duration. The lower dose of etoricoxib (5 mg/kg) had no significant effects on absence seizures. Note, no obvious statistical dose–response relationship was evidenced at these tested dose levels. *Significantly different (P < 0.01) from age-matched untreated control WAG/Rij rats.

this strain of rats (Kovacs et al., 2011; Russo et al., 2013, 2014a). LPS and pro-inflammatory cytokines can also induce the expression of COX-2 (Vezzani and Granata, 2005); prostaglandins may have a role in decreasing seizure threshold to LPS and have proconvulsant effects (Sayyah et al., 2003). Our data together with previous reports indicate that COX inhibition (i.e. with indomethacin and/or etoricoxib) can both reduce absence seizures in some animal models (i.e. WAG/Rij, GAERS and Long-Evans rats) and also block LPSdependent proconvulsant effects (Kovacs et al., 2014; Rimoli et al., 2009). Therefore, the acute effects of etoricoxib might well be due to both inflammatory modulation and other effects on cell membrane excitability (e.g. block of T-type calcium channels). On the other hand, etoricoxib has shown long-lasting antiepileptogenic effects, which might be due to the reduction of a possible background inflammatory process present in this strain of rats (Russo et al., 2013, 2014a,b); however, another reasonable explanation might be the possibility of a “seizure begets seizure” process underlying the development of absence seizures in WAG/Rij rats.

According to this process, any drug with anti-absence effects will reduce the age-dependent increase found in these animals. In other words, if we give a drug before seizure onset, it will reduce the daily number of seizures, thereby blocking the subsequent-dependent increase over time. In this light, etoricoxib as well as ethosuximide, might have an indirect antiepileptogenic effect not by acting on mechanisms involved in network re-organization but only by inhibiting seizures through other antiseizure mechanisms. 5. Conclusions In conclusion, we found that etoricoxib has some limited antiabsence seizure properties in the WAG/Rij rat model, considering that its effects are not obviously dose-dependent; whereas, it showed significant long-lasting antiepileptogenic effects. All these effects might be due to COX-dependent mechanisms such as reduced synthesis of prostaglandins and the resulting reduction in neuroinflammation-dependent adaptive processes. In

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any case, other mechanisms such as T-type calcium channel inhibition cannot be excluded. Further experiments are needed to clarify the processes underlying the development of absence seizures in WAG/Rij rats focusing on neuroinflammation, while the present results further support the antiepileptogenic properties of COX-2 inhibitors. Conflicts of interest The authors declare that there are no conflicts of interest. References Adams, J., Collaco-Moraes, Y., de Belleroche, J., 1996. Cyclooxygenase-2 induction in cerebral cortex: an intracellular response to synaptic excitation. J. Neurochem. 66, 6–13. Aid, S., Bosetti, F., 2011. Targeting cyclooxygenases-1 and -2 in neuroinflammation: therapeutic implications. Biochimie 93, 46–51, http://dx.doi.org/10.1016/ j.biochi.2010.09.009. Akin, D., Ravizza, T., Maroso, M., Carcak, N., Eryigit, T., Vanzulli, I., Aker, R.G., Vezzani, A., Onat, F.Y., 2011. 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Rij rats.

Different data suggest the involvement of specific inflammatory pathways in the pathogenesis of epilepsy. Cyclooxygenase (COX), which catalyses the pr...
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