Effect of Phenylbutazone on Pentylenetetrazole-Induced Seizure in Mice

Problem statement: The Cyclooxygenases (COXs), the key enzymes that co nvert arachidonic acid to Prostaglandins (PGs), had been implicated in physiological and pathophysiological functions in the CNS. Non-Stesoidal Anti-Inflammato ry Drugs (NSAIDs), COX inhibitors, were used largely to treat febrile condition, pain state and for prevention of and therapeutics of many diseases . However the role of PGs and NSAIDs in the seizure a ctivity has been disputed. The aim of this study was to evaluation the effect of intraperitoneally i njection of different doses of Phenylbutazone on PT Zinduced seizure threshold in mice. Approach: Mice were divided into 9 groups randomly: The first group received saline normal (ip) (control group); the second group received Carboxy Methyl Cellulose (CMC) 0.5% (ip) (vehicle group) and the n ext groups received respectively different doses of Phenylbutazone (5, 10, 20, 40, 60, 80 and 100 mg kg ip) 45 min before determination of seizure threshold induced by PTZ. Results: Results showed that PTZ-induced seizure threshold i n control mice was 34.75±1.54 mg kg . Intraperitoneal injection of Phenylbutazone showe d significant (p<0.05) increase of PTZ-induced seizure threshold in a dose ep ndently manner compared with control group. Conclusion: Phenylbutazone has anticonvulsant effects on mice. Nevertheless, new studies must be carried out in order to determine the beneficial ef fects of NSAIDs in treatment of epilepsy.


INTRODUCTION
Epilepsy is one of the major neurological diseases in humans and about one percent of the population is involved. Neuroinflammation is an important mechanism in the defense response to pathogenic events, traumatic injury and environmental toxins, but it is also recognized as a major contributor to various neurological and neurodegenerative diseases such as seizure. An innate immune response is mediated by microglia that contributes to the progression of the diseases. Activated microglia produce several proinflammatory and neurotoxic mediators including complement, cytokines, chemokines, acid arashidonic and its metabolites and reactive oxygen and nitrogen species, several of which contribute directly to neuronal injury (Perrone et al., 2010;Mirhadi, 2011). Alterations in the microenvironment such as microglial inflammation and the release of proinflammatory cytokines may affect normal cell proliferation and differentiation, which could cause ectopic neurogenesis, astrogliosis and ectopic synaptic reorganization (Haiju et al., 2009). Inflammatory processes have been implicated in both acute and chronic neurodegenerative conditions such as epilepsy (Dhir et al., 2008).
The Cyclooxygenases (COX) are the principle and obligatory enzymes in the synthesis of Prostaglandins (PGs) and other prostanoids and also the key targets for anti-inflammatory drugs (Dhir et al., 2008). Although cyclooxygenases, the key enzymes that convert arachidonic acid to prostaglandins (including PGE2, PGD2, PGF2α, PGI2 and thromboxane A2), have been implicated in physiological and pathophysiological functions in the CNS, the cellular mechanisms by which COX reaction products are involved have yet to be elucidated (Chen and Bazan, 2005). These enzymes have shown to be expressed in different areas of brain besides peripheral organs and hence speculated to play an important role in neurological disorders (Dhir et al., 2008). Non-Stesoidal Anti-Inflammatory Drugs (NSAIDs), COX inhibitors, are used largely to treat febrile condition, pain state and for prevention of and therapeutics of many diseases. However the role of PGs and NSAIDs in the seizure activity has been disputed (Kim et al., 2008). The enzyme exists as constitutive (COX-1) and inducible (COX-2) isoforms, being the latter a major player in inflammation (Minghetti and Pocchiari, 2007). In the brain, COX-2 expression has been associated with inflammatory and neurodegenerative processes of several human neurological diseases (Minghetti and Pocchiari, 2007). Reports indicated the up-regulation of cyclooxygenase enzyme following seizure activity (Takemiya et al., 2003). The potential role of COX isoforms and PGs in brain diseases has been extensively reviewed in the past years (Minghetti and Pocchiari, 2007). Interestingly, COX-2 induction appears to be characteristic of forebrain seizures, since COX-2 was not induced in midbrain seizures (Dhir et al., 2008). Transcription of COX-2, a proinflammatory mediator encoded in an early-response gene, is induced by synaptic activity; therefore, COX-2 activity could contribute to epileptic neuronal injury (Haiju et al., 2009).
Also, some trials have been performed to find out clinical and pre-clinical evidence of COX-1-mediated proinflammatory effects in neurodegenerative disorders and in models of neuroinflammation. Besides, COX-1 plays a previously unrecognized part in the neuroinflammation, a key stage in the development of several neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, traumatic brain injury, HIV-associated dementia, ischemic stroke and epilepsy, whereas COX-2, mainly localized in pyramidal neurons, is expected to predominantly contribute to increase PG synthesis in response to insults that directly challenge neurons, such as ischemia and exitotoxicity. Under these circumstances, COX-2 inhibition seems to afford protection without altering the inflammatory responce. Thus, the type of insult, the cellular target of the stimulus and whether neuroinflammation is a primary or a secondary response could determine whether COX-2 activity mediates neurotoxicity or neuroprotection. In particular, genetic ablation or pharmacological inhibition of COX-1 activity attenuates the inflammatory response and neuronal loss. This indicates that NSAIDs with higher selectivity for COX-1 rather than COX-2 are more likely to reduce neuroinflammation and should be further investigated as a potential therapeutic approach in neurodegenerative diseases with a marked inflammatory component (Choi et al., 2009). In the CNS, COX-2 is mainly expressed in glutamatergic neurons particularly with in the hippocampus and cerebral cortex, the areas that demonstrate prominent role in the onset of seizures (Choi et al., 2009). It was found that COX-2 regulates cell membrane excitability and long term synaptic plasticity in the hippocampus (Chen and Bazan, 2005), suggesting that COX-2 may play a critical role in convulsive states. However, results of previous studies about the role of COX-2 in the genesis and maintenance of convulsion are controversial; for instance both proconvulsant and anticonvulsant role for COX-2 has been reported in kainic acid-induced seizure (Kim et al., 2008). Also, overexpression of COX-2 has been associated with neurotoxiticy in acute conditions, such as hypoxia/ischemia and seizures, as well as in chronic neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (Minghetti and Pocchiari, 2007). Studies have been shown that COX-2, which is expressed in postsynaptic dendritic spines, regulates PGE2 signaling in activity-dependent long-term synaptic plasticity at hippocampal perforant path-dentate granule cell synapses suggests an important role for COX-2generated PGE2 in synaptic signaling (Zhang et al., 2009). Increased levels of PGD2 and PGE2 following PTZ-induced seizures were observed (Takemiya et al., 2003). PGE1 and PGE2 have excitatory effects on the cerebral cortex, the area that plays an important role in the onset of seizure activity. PGF2α is the predominant prostaglandin identified in the experimentally induced as well as spontaneous seizure activity (Dhir et al., 2008). Animal models of CNS injury have described a pivotal role for COX-2 in promoting neuropathology (Kyrkanides et al., 2002). Furthermore, the induction of astrocytic COX-2 was observed in epilepsy patients with hippocampal sclerosis and the concentrations of PGs increased in the cerebrospinal fluid of these patients (Desjardins et al., 2003). In one model of lithium chloride and tacrine induced status epilepticus seizures, there was an increased expression of COX-2 enzyme protein particularly, in dorsal hippocampus, further resulting in elevated brain PGE2 levels (Dhir and Kulkarni, 2006). In another study, COX-2 induction was found to be responsible for epileptic neuronal injury and that selective COX-2 inhibitors are neuroprotective (Kawaguchi et al., 2005). Studies of seizure activity in animal models indicate that PGD2 and PGE1 have seizure-inhibiting properties, In contrast, intracerebroventricular administration of PGF2α promotes chemical and electronic induced seizures, while intra-amygdaloid administration of PGs showed no significant effects on electrically kindled seizures. Effect of COX inhibitors which are suggested to prevent the production of PGs during seizure activity, were inconsistent according to seizure models, types of COX inhibitors, or time of administration. Thus, the role of COX isozymes or PGs in seizure activity is not clear (Kim et al., 2008). During kainic acid-induced seizure, the PGF2α, PGE2 and PGD2 was increased in the brain after a few minutes. Especially PGF2α was the highest concentration in hippocampus following systemic kainic acid administration and the highest level of PGF2α reaches 30 min (Yoshikawa et al., 2006). In convulsion-prone gerbil, the increase of PGD2 in the brain is significant and exogenous PGD2 inhibits pentylenetetrazole-induced convulsions. PGE2 activated on the electroencephalogram, while prostaglandin E1inhibit pentylenetetrazole-induced convulsions in rats (Kim et al., 2008). The drugs that inhibit COX-2 activity, such as indomethacin and selective COX-2 inhibitors, could reduce hippocampal cell death and seizure frequencies in several animal models of epilepsy (Jung et al., 2006;Kunz and Oliw, 2001a;Shafiq et al., 2003). Thus, drugs that reduce the production of PGs may have useful therapeutic effects in epilepsy. Most of the reports point to the anticonvulsant effects of NSAIDs focused on about orally administration of these drugs. With this background, the aim of this study was investigated the anticonvulsant effect of intraperitoneally administration of Phenylbutazone, a COX inhibitor, against PTZinduced seizure threshold in mice.

MATERIALS AND METHODS
Animals: Experiments were performed on 22-25 g adult NMRI male mice in their 8-9 week (n = 8 for each group), purchased from Razi Institute (Iran). Animals were housed 8 per cage in the Animal House of Veterinary Faculty of Tabriz Branch, Islamic Azad University in a temperature (20-22°C) and humidity (50±10%) controlled environment under a 12 h light/dark cycle (lights on at 7 am). Food and water were available ad libitum. This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of Research affairs of Tabriz University of Medical Sciences, Tabriz, Iran. All efforts were made to minimize the number of animals which were used and their suffering degree. Animals were divided into 9 groups randomly: the first group received saline normal (ip) (control group); the second group received Carboxymethylcellulose (CMC) 0.5% (ip) (vehicle group) and the next groups received respectively different doses of Phenylbutazone (5, 10, 20, 40, 60, 80 and 100 mg kg −1 ip) 45 min before PTZinduced seizure threshold.

Chemicals:
Pentylenetetrazole (PTZ) and Phenylbutazone were provided from Sigma-Aldrich. All other reagents were of analytical grade. Phenylbutazone was prepared by being suspended in 0.5% Carboxymethylcellulose (CMC) and the vehicle group was given an equal volume of vehicle.
PTZ-induced clonic seizure threshold: Behavioral experiments were done in a quiet, temperaturecontrolled (20-22°C) room between 10 and 4 pm. PTZ-induced clonic seizure threshold was determined by inserting a 30-gauge needle into the tail vein of mice and infusion of 0.5% PTZ solution at a constant rate of 1 mL min −1 to unrestrained freely moving animals. Minimal dose of PTZ (mg kg −1 ) needed to induce forelimb clonus followed by full clonus of the body was recorded as an index of clonic seizure threshold (Samini et al., 2005;Shafaroodi et al., 2004).

Data analysis:
Group data are presented as mean±SEM and analyzed statistically using student test. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test. The level for statistical significance was set at a p<0.05.

RESULTS
PTZ-induced seizure threshold in control mice was 34.75±1.54 mg kg −1 . In vehicle group, CMC have not shown significant change on PTZ-induced seizure threshold compared with control group (Fig. 1). Intraperitoneal injection of Phenylbutazone showed significant (p<0.05) increase of PTZ-induced seizure threshold in a dose dependently manner compared with control and vehicle groups ( Fig. 1). Pentylenetetrazole (PTZ) has been used widely to produce the animal model of chemically induced seizure because this model is highly sensitivity for comparing different chemical under standardized conditions (Samini et al., 2005;Shafaroodi et al., 2004). In this study PTZ-induced seizure threshold in control mice was 34.75±1.54 mg kg −1 and pretreatment with Phenylbutazone significantly increased the PTZinduced seizure threshold in a dose dependently manner in mice. Previous studies have been demonstrated the protective effect of COX-inhibitors in various models of epilepsy in animals (Dhir and Kulkarni, 2006;Dhir, 2005;, but effects of intraperitoneally injection of different doses of NSAIDs have not shown. The Effect of COX inhibitors on neuronal death also is disputed. It has been reported that the pretreatment of COX-2 inhibitors including celecoxib aggravated kainic acidinduced seizure activity in rodents and aggravated kainic acid-induced neuronal death in the hippocampus (Baik et al., 1999). The broad COX inhibitor such as ibuprofen caused deficits in spatial learning in a water maze (Shaw et al., 2003), whereas several reports showed the post-treatment of COX-2 inhibitors restored the memory deficit and learning behaviors and prevented seizure-induced neuronal death (Gobbo and O'Mara, 2004;Kunz and Oliw, 2001a;2001b). The effects of COX-2 inhibitors on the PTZ-induced seizures are also controversial; Dhir and coauthors reported the anticonvulsant effect of COX-2 inhibitors (Dhir and Kulkarni 2006;Dhir et al., 2006a), while Akasura and coauthors showed that COX-2 inhibitors have neither anticonvulsant nor proconvulsant effects on PTZ-induced seizures (Akarsu et al., 2006). Treatment with indomethacin, a COX-inhibitor offered full recovery in the E1 mouse, a genetically prone mouse (Okada et al., 2001). In one other report, nonselective COX-inhibitor such as indomethacin, aggravated kainic acid-induced seizure activity and the following hippocampal neuronal death (Baik et al., 1999). Dhir et al. (2006a) also reported that COX inhibitors, viz. nimesulide and rofecoxib, administered 45 min prior to an epileptic challenge prolonged mean onset time of convulsions, decreased duration of clonus and decreased mortality rate against bicuculline-and picrotoxin-induced convulsions in mice (Dhir et al., 2006b;2006c). Different classes of NSAIDs like indomethacin, flurbiprofen and diclofenac were shown to decrease the LD50 and threshold for the PTZinduced convulsion in mice. Based on these observations and the evidence that peripheral or intracerebral administration of PGs antagonized chemically-and electrically induced convulsions, it was suggested that endogenous PGs may exert anticonvulsant effect. In contrast, findings from other studies indicated that PGs may have proconvulsant effect as some NSAIDs, like paracetamol and diclofenac were found to increase the latency to onset of PTZ-induced seizures in mice as a result of blockade of PGs synthesis. Confirming this line of thinking, some studies have reported a potentiating effect of certain NSAIDs on concomitantly administered antiepileptic drugs in MES and PTZ seizure tests (Reeta et al., 2003). COX-2 inhibitors such NS-398, indomethacin, diclofenac and celecoxib aggravated significantly seizure activity in animal model, especially kainic acid-induced seizure model (Baik et al., 1999). There were some evidences, in which Shafiq et al. (2003) showed that there was an increase in percentage protection when celecoxib was combined with standard antiepileptic drug such as phenytoin against electroshock-induced convulsions (Samini et al., 2005). Tu and Bazan (2003) also demonstrated that the COX-2-selective inhibitor nimesulide attenuated kindling development (Tu and Bazan, 2005). In the other studies, effects of COX2 inhibitor on seizure activity are different according to seizure models, types of NSAIDs and methods of administration (Dhir et al., 2005;Yuhas et al., 2005). In electroshock convulsion, celecoxib showed anticonvulsant action (Shafiq et al., 2003) and also posttreatment of celecoxib improved learning and memory deficit (Gobbo and O'Mara, 2004). Nimesulide and rofecoxib were reported to aggravate kainic acid-induced seizures and reduce the cell loss. However those authors reported rofecoxib did not reduce memory deficit and delayed neuronal death (Kunz et al., 2005). In associated with febrile seizure, the potent COX inhibitor has more disadvantage of seizure activity than weak COX inhibitor (Rantala et al., 2001). However the preventive use of ibuprofen as a safe NSAID used largely worldwide for recurrent febrile seizure was not recommended. Ibuprofen also induced the learning and memory deficit. These cumulated data shows that NSAID including COX-2 inhibitors might be harmful especially in seizure conditions (Kim et al., 2008).
Protective effect of COX-1 inhibitors have been shown in other neuroinflammation condition in human and animal models. Alzheimer's Disease (AD), the most common cause of dementia associated with neurodegeneration in the elderly, is clinically characterized by a progressive memory loss and other cognitive impairments. The prevalence of AD increases exponentially with age. In aged rats, COX-1 mRNA expression is selectively increased in the hippocampus, possibly causing an increased susceptibility to neuroinflammation and COX-1-expressing microglia are found surrounding amyloid plaques in the AD brain, indicating a role of this isoform in the pathophysiology of the disease. Supporting this concept, a 6-month, double-blind, placebocontrolled study with indomethacin, a preferential COX-1 inhibitor, seemed to protect AD patients from cognitive decline and reduced levels of β-amyloid (Aβ) in the hippocampus and cortex in a transgenic mouse model of AD. The use of aspirin, a nonselective Aβ42-lowering agent that preferentially inhibits COX-1, has been associated with a reduced risk of AD in humans (Perrone et al., 2010). In the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) model of Parkinson's Disease (PD), in which dopaminergic neurons in the substantia nigra are selectively injured, >65% of ventral midbrain PGE2 originated from COX-1 without any change in COX-1 expression; this indicates a predominant role of this isoform in PGE2 synthesis during MPTP-induced neurodegeneration (Perrone et al., 2010). In Traumatic Brain Injury (TBI), COX-1 involvement has not been extensively studied. There are evidences of accumulation of COX-1-expressing microglia and/or macrophages at peri-lesional areas and in the developing core in patients with TBI, indicating that further investigation of the potential beneficial effects of COX-1 inhibition is necessary (Perrone et al., 2010). Neuronal degeneration in patients with prion diseases is well documented, little is known about downstream signaling cascades to enable targeted therapeutic strategies. Coexpression of CD68 (phagocytic macrophages and microglial cells) in COX-1expressing cells is found in areas of severe tissue damage and also around vacuoles in post-mortem brains from patients with Creutzfeldt-Jacob Disease (CJD). Therefore, activated COX-1-expressing microglia in CJD patients could account for increased production of PGE2 and cytokines and might contribute to the complex process of neurodegeneration (Perrone et al., 2010). Elevated levels of PGE2, PGF2α and TXB2 have been found in the cerebrospinal fluid of patient with HIV associated dementia and have been linked with the severity of cognitive impairment. Because COX-1 mRNA is upregulated approximately two-fold in HIV-demented compared with the nondemented patients, whereas COX-2 mRNA expression is unchanged, the increase in the PG levels is likely to be selectively mediated by COX-1. Thus, the roles of microglia and COX-1 in the host defense system against viral pathogens deserve further investigation (Perrone et al., 2010). In humans and in animals subjected to transient global cerebral ischemia, specific neurons degenerate following the ischemic episode. In determining brain injury there is the primary involvement of COX-2-expressing neurons and the secondary involvement of COX-1-expressing microglia (Perrone et al., 2010). Although, mice lacking COX-1 are more vulnerable to focal cerebral ischemia, probably owing to a more severe cerebral blood-flow reduction in vulnerable regions, pharmacological inhibition of COX-1 potently reduced neuronal injury and oxidative stress in the hippocampus during transient global ischemia. Interestingly, in human focal ischemic brains, COX-1-positive microglia accumulation was observed in peri-infarctional regions and in the developing necrotic core early after infarction. Therefore, the potential therapeutic effects of COX-1 inhibition should be further investigated, particularly in the secondary post-ischemic neuroinflammatory phase (Perrone et al., 2010). COX-1 inhibition also seems to attenuate neuroinflammationinduced Blood-Brain Barrier (BBB) disruption. Selective pharmacological inhibition of COX-1, but not of COX-2, reduced TNF-α-induced BBB disruption and free radical production and indomethacin completely blocked LPS induced permeability changes in cultured monolayers of brain microvessel endothelial cells (Perrone et al., 2010). In stroke, neurons which express COX-2 are directly injured, whereas COX-1-expressing microglia only contribute to the secondary neuroinflammatory response. In a study on electrical amygdala kindling mouse, an animal model of human temporal lobe epilepsy, it was demonstrated the involvement of activate microglial cells in hippocampus during the epileptogenesis, the kindling development and concurrent COX-1 enhancement in this region. Stage and region specific cyclooxygenase expression were identified in the kindled brain and changes in COX-1 immunoreactive cells were found. Treatment with SC-560 dose-dependently delayed the advancement of seizure stage with its anti-seizure effect. Indomethacin at lower doses than SC-560 is more potent, but at higher doses all animals died. Furthermore, in vitro experiment demonstrated that SC-560 reduced also PGE2 and 8-isoPGF2α productions by LPS-activated microglia leading to suppress the kindling development. This result implies that COX-1 and COX-2 are involved in manifestation of epilepsy and occurs a cooperation between the two isoforms during the kindling process (Perrone et al., 2010).
The molecular mechanisms of the anticonvulsant and neuroprotective effects of NSAIDs (COXinhibitors) have not been fully clarified, but evidences tend to suggest the possible involvement of γ-Amino Butyric Acid (GABA) because rofecoxib and nimesulide (both COX-2 inhibitors) potentiated the anticonvulsant effects of subprotective dose of diazepam and muscimol, both GABAergic modulators (Dhir et al., 2008). Similarly the effect of tiagabine, a GABA reuptake inhibitor and a newer antiepileptic was also potentiated by rofecoxib (Dhir and Kulkarni, 2006). Such similar studies have been reported by Tandon et al. (2003) against electro-and chemoconvulsions, respectively (Tandon et al., 2003). In other study, the combined effect of rofecoxib and topiramate had a synergistic action in protecting against PTZ-induced convulsions, has been reported. Topiramate is a antiepileptic agent and is reported to enhance GABA-evoked whole cell Cl¯ currents in mouse cerebral cortical neurons in culture. Besides enhancement of GABA-mediated Cl¯ fluxes into neurons, topiramate is also considered to produce its antiepileptic effect through several other distinct mechanisms, including modification of Na+ and/or Ca2+ dependent action potentials and inhibition of kainate-mediated conductance at glutamate receptors of the AMPA/kainate type. Therefore, rofecoxib when administered concurrently with topiramate displayed synergistic effect, indicating that both the drugs acted through the same mechanism (Dhir et al., 2008). In study of Haiju et al. (2009), NS398, a selective cox-2 inhibitor, increased the frequency of mIPSCs and the decay-time constant. Bicuculline completely blocked mIPSCs, indicating that these synaptic events were mediated by GABAA receptors. These results indicate that NS398 clearly enhances GABAergic transmission in the hippocampus. Also the Western blotting analysis results showed that celecoxib upregulated the expression of GABAA receptors protein. COX inhibitors might be acting through GABAergic neurons, thus increasing the inhibitory neurotransmitter and the expression of GABAA receptor protein (Haiju et al., 2009). Furthermore, arachidonic acid which is metabolized to prostaglandins by cyclooxygenases and has been proposed to be a diffusible second messenger in the CNS with a pathophysiological role in epilepsy. This is possibly due to the ability of arachidonic acid to enhance extra-neuronal glutamate concentration. Cyclooxygenase induction lead to the increase in prostaglandins levels particularly PGE2, which may facilitates glutamate release from the nerve terminal and astrocytes. Glutamate is anc excitatory neurotransmitter that can leads to decrease in GABA input, results in convulsions. Therefore, it may be conceived that COX inhibitors is acting through glutamate and GABAergic modulation (Dhir and Kulkarni, 2006). Other mechanisms by which PGE2 could indirectly contribute to synaptic plasticity include modulation of adrenergic, noradrenegic and glutamatergic neurotransmission and regulation of membrane excitability (Minghetti and Pocchiari, 2007). The possibility is that activation of cyclooxygenases causes increases in free-radical production, leading to oxidative stress and apoptosis of GABAergic neurons, thus increasing glutamate and causing epileptic discharges. Also COX-2 may play a role in excitatory synapses, as excitatory amino acid agonists such as NMDA induce strong COX-2 immunostaining in many regions of the limbic cortex and isocortex, hippocampus and amygdale (Haiju et al., 2009). Furthermore, NMDA-receptor-induced c-fos expression is prostaglandin-dependent and the calcium-dependent activation of cyclooxygenases results in superoxide production (Bidmon et al., 2009).
For PTZ models, it is known that COX inhibitors attenuate seizure activity. Although not as strong as in rat models for cerebral ischaemia, COX-2 expression is induced in human patients with epilepsy and in the model of repeated PTZ induced seizures. COX-2expressing neurons are shown to be closely associated with the processes of neurons expressing nNOS, thus revealing a direct topographical link between COX-2related superoxide and NOS-I-related NO production. As the normal constitutive neuronal expression of COX-2 and the seizure-induced induction of COX-2 take place in a region-specific manner, COX-2-related production of oxygen radicals may represent the basis for region-specific pathological changes attributed to seizure-induced oxidative stress. Some of the most affected cortical regions are the piriform and entorhinal cortices, which exhibit not only high COX-2 expression, but also one of the highest packing densities of NOSI-expressing cortical neurons. This association of neurons responding with the production of radicals such as superoxide and nitric oxide, which results in the formation of peroxynitrite, may be one reason for the fact that seizure-induced protein nitration and glutamine synthetase inhibition are much more easily detectable in piriform and entorhinal cortices than in other regions (Bidmon et al., 2009).
The results of the Zandieh et al. (2010) study indicated that the selective COX-2 inhibitor, celecoxib attenuates seizure induced by PTZ. Co-administration of sub-effective doses of L-NAME and celecoxib protected the animals against PTZ. In addition, L-NAME improved the anticonvulsant activity of celecoxib significantly. L-arginine at the dose which was not able to influence PTZ-induced convulsion, blunted the anticonvulsant effect of celecoxib. In the CNS, NO behaves as a multifunctional messenger and neurotransmitter, influences various physiological and pathological functions. The main intracellular action of NO is activation of the soluble guanylate cyclase which leads to the formation of cycline Guanine Monophosphate (cGMP) in the CNS. An increase in cGMP follows stimulation of L-glutamate receptors mainly of the NMDA type. It has been shown that NO as a retrograde messenger that is synthesized postsynaptically and acts on presynaptic terminals, plays an important role in hippocampal LTP. Further, there is direct evidence from hippocampal cultures that NO can potentiate synaptic transmission. In addition, It has been reported that PTZ kindling in mice is associated with an increase in the amount of neuronal NOS. In concordance with aforementioned findings, NO is considered to be involved in the pathophysiology of epilepsy, although the results of experiments carried out by several authors are often conflicting. Different findings may arise as a consequence of discrepancies in the kinds of drugs, the model of seizures and the species of animals used in experiments. In study of Zandieh et al. (2010) showed that L-NAME, a drug that inhibits all subtypes of NOS non-specifically, attenuates PTZ-induced convulsion dose-dependently which is consistent with previous studies. There are evidences implying the possible interaction between NO and COX pathways in some pathophysiological states including osteoarthritis, angiogenesis, renal perfusion and endotoxin-induced cardiomyopathy (Zandieh et al., 2010). Salvemini (1997) reported that COX activity is regulated by NO. They found that NO directly increases COX-1 activity which leads to increase in prostaglandin E2 production. Similarly, it has been reported that inhibition of NOS inhibits not only NO but also prostaglandin production, suggesting that COX enzymes are targets for pathophysiological roles of NO (Salvemini, 1997). It has been reported that apart from activation of COX-2 by free NO molecules, inducible NOS binds to COX-2 and activates it. There are evidences regarding that the interaction between NO and prostaglandin production pathways occurs at multilevels. For example, COX-2 expression is also modulated and up-regulated by NO. In addition, it has been shown that inducible NOS inhibitors ameliorate the antihyperalgesic effect of COX-2 inhibitors. Most of these findings indicate the interaction between NOS and COX-2; or NO and prostaglandin biosynthetic pathways in inflammatory responses and seizure model (Zandieh et al., 2010).
The latter may also explain why certain other changes affecting neuro-transmitter metabolism seen in other rodent models of epilepsy seem to occur in a most pronounced manner in these cortices. However, the question of why a neuronal induction of the Heat Shock Protein 70 (HSP-70) family does not occur in PTZ models, whereas it is a normal finding in other rodent models of epilepsy, still has to be explained, especially as HSP-70-overexpressing mice seem to be partly protected against PTZ-induced seizure activity (Bidmon et al., 2009). As oxidative and nitrosative stress are not restricted to neurons that are functionally connected with glial cells and blood vessels, it may not be surprising that concomitant glial and endothelial changes do occur. The latter are seen by strong regionspecific focal glial HSP-27 expression. HSP-27 is a well-established marker for oxidative stress. As glial cells are well equipped to cope with pathological alterations and ongoing processes such as during scar formation, it is understandable that glial cells respond in a more delayed manner. When focusing on currently used experimental models of epilepsy, glial responses have to be viewed as a secondary consequence of neuronal seizure activity rather than an initial cause of seizures, as is sometimes suggested. Nevertheless, these secondary glial responses may subsequently contribute to a continuation or progression of epileptic seizures, for example during successful kindling (Bidmon et al., 2009). Rimoli et al. (2009) have been shown that two imidazo[1,2-b]pyridazine derivatives, namely DM1 and DM2 are completely devoid of COX-1 and COX-2 inhibitory activity, but are effective in suppressing Spike and Wave Discharges (SWDs) in WAG/Rij rats, a genetic rodent model of absence epilepsy, similar to what was described for their structural congener Indomethacin (IDM), which significantly decreased SWDs in these rats. As T-type channel blockade has been considered as an electrophysiological feature common to antiabsence drugs, they also investigated whether DM1 and DM2 COX-independent antiseizure effect could depend on T-type channel blockade and they found that these compounds are indeed powerful T-type channel blockers and that this property is displayed by IDM as well. The Rimoli et al. (2009) study showed that IDM suppresses SWDs in vivo in a rat model of absence epilepsy and blocks CaV3.1 channels in vitro (Rimoli et al., 2009). In addition they report evidence suggesting that both these effects are at least in part independent from COX inhibition as they can be also observed with two indomethacin-like imidazopyridazines DM1 and DM2, which, are ineffective in blocking COXs. This suggests that COX inhibition is neither the only nor the major mechanism responsible for the antiseizure activity of NSAIDs. A large body of experimental data accumulated in the past show that COX inhibitors possess antiseizure activity in different forms of epilepsy, including absence epilepsy. However, doubts still exist on the intimate mechanism responsible for these effects (Rimoli et al., 2009). As the blockade of voltage gated T-type Ca2+ channels has been traditionally considered as a major mechanism of action of antiabsence drugs on the basis of classical electrophysiological evidence and of more work on CaV3.1 knockout mice, in the study of Rimoli et al. (2009) investigated whether IDM was effective in blocking these ion channels. In particular, they investigated IDM effect on CaV3.1 channels, which represent the main T-type isoform responsible for the firing of thalamic relay neurons and whose dysfunction is considered crucial in the genesis of absence epilepsy. Their finding that IDM potently blocks CaV3.1 channels suggests that the antiabsence effect of this NSAID could be at least in part related to T-type channel blockade. To the best of their knowledge no previous study has specifically addressed the effect of PGs on T-type channel currents and, thus, the hypothesis that IDM and other NSAIDs could affect the activity of these channels by decreasing PG synthesis remains speculative and could represent an interesting subject of future investigation (Rimoli et al., 2009). A second possible mechanism that could account for IDM-induced decrease in CaV3.1 channel activity could be the accumulation of arachidonic acid that NSAIDs induce by blocking the conversion of this fatty acid in PGs. Arachidonic acid has been shown, indeed, to potently inhibit CaV3.1 and CaV3.2 channel activity in vitro. Interestingly, a significant increase in the expression and activity of PLA2 and in free fatty acid generation have been observed in epilepsy. On the basis of these considerations, Rimoli et al. (2009) cannot exclude that COX-dependent mechanisms such as a decrease in PG synthesis and an increase in plasma membrane arachidonic acid concentration could have a role in determining IDM-induced T-type channel blockade. However, the study of Rimoli et al. (2009) provides strong evidence suggesting that this effect is at least in part independent from COX inhibition. The main argument to support this conclusion is their finding that the ability to block CaV3.1 channels is preserved in two imidazopyridazines, DM1 and DM2, structurally related to IDM but unable to block COXs; importantly, these compounds showed a relevant antiabsence activity in vivo as well (Rimoli et al., 2009). Also, as the structures of DM1 and DM2 have significant similarities with the GABAA binding drug zolpidem, which is known to suppress SWDs in WAG/Rij an interesting possibility is that these drugs could act on GABAA receptors. However, preliminary binding studies performed in their laboratories did not show any affinity of either DM1 or DM2 for GABAergic receptors in vitro (Rimoli et al., 2009).
Also, in the other study an attempt has been made to investigate the effects of three COX-2 preferring inhibitors, namely, nimesulide, celecoxib and rofecoxib in two experimental models of convulsion, Maximal Electroshock (MES)-and PTZ-induced seizures in mice. Since Benzodiazepine (BZD) GABA-ergic and Ca2+ channel neuronal activities have been intimately involved in the process of epileptogenesis, an interaction of nimesulide, the most commonly used COX-2 preferring inhibitor, with diazepam and nimodipine has also been studied in order to elucidate the nature of interaction of preferential COX-2 inhibitors with established antiepileptic drugs. The results of this studies shown that preferential COX-2 inhibitors have opposite effects on MES and PTZinduced convulsions in mice. In MES test, all the three agents protected against THLE, where as in PTZ convulsions, these drugs reduced the latency to preclonic jerks and increased the duration of clonus. There is evidence of an increase in levels of different PGs in brain during/after induction of seizure by MES and PTZ. Out of various PGs, while PGF2α and TxA2 are implicated in promoting, the PGEs, PGI2 and PGD2 have been suggested to suppress seizure activity. Further PGF2α has been reported to antagonize the seizure suppressing activity of PGE1, which, like PGE2 also inhibits PTZ-induced seizures. It thus appears that inhibitory effect on THLE in MES seizure of three studied preferential COX-2 inhibitors may be due to a decrease in synthesis of proconvulsive PGs, i.e., PGF2α and TxA2 as a result of blockade of COX-2 isoform which is not only constitutively expressed in the brain but also gets upregulated during MES and other seizurogenic stimuli (Reeta et al., 2003). In contrast to anticonvulsive effect produced by the three preferential COX-2 inhibitors in the MES test, all of these compounds displayed a proconvulsive activity in PTZ seizure model. Although all drugs showed a decrease in the latency to preclonic jerk and increased the duration of clonic convulsions, this effect was more marked on the latter parameter, since it achieved the minimal level of significance with all the studied agents. The explanation for this differential effect in two models of seizure lies in the nature of convulsive sequences in the two test paradigms. In MES, THLE occurs first followed by clonic episodes, whereas in the PTZ test, latter events precede the phase of THLE. In the PTZ test, besides a decrease in GABA activity due to PTZ, there is evidence of an increased excitatory amino acid activity, which is relative to GABA in the beginning but is further exaggerated as a result of nitric oxide-induced upregulated excitatory amino acid release. The latter is mainly responsible for prolonged/continuous phases of clonic or even clonic-tonic seizure activity. Due to blockade of upregulated COX-2 activity, all the three COX-2 preferring drugs would inhibit the enhanced activity of PGs which may be proconvulsant (PGF2α/TxA2) or anticonvulsant (PGE2), but since these compounds had no effect on excitatory amino acid activity were observed to enhance the PTZ convulsive effect. An antagonism of anti-PTZ effect of nimodipine and diazepam also points to such a possibility of unabated excitatory amino acid activity as a result of blockade of anticonvulsive PG synthesis by the COX-2 preferring inhibitor during the PTZ seizure. In this context, it is pertinent to note that an accumulated arachidonic acid due to its inhibited metabolism to PGs have in earlier studies been shown to block GABAgated chloride channel. Besides an absence of PGs may enhance the excitatory amino acid activity as the latter has been reported to be inhibited by PGE2. Thus, in this study, both of these effects may be contributing to the observed exaggerated PTZ response in animals pretreated with COX-2 preferring inhibitory agents (Reeta et al., 2003).
Ion channel blockade is probably one of the best examples of such a COX-independent action of NSAIDs.
Several published studies already demonstrated that other members of the NSAID superfamily do affect the activity of ion channels by a direct interaction with channel subunits as it has been shown, for instance, for acid sensitive channels (Voilley et al., 2001) are directly blocked by aspirin, diclofenac and flurbiprofen; high voltage activated Ca2+ channels (Zhang et al., 2007), voltage gated Na+ channels (Park et al., 2007) activity is increased by celecoxib; chloride channels (Liantonio et al., 2007), KCNQ channels (Peretz et al., 2005) and hERG channels (Malykhina et al., 2002) are activated by fenamates. Therefore, COX inhibitors may have useful therapeutic effects in seizure. Nevertheless, new and completely studies must be carried out in order to determine in more detail the beneficial actions of NSAIDs regarding the reduction of epilepsy.

CONCLUSION
According to our results, Phenylbutazone has anticonvulsant effects on mice. Probably any tendency to inhibit the COX-2 enzyme by NSAIDs to be more, neuroprotective and anticonvulsant effect of drugs will be higher. Nevertheless, new studies must be carried out in order to determine the beneficial effects of NSAIDs in treatment of epilepsy.