Montelukast reduces seizures in pentylenetetrazol-kindled mice

Brazilian Journal of Medical and Biological Research (2016) 49(4): e5031, http://dx.doi.org/10.1590/1414-431X20155031 ISSN 1414-431X 1/7 Montelukast reduces seizures in pentylenetetrazol-kindled mice J. Fleck1,2,3, F.R. Temp1, J.R. Marafiga1, A.C. Jesse1, L.H. Milanesi1, L.M. Rambo1 and C.F. Mello1 1 Departamento de Fisiologia e Farmacologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil 2 Hospital Universitário de Santa Maria, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil 3 Departamento de Farmácia, Centro de Ciências da Saúde, Centro Universitário Franciscano, Santa Maria, RS, Brasil Abstract Cysteinyl leukotrienes (CysLTs) have been implicated in seizures and kindling; however, the effect of CysLT receptor antagonists on seizure frequency in kindled animals and changes in CysLT receptor expression after pentylenetetrazol (PTZ)-induced kindling have not been investigated. In this study, we evaluated whether the CysLT1 inverse agonist montelukast, and a classical anticonvulsant, phenobarbital, were able to reduce seizures in PTZ-kindled mice and alter CysLT receptor expression. Montelukast (10 mg/kg, sc) and phenobarbital (20 mg/kg, sc) increased the latency to generalized seizures in kindled mice. Montelukast increased CysLT1 immunoreactivity only in non-kindled, PTZ-challenged mice. Interestingly, PTZ challenge decreased CysLT2 immunoreactivity only in kindled mice. CysLT1 antagonists appear to emerge as a promising adjunctive treatment for refractory seizures. Nevertheless, additional studies are necessary to evaluate the clinical implications of this research. Key words: Montelukast; CysLT1R; CysLT2R; Seizure; PTZ; Kindling Introduction Epilepsy is a chronic neurological disease characterized by recurrent seizures due to excessive discharge of cerebral neurons, and by emotional and cognitive dysfunction (1). This disorder affects approximately 50 million individuals worldwide and at least 30% of patients remain refractory, despite the use of antiepileptic drugs (2). Considering the high proportion of patients who do not respond to available treatment, it is essential to search for novel therapeutic targets and to identify seizure mechanisms. Several lines of evidence indicate that inflammation plays a role in epilepsy. Experimental and clinical studies have shown that seizures induce brain inflammation and recurrent seizures perpetuate chronic inflammation (3,4). Indeed, arachidonic acid (AA) is released from membrane phospholipids during seizures, and oxidized by COX (cyclooxygenase) and LOX (lipoxygenase), generating AA proinflammatory products (5). The products of this "uncontrolled arachidonic acid cascade" include prostaglandins, thromboxanes and leukotrienes. Levels of prostaglandin, and leukotriene B4 and C4 are increased in the hippocampus of epileptic patients and in the cerebrospinal fluid of children with febrile seizures (6,7). In addition, kainic acidinduced seizures are associated with increased brain levels Correspondence: C.F. Mello: <> Received July 31, 2015 | Accepted December 1, 2015 Braz J Med Biol Res | doi: 10.1590/1414-431X20155031 of leukotrienes and PGF2a in the cortex, hippocampus and hypothalamus of rats (8). In accordance with these findings, a role for leukotriene receptors, particularly of the CysLT1 subtype, has been proposed in seizure/epilepsy (8–11). Although LTD4 (a CysLT1 receptor agonist) facilitates pentylenetetrazol (PTZ)-induced seizures, intracerebroventricular (icv) injection of montelukast (a CysLT1 receptor inverse agonist) decreases PTZ-induced seizures. In addition, icv montelukast prevents PTZ-induced blood-brain barrier (BBB) disruption and leukocyte infiltration (10), and potentiates the anticonvulsant effect of phenobarbital on PTZ seizures and decreases sedation, a major side effect of phenobarbital (11). Montelukast attenuates PTZ-induced myoclonic jerks and increases oxidative stress markers in rats (12). However, it is still unknown whether CysLT1 receptor antagonism reduces seizures in animals with established seizure susceptibility, such as kindled animals. Therefore, the aim of the current investigation was to evaluate whether montelukast (a CysLT1 inverse agonist) reduces seizures in PTZ-kindled mice. The effects of pharmacological treatment, kindling, and challenge with PTZ on CysLT1 and CysLT2 receptor immunoreactivity in the cerebral cortex of mice were also examined. Montelukast reduces seizures in PTZ-kindled mice 2/7 Material and Methods Animals Young male Swiss mice (25–28 g, 42 days old) from the Animal House of the Universidade Federal de Santa Maria, Santa Maria, RS, Brazil, were used. Animals were housed 12 in an acrylic cage (35  52  17 cm) under controlled light and environmental conditions (12/12 h light/ dark cycle, 22±1°C, 55% relative humidity). Food (Supra, Brazil) and drinking water were provided ad libitum. Behavioral tests were carried out during the light cycle from 9:00 to 17:00 h, in accordance with the national and international legislation (Guidelines of the Brazilian Council of Animal Experimentation – CONCEA – and the EU Directive 2010/63/EU for animal experiments). The protocols were designed to minimize the number of animals used, as well as their suffering, and were approved by the Committee on Care and Use of Experimental Animal Resources of the Universidade Federal de Santa Maria (authorization No. 084/2013). Reagents PTZ was purchased from Sigma-Aldrich (USA), LTD4 and montelukast were from Cayman Chemical (USA), and phenobarbital was from Cristália Pharmaceutical Co. (Brazil). PTZ was dissolved in isotonic saline (0.9% NaCl). Phenobarbital and montelukast were dissolved in 0.5% dimethyl sulfoxide and sterile apyrogenic saline containing 10% propylene glycol. Fresh drug solutions were prepared immediately before use. Kindling induction and seizure observation Mice were intraperitoneally (ip) injected with saline (10 ml/kg) or PTZ (35 mg/kg) three times a week (Monday, Wednesday, and Friday) for 5 weeks, followed by an application-free interval of 1 week (13). After each PTZ injection, convulsive behavior was observed for 20 min and classified into the following stages, as described by Ferraro et al. (14): stage 0, no behavioral change; stage 1, hypoactivity and immobility; stage 2, two or more isolated, myoclonic jerks; stage 3, generalized clonic convulsions with preservation of righting reflex; and stage 4, generalized clonic or tonic-clonic convulsions with loss of righting reflex. An animal was considered kindled when it displayed stage 3 or 4 seizures in three consecutive sessions. The mean time to kindling was 11.2±1.3 days. Overall, 70% of the mice were kindled, 20% were not, and 7% died. Figure 1A and B shows the time-course for effective induction of kindling. The animals that reached kindling criterion were kept drug-free for 1 week and injected subcutaneously (sc) with montelukast (10 mg/kg, sc), phen (...truncated)