Turkish Journal of Medical Sciences Turk J Med Sci (2013) 43: 245-250 http://journals.tubitak.gov.tr/medical/ © TÜBİTAK Research Article doi:10.3906/sag-1204-7 The effect of combined treatment of alpha-tocopherol, ascorbic acid, and pyridoxine with NMDA blocker memantine on penicillin-induced epileptiform activity in rats 1, 2 3 3 Duygu AYDIN *, Mehmet YILDIRIM , Mustafa AYYILDIZ , Erdal AĞAR 1 Department of Physiology, Faculty of Medicine, Turgut Özal University, Ankara, Turkey 2 Department of Physiology, Faculty of Medicine, Karadeniz Technical University, Trabzon, Turkey 3 Department of Physiology, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey Received: 02.04.2012 Accepted: 25.05.2012 Published Online: 15.03.2013 Printed: 15.04.2013 Aim: To evaluate the effects of coadministration of vitamins alpha-tocopherol, ascorbic acid, and pyridoxine with memantine on a penicillin-induced experimental epilepsy model in rats so as to clarify the eventual interaction between these vitamins and the N-methyl- D-aspartate (NMDA) system. Materials and methods: The epileptic focus was produced by intracortical penicillin G potassium injection. The effects of intraperitoneal injections of memantine, memantine + alpha-tocopherol, memantine + ascorbic acid, and memantine + pyridoxine combinations on epileptiform activity were evaluated in electrocorticogram recordings. Results: The antiepileptiform effects appeared earlier in all memantine and vitamin coadministrated groups compared to the memantine-alone group, but the difference between the groups was not statistically significant considering the frequency and amplitude of the epileptiform activity. Conclusion: Coadministration of vitamins does not enhance the antiepileptiform activity of memantine in penicillin-induced epilepsy in rats. However, this coadministration causes the earlier appearance of antiseizure effects. Since moderate doses of these vitamins have no side effects, it might be a good idea to use them with NMDA blockers to provide an earlier antiepileptic effect. Key words: Experimental epilepsy, NMDA, memantine, ascorbic acid, pyridoxine, alpha-tocopherol 1. Introduction effects of free radicals are controlled by a wide range of Epileptogenesis is associated with an imbalance between antioxidants, such as ascorbic acid, alpha-tocopherol, excitatory and inhibitory control systems in selective and pyridoxine (5–7). It has also been suggested that regions of the brain. Particularly, excessive activation of such vitamins have neuroprotective properties in some N-methyl-D-aspartate (NMDA)-type glutamate receptors, experimental models of epilepsy (7–11). Alpha-tocopherol manifested as neuronal excitotoxicity, has been linked protects cell membranes against oxidative damage by to several neurological disorders including epilepsy (1). regulating the production of reactive oxygen species The therapeutic potential of various NMDA antagonists (ROS) and maintaining oxidative phosphorylation in has been investigated in different experimental models of mitochondria, thus accelerating the regeneration of high epilepsy (1,2), but none have proven to be both effective and energy phosphates (6), whereas ascorbic acid scavenges safe. Memantine is a low-affinity, uncompetitive antagonist the aqueous ROS by very rapid electron transfer and on NMDA receptors. It is approved for treatment of thus inhibits lipid peroxidation (5). Ascorbate has also Alzheimer disease and now has potentially wide-ranging been suggested to decrease NMDA-mediated currents by applications, from neuroprotection to the treatment of acting at the redox modulatory site of the NMDA receptor. experimental epilepsy, without the undesirable side effects Pyridoxine is the active coenzyme for many enzymes, of many high-affinity NMDA receptor antagonists (3). including glutamic acid decarboxylase, an enzyme that has On the other hand, it was reported that free radicals a role in gamma-aminobutyric acid (GABA) synthesis in could inactivate glutamine synthase and could induce mammalian cells (12). GABA is a neurotransmitter with epileptic seizures by abnormal expression of the excitatory an important role in the pathophysiology and treatment neurotransmitter, glutamic acid (4). The biological of epilepsy. * Correspondence: [email protected] 245 AYDIN et al. / Turk J Med Sci Therefore, we can say that all 3 vitamins chosen for this WI, USA), infusion rate 0.5 µL/min) (9). Memantine was study are related to 1 or more antiepileptogenic pathways. dissolved in distilled water and administrated 30 min However, to our knowledge, no published data are available after the penicillin injection (5 mg/kg, i.p.). Ascorbic about the effects of these vitamins on the antiepileptic acid, alpha-tocopherol, or pyridoxine were administrated potential of any drugs in experimental models of epilepsy. intraperitoneally 15 min after the memantine injection. Consequently, the objectives of the present study were 2.5. Electrocorticography recordings to compara tively study the effects of coadministration of The electrocorticography (ECoG) activity was memantine with ascorbic acid, pyridoxine, and alpha- continuously monitored on a recorder (PowerLab, 4/SP, tocopherol on penicillin-induced epileptiform activity in AD Instruments, Castle Hill, Australia) and was stored rats in order to clarify their interaction with the NMDA on a computer. The frequency and amplitude of the system. epileptiform ECoG activities were analyzed offline. 2.6. Data analysis 2. Materials and methods All statistical procedures were performed using SPSS 12.0. 2.1. Subjects The differences between the groups were analyzed with Adult male Wistar rats weighing 200–250 g were used one-way ANOVA. Significant differences were further throughout this study. All described procedures were evaluated using the Tamhane post hoc test. Data were approved by the local ethics committee. The animals were expressed as mean ± standard error of the mean (SEM). housed in standard cages and were allowed free access to Statistical significance was set at P < 0.05. food and water. They were kept in a temperature-controlled (22 ± 1 °C) environment on a 12-h light/dark cycle. 3. Results 2.2. Experimental groups The epileptiform activity characterized by spikes and The rats were assigned to the following experimental spike–wave complexes began 2–5 min after the penicillin groups and each group comprised 7 rats: injection, reached a constant level of frequency and (1) Penicillin (500 IU, 2.5 µL, intracortical); amplitude in 30 min, and lasted for 3–5 h (Figure 1). We (2) Penicillin + memantine (5 mg/kg, intraperitoneal used the most effective doses of memantine (5 mg/kg, i.p.) [i.p.]); (9), ascorbic acid (100 mg/kg, i.p.) (2), alpha-tocopherol (3) Penicillin + memantine + pyridoxine (40 mg/kg, (500 mg/kg, i.p.) (3), and pyridoxine (40 mg/kg, i.p.) (7) i.p.); for penicillin-induced epileptiform activity, which were all (4) Penicillin + memantine + alpha-tocopherol (500 determined in previous studies. Memantine significantly mg/kg, i.p.); decreased the frequency of epileptiform activity 50 min (5) Penicillin + memantine + ascorbic acid (100 mg/ after injection (Figure 2). The mean spike frequency of kg, i.p.) ECoG activity was 14 ± 2 spikes/min in the memantine- administrated group at 60 min after injection. Significant 2.3. Surgical procedure anticonvulsant effects were seen at 40 min, 40 min, and 30 The animals were anesthetized with a single injection of min after memantine injection in the groups administered urethane (1.25 g/kg, i.p.) and placed in a stereotaxic frame. memantine + ascorbic acid, memantine + alpha-tocopherol, Rectal temperature was maintained between 36–37 °C and memantine + pyridoxine, respectively (Figure 2). The using a feedback-controlled heating system. A polyethylene mean spike frequencies of epileptiform activities were 13 ± cannula was introduced into the right femoral artery 3, 13 ± 3, and 12 ± 2 spikes/min at 60 min after memantine to monitor blood pressure, which was kept above 110 injection in the memantine + ascorbic acid, memantine + mmHg during the experiments (mean: 120 ± 5 mmHg). alpha-tocopherol, and memantine + pyridoxine groups, The left cerebral cortex was exposed by craniotomy (5 mm respectively. The earliest antiepileptiform effect appeared posterior to bregma and 3 mm lateral to sagittal sutures). in the memantine + pyridoxine group as compared to the Two Ag-AgCl ball electrodes were placed over the left other groups. No significant difference was found regarding somatomotor cortex (electrode coordinates: first electrode the mean spike amplitude of epileptiform activity in any of 2 mm lateral to sagittal suture and 1 mm anterior to the groups (Figure 3). The mean spike amplitudes of ECoG bregma, second electrode 2 mm lateral to sagittal suture activities were 783 ± 50 µV, 1014 ± 146 µV, and 1000 ± 258 and 5 mm posterior to bregma). The common reference µV in the memantine + ascorbic acid, memantine + alpha- electrode was fixed on the pinna. tocopherol, and memantine + pyridoxine groups 60 min 2.4. Drug and drug administration after the memantine injection, respectively. The epileptic focus was produced by 500 IU of intracortical penicillin G potassium injection (short-term experimental 4. Discussion model of focal epilepsy; 1 mm beneath the brain surface by Previous experiments have shown that memantine, a Hamilton microsyringe, type 701N (Aldrich, Milwaukee, pyridoxine, alpha-tocopherol, and ascorbic acid have 246 ÇAKIL et al. / Turk J Med