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The effects of ethanol intake and withdrawal on penicillin-induced epileptiform activity in rats
Brain Research Bulletin 71 (2006) 111–115
The effects of ethanol intake and withdrawal on penicillin-induced epileptiform activity in rats Ramazan Kozan, Mustafa Ayyildiz, Mehmet Yildirim, Erdal Agar ∗ Department of Physiology, Faculty of Medicine, University of Ondokuz Mayis, 55139 Samsun, Turkey Received 11 April 2006; received in revised form 16 August 2006; accepted 16 August 2006 Available online 7 September 2006
Abstract Previous experiments have shown that ethanol may have either pro-convulsive or anti-convulsive effects on epileptic activity in different experimental epilepsy models. In this study, the effect of low dose ethanol and its withdrawal on penicillin-induced epileptiform activity in rat was investigated. Eight groups of adult, male Wistar rats were studied: (1) control, (2) penicillin pretreated (500 units), (3) ␣-tocopherol (500 mg/kg, i.m.), (4) penicillin pretreated + ␣-tocopherol, (5) ethanol-treated (3 g/kg, per day, for 15 days, intragastrically) + penicillin, (6) ethanoltreated + penicillin + ␣-tocopherol, (7) ethanol withdrawal + penicillin, (8) ethanol withdrawal + penicillin + ␣-tocopherol. Each animal group was composed of seven rats. The epileptiform activity was verified by electrocorticographic (ECoG) recordings. The epileptiform activity was induced by microinjection of penicillin into the left sensorimotor cortex. Administration of ethanol (3 g/kg, per day, for 15 days, intragastrically) did not change either frequency or amplitude of penicillin-induced epileptiform activity. The frequency and amplitude of epileptiform activity were evaluated 40 h after the last ethanol administration in withdrawal groups. There was no significant change in the mean frequency and amplitude of epileptiform activity compared with penicillin pretreated and ethanol-treated groups. The effective dose of ␣-tocopherol (500 mg/kg, i.m.) significantly decreased the mean frequency of epileptiform activity in the 60, 70, and 120 min after ␣-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively. However, ␣-tocopherol did not affect the amplitude of epileptiform activity in all groups. In conclusion, the present results indicate that low dose of ethanol does not have either anticonvulsive or proconvulsive effect on penicillin-induced epileptiform activity. ␣-Tocopherol has anti-convulsive effect on penicillin-induced epileptiform activity in effective dose. © 2006 Published by Elsevier Inc. Keywords: EcoG; Epileptiform activity; Ethanol; Penicillin; Rats; ␣-Tocopherol
1. Introduction Epilepsy is characterized by chronic recurrent paroxysmal changes in neurologic functions caused by abnormalities in the electrical activity of the brain [6]. Epileptogenic processes have been associated with imbalance between excitatory and inhibitory control systems in selective regions of the brain [4,16]. Ethanol and epilepsy are complexly interrelated and have been linked since Hippocrates [11]. In recent reviews, it was also shown that there is a relationship between alcoholism and epilepsy [7,11,21,28]. Experimental models of epilepsy provide evidence of a depressant [5,9] as well as of an excitatory [10] effect of ethanol upon seizure. A number of previous in vivo studies has revealed
∗
Corresponding author. Tel.: +90 312 1919x2223; fax: +90 457 6041. E-mail address: [email protected] (E. Agar).
0361-9230/$ – see front matter © 2006 Published by Elsevier Inc. doi:10.1016/j.brainresbull.2006.08.008
anticonvulsant effects of ethanol in various electrically- and chemically-induced seizures tests [9,15,29]. Scorza et al. [25] reported that the ethanol administration induces behavioral (frequency of seizures) and neuropathological changes during chronic period of pilocarpine model of epilepsy. In addition, they noted that the ethanol withdrawal syndrome is a crucial event for the development of functional and neuropathological alterations associated with epilepsy. The anticonvulsant activity of ethanol was investigated in two representative models of experimental epilepsy in a recent paper [7]. It was shown that in the maximal electroshock seizure (MES) threshold test in mice, ethanol (0.5–2 g/kg i.p.) dose-dependently raised the electroconvulsive threshold for tonic seizures [7]. In co-medication with valproate and carbamazepine, ethanol significantly increased the anticonvulsant effectiveness of both antiepileptic drugs [7]. According to Freeman, the depressant effect of ethanol on kindling could be due to its depressant action on the
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electrical activity of the reticular formation [9]. On the other hand, McQuarrie and Fingl [20] did not find changes of the mean seizure threshold during the chronic administration of ethanol (2.0 g/kg, orally) in the low frequency electroshock seizure threshold test in mice. Furthermore, the subchronic administration of a higher dose of ethanol revealed neither a significant decrease of the MES seizure threshold nor a marked tolarence to the anticonvulsant activity tested 24 h after withdrawal in the MES threshold test [7]. This question seems to be matter of special interest, since ethanol is considered to be a high risk factor for epileptic patients. However, there is no evidence that ethanol affects the epileptiform activity in the penicillin-induced epilepsy. Therefore, in the present experiments, the effects of subchronic treatment with ethanol, and its withdrawal on epileptiform activity were investigated in the penicillin-induced epilepsy in the rats. In addition, we have analyzed the effects of ␣-tocopherol (vitamin E) on epileptiform activity in the penicillin model of epilepsy after subchronic premedication of ethanol and its withdrawal in the rats. 2. Materials and methods The animal studies were conducted with governmental approval according to local guidelines for the care and use of laboratory animals. Fifty-six male Wistar rats (240–270 g) were housed individually on a 12-h light:12h dark cycle (lights on at 07.00 h), at a temperature of 21 ± 2 ◦ C and 50% humidity. Rats were assigned to the following experiments and groups: intracortical (i.c.) delivery of (1) 2 l artificial cerebrospinal fluid [aCSF containing (mM): NaCl, 124; KCl, 5; KH2 PO4 , 1.2; CaCl2 , 2.4; MgSO4 , 1.3; NaHCO3 , 26; glucose, 10; HEPES, 10; pH 7.4 when saturated with 95% O2 and 5% CO2 ] (i.c.); (2) 500 IU (2 l, i.c.); (3) 500 mg/kg ␣-tocopherol (i.m.); (4) penicillin pretreated + 500 mg/kg ␣-tocopherol (i.m.); (5) ethanoltreated (3 g/kg, per day, for 15 days, intragastrically) + penicillin; (6) ethanoltreated (3 g/kg, per day, for 15 days, intragastrically) + penicillin + 500 mg/kg ␣-tocopherol (i.m.); (7) ethanol withdrawal (3 g/kg, per day, for 15 days, intragastrically) + penicillin; (8) ethanol withdrawal (3 g/kg, per day, for 15 days, intragastrically) + penicillin + 500 mg/kg ␣-tocopherol (i.m.). Each animal group was composed of seven rats.
2.1. Induction of epileptiform activity The animals were anesthetized with urethane (1.25 g/kg−1 , i.p.) and placed in a stereotaxic frame (Harvard Stereotaxic Instrument). Rectal temperature was maintained between 36.5 and 37.0 ◦ C using a feedback controlled heating system (Harvard Apparatus Limited). A polyethylene cannula (Bicakcilar) was introduced into the right femoral artery to monitor blood pressure, which was kept above 100 mmHg during the experiments (mean 114 ± 7 mmHg). All contact and incision points were infiltrated with procaine hydrochloride to minimize possible sources of pain. The left cerebral cortex was exposed by craniotomy. The epileptic focus was produced by 500 units penicillin G potassium injection (2 mm posterior to bregma and 3 mm lateral to sagittal sutures, 1 mm beneath the brain surface by a Hamilton microsyringe type 701N; infusion rate 0.5 l/min) [2,3,19].
2.2. Drug and drug administration Ethanol treated rats, received a daily dose of 3.0 g/kg of 30% ethanol solution via an oesophageal probe for 15 days. Ethanol withdrawal rats, received a daily dose of 3.0 g/kg of 30% ethanol solution via an oesophageal probe for 15 days. At the end of this period, ethanol administration was stopped and 40 h after the last administration of ethanol, and all rats were anesthetized for induction of epileptiform activity. Ethanol intake was administrated at approximately the same time (between 9:00 and 10:00 h) during the whole procedure. Blood ethanol
concentrations were measured 90 min after ethanol administration in different groups (n = 35). d␣-tocopherol acetate (Aksu Farma Chemical Co.) was used in the experiments. Precautions were taken to minimize exposure to the light. A dose of 500 mg/kg ␣-tocopherol was administrated (i.m.) 30 min after penicillin (i.c.) application.
2.3. ECoG recordings Two Ag–AgCl ball electrodes were placed over the left somatomotor cortex (electrode coordinates: first electrode; 2 mm lateral to sagittal suture and 1 mm anterior to bregma; second electrode; 2 mm lateral to sagittal suture, 5 mm posterior to bregma). The common reference electrode was fixed on the pinna. The ECoG activity was continuously monitored on a four channel recorder (PowerLab,4/SP). All recordings were stored on a computer. The frequency and amplitude of epileptic activity was analyzed off line.
2.4. Statistical analysis All statistical procedures were performed using SPSS statistical software package (version: 12.0). Data analysis was performed using one way ANOVA and Bonferroni post hoc tests for comparisons. Data are expressed as the means ± SEM. Statistical significance was set at P < 0.05.
3. Results Baseline activities of each animal were recorded before the administration of intracortical injection of penicillin (Fig. 1a). Intracortical injection of penicillin (500 units) induced an epileptiform ECoG activity characterized by bilateral spikes and spikewave complexes (Fig. 1b). This ECoG activity began within 3–5 min after penicillin application and lasted for 3–5 h. It reached a constant level as a frequency and amplitude in 30 min. The mean of spike frequency and amplitude were 28.3 ± 3, 981 ± 168 V; 31.6 ± 4, 1,018 ± 181 V and 34.2 ± 2 spike/min, 1256 ± 211 V in penicillin pretreated, ethanoltreated, and ethanol withdrawal groups in 30 min from penicillin injection (i.c.), respectively (Fig. 1b,d,f). There was no significant difference in the mean frequency and amplitude of epileptiform activity during experiments between these groups. The dose of 500 mg/kg ␣-tocopherol significantly decreased the mean frequency of epileptiform activity to 10.4 ± 1.8, 13.8 ± 2.3, 14.7 ± 2.1 spike/min in 60, 70, and 120 min after ␣-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively (Fig. 2). ␣-Tocopherol was less effective to decrease the mean frequency of epileptiform activity in ethanol-treated group than ␣-tocopherol administrated group alone 90 min after ␣-tocopherol injection (Fig. 2). ␣-Tocopherol did not significantly change the amplitude of epileptiform activity in all groups during experiments. The amplitudes of epileptiform activity were 1,001 ± 97 V, 1,018 ± 91 V, 991 ± 87 V, after 80 min from ␣-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively (Fig. 1c,e,g). There was no change in the mean of frequency and amplitude in aCSF injected animals (Fig. 1h). Intramuscular injection of ␣-tocopherol did not cause any change in the frequency or amplitude of ECoG activity with respect to control baseline in non-penicillin injected animals.
R. Kozan et al. / Brain Research Bulletin 71 (2006) 111–115
Fig. 1. The baseline ECoG activities (a). Intracortical injection of penicillin (500 units) induces an epileptiform ECoG activity characterized by bilateral spikes and spike-wave complexes (b). ␣-Tocopherol of 500 mg/kg significantly decreases the frequency of epileptiform activity in penicillin pretreated (c), ethanol-treated (e), ethanol withdrawal groups (g). There is no significant change in the mean of frequency and amplitude of epileptiform activity compare to penicillin pretreated and ethanol-treated (3 g/kg, per day, for 15 days, intragastrically) groups (d, f). There is also no change in the mean of frequency and amplitude in aCSF injected animals (h). Representative ECoGs are presented 80 min after ␣-tocopherol or 110 min after penicillin administration (Bar 1 mV, 30 s; Bar 500 V, 10 s).
Blood ethanol concentrations (BEC) were measured 90 min after ethanol administration in different groups (n = 35). Blood ethanol concentration were 0, 245.7, 258.4, 247.3, and 252.8 mg/dl, in the control, ethanol-treated, ethanoltreated + ␣-tocopherol, ethanol withdrawal, ethanol withdrawal + ␣-tocopherol groups, respectively. The mean of BEC was 248.5 mg/dl in ethanol-administered groups. There was no significant difference found between ethanol-administrated groups. 4. Discussion Epilepsies are complex neurobehavioral disorders resulting from increased excitability of neurones in various brain regions [24]. Prolonged ingestion of ethanol or its withdrawal results in a number of signs and symptoms consistent with enhanced
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excitability, including increased sensitivity to convulsants, hypervigilance, anxiety and possibly seizure activity [26,27]. Epileptic seizures have been studied in genetic, electrophysiological, and chemical models [7,18,19]. In the present study, we provide further evidence for the effects of ethanol intake, and its withdrawal on epileptiform activity in the penicillin-induced epilepsy in the rats. A number of previous in vivo studies in the pentylenetetrazol (PTZ)-kindling and ferric chloride models in rats provide evidence for anticonvulsant and antiepileptic effects of ethanol after acute application, whereas repeated administration of high doses with abrupt withdrawal periods tend to proconvulsant actions [8,13]. Scorza et al. [25] showed that there is an increase in the frequency of seizures in the first 15 days of ethanol treatment by using the pilocarpine model of epilepsy in rats. Furthermore, they noted that the increase of seizure frequency was not evenly distributed, as there were clusters of seizures at characteristic time points after daily ethanol administration [25]. However, after 2 weeks of daily administration of ethanol they observed a decrease in the frequency of seizures, which suggests a tolerance effect [25]. Present study showed that administration of ethanol (3 g/kg, for 15 days) or its withdrawal did not change either frequency or amplitude of penicillin-induced epileptiform activity. This result confirms and extends previous observations by Fisher [7] and Hoppener et al. [12]. It has been previously reported that the subchronic administration of higher dose of ethanol revealed, neither a significant decrease of the MES seizure threshold nor a marked tolerance to the anticonvulsant activity tested 24 h after withdrawal in the MES threshold test. In addition, intake of small to moderate quantities of ethanol appears not to increase seizure frequency in non-alcoholic patients [12]. McQuarrie and Fingl [20] also did not find changes of the mean seizure threshold during the chronic administration of ethanol (2.0 g/kg, for 14 days) in the low-frequency electroshock seizure threshold test in mice. However, Kokka et al. [15] showed in the PTZ seizure threshold test in rats that the anticonvulsant activity of ethanol was only slightly decreased by chronic ethanol treatment (5.0 g/kg for 28 days). The reasons for these inconsistent results are unclear. They may be attributed to the differences in experimental design, e.g., the dose and duration of ethanol exposure, the differences among epilepsy models and tested animals strains. Unfortunately, there is a lack of data to make more complete comparison in the effects of ethanol on penicillin-induced epileptiform activity in rat, since it has not been reported before. Active oxygen free radical scavengers such as ascorbic acid or ␣-tocopherol (vitamin E) might have a role for protection against epilepsy and ethanol-induced toxicity [1,17]. In our previous work, it was found that ␣-tocopherol of 500 mg/kg (i.m.) was the most effective dose in changing of frequency on penicillininduced epileptiform activity in rat [3]. Therefore, the dose of 500 mg/kg ␣-tocopherol was used in all experiment groups in the present study and it significantly decreased the mean frequency of epileptiform activity in all groups. The anticonvulsant effects of ␣-tocopherol appeared in the 60, 70, and 120 min after ␣-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively. This result confirms previous studies which indicated that ␣-tocopherol has anti-
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Fig. 2. The effect of ␣-tocopherol on the mean frequency of penicillin-induced epileptiform activity in rats. The dose of 500 mg/kg ␣-tocopherol significantly decreases the mean of frequency of epileptiform activity. The significant effects appeared in the 60, 70, and 120 min after ␣-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively. ()P < 0.05, ( )P <0.01, ()P < 0.001, ( ) intracortical (i.c.) delivery of 500 units penicillin (2 l); ( ) penicillin pretreated (500 units) + 500 mg/kg ␣-tocopherol (i.m.); ( ) ethanol treated rats received a daily dose of 3.0 g/kg ethanol for 15 days (intragastrically); ( ) ethanol withdrawal rats were anesthetized for induction of epileptiform activity 40 h after the last administration of ethanol (3.0 g/kg ethanol, per day, for 15 days, intragastrically); ( ) ethanol-treated (3 g/kg ethanol, per day, for 15 days, intragastrically) + penicilin (500 units, i.c.) + 500 mg/kg ␣-tocopherol (i.m.); ( ) 40 h after the last administration of ethanol (3.0 g/kg ethanol, per day, for 15 days, intragastrically) + penicillin (500 units, i.c.) + 500 mg/kg ␣-tocopherol (i.m.).
seizure activity in several animal models, including the ferrous chloride [17], the hyperbaricoxygen [14], kindling, PTZ [24], and penicillin-induced [3] models. The anticonvulsant effects of ␣-tocopherol was less effective in ethanol-treated group than ␣tocopherol-treated group alone in the present study. This result is no surprise since it is known that ethanol can stimulate the formation of free radicals or exhaust the antioxidant defense system, leading to oxidative stress in the brain [1,22,23]. In conclusion, low dose of ethanol (3 g/kg) does not have either anticonvulsive or proconvulsive effect on penicillininduced epileptiform activity in rat. Effective dose of ␣tocopherol (500 mg/kg) has free radical scavenging activities [1], suggesting the anticonvulsive action of ␣-tocopherol may result from its free radical scavenging activities. However, molecular events that take place during epilepsy and the exact mechanism of ␣-tocopherol in the ethanol toxicity and epilepsy are poorly understood. These will need to be further investigated electrophysiologically.
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[25] F.A. Scorza, R.M. Arida, R.M. Cysneiros, M.R. Priel, M. de Albuquerque, E.A. Cavalheiro, The effects of alcohol intake and withdrawal on the seizures frequency and hippocampal morphology in rats with epilepsy, Neurosci. Res. 47 (2003) 323–328. [26] D.N. Stephens, G. Brown, T. Duka, T.L. Ripley, Impaired fear conditioning but enhanced seizure sensitivity in rats given repeated experience of withdrawal from alcohol, Eur. J. Neurosci. 14 (2001) 2023–2031. [27] G. Tsai, D.R. Gastfriend, J.T. Coyle, The glutamatergic basis of human alcoholism, Am. J. Psychiatry 152 (1995) 332–340. [28] L. Yang, C. Long, M.S. Evans, C. Faingold, Ethanol withdrawal results in aberrant membrane properties and synaptic responses in periaqueductal gray neurons associated with seizure susceptibility, Brain Res. 957 (2002) 99–108. [29] O.V. Zuk, V.G. Zinkovsky, N.Y. Golovenko, The pharmacodynamics of anticonvulsant and subconvulsant effects of ethanol in CBA and C57BL/6 mice, Alcohol 23 (2001) 23–28.
The effects of ethanol intake and withdrawal on penicillin-induced epileptiform activity in rats Ramazan Kozan, Mustafa Ayyildiz, Mehmet Yildirim, Erdal Agar ∗ Department of Physiology, Faculty of Medicine, University of Ondokuz Mayis, 55139 Samsun, Turkey Received 11 April 2006; received in revised form 16 August 2006; accepted 16 August 2006 Available online 7 September 2006
Abstract Previous experiments have shown that ethanol may have either pro-convulsive or anti-convulsive effects on epileptic activity in different experimental epilepsy models. In this study, the effect of low dose ethanol and its withdrawal on penicillin-induced epileptiform activity in rat was investigated. Eight groups of adult, male Wistar rats were studied: (1) control, (2) penicillin pretreated (500 units), (3) ␣-tocopherol (500 mg/kg, i.m.), (4) penicillin pretreated + ␣-tocopherol, (5) ethanol-treated (3 g/kg, per day, for 15 days, intragastrically) + penicillin, (6) ethanoltreated + penicillin + ␣-tocopherol, (7) ethanol withdrawal + penicillin, (8) ethanol withdrawal + penicillin + ␣-tocopherol. Each animal group was composed of seven rats. The epileptiform activity was verified by electrocorticographic (ECoG) recordings. The epileptiform activity was induced by microinjection of penicillin into the left sensorimotor cortex. Administration of ethanol (3 g/kg, per day, for 15 days, intragastrically) did not change either frequency or amplitude of penicillin-induced epileptiform activity. The frequency and amplitude of epileptiform activity were evaluated 40 h after the last ethanol administration in withdrawal groups. There was no significant change in the mean frequency and amplitude of epileptiform activity compared with penicillin pretreated and ethanol-treated groups. The effective dose of ␣-tocopherol (500 mg/kg, i.m.) significantly decreased the mean frequency of epileptiform activity in the 60, 70, and 120 min after ␣-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively. However, ␣-tocopherol did not affect the amplitude of epileptiform activity in all groups. In conclusion, the present results indicate that low dose of ethanol does not have either anticonvulsive or proconvulsive effect on penicillin-induced epileptiform activity. ␣-Tocopherol has anti-convulsive effect on penicillin-induced epileptiform activity in effective dose. © 2006 Published by Elsevier Inc. Keywords: EcoG; Epileptiform activity; Ethanol; Penicillin; Rats; ␣-Tocopherol
1. Introduction Epilepsy is characterized by chronic recurrent paroxysmal changes in neurologic functions caused by abnormalities in the electrical activity of the brain [6]. Epileptogenic processes have been associated with imbalance between excitatory and inhibitory control systems in selective regions of the brain [4,16]. Ethanol and epilepsy are complexly interrelated and have been linked since Hippocrates [11]. In recent reviews, it was also shown that there is a relationship between alcoholism and epilepsy [7,11,21,28]. Experimental models of epilepsy provide evidence of a depressant [5,9] as well as of an excitatory [10] effect of ethanol upon seizure. A number of previous in vivo studies has revealed
∗
Corresponding author. Tel.: +90 312 1919x2223; fax: +90 457 6041. E-mail address: [email protected] (E. Agar).
0361-9230/$ – see front matter © 2006 Published by Elsevier Inc. doi:10.1016/j.brainresbull.2006.08.008
anticonvulsant effects of ethanol in various electrically- and chemically-induced seizures tests [9,15,29]. Scorza et al. [25] reported that the ethanol administration induces behavioral (frequency of seizures) and neuropathological changes during chronic period of pilocarpine model of epilepsy. In addition, they noted that the ethanol withdrawal syndrome is a crucial event for the development of functional and neuropathological alterations associated with epilepsy. The anticonvulsant activity of ethanol was investigated in two representative models of experimental epilepsy in a recent paper [7]. It was shown that in the maximal electroshock seizure (MES) threshold test in mice, ethanol (0.5–2 g/kg i.p.) dose-dependently raised the electroconvulsive threshold for tonic seizures [7]. In co-medication with valproate and carbamazepine, ethanol significantly increased the anticonvulsant effectiveness of both antiepileptic drugs [7]. According to Freeman, the depressant effect of ethanol on kindling could be due to its depressant action on the
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electrical activity of the reticular formation [9]. On the other hand, McQuarrie and Fingl [20] did not find changes of the mean seizure threshold during the chronic administration of ethanol (2.0 g/kg, orally) in the low frequency electroshock seizure threshold test in mice. Furthermore, the subchronic administration of a higher dose of ethanol revealed neither a significant decrease of the MES seizure threshold nor a marked tolarence to the anticonvulsant activity tested 24 h after withdrawal in the MES threshold test [7]. This question seems to be matter of special interest, since ethanol is considered to be a high risk factor for epileptic patients. However, there is no evidence that ethanol affects the epileptiform activity in the penicillin-induced epilepsy. Therefore, in the present experiments, the effects of subchronic treatment with ethanol, and its withdrawal on epileptiform activity were investigated in the penicillin-induced epilepsy in the rats. In addition, we have analyzed the effects of ␣-tocopherol (vitamin E) on epileptiform activity in the penicillin model of epilepsy after subchronic premedication of ethanol and its withdrawal in the rats. 2. Materials and methods The animal studies were conducted with governmental approval according to local guidelines for the care and use of laboratory animals. Fifty-six male Wistar rats (240–270 g) were housed individually on a 12-h light:12h dark cycle (lights on at 07.00 h), at a temperature of 21 ± 2 ◦ C and 50% humidity. Rats were assigned to the following experiments and groups: intracortical (i.c.) delivery of (1) 2 l artificial cerebrospinal fluid [aCSF containing (mM): NaCl, 124; KCl, 5; KH2 PO4 , 1.2; CaCl2 , 2.4; MgSO4 , 1.3; NaHCO3 , 26; glucose, 10; HEPES, 10; pH 7.4 when saturated with 95% O2 and 5% CO2 ] (i.c.); (2) 500 IU (2 l, i.c.); (3) 500 mg/kg ␣-tocopherol (i.m.); (4) penicillin pretreated + 500 mg/kg ␣-tocopherol (i.m.); (5) ethanoltreated (3 g/kg, per day, for 15 days, intragastrically) + penicillin; (6) ethanoltreated (3 g/kg, per day, for 15 days, intragastrically) + penicillin + 500 mg/kg ␣-tocopherol (i.m.); (7) ethanol withdrawal (3 g/kg, per day, for 15 days, intragastrically) + penicillin; (8) ethanol withdrawal (3 g/kg, per day, for 15 days, intragastrically) + penicillin + 500 mg/kg ␣-tocopherol (i.m.). Each animal group was composed of seven rats.
2.1. Induction of epileptiform activity The animals were anesthetized with urethane (1.25 g/kg−1 , i.p.) and placed in a stereotaxic frame (Harvard Stereotaxic Instrument). Rectal temperature was maintained between 36.5 and 37.0 ◦ C using a feedback controlled heating system (Harvard Apparatus Limited). A polyethylene cannula (Bicakcilar) was introduced into the right femoral artery to monitor blood pressure, which was kept above 100 mmHg during the experiments (mean 114 ± 7 mmHg). All contact and incision points were infiltrated with procaine hydrochloride to minimize possible sources of pain. The left cerebral cortex was exposed by craniotomy. The epileptic focus was produced by 500 units penicillin G potassium injection (2 mm posterior to bregma and 3 mm lateral to sagittal sutures, 1 mm beneath the brain surface by a Hamilton microsyringe type 701N; infusion rate 0.5 l/min) [2,3,19].
2.2. Drug and drug administration Ethanol treated rats, received a daily dose of 3.0 g/kg of 30% ethanol solution via an oesophageal probe for 15 days. Ethanol withdrawal rats, received a daily dose of 3.0 g/kg of 30% ethanol solution via an oesophageal probe for 15 days. At the end of this period, ethanol administration was stopped and 40 h after the last administration of ethanol, and all rats were anesthetized for induction of epileptiform activity. Ethanol intake was administrated at approximately the same time (between 9:00 and 10:00 h) during the whole procedure. Blood ethanol
concentrations were measured 90 min after ethanol administration in different groups (n = 35). d␣-tocopherol acetate (Aksu Farma Chemical Co.) was used in the experiments. Precautions were taken to minimize exposure to the light. A dose of 500 mg/kg ␣-tocopherol was administrated (i.m.) 30 min after penicillin (i.c.) application.
2.3. ECoG recordings Two Ag–AgCl ball electrodes were placed over the left somatomotor cortex (electrode coordinates: first electrode; 2 mm lateral to sagittal suture and 1 mm anterior to bregma; second electrode; 2 mm lateral to sagittal suture, 5 mm posterior to bregma). The common reference electrode was fixed on the pinna. The ECoG activity was continuously monitored on a four channel recorder (PowerLab,4/SP). All recordings were stored on a computer. The frequency and amplitude of epileptic activity was analyzed off line.
2.4. Statistical analysis All statistical procedures were performed using SPSS statistical software package (version: 12.0). Data analysis was performed using one way ANOVA and Bonferroni post hoc tests for comparisons. Data are expressed as the means ± SEM. Statistical significance was set at P < 0.05.
3. Results Baseline activities of each animal were recorded before the administration of intracortical injection of penicillin (Fig. 1a). Intracortical injection of penicillin (500 units) induced an epileptiform ECoG activity characterized by bilateral spikes and spikewave complexes (Fig. 1b). This ECoG activity began within 3–5 min after penicillin application and lasted for 3–5 h. It reached a constant level as a frequency and amplitude in 30 min. The mean of spike frequency and amplitude were 28.3 ± 3, 981 ± 168 V; 31.6 ± 4, 1,018 ± 181 V and 34.2 ± 2 spike/min, 1256 ± 211 V in penicillin pretreated, ethanoltreated, and ethanol withdrawal groups in 30 min from penicillin injection (i.c.), respectively (Fig. 1b,d,f). There was no significant difference in the mean frequency and amplitude of epileptiform activity during experiments between these groups. The dose of 500 mg/kg ␣-tocopherol significantly decreased the mean frequency of epileptiform activity to 10.4 ± 1.8, 13.8 ± 2.3, 14.7 ± 2.1 spike/min in 60, 70, and 120 min after ␣-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively (Fig. 2). ␣-Tocopherol was less effective to decrease the mean frequency of epileptiform activity in ethanol-treated group than ␣-tocopherol administrated group alone 90 min after ␣-tocopherol injection (Fig. 2). ␣-Tocopherol did not significantly change the amplitude of epileptiform activity in all groups during experiments. The amplitudes of epileptiform activity were 1,001 ± 97 V, 1,018 ± 91 V, 991 ± 87 V, after 80 min from ␣-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively (Fig. 1c,e,g). There was no change in the mean of frequency and amplitude in aCSF injected animals (Fig. 1h). Intramuscular injection of ␣-tocopherol did not cause any change in the frequency or amplitude of ECoG activity with respect to control baseline in non-penicillin injected animals.
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Fig. 1. The baseline ECoG activities (a). Intracortical injection of penicillin (500 units) induces an epileptiform ECoG activity characterized by bilateral spikes and spike-wave complexes (b). ␣-Tocopherol of 500 mg/kg significantly decreases the frequency of epileptiform activity in penicillin pretreated (c), ethanol-treated (e), ethanol withdrawal groups (g). There is no significant change in the mean of frequency and amplitude of epileptiform activity compare to penicillin pretreated and ethanol-treated (3 g/kg, per day, for 15 days, intragastrically) groups (d, f). There is also no change in the mean of frequency and amplitude in aCSF injected animals (h). Representative ECoGs are presented 80 min after ␣-tocopherol or 110 min after penicillin administration (Bar 1 mV, 30 s; Bar 500 V, 10 s).
Blood ethanol concentrations (BEC) were measured 90 min after ethanol administration in different groups (n = 35). Blood ethanol concentration were 0, 245.7, 258.4, 247.3, and 252.8 mg/dl, in the control, ethanol-treated, ethanoltreated + ␣-tocopherol, ethanol withdrawal, ethanol withdrawal + ␣-tocopherol groups, respectively. The mean of BEC was 248.5 mg/dl in ethanol-administered groups. There was no significant difference found between ethanol-administrated groups. 4. Discussion Epilepsies are complex neurobehavioral disorders resulting from increased excitability of neurones in various brain regions [24]. Prolonged ingestion of ethanol or its withdrawal results in a number of signs and symptoms consistent with enhanced
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excitability, including increased sensitivity to convulsants, hypervigilance, anxiety and possibly seizure activity [26,27]. Epileptic seizures have been studied in genetic, electrophysiological, and chemical models [7,18,19]. In the present study, we provide further evidence for the effects of ethanol intake, and its withdrawal on epileptiform activity in the penicillin-induced epilepsy in the rats. A number of previous in vivo studies in the pentylenetetrazol (PTZ)-kindling and ferric chloride models in rats provide evidence for anticonvulsant and antiepileptic effects of ethanol after acute application, whereas repeated administration of high doses with abrupt withdrawal periods tend to proconvulsant actions [8,13]. Scorza et al. [25] showed that there is an increase in the frequency of seizures in the first 15 days of ethanol treatment by using the pilocarpine model of epilepsy in rats. Furthermore, they noted that the increase of seizure frequency was not evenly distributed, as there were clusters of seizures at characteristic time points after daily ethanol administration [25]. However, after 2 weeks of daily administration of ethanol they observed a decrease in the frequency of seizures, which suggests a tolerance effect [25]. Present study showed that administration of ethanol (3 g/kg, for 15 days) or its withdrawal did not change either frequency or amplitude of penicillin-induced epileptiform activity. This result confirms and extends previous observations by Fisher [7] and Hoppener et al. [12]. It has been previously reported that the subchronic administration of higher dose of ethanol revealed, neither a significant decrease of the MES seizure threshold nor a marked tolerance to the anticonvulsant activity tested 24 h after withdrawal in the MES threshold test. In addition, intake of small to moderate quantities of ethanol appears not to increase seizure frequency in non-alcoholic patients [12]. McQuarrie and Fingl [20] also did not find changes of the mean seizure threshold during the chronic administration of ethanol (2.0 g/kg, for 14 days) in the low-frequency electroshock seizure threshold test in mice. However, Kokka et al. [15] showed in the PTZ seizure threshold test in rats that the anticonvulsant activity of ethanol was only slightly decreased by chronic ethanol treatment (5.0 g/kg for 28 days). The reasons for these inconsistent results are unclear. They may be attributed to the differences in experimental design, e.g., the dose and duration of ethanol exposure, the differences among epilepsy models and tested animals strains. Unfortunately, there is a lack of data to make more complete comparison in the effects of ethanol on penicillin-induced epileptiform activity in rat, since it has not been reported before. Active oxygen free radical scavengers such as ascorbic acid or ␣-tocopherol (vitamin E) might have a role for protection against epilepsy and ethanol-induced toxicity [1,17]. In our previous work, it was found that ␣-tocopherol of 500 mg/kg (i.m.) was the most effective dose in changing of frequency on penicillininduced epileptiform activity in rat [3]. Therefore, the dose of 500 mg/kg ␣-tocopherol was used in all experiment groups in the present study and it significantly decreased the mean frequency of epileptiform activity in all groups. The anticonvulsant effects of ␣-tocopherol appeared in the 60, 70, and 120 min after ␣-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively. This result confirms previous studies which indicated that ␣-tocopherol has anti-
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Fig. 2. The effect of ␣-tocopherol on the mean frequency of penicillin-induced epileptiform activity in rats. The dose of 500 mg/kg ␣-tocopherol significantly decreases the mean of frequency of epileptiform activity. The significant effects appeared in the 60, 70, and 120 min after ␣-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively. ()P < 0.05, ( )P <0.01, ()P < 0.001, ( ) intracortical (i.c.) delivery of 500 units penicillin (2 l); ( ) penicillin pretreated (500 units) + 500 mg/kg ␣-tocopherol (i.m.); ( ) ethanol treated rats received a daily dose of 3.0 g/kg ethanol for 15 days (intragastrically); ( ) ethanol withdrawal rats were anesthetized for induction of epileptiform activity 40 h after the last administration of ethanol (3.0 g/kg ethanol, per day, for 15 days, intragastrically); ( ) ethanol-treated (3 g/kg ethanol, per day, for 15 days, intragastrically) + penicilin (500 units, i.c.) + 500 mg/kg ␣-tocopherol (i.m.); ( ) 40 h after the last administration of ethanol (3.0 g/kg ethanol, per day, for 15 days, intragastrically) + penicillin (500 units, i.c.) + 500 mg/kg ␣-tocopherol (i.m.).
seizure activity in several animal models, including the ferrous chloride [17], the hyperbaricoxygen [14], kindling, PTZ [24], and penicillin-induced [3] models. The anticonvulsant effects of ␣-tocopherol was less effective in ethanol-treated group than ␣tocopherol-treated group alone in the present study. This result is no surprise since it is known that ethanol can stimulate the formation of free radicals or exhaust the antioxidant defense system, leading to oxidative stress in the brain [1,22,23]. In conclusion, low dose of ethanol (3 g/kg) does not have either anticonvulsive or proconvulsive effect on penicillininduced epileptiform activity in rat. Effective dose of ␣tocopherol (500 mg/kg) has free radical scavenging activities [1], suggesting the anticonvulsive action of ␣-tocopherol may result from its free radical scavenging activities. However, molecular events that take place during epilepsy and the exact mechanism of ␣-tocopherol in the ethanol toxicity and epilepsy are poorly understood. These will need to be further investigated electrophysiologically.
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