Journal of Pharmacological Sciences
J Pharmacol Sci 104, 56 – 60 (2007)
©2007 The Japanese Pharmacological Society
Full Paper
Effects of Certain Antiepileptics on Behavioral and Electromyographic Seizure Patterns Induced by Maximal Electroshock in Mice Aya Murakami1, Takashi Ishikawa1, Kenshi Takechi1, Jun Ago1, Naotaka Matsumoto1, and Chiaki Kamei1,* 1
Department of Medicinal Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan
Received November 24, 2006; Accepted March 14, 2007
Abstract. The changes of electromyographic activity (EMG seizure) induced by maximal electroshock were studied in comparison with those of behavioral seizures in mice. In addition, the effects of certain antiepileptics on behavioral seizures and EMG seizure induced by maximal electroshock were also studied. High amplitude with high frequency EMG seizure was observed in parallel with the appearance of tonic extensor (TE) seizure and an intimate relationship was observed between the two parameters. On the other hand, to investigate the intensity of TE seizure, the product of the amplitude and the duration in EMG seizure was calculated, and the effects of antiepileptics on the magnitude of EMG seizure were investigated. As a result, a significant difference was observed at the doses of antiepileptics that showed no significant effects on the durations of TE and EMG seizures; that is, phenytoin, phenobarbital, topiramate, and carbamazepine showed significant effects on the magnitude of EMG seizure at doses of 5, 2, 10, and 5 mg / kg, respectively. From these findings, it may be concluded that this index, that is, the magnitude of EMG seizure induced by maximal electroshock, is a more reliable and highly sensitive method for the assessment of the potential activity of antiepileptics. Keywords: maximal electroshock, tonic extensor (TE) seizure, clonic (CL) seizure, electromyographic (EMG) activity, EMG seizure
method to find a new drug that is effective in generalized tonic-clonic seizures (5). In many studies, the duration of the TE seizure is measured with the naked eye. Although such manual observation is often used in the field of behavioral pharmacology, its main disadvantage is the lack of objectivity. In addition, in this method, it is difficult to estimate the extent of the intensity of TE seizure. On the other hand, the electromyogram (EMG) is well known to show the electric activity of the muscle; and when muscles are active, they produce an electrical current that is usually proportional to the level of muscle activity. From these findings, it can be assumed that an increase of muscle activity will be observed when TE seizure occurs in the hind legs by maximal electroshock. In the present study, therefore, the changes of electromyographic activity (EMG seizure), as well as behavioral seizures induced by maximal electroshock, were studied. In addition, the effects of certain antiepileptics on both parameters induced by maximal electroshock were also studied.
Introduction Epilepsy is a neurological disorder consisting of recurrent seizures, resulting from excessive, uncontrolled electrical activity in the brain. Patients with epilepsy are treated with antiepileptics to inhibit the epileptic seizures (1). The maximal electroshock seizure test has been widely used as a method for the assessment of the potential activity of antiepileptics (2, 3). After the maximal electroshock, the animals experienced the flexor component of the tonic phase (tonic flexor, TF), extensor component of the tonic phase (tonic extensor, TE), clonic phase (clonic convulsion, CL), and period of post-seizure depression (come, CO), in order (4). Among these convulsive behaviors, TE seizure of the hind legs in animals has been recognized as a screening *Corresponding author.
[email protected] Published online in J-STAGE: April 24, 2007 doi: 10.1254/jphs.FP0061487
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Materials and Methods Animals Male ddY mice, 4-week-old (body weight, 20 – 24 g) were purchased from Japan SLC, Shizuoka. Animals were maintained in an air-conditioned room with controlled temperature (24 ± 2°C) and humidity (55 ± 15%). They were housed in plastic cages with sawdust under a light-dark cycle (lights on from 07:00 to 19:00). The mice were allowed free access to food and water, except during the experiments. All procedures involving animals were carried out in accordance with the Guidelines for Animal Experiments at Okayama University Advanced Science Research Center. Groups of 8 mice each were used for each dose of the drugs in the study.
Fig. 1. Representative example of the behavioral and electromyographic changes induced by maximal electroshock. Abbreviations: E.S., electroshock stimulation; EMG, electromyogram; TF, tonic flexion; TE, tonic extension; CL, clonic convulsion.
Experimental procedure Electroconvulsion was induced by stimulating the animals with 50 Hz, 200 V for 1 s (SEN-3301; NihonKohden, Tokyo) using ear-clip electrodes. The EMG was amplified with a bioelectric amplifier (AB-651J, Nihon-Kohden) and recorded with a thermal arraycorder (WR-300; Graphtec, Tokyo). For the measurement of the EMG, bipolar needle electrodes were inserted into the both hind legs in the mice. In the present study, the duration of EMG seizure was defined as the time that restored the amplitude before electroshock. In addition, to calculate the magnitude of the EMG seizure (cm2), Scion Image (Scion Corporation, Frederick, MD, USA) was used to perform an image analysis using the area based on the amplitude and the duration of EMG seizure.
ately after the electroshock, TF seizure, characterized by extreme tonic flexion of all limb joints with a slight superimposed tremor lasting approximately 2.0 s was observed. The TE seizure that appeared subsequent to TF seizure is extreme tonic extension at all limb joints, with little or no tremor, and lasted for approximately 15.0 s, followed by abrupt relaxation. One or more extensor thrusts, followed by relaxation, were observed. This behavior resembles CL; therefore, the time from the end of TE seizure to complete relaxation was defined as the duration of CL seizure. Simultaneously with the appearance of TE seizure, high amplitude with high frequency electromyographic activity was observed and gradually attenuated. This is designated as EMG seizure.
Drugs The following drugs were used: phenytoin and phenobarbital sodium (Wako, Osaka); carbamazepine, ethosuximide, and topiramate (Sigma, St. Louis, MO, USA). These drugs were suspended in 0.5% carboxymethyl cellulose (CMC) solution and orally administered 1.0 h before electrical stimulation at a volume of 5 ml / kg of body weight. Statistical analysis All data are expressed as the means ± S.E.M. One way-analysis of variance (ANOVA) with Dunnett’s test was used for statistical analysis of all data, and a P-value less than 0.05 was considered significant. Results Behavioral and electromyographic changes induced by maximal electroshock A representative example is shown in Fig. 1. Immedi-
Correlation between the duration of TE seizure and EMG seizure To investigate the relationship between the durations of TE seizure and the EMG seizure after electroshock, the regression line of Y (duration of TE seizure) and X (duration of EMG seizure) and the correlation coefficient (r) was calculated. As shown in Fig. 2, the regression line of the durations of TE seizure and EMG seizure is Y = 0.903X + 1.458. In addition, a high correlation coefficient (r = 0.86) was also obtained. Effects of some antiepileptics on the duration of TE A representative example of the effect of phenobarbital on the behavioral and EMG seizures induced by maximal electroshock is shown in Fig. 3. Both the behavioral and EMG seizures were suppressed by phenobarbital. Figure 4 shows the effects of some antiepileptics on the duration of TE seizure. The duration of TE seizure in the control group was 15.9 ± 0.40 s (n = 8). The duration of TE seizure induced by maximal
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Fig. 2. Correlation between the duration of TE seizure and EMG seizure.
electroshock was dose-dependently shortened by phenytoin, phenobarbital, topiramate, and carbamazepine. A significant difference in the control group was observed with phenytoin at doses of 10 and 20 mg / kg, phenobarbital at doses of 5 and 10 mg / kg, topiramate at doses of 20 and 50 m / kg, and carbamazepine at a dose of 20 mg / kg. On the other hand, ethosuximide caused no significant inhibition, even at a dose of 1000 mg / kg. Effects of some antiepileptics on the EMG seizure The duration of EMG seizure in the control mice was 15.0 ± 0.61 s (n = 8) (Fig. 5). The duration of EMG seizure was defined as the time that restored the
amplitude before electroshock. The duration of EMG seizure was shortened by phenytoin, phenobarbital, topiramate, and carbamazepine. A significant difference was observed with phenytoin at doses of 10 and 20 mg / kg, phenobarbital at doses of 5 and 10 mg / kg, topiramate at doses of 20 and 50 mg / kg, and carbamazepine at a dose of 20 mg / kg. Ethosuximide caused no significant inhibition, even at a dose of 1000 mg / kg. The effects on the duration of EMG seizure for some antiepileptics were almost the same as those on the duration of TE seizure (Figs. 4 and 5). Figure 6 shows the effects of some antiepileptics on the magnitude of EMG seizure by measuring the product of amplitude and duration of electromyographic activity. In this case, significant effects were observed at the doses that showed no significant effect on the duration of TE seizure (Fig. 4) and EMG seizure (Fig. 5). That is, phenytoin, phnobarbital, topiramate, and carbamazepine showed significant effects at a dose of 5, 2, 10, and 5 mg / kg, respectively. On the other hand, ethosuximide caused no significant inhibition, even at a dose of 1000 mg / kg (Fig. 6). Effects of some antiepileptics on the duration of CL seizure The duration of CL seizure in the control mice was 20.1 ± 1.82 s (n = 8). No significant effects were observed on the duration of CL seizure by the antiepileptics used (Fig. 7). Discussion In the present study, it was found that high amplitude
Fig. 3. Representative example of the effect of phenobarbital on the behavioral and EMG seizures induced by maximal electroshock. Abbreviations: E.S., electroshock stimulation; EMG, electromyogram; TF, tonic flexion; TE, tonic extension; CL, clonic convulsion.
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Fig. 4. Effects of some antiepileptics on the duration of TE seizure. Each value represents the mean ± S.E.M of 8 mice. *, **: Significantly different from the control group with P<0.05 and P<0.01, respectively.
Fig. 5. Effects of some antiepileptics on the duration of EMG seizure. The duration of EMG seizure was defined as the time that restored the amplitude before electroshock. Each value represents the mean ± S.E.M of 8 mice. *, **: Significantly different from the control group with P<0.05 and P<0.01, respectively.
Fig. 6. Effects of some antiepileptics on the magnitude of EMG seizure. The magnitude of EMG seizure was estimated by the product of amplitude and duration of electromyographic activity. Each value represents the mean ± S.E.M of 8 mice. *, **: Significantly different from the control group with P<0.05 and P<0.01, respectively.
Fig. 7. Effects of some antiepileptics on the duration of CL seizure. Each value represents the mean ± S.E.M of 8 mice.
and high frequency electromyographic activity (EMG seizure) was observed after maximal electroshock in mice. The duration of EMG seizure was parallel to the duration of TE seizure. In the present study, the duration of TE seizure was 15.9 ± 0.40 s. Su et al. (6) reported that the duration of TE seizure after electroshock in Kunming mice was 14.8 ± 0.4 s. Vohora et al. (7) also demonstrated that the duration of TE seizure in male
Swiss-strain mice was 14.0 ± 0.7 s. Previously, we (8) have also found that the duration of TE seizure in male ddY mice was 15.2 ± 0.5 s. These values in the duration of TE seizure were almost the same. Therefore, it can be regarded that observing TE seizures induced by maximal electroshock by the naked eye is not always an unreliable method. However, it goes without saying that measuring the duration of EMG seizure is a more
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reliable method than measuring the duration of TE seizures. As a matter of course, almost the same findings were observed in the results concerning the effects of some antiepileptics on the durations of EMG and TE seizures. In both tests, significant effects were observed with phenytoin at a dose of 10 mg / kg, phenobarital at a dose of 5 mg/ kg, topiramate at a dose of 20 mg/ kg, and carbamazepine at a dose of 20 mg / kg. In the maximal electroshock test using mice, almost the same effects were observed with phenytoin, phenobarbital, and carbamazepine when given to animals by oral administration. For instance, Masuda et al. (9) reported that the ED50 values of phenytoin, phenobarbital, and carbamazepine were 7.9, 11.9, and 13.3 mg / kg, respectively, in male ddY mice. Vohora et al. (7) also demonstrated that phenytoin and carbamazepine at doses of 10 and 3.5 mg / kg, respectively, caused no significant effects on TE seizure in Swiss-strain mice. Yamashita et al. (10) recently reported that the ED50 values of phenytoin, phenobarbital, and carbamazepine were 16.6, 15.6, and 17.9 mg/ kg, respectively. That is to say, it can be said that the effects of these antiepileptics obtained in the present study were common values. Next, we tried to examine the effects of some antiepileptics on the intensity of TE seizures. For this purpose, the product of the amplitude and duration of the EMG seizure, that is, the magnitude of EMG seizure, was calculated. As a result, it was found that this method is highly sensitive for estimating the efficacy of antiepileptics. As already mentioned, in the durations of both TE seizure and EMG seizures, phenytoin, phenobarbital, topiramate, and carbamazepine showed significant effects at doses of 10, 5, 20, and 20 mg / kg, respectively. On the other hand, regarding the magnitude of the EMG seizure, significant effects were obtained with smaller doses; that is, phenytoin, phenobarbital, topiramate, and carbamazepine caused significant effects at doses of 5, 2, 10, and 5 mg / kg, respectively. As shown in the present data, CL seizure after electroshock was not significantly inhibited by any of the antiepileptics used. In the present study, the duration of CL seizure was 20.1 ± 1.82 s. Su et al. (6) also reported that the duration of CL was 23.7 ± 2.5 s. On the other hand, Vohora et al. (7) and Yokoyama et al. (11) demonstrated that the duration of CL seizure was 5.0 ± 0.7 s and 9 – 10 s, respectively. From these findings, a question has been raised about the measurement of CL seizure after electroshock. In addition, it seems likely that there is a great difference between the CL seizure induced by maximal electroshock and the CL seizure induced by pentetrazol. When pentetrazol at a dose of 100 mg / kg was injected subcutaneously in mice, wild running and CL seizures were observed for 30 min (10). In addition,
it has been considered that ethosuximide is effective in CL seizure. In fact, there are some reports that ethosuximde caused an inhibition of pentetrazol-induced CL seizure at a dose of 267 and 344 mg / kg, respectively (9, 10). However, as shown in the present study, ethosuximide caused no inhibition of the CL seizure induced by maximal electroshock. According to these findings, it may be that the CL seizure-induced by maximal electroshock is not a reliable indicator for the assessment of the efficacy of antiepileptics. In summary, it may be concluded that this method, that is, the magnitude of EMG seizure induced by maximal electroshock, is a more reliable and highly sensitive method for the assessment of the potential activity of antiepileptics.
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