Characterization of phenytoin-resistant kindled rats, a new model of drug-resistant partial epilepsy: influence of experimental and environmental factors
Introduction
Amygdala-kindled seizures in rats are the most widely used model for partial seizures, particularly of the complex partial type (Löscher and Schmidt, 1988, McNamara, 1989). Generally, there is an excellent correlation between activity of anticonvulsant drugs in this model and their clinical efficacy against complex partial seizures (Löscher and Schmidt, 1988, McNamara, 1989). One striking exception to this correlation is phenytoin which is one of the major non-sedating antiepileptic drugs for treatment of generalized tonic-clonic and partial seizures (Rogawski and Porter, 1990), but has been reported to be ineffective or even proconvulsant in the kindling model (Callaghan and Schwark, 1980, Baimbridge and Miller, 1984). Other groups, however, found that phenytoin exerts potent anticonvulsant effects in this model (Albright and Burnham, 1980, Ehle, 1980, Löscher et al., 1986, Rundfeldt et al., 1990, Voits and Frey, 1994, Ebert et al., 1997). This discrepancy led several groups to re-examine the anticonvulsant activity of phenytoin and the factors which may contribute to its activity in kindled animals. By these more recent studies, it was shown that phenytoin exerts potent anticonvulsant effects in the kindling model provided that certain pharmacokinetic and technical factors are dealt with (McNamara et al., 1989, Rundfeldt et al., 1990, Lothman et al., 1991). However, even if such factors are considered, there is a marked individual and inter-individual variation in the response to phenytoin within a group of kindled rats (Rundfeldt et al., 1990). We have recently found that part of this variation is due to the existence of subgroups of kindled rats with different sensitivities to phenytoin, i.e. some animals consistently respond and others never respond to phenytoin (Löscher and Rundfeldt, 1991, Löscher et al., 1993b). In view of the fact that only about 30% of patients with complex partial seizures can be controlled with high-dose antiepileptic therapy (Dam, 1986), kindled rats with phenytoin resistance would be a unique resource for the investigation of mechanisms of drug resistance in this type of epilepsy. Indeed, phenytoin non-responders are not only resistant to phenytoin but also to other major antiepileptic drugs (Löscher et al., 1993b).
Responsiveness to phenytoin appears to be an individual characteristic of the kindled rat, and this led to a reproducible amount of some 20% responders and 20% non-responders in a large group of female Wistar rats (Löscher and Rundfeldt, 1991, Löscher et al., 1993b, Ebert et al., 1994). The reasons for a rat being or becoming a responder or non-responder are unclear. In a recent selection of male and female rats, we found non-responders more frequently in the group of males compared to the group of females (Ebert et al., 1994). These data suggest the influence of sex hormones on anticonvulsant efficacy of phenytoin. With respect to female rats, however, we have previously demonstrated that the individual variation in anticonvulsant activity of phenytoin is not related to variation in sex hormone levels during the estrous cycle (Rundfeldt et al., 1990). We therefore asked whether the observed variation of responders and non-responders may originate from systematical differences in individual technical factors, such as precise electrode location and kindling parameters. Furthermore, the selection procedure of phenytoin-resistant rats in the kindling model lasts many weeks, so that environmental factors, such as seasonal influences or atmospheric pressure, may have changed the amount of responders and non-responders in different groups of rats during subsequent selection experiments. We therefore studied the influence of these parameters in a great number of Wistar rats which have been tested for their anticonvulsant response to phenytoin in our laboratory during the past 4 years. Although some of the technical factors have been shown to have no influence on the anticonvulsant response to phenytoin (Löscher and Rundfeldt, 1991, Löscher et al., 1993b, Ebert et al., 1994), the large database of the present study offers a unique possibility to study the influence of single parameters on the response type in a statistically relevant number of experiments.
Section snippets
Animals and surgery
Female and male Wistar rats were either purchased at a body weight of 200–220 g (males: 8 weeks old; females: 12 weeks old; Harlan-Winkelmann Versuchstierzucht, Borchen, Germany) or bred in our animal colony. Following arrival, the purchased animals were kept in our animal colony under controlled environmental conditions (ambient temperature 24–25°C, humidity 50–60%, 12-h light/dark cycle, light on at 06:00 h) for at least 1 week before being used in the experiments. At the time of electrode
Individual and technical factors
The present study includes the data of 114 female and 44 male Wistar rats which have been kindled and tested for the anticonvulsant response to phenytoin in our laboratory during the last 4 years. The response to 75 mg/kg phenytoin (measured as increase in individual ADT) was determined three times in each rat. As seizure parameters (seizure severity and duration) and afterdischarge duration did not significantly change after phenytoin application (see Ebert et al., 1997for details), these data
Discussion
Repeated applications of phenytoin in amygdala-kindled Wistar rats performed in our laboratory during the past few years demonstrated that the ability to respond to phenytoin is an individual characteristic of each rat. The response to phenytoin was characterized by a significant increase of ADT, i.e. the most sensitive measure of anticonvulsant efficacy of phenytoin in kindled seizures (Rundfeldt et al., 1990). Other seizure parameters did not significantly change after phenytoin application,
Acknowledgements
We thank Professor W. Lehmacher (previously of the Department of Biometrics and Epidemiology of this school) for advice on the statistical evaluation of data, Professor G. Gross (Institute of Meteorology, University of Hannover) for providing data and information concerning the evaluation of seasonal influences and atmospheric pressure on our data, and C. Bartling, M. Halves and D. Pieper for skilful technical assistance. This study was supported by a grant from the Deutsche
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- 1
Present address: Arzneimittelwerk Dresden, Radebeul, Germany.
- 2
Present address: Bayer AG, Leverkusen, Germany.