Repeated low-dose treatment of rats with pilocarpine: low mortality but high proportion of rats developing epilepsy
Introduction
Systemic administration of the cholinergic muscarinic agonist, pilocarpine, in rats is widely used as an experimental model of status epilepticus (SE; cf. Goodman, 1998). Furthermore, more importantly, the pilocarpine seizure model is used to study temporal lobe epilepsy (TLE), because anatomical changes resembling those seen in human mesial temporal sclerosis develop after the initially induced SE, and spontaneous recurrent focal and secondary generalized seizures as well as synaptic reorganization arise following a latent period after the SE (Turski et al., 1989, Goodman, 1998). While initiation of SE by pilocarpine is due to activation of the cholinergic system, the histopathology, neuronal loss, and spontaneous seizure activity is thought to be a result of seizure-induced glutamate release (Goodman, 1998).
Among the drawbacks of the pilocarpine model is that pilocarpine at the high doses used (320–400 mg/kg i.p. or s.c.) does not always induce SE. Furthermore, the use of such high doses is associated with a high mortality rate (Turski et al., 1989, Goodman, 1998). Pretreatment of rats with lithium chloride allows the dose of pilocarpine to be decreased to 30 mg/kg which results in a lower mortality and a higher percentage of animals exhibiting SE (Clifford et al., 1987). Despite these observations, both high-dose pilocarpine and the lithium plus pilocarpine combination are popular experimental models of SE and TLE.
Prompted by a recent study by Hellier et al. (1998) on the kainate model of TLE showing that mortality in this model can be substantially reduced by repeated low-dose treatment compared to administration of one high single dose, we studied whether the pilocarpine model can be improved by such a repeated low-dose administration technique. Furthermore, since the duration of SE is related to the mortality rate (Goodman, 1998), we limited SE duration to different periods of time by injecting diazepam with the aim to minimize mortality rate as much as possible. For a comparison of the different experimental protocols, we determined the percentage of rats exhibiting SE, the mortality rate, the proportion of rats developing spontaneous seizures, the latency for the first spontaneous motor seizure, and the frequency of the spontaneous recurrent seizures.
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Animals
Female Wistar rats were purchased at a body weight of 200–220 g (Harlan-Winkelmann Versuchstierzucht, Borchen, Germany) and were then kept under controlled environmental conditions (24–25 °C, 50–60% humidity, 12 h light/dark cycle, light on at 06:00 h) with free access to standard laboratory chow (Altromin 1324 standard diet) and tap water. Female Wistar rats were used to allow direct comparison with various other seizure models, including kindling and kainate, for which we used this strain and
Induction of SE with pilocarpine in lithium-free rats
In some preliminary experiments, pilocarpine was injected in a high dose of 370 mg/kg i.p. in lithium-free rats and SE was interrupted by diazepam after 90 min, resulting in 50% mortality rate (not illustrated), which is in the range of mortality rates reported by other groups for such doses of pilocarpine (cf. Turski et al., 1989, Goodman, 1998). In order to prove whether mortality can be decreased by multiple low-dose i.p. injections of pilocarpine in lithium-free rats, an initial dose of 200
Discussion
An ideal model of TLE should show chronically recurring, spontaneous seizures that behaviorally resemble complex partial seizures associated with mesial temporal sclerosis. In the pilocarpine model, behavioral, EEG, pathological, and pharmacological features resemble those in patients with TLE, suggesting that it could be a useful model for studying the mechanisms of TLE (Turski et al., 1989, Cavalheiro et al., 1991, Leite and Cavalheiro, 1995, Goodman, 1998). For this purpose, pilocarpine is
Acknowledgements
The study was supported by grants from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Bonn, Germany), the Deutsche Forschungsgemeinschaft (Bonn, Germany), and UCB Pharma (Braine-l'Alleud, Belgium).
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