Intrastrain differences in seizure susceptibility, pharmacological response and basal neurochemistry of Wistar rats
Introduction
A great variety of animal models of seizures and epilepsy have played and still play a major role in unravelling the pathophysiology of human epilepsies (Pitkänen et al., 2006). In general, animal models reproduce several components of human epilepsies. They are used to study the mechanisms underlying seizures and for the screening of potential anticonvulsant drugs. The model of choice depends upon several factors, including the question to be studied, the type of epilepsy to be modelled, and convenience. The pilocarpine seizure model is one of the most commonly used chemoconvulsant-induced models for temporal lobe epilepsy (TLE) (Sarkisian, 2001). The muscarinic agonist pilocarpine is administered systemically or focally to induce limbic seizures and status epilepticus.
Typically, rodents are used to conduct these epilepsy studies and the type of rodent strain used seems crucial. Most pilocarpine-induced seizure studies have been carried out on Wistar rats (Curia et al., 2008). While certain strains, such as Sprague–Dawley, show no major differences in the development of behavioural and electrographic alterations in comparison to Wistar rats (Honchar et al., 1983, Jope et al., 1986), use of Long-Evans rats showed larger hippocampal damage, worse behavioural outcome as well as a higher mortality rate following intraperitoneal administration of pilocarpine (Hort et al., 2000). Substantial interstrain differences have also been reported in animal models for other central nervous system diseases (Hort et al., 2000, Lucki et al., 2001, Mills et al., 2001, Alahmed and Herbert, 2008, Siller-Matula and Jilma, 2008). By systematically including the same rodent strain in their epilepsy studies, scientists hope to minimize experimental bias. A number of reports have demonstrated fundamentally different experimental outcomes when using the same strain of rats but from different vendors and/or different breeding locations (Pare and Kluczynski, 1997, Ciccotosto et al., 2000, Alemayehu et al., 2002, Baars et al., 2006, Rex et al., 2007, Lamon et al., 2008). Clear phenotype differences have been described between Sprague–Dawley rats purchased from Charles River Laboratories and Harlan Laboratories (Pare and Kluczynski, 1997, Pollock and Rekito, 1998, Turnbull and Rivier, 1999, Kitzmann et al., 2002, Sampaio-Maia et al., 2003, Diaz-Silva et al., 2004, Pecoraro et al., 2006). Focusing on the Wistar rat, differences noted in rats obtained from different vendors or breeding locations include: different patterns of melatonin secretion (Barassin et al., 1999), differences in hippocampal CA1 responses to hypoperfusion induced by two-vessel occlusion (Marosi et al., 2006), discrepancies in motor and sensory outcome after spinal compression (Lonjon et al., 2009), and variability in stress response of Wistar–Kyoto rats (Pare and Kluczynski, 1997).
Over the past decade, the focal pilocarpine model has been the standard seizure model in our laboratory for pharmacological screening of potential anticonvulsants and for determining the mechanism of action of antiepileptic drugs (Smolders et al., 2004, Smolders et al., 2008, Clinckers et al., 2005a, Clinckers et al., 2005b, Meurs et al., 2007, Meurs et al., 2008). To focally induce seizures, pilocarpine is perfused intrahippocampally via a microdialysis probe. Pilocarpine-induced seizures are characterised by a sequential development of typical behavioural patterns and electrographic activity. Typical behavioural changes include hyperactivity, tremor, scratching, head bobbing, and myoclonic movements of the limbs, progressing to recurrent myoclonic convulsions with rearing, salivation, and falling. In our laboratory, pilocarpine control experiments are conducted on a regular basis and are used as a reference to investigate the anticonvulsant potential of a pharmacological treatment. From a practical point of view, one of the main advantages of this acute seizure model lies in the reproducibility of the induced seizures enabling statistical evaluation of intergroup differences with relative small experimental groups (4–8 animals on average).
We consistently use the Wistar rat strain to perform these studies. Recently we were forced to consecutively change vendor (Charles River to Harlan) and breeding location (Charles River France to Charles River Germany) over a period of a few months. The first change was due to breeding problems with the company, and subsequent changes were the consequence of apparent changes in the focal pilocarpine-induced seizure susceptibility. The present retrospective study was undertaken to investigate how the vendor and breeding location changes affected the outcome of the epilepsy experiments conducted within our laboratory. The pharmacological response to anaesthetics was evaluated by comparison of the ketamine dose required to establish full pre-operative sedation necessary for stereotactic microdialysis probe implantation. Additionally, the intrastrain differences in Wistar rats concerning seizure susceptibility were evaluated by comparing behaviourally assessed seizure severity following intrahippocampal pilocarpine perfusion. As extracellular neurotransmitter levels are important modulators of neuronal excitability and hence epileptic activity, we also looked for intrastrain differences in basal hippocampal neurotransmitter levels. Significant differences were found in all of the parameters analyzed. To the best of our knowledge, we are the first to report intrastrain differences in seizure susceptibility and related neurochemical outcome.
Section snippets
Chemicals and reagents
Neuroactive substance standards and pilocarpine were supplied by Sigma–Aldrich (Bornem, Belgium). Ketamine HCl (100 mg/1 mL; Ceva Sante Animale, Belgium), diazepam (10 mg/2 mL; Roche, Belgium), and ketoprofen (1 g/100 mL; Merial, Belgium) were purchased from the hospital pharmacy (University Hospital Brussels). All other chemicals were analytical reagent grade or better and were supplied by Merck (Darmstadt, Germany). Aqueous solutions were made with purified water (Seralpur pro 90 CN; Belgolabo,
Pharmacological response to anaesthesia
The body weight-corrected dose of ketamine required to establish full pre-operative anaesthesia necessary for the implantation of the microdialysis probe was calculated for each rat and compared between groups. The total amount of ketamine required for adequate sedation differs significantly between the groups (Fig. 1a). Significantly higher ketamine doses are required for the CRL2 (18%, p < 0.01; n = 82) and HAR HAN (37%, p < 0.001; n = 83) when compared to CRL1 rats (n = 115). The HAR HAN group
Discussion
In the past decade our laboratory has gained extensive experience with the focal pilocarpine model, a highly reproducible acute limbic seizure model. We have performed numerous studies to characterise intrahippocampal neurochemical, pharmacological and pharmacokinetic changes during pilocarpine-induced seizure activity (Smolders et al., 2004, Smolders et al., 2008, Clinckers et al., 2005a, Clinckers et al., 2005b, Meurs et al., 2007, Meurs et al., 2008). The present retrospective study brings
Conclusion
This retrospective study clearly indicates that several pharmacological, behavioural and neurochemical factors are significantly different from one vendor/breeding location to another and can even vary in time between rats coming from the same breeding location. Intrastrain differences can have a substantial impact on the outcome of animal epilepsy studies and basic preclinical scientists should pay attention to this issue when designing studies. Indeed, intrastrain differences in epilepsy may
Acknowledgements
The authors wish to acknowledge the technical assistance of R. Berckmans, G. De Smet, C. De Rijck, and R.M. Geens. We thank the Vrije Universiteit Brussel for financial support. Jeanelle Portelli and Najat Aourz are doctoral research fellows supported by the Research Council (Onderzoeksraad—OZR) of the Vrije Universiteit Brussel. Dimitri de Bundel is a doctoral research fellow of the Research Foundation Flanders (FWO). Ralph Clinckers is a research and postdoctoral fellow of the Research
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