Refractory atypical absence seizures in rat: a two hit model
Introduction
Although the prognosis for the majority of patients with epilepsy is favorable, up to 30% have medically refractory epilepsy, i.e. recurrent, spontaneous seizures that continue relentlessly in the face of appropriate therapy with antiepileptic drugs (Kwan and Brodie, 2000). The cognitive and psychosocial consequences of medically refractory seizures are onerous (Devinsky, 1999), particularly in children where continuous uncontrolled seizures usually herald a poor developmental outcome. For example, seizures are drug resistant in the vast majority of patients with Lennox–Gastaut syndrome (Sillinpää, 1995), an epilepsy syndrome notorious for its poor neurodevelopmental outcome.
The neurobiological events that result in medically refractory epilepsy are not known, but intractable seizures often are associated with the presence of cortical malformations (Lee et al., 1997) which may occur in 30–40% of patients with refractory epilepsy (Farrell et al., 1992, Lee et al., 1997), as compared to 7–15% of all epilepsy patients (Brodtkorb et al., 1992, Lee et al., 1997) and 1% of the general population (Lee et al., 1997). These data raise the possibility that disorders of cortical development are a major contributing factor to the intractability of seizures in medically refractory epilepsy (Porter et al., 2002). For example, cortical malformations are particularly common in pediatric epilepsy syndromes notable for abysmal neurodevelopmental outcomes and intractable epilepsy, such as West's syndrome (Meencke and Janz, 1984, Blume, 1988) and the Lennox–Gastaut syndrome (Blume, 1988). Recurrent seizures were found in 82% of 33 patients with cortical or subcortical heterotopias that led to unilateral or bilateral independent epileptiform discharges (Dubeau et al., 1995).
Most cortical malformations seen in children with epilepsy are caused by disruptions in cell proliferation and neuronal migration (Mischel et al., 1995, Lee et al., 1997, Germano and Sperber, 1998), and include loss of cerebral lamination, clusters of ectopic neurons, and heterotopias (Dubeau et al., 1995, Germano and Sperber, 1998). In rats, prenatal treatment of the mother on gestational day 15 (G15) with the antimitotic agent methylazoxymethanol (MAM) produces a brain dysgenesis in the pups similar to that seen in human neuronal migration disorders. MAM acts by methylating pyrimidine bases within 2–24 h of administration (Colacitti et al., 1998) and affects selected neuronal populations depending on the time of administration (Johnston and Coyle, 1979). Rat pups born to mothers exposed to MAM on G15 exhibit ataxia, tremors, learning and memory impairments (Ramakers et al., 1993, Di Luca et al., 1995). These MAM-treated offspring are more susceptible to kainic acid, bicuculline, fluorothyl, and metrazol-induced seizures (de Feo et al., 1995, Baraban and Schwartzkroin, 1996, Chevassus-Au-Louis et al., 1998, Germano and Sperber, 1998) and they show abnormal neuronal activity in the hippocampus and neocortex (Baraban and Schwartzkroin, 1995, Colacitti et al., 1999). However, no spontaneous seizures have been documented either by direct observation or by EEG in MAM-treated rats (Germano and Sperber, 1998).
We sought to induce chronic, recurrent, medically refractory atypical absence seizures, in MAM-exposed rats by administering the cholesterol biosynthesis inhibitor AY-9944 (AY) (Fischer et al., 1972, Smith and Bierkamper, 1990, Cortez et al., 2001). We hypothesized that fetal treatment with MAM would increase the severity of the AY-induced seizures and make them refractory to the drugs that normally suppress them. We investigated the electrocorticographic (ECoG) changes in the MAM–AY-treated rats and conducted a pharmacological characterization of this animal model. We as well examined the histopathology of brains in MAM–AY-treated rats and compared those findings to the brain histology in control animals.
Section snippets
Animals
All these experiments were approved by the Ethics Committee of Lab Animal Services at the Hospital for Sick Children. Animals involved in this experimental design were treated according to guidelines for invasive surgical procedures. Timed, pregnant Long Evans hooded rats (G12) (n = 4) were obtained from Charles River (St. Constant, Quebec, Canada) and housed in the lab animal services facility of the Hospital for Sick Children in Toronto. The suckling rats were weaned at postnatal day (P) 21 and
ECoG and behavioral characteristics of seizures in the MAM–AY-treated rat
As indicated the total SWD duration was quantified in 20-min epoch over 1-h ECoG recordings. The ECoG recordings in C–C rats showed spontaneously recurrent SWD at 9 Hz lasting 1–2 s in duration. AY treatment during postnatal development both in C–AY and in MAM–AY animals resulted in a marked exacerbation of SWD of slower frequency at 5–6 Hz. ECoG recordings in AY-treated animals were characterized by spontaneous, recurrent, bilaterally synchronous 5–6 Hz SWD that were significantly longer in
Discussion
We report here the ECoG, pharmacological and histopathological characterization of the MAM–AY rat model with chronic and medically refractory atypical absence seizures. This model was developed by exposing Long Evans hooded rats to MAM on G15, followed by treatment with AY-9944 during the first month of postnatal life. Our data indicate that (1) prenatal MAM treatment produces a neuronal migration disorder, which seems to target the hippocampus, as previously reported (Spatz and Laqueur, 1968,
Acknowledgements
This project was performed in part using MAM, a compound provided by the National Cancer Institute's Chemical Carcinogen Reference Standards Repository operated under contract by Midwest Research Institute No. N02-CB-07008. The authors thank the technical staff of Laboratory Animal Services for the care taken during the chronic experiments, Dick Liu for excellent technical support, Craig Fleming for recovery and preparation of tissue for histological evaluation, and Marilyn McLaughlin for
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