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Summary: Purpose: We developed a new in vitro preparation of immature rats, in which intact corticohippocampal formations (CHFs) depleted in magnesium ions become progressively epileptic. The better to characterize this model, we examined the effects of 14 antiepileptic drugs (AEDs) currently used in clinical practice.
Methods: Recurrent ictal-like seizures (ILEs, four per hour) were generated in intact CHFs of P7–8 rats, and extracellular recordings were performed in the hippocampus and neocortex. AEDs were applied at clinically relevant concentrations (at least two), during 30 min after the third ILE. Their ability to prevent or to delay the next ILE was examined.
Results: Valproic acid and benzodiazepines (clobazam and midazolam) but also phenobarbital and levetiracetam prevent the occurrence of seizures. In contrast, usual concentrations of carbamazepine (CBZ), phenytoin, vigabatrin, tiagabine, gabapentin, lamotrigine (LTG), topiramate, felbamate, and ethosuximide did not suppress ILEs. In addition, LTG and CBZ aggravate seizures in one third of the cases.
Conclusions: This intact in vitro preparation in immature animals appears to be quite resistant to most AEDs. Blockade of seizures was achieved with drugs acting mainly at the γ-aminobutyric acid (GABA)A-receptor site but not with those that increase the amount of GABA. Drugs with a broad spectrum of activity are efficient but not those preferentially used in partial seizures or absences. We suggest that this preparation may correspond to a model of epilepsy with generalized convulsive seizures and could be helpful to develop new AEDs for refractory infantile epilepsies.
The development of new antiepileptic drugs (AEDs) devoted to adult patients requires initial studies on adult animal models of epilepsy to select the efficient compounds and to eliminate the toxic ones (1–3). A similar strategy cannot be applied to the age-dependent infantile epilepsies because models adapted for each critical developmental stage are not available. As a consequence, most currently available AEDs to treat infantile epilepsy have been developed initially for adults. However, adult and infantile epilepsies are largely distinct, and drugs that are effective for adults are not necessarily indicated for children that display a large variety of epilepsy syndromes. AEDs developed this way may even induce adverse effects (4) or worsen infantile epilepsy syndromes (5,6). Therefore a great need exists to develop relevant animal models that could discriminate the effects of currently available AEDs and contribute to the evaluation of new drugs.
We describe the effects of a large number of AEDs in a novel rat preparation: the intact corticohippocampal formation (CHF) in vitro, a preparation that allows preservation of most of the intrinsic and extrinsic connections within and between cortical and hippocampal regions. We have previously reported that during the first postnatal week, the isolated neonatal intact preparation depleted in magnesium ions becomes progressively epileptic with recurrent ictal-like events (ILEs) recorded as soon as P1 in both cortical and hippocampal regions (7). The effects of AEDs were therefore studied at the end of the first postnatal week. This critical period of development is characterized by an intense activity-dependent construction of the network, and the depolarizing effects of γ-aminobutyric acid (GABA), allowing us to test and to compare the effects of AEDs used in clinical practice in conditions relevant to infantile epilepsy within a developmental context. We now report that recurrent seizures are suppressed by few AEDs, resistant to most of them, and particularly to those that are efficient in partial epilepsies or in absences and aggravated by some others. Therefore this model that allows the discrimination of the commonly used AEDs may be suitable to determine the effects of novel AEDs in the developing brain.
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All protocols were designed according to INSERM guidelines for the care and use of animals. Experiments were performed on intact CHFs taken from Wistar rats at postnatal (P) days 7 and 8 as previously described (7). This preparation comprised limbic (hippocampus, septum, and entorhinal cortex) and nonlimbic areas (large parts of the neocortex). This intact preparation, in which the integrity of the network is preserved within and between brain regions, offers an excellent compromise between in vivo and in vitro preparations and allows the study of epileptic processes during the first week of life (7). Rats were killed and brains were extracted from the skull at 4°C. The hemispheres were separated and dissected to obtain two CHFs containing interconnected septum, hippocampus, entorhinal cortex, and a large part of neocortex. Each CHF was placed in oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (aCSF) with the following composition (in mM): NaCl, 126; KCl, 3.5; CaCl2, 2; MgCl2, 1.3; NaHCO3, 25; NaH2PO4, 1.2; glucose, 10 (pH 7.3). After ≥1 h rest at room temperature, the CHF was transferred to the recording chamber where it was fully submerged and superfused with oxygenated aCSF at 31 ± 1°C at a flow rate of 5.0 ± 0.2 ml/min. After 15–30 min, the CHF was then superfused with an aCSF without magnesium ions (low-Mg2+ aCSF). In these conditions, the extracellular concentration of Mg2+ ions does not completely decrease to zero because of the contamination by Mg2+ of the other constituents of the aCSF (8).
In a previous study we showed that spontaneous seizures were synchronized in hippocampal and cortical regions (7). Recordings were performed with extracellular electrodes filled with aCSF, placed in the neocortex and the hippocampus. Bipolar twisted nichrome electrodes were used to stimulate Schaffer collaterals (15–30 V, 30-μs duration at 0.033 Hz), and recording electrodes were placed at a location of the highest evoked field potential response. Data were acquired by using a Digidata 1200B card (Axon Instruments, Foster City, CA, U.S.A.) and were analyzed by using Clampfit (Axon Laboratories, Foster City, CA, U.S.A.) software.
Carbamazepine (CBZ) and ethosuximide (ESM) were purchased from Sigma-Aldrich (France). Other AEDs were kindly supplied by the corresponding laboratories: clobazam (CLB), phenytoin (PHT), tiagabine hydrochloride (TGB), and sodium valproate (VPA) by Sanofi-Synthelabo, vigabatrin (VGB) and phenobarbital (PB) by Aventis, gabapentin (GBP) by Pfizer, felbamate (FBM) by Schering-Plough, midazolam maleate (MDL) by Roche, lamotrigine (LTG) by Glaxo-Smith-Kline, levetiracetam (LEV) by UCB-Pharma, and topiramate (TPM) by Janssen-Cilag. CLB and TPM were directly dissolved in aCSF. PHT was dissolved in NaOH (1N) adjusted to pH 7.3 with HCl (3N) and then diluted to the final concentration in aCSF. The other AEDs were dissolved in dimethylsulfoxide (DMSO) and diluted in aCSF to a final concentration of 0.1% DMSO. Previous studies have shown that such a concentration of DMSO has no effect on seizures. All drugs were applied by superfusion (5 ml/min).
Concentrations of AEDs
The concentrations used in this in vitro model were chosen according to the therapeutic range of each AED, when available (9–11). The therapeutic range represents the plasma concentrations at which most drugs are effective and well tolerated for most people. However, therapeutic ranges for GBP, LEV, MDL, TGB, and VGB are either unknown or not firmly established. For instance, a value >2 μg/ml of GBP (10) was thought to be effective, and 20 μg/ml appeared not to be toxic (12). For LEV, we took into account the value of 25 μg/ml reported recently in a study in which children (6–12 years old) received a single dose of LEV (20 mg/kg) as an adjunct to their stable regimen of a single concomitant AED (13). MDL is used in status epilepticus (14,15) and has been shown to be efficient in a similar in vitro model at concentrations of 50–100 μM (16,17). However, lower concentrations were also evaluated because they are more related to those required for activation of BZD receptors. Plasma concentrations for TGB were not defined in patients. We therefore selected the concentrations used by Sabau et al. (1999) on rat pup slices exposed to low-Mg2+. The situation of VGB is different because it is a noncompetitive drug, and its plasma half-life is less critically related to its duration of action than is that of other AEDs (12). We therefore used VGB concentrations that have been evaluated in a similar in vitro model but in adult animal (18) or human slices (19). Table 1 summarizes the concentrations of AEDs used in this study and their therapeutic ranges in humans.
Table 1. Therapeutic ranges of AEDs and their corresponding concentrations used in vitro
|AEDs||Therapeutic range (μg/ml)||Highest concentration (μM)||In vitro concentrations (μM)|
|CBZ||4 to 10 (a)|| 42 ||30, 100|
|CLB||0.3 to 0.8 (b)|| 3 ||0.3, 1, 3, 10|
|ESM||40 to 100 (a)||708 ||300, 1,000, 3,000|
|FBM||30 to 60 (a)||252 ||100, 300|
|GBP||>2 (c, d)f|| 58f||30, 100|
|LTG||1 to 5 (a)|| 20 ||10, 30|
|LEV||25 (e)f||253 ||30, 100, 300|
|MDL||Not known||–||1, 3, 30,100|
|PB||15 to 40 (a)||172 ||30, 100, 300|
|PHT||10 to 20 (a)|| 79 ||30, 100|
|TGB||Not known||–||10, 30|
|TPM||1 to 3|| 15 ||10, 30, 100|
|VGB||Not known||–||100, 300, 1,000|
|VPA||40 to 100 (c)||700 ||300, 1,000, 3,000|
Results were expressed as mean values ± SEM of (n) independent experiments. A paired two-tailed t test between values obtained before and after drug application was performed for statistical evaluations: a p value <0.05 was considered a significant difference between mean values.
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Acknowledgment: We thank Dr. O. Dulac for helpful discussion. We are grateful to the following laboratories for the kind gift of most of the compounds used in this study: Aventis (PB), Glaxo-Wellcome (LTG), Janssen-Cilag (TPM), Pfizer (GBP), Sanofi-Synthelabo (CLB, PHT, TGB, VPA), Schering-Plough (FBM), Roche (MDL) and UCB-Pharma (LEV). P.P.Q. was supported by a grant from Fondation Française pour la Recherche sur l'Epilepsie (FFRE).