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- MATERIAL AND METHODS
Summary: Purpose: The management of epilepsy during pregnancy entails a number of concerns. While seizures may affect adversely maternal and fetal outcome, antiepileptic drugs (AEDs) may increase the incidence of congenital abnormalities and possibly affect postnatal cognitive development in the offspring. Experimental animal studies can aid in assessing teratogenic features associated with individual AEDs and/or with seizures, and to identify the mechanisms involved. The purpose of this study was to investigate the consequences of prenatal exposure to (a) different AEDs and (b) maternal seizures on brain maturational processes in rats.
Methods: Pregnant rats received from embryonic days 14 to 19 intraperitoneal injections of carbamazepine (20 mg/kg/day), vigabatrin (200 mgkg/day), and valproate (100 mg/kg/day) at doses not widely different from those used clinically. Pups exposed to AEDs in utero were analyzed postnatally. Animals born to “kindled” pregnant animals that had experienced one generalized convulsive seizure per day during the same gestational period were analyzed in parallel.
Results: Prenatal exposure to vigabatrin and valproate, which act on GABA signaling, induced hippocampal and cortical dysplasias, which were likely to result from a neuronal migration defect and neuronal death. By contrast, offspring of rats exposed to carbamazepine (which at the dose used produced low plasma concentrations) or to generalized convulsive seizures showed no clear-cut evidence of dysplasias.
Conclusions: We suggest that AEDs that increase the extracellular concentration of GABA might induce severe neuronal migration disorders. Drugs acting through other molecular targets would also perturb cortical maturation. The potential clinical relevance of these results should be a subject of future research.
The treatment of epilepsy during pregnancy entails a number of concerns. While maternal seizures may adversely affect maternal and fetal outcome (La Joie and Moshe, 2004), prenatal exposure to antiepileptic drugs (AEDs) may increase the incidence of congenital abnormalities (Samren et al., 1999; Arpino et al., 2000; Holmes, 2002; Perucca, 2005) and possibly affect postnatal cognitive development (Koch et al., 1999; Adab et al., 2001; Gaily et al., 2004; Vinten et al., 2005; Eriksson et al., 2005; Perucca, 2005; Viinikainen et al., 2006). Clinical studies in this area are often difficult to interpret and compare due to a number of confounders (see [Barrett and Richens, 2003]), including the influence of socioeconomical factors and parental educational level on the outcome of exposed children. Moreover, the analysis of the neurodevelopmental consequences of fetal exposure to AEDs has been restricted to the most severe malformations (i.e., spina bifida, microcephaly), and the potential occurrence of subtle brain maturation abnormalities has been poorly investigated, partly because of difficulties in detecting such defects in the clinical setting. Experimental animal studies can be useful in investigating the effects of AEDs and/or seizures on neurodevelopment, and in identifying the potential mechanisms involved.
In the present study, we compared the morphological consequences of prenatal exposure to AEDs and seizures in rats. We focused our examinations on cortical dysplasias, also referred to as cortical malformations, neuronal migration disorders, or cortical dysgenesis, that are among the most frequent pathological findings in pediatric epilepsies (Barkovich and Raybaud, 2004). Our findings indicate that prenatal-exposure to vigabatrin (VGB) and valproate (VPA), which act on GABA signaling (Grant et al., 1991; Loscher, 2002), induces hippocampal and cortical dysplasias in rats. By contrast, offspring of rats exposed to kindled-seizures show no clear-cut evidence of dysplasias. Since GABA is the principal excitatory transmitter in the developing brain and acts as an epigenetic factor to control developmental processes (Represa and Ben Ari, 2005), including cortical and hippocampal neuroblast migration (Behar et al., 2000; Manent et al., 2005), we suggest that AEDs that increase the extracellular concentration of GABA may interfere with neuronal migration in the developing brain. The potential clinical relevance of these findings should be ascertained.
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- MATERIAL AND METHODS
In this article, we examined the effects of some AED on brain maturation. The choice of VPA, CBZ, and VGB in this investigation was based on a number of considerations: (a) CBZ and VPA are among the AEDs most widely used in the current treatment of epilepsy, and they are the drugs with the largest exposure data in pregnancy registries to date (Perucca, 2005; Morrow et al., 2006); (b) VPA is associated with special concerns with respect to its potential adverse effects on neurodevelopment after prenatal exposure in the clinical setting (Gaily et al., 2004; Vinten et al., 2005; Eriksson et al., 2005; Viinikainen et al., 2006); (c) CBZ, VGB, and VPA allow exploration of different mechanisms of drug action, e.g., blockade of voltage dependent sodium channels for CBZ (White et al., 1999), selective increase in brain GABA levels for VGB (Grant and Heel, 1991) and potentiation of GABAergic transmission coupled with additional modes of action for VPA (Loscher, 2002). The dosages selected were not greatly in excess of those used clinically and, at least in the case of VPA and CBZ, resulted, respectively, in plasma drug levels that were within the clinically occurring range or below. There were also limitations in our study. In particular, plasma concentration profiles after i.p. administration in rats do not mimic closely those observed after orally administered therapeutic doses in the clinical setting. In the case of VPA, in particular, the administration schedule used resulted in marked swings between peak and trough concentrations, a potentially important feature in view of the observation that high peaks in plasma VPA concentrations are critical in adversely affecting embryonic central nervous system development in rodents (Nau et al., 1991). Another limitation was the fact that only one dose was used for each drug, a decision prompted by the complex and labor-intensive nature of the methodology used. In addition, the window time of exposure to AEDs was shorter in our paradigm than in clinal conditions so that the deleterious effects reported here are likely to be more prominent in the latter.
While the above limitations should be kept in mind, our findings demonstrate that prenatal exposure to AEDs that increase brain GABA levels (VPA, VGB) during the last week of gestation in rats leads to the formation of dysplasias in the hippocampus and somatosensory cortex. Moreover, the findings suggest that these abnormalities result from neuronal migration defects as well as neuronal cell death.
Although it cannot be excluded that other pharmacological actions contributed to the adverse effects of VGB and VPA in this study, the most likely explanation is that an increase in GABA transmission played a crucial role, as suggested by different lines of evidence. First, unlike VPA, VGB is considered to be selective on producing GABA-mediated effects (Grant and Heel, 1991). Perhaps more importantly, GABA has been shown to provide most of the excitatory drive at an early stage of brain development, both in rodents and in primates (Ben Ari, 2002). GABA signaling modulates a wide range of essential developmental processes (Represa and Ben Ari, 2005) even before synapses are formed, when it acts in a paracrine manner (Demarque et al., 2002) and is able to modulate the migration of pyramidal neuroblasts (Manent et al., 2005).
The lack of comparably deleterious effects of CBZ, a drug that acts primarily by blocking voltage-dependent sodium channels (White, 1999), may be due to the low density of these channels at an early developmental stage (Manent et al., 2005). It should be noted, however, that plasma CBZ concentrations in these experiments were lower than the range found in patients receiving therapeutic doses (see Table 1) and therefore we cannot exclude that greater histological brain abnormalities could occur in animals exposed to higher CBZ concentrations. Therefore, in addition to GABA-acting drugs, drugs acting on other molecular targets, this including voltage-gated ion channels, might have deleterious effects on brain construction. Further studies are required to clarify this point.
The potential clinical applicability of our findings remains to be established, but these observations should be taken into consideration in future studies on neurodevelopmental outcome in children prenatally exposed to AEDs. In fact, there are indications from other animal studies (Vorhees et al., 1988; Phillips and Lockard, 1996) and from clinical observations (Koch et al., 1999; Dean et al., 2002; Adab et al., 2004; Gaily et al., 2004; Hirano et al., 2004; Perucca, 2005; Vinten et al., 2005; Eriksson et al., 2005) that exposure in utero to some AEDs may lead to neurological and/or cognitive alterations. In particular, while CBZ has been regarded as safe (Gaily et al., 2004), VPA has been associated with developmental delay, including lower verbal IQ (Vinten et al., 2005; Eriksson et al., 2005) and higher incidence of additional educational needs (Adab et al., 2001; Viinikainen et al., 2006) in prenatally exposed children, even though an influence of confounding factors cannot be excluded (Eriksson et al., 2005; Viinikainen et al., 2006). Several observations suggest that cortical malformations involving the hippocampus and medial temporal cortex are instrumental in mental retardation (Barkovich and Raybaud, 2004). Whether drug-induced subtle cortical and hippocampal dysplasias are involved in causing postnatal cognitive deficits in offspring exposed to VGB, VPA, or other GABAergic drugs during pregnancy should be subject for future investigations, possibly involving careful neuroimaging assessments.
Finally, a possible link between AEDs exposure and epileptogenesis in offspring has been suggested (Phillips and Lockard, 1996; Koch et al., 1999). Though our animals did not apparently displayed spontaneous seizures, it is worth to stress that focal cortical dysplasias are among the most epileptogenic lesions, associated with early onset medically refractory epilepsy (Barkovich and Raybaud, 2004). Therefore, it would be interesting to evaluate whether rats exposed to VGB or VPA during pregnancy have epileptic features or higher susceptibility to convulsant agents.
The use of animal models, such as those we developed in the present study, may also be useful to investigate the mechanisms that lead to cortical dysplasias in order to develop AEDs less deleterious when taken during pregnancy.