Please use this identifier to cite or link to this item: https://hdl.handle.net/10316/23653
Title: Effects of exposure to eslicarbazepine acetate and to other antiepileptic drugs on neurotoxicity and hippocampal development
Authors: Morte, Maria Inês Frade Marquez Varela 
Orientador: Carvalho, Caetana Angélica Ermitão Monteiro de
Issue Date: 15-Jul-2013
Citation: MORTE, Maria Inês Frade Marquez Varela - Effects of exposure to eslicarbazepine acetate and to other antiepileptic drugs on neurotoxicity and hippocampal development. Coimbra : [s.n.], 2013. Tese de doutoramento
Abstract: Eslicarbazepine acetate (ESL) is a new antiepileptic drug (AED), which is used as add-on therapy for adult patients with partial-onset seizures, with or without secondary generalization, since its approval in Europe in 2009. ESL is structurally related to carbamazepine (CBZ) and oxcarbazepine (OXC), and after oral administration to humans is rapidly and extensively hydrolyzed to its major and active metabolite, eslicarbazepine (SLic). S-Lic is responsible for the anticonvulsant action of ESL and, like CBZ and OXC, blocks voltage-gated sodium channels. In addition to S-Lic, there are two other metabolites present in the plasma after administration of ESL: R-licarbazepine (R-Lic), the R-enantiomer of S-Lic, and OXC, both without contribution to the anticonvulsant action of ESL. To date, ESL was shown to be more effective and less toxic than OXC and CBZ. To date, there are no studies regarding ESL administration during pregnancy and nursing on women with epilepsy and the potential consequences of in utero exposure to the offspring. To address some of these aspects, we used an animal model, CD1 mice, which are known to be the species most similar to humans regarding the pharmacokinetics and oral biodisposition of ESL. Thus, this study had three main goals: a) to study the impact of AED exposure during pregnancy and during nursing on the biochemical parameters of blood and serum of the females progenitors, as well as, the effects of AED exposure on the formation of new cells in the adult hippocampus; b) to study the impact of AED exposure during pregnancy and during nursing on cognitive and non-cognitive behaviour of the progeny, as well as, the effects of AED exposure on the formation of new neurons in the adult hippocampus, and c) to study the possible effects of ESL and of other AEDs on the activation of cell death/neuroprotective mechanisms in cultures of embryonic hippocampal neurons; and, as well as, the effects of AEDs on the basal proliferation and cell cycle phases of neural stem cell cultures from the subventricular region (SVZ) of rats. In vitro experiments were performed in cultured hippocampal neurons of embryonic rats, which were exposed to different concentrations of AEDs [ESL, S-Lic, R-Lic, CBZ, OXC, lamotrigine (LTG) and sodium valproate (VPA)]. We observed that neither ESL nor its metabolites reduced cell viability or induced the appearance of markers of cell death. However, exposure to OXC and VPA did induce the appearance of markers related to cell death by apoptosis, and OXC was the most toxic drug. ESL, S-Lic and OXC decreased the levels of phospho-ERK1/2 and phospho-Akt but did not change the levels of phospho- SAPK/JNK, when compared to control situations, whereas CBZ decreased phospho- SAPK/JNK and phospho-Akt levels. However, LTG, and to some extent VPA, increased the phosphorylation levels of SAPK/JNK. Altogether, the results suggest that ESL and its metabolites are less deleterious to neuronal cells than the other AEDs studied. In the studies performed in vivo, CD1 mice were used as a model. We observed that after a daily intake of the AEDs (ESL, CBZ, OXC and VPA), at therapeutic doses, during pregestation period, pregnancy and nursing, females treated with ESL and CBZ did not show changes in the evaluated biochemical parameters of blood and serum (glucose, total cholesterol, triglyceride, alanine and aspartate aminotransferase, creatinine and creatine kinase), whereas OXC and VPA decreased creatinine levels by 30%, and VPA also decreased the levels of triglycerides by 50%, when compared to untreated females. We also assessed the effects of AED exposure on the formation of newly born cells in the hippocampus of CD1 female mice. We observed that both CBZ and VPA decreased by 40% the number of cells positive for BrdU in the subgranular zone (SGZ) of the dentate gyrus (DG), while ESL and OXC did not change these values when compared to untreated females. Then we studied the effects of AED exposure in utero and during nursing on the cognitive performance (memory and learning), locomotion, the possible anxiogenic vs. anxiolytic effects, and also pro-depressive effects, in the progeny of CD1 females. The performance of males and females was evaluated separately in the behavioural tests, which were conducted twice, at one month and at four months of age. Overall, we observed that the animals exposed to ESL did not show deficits in the performance of cognitive or noncognitive tasks, but we observed a slight increase in locomotor activity of males, at one month of age. Animals exposed to CBZ, OXC and VPA showed a decrease in performance of some tasks associated with memory and learning. These effects were observed mostly in young animals, and few effects were kept into adulthood. Next, we evaluated the effects of exposure to AEDs in utero and during nursing, on the basal proliferation and formation of new neurons in the DG of the hippocampus of CD1 mice born. The basal proliferation was evaluated by incorporation of EdU into neural progenitor cells found in DG, and we observed a decrease of about 40% of EdU-positive cells in the SGZ of males, but not in females, exposed to OXC when compared to the untreated animals. Neither ESL, CBZ nor VPA induced changes in the basal proliferation of the DG. Regarding the formation of new cells/neurons, we quantified the BrdU-positive cells in the DG or those that co-localize with other neuronal markers, such as doublecortin (DCX) and the neuronal nuclei (NeuN) marker. Interestingly, we observed that in both males and females exposed to OXC the number of BrdU-positive cells in SGZ was decreased by 54% and 47%, respectively; in males exposed to VPA that number was decreased by 60%, when compared to control animals. Except for males exposed to CBZ, which increased by 16% the number of mature neurons in the SGZ, none of the other groups of males had changes in the number of mature or immature neurons in the DG. Regarding females, we observed that those who were exposed to OXC had less mature and immature neurons in the SGZ compared with the control group. ESL, CBZ or VPA did not induce changes in the formation of new neurons in the DG of CD1 females. We also performed experiments in vitro, with cultured neural stem cells isolated from the SVZ of rats, to assess the effect of AED exposure (ESL, S-Lic, R-Lic, CBZ, OXC, LTG and VPA), on the basal proliferation and on the different cell cycle phases (G0/G1, S and G2/M). We used different concentrations of the AEDs and observed that the active metabolite of ESL, S-Lic, did not induce any change in basal proliferation or on cell-cycle phases. The other AEDs tested decreased basal proliferation at different concentrations, but the highest effect was caused by exposure to VPA (1 or 3 mM), which decreased basal proliferation by 85% or 95%, respectively. Regarding the cell cycle, we observed different effects of the AEDs, at different concentrations. We observed an increase (65%) in the percentage of cells in G2/M phase, after exposure to the OXC (0.3 mM), whereas VPA (3 mM) exposure caused a decrease in the percentage of cells in G2/M phase of about 70%. We also evaluated the effect of AEDs on cell death by apoptosis in the SVZ cultures, and observed that the VPA (3 mM) and OXC (0.3 mM) significantly increased the percentage of apoptotic cells. According to the results obtained from both in the in vivo and in the in vitro approaches, ESL emerges as potentially less risky AED to be used by pregnant women with epilepsy and during nursing, since ESL did not have impact on the biochemical parameters of blood and serum of the pregnant CD1 females, and no adverse effects were detected in cognitive and noncognitive functions of their progeny. Moreover, no changes were observed in the formation of new cells and neurons in the adult hippocampal DG of the pregnant CD1 females and their progeny, respectively. In addition, S-Lic, the active metabolite of ESL was not toxic to cultured hippocampal neurons, and it did not affect basal proliferation and cell cycle phases of the neural stem cell cultures from rat SVZ. In conclusion, ESL is a safe and effective anticonvulsant with potential to be used in the treatment of pregnant women with epilepsy, while AEDs that have negative effects on cognitive performance, such as OXC, and also have a negative impact on the formation of new cells and adult neurons in the hippocampus, should be avoided during the pregestation, pregnancy and nursing periods,.
Description: Tese de doutoramento em Biociências, no ramo de especialização de Neurociências, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
URI: https://hdl.handle.net/10316/23653
Rights: openAccess
Appears in Collections:FCTUC Ciências da Vida - Teses de Doutoramento

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