The lithium/pilocarpine model of epilepsy mimics mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) in humans. Systemic injection of pilocarpine in lithium chloride (LiCL) pretreated adult rats results in an acute episode of severe continuous seizure activity (status epilepticus, SE). SE causes a latent period, whereby the animal appears neurologically normal, with subsequent development of spontaneous recurrent seizures (SRSs). Neuropathological changes that occur during the latent period are believed to contribute to the epileptic condition. The present thesis characterized the development of neuronal death and behavioural alterations in rats after SE induced by the repeated low-dose pilocarpine procedure (RLDP), and investigated the causal relationship between these two processes. Our data demonstrated that the RLDP procedure for the induction of SE results in widespread neurodegeneration and behavioural alterations comparable to the pilocarpine and low-dose pilocarpine (LDP) procedures. However, the advantage to using this protocol was strain-dependent as it reduced mortality in Wistar, but not in Long Evans Hooded (LEH), rats. Stereological analysis of neurons (stained for the neuronal specific marker [NeuN]) at various times (1 hr to 3 months) following SE showed that different brain regions within the hippocampus, amygdala, thalamus and piriform cortex exhibited differential rates of neuronal loss, with the majority of SE-induced neuronal death present by 24 hours. SE resulted in decreased exploratory behavior as assessed in the open field test, increased aggression to handling, increased hyperreactivity as assessed in the touch-response test, and anxiolytic effects as measured in the elevated-plus maze. Furthermore, deficits in search strategies used, as well as impaired spatial learning and memory, contributed to poor Morris water maze (MWM) performance. Partial neuroprotection within the hippocampus (by tat-NR2B9c) had no effect on the number of rats developing SRSs or on behavioural alterations; this argues against a causal relationship between neurodegeneration within this region, genesis of SRSs, and behavioural morbidity.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Levetiracetam (LEV) is a new antiepileptic drug effective as adjunctive therapy for partial seizures and in experimental models of seizures, including pilocarpine-induced seizures in rodents. Objectifying to clarify if anticonvulsant activity of LEV occurs near cholinergic muscarinic alterations, adult male mice received LEV injections before muscarinic agonistsâ administration. Pretreatment with LEV (30, 50, 100 or 200 mg/Kg, i.p.) increased the latency of seizures, latency of status epilepticus and latency of death on the seizure model induced by pilocarpine, 400 mg/Kg, s.c. (P400). LEV (200 mg/Kg, i.p., LEV200) pretreatment abolished seizures in 53% (20/38) of the animals; reduced status epilepticus appearance in 58% (22/38); increased deathâs latency in 116% compared to P400 group; protected 61% (23/38) of the animals from death and also reduced the intensity of tremors induced by oxotremorine (0,5 mg/kg, i.p). [3H]-N-methylscopolamine binding assays in mice hippocampus showed that LEV200 pretreatment reverts the downregulation on muscarinic acetylcholine receptors (mAChR), induced by P400 administration, bringing back these density values to control ones (0,9% NaCl, i.p.). However, subtype-specific binding assays revealed that P400 and LEV200 alone treatments results in M1 and M2 subtypes decrease, respectively. The agonist-like behavior of LEV200 on the inhibitory M2 mAChR subtype, observed in this work, could contribute to explain the reduction on oxotremorine-induced tremors and the delay on pilocarpine-induced seizures, by an increase in the attenuation of neuronal activity mediated by the M1 receptors. [3H]-spiroperidol binding assays also showed a downregulation of dopaminergic D2 receptors, induced by LEV200 alone treatment. Although this effect was reverted on P400 presence, maybe it posses some influence on LEV200 protective effect, since the stimulation of these receptors can reduce the intensity of pilocarpine induced-seizures. Objectifying to investigate if LEVâ mechanism of action also involves anti-oxidant proprieties, neurochemical analysis were carried out in hippocampus and striatum and showed that P400 produced an increased lipid peroxidation in hippocampus, main epileptogenic focus on cholinergic agents induced-seizures, demonstrating and confirming the involvement of free radical oxygen injury in the P400 induced brain injury. However, LEV200 pretreatment abolished this increase, bringing lipid peroxidation levels back to normal values, suggesting an anti-oxidative property of LEV. Our finds also showed a decrease of nitrite levels and stabilization of catalase activity in hippocampus and striatum, beyond an increase on reduced glutathione levels on hippocampus, induced by LEV200 treatment before P400 administration. In this way, the anti-oxidative action of LEV was showed in several stages involved on oxidative injury, suggesting a novel mechanism of its protector effects. / O levetiracetam (LEV) à uma nova droga antiepilÃptica, com eficÃcia na terapia adicional das convulsÃes parciais e em vÃrios modelos experimentais de convulsÃo, inclusive nas convulsÃes induzidas por pilocarpina em roedores. Objetivando investigar se o mecanismo de aÃÃo anticonvulsivante do LEV està relacionado a alteraÃÃes no sistema colinÃrgico muscarÃnico, camundongos machos adultos receberam injeÃÃes de LEV uma hora antes da administraÃÃo de agonistas muscarÃnicos. O prÃ-tratamento com LEV (30, 50, 100 ou 200 mg/Kg, i.p.) aumentou significativamente as latÃncias de convulsÃo, de estatus epilepticus e de morte no modelo de convulsÃes induzidas por pilocarpina, 400 mg/Kg, s.c. (P400). O prÃ-tratamento com LEV (200 mg/Kg, i.p., LEV200) levou à ausÃncia de convulsÃes em 53% (20/38) dos animais; reduziu a ocorrÃncia de estatus epilepticus em 58% (22/38); aumentou a latÃncia de morte em 116% comparada ao grupo P400; protegeu 61% (23/38) dos animais da morte e, ainda, reduziu a intensidade dos tremores induzidos por oxotremorina (0,5 mg/kg, i.p). Ensaios de binding com [3H]-N-metilescopolamina em hipocampo mostraram que o prÃ-tratamento com LEV200 reverte a downregulation dos receptores muscarÃnicos de acetilcolina, induzida por P400, normalizando a densidade desses receptores. Todavia, ensaios para subtipos especÃficos de receptores muscarÃnicos revelaram que a administraÃÃo de P400 ou LEV200, isoladamente, resulta em downregulation dos subtipos M1 e M2, respectivamente. A aÃÃo agonista-sÃmile do LEV nos receptores prÃ-sinÃpticos inibitÃrios M2, observada no presente estudo, poderia contribuir para explicar a reduÃÃo nos tremores induzidos por oxotremorina e o retardo na instalaÃÃo das convulsÃes induzidas por P400, atravÃs da atenuaÃÃo da atividade neuronal mediada pelos receptores M1. Ensaios de binding com [3H]-espiroperidol demonstraram uma downregulation dos receptores dopaminÃrgicos D2, induzida pela administraÃÃo isolada de LEV200. Embora esse efeito tenha sido revertido na presenÃa de P400, talvez possua alguma influÃncia na aÃÃo protetora oferecida por LEV200, considerando que a estimulaÃÃo desses receptores reduz a intensidade das convulsÃes induzidas pela pilocarpina. Objetivando investigar se o efeito protetor demonstrado pelo LEV envolve propriedades antioxidantes, anÃlises neuroquÃmicas foram realizadas em hipocampo e corpo estriado. A administraÃÃo de P400 aumentou a ocorrÃncia de peroxidaÃÃo lipÃdica no hipocampo, considerado principal foco de instalaÃÃo das convulsÃes provocadas por agentes colinÃrgicos, demonstrando e confirmando o envolvimento de espÃcies deletÃrias na injÃria cerebral produzida por esse modelo de convulsÃes. O prÃ-tratamento com LEV200 reverteu esse efeito, fornecendo indÃcios de uma atividade antioxidante dessa droga. Nossos estudos tambÃm mostraram uma diminuiÃÃo da produÃÃo de nitrito e estabilizaÃÃo da atividade da catalase no hipocampo e corpo estriado e o aumento dos nÃveis de glutationa reduzida no hipocampo, decorrentes do tratamento com LEV antes de P400. A aÃÃo antioxidante do LEV foi evidenciada em vÃrias etapas envolvidas no processo de dano oxidativo, sugerindo um novo mecanismo atravÃs do qual essa droga poderia exercer seus efeitos protetores.

Orientador: Iscia Teresinha Lopes Cendes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Médicas / Made available in DSpace on 2018-08-19T12:08:05Z (GMT). No. of bitstreams: 1 Marchesini_RafaelBreglio_D.pdf: 9525871 bytes, checksum: f90452b399007d69d5b96e4070e34d9a (MD5) Previous issue date: 2011 / Resumo: As epilepsias afetam aproximadamente 1% da população mundial, sendo que a dentre as diferentes síndromes epilépticas a epilepsia de lobo temporal (ELT) é a mais freqüente, e aquela com a maior proporção de pacientes adultos que apresentam crises refratárias ao tratamento clínico. Molelos animais induzidos têm sido já largamente utilizados para estudar a ELT, já que se considera que os mesmos apresentam epileptogenicidade similar àquela observada em tecidos "epilépticos" humanos quando estudados ex vivo. Dentre os vários modelos disponíveis, aquele induzido pela pilocarpina, além de muito bem estabelecido em nosso meio, tem já ampla caracterização, sendo dividido em três fases de acordo com os aspectos fenomenológicos, histopatológicos e moleculares: fase aguda, fase silenciosa e fase crônica. Baseado em padrões de expressão diferencial de genes na fase silenciosa do modelo, tem-se que a modificação de circuitos neuronais é um importante pré-requisito para a plasticidade funcional no sistema nervoso central sob condições patológicas. Levando-se em conta que a remodelação hipocampal ocorre durante o período livre de crises (fase silenciosa), danos estruturais causados pelo status epilepticus (SE) induzido pela pilocarpina na fase aguda acabam resultando em crises espontâneas recorrentes na fase crônica do modelo. Dessa maneira, esse trabalho teve como objetivo principal investigar a ação de genes que têm suas expressões induzidas durante a fase silenciosa do modelo da pilocarpina, e que estariam possivelmente relacionados com danos neuronais e epileptogenêse através de mecanismos associados à plasticidade e reorganização neuronal. Além disso, o trabalho investigou mais profundamente o papel do gene interleucina1-beta (Il1b), o qual foi demonstrado previamente estar envolvido no aumento da liberação de glutamato durante a fase aguda do modelo da pilocarpina. A principal estratégia experimental utilizada foi baseada no silenciamento póstranscricional pela técnica de interferência por RNA (RNAi) aplicada in vivo. Foi definido um padrão de expressão gênica para os genes TrkB e Plat, que sabidamente estão envolvidos com a fase silenciosa, e também para os genes da família dos Toll like receptors, durante o início da fase silenciosa, demonstrando suas expressões alteradas. Tais alterações parecem participar dos processos críticos que ocorrem na fase silenciosa, processos inflamatórios que associadas à remodelação sináptica geradas pelos genes estruturais (TrkB e Plat), culminam na fase crônica do modelo. A expressão dos genes Il1b e Il1ra também foram definidas, além do silenciamento dos mesmos. O silenciamento na fase aguda do modelo da pilocarpina levou a alterações fenotípicas detectáveis nos animais silenciados. Sendo assim, acreditamos que nossos resultados fornecem dados relevantes referentes ao papel crítico dos genes investigados na determinação da epileptogênese durante as fases aguda e silenciosa do modelo da pilocarpina para ELT, além de ter demonstrado a viabilidade do uso da RNAi na investigação funcional de genes envolvidos na patogênese das epilepsias in vivo / Abstract: The epilepsies affect approximately 1% of the population worldwide. Temporal lobe epilepsy (TLE) is the most frequent and commonly resistant to clinical treatment. Animal models have been widely used in order to study the mechanisms underlying TLE, especially those induced by chemical agents or electrical stimulation. It is believed that these models present similar epileptogenesis to that of human epileptic tissues studied ex vivo. Among the various animals models available we decided to use the one induced by pilocarpine injections. This model is characterized by three different phases accordingly to phenomenological, histopathological and molecular aspects: acute phase, silent phase and chronic phase. Based on the presence of genes with differential expression patterns in the silent phase, it is believed that it is in this phase that occur the modifications in neuronal circuits that are essential to functional plasticity of the central nervous system under pathological conditions. The changes in hippocampal circuits occuring during the silent phase are the result of the damage caused by the status epilepticus (SE) induced by pilocarpine in the acute phase. These events will ultimately result in spontaneous recurrent seizures in the chronic phase of the model. The main objective of this work was to explore the action of genes that are induced during the silent phase of the pilocarpine model. These would be possibly related with the neuronal damage and the development of epileptogenesis through mechanisms associated with plasticity and neuronal reorganization. In addition, we aimed to explore futher the role of Interleukin1-beta (Il1b), which had been previously demonstrated to be involved in the increase of glutamate release during the acute phase of the pilocarpine model. To achieve these objectives we used a pos-transcriptional gene silencing strategy named RNA interference (RNAi) applied in vivo. It was defined a pattern of gene expression for Trkb and Plat, that are known to be involved with the silent phase. Besides that, the Toll like receptors genes family had the pattern of expression defined during the beginning of the silent phase, demonstrating the expression altered. These alterations seem to participate in the critical processes that occur in the silent phase, inflammatory processes that associated to the synaptic reorganization generated by the structural genes (TrkB and Plat), culminate in the chronic phase of the model. The expression of the genes Il1b and Il1ra were defined too, besides the silencing of them. The silencing in the acute phase of the pilocarpine model led to phenotypic alterations detectable in the animals that had the genes silenced. We believe that our results can provide relevant new information regarding the role of genes in the determination of epileptogenesis during the acute and silent phases of the pilocarpine model. In addition, we have demonstrated the fisibility of the using RNAi to study epileptogenesis in vivo / Doutorado / Neurociencias / Doutor em Ciências

Prolonged prenatal exposure to ethanol produces long-lasting alterations in the level of endogenous brain biogenic amines in rats. To test whether there might be long-lived alterations in the reactivity to dopaminergic, serotoninergic or muscarinic agonists in rats exposed prenatally to ethanol, the following study was done. Wistar rats were given 10% (v/v) ethanol in drinking water, starting 10 days before mating and continuing to the end of pregnancy. Male offspring were tested at 3 months for characteristic behavioral effects (oral activity) known to be induced by agonists acting at central dopamine D2 (quinpirole), serotonin 5-HT2C (m-chlorophenylpiperazine, m-CPP) and muscarine (pilocarpine) receptors. Dose-effect curves demonstrated that oral activity responses to quinpirole HCl (0.05-0.40 mg/kg i.p.) and m-CPP 2HCl (0.3-6.0 mg/kg i.p.) were greatly reduced (P < .001) in rats that were exposed to ethanol in utero. Responses to pilocarpine HCl (0.1-3.0 mg/kg) remained unaltered. The findings indicate that prenatal ethanol exposure alters behavioral responses to D2 and 5-HT2C agonists in male rats tested three months after birth. We propose that prenatal ethanol diminishes reactivity of receptors for dopamine D2 and serotonin 5-HT2C receptors.

No description available.

No description available.

Le carisbamate est la 1ère molécule à avoir un effet modificateur de l’épileptogenèse, dont environ 50% des animaux traités développent des crises d’absence, au lieu des crises limbiques normalement observées dans le modèle de la pilocarpine. Le principal objectif de cette thèse a été celui d’étudier les altérations qui se produisent par le traitement par le carisbamate. Pour cela nous avons effectué une cartographie de l’activité cérébrale, avec un immunomarquage de la protéine c-Fos, et nous avons vérifié les concentrations des monoamines et acides aminés dans l’hippocampe, le thalamus et dans le cortex piriforme par HPLC, 4h après le début du status epilepticus (SE). Enfin, nous avons vérifié le profil d’expression protéique dans l’hippocampe, 2 mois après le SE par électrophorèse bidimensionnelle.Les résultats indiquent que le carisbamate augmente l’activité des noyaux MD et LD du thalamus. Les résultats suggèrent aussi que la dopamine, la noradrenaline, le GABA et la sérotonine peuvent participer à la significative neuroprotection et à l’effet modificateur de l’épileptogenèse du carisbamate. L’étude de protéomique suggère également une réduction globale du métabolisme énergétique cellulaire chez les rats traité par le carisbamate qui développent des crises d’absence. / The carisbamate is the 1st molecule showing an epileptogenesis modifying effect, that about 50% of treated animals develops absence instead limbic seizures, commonly seen in the pilocarpine model. The aim of this thesis was to study the changes that follows carisbamate treatment. Therefore, we made a brain activity cartography, by labelling c-Fos protein, and we quantified the concentrations of amino acids and monoamines in hippocampus, thalamus and piriform cortex, 4h after the status epilepticus (SE). Moreover, we studied the protein expression profile in the hippocampus, 2 month after SE, by two-dimensional electrophoresis.The results points towards an increased activity of MD and LD thalamic nuclei in carisbamate treated rats. Furthermore, dopamine, noradrenaline, GABA and serotonin appears to play a role in neuroprotection and in the epileptogenesis modifying effect of carisbamate. The proteomic study revealed a global reduction of cellular energetic metabolism of carisbamate treated rats that develops absence seizures.

Les épilepsies mésiales du lobe temporal (ELTM) sont parmi les formes les plus fréquentes d’épilepsies partielles pharmaco-résistantes de l’adulte et l’enfant. Dans ces épilepsies les études électrocliniques et expérimentales indiquent que la zone épileptogène, qui désigne un ensemble de neurones nécessaire et suffisant à l’organisation d’une décharge anormale, ne peut être réduite à la seule formation hippocampique (FH) et impliquerait une réorganisation mettant en jeu plusieurs structures au sein du système limbique. Dans ce travail de thèse, nous nous sommes intéressés à la connectivité structurale entre le noyau supramammillaire (SuM) et la FH chez le rat dans le but de déterminer l’identité neurochimique de la voie de projection supramammillaro-hippocampique qui n’avait pas été clairement identifiée et, vérifier l’hypothèse d’une éventuelle réorganisation de cette voie de projection dans le modèle d’ELTM induit par l’injection intrapéritonéale de pilocarpine chez le rat. Chez les rats naïfs, nous mettons en évidence deux voies de projection distinctes. La première a pour origine les neurones localisés dans la partie latérale du SUM (SuML) qui innervent le champ CA2-CA3a et principalement la couche supragranulaire du gyrus dentelé dorsal. Cette voie est essentiellement ipsi-latérale et a la caractéristique de présenter un profil neurochimique unique, à la fois GABAergique et glutamatergique. La seconde voie de projection a pour origine les neurones localisés dans la partie plus postérieure et médiane du SuM (SuMM) qui innervent la région CA2-CA3a et la région ventrale du gyrus dentelé exclusivement ; cette voie est purement glutamatergique. Chez les rats traités à la pilocarpine, nos résultats montrent une réorganisation structurale des afférences des noyaux SuML et SuMM qui innervent le gyrus dentelé. Cette réorganisation est caractérisée par une distribution aberrante et une augmentation du nombre de fibres et terminaisons axonales en provenance des noyaux SuML et SuMM dans la couche moléculaire interne du gyrus dentelé. Cette réorganisation commence à la fin de la période de latence, et évolue pendant l’épilepsie induite par la pilocarpine. Avec ce travail, nous montrons pour la première fois : 1) l’hétérogénéité à la fois anatomique et neurochimique des voies de projection supramammillaro-hippocampiques ; 2) dans le gyrus dentelé des animaux traités à la pilocarpine, une réorganisation structurale d’origine extra-hippocampique, en provenance des noyaux SuML et SuMM. Cette connectivité aberrante pourrait contribuer avec la réorganisation des circuits intrinsèques de l’hippocampe à l’émergence des premières crises spontanées et à l’installation de l’épilepsie. / Mesial temporal lobe epilepsies (MTLE) are among the most common forms of pharmacoresistant partial epilepsies in adults and children. In these epilepsies, spontaneous seizures likely originate from a multi-structural epileptogenic zone including several structures of the limbic system connected to the hippocampal formation (HF). In this thesis, we investigate the structural connectivity between the supramammillary nucleus (SuM) and the HF in rat, in order to determine the not yet known neurochemical identity of the supramammillaro-hippocampal pathway and, to test the hypothesis of a potential reorganization of this pathway in the rat pilocarpine model of MTLE. In naïve rats, our results highlight two distinct pathways. The first pathway originates in the lateral part of the SuM (SuML) and innervates the supragranular layer of the dorsal dentate gyrus mainly, and the CA2-CA3a pyramidal cell layer of the hippocampus. This pathway is mainly ipsilateral and displays a unique dual phenotype for GABAergic and glutamatergic neurotransmission. The second pathway originates in the most posterior and medial part of the SuM (SuMM) and innervates exclusively the inner molecular layer of the ventral dentate gyrus and the CA2-CA3a subfield and is glutamatergic only.In pilocarpine-treated animals, our findings demonstrate a structural reorganization of dentate gyrus afferents originating from the SuM nuclei. Such reorganization is characterized by an aberrant distribution and an increased number of fibers and axon terminals from neurons of the both lateral and medial regions of the SuM, invading the entire inner molecular layer of the dentate gyrus. It starts at the end of the latent period and evolves during the epilepsy induced by pilocarpine. Our findings demonstrate for the first time: 1) the anatomical and neurochemical heterogeneity of the supramammillaro-hippocampal pathways; 2) in pilocarpine-treated animals, a marked reorganization of dentate gyrus afferents originating from the SuM nuclei. This aberrant connectivity could contribute along with the reorganization of hippocampal intrinsic circuitry to the emergence of the first spontaneous seizures and epilepsy installation.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Levetiracetam (LEV), nova droga antiepilÃptica, apresenta eficÃcia na terapia adicional das convulsÃes e em modelos experimentais. Clonazepam (CNZP) à um benzodiazepÃnico utilizado no tratamento de convulsÃes mioclÃnicas e crises generalizadas. Melatonina (MEL), hormÃnio pineal, demonstra atividade anticonvulsivante em diversos modelos animais. Objetivando investigar novos mecanismos relacionados aos efeitos dessas drogas, foi realizado estudo comparativo, a partir da anÃlise da influÃncia do prÃ-tratamento com LEV, CNZP ou MEL sobre o estresse oxidativo neuronal e sobre a modulaÃÃo de sistemas de neurotransmissÃo (colinÃrgico e dopaminÃrgico) durante as convulsÃes induzidas por pilocarpina (400mg/Kg; P400). Camundongos Swiss, machos, 25-30g receberam LEV, 200 mg/Kg, CNZP, 0,5mg/Kg ou MEL, 25mg/Kg, i.p., (doses escolhidas à partir de curvas dose-resposta) 30min antes de P400. Hipocampo e corpo estriado foram removidos para as anÃlises neuroquÃmicas. Experimentos in vitro, onde homogenatos cerebrais foram incubados com as drogas em estudo (50, 100 ou 200&#61549;g/mL), tambÃm permitiram analisar alteraÃÃes no estresse oxidativo apÃs a induÃÃo de choque tÃrmico e, ainda, a densidade de receptores muscarÃnicos no hipocampo. Os estudos sobre os efeitos sobre o sistema de neurotransmissores colinÃrgicos demonstraram que o prÃ-tratamento com LEV, CNZP ou MEL causou reduÃÃo nos tremores induzidos por oxotremorina e elevou a atividade da acetilcolinesterase no hipocampo. LEV e CNZP alteraram o binding dos receptores muscarÃnicos hipocampais in vivo, revertendo a downregulation induzida por P400, e ensaios in vitro demostraram alteraÃÃo no binding muscarÃnico hipocampal pela prÃvia incubaÃÃo com LEV, CNZP ou MEL. Os ensaios de binding demonstraram, ainda, a downregulation dos receptores D2 hipocampais nos animais tratados LEV, CNZP ou prÃ-tratados com MEL antes de P400. As anÃlises para investigaÃÃo da atividade antioxidante de LEV e CNZP e do papel da aÃÃo antioxidativa da MEL na proteÃÃo contra as convulsÃes permitiram observar que a associaÃÃo com vitamina E potencializou os efeitos anticonvulsivantes de todas as drogas estudas. A administraÃÃo prÃvia de LEV, CNZP ou MEL, antes de P400, reverteu o aumento nos nÃveis de peroxidaÃÃo lipÃdica e nitrito-nitrato e normalizou a atividade da catalase e os nÃveis fisiolÃgicos do antioxidante glutationa em hipocampo e/ou corpo estriado. Nos experimentos de stress oxidativo in vitro, o aumento da peroxidaÃÃo lÃpÃdica, dos nÃveis de nitrito-nitrato e da atividade da catalase nos homogenatos cerebrais submetidos ao choque tÃrmico, foram alterados de forma significativa pela incubaÃÃo prÃvia com LEV, CNZP ou MEL, onde estas drogas foram capazes de reduzir os nÃveis de MDA, de nitrito-nitrato e, ainda, estabilizar a atividade da catalase, potencializando, assim, a atividade enzimÃtica antioxidante endÃgena e a capacidade de inativaÃÃo de radicais livres. Dessa forma, o estudo sugere uma aÃÃo moduladora, exercida por LEV, CNZP e MEL sobre o funcionamento dos sistemas muscarÃnico e dopaminÃrgico, em nÃvel central, como mecanismo alternativo para a proteÃÃo contra as convulsÃes no modelo de P400, bem como a participaÃÃo de propriedades antioxidantes diretas ou indiretas dessas drogas, atravÃs da capacidade de modificar a resposta ao estresse oxidativo neuronal. / Levetiracetam (LEV), nova droga antiepilÃptica, apresenta eficÃcia na terapia adicional das convulsÃes e em modelos experimentais. Clonazepam (CNZP) à um benzodiazepÃnico utilizado no tratamento de convulsÃes mioclÃnicas e crises generalizadas. Melatonina (MEL), hormÃnio pineal, demonstra atividade anticonvulsivante em diversos modelos animais. Objetivando investigar novos mecanismos relacionados aos efeitos dessas drogas, foi realizado estudo comparativo, a partir da anÃlise da influÃncia do prÃ-tratamento com LEV, CNZP ou MEL sobre o estresse oxidativo neuronal e sobre a modulaÃÃo de sistemas de neurotransmissÃo (colinÃrgico e dopaminÃrgico) durante as convulsÃes induzidas por pilocarpina (400mg/Kg; P400). Camundongos Swiss, machos, 25-30g receberam LEV, 200 mg/Kg, CNZP, 0,5mg/Kg ou MEL, 25mg/Kg, i.p., (doses escolhidas à partir de curvas dose-resposta) 30min antes de P400. Hipocampo e corpo estriado foram removidos para as anÃlises neuroquÃmicas. Experimentos in vitro, onde homogenatos cerebrais foram incubados com as drogas em estudo (50, 100 ou 200&#61549;g/mL), tambÃm permitiram analisar alteraÃÃes no estresse oxidativo apÃs a induÃÃo de choque tÃrmico e, ainda, a densidade de receptores muscarÃnicos no hipocampo. Os estudos sobre os efeitos sobre o sistema de neurotransmissores colinÃrgicos demonstraram que o prÃ-tratamento com LEV, CNZP ou MEL causou reduÃÃo nos tremores induzidos por oxotremorina e elevou a atividade da acetilcolinesterase no hipocampo. LEV e CNZP alteraram o binding dos receptores muscarÃnicos hipocampais in vivo, revertendo a downregulation induzida por P400, e ensaios in vitro demostraram alteraÃÃo no binding muscarÃnico hipocampal pela prÃvia incubaÃÃo com LEV, CNZP ou MEL. Os ensaios de binding demonstraram, ainda, a downregulation dos receptores D2 hipocampais nos animais tratados LEV, CNZP ou prÃ-tratados com MEL antes de P400. As anÃlises para investigaÃÃo da atividade antioxidante de LEV e CNZP e do papel da aÃÃo antioxidativa da MEL na proteÃÃo contra as convulsÃes permitiram observar que a associaÃÃo com vitamina E potencializou os efeitos anticonvulsivantes de todas as drogas estudas. A administraÃÃo prÃvia de LEV, CNZP ou MEL, antes de P400, reverteu o aumento nos nÃveis de peroxidaÃÃo lipÃdica e nitrito-nitrato e normalizou a atividade da catalase e os nÃveis fisiolÃgicos do antioxidante glutationa em hipocampo e/ou corpo estriado. Nos experimentos de stress oxidativo in vitro, o aumento da peroxidaÃÃo lÃpÃdica, dos nÃveis de nitrito-nitrato e da atividade da catalase nos homogenatos cerebrais submetidos ao choque tÃrmico, foram alterados de forma significativa pela incubaÃÃo prÃvia com LEV, CNZP ou MEL, onde estas drogas foram capazes de reduzir os nÃveis de MDA, de nitrito-nitrato e, ainda, estabilizar a atividade da catalase, potencializando, assim, a atividade enzimÃtica antioxidante endÃgena e a capacidade de inativaÃÃo de radicais livres. Dessa forma, o estudo sugere uma aÃÃo moduladora, exercida por LEV, CNZP e MEL sobre o funcionamento dos sistemas muscarÃnico e dopaminÃrgico, em nÃvel central, como mecanismo alternativo para a proteÃÃo contra as convulsÃes no modelo de P400, bem como a participaÃÃo de propriedades antioxidantes diretas ou indiretas dessas drogas, atravÃs da capacidade de modificar a resposta ao estresse oxidativo neuronal. / Levetiracetam (LEV), a new antiepileptic drug, shows efficacy in the treatment of additional seizures and in experimental models. Clonazepam (CNZP) is a benzodiazepine used to treat myoclonic seizures and generalized seizures. Melatonin (MEL), the pineal hormone, shows anticonvulsant activity in several animal models. To investigate new mechanisms related to the effects of these drugs, comparative study was conducted, from the analysis of the influence of pretreatment with LEV, CNZP or MEL on the oxidative stress and neuronal modulation of neurotransmitter systems (cholinergic and dopaminergic) during seizures induced by pilocarpine (400mg/Kg; P400). Male Swiss mice, 25-30g received LEV, 200 mg / kg, CNZP, 0.5 mg / kg or MEL, 25mg/kg, ip (doses chosen from the dose-response curves) 30min before P400. Hippocampus and striatum were removed for neurochemical analysis. In vitro experiments, where brain homogenates were incubated with drugs under study (50, 100 ou 200&#61549;g/mL) also allowed us to analyze changes in oxidative stress after induction of heat shock and also the density of muscarinic receptors in the hippocampus. Studies on the muscarinic modulation demonstrated that pretreatment with LEV, CNZP or MEL resulted in lower oxotremorina induced tremors and increased acetylcholinesterase activity in the hippocampus. LEV and CNZP altered the binding of hippocampal muscarinic receptors in vivo, reversing the P400-induced downregulation and in vitro tests showed changes in hippocampal muscarinic binding by previous incubation with LEV, CNZP or MEL. The binding assays also showed a downregulation of hippocampal D2 receptors in treated animals LEV, CNZP or pretreated with MEL before P400. Analyses to investigate the antioxidant activity of LEV and CNZP and role of antioxidative action of MEL in the protection against seizures propose that the association with vitamin E increased the anticonvulsant effects of all studied drugs. The prior administration of LEV, MEL or CNZP before P400, reversed the increased levels of lipid peroxidation and nitrite-nitrate and normalized the activity of catalase and the physiological levels of the antioxidant glutathione in the hippocampus and / or striatum. According to in vitro experiments, increased lipid peroxidation, levels of nitrite-nitrate and catalase activity in brain homogenates subjected to thermal shock were significantly altered by incubation with LEV, CNZP or MEL, where these drugs were able to reduce the levels of MDA, nitrite-nitrate, and also stabilize the activity of catalase, enhancing thus the endogenous antioxidant enzyme activity and the ability to inactivate free radicals.Thus, the study suggests a modulatory action exerted by LEV, CNZP and MEL on the functioning of muscarinic and dopaminergic systems in the central nervous system as an alternative mechanism to protect against seizures in the model P400, and the participation of direct and indirect antioxidant properties of these drugs, through the ability to modify the neuronal response to oxidative stress. / Levetiracetam (LEV), a new antiepileptic drug, shows efficacy in the treatment of additional seizures and in experimental models. Clonazepam (CNZP) is a benzodiazepine used to treat myoclonic seizures and generalized seizures. Melatonin (MEL), the pineal hormone, shows anticonvulsant activity in several animal models. To investigate new mechanisms related to the effects of these drugs, comparative study was conducted, from the analysis of the influence of pretreatment with LEV, CNZP or MEL on the oxidative stress and neuronal modulation of neurotransmitter systems (cholinergic and dopaminergic) during seizures induced by pilocarpine (400mg/Kg; P400). Male Swiss mice, 25-30g received LEV, 200 mg / kg, CNZP, 0.5 mg / kg or MEL, 25mg/kg, ip (doses chosen from the dose-response curves) 30min before P400. Hippocampus and striatum were removed for neurochemical analysis. In vitro experiments, where brain homogenates were incubated with drugs under study (50, 100 ou 200&#61549;g/mL) also allowed us to analyze changes in oxidative stress after induction of heat shock and also the density of muscarinic receptors in the hippocampus. Studies on the muscarinic modulation demonstrated that pretreatment with LEV, CNZP or MEL resulted in lower oxotremorina induced tremors and increased acetylcholinesterase activity in the hippocampus. LEV and CNZP altered the binding of hippocampal muscarinic receptors in vivo, reversing the P400-induced downregulation and in vitro tests showed changes in hippocampal muscarinic binding by previous incubation with LEV, CNZP or MEL. The binding assays also showed a downregulation of hippocampal D2 receptors in treated animals LEV, CNZP or pretreated with MEL before P400. Analyses to investigate the antioxidant activity of LEV and CNZP and role of antioxidative action of MEL in the protection against seizures propose that the association with vitamin E increased the anticonvulsant effects of all studied drugs. The prior administration of LEV, MEL or CNZP before P400, reversed the increased levels of lipid peroxidation and nitrite-nitrate and normalized the activity of catalase and the physiological levels of the antioxidant glutathione in the hippocampus and / or striatum. According to in vitro experiments, increased lipid peroxidation, levels of nitrite-nitrate and catalase activity in brain homogenates subjected to thermal shock were significantly altered by incubation with LEV, CNZP or MEL, where these drugs were able to reduce the levels of MDA, nitrite-nitrate, and also stabilize the activity of catalase, enhancing thus the endogenous antioxidant enzyme activity and the ability to inactivate free radicals.Thus, the study suggests a modulatory action exerted by LEV, CNZP and MEL on the functioning of muscarinic and dopaminergic systems in the central nervous system as an alternative mechanism to protect against seizures in the model P400, and the participation of direct and indirect antioxidant properties of these drugs, through the ability to modify the neuronal response to oxidative stress.

Patients with epilepsy often suffer debilitating cognitive and psycho-affective disorders.In some cases, epilepsy is associated to neurodegenerative processes that are the targetof certain therapeutical agents. Today, erythropoietin is considered as one of the most promising neuroprotective agents. In addition, an increased body of studies provides evidence that enrichment (or complexity) of housing decreases the cerebral vulnerabilityin the context of diverse brain insults. In this thesis, we demonstrate: 1) in a model ofepilepsy with large neuronal lesions, that erythropoietin protects the most vulnerable neuronal populations to excitotoxic injury, at the only condition that neuronal expression of its receptor is optimized prior to the primary insult causing epilepsy; 2) in a model of epilepsy associated with faint neuronal lesions that: i) erythropoietin prevents anxietyand impaired spatial learning and memory, ii) enriched housing in Marlau™ cages is moreefficient than erythropoietin, and iii) erythropoietin treatment abolishes beneficial effectsof enriched housing. These results, obtained in animal models of epilepsies associatedwith cognitive disorders establish that beneficial effects of a potential therapeutic agentmay rely on quality of life / Dans de nombreux cas, les patients avec des épilepsies présentent d’importants troubles cognitifs et psycho-affectifs. Ces épilepsies sont parfois accompagnées de phénomènes neuro-dégénératifs qui sont la cible de certains agents thérapeutiques. Aujourd’hui, l’érythropoïétine apparaît comme l’un des agents neuro-protecteurs les plus prometteurs.En outre, des études de plus en plus nombreuses montrent que l’enrichissement (ou la complexification) des conditions d’hébergement diminue la vulnérabilité cérébrale face à des agressions d’origine différente. Dans cette thèse, nous montrons : 1) dans un modèle d’épilepsie accompagné de vastes lésions neuronales, que l’érythropoïétine protège les populations neuronales les plus vulnérables aux atteintes excitotoxiques, à la condition d’optimiser l’expression neuronale de son récepteur, préalablement à l’agression cérébrale ayant initié l’épilepsie ; 2) dans un modèle d’épilepsie associé à des phénomènes neurodégénératifs très discrets, que : i) l’érythropoïétine bloque le développement de l’anxiété et des troubles de l’apprentissage et de la mémoire spatiale,ii) l’enrichissement du milieu d’hébergement en cages Marlau™ est plus efficace que l’érythropoïétine, et iii) le traitement par l’érythropoïétine annihile les effets bénéfiques de l’enrichissement du milieu d’hébergement. Les résultats de cette thèse, obtenus dans des modèles expérimentaux d’épilepsies associés à des troubles cognitifs, démontrent que les effets bénéfiques d’un potentiel agent thérapeutique peuvent dépendre de la qualité de vie