Zugl.: Berlin, Humboldt-Universiẗat, Diss., 2004.

<p>The goal of this study is to use a rat model of acquired temporal lobe epilepsy, pilocarpine-induced status epilepticus (SE), to test the hypothesis that WD exacerbates the inflammatory consequences of SE in hippocampus of male rats. Testing this hypothesis in animals consuming WD from early in life may provide insight into how a modifiable environmental factor could influence the development of seizure disorders. </p>

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-22T12:07:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_PricilaTolioMuller.pdf: 1581572 bytes, checksum: 2ee45bab6500d240c98b9a12ad2e330d (MD5) / Made available in DSpace on 2018-08-22T12:07:03Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_PricilaTolioMuller.pdf: 1581572 bytes, checksum: 2ee45bab6500d240c98b9a12ad2e330d (MD5) Previous issue date: 2018-04-20 / Epilepsy is characterized by a cerebral disorder with interruptions, recurrent and unpredictable of normal brain function. Glutamate is the major excitatory neurotransmitter in the brain and is involved in the mechanism of status epilepticus - a prolonged and self-sustaining seizure. The drug N-methyl-D-aspartate (NMDA) is a synthetic excitotoxin that acts as a specific agonist for the NMDA receptor subtype of glutamate. Pre- and post-conditioning strategies provide a proposal for molecular mechanisms responsible for endogenous neuronal protection. The aim of the study is evaluating the effect of NMDA in different doses against damage following status epilepticus induced by pilocarpine in animal model of epilepsy. For this purpose, 210 adult male mice (60 days, 30-40 g) Swiss albino were treated intraperitoneally with pilocarpine (100 mg/kg every 20 min up to a maximum of 300 mg/kg) evoking status epilepticus. All animals were exposed to behavioral tasks for motor evaluation (locomotion, balance, motor coordination) and memory, followed by evaluation of cellular viability at 30 days post-pilocarpine. The pilocarpine death in approximately 40% of the mice during 24 hours. Treatment with lower doses of NMDA (18 mg/kg) prolonged survival time without change the outcome. The other NMDA doses also had no significant effect on the number of deaths. The status epilepticus caused reduction of the locomotor activity of the mice, an effect reversed by treatment with NMDA 37 mg/kg. The pilocarpine group mice did not show expressive cognitive deficit during acquisition of memory, however reduced evocation. Cellular viability assessed 30 days after status epilepticus indicated damage by MTT test, but not by propidium iodide. Only the 18 mg/kg NMDA reduced damage. It is concluded that the dose 18 mg/kg NMDA induced neuroprotection in surviving animals. The cellular mechanisms of NMDA-induced tolerance remain unknown, but the present study reinforces the dual NMDA effect, since the same dose that increased the mortality rate was able to reduce cellular damage. / A epilepsia é caracterizada por uma desordem cerebral com interrupções, recorrentes e imprevisíveis da função cerebral normal. O glutamato é o principal neurotransmissor excitatório no cérebro e está envolvido no mecanismo de status epilepticus - estado de mal epiléptico, convulsões espontâneas. O fármaco N-metil-D-aspartato (NMDA) é uma excitotoxina sintética que age como agonista específico do subtipo de receptor NMDA do glutamato. As estratégias de pré- e pós-condicionamento fornecem uma nova proposta de mecanismos moleculares responsáveis pela proteção neuronal endógena. Diante do exposto, neste trabalho avaliamos o efeito do NMDA em diferentes doses contra danos decorrentes do status epilepticus induzido por pilocarpina em modelo animal de epilepsia. Para tal, foram utilizados 210 camundongos machos adultos (60 dias, 30-40g) da linhagem albino Swiss que foram tratados pela via intraperitoneal com pilocarpina (100 mg/kg a cada 20 min até o máximo de 300 mg/kg) para a indução do status epilepticus. Todos os animais foram submetidos a tarefas comportamentais para avaliação motora (locomoção, equilíbrio, coordenação motora) e memória, seguidas de avaliação de viabilidade celular em 30 dias pós-pilocarpina. O modelo de epilepsia induzido por pilocarpina induziu morte de aproximadamente 40% dos animais em 24 horas. O tratamento com a menor doses de NMDA (18 mg/kg) aumentou o período de sobrevivência dos animais, mas não modificou o desfecho. As demais doses de NMDA também não tiveram efeito significativo no número total de mortes. O status epilepticus causou redução da atividade locomotora dos camundongos, efeito revertido pelo tratamento com NMDA 37 mg/kg. O grupo pilocarpina não demonstrou expressivo déficit cognitivo na aquisição da memória, mas sim na evocação. A avaliação tardia, aos 30 dias, da viabilidade celular indicou efeito danoso da pilocarpina no teste do MTT, com proteção significativa de NMDA 18 mg/kg. Conclui-se que o NMDA na dose de 18 mg/kg nos animais sobreviventes induziu neuroproteção. Os mecanismos celulares da tolerância induzida por NMDA permanecem desconhecidos, mas o presente estudo reforça a função dual do NMDA, pois a mesma dose que aumentou a taxa de mortalidade foi capaz de reduzir o dano celular.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

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.

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.

Submitted by Marta Toyoda (1144061@mackenzie.br) on 2017-01-06T16:21:47Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Ana Miriã Pacifico.pdf: 777682 bytes, checksum: 45b0329e3b048868550b9ecf6fd57735 (MD5) / Approved for entry into archive by Paola Damato (repositorio@mackenzie.br) on 2017-01-06T19:01:15Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Ana Miriã Pacifico.pdf: 777682 bytes, checksum: 45b0329e3b048868550b9ecf6fd57735 (MD5) / Made available in DSpace on 2017-01-06T19:01:15Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Ana Miriã Pacifico.pdf: 777682 bytes, checksum: 45b0329e3b048868550b9ecf6fd57735 (MD5) Previous issue date: 2016-08-18 / Fundo Mackenzie de Pesquisa / Experimental studies show evidence that rats subjected to neonatal seizures have morphological and cognitive changes, also exhibiting autistic behavior characterized by low preference for social novelty, social discrimination deficits and behavior type anxious. However, the mechanisms that account for these changes are not yet known. Several lines of evidence show that Oxytocin (OT) is present in key regions for social recognition, such as the medial amygdala and the hippocampus. We postulate that the deficiency in signaling mediated by OT on neural network related to the social brain responds by impaired sociability and increased emotionality subsequent neonatal seizure. This study aims to assess the social recognition memory and gene expression of OT and its receptor (OTR) in animals with neonatal status epilepticus. Wistar rats were adult males submitted to status epilepticus on the ninth day of life (P9) the administration of pilocarpine (350 mg / kg, ip) and the controls received saline 0.9% (0.1 ml / 10 g). In P90 social memory was evaluated by habituation/deshabituation paradigm. At the end of behavioral testing, the animals were anesthetized and decapitated to remove the structures of study (amygdala, hippocampus, and hypothalamus). Thereafter it was performed gene expression analysis of oxytocin and its receptor in the aforementioned tissues by real-time PCR. In social memory test experimental animals had shorter social research, injury indicative of the motivation system / reward and loss of habituation / deshabituation, suggesting impairment in social recognition memory. The gene expression of oxytocin did not differ between the groups in the analyzed structures, but there was a small decrease in gene expression of oxytocin receptor in the hippocampus. Thus concludes the neonatal status epilepticus in rats produces memory deficits in social recognition and motivation /mesolímibic reward systems. This work demonstrated that animals subjected to neonatal status epilepticus have reduced exploration of social novelty, suggesting impairment in motivation system / mesolimbic reward and social recognition memory that may be related to reduced expression of oxytocin receptors in the hippocampus. / Estudos experimentais mostram evidências de que ratos adultos submetidos às convulsões neonatais apresentam mudanças morfológicas e cognitivas, exibindo também comportamento autista caracterizado pela baixa preferência pela novidade social, déficit de discriminação social e comportamento tipo ansioso. Porém, os mecanismos que respondem por estas alterações ainda não são conhecidos. Várias evidências demonstram que a ocitocina (OT) está presente em regiões importantes para reconhecimento social, tais como a amígdala medial e o hipocampo. Postulamos que a deficiência na sinalização mediada pela OT na rede neural relacionada com o cérebro social responda pelo prejuízo na sociabilidade e pelo aumento da emocionalidade subsequente à convulsão neonatal. Este trabalho tem como objetivo avaliar a memória de reconhecimento social e a expressão gênica da OT e do seu receptor (OTR) em animais submetidos ao status epilepticus neonatal. Foram utilizados ratos Wistar machos adultos submetidos ao status epilepticus no nono dia de vida (P9) pela administração da pilocarpina (350 mg/kg, ip) e os controles receberam salina 0.9% (0,1 mL/10 g). Em P90 foi avaliada a memória social pelo paradigma de habituação/desabituação. Ao fim dos testes comportamentais, os animais foram anestesiados e decapitados para a retirada das estruturas de estudo (amígdala, hipocampo, e hipotálamo). Posteriormente foi realizada a análise da expressão gênica da ocitocina e seu receptor nos tecidos citados, por meio da PCR em tempo real. No teste de memória social os animais experimentais apresentaram menor tempo de investigação social, indicativo de prejuízo no sistema de motivação/recompensa e prejuízo de habituação/desabituação, sugestivo de prejuízo na memória de reconhecimento social. A expressão gênica da ocitocina não diferiu entre os grupos nas estruturas analisadas, mas observou-se uma pequena redução na expressão gênica do receptor de ocitocina no hipocampo. Com isso conclui-se o status epilepticus neonatal em ratos produz déficit na memória de reconhecimento social e do sistema de motivação / recompensa mesolímibico. O presente trabalho mostrou que animais submetidos ao status epilepticus neonatal apresentam redução da exploração da novidade social, sugestivo de prejuízo no sistema de motivação/recompensa mesolímbico e da memória de reconhecimento social que pode estar relacionada a redução da expressão dos receptores da ocitocina no hipocampo.

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Introdução: A vigabatrina (VGB - Gama-Vinil-GABA) é um fármaco que eleva os níveis de GABA no organismo, por inibição irreversível da GABA transaminase, cuja eficácia foi bem demonstrada no controle dos espasmos epilépticos em lactentes, especialmente na síndrome de West secundária à esclerose tuberosa. Há escassez de estudos clínicos evidenciando um possível papel deste fármaco no controle de crises epilépticas neonatais e pouco se sabe sobre o potencial impacto do seu uso nessa faixa etária, seus possíveis efeitos adversos, ou se sua introdução teria associações positivas com controle mais adequado das crises na evolução e melhor desenvolvimento neuropsicomotor da criança. A VGB foi introduzida em nosso serviço como terapia de adição para o controle de crises neonatais refratárias, há vários anos, instigando nossa impressão sobre a eficácia deste medicamento no período neonatal. Objetivos: Avaliar a efetividade do uso da VGB como adjuvante no controle das crises eletrográficas e eletroclínicas do período neonatal e seus efeitos sobre o padrão do eletroencefalograma (EEG); Avaliar a evolução clínica e eletrográfica das crianças durante seguimento ambulatorial; Pesquisar associação entre \"controle de crises neonatais com introdução de VGB\" e diversas características demográficas, clínicas e evolutivas destes recém nascidos; Quantificar e caracterizar a ocorrência de efeitos adversos precoces e durante o seguimento. Pacientes e métodos: Estudo transversal retrospectivo, envolvendo o levantamento dos prontuários de uma amostra de recém-nascidos que receberam VGB como tratamento para crises neonatais refratárias aos fármacos convencionais e status epilepticus, no período de janeiro de 2007 a março de 2014, no Serviço de Neonatologia e Terapia Intensiva Neonatal do HCFMRP-USP, mantendo seguimento ambulatorial por pelo menos 1 ano. Foram avaliados os dados demográficos, etiologia e semiologia clínico-eletroencefalográfica das crises, esquema terapêutico prescrito, indicação da introdução da VGB, tempo de internação e tempo para atingir o controle das crises, evolução clínica e eletrencefalográfica durante a internação e no seguimento ambulatorial, época da suspensão da VGB, além de seus efeitos adversos. Resultados: De 48 recém-nascidos avaliados, 34 (79,2 %) obtiveram controle de crises eletrográficas e/ou clínicas durante o período neonatal, havendo melhora no padrão eletrográfico após a introdução da VGB em 79%. Quanto aos critérios para sua indicação, 33,3% (16 indivíduos) iniciaram VGB devido a falha terapêutica no controle das crises com fenobarbital e/ou fenitoína; 27,1% (13 recém nascidos), pela presença de estado de mal epilético e, em 12 crianças (25%), por falha terapêutica do midazolam. Ao final do primeiro ano de vida, a atividade de base do EEG mostrou-se desorganizada em 58,1% (18 de 29 pacientes que o realizaram aos 12 meses de vida). No seguimento ambulatorial de 38 pacientes, algum grau de atraso do desenvolvimento neuropsicomotor foi detectado em 20 crianças (52,6%); 19 lactentes (39,5%) mantiveram o uso da VGB em politerapia, tendo 22 crianças (57,9%) evoluído com persistência das crises epilépticas. Já 37,8% (14 pacientes) enquadraram-se em um padrão de encefalopatia epiléptica, que correspondeu à síndrome de West em 13,9% (5 de 36 crianças). Quanto ao EEG realizado em 34 crianças nessa fase, 17,6% (6 casos) demonstraram a presença de hipsarritmia, enquanto anormalidades focais ou multifocais foram detectadas em 50% (17 lactentes). A taxa de óbito ao final do primeiro ano foi de 23,3% (10 de 43 crianças analisadas quanto a este dado). Não foi possível comprovar déficit visual relacionado diretamente ao uso da VGB. A variável \"controle de crises no período neonatal com o uso da VGB\" foi associada aos seguintes desfechos clínicos favoráveis: melhora no padrão eletrográfico (92,1%), proporção menor de crianças evoluindo para síndrome de West e outras encefalopatias epilépticas (71,9% não tiveram tal desfecho); menor frequência de hipsarritmia no EEG (92,9% sem hipsarritmia), maior alcance de desenvolvimento neuropsicomotor normal (56,2% com bom desenvolvimento neurológico), menor índice de óbito neonatal (97,4% vivos nesta fase) e durante os primeiros doze meses de vida (87,9%). Conclusão: Acreditamos que a VGB seja uma opção terapêutica efetiva e com adequada relação custo-benefício, a ser implementada no controle de crises epilépticas neonatais refratárias como fármaco adjuvante aos convencionais. Entretanto, estudos randomizados e controlados são necessários para confirmar sua eficácia quando comparada a outros medicamentos disponíveis para uso nesta população, bem como para avaliar seus possíveis efeitos adversos a longo prazo. / Introduction: Vigabatrin (VGB - Gama-Vinil-GABA) is an antiepileptic drug which increases systemic GABA levels by irreversibly inhibiting GABA transaminase, with well demonstrated efficacy in the control of infantile epileptic spasms, specially related to West syndrome due to tuberous sclerosis. Clinical studies demonstrating a possible role of VGB in the control of neonatal seizures are still very scarce and very little is known on the impact of its use at this early age, as well as on its possible side effects or eventual positive associations from its use with more adequate seizure control or better neuropsychomotor development in the outcome. VGB has been used in our service as an add-on therapy for refractory neonatal seizures arising the impression that this could be an effective antiepileptic medication in the neonatal period. Objectives: To evaluate the use of VGB as an add-on medication regarding its effectiveness for the control of neonatal electrographic and electroclinical seizures, as well as its effects over the EEG pattern; To evaluate clinical and electrographic evolution of the children in follow-up; To estimate VGB efficacy on the control of neonatal seizures in relation to the demographical and clinical characteristics of those newborns; To quantify and characterize the occurrence of early and late side effects of this medication along follow-up. Patients and methods: This is a transverse retrospective study carried out through charts analysis from a sample of newborns who received VGB as add-on medication for seizures and/or status epilepticus refractory to conventional drugs, from January 2007 through March 2014, at the Neonatal Intensive Care Service of HCFMRP-USP, keeping follow-up in our institution for at least 1 year. Demographical and etiological data were analyzed, as well as clinical-electrographical semiology, VGB prescription indication, therapeutic schedule, time to reach seizure control, clinical and electrographical evolution while in hospital and at the follow-up, age at VGB withdrawal, besides adverse effects. Results: Among 48 newborns evaluated, 34 (79.2%) reached control of electrographic and/or clinical seizures during neonatal period, with improvement of the EEG pattern after VGB introduction in 79%. As for drug introduction criteria, 33.3% (16 children) were started on VGB due to therapeutic failure of phenobarbital and/or phenytoin; 27.1% (13 newborns), due to status epilepticus and, in 12 babies (25%), due to therapeutic failure of midazolam. By the end of the first year of life, EEG background activity was disorganized in 58.1% (18 out of 29 children who had EEG registered at 12 month of life). Along the one year follow-up of 38 patients, 20 infants (52.6%) showed some degree of neurodevelopmental delay; 19 children (39.5%) remained on VGB in polytherapy, with seizure persistence in 22 (57.9%). Evolution to an epileptic encephalopathy was found in 14 kids (37.8%), with West Syndrome being characterized in 13.9% (5 out of 36 kids). As for the EEG carried out in 34 children at the follow-up, 17.6% (6 cases) showed hypsarrhythmia while focal or multifocal abnormalities were seen in 50% (17 infants). Up to 12 month of life, the death rate was 23.3% (10 out of 43 children evaluated for such endpoint). Visual deficit directly related to VGB use could not be determined. The variable \"seizure control during the neonatal period after VGB use\" was associated to the following endpoints: improvement of the EEG pattern (92,1% of children with seizure control after VGB), lower proportion of children evolving into West syndrome and other epileptic encephalopathies (71.9% did not show such endpoint), lower frequency of hypsarrhythmia in the EEG (92.9% without hypsarrhythmia), better milestones reached regarding neuropsychomotor development (56.2% with good neurological outcome), lower rate of neonatal death (97.4% alive by the end of neonatal period) and along the first year of life (87.9%). Conclusion: VGB is an effective therapeutic option with adequate cost-benefit relationship which should be implemented for the control of refractory neonatal seizures as add-on therapy to conventional drugs. However, controlled randomized studies are necessary to confirm such efficacy as compared to other drugs available for use in the neonatal period, as well as to evaluate its possible long term side effects.