O Mapeamento do padrão de expressão de c-Fos e definição de seu período refratário no cérebro de ratos e macacos (Callithrix jacchus) após estimulação com pentilenotetrazol (PTZ)
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Data
2014
Autores
Barros, Vanessa Novaes [UNIFESP]
Orientadores
Mello, Luiz Eugênio Araújo de Moraes [UNIFESP]
Tipo
Dissertação de mestrado
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Introdução: Quando um neuronio e ativado de forma intensa, desencadeia-se o aparecimento de genes de expressao imediata, dentre eles o c-fos. Este gene codifica uma proteina nuclear c-Fos, relacionada a diversas cascatas de sinais envolvidos em importantes processos bioquimicos como plasticidade neuronal, crescimento celular e mitose. Objetivos: Neste trabalho investigamos o padrao de expressao e do periodo refratario de c-Fos no cerebro de ratos e macacos apos estimulacao com pentilenotetrazol. Materiais e metodos: Foram utilizados 40 ratos (Wistar) e 40 macacos (Callithrix jacchus) sacrificados em diferentes tempos apos crise convulsiva. Os grupos para o mapeamento do padrao de expressao de c-Fos foram: grupo controle (animais sem crise); grupo PTZ 0,5h; 1, 2, 3, 6, 9 e 12h. Ja os grupos para o mapeamento do periodo refratario de c-Fos foram: PTZ1h/1h, PTZ3h/1h e PTZ6h/1h em ratos e PTZ6h/3h, PTZ 9h/3h e PTZ12h/3h nos macacos onde a primeira hora corresponde ao momento da aplicacao da 2ª dose de PTZ (apos a 1ª crise), e a 2ª hora correspondendo ao momento em que os animais foram sacrificados apos a 2ª crise. Foi feita analise do gene e proteina c-fos por meio do PCR em tempo real e imunohistoquimica no cortex motor, piriforme e cingulado. Resultados: Tantos ratos quanto macacos apresentaram inicio de expressao de c-Fos em 0,5h apos crise. No entanto, o padrao de expressao proteica entre esses animais foram distintos: em ratos, o pico de expressao se deu entre 1h e 2h apos a crise, retornando aos niveis basais apos 6h em todas as regioes. Nos macacos o pico de expressao foi de 1h a 3h apos a crise e retorno aos niveis basais de 9 a 12h. Em relacao ao mapeamento do periodo refratario de c-Fos, observamos: nos ratos um periodo refratario de expressao proteica com duracao de ate 6h, enquanto que os macacos exibem refratariedade em ate 12h apos o primeiro estimulo. Porem, nao houve diferenca de expressao genica de c-fos entre esses grupos, demonstrando um controle pos-transcricional do mecanismo de inducao da refratariedade.Conclusao: A diferenca do padrao de expressao da proteina nos roedores e primatas demonstra um aspecto funcional da bioquimica cerebral diferenciada entre as especies que pode nos ajudar a entender a maior complexidade cognitiva dos primatas em relacao aos roedores em vista da ampla relacao de c-Fos com tarefas cognitivas e de aprendizagem
Introduction: Intense activation of neurons triggers the appearance of immediate genes, including c-fos. This gene encodes a nuclear protein, c-Fos, that isrelated to various signal cascades involved in biochemical processes such as neuronal plasticity, cell growth and mitosis. Objective: Here we investigated the expression pattern and the refractory period of c-Fos in rats ́ and monkeys ́ brains, after stimulation with pentylenetetrazol. Methods: 55 rats (Wistar) and 50 monkeys (Callithrix jacchus) were sacrificed at various times after PTZ - induced seizure. The groups for mapping the c-fos expression pattern were: control group (animals without seizure); PTZ group: 0.5 h, 1, 2, 3, 6, 9 and 12h. The groups for mapping the c-fos refractory period were: PTZ1h/1h, PTZ3h/1h and PTZ6h/1h in rats and PTZ6h/3h, PTZ 9h/3h and PTZ12h/3h in monkeys. The first hour refers to the application time of the 2nd dose of PTZ after the first seizure and the 2nd hour refers to the time when the animals were sacrificed after the 2nd seizure. RNA and protein expression were assessed by real- time PCR and immunohistochemistry in motor, cingulate and piriform cortex. Results: Rats and monkeys already showed c-Fos expression at 0.5 h after seizure. Yet, the pattern of protein expression differed between rats and monkeys: in rats, the expression peaked in 1 to 2h after the seizure, returning to baseline in 6 hours in all regions. In monkeys, the expression peaked in 1 to 3h after the seizure and returned to basal levels only in 9 to 12h. Regarding the refractory period of c-Fos, we observed: in rats, a refractory period of protein expression during up to 6h, while monkeys exhibit refractoriness within 12 hours after the first stimulus. However, there were no differences in c-fos gene expression among these groups, which indicates a post - transcriptional control mechanism underlying the refractory period. Conclusion: The difference in the protein expression pattern in rodents and primates characterizes a functional aspect of brain biochemistry between these orders and my contribute for the more developed primate cognitive complexity as compared to rodents because of the relation of c-Fos with cognitive and learning tasks.
Introduction: Intense activation of neurons triggers the appearance of immediate genes, including c-fos. This gene encodes a nuclear protein, c-Fos, that isrelated to various signal cascades involved in biochemical processes such as neuronal plasticity, cell growth and mitosis. Objective: Here we investigated the expression pattern and the refractory period of c-Fos in rats ́ and monkeys ́ brains, after stimulation with pentylenetetrazol. Methods: 55 rats (Wistar) and 50 monkeys (Callithrix jacchus) were sacrificed at various times after PTZ - induced seizure. The groups for mapping the c-fos expression pattern were: control group (animals without seizure); PTZ group: 0.5 h, 1, 2, 3, 6, 9 and 12h. The groups for mapping the c-fos refractory period were: PTZ1h/1h, PTZ3h/1h and PTZ6h/1h in rats and PTZ6h/3h, PTZ 9h/3h and PTZ12h/3h in monkeys. The first hour refers to the application time of the 2nd dose of PTZ after the first seizure and the 2nd hour refers to the time when the animals were sacrificed after the 2nd seizure. RNA and protein expression were assessed by real- time PCR and immunohistochemistry in motor, cingulate and piriform cortex. Results: Rats and monkeys already showed c-Fos expression at 0.5 h after seizure. Yet, the pattern of protein expression differed between rats and monkeys: in rats, the expression peaked in 1 to 2h after the seizure, returning to baseline in 6 hours in all regions. In monkeys, the expression peaked in 1 to 3h after the seizure and returned to basal levels only in 9 to 12h. Regarding the refractory period of c-Fos, we observed: in rats, a refractory period of protein expression during up to 6h, while monkeys exhibit refractoriness within 12 hours after the first stimulus. However, there were no differences in c-fos gene expression among these groups, which indicates a post - transcriptional control mechanism underlying the refractory period. Conclusion: The difference in the protein expression pattern in rodents and primates characterizes a functional aspect of brain biochemistry between these orders and my contribute for the more developed primate cognitive complexity as compared to rodents because of the relation of c-Fos with cognitive and learning tasks.
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BARROS, Vanessa Novaes. O Mapeamento do Padrão de Expressão de c-Fos e Definição de seu Período Refratário no Cérebro de Ratos e Macacos (Callithrix jacchus) Após Estimulação com Pentilenotetrazol (PTZ). 2014. 89 f. Dissertação (Mestrado em Ciências) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2014.