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Advisor(s)
Abstract(s)
A expectativa de vida aumentou consideravelmente nas últimas décadas. Relatórios das
Nações Unidas revelam que o número de pessoas com mais de sessenta anos de idade
está a aumentar mais rapidamente que em grupos etários mais jovens em todo o
mundo. O envelhecimento é um processo fisiológico, com diversos mecanismos que
podem desencadear efeitos cruciais no surgimento de doenças neurodegenerativas. A
principal característica do envelhecimento é a acumulação de células senescentes, no
qual, o stress oxidativo, está implicado diretamente. Diante da complexidade do
envelhecimento, é imprescindível a compreensão dos mecanismos moleculares
relativos a esse processo. Atualmente, os modelos experimentais utilizados para
investigar o envelhecimento cerebral são in vitro e in vivo, que por sua vez, trazem
algumas desvantagens. Como tal, o objetivo deste trabalho foi estabelecer um novo
modelo ex vivo de envelhecimento cerebral, utilizando o composto terc-butil
hidroperóxido (t-BHP) em culturas organotípicas de hipocampo de murganhos
C57BL/6J com 6 a 9 dias de idade. As culturas foram expostas a um tratamento subcrónico com t-BHP com o objetivo de induzir stress oxidativo, e consequentemente, a
senescência. A indução de senescência celular foi avaliada através do ensaio da ßgalactosidase, enquanto a viabilidade celular foi avaliada através do ensaio de lactato
desidrogenase (LDH). A expressão dos marcadores de senescência (SA-ß-gal), stress
oxidativo (SOD2), sobrevivência e atividade neuronal (NeuN e DCX), foram analisados
por Western blotting. Verificou-se que o t-BHP (10µM e 25µM) não induziu um
aumento na atividade da ß-galactosidase, sugerindo que este tratamento não foi capaz
de induzir senescência. Contudo, o tratamento com 25µM t-BHP induziu morte celular
avaliada através do ensaio LDH. Foi verificada uma tendência no aumento da
expressão de SOD2 (com a concentração de 25µM t-BHP), e quanto à sobrevivência e
dinâmica neuronal, não foram observadas alterações significativas. Em suma, os
resultados sugerem que a indução da senescência, nos parâmetros utilizados neste
estudo, não foi suficiente para o estabelecimento do modelo. Para se alcançarem
conclusões mais robustas, será necessário realizar-se estudos adicionais de mecanismos
de stress oxidativo e senescência celular em culturas organotípicas de hipocampo.
Life expectancy has increased considerably in recent decades. United Nations Reports reveal that the number of people over sixty years of age is increasing faster than younger age groups across the world. Aging is a physiological process, with several mechanisms that can trigger crucial effects in the etiology of neurodegenerative diseases. The main characteristic of aging is the accumulation of senescent cells. However, oxidative stress is directly implicated. Given the complexity of aging, it is necessary to understand the molecular mechanisms related to this process. Currently, the experimental models used to investigate brain aging are in vitro and in vivo, which in turn, bring some disadvantages. Therefore, the objective of this work was to establish a new ex vivo model of brain aging, using the compound tert-butyl hydroperoxide (tBHP) in organotypic hippocampal cultures of C57BL/6J mice aged 6 to 9 days. The cultures were exposed to a sub chronic treatment with t-BHP with the aim of inducing oxidative stress, and consequently senescence. The induction of cellular senescence was assessed using the ß-galactosidase assay, while cellular viability was assessed using the LDH assay. The expression of senescence markers (SA-ß-gal), oxidative stress (SOD2), survival and neuronal activity (NeuN and DCX), were assessed by Western blotting. It was found that t-BHP (10uM and 25µM) did not induce an increase in ß-galactosidase activity, indicating that this treatment was not capable of inducing senescence. However, treatment with 25µM t-BHP induced cell death assessed through the LDH assay. A trend of increased SOD2 expression was obtained at the concentration of 25µM and regarding neuronal survival and dynamics, no significant changes were observed. In short, the results suggest that the induction of senescence, in the parameters used in this study, were not sufficient to establish the model. To achieve more robust lessons, more studies of mechanisms of oxidative stress and cellular senescence in organotypic hippocampal cultures will be necessary.
Life expectancy has increased considerably in recent decades. United Nations Reports reveal that the number of people over sixty years of age is increasing faster than younger age groups across the world. Aging is a physiological process, with several mechanisms that can trigger crucial effects in the etiology of neurodegenerative diseases. The main characteristic of aging is the accumulation of senescent cells. However, oxidative stress is directly implicated. Given the complexity of aging, it is necessary to understand the molecular mechanisms related to this process. Currently, the experimental models used to investigate brain aging are in vitro and in vivo, which in turn, bring some disadvantages. Therefore, the objective of this work was to establish a new ex vivo model of brain aging, using the compound tert-butyl hydroperoxide (tBHP) in organotypic hippocampal cultures of C57BL/6J mice aged 6 to 9 days. The cultures were exposed to a sub chronic treatment with t-BHP with the aim of inducing oxidative stress, and consequently senescence. The induction of cellular senescence was assessed using the ß-galactosidase assay, while cellular viability was assessed using the LDH assay. The expression of senescence markers (SA-ß-gal), oxidative stress (SOD2), survival and neuronal activity (NeuN and DCX), were assessed by Western blotting. It was found that t-BHP (10uM and 25µM) did not induce an increase in ß-galactosidase activity, indicating that this treatment was not capable of inducing senescence. However, treatment with 25µM t-BHP induced cell death assessed through the LDH assay. A trend of increased SOD2 expression was obtained at the concentration of 25µM and regarding neuronal survival and dynamics, no significant changes were observed. In short, the results suggest that the induction of senescence, in the parameters used in this study, were not sufficient to establish the model. To achieve more robust lessons, more studies of mechanisms of oxidative stress and cellular senescence in organotypic hippocampal cultures will be necessary.
Description
Keywords
Culturas Organotípicas de
Hipocampo Nvelhecimento Senescência Celular Stress Oxidativo