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Exploring the activation of NLRP3 inflammasome by reactive oxygen species (ROS) in a model of epileptogenesis
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Abstract Epilepsy is a prevalent neurological disorder that affects 65 million people worldwide, according to the World Health Organization. It is characterized by the occurrence of seizures, due to an excessive and deregulated neuronal activity. In the last decades, many researches proved a correlation between epilepsy and neuroinflammation. Temporal lobe epilepsy, one of the most common and severe forms of epilepsy, affects mainly the hippocampus and is associated to inflammatory events that promote neuronal death and and increase in the expression of inflammatory markers. Interleukin (IL)-1β has a key role, promoting synaptic dysfunction, hyperexcitabillity and neuronal death. Taking this into account, neuroinflammation is considered a potential therapeutic target for epilepsy. Recently, an inflammatory mediator, the NLRP3 inflammasome was found to be related to the epileptogenic process. This cytoplasmic protein complex is composed of 3 domains: the sensor (NLRP3) that detects stress or infection signs, the adaptor, ASC, and the effector, Caspase-1 which ensures the conversion of pro IL-1β into IL-1β, when activated. For this epileptogenesis model, rhinal-cortex-hippocampus organotypic slices were used, in order to simulate hippocampus environmental features in vivo, as the neuronal connectivity and the synaptic plasticity, allowing the epileptiform activity. Previous studies showed NLRP3 domains expression with this model, as well as an increase in the expression of intracellular IL-1β throughout the culture. This work aims to explore reactive oxygen species (ROS) as potential activators of NLRP3 inflammasome, in this ex vivo model of epileptogenesis. Control (CTL) and epileptic-like (EL) slices were maintained under a serum reduction protocol throughout the time. Samples of CTL and EL organotypic slices were collected at 3, 7, 14 and 21 days in vitro (DIV), and assays of ROS quantification, immunoprecipitation and ELISA were performed. In the second phase of this project, slices were maintained in the presence of an antioxidant and its impact in the ROS production, NLRP3 inflammasome assembly and IL-1β release was later evaluated. ROS production was evaluated through a fluorescence detection assay. An immunoprecipitation assay, followed by a western blot analysis, was carried out in order to assess NLRP3 inflammasome assembly, throughout the detection of its components: NLRP3 protein and ASC. The secretion of the pro-inflammatory cytokine IL-1β was assessed by ELISA. All the assays were performed in samples of CTL and EL slices from all time points. After that, the process was repeated with an antioxidant treatment applied to the cultures. Statistical analysis was performed in GraphPad Prism with Two-way Analysis of Variance (ANOVA) and statistical significance was considered when p…
Descrição
Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2021
Palavras-chave
Epilepsia Inflamassoma NLRP3 Espécies Reativas de Oxigénio Neuroinflamação Stress Oxidativo Teses de mestrado - 2021