An in vitro model of microglia-induced neurotoxicity /
Microglia are the resident macrophages of the brain and spinal cord. Excessive inflammatory reactions by microglia lead to detrimental effects in the central nervous system (CNS). Thus, a model of microglia-induced neurotoxicity is important to develop as it provides a tool to explore the pathophysi...
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| Κύριος συγγραφέας: | |
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| Μορφή: | Thesis Βιβλίο |
| Γλώσσα: | English |
| Έκδοση: |
2017.
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| Ετικέτες: |
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| Περίληψη: | Microglia are the resident macrophages of the brain and spinal cord. Excessive inflammatory reactions by microglia lead to detrimental effects in the central nervous system (CNS). Thus, a model of microglia-induced neurotoxicity is important to develop as it provides a tool to explore the pathophysiology of neuroinflammation and a platform to test various anti-inflammatory therapies. We aimed to establish a culture model that represents microglia-induced neurotoxicity. In setting up this model, we addressed several important considerations including to use microglia stimulators that are more representative of generic inflammation, to ensure the microglia remain viable after stimulation and that there were no direct neurotoxic effects from the microglia stimulators, as well as the resulting neurotoxicity was not due to the lack of nutrients in the media. The first focus of the project was to develop the microglia stimulation model. The immortalised murine microglial cell line BV2 was treated with three different inflammatory stimuli, namely lipopolysaccharide (LPS; used as a positive control), interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α). LPS is a bacterial endotoxin and has been considered a gold standard for inducing microglia activation while IFN-γ and TNF-α both are proinflammatory mediators that represent an endogenous stimulation of inflammation. Microglia stimulation was evaluated by determine the level of nitric oxide (NO) and viability. It was observed that 0.2 and 0.1 x 10⁶ cells/well was an ideal seeding densities as they demonstrated good NO production coupled with high viability at particular time points. With this, the model of microglia-induced neurotoxicity was then set-up using a conditioned-media transfer approach. Briefly, media from stimulated BV2 cell cultures were transferred to N2a neuroblastoma cultures at a 1:1 ratio. MTS (3-(4,s-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrozolium inner salt) assay performed on N2a cells revealed a significant reduction in viability to 83 ± 0.007% 24 hours post conditioned-media transfer. Therefore, a model of microglia-induced neurotoxicity based on conditioned-media transfer from BV2 microglia stimulated with 10 ng/ml IFN-γ for 24 hours onto N2a cells appears to have been established. To further characterise this model, we will differentiate the N2a cells and evaluate the neuron viability using Annexin V/P1 (propidium iodide). |
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| Φυσική περιγραφή: | 75 leaves : ill. ; 30cm. |