Sivaram, Anjali
(2024)
Evaluating The effects of lipopolysaccharides and hydrogen peroxide on human and mouse astrocytes in vivo and in vitro, in relation to Alzheimer’s Disease.
MRes thesis, University of Nottingham.
Abstract
Alzheimer’s disease is a neurodegenerative disease characterised by gliosis, decreased neuronal cell viability, cognitive impairment, as well as increased amyloid beta levels, cytokine expression and oxidative stress. These symptoms have been seen in in vivo and in vitro studies that have utilised LPS, inflammatory endotoxins that are derived from gram-negative bacteria. Moreover, these are found at elevated levels co-localised with amyloid plaques and astrocytes in the brains of AD patients. Given its ability to mimic or exacberate such neuroinflammatory changes, and its relevance in AD pathology, this study looks at leveraging LPS to stimulate astrocytes in vivo using intranasal LPS, and in vitro, using the astrocytic SVG-A cells. These models can then be used in the future to test novel anti-inflammatory therapies against AD.
The first part of this study determined the efficiency of delivering LPS intranasally to the hippocampus of C57BL/6 mice. In this double-blind experiment, wild-type C57BL/6 mice were stimulated with 60 µg/ml of LPS given bilaterally in three doses over 24 hours. The effect of this LPS challenge on the hippocampal astrocyte population was analysed 14 days post-treatment, by quantifying and comparing the percentage area covered by DAB-labelled GFAP-positive astrocytes in LPS versus PBS treated mice.
The second part of this study investigated the effects of LPS in-vitro, by stimulating immortalised astrocytic SVG-A cells with 0 µg/ml to 10 µg/ml of LPS. The levels of pro-inflammatory IL-6 and IL-1β secreted after 3 hours were quantified by ELISA, and their cell viability after 24 hours was analysed using a MTT assay. Furthermore, the presence of key LPS receptors, TLR-4 and CD-14 receptors were visualised using immunocytochemistry.
In the last part, the effect of oxidative stress on the cell viability of astrocytes was also analysed. This was done by stimulating the SVG-A cells with 0 µM to 1000 µM of hydrogen peroxide, a pro-oxidant, using MTT assay.
The results showed no difference (p>0.999) in the total hippocampal area occupied by the DAB labelled GFAP-positive astrocytes in the mice treated intranasally with LPS (median = 4.617) or PBS (median = 4.979). In the in vitro studies, 0 µg/ml to 1µg/ml of LPS did not affect the cell viability of the astrocytes. While the SVG-A cells did express TLR-4 and CD-14 receptors, it was seemingly hyporesponsive to LPS. Only a maximum dose of LPS at 10 µg/ml resulted in a significant increase in the IL-6, however, even at this dose there were no detectable levels of IL-1β that were secreted. Alternatively, stimulating the SVG-A cells with hydrogen peroxide dose-dependently decreased their cell viability.
The results indicate that the intranasal administration of LPS did not have any long-term effects on mouse astrocytes. Further studies are required to see if LPS can be delivered successfully into the hippocampus using this protocol. The in vitro stimulation of SVG-A cells with LPS indicates that the SVG-A cells are hypo-responsive to LPS. In contrast, hydrogen peroxide might be a better stimulator in these cells. In conclusion, the SVG-A cells cannot be used with LPS to study AD, rather they are a better model to study hydrogen peroxide-induced oxidative toxicity, in relation to AD.
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