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In vitro cell culture to study microglial inflammation

Inflammation of the central nervous system (CNS) characterised by release of cytokines such as tumour necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1) is an event that is often detrimental to neuronal cells and is thought to contribute to the neuronal loss observed during neurodegeneration. One objective of this study was to investigate the potential anti-inflammatory properties of kawakawa leaf extracts using in vitro model cell systems. Kawakawa leaf extracts anti-inflammatory properties have anecdotally been documented in traditional Maori medicinal practices (Rangoa). However, before this aspect could be carried out, experiments were undertaken to investigate the effects the kawakawa leaf extracts had on the growth rates of two mammalian cell lines (HeLa and THP-1 cells). Interestingly, the growth assays showed that the extracts to have both a stimulatory and inhibitory effect on cell growth. Toxicity of cells can be categorised as either necrotic or apoptotic cell death. In this study, HeLa cells and THP-1 cells were incubated under standard growth conditions with various concentrations (10 to 100ug/ml) of Kawakawa leave extracts prepared at room temperature or 60OC. Growth data as seen in figures 2 and 3 typically have a long lag phase before they start to reach exponential growth between days 4 and 5. The extract having the largest stimulatory effect on HeLa cells as shown by figure 6 was 25 ug/ml at 60OC and the largest inhibitory effect seen on HeLa cells was seen at 50 ug/ml RT shown by figure 6. The extract which has the largest stimulatory effect on THP-1 cells is 25 ug/ml 60OC as shown by figure 11 whereas the extract producing the largest inhibitory effect is 25 ug/ml RT. Apoptotic and necrotic cell death mechanisms were examined by using in vitro biochemistry techniques. Assessments were largely made based on biochemical changes of HeLa cells and THP-1 cells post treatment of Kawakawa leave extracts. Measurement of lactate dehydrogenase (LDH assays) in the growth media of cells that were shown to be growth inhibited during exposure to Kawakawa extracts indicated that the growth inhibition resulting from exposure to the extracts was not due to necrotic cell death processes. (ie that cells maintained their membrane integrity) When compared to the control, a lower amount of cytosolic LDH was seen in THP-1 cell lines. However, HeLa cell lines produced a high level of cytosolic LDH which may be due to mycoplasma contamination. On average, Kawakawa concentrations at 12.5ug/ml and 25ug/ml yielded less lysed cells percentage across both cell lines. The demonstrated cell growth inhibition was unlikely to be due to necrosis and because of this it was decided to see if cell death was caused by mitochondrial processes that would further support apoptotic cell death. The biochemical assay MTT indirectly measures mitochondrial dehydrogenase activity in the presence of kawakawa leave extracts. The MTT data illustrated mitochondrial impairment where HeLa cells exposed to Kawakawa 60OC concentrations 6.25 ug/ml and 12.5 ug/ml had the lowest average mitochondrial activity. THP-1 cells exposed to Kawakawa 60OC concentrations 12.5 ug/ml and 50 ug/ml had the lowest average mitochondrial activity. The main results of this study include, the treatment of HeLa and THP-1 cells with kawakawa extracts demonstrated that cytotoxicity was not due to necrosis. The most interesting finding of this study is when kawakawa extracts inhibit cell growth there is no increase in LDH. This is suggestive that the cellular membrane remains intact when exposed to the extract.
Type of thesis
Newland, J. (2016). In vitro cell culture to study microglial inflammation (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/12053
University of Waikato
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