Smith, Andrew John
Excitotoxicity, oxidative stress and neuroprotection in cerebellar granule neurones.
PhD thesis, University of Glasgow.
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Neuronal death due to excitotoxicity and oxidative stress is a critical part of several major disease processes, including ischaemic brain damage and Alzheimer’s disease. This study used cultures of cerebellar granule neurones as a model for investigation of these processes, considering both their pharmacological and molecular aspects. Another important part of the study was the development and investigation of a mode of neuroprotection which was effective in protecting against these factors. Additionally, the optimal culturing conditions for neurone survival were determined and the efficacy of two cell viability assays established.
Examination of excitotoxicity and oxidative stress considered the effects of glutamate, N-methyl-D-aspartate, 3-nitropropionic acid and oxygen-glucose deprivation. Additionally, extensive study was carried out of the actions of increased glucose concentration and a range of metabolites of the essential amino acid tryptophan. It was demonstrated that several tryptophan metabolites induced neurotoxic effects, including 5-hydroxyanthranilic acid, which caused neuronal death via oxidative damage mediated by generation of reactive oxygen species and prevented by catalase but not superoxide dismutase. 5-Hydroxyanthranilic acid treatment also led to activation of the p38 signalling pathway, although the cell death caused was independent of caspase-3 activation.
Investigation of neuroprotection was concerned with establishing an effective method of protection, with a range of stimuli used to precondition neurones, such as N-methyl-D-aspartate, 3-nitropropionic acid, hydrogen peroxide, bicuculline and 4-aminopyridine. Preconditioning with 100µM N-methyl-D-aspartate at 8 DIV was effective in protecting against the neurotoxic effects of glutamate or 3-nitropropionic acid applied 24 hours after the commencement of preconditioning, but was not effective against oxygen-glucose deprivation of 4 hours duration. Preconditioning with 2.5mM 4-aminopyridine was effective in providing protection against a range of insults (glutamate, N-methyl-D-aspartate, 3-nitropropionic acid). This protection was independent of N-methyl-D-aspartate receptor activation, reduced by blockade of depolarisation and was effective in protecting against caspase-3-independent cell death.
||Results from one chapter previously published in: Smith AJ, Stone TW, Smith RA (2008). Preconditioning with NMDA protects against toxicity of glutamate or 3-nitropropionic acid in cultured cerebellar granule neurones. Neuroscience Letters, in press, doi:10.1016/j.neulet.2008.05.066
||excitotoxicity, oxidative stress, neuroprotection, preconditioning, cerebellar granule neurones, neuroscience, neurotoxicity
||College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
||Stone, Prof Trevor W and Smith, Prof Robert A
|Date of Award:
Dr Andrew J Smith
||Copyright of this thesis is held by the author.
||24 Jun 2008
||10 Dec 2012 13:17
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