An investigation into the role of tumour necrosis factor-a in ischaemic neuronal damage in-vitro

Wilde, Geraint John Colston (1997) An investigation into the role of tumour necrosis factor-a in ischaemic neuronal damage in-vitro. University of Southampton, Doctoral Thesis.

Abstract

We have developed an in-vitro model of ischaemia related neuronal damage using organotypic hippocampal slice cultures. We established that this culture system provided us with an in-vitro model closely resembling the in-vivo situation following cerebral ischaemia, without the complication of a BBB. We witnessed a reduction in neuronal damage, provided TNF-α was added 24 hours prior to the onset of combined oxygen and glucose deprivation, but in contrast TNF-α exacerbated neurodegeneration when added following the same insult, without prior exposure to the cytokine. We established through which mechanism TNF-α mediated this dual effect, focusing on glutamate toxicity and excessive levels of free radicals. TNF-α had little effect on the excitotoxic component of ischaemic damage, affording slight protection whether present before or after the insult. In investigating a suitable model for free radical generation, we utilized free intracellular iron, so producing the hydroxyl radical, and developed a new neuronal model using duroquinone, a mitochondrial electron transport chain inhibitor, to generate the superoxide anion. In using the organotypic hippocampal cultures, grossly retaining their in-vivo stoichimetry, we discovered that iron-induced radical generation does not mimic that of ischaemia, producing a selective lesion of the CA3 hippocampal subfield. In contrast, duroquinone induced neurodegeneration specifically within the CA1 subfield comparable to an ischaemic insult. In accordance with these observations, TNF-α was found to have the same pre- and post-insult actions with generated superoxide, as those witnessed in the combined hypoxia/hypoglycaemia, but differed with the hydroxyl radical-mediated damage. Both a sublethal dose of duroquinone, and TNF-α, were found to induce the endogenous superoxide radical scavenger, manganese superoxide dismutase (MnSOD), a protein believed to protect cells against oxidative damage when elevated above normal levels. It is our conclusion that TNF-α co-ordinates a damage limitation response following ischaemia.

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