Cellular Effects of Cerebral Ischaemia In Vitro’. Cerebroprotective Actions of GABAmimetic agents

Stroke is a major cause of death and disability, with more than 80% of all strokes being the result of cerebral ischaemia. There is substantial evidence that during an ischaemic insult extracellular glutamate levels dramatically increase and this increase is strongly implicated in neuronal death. Decreased function of inhibitory mechanisms may also contribute to ischaemia-induced cell death. The mechanisms involved in ischaemia-induced glutamate efflux are contentious so a major aim of this study was to develop an in vitro model of ischaemia to examine the factors involved in the glutamate efflux and also to study the actions of selected GABAmimetics in order to explain why some GABAmimetic agents are neuroprotective but others are not. In parallel electrophysiological studies, the actions of clomethiazole were analysed further. Incubating rat cortical prisms in an ischaemic medium produced a significant enhancement of glutamate efflux that was not due to cell lysis. This efflux was partially Ca^^-dependent over the first 25 min of a 45 min incubation period, with volume activated anion channels becoming involved after this time and reversal of excitatory amino acid transporters playing a role throughout. Although tetrodotoxin had no effect on glutamate efflux, the Ca"* channel blockers ro-Conotoxin MVIIC and sipatrigine inhibited ischaemic glutamate efflux. NMDA and AMPA/kainate receptor antagonists also inhibited glutamate efflux, fhc group II metabotropic glutamate receptor agonist DCG-IV and the endogenous nociceptin receptor agonist nociceptin inhibited glutamate efflux whilst the group 1 mGluR antagonists MI’EP and CPCCOEt did not. These results demonstrate a good correlation between the effect of compounds on ischaemic glutamate efflux in vitro and their ncuroprotectivc efficacy in vivo, suggesting this model may be useful for predicting efficacy in animal models of stroke. In the glutamate efflux assay, elomethiazole inhibited ischaemic glutamate efflux in a bicuculline-insensitive manner whilst pentobarbitone did not, indicating that clomethiazolc may exert its neuroprotective action by direct activation of the GABAa receptor. Electrophysiological studies using cells stably transfected with al:pl:Y2L or al:P2:y2S GABAa receptor subunits found that clomethiazole potentiated responses to GABA but also directly activated the GABAa receptor. In contrast to the glutamate efflux data. however, this direct effect of clomethiazole was inhibited by bicuculline. This discrepancy requires further investigation.

Name dc:type.qualificationname

PhD

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

De Montfort University

Year dc:date.issued

2002

Author dc:creator

• Nelson, Rachael M.

Licence

• https://dora.dmu.ac.uk/bitstreams/b8005560-07c4-4918-8a84-f0f575c1f5bd/download