Excitotoxic ATP and Glutamate Signalling during Central Nervous System Ischaemia
thesisposted on 09.12.2010, 09:49 by Philipp Julio Vermehren
Neural cell death plays a crucial role in the pathogenesis of various ischaemic disorders of the central nervous system (CNS), most prominently stroke, causing very significant mortality and morbidity. Severe ischaemia rapidly kills both neurons and astrocytes, two CNS cell types whose interactions are essential to normal brain functioning, so ideally a target needs to be found which will protect both. Glutamate mediated excitotoxicity, the process whereby excessive extracellular glutamate causes cell death via the over-activation of ionotropic glutamate receptors, is known to operate during ischaemia. However, there is increasing evidence that ATP mediated excitotoxicity may also occur. Using an in vitro model of ischaemia (oxygen-glucose deprivation: OGD) and various murine primary cortical cell cultures, I investigated the hypothesis that parallel pathways of ATP and glutamate mediated excitotoxicity contribute to the death of both astrocytes and neurons during ischaemia. OGD produced rapid and significant ATP and glutamate release from co-cultures of astrocytes and neurons, as measured using microelectrode biosensors. Glutamate release was mainly from astrocytes, whereas the cellular origin of ATP was less clear. Ca2+ imaging of Fura-2 loaded cells confirmed functional P2 receptor expression in all astrocytes and 60% of neurons along with glutamate receptor expression in all neurons but only a small proportion of astrocytes. During OGD, blocking NMDA and AMPA/kainate receptors significantly reduced neuronal death, while non-selective P2 receptor antagonists as well as selective P2Y1 (but not P2X7) receptor antagonists prevented the death of both neurons and astrocytes. Furthermore, a synergistic protective effect was produced by combining low concentrations of P2 and glutamate receptor antagonists, reducing cell death to control levels. These results suggest that ATP and glutamate, acting at P2 and ionotropic glutamate receptors, are the main mediators of early cell death during severe CNS ischaemia, and thus represent a potential target for powerful neuroprotective strategies.