Towards a representative in vitro model for testing putative neuroprotective drugs in stroke : dissecting factors that govern the selectivity and time cource of OGD-induced damage

Abstract

A stroke occurs when the blood flow to a brain region is disturbed causing oxygen and glucose deprivation (OGD) and the initiation of an ischemic signalling cascade leading to cell death. In industrialized countries stroke is the third leading cause of death and the leading cause of disability. The only approved treatment for stroke patients is administration of the thrombolytic tissue plasminogen activator (t-PA) to dissolve the clot and restore the blood flow. However, t-PA can be given only within ~3 hours after onset of the insult. Since the the discovery of the involvement of glutamate excitotoxicity in ischemic cell death, there has been extensive search for neuroprotective drugs in stroke. Many have proved neuroprotective in in vitro and in vivo models, but so far all of them have failed in clinical trials. Good in vitro and in vivo models for ischemia are a prerequiste both for improving the understanding of stroke pathology and for promoting drug development. This thesis focuses on an in vitro ischemia model based on organotypical hippocampal slice cultures exposed to OGD. The major aim was to optimize this model with regard to the important features of ischemic cell death seen in vivo and in stroke patients namely delayed development of cell death and selective vulnerability of the CA1 pyramidal cells of the hippocampus. We also wanted to use the improved slice culture protocol to test bumetanide (selective antagonist of the Na+/K+/Cl- co-transporter NKCC1) for cytoprotective effects after OGD in the hippocampus. We found that by subjecting the slice cultures to immediate onset hypoxia (resembling the abrupt interruption of the blood flow in vivo) in combination with an incubation buffer resembling the ionic concentration of the cerebral spinal fluid during a stroke, a delayed cell death pattern characterised by selectivity for the CA1 region was obtained. We also found that the vehicle ethanol potentiated OGD induced cell death which could be attenuated by administration of bumetanide. However, bumetanide failed to provide neuroprotection after OGD when compared to a control group not exposed to vehicle ethanol. NMDA induced excitotoxicity could be blocked by the NMDA receptor antagonist MK-801 but not by bumetanide. In conclusion the results indicate that the role of NKCC1 in ischemic cell death is not directly coupled to excitotoxic cell death signalling.