Ischemic Cell Death in the CNS - applications of a new in vitro model

Sammanfattning: Ischemic brain damage is a common cause of death and disability. A global ischemic insult is usually the result of a transient cardiac arrest while occlusion of cerebral blood vessels leads to focal ischemic lesions, commonly termed stroke. During the last decades our knowledge about the metabolic and cellular events leading to cell death following ischemia has expanded mainly due to experimental studies in vivo and in vitro. Important findings concerning the relevance of body temperature and blood glucose levels have been confirmed in humans. However, a vast number of pharmacological agents with protective effects in animal models of ischemia have failed in subsequent clinical trials. This illustrates that our knowledge of the mechanisms of ischemic cell death is still incomplete and that we need to question the models we use to mimic the human disorders. We have used the organotypic tissue culture from mouse hippocampus to establish a new model of in vitro ischemia (IVI). Similar to previous models we combine anoxia and aglycemia but in addition we apply a combination of ions similar to what is found in the brain extracellular fluid during ischemia. We found that the combination of a high potassium level (70mM), a low calcium level (0.3mM) and acidosis (pH 6.8) during IVI made the pattern of cell death more similar to what is found following global ischemia in vivo in that became more delayed and selective. A high level of glucose was found to increase cell death in contrast to what had previously been found in other cell culture models of ischemia but in similarity to what is found in vivo. While cell death following IVI could be completely prevented by the withdrawal of extracellular calcium during the insult or antagonists of glutamatergic NMDA-receptors, no effect of either was found in the hyperglycaemic IVI paradigm. On the other hand, intracellular calcium chelation prevented against cell death following hyperglycaemic IVI but not IVI. Inhibition of free radicals was ineffective in both paradigms. These findings illustrates that IVI and hyperglycaemic IVI induces two different patterns of cell death both of which may be important during ischemia in vivo. Intraischemic acidosis protected neurons in the CA3-subregion of hippocampus more than in CA1. NMDA-receptors in both regions were inhibited by acidosis but they recovered significantly slower in the CA3-region. This prolonged inhibition could explain the sparing of the CA3-neurons following IVI and global ischemia. The neuromodulator adenosine inhibits glutamate release through presynaptic A1-receptors. We used transgenic A1-receptor knock-out mice to study the importance of this receptor for the development of cell death following IVI and global ischemia in vivo. No effect of the knock-out was found in any of the two paradigms. The A1-receptor antagonist, 8-CPT increased damage in vivo but had no effect in vitro. This discrepancy between the models could be explained by a less importance of vesicular glutamate release in vitro or an undiscovered systemic side-effect of 8-CPT in vivo. The described models of IVI and hyperglycaemic IVI are well suited for further studies on the pathophysiology of cerebral ischemia using transgenic, pharmacological, electrophysiological and imaging techniques.