Mechanisms of Ischemic Brain Injury- studies in murine hippocampal slice cultures

Sammanfattning: Cerebral ischemia is a major cause of mortality and morbidity in the western world. Even though much effort has been put into research and development of effective drugs against ischemic damage there is to date no effective pharmacological brain protective treatment. In order to study particular molecular mechanisms, isolated cellular events and the contribution of individual factors in ischemic damage, in vitro models resembling the in vivo situation are needed. The aim of this thesis was to establish and describe an in vitro model of ischemia that reproduces the cell death pattern following ischemia in vivo, using mouse organotypic hippocampal slice cultures. In most models, in vitro ischemia is mimicked by the deprivation of oxygen and glucose (OGD) in a medium with an ion composition similar to that of the extra cellular fluid of the normal brain (2-4 mM K+, 2-3 mM Ca2+ and pH 7.4). During in vivo ischemia the distribution of ions across cell membranes shifts. We therefore exposed cultures to OGD in a medium with 70 mM K+, 0.3 mM Ca2+ and pH 6.5-6.8, similar to the extracellular fluid of the brain during ischemia in vivo. Damage induced by 12-15 minutes of OGD in this medium is delayed and observed only in the CA1 region, similar as in in vivo models of ischemia. Another feature of in vivo ischemia is the aggravating effect of glucose on damage. In our model high levels of glucose during the insult delayed and aggravated damage. Our results demonstrate that glucose in combination with acidosis mediates the detrimental effect. The cell death caused by glucose-free ischemia was inhibited by antagonists of ionotropic glutamate receptors, but when glucose was present during ischemia the same antagonists had no effect. Hypothermia is the most powerful method for protecting the brain from ischemic damage. In our model hypothermia of 31°C during both IVI and hyperglycemic IVI provided profound protection, whereas hypothermia only after the insult did not affect the development of damage in either of the paradigms. Following glucose-free ischemia no immunoreactivity of activated caspase-3 could be seen and neither was there any effect of a pancaspase inhibitor, instead activation of the MPTP was induced. In the hyperglycemic paradigm the dentate gyrus displayed active caspase-3 and cell death in this region was abolished by the caspase inhibitor. Cell death in the CA1 region following hyperglycemic IVI could have a component of caspase activity, but neither the MPTP nor caspases play critical roles. Activation of adenosine A1 receptors (A1Rs) is thought to be protective. But the deletion of the A1R-gene did not influence the outcome neither following ischemia in a mouse global ischemia model nor following in vitro ischemia. The results suggest that some effects of A1 receptors are compensated for in knockout animals. In conclusion this model of in vitro ischemia mimics central features of in vivo ischemia and can be useful in future studies of the mechanisms and treatment of ischemic cell death.