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- Rytter, Anna
(författare)
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Mechanisms of Ischemic Brain Injury- studies in murine hippocampal slice cultures
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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- Rytter, Anna, et al.
(författare)
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Mouse hippocampal organotypic tissue cultures exposed to in vitro "ischemia" show selective and delayed CA1 damage that is aggravated by glucose
- 2003
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Ingår i: Journal of Cerebral Blood Flow and Metabolism. - : SAGE Publications. - 1559-7016 .- 0271-678X. ; 23:1, s. 23-33
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Tidskriftsartikel (refereegranskat)abstract
- Oxygen and glucose deprivation (OGD) in cell cultures is generally studied in a medium, such as artificial cerebrospinal fluid (CSF), with an ion composition similar to that of the extracellular fluid of the normal brain (2 to 4 mmol/L K+, 2 to 3 mmol/L Ca2+; pH 7.4). Because the distribution of ions across cell membranes dramatically shifts during ischemia, the authors exposed mouse organotypic hippocampal tissue cultures to OGD in a medium, an ischemic cerebrospinal fluid, with an ion composition similar to the extracellular fluid of the brain during ischemia in vivo (70 mmol/L K+, 0.3 mmol/L Ca2+; pH 6.8). In ischemic CSF, OGD induced a selective and delayed cell death in the CA1 region, as assessed by propidium iodide uptake. Cell death was glutamate receptor dependent since blockade of the N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors mitigated cell damage. Hyperglycemia aggravates ischemic brain damage in vivo, whereas in vitro glucose in artificial CSF prevents oxygen deprivation-induced damage. The authors demonstrate that glucose in ischemic CSF significantly exacerbates cell damage after oxygen deprivation. This new model of in vitro "ischemia" can be useful in future studies of the mechanisms and treatment of ischemic cell death, including studies using genetically modified mice.
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- Rytter, Anna, et al.
(författare)
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The temperature dependence and involvement of mitochondria permeability transition and caspase activation in damage to organotypic hippocampal slices following in vitro ischemia.
- 2005
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Ingår i: Journal of Neurochemistry. - : Wiley. - 1471-4159 .- 0022-3042. ; 95:4, s. 1108-1117
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Tidskriftsartikel (refereegranskat)abstract
- The aggravating effect of hyperglycemia on ischemic brain injury can be mimicked in a model of in vitro ischemia (IVI) using murine hippocampal slice cultures. Using this model, we found that the damage in the CA1 region following IVI in the absence or presence of 40 mM glucose (hyperglycemia) is highly temperature dependent. Decreasing the temperature from 35 to 31 degrees C during IVI prevented cell death, whereas increasing the temperature by 2 degrees C markedly aggravated damage. As blockade of the mitochondrial permeability transition (MPT) is equally effective as hypothermia in preventing ischemic cell death in vivo, we investigated whether inhibition of MPT or of caspases was protective following IVI. In the absence of glucose, the MPT blockers cyclosporin A and MeIle(4)-CsA but not the immunosuppressive compound FK506 diminished cell death. In contrast, following hyperglycemic IVI, MPT blockade was ineffective. Also, the pan-caspase inhibitor Boc-Asp(OMe)fluoromethyl ketone did not decrease cell death in the CA1 region following IVI or hyperglycemic IVI. We conclude that cell death in the CA1 region of organotypic murine hippocampal slices following IVI is highly temperature dependent and involves MPT. In contrast, cell death following hyperglycemic IVI, although completely prevented by hypothermia, is not mediated by mechanisms that involve MPT or caspase activation
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