Browsing School, Graduate by Author "Danilov, Camelia A."
Protection by normoxic reoxygenation and sulforaphane against astrocyte oxidative stress and death after oxygen and glucose deprivationDanilov, Camelia A.; Fiskum, Gary (2008)Astrocyte dysfunction and death accompany cerebral ischemia and reperfusion, thus compromising neuronal survival. Animal models of global ischemic brain injury indicate that neuronal death and neurologic injury are worse in animals exposed to hyperoxic compared to normoxic reperfusion, supporting the pathogenic role of oxidative stress. One objective of this research was to test the hypothesis that compared to the level of 20% O2 normally used in cell culture, lower, more physiological O2 levels protect astrocytes from delayed death following oxygen and glucose deprivation (OGD). Primary rat cortical astrocytes were cultured under either 7 or 20% O 2, exposed to O2 and glucose deprivation for 4 h, and then exposed to normal medium under either 7 or 20% O2. Cell death and both protein and DNA/RNA oxidation (3-nitrotyrosine and 8-hydroxy-2-deoxyguanosine immunoreactivities) were assessed at different periods of reoxygenation. Compared to 20% O2, astrocytes exposed to 7% ambient O2 during reoxygenation underwent less early protein nitration and nucleic acid oxidation and less delayed cell death. A second objective was to test the hypothesis that treatment of cultured astrocytes with sulforaphane, an inducer of NRF2-dependent antioxidant gene expression, protects astrocytes against delayed death following OGD. Exposure to sulforaphane either before or after OGD significantly reduced cell death at 48 hr after OGD. Immunostaining for 8-hydroxy-2-deoxyguanosine was reduced at 4 hr reoxygenation with sulforaphane pretreatment. Sulforaphane exposure was followed by an increase in cellular and nuclear NRF2 immunoreactivity. Sulforaphane also increased the mRNA, protein level, and enzyme activity of NADPH/Quinone Oxidoreductase 1, a known target of NRF2 transcriptional activation. These results demonstrate for the first time that compared to the 20% O2 normally used in cell culture, a lower level of 7% O2 that is closer to what exists in vivo protects against astrocyte death and oxidative stress in an in vitro ischemia/reperfusion model. Another important conclusion is that sulforaphane stimulates the NRF2/ARE pathway of antioxidant gene expression in astrocytes and protects them from cell death following OGD. These findings support the development of normoxic resuscitation and the pharmacologic use of sulforphane as neuroprotective interventions following global cerebral ischemia, e.g., that caused by cardiac arrest.