Effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transfer activity in rat brain slices assessed by gas–tissue autoradiography using [ 15O] molecular oxygen

Abstract

We have investigated the effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transport function in brain slices using gas–tissue autoradiography technique with [ 15O]O 2. Brain slices were preincubated in Krebs-Ringer phosphate medium bubbled with 100% O 2 for 30 min at 37°C. (1) Control culture was incubated in the same medium bubbled with 100% O 2 for 5–40 min at 37°C, then for another 30 min under the same conditions. (2) In vitro ischemia was induced by placing the culture in the medium deprived of glucose and bubbled with 100% N 2 for 5–40 min, then returning it to control conditions and culturing for another 30 min. (3) In vitro hypoxia was induced by placing the culture in the medium with glucose and bubbled with 100% N 2 for 5–40 min, then returning it to the control conditions for 30 min. After the three different treatments, the [ 15O]O 2 fixation by brain slices reflect to mitochondrial electron transport function was determined using gas–tissue autoradiography technique with [ 15O]O 2. The fixation of [ 15O]O 2 by striatum, cerebral cortex and hippocampus was reduced dependent upon the period of in vitro ischemic treatment. In contrast, the [ 15O]O 2 fixation by those brain regions was only slightly reduced by hypoxia treatment. The reduction in [ 15O]O 2 fixation induced by ischemic treatment was prevented by an antioxidant: glutathione, glutathione monoethyl ester or acetylsalicylic acid. The preventive effect of antioxidants on the mitochondrial damage induced by ischemia was more remarkable in the striatum than in the cerebral cortex and hippocampus. In the comparison of [ 15O]O 2 fixation between ischemia-treated young and senescent brain slices, reduction of 15O fixation by every brain region examined was more prominent in senescence than in the young. These results suggest that gas–tissue autoradiography using [ 15O]O 2 is useful to assess mitochondrial electron transport dysfunction induced by ischemia treatment in brain slices and that the oxidative stress participates in the mechanism of ischemia-induced dysfunction in mitochondria.