The isolated canine brain preparation will be utilized in studies to: a) correlate the magnitude of hypoxic brain damage with changes in mitochondrial function and energy metabolism and b) identify the pathways involved in brain glucose utilization during hypoxia and post-hypoxic reoxygenation. a) Brains will be perfused at PaO2's ranging from 10 to 50 mmHg for periods of 16 to 125 min to vary O2 deficit ((Normal CMRO2 - Experimental CMRO2) x Time) and time to achieve a specific O2 deficit. Fresh cerebral cortex with O2 deficits totaling 10 to 80 Mumol/g and 0, 30, or 60 min of reoxygenation will be exercised and the mitochondria isolated for measurement of respiration. Similar samples of cortex will be frozen in situ and examined for ATP, ADP, AMP, PCr, FFA, glucose, G-6-P, G-1-P, glycogen and lactate. Extracellular Ca++ will be measured continuously and CMRO2 at 3 min intervals throughout the study. A two level factorial analysis will be made to determine whether: i) CMRO2 reflects the status of variables aassociated with energy metabolism, ii) O2 deficit is a factor in producing mitochondrial damage and iii) the decrease in extracellular Ca++ correlates with the onset of irreversible changes in cerebral energy metabolism. b) The metabolic fate of U14C-glucose will be studied by pulse labeling brain and measuring 14CO2 efflux before, during or after 30 min of either PaO2 30 or 40 mmHg hypoxia. In addition, cerebral cortex will be frozen in situ, fractionated into constituent FFA, lipids, amino acids, proteins and polysaccharides, quantified and analyzed for radioactivity. To quantify the amount of glucose entering the various pathways, the U14C-glucose experiments will be repeated with variously labeled (1-14C, 6-14C, and 3,4-14C)-glucose. If CMRglu is known one can calculate the contribution of each pathway based on the percent of total glucose radioactivity entering brain which appears as 14CO2 with each of the specific labels. Pathways of glucose utilization together with labeling and tissue levels of the constituents at PaO2 30 mmHg will be compared with corresponding PaO2 40 mmHg values to determine how glucose metabolism is altered by a further decrease in O2 availability and whether the changes can explain the inability of the PaO2 30 mmHg group to recover from hypoxia.
