This study is the first to report direct evidence that hyperoxic resuscitation and reperfusion after cardiac arrest impairs cerebral aerobic energy metabolism. Decreased incorporation of [1-13
C]glucose into [4-13
C]glutamate (and subsequently into [3-13
C] and [1-13
C] glutamate) in the hippocampus indicates altered metabolism via a pathway that uses the PDHC for entry into the TCA cycle. Because incorporation of label from infused glucose first appears in brain glutamate and later in glutamine16
and because acetyl CoA derived from glucose predominantly enters the neuronal TCA cycle, the decreased incorporation of 13
C into [4-13
C]glutamate primarily reflects an impairment in neuronal, rather than astrocytic, metabolism. Moreover, hippocampal 13
C enrichment of all isotopomers of glutamate (), but not glutamine, was reduced after hyperoxic reperfusion, providing further evidence that neuronal metabolism is impaired. In addition, because the total amounts of glutamate and glutamine analyzed by HPLC were unchanged in the different experimental groups, the reduced 13
C glutamate enrichment can be attributed to decreased energy production and not other metabolic processes, such as changes in glutamate utilization. For several glutamate isotopomers, there was also a trend toward lower incorporation in the normoxic animals compared with nonischemics and a trend toward higher incorporation after normoxic compared with hyperoxic resuscitation. The variability in these results is not surprising, considering the large number of enzymatic and transport activities required for conversion of systemically administered glucose into glutamate and other metabolites within the brain. Future studies using this approach will therefore require a larger number of animals per group to reach a power sufficient to detect significant differences.
Postischemic impairment of PDHC enzyme activity and reduction in subunit immunoreactivity have been documented.2,3,6,17–20
In particular, previous results from our laboratory showed a 33% decrease in total hippocampal PDHC activity after hyperoxic reperfusion compared with nonischemic control animals.3
Cardell et al19
reported a 50% reduction in PDHC activity within 15 minutes of recirculation in a rat model of cerebral ischemia, attributable most likely to increased protein phosphorylation rather than a loss of total activity. The decreased incorporation of 13
C into the C4 position of glutamate in the present study is consistent with the reduction in PDHC activity, but it could be caused by impairment of other metabolic enzymes.
Hyperoxic reperfusion after cardiac arrest exacerbates oxidative stress leading to increased lipid oxidation1
and elevated hippocampal nitrotyrosine immunoreactivity.6
Prooxidant reactive species, eg, the hydroxyl radical and peroxynitrite that cause these molecular alterations, can also inactivate important enzymes in energy metabolism, like PDHC3
or components of the electron transport chain. The findings that the brain NAD(P)H redox state is hyperoxidized during the first hour after ischemia and that this hyperoxidized state is exacerbated by hyperoxia suggest that reactions prior to the electron transport chain limit postischemic aerobic energy metabolism.21
The increased percent enrichment of [3-13
C]lactate after hyperoxic reperfusion in the present study shows that glycolysis is not impaired.
In the present study, hyperoxic reperfusion decreased the incorporation of label from the metabolism of [1-13
C]glucose into [4-13
C]glutamate to 51% of that observed in nonischemic controls (). Studies of focal cerebral ischemia in rat models have also shown decreased incorporation of [1-13
C]glucose into [4-13
C]glutamate, indicating alterations in neuronal TCA cycle metabolism.15,22
Oxidation of glutamate via a partial TCA cycle is known to occur in neurons under various pathologic conditions13
in response to impaired aerobic glucose metabolism.23
Pascual et al23
showed increased glutamate oxidation after focal cerebral ischemia in the rat, consistent with intact metabolism via α-ketoglutarate dehydrogenase and use of glutamate as an alternative energy substrate in brain.
Although multiple turns of the TCA cycle lead to incorporation of label from [1-13
C]glucose into [2-13
C]glutamate, this isotopomer is primarily labeled by metabolism via the pyruvate carboxylase pathway in astrocytes.24
This pathway, which adds net carbon to the astrocytic TCA cycle, leads to de novo synthesis of glutamine in astrocytes and has an important role in providing glutamine to neurons to replenish the glutamate released during neurotransmission.15,25
Therefore, the decreased [2-13
C]glutamate observed in the present study after hyperoxic reperfusion reflects both decreased metabolism via pyruvate carboxylase (astrocytic) and decreased labeling from subsequent turns of the TCA cycle after initial metabolism via PDHC (predominantly neuronal), because both processes lead to labeling in the C2 position.
To discern the relative astrocytic and neuronal contributions to the alterations in metabolism after hyperoxic reperfusion, a study using both 13
C-glucose and 13
C-acetate is warranted. Labeled acetate is taken up and metabolized exclusively by astrocytes, allowing for a more direct comparison of changes in glial metabolism.4
Because reperfusion improves astrocytic metabolism after focal cerebral ischemia,22
monitoring astrocytic metabolism at multiple time points may be useful in determining the precise role of astrocytes in reperfusion-mediated changes in metabolism after global ischemia.
Although significant differences in incorporation of 13
C label into glutamate in the hippocampus were found, no difference in incorporation of [1-13
C]glucose into isotopomers of glutamine occurred during either hyperoxic or normoxic reperfusion compared with nonischemic controls. Whereas Haberg et al22
reported changes in 13
C incorporation into glutamine after focal cerebral ischemia, these differences were detected in the rat brain after 120 minutes of ischemia. In addition, alterations in glutamine are generally attenuated compared with the striking decrease in 13
C incorporation into glutamate.22
Because considerably less label from glucose is incorporated into glutamine than glutamate, it is more difficult to determine alterations in labeling of 13
C-glutamine in anesthetized brain after a short period of ischemia.
A significant increase in percent enrichment of lactate and a trend toward elevated incorporation of [1-13
C]glucose into [3-13
=0.06) were observed in the hippocampus of animals resuscitated under hyperoxic but not normoxic conditions compared with nonischemic controls. Similarly, in a rat focal cerebral ischemia model, Haberg et al22
reported decreased 13
C-glucose metabolism in the penumbra but did not observe increased 13
C incorporation into lactate.22
In the current study, there was no change in enrichment or 13
C-lactate labeling in the cortex, providing further evidence that hippocampal metabolism is particularly vulnerable in this clinically relevant model of global ischemia/reperfusion. The recent report by Richards et al3
that there was no change in PDHC activity in the cortex, regardless of the reperfusion paradigm, is consistent with this concept.
Significantly elevated amounts of unmetabolized 13
C-glucose were observed in both the hippocampus and cortex of animals resuscitated under hyperoxic conditions after ischemia/reperfusion, with a trend toward greater unmetabolized glucose in animals resuscitated under normoxic conditions. These findings are consistent with impaired postischemic metabolism of [1-13
C]glucose in rat models of focal cerebral ischemia.15,22,23
In the present study impaired metabolism of [1-13
C]glucose to 13
C-glutamate was only observed in the hippocampus after hyperoxic resuscitation. This finding indicates that the canine hippocampus is selectively vulnerable to the high oxygen tension used during hyperoxic reperfusion, which is consistent with earlier findings in our model3,6
and in rodent models of cerebral ischemia.10,26