Ivan T. Demchenko

Transient hypoxia stimulates mitochondrial biogenesis in brain subcortex by a neuronal nitric oxide synthase-dependent mechanism.

The adaptive mechanisms that protect brain metabolism during and after hypoxia, for instance, during hypoxic preconditioning, are coordinated in part by nitric oxide (NO). We tested the hypothesis that acute transient hypoxia stimulates NO synthase (NOS)-activated mechanisms of mitochondrial biogenesis in the hypoxia-sensitive subcortex of wild-type (Wt) and neuronal NOS (nNOS) and endothelial NOS (eNOS)-deficient mice. Mice were exposed to hypobaric hypoxia for 6 h, and changes in immediate hypoxic transcriptional regulation of mitochondrial biogenesis was assessed in relation to mitochondrial DNA (mtDNA) content and mitochondrial density. There were no differences in cerebral blood flow or hippocampal PO2 responses to acute hypoxia among these strains of mice. In Wt mice, hypoxia increased mRNA levels for peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1 α), nuclear respiratory factor-1, and mitochondrial transcription factor A. After 24 h, new mitochondria, localized in reporter mice expressing mitochondrial green fluorescence protein, were seen primarily in hippocampal neurons. eNOS−/− mice displayed lower basal levels but maintained hypoxic induction of these transcripts. In contrast, nuclear transcriptional regulation of mitochondrial biogenesis in nNOS−/− mice was normal at baseline but did not respond to hypoxia. After hypoxia, subcortical mtDNA content increased in Wt and eNOS−/− mice but not in nNOS−/− mice. Hypoxia stimulated PGC-1α protein expression and phosphorylation of protein kinase A and cAMP response element binding (CREB) protein in Wt mice, but CREB only was activated in eNOS−/− mice and not in nNOS−/− mice. These findings demonstrate that hypoxic preconditioning elicits subcortical mitochondrial biogenesis by a novel mechanism that requires nNOS regulation of PGC-1α and CREB.

Regulation of the Brain’s Vascular Responses to Oxygen

Abstract— The mechanism of oxygen-induced cerebral vasoconstriction has been sought for more than a century. Using genetically altered mice to enhance or disrupt extracellular superoxide dismutase (EC-SOD, SOD3), we tested the hypothesis that this enzyme plays a critical role in the physiological response to oxygen in the brain by regulating nitric oxide (NO·) availability. Cerebral blood flow responses in these genetically altered mice to changes in Po2 demonstrate that SOD3 regulates equilibrium between superoxide (·O2−) and NO·, thereby controlling vascular tone and reactivity in the brain. That SOD3 opposes inactivation of NO·is shown by absence of vasoconstriction in response to Po2 in the hyperbaric range in SOD3+/+ mice, whereas NO-dependent relaxation is attenuated in SOD3−/− mutants. Thus, EC-SOD promotes NO·vasodilation by scavenging ·O2− while hyperoxia opposes NO·and promotes constriction by enhancing endogenous ·O2− generation and decreasing basal vasodilator effects of NO·.

Rapid Cerebral Ischemic Preconditioning in Mice Deficient in Endothelial and Neuronal Nitric Oxide Synthases

Background and Purpose— The purpose of this study was to test the hypothesis that nitric oxide is required for preconditioning in an intact animal model of focal ischemia using neuronal and endothelial nitric oxide synthase (nNOS and eNOS) knockout mice. Methods— Cerebral blood flow was measured in wild-type, nNOS knockout, and eNOS knockout mice by hydrogen clearance (absolute) and laser Doppler flowmetry (relative). Mice were preconditioned by three 5-minute episodes of transient middle cerebral artery occlusion (MCAO) and subjected to permanent MCAO. Neurological deficit and infarct size were determined 24 hours later. Results— Although wild-type mice showed protection from ischemic preconditioning, neither eNOS nor nNOS knockout mice showed protection. Laser Doppler measurements indicated that the relative blood flow decreases in core ischemic areas were the same in all groups. Conclusions— Neither eNOS nor nNOS knockout mice show protection from rapid ischemic preconditioning, suggesting that nitric oxide may play a role in the molecular mechanisms of protection.

Two faces of nitric oxide: implications for cellular mechanisms of oxygen toxicity

Recent investigations have elucidated some of the diverse roles played by reactive oxygen and nitrogen species in events that lead to oxygen toxicity and defend against it. The focus of this review is on toxic and protective mechanisms in hyperoxia that have been investigated in our laboratories, with an emphasis on interactions of nitric oxide (NO) with other endogenous chemical species and with different physiological systems. It is now emerging from these studies that the anatomical localization of NO release, which depends, in part, on whether the oxygen exposure is normobaric or hyperbaric, strongly influences whether toxicity emerges and what form it takes, for example, acute lung injury, central nervous system excitation, or both. Spatial effects also contribute to differences in the susceptibility of different cells in organs at risk from hyperoxia, especially in the brain and lungs. As additional nodes are identified in this interactive network of toxic and protective responses, future advances may open up the possibility of novel pharmacological interventions to extend both the time and partial pressures of oxygen exposures that can be safely tolerated. The implications of a better understanding of the mechanisms by which NO contributes to central nervous system oxygen toxicity may include new insights into the pathogenesis of seizures of diverse etiologies. Likewise, improved knowledge of NO-based mechanisms of pulmonary oxygen toxicity may enhance our understanding of other types of lung injury associated with oxidative or nitrosative stress.

Nitric oxide production is enhanced in rat brain before oxygen-induced convulsions.

Central nervous system oxygen toxicity (CNS O2 toxicity) is preceded by release of hyperoxic vasoconstriction, which increases regional cerebral blood flow (rCBF). These increases in rCBF precede the onset of O2-induced convulsions. We have tested the hypothesis that hyperbaric oxygen (HBO2) stimulates NO* production in the brain that leads to hyperemia and anticipates electrical signs of neurotoxicity. We measured rCBF and EEG responses in rats exposed at 4 to 6 atmospheres (ATA) of HBO2 and correlated them with brain interstitial NO* metabolites (NO(x)) as an index of NO* production. During exposures to hyperbaric oxygen rCBF decreased at 4 ATA, decreased for the initial 30 min at 5 ATA then gradually increased, and increased within 30 min at 6 ATA. Changes in rCBF correlated positively with NO(x) production; increases in rCBF during HBO2 exposure were associated with large increases in NO(x) at 5 and 6 ATA and always preceded EEG discharges as a sign of CNS O2 toxicity. In rats pretreated with L-NAME, rCBF remained maximally decreased throughout 75 min of HBO2 at 4, 5 and 6 ATA. These data provide the first direct evidence that increased NO* production during prolonged HBO2 exposure is responsible for escape from hyperoxic vasoconstriction. The finding suggests that NO* overproduction initiates CNS O2 toxicity by increasing rCBF, which allows excessive O2 to be delivered to the brain.

Contributions of endothelial and neuronal nitric oxide synthases to cerebrovascular responses to hyperoxia.

Hyperoxia causes a transient decrease in CBF, followed by a later rise. The mediators of these effects are not known. We used mice lacking endothelial or neuronal nitric oxide synthase (NOS) isoforms (eNOS−/− and nNOS−/− mice) to study the roles of the NOS isoforms in mediating changes in cerebral vascular tone in response to hyperoxia. Resting regional cerebral blood flow (rCBF) did not differ between wild type (WT), eNOS−/− mice, and nNOS−/− mice. eNOS−/− mice showed decreased cerebrovascular reactivities to NG-nitro-L-arginine methyl ester (L-NAME), PAPA NONOate, acetylcholine (Ach), and SOD1. In response to hyperbaric oxygen (HBO2) at 5 ATA, WT and nNOS−/− mice showed decreases in rCBF over 30 minutes, but eNOS−/− mice did not. After 60 minutes HBO2, rCBF increased more in WT mice than in eNOS−/− or nNOS−/− mice. Brain NO-metabolites (NOx) decreased in WT and eNOS−/− mice within 30 minutes of HBO2, but after 45 minutes, NOx rose above control levels, whereas they did not change in nNOS−/− mice. Brain 3NT increased during HBO2 in WT and eNOS−/− but did not change in nNOS−/− mice. These results suggest that modulation of eNOS-derived NO by HBO2 is responsible for the early vasoconstriction responses, whereas late HBO2-induced vasodilation depends upon both eNOS and nNOS.

Oxygen seizure latency and peroxynitrite formation in mice lacking neuronal or endothelial nitric oxide synthases.

Nitric oxide (NO) from endothelial or neuronal NO synthases (eNOS or nNOS) may contribute both to the cerebrovascular responses to oxygen and potentially to the peroxynitrite-mediated toxic effects of hyperbaric oxygen (HBO(2)) on the central nervous system (CNS O(2) toxicity). In mice lacking eNOS or nNOS (-/-), regional cerebral blood flow (rCBF) and 3-nitrotyrosine (3-NT), a biochemical marker for peroxynitrite (ONOO(-)) formation, were measured in the brain during HBO(2) exposure. These variables were then correlated with EEG spiking activity related to CNS O(2) toxicity. In wild-type (WT) mice, HBO(2) exposure transiently reduced rCBF, but by 60 min rCBF was restored to baseline levels and above, followed by EEG spikes. Mice lacking nNOS also showed initial depression of rCBF followed by hyperemia but the delay in the onset of EEG discharges was greater. In contrast, in eNOS-deficient mice rCBF did not decrease and hyperemia was less pronounced during HBO(2). EEG spike latency was longer in eNOS(-/-) compared to WT or nNOS(-/-) mice. 3-NT gradually increased in all strains during HBO(2) but accumulation was slower in nNOS(-/-) mice, consistent with less ONOO(-) production. These results indicate that NOS-deficient mice have different cerebrovascular responses and tolerance to HBO(2) depending on which enzyme isoform is affected. The data suggest a key role for eNOS-dependent NO production in cerebral vasoconstriction and in the development of hyperoxic hyperemia preceding O(2) seizures, whereas neuronal NO may mediate toxic effects of HBO(2) mainly by its reaction with superoxide to generate the stronger oxidant, peroxynitrite.

Production of Hydroxyl Radical in the Hippocampus After CO Hypoxia or Hypoxic Hypoxia in the Rat

Carbon monoxide poisoning produces both immediate and delayed neuronal injury in selective regions of the brain that is not readily explained on the basis of tissue hypoxia. One possibility is that cellular injury during and after CO poisoning is related to the production of reactive oxygen species (ROS) by the brain. In this study, we hypothesized that the extent of ROS generation in the brain would be greater after CO than after hypoxic hypoxia due to intracellular uptake of CO. We assessed hydroxyl radical (OH.) production by comparing the nonenzymatic hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid (2,3-DHBA) in the hippocampus of the rat by microdialysis during either CO hypoxia or an exposure to hypoxic hypoxia that produced similar PO2 and cerebral blood flow (CBF) values in the region of microdialysis. We found neither control animals nor animals exposed to 30 min of hypoxic hypoxia at a mean tissue PO2 of 15 mmHg demonstrated significant increases in 2,3-DHBA production in the hippocampus over the 2-h the exposure. In contrast, CO exposed rats which also developed brain PO2 values in the range of 15 mmHg showed highly significant increases in 2,3-DHBA production. We conclude that cerebral oxidative stress in the hippocampus of the rat during CO hypoxia in vivo is not a direct effect of decreased tissue oxygen concentration.

Cerebral blood flow and brain oxygenation in rats breathing oxygen under pressure.

Hyperbaric oxygen (HBO2) increases oxygen tension (PO2) in blood but reduces blood flow by means of O2-induced vasoconstriction. Here we report the first quantitative evaluation of these opposing effects on tissue PO2 in brain, using anesthetized rats exposed to HBO2 at 2 to 6 atmospheres absolute (ATA). We assessed the contribution of regional cerebral blood flow (rCBF) to brain PO2 as inspired PO2 (PiO2) exceeds 1 ATA. We measured rCBF and local PO2 simultaneously in striatum using collocated platinum electrodes. Cerebral blood flow was computed from H2 clearance curves in vivo and PO2 from electrodes calibrated in vitro, before and after insertion. Arterial PCO2 was controlled, and body temperature, blood pressure, and EEG were monitored. Scatter plots of rCBF versus pO2 were nonlinear (R2 = 0.75) for rats breathing room air but nearly linear (R2 = 0.88–0.91) for O2 at 2 to 6 ATA. The contribution of rCBF to brain PO2 was estimated at constant inspired PO2, by increasing rCBF with acetazolamide (AZA) or decreasing it with N-nitro-l-arginine methyl ester (l-NAME). At basal rCBF (78 mL/100 g min), local PO2 increased 7- to 33-fold at 2 to 6 ATA, compared with room air. A doubling of rCBF increased striatal PO2 not quite two-fold in rats breathing room air but 13- to 64-fold in those breathing HBO2 at 2 to 6 ATA. These findings support our hypothesis that HBO2 increases PO2 in brain in direct proportion to rCBF.

Oxygen-induced mitochondrial biogenesis in the rat hippocampus.

The hypothesis that damage to mitochondrial DNA by reactive oxygen species increases the activity of nuclear and mitochondrial transcription factors for mitochondrial DNA replication was tested in the in vivo rat brain. Mitochondrial reactive oxygen species generation was stimulated using pre-convulsive doses of hyperbaric oxygen and hippocampal mitochondrial DNA content and neuronal and mitochondrial morphology and cell proliferation were evaluated at 1, 5 and 10 days. Gene expression was subsequently evaluated to assess nuclear and mitochondrial-encoded respiratory genes, mitochondrial transcription factor A, and nuclear respiratory transcription factors-1 and -2. After 1 day, a mitochondrial DNA deletion emerged involving Complex I and IV subunit-encoding regions that was independent of overt neurological or cytological O(2) toxicity, and resolved before the onset of cell proliferation. This damage was attenuated by blockade of neuronal nitric oxide synthase. Compensatory responses were found in nuclear gene expression for manganese superoxide dismutase, mitochondrial transcription factor A, and nuclear respiratory transcription factor-2. Enhanced nuclear respiratory transcription factor-2 binding activity in hippocampus was accompanied by a nearly three-fold boost in mitochondrial DNA content over 5 days. The finding that O(2) activates regional mitochondrial DNA transcription, replication, and mitochondrial biogenesis in the hippocampus may have important implications for maintaining neuronal viability after brain injury.