Exposure to LTIH results in an array of significant oxidative injuries in sleep-wake regions of the brain, and these biochemical changes are associated with marked hypersomnolence and increased susceptibility to short-term sleep loss. The residual forebrain redox alterations in wake-promoting brain regions may contribute to persistent sleepiness in a prevalent disorder, obstructive sleep apnea.

Rationale:Persons with obstructive sleep apnea may have significant residual hypersomnolence, despite therapy. Long-term hypoxia/ reoxygenation events in adult mice, simulating oxygenation patterns of moderate-severe sleep apnea, result in lasting hypersomnolence, oxidative injury, and proinflammatory responses in wakeactive brain regions. We hypothesized that long-term intermittent hypoxia activates brain NADPH oxidase and that this enzyme serves as a critical source of superoxide in the oxidation injury and in hypersomnolence. Objectives: We sought to determine whether long-term hypoxia/ reoxygenation events in mice result in NADPH oxidase activation and whether NADPH oxidase is essential for the proinflammatory response and hypersomnolence. Methods: NADPH oxidase gene and protein responses were measured in wake-active brain regions in wild-type mice exposed to long-term hypoxia/reoxygenation. Sleep and oxidative and proinflammatory responses were measured in adult mice either devoid of NADPH oxidase activity (gp91 phox -null mice) or in which NADPH oxidase activity was systemically inhibited with apocynin osmotic pumps throughout hypoxia/reoxygenation. Main Results: Long-term intermittent hypoxia increased NADPH oxidase gene and protein responses in wake-active brain regions. Both transgenic absence and pharmacologic inhibition of NADPH oxidase activity throughout long-term hypoxia/reoxygenation conferred resistance to not only long-term hypoxia/reoxygenation hypersomnolence but also to carbonylation, lipid peroxidation injury, and the proinflammatory response, including inducible nitric oxide synthase activity in wake-active brain regions. Conclusions: Collectively, these findings strongly support a critical role for NADPH oxidase in the lasting hypersomnolence and oxidative and proinflammatory responses after hypoxia/reoxygenation patterns simulating severe obstructive sleep apnea oxygenation, highlighting the potential of inhibiting NADPH oxidase to prevent oxidation-mediated morbidities in obstructive sleep apnea.Keywords: intermittent hypoxia; non-REM sleep; oxidation; peroxynitrite Obstructive sleep apnea (OSA) with daytime hypersomnolence is present in at least 2 to 4% of adults in developed countries (1). This disorder manifests as repeated events of sleep statedependent reductions in upper airway dilator motoneuronal activity, with consequent upper airway occlusions and oxyhemoglobin desaturations, each terminating with abrupt arousal and reoxygenation (2). The hypoxia and reoxygenation events may occur as frequently as once every minute of sleep. Despite therapy to alleviate OSA events, many individuals with OSA have residual sleepiness (3, 4). Mechanisms of the residual hypersomnolence in persons with OSA are not understood, but severity of hypoxemia in OSA predicts, in part, the severity of hypersomnolence (5, 6).Long-term intermittent hypoxia (LTIH) in mice, modeling the patterns of hypoxia/reoxygenation observed in moderate to severe sleep apnea, results in protracted hypersomnolence (7,8) and h...

Modern society enables a shortening of sleep times, yet long-term consequences of extended wakefulness on the brain are largely unknown. Essential for optimal alertness, locus ceruleus neurons (LCns) are metabolically active neurons that fire at increased rates across sustained wakefulness. We hypothesized that wakefulness is a metabolic stressor to LCns and that, with extended wakefulness, adaptive mitochondrial metabolic responses fail and injury ensues. The nicotinamide adenine dinucleotide-dependent deacetylase sirtuin type 3 (SirT3) coordinates mitochondrial energy production and redox homeostasis. We find that brief wakefulness upregulates SirT3 and antioxidants in LCns, protecting metabolic homeostasis. Strikingly, mice lacking SirT3 lose the adaptive antioxidant response and incur oxidative injury in LCns across brief wakefulness. When wakefulness is extended for longer durations in wild-type mice, SirT3 protein declines in LCns, while oxidative stress and acetylation of mitochondrial proteins, including electron transport chain complex I proteins, increase. In parallel with metabolic dyshomeostasis, apoptosis is activated and LCns are lost. This work identifies mitochondrial stress in LCns upon wakefulness, highlights an essential role for SirT3 activation in maintaining metabolic homeostasis in LCns across wakefulness, and demonstrates that extended wakefulness results in reduced SirT3 activity and, ultimately, degeneration of LCns.

The presence of refractory wake impairments in many individuals with severe sleep apnea led us to hypothesize that the hypoxia/ reoxygenation events in sleep apnea permanently damage wake-active neurons. We now confirm that long-term exposure to hypoxia/ reoxygenation in adult mice results in irreversible wake impairments. Functionality and injury were next assessed in major wake-active neural groups. Hypoxia/reoxygenation exposure for 8 weeks resulted in vacuolization in the perikarya and dendrites and markedly impaired c-fos activation response to enforced wakefulness in both noradrenergic locus ceruleus and dopaminergic ventral periaqueductal gray wake neurons. In contrast, cholinergic, histaminergic, orexinergic, and serotonergic wake neurons appeared unperturbed. Six month exposure to hypoxia/reoxygenation resulted in a 40% loss of catecholaminergic wake neurons. Having previously identified NADPH oxidase as a major contributor to wake impairments in hypoxia/reoxygenation, the role of NADPH oxidase in catecholaminergic vulnerability was next addressed. NADPH oxidase catalytic and cytosolic subunits were evident in catecholaminergic wake neurons, where hypoxia/reoxygenation resulted in translocation of p67 phox to mitochondria, endoplasmic reticulum, and membranes. Treatment with a NADPH oxidase inhibitor, apocynin, throughout hypoxia/reoxygenation exposures conferred protection of catecholaminergic neurons. Collectively, these data show that select wake neurons, specifically the two catecholaminergic groups, can be rendered persistently impaired after long-term exposure to hypoxia/reoxygenation, modeling sleep apnea; wake impairments are irreversible; catecholaminergic neurons are lost; and neuronal NADPH oxidase contributes to this injury. It is anticipated that severe obstructive sleep apnea in humans destroys catecholaminergic wake neurons.

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