Effects of intermittent hypoxia on oxidative stress-induced myocardial damage in mice.

Obstructive sleep apnea is associated with increased risk for cardiovascular diseases. As obstructive sleep apnea is characterized by episodic cycles of hypoxia and normoxia during sleep, we investigated effects of intermittent hypoxia (IH) on ischemia-reperfusion-induced myocardial injury. C57BL/6 mice were subjected to IH (2 min 6% O(2) and 2 min 21% O(2)) for 8 h/day for 1, 2, or 4 wk; isolated hearts were then subjected to ischemia-reperfusion. IH for 1 or 2 wk significantly enhanced ischemia-reperfusion-induced myocardial injury. However, enhanced cardiac damage was not seen in mice treated with 4 wk of IH, suggesting that the heart has adapted to chronic IH. Ischemia-reperfusion-induced lipid peroxidation and protein carbonylation were enhanced with 2 wk of IH, while, with 4 wk, oxidative stress was normalized to levels in animals without IH. H(2)O(2) scavenging activity in adapted hearts was higher after ischemia-reperfusion, suggesting the increased antioxidant capacity. This might be due to the involvement of thioredoxin, as the expression level of this protein was increased, while levels of other antioxidant enzymes were unchanged. In the heart from mice treated with 2 wk of IH, ischemia-reperfusion was found to decrease thioredoxin. Ischemia-reperfusion injury can also be enhanced when thioredoxin reductase was inhibited in control hearts. These results demonstrate that IH changes the susceptibility of the heart to oxidative stress in part via alteration of thioredoxin.     

Selected Contribution: Pulmonary hypertension in mice following intermittent hypoxia.

Sleep apnea (intermittent periods of hypoxia with or without hypercapnia) is associated with systemic hypertension and increased mortality from cardiovascular disease, but the relationship to pulmonary hypertension is uncertain. Previous studies on intermittent hypoxia (IH) in rats that demonstrated pulmonary hypertension utilized relatively long periods of hypoxia. Recent studies that utilized brief periods of hypoxia have conflicting reports of right ventricular (RV) hypertrophy. In addition, many studies have not measured pulmonary hemodynamics to asses the severity of pulmonary hypertension in vivo. Given the increasing availability of genetically engineered mice and the need to establish a rodent model of IH-induced pulmonary hypertension, we studied the effect of IH (2-min cycles of 10% and 21% O2, 8 h/day, 4 wk) on wild-type mice, correlating in vivo measurements of pulmonary hypertension with RV mass and pulmonary vascular remodeling. RV systolic pressure was increased after IH (36 +/- 0.9 mmHg) compared with normoxia (29.5 +/- 0.6) but was lower than continuous hypoxia (44.2 +/- 3.4). RV mass [RV-to-(left ventricle plus septum) ratio] correlated with pressure measurements (IH = 0.27 +/- 0.02, normoxia = 0.22 +/- 0.01, and continuous hypoxia = 0.34 +/- 0.01). Hematocrits were also elevated after IH and continuous hypoxia (56 +/- 1.6 and 54 +/- 1.1 vs. 44.3 +/- 0.5%). Evidence of neomuscularization of the distal pulmonary circulation was found after IH and continuous hypoxia. We conclude that mice develop pulmonary hypertension following IH, representing a possible animal model of pulmonary hypertension in response to the repetitive hypoxia-reoxygenation of sleep apnea.     

Intermittent hypoxia has organ-specific effects on oxidative stress

Obstructive sleep apnea is characterized by upper airway collapse, leading to intermittent hypoxia (IH). It has been postulated that IH-induced oxidative stress may contribute to several chronic diseases associated with obstructive sleep apnea. We hypothesize that IH induces systemic oxidative stress by upregulating NADPH oxidase, a superoxide-generating enzyme. NADPH oxidase is regulated by a cytosolic p47(phox) subunit, which becomes phosphorylated during enzyme activation. Male C57BL/6J mice were exposed to IH with an inspired O(2) fraction nadir of 5% 60 times/h during the 12-h light phase (9 AM-9 PM) for 1 or 4 wk. In the aorta and heart, IH did not affect lipid peroxidation [malondialdehyde (MDA) level], nitrotyrosine level, or p47(phox) expression and phosphorylation. In contrast, in the liver, exposure to IH for 1 wk resulted in a trend to an increase in MDA levels, whereas IH for 4 wk resulted in a 38% increase in MDA levels accompanied by upregulation of p47(phox) expression and phosphorylation. Administration of an NADPH oxidase inhibitor, apocynin, during IH exposure attenuated IH-induced increases in hepatic MDA. In p47(phox)-deficient mice, MDA levels were higher at baseline and, unexpectedly, decreased during IH. In conclusion, oxidative stress levels and pathways under IH conditions are organ and duration specific.     

Carotid body function and ventilatory responses in intermittent hypoxia. Evidence for anomalous brainstem integration of arterial chemoreceptor input

Obstructive sleep apnea is a frequent medical condition consisting in repetitive sleep-related episodes of upper airways obstruction and concurrent events of arterial blood hypoxia. There is a frequent association of cardiovascular diseases and other pathologies to this condition conforming the obstructive sleep apnea syndrome (OSAS). Laboratory models of OSAS consist in animals exposed to repetitive episodes of intermittent hypoxia (IH) which also develop cardiovascular pathologies, mostly hypertension. The overall OSAS pathophysiology appears to be linked to the repetitive hypoxia, which would cause a sensitization of carotid body (CB) chemoreflex and chemoreflex-driven hyperreactivity of the sympathetic nervous system. However, this proposal is uncertain because hyperventilation, reflecting the CB sensitization, and increased plasma CA levels, reflecting sympathetic hyperreactivity, are not constant findings in patients with OSAS and IH animals. Aiming to solve these uncertainties we have studied the entire CB chemoreflex arch in a rat model of IH, including activity of chemoreceptor cells and CB generated afferent activity to brainstem. The efferent activity was measured as ventilation in normoxia, hypoxia, and hypercapnia. Norepinephrine turnover in renal artery sympathetic endings was also assessed. Findings indicate a sensitization of the CB function to hypoxia evidenced by exaggerated chemoreceptor cell and CB afferent activity. Yet, IH rats exhibited marked hypoventilation in all studied conditions and increased turnover of norepinephrine in sympathetic endings. We conclude that IH produces a bias in the integration of the input arising from the CB with a diminished drive of ventilation and an exaggerated activation of brainstem sympathetic neurons.     

Role for PKCβ in enhanced endothelin-1-induced pulmonary vasoconstrictor reactivity following intermittent hypoxia

Intermittent hypoxia (IH) resulting from sleep apnea causes both systemic and pulmonary hypertension. Enhanced endothelin-1 (ET-1)-induced vasoconstrictor reactivity is thought to play a central role in the systemic hypertensive response to IH. However, whether IH similarly increases pulmonary vasoreactivity and the signaling mechanisms involved are unknown. The objective of the present study was to test the hypothesis that IH augments ET-1-induced pulmonary vasoconstrictor reactivity through a PKCβ-dependent signaling pathway. Responses to ET-1 were assessed in endothelium-disrupted, pressurized pulmonary arteries (～150 μm inner diameter) from eucapnic-IH [(E-IH) 3 min cycles, 5% O(2)-5% CO(2)/air flush, 7 h/day; 4 wk] and sham (air-cycled) rats. Arteries were loaded with fura-2 AM to monitor vascular smooth muscle (VSM) intracellular Ca(2+) concentration ([Ca(2+)](i)). E-IH increased vasoconstrictor reactivity without altering Ca(2+) responses, suggestive of myofilament Ca(2+) sensitization. Consistent with our hypothesis, inhibitors of both PKCα/β (myr-PKC) and PKCβ (LY-333-531) selectively decreased vasoconstriction to ET-1 in arteries from E-IH rats and normalized responses between groups, whereas Rho kinase (fasudil) and PKCδ (rottlerin) inhibition were without effect. Although E-IH did not alter arterial PKCα/β mRNA or protein expression, E-IH increased basal PKCβI/II membrane localization and caused ET-1-induced translocation of these isoforms away from the membrane fraction. We conclude that E-IH augments pulmonary vasoconstrictor reactivity to ET-1 through a novel PKCβ-dependent mechanism that is independent of altered PKC expression. These findings provide new insights into signaling mechanisms that contribute to vasoconstriction in the hypertensive pulmonary circulation.     

Leukotriene B4 pathway activation and atherosclerosis in obstructive sleep apnea

Leukotriene B(4) (LTB(4)) production increases in obstructive sleep apnea syndrome (OSA) and is linked to early vascular remodeling, the mechanism of which is unknown. The objective of this study was to to determine the molecular mechanisms of LTB(4) pathway activation in polymorphonuclear cells (PMNs) and early vascular remodeling in OSA and the specific contribution of intermittent hypoxia (IH). PMNs were isolated from 120 OSA patients and 33 healthy subjects and used for measurements of LTB(4) production, determination of mRNA and protein expression levels, or exposed for four cycles of in vitro IH. PMNs derived from OSA patients exhibited increased LTB(4) production, for which apnea-hypopnea index was an independent predictor (P=0.042). 5-Lipoxygenase-activating protein (FLAP) mRNA and protein increased significantly in PMNs from OSA patients versus controls and were associated with carotid luminal diameter and intima-media thickness. LTB(4) (10 ng/ml) increased IL-6 (P=0.006) and MCP-1 (P=0.002) production in OSA patient monocytes. In vitro exposure of PMNs from controls to IH enhanced FLAP mRNA levels (P= 0.027) and induced a 2.7-fold increase (P=0.028) in LTB(4) secretion compared with PMNs exposed to normoxia. In conclusion, upregulation of FLAP in PMNs in response to IH may participate in early vascular remodeling in OSA patients, suggesting FLAP as a potential therapeutic target for the cardiovascular morbidity associated with OSA.     

Intermittent hypoxia induces transient arousal delay in newborn mice.

Previous studies suggested that defective arousal might be a major mechanism in sleep-disordered breathing such as sudden infant death syndrome and obstructive sleep apnea. In this study, we examined the effects of intermittent hypoxia (IH) on the arousal response to hypoxia in 4-day-old mice. We hypothesized that IH would increase arousal latency, as previously reported in other species, and we measured the concomitant changes in ventilation to shed light on the relationship between breathing and arousal. Arousal was scored according to behavioral criteria. Breathing variables were measured noninvasively by use of whole-body flow plethysmography. In the hypoxic group (n = 14), the pups were exposed to 5% O(2) in N(2) for 3 min and returned to air for 6 min. This test was repeated eight times. The normoxic mice (n = 14) were constantly exposed to normoxia. The hypoxic mice showed a 60% increase in arousal latency (P < 0.0001). Normoxic controls showed virtually no arousals. IH depressed normoxic ventilation below baseline prehypoxic levels, while preserving the ventilatory response to hypoxia. The breathing pattern and arousal responses recovered fully after 2 h of normoxia. We conclude that IH rapidly and reversibly depressed breathing and delayed arousal in newborn mice. Both effects may be due to hypoxia-induced release of inhibitory neurotransmitters acting concomitantly on both functions.     

Intermittent Hypoxia-Induced NF-κB and HO-1 Regulation in Human Endothelial EA.hy926 Cells

Intermittent hypoxia (IH) is a hallmark feature in obstructive sleep apnea (OSA) which is increasingly recognized as an independent risk factor for atherosclerosis. Oxidative stress, inflammation, and cell apoptosis are major pathological events initiating or accelerating atherogenesis. This study addressed whether IH would affect these proatherogenic factors in endothelial cells and the mechanistic pathways involved. EA.hy926 cells were exposed to intermittent normoxia or IH for different numbers of cycles (32, 64, or 96). IH exposure time-dependently raised cellular GSSG/GSH ratio, increased production of IL-6 and IL-8, and accelerated cell apoptosis and death, concurrent with activation of NF-κB and inhibition of Nrf2/HO-1 pathways. At 64 cycles, inhibition of NF-κB attenuated IH-induced cellular oxidative stress and accumulation of inflammatory cytokines in cell culture medium but aggravated IH-induced cell apoptosis, while stimulation of HO-1 suppressed IH-induced cellular oxidative stress and cell apoptosis without affecting accumulation of inflammatory cytokines in cell culture medium. We demonstrated that early stage of exposure to IH-induced oxidative and inflammatory stresses leading to acceleration of cell apoptosis via NF-κB and Nrf2/HO-1 pathways in endothelial cells, suggesting the potential mechanisms for IH-induced vascular pathogenesis, in resemblance to OSA.     

Selected Contribution: Improved anoxic tolerance in rat diaphragm following intermittent hypoxia.

Intermittent hypoxia (IH), associated with obstructive sleep apnea, initiates adaptive physiological responses in a variety of organs. Little is known about its influence on diaphragm. IH was simulated by exposing rats to alternating 15-s cycles of 5% O2 and 21% O2 for 5 min, 9 sets/h, 8 h/day, for 10 days. Controls did not experience IH. Diaphragms were excised 20-36 h after IH. Diaphragm bundles were studied in vitro or analyzed for myosin heavy chain isoform composition. No differences in maximum tetanic stress were observed between groups. However, peak twitch stress (P < 0.005), twitch half-relaxation time (P < 0.02), and tetanic stress at 20 or 30 Hz (P < 0.05) were elevated in IH. No differences in expression of myosin heavy chain isoforms or susceptibility to fatigue were seen. Contractile function after 30 min of anoxia (95% N2-5% CO2) was markedly preserved at all stimulation frequencies during IH and at low frequencies after 15 min of reoxygenation. Anoxia-induced increases in passive muscle force were eliminated in the IH animals (P < 0.01). These results demonstrate that IH induces adaptive responses in the diaphragm that preserve its function in anoxia.     

Role of high-fat diet in stress response of Drosophila

Obesity is associated with many diseases, one of the most common being obstructive sleep apnea (OSA), which in turn leads to blood gas disturbances, including intermittent hypoxia (IH). Obesity, OSA and IH are associated with metabolic changes, and while much mammalian work has been done, mechanisms underlying the response to IH, the role of obesity and the interaction of obesity and hypoxia remain unknown. As a model organism, Drosophila offers tremendous power to study a specific phenotype and, at a subsequent stage, to uncover and study fundamental mechanisms, given the conservation of molecular pathways. Herein, we characterize the phenotype of Drosophila on a high-fat diet in normoxia, IH and constant hypoxia (CH) using triglyceride and glucose levels, response to stress and lifespan. We found that female flies on a high-fat diet show increased triglyceride levels (p<0.001) and a shortened lifespan in normoxia, IH and CH. Furthermore, flies on a high-fat diet in normoxia and CH show diminished tolerance to stress, with decreased survival after exposure to extreme cold or anoxia (p<0.001). Of interest, IH seems to rescue this decreased cold tolerance, as flies on a high-fat diet almost completely recovered from cold stress following IH. We conclude that the cross talk between hypoxia and a high-fat diet can be either deleterious or compensatory, depending on the nature of the hypoxic treatment.     

Differential effects of chronic hypoxia and intermittent hypocapnic and eucapnic hypoxia on pulmonary vasoreactivity.

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension (PH) and right heart failure, similar to chronic sustained hypoxia (CH). Supplemental CO(2), however, attenuates hypoxic PH. We therefore hypothesized that, similar to CH, IH elicits PH and associated increases in arterial endothelial nitric oxide synthase (eNOS) expression, ionomycin-dependent vasodilation, and receptor-mediated pulmonary vasoconstriction. We further hypothesized that supplemental CO(2) inhibits these responses to IH. To test these hypotheses, we measured eNOS expression by Western blot in intrapulmonary arteries from CH (2 wk, 0.5 atm), hypocapnic IH (H-IH) (3 min cycles of 5% O(2)/air flush, 7 h/day, 2 wk), and eucapnic IH (E-IH) (3 min cycles of 5% O(2), 5% CO(2)/air flush, 7 h/day, 2 wk) rats and their respective controls. Furthermore, vasodilatory responses to the calcium ionophore ionomycin and vasoconstrictor responses to the thromboxane mimetic U-46619 were measured in isolated saline-perfused lungs from each group. Hematocrit, arterial wall thickness, and right ventricle-to-total ventricle weight ratios were additionally assessed as indexes of polycythemia, arterial remodeling, and PH, respectively. Consistent with our hypotheses, E-IH resulted in attenuated polycythemia, arterial remodeling, RV hypertrophy, and eNOS upregulation compared with H-IH. However, in contrast to CH, neither H-IH nor E-IH increased ionomycin-dependent vasodilation. Furthermore, H-IH and E-IH similarly augmented U-46619-induced pulmonary vasoconstriction but to a lesser degree than CH. We conclude that maintenance of eucapnia decreases IH-induced PH and upregulation of arterial eNOS. In contrast, increases in pulmonary vasoconstrictor reactivity following H-IH are unaltered by exposure to supplemental CO(2).     

Effects of different acute hypoxic regimens on tissue oxygen profiles and metabolic outcomes

Obstructive sleep apnea (OSA) causes intermittent hypoxia (IH) during sleep. Both obesity and OSA are associated with insulin resistance and systemic inflammation, which may be attributable to tissue hypoxia. We hypothesized that a pattern of hypoxic exposure determines both oxygen profiles in peripheral tissues and systemic metabolic outcomes, and that obesity has a modifying effect. Lean and obese C57BL6 mice were exposed to 12 h of intermittent hypoxia 60 times/h (IH60) [inspired O? fraction (Fi(O?)) 21-5%, 60/h], IH 12 times/h (Fi(O?) 5% for 15 s, 12/h), sustained hypoxia (SH; Fi(O?) 10%), or normoxia while fasting. Tissue oxygen partial pressure (Pti(O?)) in liver, skeletal muscle and epididymal fat, plasma leptin, adiponectin, insulin, blood glucose, and adipose tumor necrosis factor-α (TNF-α) were measured. In lean mice, IH60 caused oxygen swings in the liver, whereas fluctuations of Pti(O?) were attenuated in muscle and abolished in fat. In obese mice, baseline liver Pti(O?) was lower than in lean mice, whereas muscle and fat Pti(O?) did not differ. During IH, Pti(O?) was similar in obese and lean mice. All hypoxic regimens caused insulin resistance. In lean mice, hypoxia significantly increased leptin, especially during SH (44-fold); IH60, but not SH, induced a 2.5- to 3-fold increase in TNF-α secretion by fat. Obesity was associated with striking increases in leptin and TNF-α, which overwhelmed effects of hypoxia. In conclusion, IH60 led to oxygen fluctuations in liver and muscle and steady hypoxia in fat. IH and SH induced insulin resistance, but inflammation was increased only by IH60 in lean mice. Obesity caused severe inflammation, which was not augmented by acute hypoxic regimens.     

Influence of arterial O2 on the susceptibility to posthyperventilation apnea during sleep.

To investigate the contribution of the peripheral chemoreceptors to the susceptibility to posthyperventilation apnea, we evaluated the time course and magnitude of hypocapnia required to produce apnea at different levels of peripheral chemoreceptor activation produced by exposure to three levels of inspired P(O2). We measured the apneic threshold and the apnea latency in nine normal sleeping subjects in response to augmented breaths during normoxia (room air), hypoxia (arterial O2 saturation = 78-80%), and hyperoxia (inspired O2 fraction = 50-52%). Pressure support mechanical ventilation in the assist mode was employed to introduce a single or multiple numbers of consecutive, sigh-like breaths to cause apnea. The apnea latency was measured from the end inspiration of the first augmented breath to the onset of apnea. It was 12.2 +/- 1.1 s during normoxia, which was similar to the lung-to-ear circulation delay of 11.7 s in these subjects. Hypoxia shortened the apnea latency (6.3 +/- 0.8 s; P < 0.05), whereas hyperoxia prolonged it (71.5 +/- 13.8 s; P < 0.01). The apneic threshold end-tidal P(CO2) (Pet(CO2)) was defined as the Pet(CO2)) at the onset of apnea. During hypoxia, the apneic threshold Pet(CO2) was higher (38.9 +/- 1.7 Torr; P < 0.01) compared with normoxia (35.8 +/- 1.1; Torr); during hyperoxia, it was lower (33.0 +/- 0.8 Torr; P < 0.05). Furthermore, the difference between the eupneic Pet(CO2) and apneic threshold Pet(CO2) was smaller during hypoxia (3.0 +/- 1.0 Torr P < 001) and greater during hyperoxia (10.6 +/- 0.8 Torr; P < 0.05) compared with normoxia (8.0 +/- 0.6 Torr). Correspondingly, the hypocapnic ventilatory response to CO2 below the eupneic Pet(CO2) was increased by hypoxia (3.44 +/- 0.63 l.min(-1).Torr(-1); P < 0.05) and decreased by hyperoxia (0.63 +/- 0.04 l.min(-1).Torr(-1); P < 0.05) compared with normoxia (0.79 +/- 0.05 l.min(-1).Torr(-1)). These findings indicate that posthyperventilation apnea is initiated by the peripheral chemoreceptors and that the varying susceptibility to apnea during hypoxia vs. hyperoxia is influenced by the relative activity of these receptors.     

Chronic intermittent hypoxia exposure improves left ventricular contractility in transgenic mice with heart failure

We previously reported the unexpected finding that 4 wk of exposure to intermittent hypoxia (IH), which simulates the hypoxic stress of obstructive sleep apnea, improved LV cardiac function in healthy, lean C57BL/6J mice. The purpose of the present study was to assess the impact of 4 wk of IH on cardiac function in a transgenic murine model that exhibits a natural history of heart failure. We hypothesized that IH exposure would exacerbate cardiac decompensation in heart failure. Adult male FVB (wild type) and transgenic mice with cardiac overexpression of tumor necrosis factor α (TNF-αTG) at 10-12 wk of age were exposed to 4 wk of IH (nadir inspired oxygen 5-6% at 60 cycles/h for 12 h during light period) or intermittent air (IA) as control. Cardiac function was assessed by echocardiography and pressure-volume loop analyses, and mRNA and protein expression were performed on ventricular homogenates. TNF-αTG mice exposed to IA exhibited impaired LV contractility and increased LV dilation associated with markedly elevated cardiac expression of atrial natriuretic peptide and brain natriuretic peptide compared with wild-type mice. When wild-type FVB mice were exposed to IH, they exhibited increases in arterial pressure and dP/dt(max), consistent with our previous report in C57BL/6J mice. Surprisingly, we found that TNF-αTG mice exposed to IH showed a reduction in end-diastolic volume (38.7 ± 3.8 to 22.2 ± 2.1 ul; P < 0.01) and an increase in ejection fraction (29.4 ± 2.5 to 41.9 ± 3.1%; P < 0.05). In contrast to our previous study in C56Bl/6J mice, neither FVB nor TNF-αTG mice exhibited an upregulation in β-adrenergic expression or cAMP in response to IH exposure. We conclude that 4 wk of exposure to IH in mice induces adaptive responses that improve cardiac function in not only healthy animals but also in animals with underlying heart failure.     

Vascular and Hepatic Impact of Short-Term Intermittent Hypoxia in a Mouse Model of Metabolic Syndrome

BackgroundExperimental models of intermittent hypoxia (IH) have been developed during the last decade to investigate the consequences of obstructive sleep apnea. IH is usually associated with detrimental metabolic and vascular outcomes. However, paradoxical protective effects have also been described depending of IH patterns and durations applied in studies. We evaluated the impact of short-term IH on vascular and metabolic function in a diet-induced model of metabolic syndrome (MS).MethodsMice were fed either a standard diet or a high fat diet (HFD) for 8 weeks. During the final 14 days of each diet, animals were exposed to either IH (1 min cycle, FiO₂ 5% for 30s, FiO₂ 21% for 30s; 8 h/day) or intermittent air (FiO₂ 21%). Ex-vivo vascular reactivity in response to acetylcholine was assessed in aorta rings by myography. Glucose, insulin and leptin levels were assessed, as well as serum lipid profile, hepatic mitochondrial activity and tissue nitric oxide (NO) release.ResultsMice fed with HFD developed moderate markers of dysmetabolism mimicking MS, including increased epididymal fat, dyslipidemia, hepatic steatosis and endothelial dysfunction. HFD decreased mitochondrial complex I, II and IV activities and increased lactate dehydrogenase (LDH) activity in liver. IH applied to HFD mice induced a major increase in insulin and leptin levels and prevented endothelial dysfunction by restoring NO production. IH also restored mitochondrial complex I and IV activities, moderated the increase in LDH activity and liver triglyceride accumulation in HFD mice.ConclusionIn a mouse model of MS, short-term IH increases insulin and leptin levels, restores endothelial function and mitochondrial activity and limits liver lipid accumulation.     

Ventilatory long-term facilitation in mice can be observed during both sleep and wake periods and depends on orexin.

Respiratory long-term facilitation (LTF) is a long-lasting (>1 h) augmentation of respiratory motor output that occurs even after cessation of hypoxic stimuli, is serotonin-dependent, and is thought to prevent sleep-disordered breathing such as sleep apnea. Raphe nuclei, which modulate several physiological functions through serotonin, receive dense projections from orexin-containing neurons in the hypothalamus. We examined possible contributions of orexin to ventilatory LTF by measuring respiration in freely moving prepro-orexin knockout mice (ORX-KO) and wild-type (WT) littermates before, during, and after exposure to intermittent hypoxia (IH; 5 x 5 min at 10% O2), sustained hypoxia (SH; 25 min at 10% O2), or sham stimulation. Respiratory data during quiet wakefulness (QW), slow wave sleep (SWS), and rapid-eye-movement sleep were separately calculated. Baseline ventilation before hypoxic stimulation and acute responses during stimulation did not differ between the ORX-KO and WT mice, although ventilation depended on vigilance state. Whereas the WT showed augmented minute ventilation (by 20.0 +/- 4.5% during QW and 26.5 +/- 5.3% during SWS; n = 8) for 2 h following IH, ORX-KO showed no significant increase (by -3.1 +/- 4.6% during QW and 0.3 +/- 5.2% during SWS; n = 8). Both genotypes showed no LTF after SH or sham stimulation. Sleep apnea indexes did not change following IH, even when LTF appeared in the WT mice. We conclude that LTF occurs during both sleep and wake periods, that orexin is necessary for eliciting LTF, and that LTF cannot prevent sleep apnea, at least in mice.     

Intermittent hypoxia augments pulmonary vascular smooth muscle reactivity to NO: regulation by reactive oxygen species

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension. IH causes oxidative stress that may limit bioavailability of the endothelium-derived vasodilator nitric oxide (NO) and thus contribute to this hypertensive response. We therefore hypothesized that increased vascular superoxide anion (O(2)(-)) generation reduces NO-dependent pulmonary vasodilation following IH. To test this hypothesis, we examined effects of the O(2)(-) scavenger tiron on vasodilatory responses to the endothelium-dependent vasodilator ionomycin and the NO donor S-nitroso-N-acetylpenicillamine in isolated lungs from hypocapnic-IH (H-IH; 3 min cycles of 5% O(2)/air flush, 7 h/day, 4 wk), eucapnic-IH (E-IH; cycles of 5% O(2), 5% CO(2)/air flush), and sham-treated (air/air cycled) rats. Next, we assessed effects of endogenous O(2)(-) on NO- and cGMP-dependent vasoreactivity and measured O(2)(-) levels using the fluorescent indicator dihydroethidium (DHE) in isolated, endothelium-disrupted small pulmonary arteries from each group. Both E-IH and H-IH augmented NO-dependent vasodilation; however, enhanced vascular smooth muscle (VSM) reactivity to NO following H-IH was masked by an effect of endogenous O(2)(-). Furthermore, H-IH and E-IH similarly increased VSM sensitivity to cGMP, but this response was independent of either O(2)(-) generation or altered arterial protein kinase G expression. Finally, both H-IH and E-IH increased arterial O(2)(-) levels, although this response was more pronounced following H-IH, and H-IH exposure resulted in greater protein tyrosine nitration indicative of increased NO scavenging by O(2)(-). We conclude that IH increases pulmonary VSM sensitivity to NO and cGMP. Furthermore, endogenous O(2)(-) limits NO-dependent vasodilation following H-IH through an apparent reduction in bioavailable NO.     

Neonatal intermittent hypoxia and hypertension

Obstructive apnea during sleep is accompanied by intermittent hypoxia (IH) leading to hypertension and other cardiovascular disturbances. A comparative evaluation of long-term effects of the neonatal IH on the cardiovascular functions was performed in normotensive Sprague-Dawley and spontaneously hypertensive rats (SHR). The newborn rats were placed for 30 days to conditions of IH (8% and 21% O₂, alternating every 90 s for 12 h/day). Control groups of rats were constantly kept in normoxia. By 6 months, in the spontaneously hypertensive rats exposed to IH at the period of wakefulness there was a statistically significant increase (as compared with control) of the systolic (185.8 ± 1.7 and 169.9 ± 1.4 mm Hg, correspondingly, p < 0.010 and the diastolic pressure (96.2 ± 4.9 and 86.0 ± 2.6 mm Hg, correspondingly, p < 0.01). During sleep, the systolic and diastolic pressure in these rats was higher than in control animals by 10 mm Hg (p < 0.01) and 12 mm Hg (p < 0.01), its decrease during sleep being absent. In SHR submitted to IH there was an increase in the power ratio of the heart rate variability from 0.9 ± 0.15 to 1.5 ± 0.17, which indicates a shift of the sympathico-parasympathetic balance in this group towards predominance of the sympathetic component. In the Sprague-Dawley rats exposed to neonatal hypoxia, the above-indicated changes were not prominent. These peculiarities of the hypertensive rats allow establishing connection of the genetic factor with the sympathetic mechanism providing long-term consequences of the neonatal IH for the cardiovascular control in the SHR.     

Chronic Intermittent Hypoxia Exerts CNS Region-Specific Effects on Rat Microglial Inflammatory and TLR4 Gene Expression

Intermittent hypoxia (IH) during sleep is a hallmark of sleep apnea, causing significant neuronal apoptosis, and cognitive and behavioral deficits in CNS regions underlying memory processing and executive functions. IH-induced neuroinflammation is thought to contribute to cognitive deficits after IH. In the present studies, we tested the hypothesis that IH would differentially induce inflammatory factor gene expression in microglia in a CNS region-dependent manner, and that the effects of IH would differ temporally. To test this hypothesis, adult rats were exposed to intermittent hypoxia (2 min intervals of 10.5% O₂) for 8 hours/day during their respective sleep cycles for 1, 3 or 14 days. Cortex, medulla and spinal cord tissues were dissected, microglia were immunomagnetically isolated and mRNA levels of the inflammatory genes iNOS, COX-2, TNFα, IL-1β and IL-6 and the innate immune receptor TLR4 were compared to levels in normoxia. Inflammatory gene expression was also assessed in tissue homogenates (containing all CNS cells). We found that microglia from different CNS regions responded to IH differently. Cortical microglia had longer lasting inflammatory gene expression whereas spinal microglial gene expression was rapid and transient. We also observed that inflammatory gene expression in microglia frequently differed from that in tissue homogenates from the same region, indicating that cells other than microglia also contribute to IH-induced neuroinflammation. Lastly, microglial TLR4 mRNA levels were strongly upregulated by IH in a region- and time-dependent manner, and the increase in TLR4 expression appeared to coincide with timing of peak inflammatory gene expression, suggesting that TLR4 may play a role in IH-induced neuroinflammation. Together, these data indicate that microglial-specific neuroinflammation may play distinct roles in the effects of intermittent hypoxia in different CNS regions.     

Hypoxemia alone does not explain blood pressure elevations after obstructive apneas

In patients with obstructive sleep apnea (OSA), substantial elevations of systemic blood pressure (BP) and depressions of oxyhemoglobin saturation (SaO2) accompany apnea termination. The causes of the BP elevations, which contribute significantly to nocturnal hypertension in OSA, have not been defined precisely. To assess the relative contribution of arterial hypoxemia, we observed mean arterial pressure (MAP) changes following obstructive apneas in 11 OSA patients during non-rapid-eye-movement (NREM) sleep and then under three experimental conditions: 1) apnea with O2 supplementation; 2) hypoxemia (SaO2 80%) without apnea; and 3) arousal from sleep with neither hypoxemia nor apnea. We found that apneas recorded during O2 supplementation (SaO2 nadir 93.6% +/- 2.4; mean +/- SD) in six subjects were associated with equivalent postapneic MAP elevations compared with unsupplemented apneas (SaO2 nadir 79-82%): 18.8 +/- 7.1 vs. 21.3 +/- 9.2 mmHg (mean change MAP +/- SD); in the absence of respiratory and sleep disruption in eight subjects, hypoxemia was not associated with the BP elevations observed following apneas: -5.4 +/- 19 vs. 19.1 +/- 7.8 mmHg (P less than 0.01); and in five subjects, auditory arousal alone was associated with MAP elevation similar to that observed following apneas: 24.0 +/- 8.1 vs. 22.0 +/- 6.9 mmHg. We conclude that in NREM sleep postapneic BP elevations are not primarily attributable to arterial hypoxemia. Other factors associated with apnea termination, including arousal from sleep, reinflation of the lungs, and changes of intrathoracic pressure, may be responsible for these elevations.