Obstructive sleep apnea and chronic intermittent hypoxia: a review

Hypoxia is an important topic both physiologically and clinically. Traditionally, physiology research has been focusing on the effect of acute and chronic sustained hypoxia and human adaptive response to high altitude. In the past 20 years, genetic studies by many have expanded our understanding of hypoxia to the molecular level. However, in contrast to our extensive knowledge about acute and chronic sustained hypoxia, we know relatively little about the effect of chronic intermittent hypoxia (CIH). In recent years, CIH has attracted more research attention because of the increasing prevalence of obesity and obstructive sleep apnea (OSA) in the western countries. Clinically, CIH is commonly seen in patients with sleep-disordered breathing including OSA, Cheyne-Stokes respiration and nocturnal hypoventilation. It was estimated that for OSA of at least mild severity prevalence estimates range from 3 to 28% in the general population. OSA is characterized by recurrent upper airway collapse during sleep leading to intermittent nocturnal hypoxia and sleep fragmentation. OSA is associated with significant mortality and morbidity including neurocognitive dysfunction, hypertension, many cardiovascular disorders and metabolic disorders such as diabetes and metabolic syndrome. The intermittent hypoxia in OSA closely mimics what is seen in the ischemia-reperfusion injury. Experimentally, there is no universally accepted definition for CIH. Laboratory protocols vary greatly in duration of hypoxia exposure, numbers of hypoxia episodes per day and the total number of days of exposure. Despite the lack of a uniform definition, recent data suggest that CIH may lead to multiple long-term pathophysiologic consequences similar to what we see in patients with OSA. Recent evidences also demonstrate that there are remarkable differences in the response of the physiologic systems to sustained hypoxia and intermittent hypoxia. This review is aimed to briefly discuss the clinical significance of sleep-disordered breathing and our current understanding of CIH.     

Antioxidant responses to chronic hypoxia in the rat cerebellum and pons

Obstructive sleep apnea (OSA) is characterized by chronic intermittent hypoxia (CIH) and sleep fragmentation and deprivation. Exposure to CIH results in oxidative stress in the cortex, hippocampus and basal forebrain of rats and mice. We show that sustained and intermittent hypoxia induces antioxidant responses, an indicator of oxidative stress, in the rat cerebellum and pons. Increased glutathione reductase (GR) activity and thiobarbituric acid reactive substance (TBARS) levels were observed in the pons and cerebellum of rats exposed to CIH or chronic sustained hypoxia (CSH) compared with room air (RA) controls. Exposure to CIH or CSH increased GR activity in the pons, while exposure to CSH increased the level of TBARS in the cerebellum. The level of TBARS was increased to a greater extent after exposure to CSH than to CIH in the cerebellum and pons. Increased superoxide dismutase activity (SOD) and decreased total glutathione (GSHt) levels were observed after exposure to CIH compared with CSH only in the pons. We have previously shown that prolonged sleep deprivation decreased SOD activity in the rat hippocampus and brainstem, without affecting the cerebellum, cortex or hypothalamus. We therefore conclude that sleep deprivation and hypoxia differentially affect antioxidant responses in different brain regions.     

Effects of Cyclic Intermittent Hypoxia on ET-1 Responsiveness and Endothelial Dysfunction of Pulmonary Arteries in Rats

Obstructive sleep apnoea (OSA) is a risk factor for cardiovascular disorders and in some cases is complication of pulmonary hypertension. We simulated OSA by exposing rats to cyclic intermittent hypoxia (CIH) to investigate its effect on pulmonary vascular endothelial dysfunction. Sprague-Dawley Rats were exposed to CIH (FiO₂ 9% for 1 min, repeated every 2 min for 8 h/day, 7 days/wk for 3 wk), and the pulmonary arteries of normoxia and CIH treated rats were analyzed for expression of endothelin-1 (ET-1) and ET receptors by histological, immunohistochemical, RT-PCR and Western Blot analyses, as well as for contractility in response to ET-1. In the pulmonary arteries, ET-1 expression was increased, and ET-1 more potently elicited constriction of the pulmonary artery in CIH rats than in normoxic rats. Exposure to CIH induced marked endothelial cell damage associated with a functional decrease of endothelium-dependent vasodilatation in the pulmonary artery. Compared with normoxic rats, ET_A receptor expression was increased in smooth muscle cells of the CIH rats, while the expression of ET_B receptors was decreased in endothelial cells. These results demonstrated endothelium-dependent vasodilation was impaired and the vasoconstrictor responsiveness increased by CIH. The increased responsiveness to ET-1 induced by intermittent hypoxia in pulmonary arteries of rats was due to increased expression of ET_A receptors predominantly, meanwhile, decreased expression of ET_B receptors in the endothelium may also participate in it.     

Impact of chronic intermittent hypoxia on the long non-coding RNA and mRNA expression profiles in myocardial infarction

Chronic intermittent hypoxia (CIH) is the primary feature of obstructive sleep apnoea (OSA), a crucial risk factor for cardiovascular diseases. Long non-coding RNAs (lncRNAs) in myocardial infarction (MI) pathogenesis have drawn considerable attention. However, whether CIH participates in the modulation of lncRNA profiles during MI is yet unclear. To investigate the influence of CIH on MI, cardiac damage was assessed by histology and echocardiography, and lncRNA and mRNA integrated microarrays were screened. MI mouse model showed myocardial hypertrophy, aggravated inflammation and fibrosis, and compromised left ventricle function under CIH. Compared with normoxia, 644 lncRNAs and 1084 differentially expressed mRNAs were identified following CIH for 4 weeks, whereas 1482 lncRNAs and 990 mRNAs were altered at 8 weeks. Strikingly, reoxygenation after CIH markedly affected 1759 lncRNAs and 778 mRNAs. Of these, 11 lncRNAs modulated by CIH were restored after reoxygenation and were validated by qPCR. The GO terms and KEGG pathways of genes varied significantly by CIH. lncRNA-mRNA correlation further showed that lncRNAs, NONMMUT032513 and NONMMUT074571 were positively correlated with ZEB1 and negatively correlated with Cmbl. The current results demonstrated a causal correlation between CIH and lncRNA alternations during MI, suggesting that lncRNAs might be responsible for MI aggravation under CIH.     

Chronic intermittent hypoxia causes hepatitis in a mouse model of diet-induced fatty liver

Obstructive sleep apnea (OSA) causes chronic intermittent hypoxia (CIH) during sleep. OSA is associated with nonalcoholic steatohepatitis (NASH) in obese individuals and may contribute to progression of nonalcoholic fatty liver disease from steatosis to NASH. The purpose of this study was to examine whether CIH induces inflammatory changes in the liver in mice with diet-induced hepatic steatosis. C57BL/6J mice (n = 8) on a high-fat, high-cholesterol diet were exposed to CIH for 6 mo and were compared with mice on the same diet exposed to intermittent air (control; n = 8). CIH caused liver injury with an increase in serum ALT (461 +/- 58 U/l vs. 103 +/- 16 U/l in the control group; P < 0.01) and AST (637 +/- 37 U/l vs. 175 +/- 13 U/l in the control group; P < 0.001), whereas alkaline phosphatase and total bilirubin levels were unchanged. Histology revealed hepatic steatosis in both groups, with mild accentuation of fat staining in the zone 3 hepatocytes in mice exposed to CIH. Animals exposed to CIH exhibited lobular inflammation and fibrosis in the liver, which were not evident in control mice. CIH caused significant increases in lipid peroxidation in serum and liver tissue; significant increases in hepatic levels of myeloperoxidase and proinflammatory cytokines IL-1beta, IL-6, and CXC chemokine MIP-2; a trend toward an increase in TNF-alpha; and an increase in alpha1(I)-collagen mRNA. We conclude that CIH induces lipid peroxidation and inflammation in the livers of mice on a high-fat, high-cholesterol diet.     

Cardiovascular and ventilatory acclimatization induced by chronic intermittent hypoxia: a role for the carotid body in the pathophysiology of sleep apnea

Patients with obstructive sleep apnea (OSA) show augmented ventilatory, sympathetic and cardiovascular responses to hypoxia. The facilitatory effect of chronic intermittent hypoxia (CIH) on the hypoxic ventilatory response has been attributed to a potentiation of the carotid body (CB) chemosensory response to hypoxia. However, it is a matter of debate whether the effects induced by CIH on ventilatory responses to hypoxia are due to an enhanced CB activity. Recently, we studied the effects of short cyclic hypoxic episodes on cat cardiorespiratory reflexes, heart rate variability, and CB chemosensory activity. Cats were exposed to cyclic hypoxic episodes repeated during 8 hours for 4 days. Our results showed that CIH selectively enhanced ventilatory and carotid chemosensory responses to acute hypoxia. Exposure to CIH did not increase basal arterial pressure, heart rate, or their changes induced by acute hypoxia. However, the spectral analysis of heart rate variability of CIH cats showed a marked increase of the low/high frequency ratio and an increased variability in the low frequency band of heart rate variability, similar to what is observed in OSA patients. Thus, it is likely that the enhanced CB reactivity to hypoxia may contribute to the augmented ventilatory response to hypoxia.     

Gene expression in mouse brain following chronic hypoxia: role of sarcospan in glial cell death.

Hypoxia is a hallmark of respiratory, neurological, or hematological diseases as well as life at high altitude. For example, chronic constant hypoxia (CCH) occurs in chronic lung diseases or at high altitude, whereas chronic intermittent hypoxia (CIH) occurs in diseases such as sleep apnea or sickle cell disease. Despite the fact that such conditions are frequent, the cellular and molecular mechanisms underlying the effect of hypoxia, whether constant or intermittent, are not well understood. In this study, we first determined the effect of CCH and CIH on global gene expression in different regions of mouse brain using microarrays and then investigated the biological role of genes of interest. We found that: 1) in the cortical region, the expression level of 80 genes was significantly altered by CIH (16 up- and 64 downregulated), and this number increased to 137 genes following CCH (34 up- and 103 downregulated); 2) a similar number of gene alterations was identified in the hippocampal area, and the majority of the changes in this region were upregulations; 3) two genes (Sspn and Ttc27) were downregulated in both brain regions and following both treatments; and 4) RNA interference-mediated knockdown of Sspn increased cell death in hypoxia in a cell culture system. We conclude that CIH or CCH induced significant and distinguishable alterations in gene expression in cortex and hippocampus and that Sspn seems to play a critical role in inducing cell death under hypoxic conditions.     

Endothelin-1-Mediated Mechanisms in the Carotid Body Modulates Cardiovascular Responses in Rats Exposed to Chronic Intermittent Hypoxia

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea (OSA), is associated with hypertension. The increased of carotid body (CB) sensitivity due to enhanced sympathetic efferent may be mainly responsible for the elevation of blood pressure. Accordingly, we studied this effect of Endothelin-1 (ET-1)-induced CB chemosensory response to CIH, as a vasoactive peptide expressed in CB. The purpose of this study was to investigate the mean arterial blood pressure (MAP) and renal sympathetic nerve activity (RSNA) responses in CIH group by injecting ET-1 to directly stimulate CB chemoreceptor. Furthermore, whether ET receptor-mediated PKC and p³⁸MAPK signaling pathway was involved in CIH-induced CB activation was also studied. Male Sprague–Dawley rats were exposed to CIH (8 h/day for 3 weeks) and the MAP and RSNA were recorded in CIH rats and Sham rats. Our results demonstrated that ET-1-induced MAP and RSNA increase were mainly mediated by ET_A receptor activation in CB chemosensory after CIH exposure. Moreover, P³⁸MAPK and PKC signaling pathway might be involved in ET-1-induced increase of MAP and RSNA in CIH group, which provided a potential therapeutic target of OSA.

     

Diaphragm Muscle Remodeling in a Rat Model of Chronic Intermittent Hypoxia

Respiratory muscle remodeling occurs in human sleep apnea—a common respiratory disorder characterized by chronic intermittent hypoxia (CIH) due to recurrent apnea during sleep. We sought to determine if CIH causes remodeling in rat sternohyoid (upper airway dilator) and diaphragm muscles. Adult male Wistar rats were exposed to CIH (n=8), consisting of 90 sec of hypoxia (5% at the nadir; SaO₂ ~80%)/90 sec of normoxia, 8 hr per day, for 7 consecutive days. Sham animals (n=8) were exposed to alternating air/air cycles in parallel. The effect of CIH on myosin heavy-chain (MHC) isoform (1, 2a, 2x, 2b) distribution, sarcoplasmic reticulum calcium ATPase (SERCA) isoform distribution, succinate dehydrogenase activity, glycerol phosphate dehydrogenase activity, and Na⁺/K⁺ ATPase pump content was determined. Sternohyoid muscle structure was unaffected by CIH treatment. CIH did not alter oxidative/glycolytic capacity or the Na⁺/K⁺-ATPase pump content of the diaphragm. CIH significantly increased the areal density of MHC 2b fibers in the rat diaphragm, and this was associated with a shift in SERCA proteins from SERCA2 to SERCA1. We conclude that CIH causes a slow-to-fast fiber transition in the rat diaphragm after just 7 days of treatment. Respiratory muscle functional remodeling may drive aberrant functional plasticity such as decreased muscle endurance, which is a feature of human sleep apnea.     

2-Methoxyestradiol attenuates chronic-intermittent-hypoxia-induced pulmonary hypertension through regulating microRNA-223

Pulmonary hypertension (PH) is prevalent in patients with obstructive sleep apnea (OSA) syndrome, and coexistence of PH and OSA indicates a worse prognosis and higher mortality. Chronic intermittent hypoxia (CIH) is the key pathogenesis of OSA. Also, microRNA-223 (miR-223) plays a role in the regulation of CIH-induced PH process. However, the detailed mechanism of CIH inducing PH is still unclear. This study aimed to investigate the pathological process of CIH associated PH and explore the potential therapeutic methods. In this study, adult Sprague–Dawley rats were exposed to CIH or normoxic (N) conditions with 2-methoxyestradiol (2-Me) or vehicle treatment for 6 weeks. The results showed that 2-Me treatment reduced the progression of pulmonary angiogenesis in CIH rats, and alleviated proliferation, cellular migration, and reactive oxygen species formation was induced by CIH in pulmonary artery smooth muscle cells (PASMCs). CIH decreased the expression of miR-223, whereas 2-Me reversed the downregulation of miR-223 both in vivo and in vitro. Furthermore, the antiangiogenic effect of 2-Me observed in PASMCs was abrogated by miR-223 inhibitor, while enhanced by miR-223 mimic. These findings suggested that miR-223 played an important role in the process of CIH inducing PH, and 2-Me might reverse CIH-induced PH via upregulating miR-223.     

慢性间歇性缺氧对大鼠肺组织凋亡相关基因Bcl-2、Bax表达的影响

目的探讨凋亡(apoptosis)相关基因Bcl-2、Bax在慢性间歇性缺氧(chronic intermittent hypoxia,CIH)大鼠肺组织中的表达,明确其与阻塞性睡眠呼吸暂停低通气综合征(obstructive sleep apnea hypopnea syndrome,OSAHS)肺部病变的关系.方法取健康雄性Sprague-Dawley(S-D)大鼠20只,随机分为正常对照组(A组)和CIH组(B组),每组10只,根据实验要求,将B组大鼠暴露于CIH的环境中,8h/d(上午9时至下午5时),共5w,以模拟OSAHS患者CIH的特征.A组大鼠常规饲养,不予以任何处理.采用免疫组织化学方法(SP法)检测大鼠肺组织中Bcl-2、Bax蛋白的表达.结果大鼠肺组织中阳性细胞表达部位主要集中于支气管上皮细胞和肺泡上皮细胞.正常大鼠Bcl-2、Bax蛋白均有基础低表达,经CIH处理后可见大鼠支气管上皮细胞和肺泡上皮细胞Bcl-2、Bax蛋白表达的上调和Bax/bcl-2比值的升高.结论 CIH可导致大鼠肺组织细胞凋亡的增加,凋亡参与了CIH大鼠肺部病变的病理生理过程;Bcl-2和Bax这一对凋亡抑制和促进基因参与了CIH过程中大鼠肺组织细胞凋亡的调控.     

Hyperlipidemia and lipid peroxidation are dependent on the severity of chronic intermittent hypoxia.

Obstructive sleep apnea (OSA) is characterized by chronic intermittent hypoxia (CIH) and associated with dysregulation of lipid metabolisms and atherosclerosis. Causal relationships between OSA and metabolic abnormalities have not been established because of confounding effects of underlying obesity. The goal of the study was to determine if CIH causes lipid peroxidation and dyslipidemia in the absence of obesity and whether the degrees of dyslipidemia and lipid peroxidation depend on the severity of hypoxia. Lean C57BL/6J mice were exposed to CIH for 4 wk with a fractional inspired O2 (FI(O2)) nadir of either 10% (moderate CIH) or 5% (severe CIH). Mice exposed to severe CIH exhibited significant increases in fasting serum levels of total cholesterol (129 +/- 2.9 vs. 113 +/- 2.8 mg/dl in control mice, P < 0.05) and low-density lipoprotein cholesterol (85.7 +/- 8.9 vs. 56.4 +/- 9.7 mg/dl, P < 0.05) in conjunction with a 1.5- to 2-fold increase in lipoprotein secretion, and upregulation of hepatic stearoyl coenzyme A desaturase 1 (SCD-1). Severe CIH also markedly increased lipid peroxidation in the liver (malondialdehyde levels of 94.4 +/- 5.4 vs. 57.4 +/- 5.2 nmol/mg in control mice, P < 0.001). In contrast, moderate CIH did not induce hyperlipidemia or change in hepatic SCD-1 levels but did cause lipid peroxidation in the liver at a reduced level relative to severe CIH. In conclusion, CIH leads to hypercholesterolemia and lipid peroxidation in the absence of obesity, and the degree of metabolic dysregulation is dependent on the severity of the hypoxic stimulus.     

Left ventricular dysfunction and associated cellular injury in rats exposed to chronic intermittent hypoxia.

Obstructive sleep apnea (OSA) increases cardiovascular morbidity and mortality. We have reported that chronic intermittent hypoxia (CIH), a direct consequence during OSA, leads to left ventricular (LV) remodeling and dysfunction in rats. The present study is to determine LV myocardial cellular injury that is possibly associated with LV global dysfunction. Fifty-six rats were exposed either to CIH (nadir O(2) 4-5%) or sham (handled normoxic controls, HC), 8 h/day for 6 wk. At the end of the exposure, we studied LV global function by cardiac catheterization, and LV myocardial cellular injury by in vitro analyses. Compared with HC, CIH animals demonstrated elevations in mean arterial pressure and LV end-diastolic pressure, but reductions in cardiac output (CIH 141.3 +/- 33.1 vs. HC 184.4 +/- 21.2 ml x min(-1) x kg(-1), P < 0.01), maximal rate of LV pressure rise in systole (+dP/dt), and maximal rate of LV pressure fall in diastole (-dP/dt). CIH led to significant cell injury in the left myocardium, including elevated LV myocyte size, measured by cell surface area (CIH 3,564 +/- 354 vs. HC 2,628 +/- 242 microm(2), P < 0.05) and cell length (CIH 148 +/- 23 vs. HC 115 +/- 16 microm, P < 0.05), elevated terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-stained positive cell number (CIH 98 +/- 45 vs. HC 15 +/- 13, P < 0.01), elevated caspase-3 activity (906 +/- 249 vs. 2,275 +/- 1,169 pmol x min(-1) x mg(-1), P < 0.05), and elevated expression of several remodeling gene markers, including c-fos, atrial natriuretic peptide, beta-myosin heavy chain, and myosin light chain-2. However, there was no difference between groups in sarcomere contractility of isolated LV myocytes, or in LV collagen deposition on trichrome-stained slices. In conclusion, CIH-mediated LV global dysfunction is associated with myocyte hypertrophy and apoptosis at the cellular level.     

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.     

翘嘴鳜per1 mRNA表达量分析中采用的内参基因稳定性比较

为筛选生物钟核心基因per1表达定量中的相对稳定性最好的内参基因,本研究取翘嘴鳜成鱼心脏、肝脏、肾脏、脑、红肌、白肌、肠、眼和脾等九个组织为研究对象,选取GAPDH、18S rRNA、β-actin、rps29、RPL13a、B2M和EF1a为内参基因,采用实时荧光定量PCR(qRT-PCR)对per1基因mRNA表达水平进行检测分析。研究结果表明18S rRNA和GAPDH的平均稳定值M最低,相对表达量最稳定。以18S rRNA和GAPDH为内参基因时分析发现per1基因表达量在肝脏中最高。本研究为在鱼类per1 mRNA表达检测过程中选用稳定的内参基因提供了实验和理论参考。     

Increased C-C Chemokine Receptor 2 Gene Expression in Monocytes of Severe Obstructive Sleep Apnea Patients and under Intermittent Hypoxia

Background

Obstructive sleep apnea (OSA) is known to be a risk factor of coronary artery disease. The chemotaxis and adhesion of monocytes to the endothelium in the early atherosclerosis is important. This study aimed to investigate the effect of intermittent hypoxia, the hallmark of OSA, on the chemotaxis and adhesion of monocytes.

Methods

Peripheral blood was sampled from 54 adults enrolled for suspected OSA. RNA was prepared from the isolated monocytes for the analysis of C-C chemokine receptor 2 (CCR2). The effect of intermittent hypoxia on the regulation and function of CCR2 was investigated on THP-1 monocytic cells and monocytes. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. Transwell filter migration assay and cell adhesion assay were performed to study the chemotaxis and adhesion of monocytes.

Results

Monocytic CCR2 gene expression was found to be increased in severe OSA patients and higher levels were detected after sleep. Intermittent hypoxia increased the CCR2 expression in THP-1 monocytic cells even in the presence of TNF-α and CRP. Intermittent hypoxia also promoted the MCP-1-mediated chemotaxis and adhesion of monocytes to endothelial cells. Furthermore, inhibitor for p42/44 MAPK or p38 MAPK suppressed the activation of monocytic CCR2 expression by intermittent hypoxia.

Conclusions

This is the first study to demonstrate the increase of CCR2 gene expression in monocytes of severe OSA patients. Monocytic CCR2 gene expression can be induced under intermittent hypoxia which contributes to the chemotaxis and adhesion of monocytes.     

Stability of Expression of Reference Genes Among Different Lentil (Lens culinaris) Genotypes Subjected to Cold Stress, White Mold Disease, and Aphanomyces Root Rot

Precise quantification of differences in gene expression between plants requires the use of “reference” genes, which are stably expressed across different lines and treatments and serve as endogenous controls for normalizing gene expression data. The objectives of this study were to determine the expression stability of several reference genes across five different lentil varieties subjected to either cold stress, inoculation with Sclerotinia sclerotiorum, the causal agent of white mold disease, or inoculation with Aphanomyces euteiches, the causal agent of Aphanomyces root rot. Expression stability was examined in the stems and leaves of plants subjected to cold stress or inoculation with S. sclerotiorum and in the roots of plants inoculated with A. euteiches. Real-time PCR assays (SYBR Green) were designed for six different genes: translation initiation factor (TIF), 18S rRNA, actin, β-tubulin-2, β-tubulin-3, and glyceraldehyde 3-phosphate dehydrogenase. TIF, actin, and 18S rRNA tended to be the most stably expressed genes, with expression stability (M) values less than 0.5 during cold stress and inoculation with A. euteiches. Two reference genes were required to normalize data from plants exposed to cold stress or inoculated with A. euteiches. The reference genes exhibited the lowest expression stability in plants inoculated with S. sclerotiorum, for which five reference genes were required to normalize data. The reference genes reported in this study appear to have a promise for examining gene expression in lentil foliar and root tissues in response to diverse abiotic and biotic factors.     

Selected Contribution: Regulation of sleep-wake states in response to intermittent hypoxic stimuli applied only in sleep.

Recurrent sleep-related hypoxia occurs in common disorders such as obstructive sleep apnea (OSA). The marked changes in sleep after treatment suggest that stimuli associated with OSA (e.g., intermittent hypoxia) may significantly modulate sleep regulation. However, no studies have investigated the independent effects of intermittent sleep-related hypoxia on sleep regulation and recovery sleep after removal of intermittent hypoxia. Ten rats were implanted with telemetry units to record the electroencephalogram (EEG), neck electromyogram, and body temperature. After >7 days recovery, a computer algorithm detected sleep-wake states and triggered hypoxic stimuli (10% O2) or room air stimuli only during sleep for a 3-h period. Sleep-wake states were also recorded for a 3-h recovery period after the stimuli. Each rat received an average of 69.0 +/- 6.9 hypoxic stimuli during sleep. The non-rapid eye movement (non-REM) and rapid-eye-movement (REM) sleep episodes averaged 50.1 +/- 3.2 and 58.9 +/- 6.6 s, respectively, with the hypoxic stimuli, with 32.3 +/- 3.2 and 58.6 +/- 4.8 s of these periods being spent in hypoxia. Compared with results for room air controls, hypoxic stimuli led to increased wakefulness (P < 0.005), nonsignificant changes in non-REM sleep, and reduced REM sleep (P < 0.001). With hypoxic stimuli, wakefulness episodes were longer and more frequent, non-REM periods were shorter and more frequent, and REM episodes were shorter and less frequent (P < 0.015). Hypoxic stimuli also increased faster frequencies in the EEG (P < 0.005). These effects of hypoxic stimuli were reversed on return to room air. There was a rebound increase in REM sleep, increased slower non-REM EEG frequencies, and decreased wakefulness (P < 0.001). The results show that sleep-specific hypoxia leads to significant modulation of sleep-wake regulation both during and after application of the intermittent hypoxic stimuli. This study is the first to determine the independent effects of sleep-related hypoxia on sleep regulation that approximates OSA before and after treatment.     

Hypoxia-mediated prolonged elevation of sympathetic nerve activity after periods of intermittent hypoxic apnea.

Obstructive sleep apnea (OSA) is associated with transient elevation of muscle sympathetic nerve activity (MSNA) during apneic events, which often produces elevated daytime MSNA in OSA patients. Hypoxia is postulated to be the primary stimulus for elevated daytime MSNA in OSA patients. Therefore, we studied the effects of 20 min of intermittent voluntary hypoxic apneas on MSNA during 180 min of recovery. Also, we compared MSNA during recovery after either 20 min of intermittent voluntary hypoxic apneas, hypercapnic hypoxia, or isocapnic hypoxia. Consistent with our hypothesis, both total MSNA and MSNA burst frequency were elevated after 20 min of intermittent hypoxic apnea compared with baseline (P < 0.05). Both total MSNA and MSNA burst frequency remained elevated throughout the 180-min recovery period and were statistically different from time control subjects throughout this period (P < 0.05). Finally, MSNA during recovery from intermittent hypoxic apnea, hypercapnic hypoxia, and isocapnic hypoxia were not different (P = 0.50). Therefore, these data support the hypothesis that short-term exposure to intermittent hypoxic apnea results in sustained elevation of MSNA and that hypoxia is the primary mediator of this response.