Intermittent Normoxia Research Articles

Effect of intermittent hypoxia on glucose tolerance in adults with type 2 diabetes

Hypoxia stimulates glucose uptake through an insulin-independent pathway. The purpose of this single-blind randomized study was to determine the acute effect of intermittent hypoxia, consisting of alternating short bouts of breathing hypoxic and normoxic air, on glucose and insulin concentrations during an oral glucose tolerance test in adults with type 2 diabetes. It was hypothesized that intermittent hypoxia would attenuate the increase in glucose and insulin concentrations during an oral glucose tolerance test. Six adults with type 2 diabetes (5 men, age: 51±15 years, HbA1c: 7.3±1.5%) visited the laboratory on two occasions. On both visits, a 2-hour oral glucose tolerance test was performed, with venous blood samples collected 0, 30, 60, 90, and 120 min after ingestion of a high-glucose drink. Following ingestion of the drink, participants were exposed to either an intermittent hypoxia (IH) protocol, consisting of eight 4-min hypoxic cycles at a targeted arterial oxygen saturation of 80% interspersed with breathing room air to resaturation, or an intermittent normoxia (IN) protocol consisting of eight 4-min normoxic cycles interspersed with breathing room air. By design, oxygen saturation was lower during intermittent hypoxia than intermittent normoxia (81±3 vs. 97±1%, p<0.01). Relative changes in plasma glucose concentrations in response to the oral glucose tolerance tests were not different between conditions (IH vs. IN: 30: 42±15 vs. 35±15; 60: 57±26 vs. 65±30; 90: 73±32 vs. 88±30; and 120: 78±26 vs. 83±29 mg/dl, interaction effect: p=0.13). Similarly, the relative changes in insulin concentrations in response to the oral glucose tolerance tests were not different between conditions (IH vs. IN: 30: 14±17 vs. 25±17; 60: 42±36 vs. 48±35; 90: 70±58 vs. 86±60; and 120: 103±72 vs. 125±94 ulU/ml, main effect for condition: p=0.12). While these preliminary results did not reach statistical significance, the observed trends for reduced glucose concentrations in combination with lower insulin concentrations during intermittent hypoxia suggest that short bouts of hypoxia improve glucose tolerance by stimulating glucose uptake independently from the action of insulin. Data from a larger sample size of adults with type 2 diabetes are needed to confirm these preliminary findings. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Brain Derived Neurotrophic Factor (BDNF) Immunolabeling in C4-C5 Spinal Cord of Adult Female Rats Following Acute Intermittent Hypoxia (AIH) Exposure Composed of 5,3-min Hypoxic Episodes (AIH5x3min) at 4-weeks After mid-thoracic SCI

Acute intermittent hypoxia (AIH) induces spinal neural plasticity in both respiratory and non-respiratory somatic motor systems. AIH-induced neural plasticity has been best studied in the respiratory neural control system, where exposure to moderate AIH (9-15% inspired O2), typically composed of 3,5-min hypoxic episodes (AIH3x5min), has been shown to consistently elicit a robust increase in phrenic motor activity due to a serotonin-dependent mechanism that promotes de novo synthesis of BDNF and activation of high affnity BDNF-TrkB receptors to initiate ERK MAP kinase signaling in phrenic motoneurons. Whether other protocols that use a total duration of 15 min of hypoxic exposure, but with an increased number of shorter duration hypoxic episodes ( e.g, AIH5x3min) similarly elicit serotonin-dependent BDNF pathway-mediated neural plasticity mechanisms in this region remains to be determined. To begin to assess this possibility, we evaluated BDNF expression in C4-C5 spinal cord segments containing the phrenic motor nucleus following single exposure to AIH5x3min (12% O2) in urethane-anesthetized spontaneously breathing adult female rats at 4-weeks after mid-thoracic SCI. Experiments using AIH3x5min (12% O2) exposure and intermittent normoxia (Nx, 21% O2) exposure served as controls. At ~60-90 min after the AIH or Nx exposure protocol, the rats were transcardially perfused with saline followed by 4% PFA, and various regions of the CNS, including the C3-C5 spinal cord were removed, post-fixed, and cryoprotected. 20 μm thick transverse sections were then cut to obtain sets of slides for single- or double-label fluorescence IHC staining to localize BDNF protein expression in the C4-C5 spinal cord, including the region containing the phrenic motor nucleus. Preliminary analyses show that both AIH5x3min and AIH3x5min exposures elicit increased BDNF expression when compared to Nx exposure, with both diffuse cytosolic and punctate immunofluorescence labeling in and around neurons within the phrenic motor nucleus region, including labeling of presumptive phrenic motoneurons. No labeling was seen in adjacent tissue sections that were incubated without the primary antibody. Immunofluorescence labeling was also seen in the more medial regions of lamina VIII in the ventral horn, the medial regions of laminae VI and VII, and throughout lamina X; these regions have been reported to contain the V0-V3 classes of spinal interneurons (SpINs) albeit we did not verify BDNF labeling of specific classes of presumptive SpINs. These preliminary observations suggest that AIH5x3min exposure likely recruits a serotonin-dependent BDNF pathway-mediated mechanism for neural plasticity in a manner similar to that seen for AIH3x5min exposure. APS Summer Undergraduate Research Fellowship; DOD CDMRP W81XWH-17-1-0260; NYS DOH SCIRB C37711GG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

LncRNA NEAT1 aggravates human microvascular endothelial cell injury by inhibiting the Apelin/Nrf2/HO-1 signalling pathway in type 2 diabetes mellitus with obstructive sleep apnoea

ABSTRACT Long noncoding RNAs (lncRNAs) regulate the progression of type 2 diabetes mellitus complicated with obstructive sleep apnoea (T2DM-OSA). However, the role of the lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in T2DM-OSA remains unknown. This study aimed to reveal the function of NEAT1 in T2DM-OSA and the underlying mechanism. KKAy mice were exposed to intermittent hypoxia (IH) or intermittent normoxia to generate a T2DM-OSA mouse model. HMEC-1 cells were treated with high glucose (HG) and IH to construct a T2DM-OSA cell model. RNA expression was detected by qRT-PCR. The protein expression of Apelin, NF-E2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1), and up-frameshift suppressor 1 (UPF1) was assessed using western blot. Cell injury was evaluated using flow cytometry, enzyme-linked immunosorbent assay, and oxidative stress kit assays. RIP, RNA pull-down, and actinomycin D assays were performed to determine the associations between NEAT1, UPF1, and Apelin. NEAT1 expression was upregulated in the aortic vascular tissues of mice with T2DM exposed to IH and HMEC-1 cells stimulated with HG and IH, whereas Apelin expression was downregulated. The absence of NEAT1 protected HMEC-1 cells from HG- and IH-induced damage. Furthermore, NEAT1 destabilized Apelin mRNA by recruiting UPF1. Apelin overexpression decreased HG- and IH-induced injury to HMEC-1 cells by activating the Nrf2/HO-1 pathway. Moreover, NEAT1 knockdown reduced HG- and IH-induced injury to HMEC-1 cells through Apelin. NEAT1 silencing reduced HMEC-1 cell injury through the Apelin/Nrf2/HO-1 signalling pathway in T2DM-OSA. Abbreviations: LncRNAs, long non-coding RNAs; T2DM, type 2 diabetes mellitus; OSA, obstructive sleep apnoea; NEAT1, nuclear paraspeckle assembly transcript 1; IH, intermittent hypoxia; HMEC-1, human microvascular endothelial cells; HG, high glucose; Nrf2, NF-E2-related factor 2; UPF1, up-frameshift suppressor 1; HO-1, haem oxygenase-1; qRT-PCR, quantitative real-time polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TNF-α, tumour necrosis factor-α; CCK-8, Cell Counting Kit-8; IL-1β, interleukin-1β; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase; RIP, RNA immunoprecipitation; SD, standard deviations; GSH, glutathione; AIS, acute ischaemic stroke; HMGB1, high mobility group box-1 protein; TLR4, toll-like receptor 4.

Gestational intermittent hypoxia increases FosB-immunoreactive perikaryas in the paraventricular nucleus of the hypothalamus of adult male (but not female) rats.

Sleep-disordered breathing is a respiratory disorder commonly experienced by pregnant women. The recurrent hypoxaemic events associated with sleep-disordered breathing have deleterious consequences for the mother and fetus. Adult male (but not female) rats born to dams subjected to gestational intermittent hypoxia (GIH) have a higher resting blood pressure than control animals and show behavioural/neurodevelopmental disorders. The origin of this persistent, sex-specific effect of GIH in offspring is unknown, but disruption of the neuroendocrine stress pathways is a key mechanism by which gestational stress increases disease risk in progeny. Using FosB immunolabelling as a chronic marker of neuronal activation, we determined whether GIH augments basal expression of FosB in the perikaryas of cells in the paraventricular nucleus of the hypothalamus (PVN), a key structure in the regulation of the stress response and blood pressure. From gestational day 10, female rats were subjected to GIH for 8h/day (light phase) until the day before delivery (gestational day 21); GIH consisted of 2min hypoxic bouts (10.5% O2 ) alternating with normoxia. Control rats were exposed to intermittent normoxia over the same period (GNX). At adulthood (10-15weeks), the brains of male and female rats were harvested for FosB immunohistochemistry. In males, GIH augmented PVN FosB labelling density by 30%. Conversely, PVN FosB density in GIH females was 28% lower than that of GNX females. We conclude that GIH has persistent and sex-specific impacts on the development of stress pathways, thereby offering a plausible mechanism by which GIH can disturb neural development and blood pressure homeostasis in adulthood. NEW FINDINGS: What is the central question of this study? In pregnant women, sleep apnoea increases the risk of disease for the offspring at various life stages. Given that gestational stress disrupts the programming of the stress pathways, we determined whether exposing female rats to gestational intermittent hypoxia (GIH) activates hypothalamic neurons regulating the stress response in adult rats. What is the main finding and its importance? Using FosB immunolabelling as a marker of marker of neuronal activation, we showed that GIH augmented basal activation of the paraventricular nucleus of the hypothalamus in males, but not females. Disruption of the stress pathways is a new hypothesis to explain the persistent and sex-specific impacts of GIH on offspring health.

Brief exposure to intermittent hypoxia increases erythropoietin levels in older adults.

Eight 4-min cycles of intermittent hypoxia represent the shortest hypoxic exposure to increase erythropoietin (EPO) levels in young adults. The impact of aging on the EPO response to a hypoxic stimulus remains equivocal. Thus, the objective of this study was to determine the effect of the same intermittent hypoxia protocol on EPO levels in older adults. Twenty-two participants (12 women, age: 53 ± 7 yr) were randomly assigned to an intermittent hypoxia group (IH, n = 11) or an intermittent normoxia group (IN, n = 11). Intermittent hypoxia consisted of eight 4-min cycles at a targeted oxygen saturation of 80% interspersed with normoxic cycles to resaturation. Air was made hypoxic by titrating nitrogen into a breathing circuit. Intermittent normoxia consisted of the same protocol, but nitrogen was not added to the breathing circuit. EPO levels were measured before and 4.5 h after the beginning of each protocol. Intermittent hypoxia lowered oxygen saturation to 82 ± 3%, which corresponded to a fraction of inspired oxygen of 10.9 ± 1.0%. There was a greater increase in EPO levels following intermittent hypoxia than intermittent normoxia (IH: 3.2 ± 2.2 vs. IN: 0.7 ± 0.8 mU/mL, P < 0.01). A single session of eight 4-min cycles of hypoxia increased EPO levels, the glycoprotein stimulating red blood cell production, in older adults. Exposure to intermittent hypoxia has therefore the potential to increase oxygen-carrying capacity in a population with reduced red blood cell volume.NEW & NOTEWORTHY We previously identified the shortest intermittent hypoxia protocol necessary to increase erythropoietin levels in young adults. The objective of this study was to determine whether the same intermittent hypoxia protocol increases erythropoietin levels in older adults. Eight 4-min bouts of hypoxia, representing a hypoxic duration of 32 min at a targeted oxygen saturation of 80%, increased erythropoietin levels in older adults, suggesting that exposure to intermittent hypoxia has the potential to increase oxygen-carrying capacity in an aging population.

Respiratory instability in neonatal rats after exposure to gestational intermittent hypoxia

Sleep disordered breathing (SDB) during pregnancy is a growing concern because it causes adverse outcomes for infant offspring. Recently, our lab developed a rat model of SDB during pregnancy in which pregnant dams were exposed to gestational intermittent hypoxia (GIH; 15 episodes/hr of 10.5% oxygen for 8h daily from days 10-20 of pregnancy). Control dams were exposed in parallel to gestational intermittent normoxia (GNX). We found that male (but not female) GIH offspring had increased apneas in adulthood compared to their GNX control counterparts. Thus, in this study, we tested the hypothesis that GIH would increase respiratory instability (i.e. increase the number of apneas) in neonatal male, not female, offspring. Plethysmography was performed on GIH and GNX male and female pups at postnatal (P) days 0, 10, and 15, and the number of apneas was quantified. An apnea was defined as two missed breaths (2x the average period between breaths). Preliminary data suggest that apnea frequency was sexually dimorphic and changed both with development and treatment. At P10 and P15 male GIH offspring had more apneas compared to GNX offspring (p&lt;0.05). However, at P0, male GIH offspring (16 ± 2.4 apneas/10m) had surprisingly fewer apneas compared to GNX offspring (28 ± 1.9 apneas/10m). In female offspring in contrast, there was no difference in apnea frequency between GIH and GNX treatment at P0, but at P10 and P15 respectively, female GIH offspring (17 ± 2.8 and 9 ± 2.3 apneas/10m) had more apneas compared to their GNX counterparts (8 ± 1.2 and 2.9 ± 0.57 apneas/10m). Apnea frequency decreased with increasing age in both sexes, regardless of GIH and GNX treatment. Together, as expected, these results suggest that the increased apnea phenotype observed in the adult GIH male emerges as early as postnatal day 10. Unexpected however is the observation that female neonatal GIH offspring also experience respiratory instability in during the postnatal period despite its absence in adult female GIH offspring. Studies are currently underway to better understand the mechanisms underlying these sex differences in respiratory neural control, and the protective responses utilized by adult female GIH offspring to mitigate these respiratory deficits induced by intermittent hypoxia exposure in utero. R01 HL142752 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Adequacy of an Altitude Fitness Program (Living and Training) plus Intermittent Exposure to Hypoxia for Improving Hematological Biomarkers and Sports Performance of Elite Athletes: A Single-Blind Randomized Clinical Trial.

Athletes incorporate altitude training programs into their conventional training to improve their performance. The purpose of this study was to determine the effects of an 8-week altitude training program that was supplemented with intermittent hypoxic training (IHE) on the blood biomarkers, sports performance, and safety profiles of elite athletes. In a single-blind randomized clinical trial that followed the CONSORT recommendations, 24 male athletes were randomized to an IHE group (HA, n = 12) or an intermittent normoxia group (NA, n = 12). The IHE consisted of 5-min cycles of hypoxia–normoxia with an FIO2 of between 10–13% for 90 min every day for 8 weeks. Hematological (red blood cells, hemoglobin, hematocrit, hematocrit, reticulated hemoglobin, reticulocytes, and erythropoietin), immunological (leukocytes, monocytes, and lymphocytes), and renal (urea, creatinine, glomerular filtrate, and total protein) biomarkers were assessed at the baseline (T1), day 28 (T2), and day 56 (T3). Sports performance was evaluated at T1 and T3 by measuring quadriceps strength and using three-time trials over the distances of 60, 400, and 1000 m on an athletics track. Statistically significant increases (p < 0.05) in erythropoietin, reticulocytes, hemoglobin, and reticulocyte hemoglobin were observed in the HA group at T3 with respect to T1 and the NA group. In addition, statistically significant improvements (p < 0.05) were achieved in all performance tests. No variations were observed in the immunological or renal biomarkers. The athletes who were living and training at 1065 m and were supplemented with IHE produced significant improvements in their hematological behavior and sports performance with optimal safety profiles.

Acute Effect of Intermittent Hypoxia on Peripheral Vascular Function in Young Healthy Adults

Continuous exposure to hypoxia elicits a chemoreceptor‐mediated increase in sympathetic nerve activity and a simultaneous peripheral vasodilation due to an increase in nitric oxide production. The aim of the present study was to determine whether short, repeated bouts of hypoxia induce brachial artery vasodilation and acutely improve endothelial function. Ten young healthy adults (4 women, age: 24±3 years, height: 176±9 cm, body weight: 80±13 kg) visited the laboratory on two separate occasions. Endothelium‐dependent vasodilation was assessed by brachial artery flow‐mediated dilation using a semiautomated diagnostic ultrasound system before and after exposure to either intermittent hypoxia (IH) or intermittent normoxia (IN). Intermittent hypoxia consisted of eight 4‐min hypoxic cycles at a targeted arterial oxygen saturation of 80% interspersed with breathing room air to resaturation, and intermittent normoxia consisted of eight 4‐min normoxic cycles separated by one minute of breathing room air. Air was made hypoxic by titrating nitrogen into a breathing circuit. Brachial artery diameter was assessed via ultrasound throughout the protocols. Hemodynamics, arterial oxygen saturation, and pulmonary gas exchange were also continuously assessed during the protocols. By design, intermittent hypoxia resulted in an arterial oxygen saturation of 81±2%, corresponding to oxygen levels of 11.9±2.2%. Exposure to intermittent hypoxia, but not intermittent normoxia, elicited a brachial artery vasodilation (IH: 0.38±0.06 to 0.40±0.06 vs. IN: 0.38±0.06 to 0.38±0.06 cm, p&lt;0.01). However, neither intermittent hypoxia nor intermittent normoxia acutely affected brachial artery flow‐mediated dilation (IH: 5.8±1.9 to 5.3±1.6% vs. IN: 6.1±2.4 to 6.5±1.7%, p=0.96). Intermittent hypoxia increased cardiac output (5.2±0.9 to 5.9±1.4 L·min‐1, p=0.03) mainly due to an increase in heart rate (57±9 to 68±11 bpm, p&lt;0.01). In conclusion, exposure to intermittent hypoxia triggered a brachial artery vasodilation but did not affect endothelium‐dependent vasodilation in young healthy adults. Multiple sessions of intermittent hypoxia may be necessary to improve nitric oxide synthesis and endothelial function.

Hypoxic Preconditioning Attenuates Ischemia‐Reperfusion Injury in Older Adults

Sudden restoration of blood flow to an ischemic vessel paradoxically damages endothelial cells. In young healthy adults, ischemic preconditioning, caused by repeated periods of brief ischemia induced by local cuff inflation prior to reperfusion, attenuates endothelial dysfunction following an ischemia‐reperfusion injury. However, ischemic preconditioning does not consistently protect against ischemia‐reperfusion injury in older adults. Intermittent systemic hypoxemia, induced via brief bouts of breathing low levels of oxygen, attenuates endothelial dysfunction following an ischemia‐reperfusion injury in young healthy adults. Therefore, the aim of the present study was to determine whether intermittent hypoxia protects against ischemia‐reperfusion injury in older adults. Eleven older adults (4 women, age: 57±9 years, height: 173±8 cm, body weight: 76±13 kg) visited the laboratory on two separate occasions. Endothelium‐dependent vasodilation was assessed by brachial artery flow‐mediated dilation using a semiautomated diagnostic ultrasound system before and after 20 minutes of blood flow occlusion to induce an ischemia‐reperfusion injury. Blood flow occlusion was preceded by either intermittent hypoxia (IH), consisting of three 4‐min hypoxic cycles at a targeted arterial oxygen saturation of 80% interspersed with 4‐min room air cycles, or intermittent normoxia (IN), consisting of three 4‐min normoxic cycles separated by 4‐min room air cycles. Intermittent hypoxia resulted in an arterial oxygen saturation of 80±2%, which corresponded to oxygen levels of 11.6±1.0%. When preceded by intermittent normoxia, blood flow occlusion reduced flow‐mediated dilation by 4.0±2.6% (6.4±1.7 to 2.4±1.8%). In contrast, flow‐mediated dilation was reduced by 1.8±1.5% when blood flow occlusion was preceded by intermittent hypoxia (5.6±1.8 to 3.7±2.4%: p=0.05). Intermittent hypoxia increased cardiac output (5.2±1.5 to 5.7±1.8 L·min‐1, p=0.03) mainly due to an increase in heart rate (61±12 to 69±10 bpm, p&lt;0.01). In conclusion, hypoxic preconditioning attenuated the reduction in flow‐mediated dilation induced by blood flow occlusion in older adults. Thus, exposure to intermittent hypoxia represents a potential strategy to protect against ischemia‐reperfusion injury in populations at risk for ischemic events.

Impact of Intermittent Hypoxia Exposure on Erythropoietin Levels in Older Individuals

Few minutes of hypoxia exposure stabilizes hypoxia‐inducible factors, resulting in erythropoietin (EPO) gene transcription and production. A brief intermittent hypoxia exposure increased EPO levels in young healthy individuals, suggesting that a single session of intermittent hypoxia has the potential to increase oxygen‐carrying capacity. Thus, the objective of this study was to determine the effect of a single session of intermittent hypoxia on serum EPO levels and hemoglobin mass among older individuals. We hypothesized that a single session of intermittent hypoxia would raise serum EPO levels and lead to an increase in hemoglobin mass in older individuals. Seventeen participants (8 women, age: 54 ± 8 years, height: 177 ± 10 cm, weight: 76 ± 14 kg, BMI: 24 ± 4 kg/m2) were randomly assigned to an intermittent hypoxia group (IH, n=11) or an intermittent normoxia group (IN, n=6). Intermittent hypoxia consisted of eight 4‐minute cycles at a targeted arterial oxygen saturation of 80% interspersed with normoxic cycles to resaturation. Air was made hypoxic by titrating nitrogen into the breathing circuit. Intermittent normoxia consisted of the same protocol, but nitrogen was not added to the breathing circuit. Pulmonary gas exchange, arterial oxygen saturation, and hemodynamics were continuously measured throughout both protocols. EPO levels were measured before and 4.5 hours after the beginning of each protocol. Hemoglobin mass was assessed via carbon monoxide rebreathing the day before and seven following intermittent hypoxia or normoxia. Intermittent hypoxia lowered arterial oxygen saturation (­­98 ±­ 1 to 82 ± 3 %, p&lt;0.01), which resulted in a lower fraction of inspired oxygen (20.8 ±­ 0.1 to 10.9 ± 1.0 %, p&lt;0.01). There was no significant change in EPO levels in either condition (IH:10.4 ±­ 2.9 to 13.3 ± 4.2; IN: 5.6 ±­ 2.4 to 6.5 ± 2.9 mU/ml, main effect for time p=0.12). Similarly, there was no change in hemoglobin mass in response to both conditions (IH: 752 ±­ 189 to 754 ± 189; IN: 858 ± 177 to 879 ± 157 g, main effect for time p=0.87). Intermittent hypoxia did not affect mean arterial pressure (87 ± 15 to 88 ± 14 mmHg, p=0.18) or cardiac output (5.5 ± 1.5 to 5.7 ± 1.5 L/min, p=0.22), but increased heart rate (62 ± 9 to 68 ± 9 bpm, p&lt;0.01). In conclusion, a single session of eight 4‐minute cycles of intermittent hypoxia did not increase serum EPO levels in older individuals.

Butyrate supplementation rescues sex‐specific deficits in respiratory plasticity and mean arterial pressure in adult offspring exposed to gestational intermittent hypoxia

Sleep disordered breathing (SDB) in pregnancy is increasing in parallel with the obesity epidemic, and is linked to adverse perinatal outcomes in the offspring. Consequences of maternal SDB during pregnancy on the adult offspring are not well‐understood. Preliminary data indicate that adult male, but not female, offspring of dams exposed to intermittent hypoxia during gestation (GIH ‐ gestational intermittent hypoxia), a rat model of SDB during pregnancy, have elevated mean arterial pressure (MAP), increased neuroinflammation, inflammation‐induced impairment of homeostatic plasticity in the respiratory control system, and gut dysbiosis. Specifically, adult male GIH offspring have decreased relative abundance of gut bacteria that produce butyrate, a short‐chain fatty acid that has potent anti‐inflammatory properties and regulates immune function in the central nervous system. Since literature suggests that gut dysbiosis, neuroinflammation, and hypertension exist in a complicated vicious cycle, we treated male GIH offspring with tributyrin, a butyrate prodrug. Prior to tributyrin treatment, MAP in male GIH offspring was 96±3 mmHg, versus 76±1 mmHg in control rats receiving gestational intermittent normoxia (GNX). Similarly, male GIH offspring exhibited deficits in respiratory plasticity in response to recurrent central apneas (3.6±6.8% plasticity), when compared to GNX offspring (59.3±4.8% plasticity). GIH offspring exhibited evidence of elevated neuroinflammation in brain regions underlying cardiorespiratory control (e.g., IL‐1β expression 1.6±0.2‐fold higher than GNX controls (1.0±0.2)). Following three weeks of tributyrin treatment, MAP had normalized (80±2 mmHg), neuroinflammation was reduced to control levels, and the ability to express respiratory neuroplasticity was rescued (78±30%). These data indicate that gut dysbiosis may contribute to sex‐specific impairments induced by maternal exposure to intermittent hypoxia during gestation that can be rescued in adulthood by supplementing with butyrate.

Effects of Intermittent Normoxia on Chronic Hypoxic Pulmonary Hypertension and Right Ventricular Hypertrophy in Rats.

Liu, Chunlei, Xu Chen, Ge Guo, Xiang Xu, Xin Li, Qingxia Wei, Yanying Shen, Hanlu Li, Jianxiu Hao, Ya Ping Tian, and Kunlun He. Effects of intermittent normoxia on chronic hypoxic pulmonary hypertension and right ventricular hypertrophy in rats. High Alt Med Biol. 22: 184-192, 2021. Background: Individuals with chronically low arterial oxygen tension owing to high altitude develop elevated rates of pulmonary hypertension (PH) and right ventricular (RV) hypertrophy. However, the effects of the frequency and duration of normoxic exposure on PH and RV hypertrophy have not been adequately assessed; thus, we aimed to analyze the same. Materials and Methods: PH and RV hypertrophy were induced in 60 rats using a hypobaric chamber. Of these 60 rats, every 10 were exposed to normoxic conditions for 30 minutes once (1T/D), three times (3T/D), or five times daily (5T/D), or for one 150-minute recovery daily (1LT/D). Furthermore, 10 rats were housed in a normoxic environment, and another 10 were subjected to continuous hypoxia. After 4 weeks, hemodynamic measurements were recorded, and the hearts were harvested for pathomorphological observations. Results: Average pulmonary arterial pressures (PAP) of control rats and those exposed to hypobaric hypoxia were 14.1 and 32.3 mmHg, respectively. After 30 minutes of exposure to normoxia 3T/D, 5T/D, or 1LT/D, PAP values were reduced to 27.1, 27.9, or 26.8 mmHg, respectively. Four weeks of hypoxic exposure elevated the RV/heart weight (HW) ratios, while exposure to normoxia 3T/D, 5T/D, and 1LT/D significantly reduced RV/HW. In addition, exposure to normoxia 3T/D, 5T/D, 1LT/D reduced the percentage wall thickness of the pulmonary artery as well as the hypertrophy indices of atrial natriuretic peptide, brain natriuretic peptide, and myosin heavy chain 7 (MYH-7). Conclusions: Thirty-minute exposure to normoxic conditions of 3T/D, 5T/D, or 1LT/D effectively ameliorates PH and RV thickening.

Butyrate Supplementation Restores Compensatory Plasticity Induced by Recurrent Reductions in Respiratory Neural Activity in Offspring Exposed to Gestational Intermittent Hypoxia

Sleep disordered breathing (SDB) during pregnancy is increasing in parallel with the obesity epidemic, yet the long-term effect on the adult offspring are unknown. In our animal model of SDB during pregnancy, pregnant rats are exposed to chronic intermittent hypoxia (21/10.5% O2, 15 episodes/hr, 8 hrs/day) or intermittent normoxia from gestational days 10-21. Preliminary data show that adult male (but not female) offspring exposed to gestational intermittent hypoxia (GIH) show deficits in respiratory control that manifest as increased spontaneous central apneas during presumptive sleep and impaired compensatory responses to recurrent reductions in respiratory neural activity, a form of plasticity known as inactivity-induced inspiratory motor facilitation (iMF). Further, our data show that microglia isolated from male GIH offspring show primed inflammatory responses in respiratory control regions, and that microglial depletion or inhibition of inflammation in the region of the phrenic motor pool rescues deficits in respiratory control. Our preliminary data show that male GIH offspring suffer from gut dysbiosis. Most notably, they exhibit decreased levels of bacteria that produce butyrate, a short-chain fatty acid (SCFA) necessary for the proper development of microglia. Because butyrate supplementation reduces microglial inflammation, we tested the hypothesis that adult butyrate supplementation restores the capacity to elicit iMF in adult male GIH offspring. Vehicle or tributyrin was delivered by oral gavage in 8 doses over 22 days (2g/kg). Phrenic inspiratory output was measured in urethane-anesthetized, vagotomized, mechanically ventilated GIH offspring. As expected, recurrent neural apnea (5, ~1 min neural apnea episodes, separated by 5 minutes) elicited an increase in phrenic inspiratory output in GIH offspring treated with tributyrin compared to vehicle treated GIH offspring (78±30% and 15±2%, respectively, n = 3-4, p = 0.057), indicating that butyrate supplementation rescues the capacity to elicit iMF in GIH offspring. These data indicate that gut dysbiosis may contribute to impaired capacity for recurrent reductions in respiratory neural activity to trigger compensatory forms of neuroplasticity.

Endothelial Dysfunction in a Cell Culture Model Exposed to Various Intermittent Hypoxia Modes.

Wang, Juan, Jiahui Wang, Xin Li, Wanju Hou, Jie Cao, and Jing Feng. Endothelial dysfunction in a cell culture model exposed to various intermittent hypoxia modes. High Alt Med Biol. 21:388-395, 2020. Objective: To construct an in vitro model of endothelial cells exposed to various intermittent hypoxia (IH) modes, and determine whether different frequencies and degrees can cause different effects on endothelial cells. Methods: EA.hy926 cells were used to set up the cell model. A program-controlled gas delivery system was designed to regulate the flow of premixed air into the cell culture chamber. The cells were divided into eight groups exposed to various IH modes: standard cell culture group, intermittent normoxia (IN) group (21% O2 15 seconds/21% O2 3 minutes 45 seconds for 12 cycles/h), IH1 group (1.5% O2 15 seconds/21% O2 8 minutes 15 seconds for 6.32 cycles/h), IH2 group (1.5% O2 15 seconds/21% O2 5 minutes 15 seconds for 9.23 cycles/h), IH3 group (1.5% O2 15 seconds/21% O2 3 minutes 45 seconds for 12 cycles/h), IH4 group (1.5% O2 15 seconds/21% O2 1 minute 45 seconds for 20 cycles/h), IH5 group (1.5% O2 15 seconds/21% O2 15 seconds for 40 cycles/h), and IH6 group (10% O2 15 seconds/21% O2 3 minutes 45 seconds for 12 cycles/h). Results: Nuclear factor κB (NFκB) p65, c-fos, tumor necrosis factor-alpha (TNFα), malondialdehyde (MDA), and endothelin-1 (ET-1) were higher in the IH3 group or IH6 group than those in IN group, and they were much higher in IH3 group than those in IH6 group. Superoxide dismutase (SOD) and nitric oxide (NO) were the opposite results. In IH1, IH2, IH3, IH4, and IH5 groups, the frequencies increased gradually. NFκB p65, TNFα, and c-fos were the highest in IH3 group. MDA and ET-1 were the highest in IH2 group. SOD and NO were the lowest in IH2 group. Conclusions: Different IH frequencies and degrees could cause different effects on endothelial cells. The endothelial responses varied with the duration of reoxygenation. So, the duration of reoxygenation was the key phase for endothelial dysfunction.

Effect of a Single Session of Intermittent Hypoxia on Erythropoietin and Oxygen-Carrying Capacity.

Intermittent hypoxia, defined as alternating bouts of breathing hypoxic and normoxic air, has the potential to improve oxygen-carrying capacity through an erythropoietin-mediated increase in hemoglobin mass. The purpose of this study was to determine the effect of a single session of intermittent hypoxia on erythropoietin levels and hemoglobin mass in young healthy individuals. Nineteen participants were randomly assigned to an intermittent hypoxia group (Hyp, n = 10) or an intermittent normoxia group (Norm, n = 9). Intermittent hypoxia consisted of five 4-min hypoxic cycles at a targeted arterial oxygen saturation of 90% interspersed with 4-min normoxic cycles. Erythropoietin levels were measured before and two hours following completion of the protocol. Hemoglobin mass was assessed the day before and seven days after exposure to intermittent hypoxia or normoxia. As expected, the intermittent hypoxia group had a lower arterial oxygen saturation than the intermittent normoxia group during the intervention (Hyp: 89 ± 1 vs. Norm: 99 ± 1%, p < 0.01). Erythropoietin levels did not significantly increase following exposure to intermittent hypoxia (Hyp: 8.2 ± 4.5 to 9.0 ± 4.8, Norm: 8.9 ± 1.7 to 11.1 ± 2.1 mU·mL−1, p = 0.15). Hemoglobin mass did not change following exposure to intermittent hypoxia. This single session of intermittent hypoxia was not sufficient to elicit a significant rise in erythropoietin levels or hemoglobin mass in young healthy individuals.

Effect of a Single Session of Intermittent Hypoxia on Erythropoietin and Oxygen‐Carrying Capacity

Intermittent hypoxia, defined as alternating bouts of breathing hypoxic and normoxic air, has the potential to improve oxygen‐carrying capacity through an erythropoietin‐mediated increase in hemoglobin mass. The purpose of this study was to determine the effect of a single exposure of intermittent normobaric hypoxia on erythropoietin levels and hemoglobin mass in young healthy individuals. Nineteen healthy individuals (10 women and 9 men, age: 24 ± 4 years, height: 174 ± 11 cm, weight: 72.2 ± 12.2 kg) participated in the study. Participants were randomly assigned to an intermittent hypoxia group (Hyp, n = 10) or a placebo intermittent normoxia group (Norm, n = 9). Intermittent hypoxia consisted of five 4‐min hypoxic cycles at a targeted arterial oxygen saturation of 90% interspersed with 4‐min normoxic cycles. Air was made hypoxic by titrating nitrogen to a breathing circuit connected to a tank of compressed air. Nitrogen was not added to the breathing circuit in the intermittent normoxia condition. Pulmonary gas exchange, arterial oxygen saturation, and hemodynamics, using finger plethysmography, were continuously assessed during the intervention. Erythropoietin levels were measured before and two hours following the completion of the protocol. Hemoglobin mass was assessed using the carbon monoxide rebreathing technique the day before and seven days after exposure to intermittent hypoxia or normoxia. As anticipated, the intermittent hypoxia group had a lower arterial oxygen saturation than the intermittent normoxia group during the intervention (Hyp: 89 ± 1 vs. Norm: 98 ± 1%, p &lt; 0.01), which was equivalent to a lower fraction of inspired oxygen (Hyp: 0.119 ± 0.008, Norm: 0.209 ± 0.001, p &lt; 0.01). Erythropoietin levels did not significantly increase following exposure to intermittent hypoxia (Hyp: 8.2 ± 4.5 to 9.0 ± 4.8, Norm: 8.9 ± 1.7 to 11.1 ± 2.1 mU/ml, p = 0.56). Hemoglobin mass did not change following exposure to intermittent hypoxia (Hyp: 10.6 ± 1.4 to 10.2 ± 1.4, Norm: 9.7 ± 1.6 to 9.4 ± 1.5 g/kg, p = 0.48). Exposure to intermittent hypoxia did not affect mean arterial pressure (Hyp: 91 ± 6 to 90 ± 6, Norm: 93 ± 12 to 93 ± 12 mmHg, p = 0.84) or heart rate (Hyp: 68 ± 8 to 74 ± 9, Norm: 68 ± 8 to 68 ± 8 mmHg, p = 0.27). Respiratory rate, tidal volume, end‐tidal CO2 and total minute ventilation were not affected by intermittent hypoxia. Thus, a 40‐min session of intermittent hypoxia was not sufficient to elicit a rise in erythropoietin levels or oxygen‐carrying capacity in young healthy individuals. A longer exposure to intermittent hypoxia at a lower arterial oxygen saturation may be necessary to trigger an erythropoietin‐mediated increase in hemoglobin mass in young healthy individuals.

Effect of Intermittent Hypoxia on Ischemic‐Reperfusion Injury in Healthy Individuals

Brief periods of ischemia preceding an ischemic‐reperfusion injury attenuate the reduction in brachial artery endothelial function. It has remained unknown whether brief bouts of systemic hypoxemia would similarly mitigate the blunted vasodilatory response induced by an ischemic‐reperfusion injury. Therefore, the purpose of this study was to determine whether intermittent hypoxia protects against an ischemic‐reperfusion injury in young healthy adults. Ten healthy individuals, 7 men and 3 women (age: 24 ± 2 years, height: 176 ± 9 cm, weight: 77.1 ± 13.9 kg), participated in the study. Brachial artery endothelial function was assessed by flow‐mediated dilation before and after an ischemic‐reperfusion injury induced by a 20‐minute blood flow occlusion. The ischemic‐reperfusion injury was preceded by either intermittent hypoxia (Hyp) or intermittent normoxia (Norm). Both visits were separated by a period of seven days, whereas women who had regular menstrual cycles were scheduled in the early follicular phase. Intermittent hypoxia was created by titrating nitrogen into a breathing system to achieve an arterial oxygen saturation of 90%. Intermittent hypoxia consisted of three 4‐minute hypoxic cycles separated by 4‐minute normoxic cycles. As expected, intermittent hypoxia resulted in a lower arterial oxygen saturation (Hyp: 88 ± 4 vs. Norm: 98 ± 2%, p &lt; 0.01), which was equivalent to a lower fraction of inspired oxygen (Hyp: 0.13 ± 0.04, Norm: 0.21 ± 0.03, p &lt; 0.01). The reduction in flow‐mediated dilation resulting from the ischemic‐reperfusion injury was attenuated by intermittent hypoxia (Hyp: 5.9 ± 0.7 to 4.5 ± 0.7%, Norm: 6.0 ± 0.7 to 3.6 ± 0.7%, p = 0.07). Exposure to intermittent hypoxia did not affect mean arterial blood pressure (Hyp: 97 ± 7 to 99 ± 8 mmHg, Norm: 94 ± 8 to 95 ± 10 mmHg, p = 0.52) but significantly increased heart rate (Hyp: 63 ± 6 to 70 ± 7 bpm, Norm: 61 ± 7 to 61 ± 6 bpm, p &lt; 0.01). Thus, intermittent hypoxia seems to prevent the reduction in flow‐mediated dilation induced by an ischemic‐reperfusion injury in young healthy individuals.

Inhibition of Spinal Inflammation Restores Compensatory Plasticity Induced by Recurrent Reductions in Respiratory Neural Activity in Offspring Exposed to Gestational Intermittent Hypoxia

Sleep disordered breathing (SDB) during pregnancy is increasing in parallel with the obesity epidemic, yet the long‐term effects on the adult offspring are unknown. In our animal model of SDB during pregnancy, pregnant rats are exposed to chronic intermittent hypoxia (8 hrs/day, 2 min 10.5% O2 separated by 2 min of 21% O2) or intermittent normoxia from gestational days 10–21. Preliminary data show that adult male (but not female) offspring exposed to gestational intermittent hypoxia (GIH) show deficits in respiratory control that manifest as increased spontaneous central apneas during presumptive sleep and impaired compensatory responses to recurrent reductions in respiratory neural activity, a form of plasticity known as inactivity‐induced inspiratory motor facilitation (iMF). Preliminary data also show that microglia isolated from male GIH offspring show primed inflammatory responses in respiratory control regions. Since neuroinflammation impairs respiratory plasticity, we tested the hypothesis that inhibiting NF‐kappaB and STAT3 inflammatory transcription factor activation restores the capacity to elicit iMF in GIH offspring. Phrenic inspiratory output was measured in urethane‐anesthetized, vagotomized, mechanically ventilated GIH or gestational intermittent normoxia (GNX) offspring. Vehicle or 2‐[(aminocarbonyl)amino]‐5 ‐(4‐fluorophenyl)‐3‐ thiophenecarboxamide (TPCA‐1) was delivered intrathecally over the phrenic motor pool ~1 hour before exposure to recurrent reductions in neural activity (5, ~1 min neural apnea episodes, separated by 5 min). As expected, recurrent neural apnea elicited a significant increase in phrenic inspiratory output in vehicle‐ and TPCA‐treated GNX offspring (54±17% and 62±12% baseline, n=6, 5, respectively, p&lt;0.05) when compared to time controls not receiving activity deprivation (11±4% baseline, n=4). By contrast, recurrent neural apnea did not elicit iMF in vehicle‐treated GIH offspring (25±3%, n=7, p&gt;0.05). However, in GIH offspring receiving intrathecal TPCA‐1, recurrent neural apnea induced a significant increase in phrenic inspiratory output (75+/−10% baseline, n=7, p&lt;0.05), indicating that NF‐kappaB and STAT3 inflammatory transcription factor inhibition rescues the capacity to elicit iMF in GIH offspring. These data indicate that increased neuroinflammation in male GIH offspring impairs the capacity for recurrent reductions in respiratory neural activity to trigger compensatory forms of neuroplasticity.Support or Funding InformationSupported by R01HL142752 (JJW and TLB)

HMGB1/TLR4 promotes apoptosis and reduces autophagy of hippocampal neurons in diabetes combined with OSA

AimsObstructive sleep apnea (OSA) combined with type 2 diabetes (T2DM) may lead to cognitive dysfunction. We previously reported that cognitive impairment is exacerbated in KKAy mice exposed to intermittent hypoxia (IH), during which the DNA binding protein HMGB1 mediates hippocampal neuronal apoptosis by maintaining microglia-associated neuroinflammation, but the underlying mechanism remains largely unknown. Materials and methodsWe performed immunofluorescence, Western blotting, and immunohistochemistry experiments in mouse hippocampal tissues and HT22 cells. KKAy type 2 diabetes model mice and normal C57BL/6J mice were exposed to IH or intermittent normoxia. HT22 cells were cultured in high glucose medium and exposed to IH or intermittent normoxia. We transfected HMGB1 siRNA into HT22 cells and then treated them with high glucose combined with intermittent hypoxia. Key findingsIn conclusion, IH aggravated apoptosis and autophagy defects in T2DM mice, and increased the protein expression of HMGB1 and TLR4. This was also confirmed in HG + IH-treated hippocampal HT22 cells. HMGB1 siRNA can significantly reduce the protein expression of HMGB1 and TLR4, reverse neuronal apoptosis and enhance autophagy. SignificanceWe believe that HMGB1 is a key factor in the regulation of hippocampal neuronal apoptosis and autophagy defects in T2DM combined with OSA. Targeting HMGB1/TLR4 signaling as a novel approach may delay or prevent the increased apoptosis and decreased autophagy induced by T2DM combined with OSA, and may ultimately improve cognitive dysfunction.

HMGB1/TLR4 Promotes Apoptosis and Weakens Autophagy of Hippocampal Neurons in Diabetes Combined with OSA

Background: Obstructive sleep apnea (OSA) combined with type 2 diabetes (T2DM) may lead to cognitive dysfunction, but the underlying mechanism remain largely unknown. Methods: We performed experiments in mice hippocampal tissues and HT22 cells by immunofluorescence and western blot. Type 2 diabetes model KKAy mice and normal C57BL / 6J mice were exposed to IH or intermittent normoxia. HT22 cells were cultured in high glucose medium and exposed to IH or intermittent normoxia. We then transfected siRNA HMGB1 into HT22 cells then treated with high glucose combined with intermittent hypoxia. Findings: In conclusion, IH aggravated apoptosis and autophagy defects in T2DM mice, and increased protein expression of HMGB1 and TLR4. It was also confirmed in HG combined with IH-treated hippocampal HT22 cells. HMGB1 siRNA can significantly reduce the protein expression of HMGB1 and TLR4, reverse neuronal apoptosis and enhance autophagy. Interpretation: HMGB1-TLR4 signaling regulates autophagy dysfunction and promotes apoptosis in hippocampal neurons of OSA complicated with T2DM. Targeting HMGB1/TLR4 signaling as a novel approach may delay or prevent the increased apoptosis and decreased autophagy induced by T2DM combined with OSA, and ultimately hope to improve cognitive dysfunction. Funding Statement: This work was partly supported by the National Natural Science Foundation of China (81570084 and 81270144 to J.F.), the China Postdoctoral Science Foundation Grant (2015M581309 to B.S.). Declaration of Interests: The author declares that the study does not have any potential conflicts of interest. Ethics Approval Statement: The animal research program follows the Animal Ethics and Use Committee of Tianjin Medical University.