Min Of Normoxia Research Articles

Randomized controlled trial of intermittent hypoxia in Parkinson’s disease: study rationale and protocol

BackgroundParkinson’s disease (PD) is a neurodegenerative disease for which no disease-modifying therapies exist. Preclinical and clinical evidence suggest that repeated exposure to intermittent hypoxia might have short- and long-term benefits in PD. In a previous exploratory phase I trial, we demonstrated that in-clinic intermittent hypoxia exposure is safe and feasible with short-term symptomatic effects on PD symptoms. The current study aims to explore the safety, tolerability, feasibility, and net symptomatic effects of a four-week intermittent hypoxia protocol, administered at home, in individuals with PD.Methods/Design: This is a two-armed double-blinded randomized controlled trial involving 40 individuals with mild to moderate PD. Participants will receive 45 min of normobaric intermittent hypoxia (fraction of inspired oxygen 0.16 for 5 min interspersed with 5 min normoxia), 3 times a week for 4 weeks. Co-primary endpoints include nature and total number of adverse events, and a feasibility-tolerability questionnaire. Secondary endpoints include Movement Disorders Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) part II and III scores, gait tests and biomarkers indicative of hypoxic dose and neuroprotective pathway induction.DiscussionThis trial builds on the previous phase I trial and aims to investigate the safety, tolerability, feasibility, and net symptomatic effects of intermittent hypoxia in individuals with PD. Additionally, the study aims to explore induction of relevant neuroprotective pathways as measured in plasma. The results of this trial could provide further insight into the potential of hypoxia-based therapy as a novel treatment approach for PD.Trial registrationClinicalTrials.gov Identifier: NCT05948761 (registered June 20th, 2023).

Non-neuronal acetylcholine is causal for cardioprotection by hypoxic preconditioning in adult rat cardiomyocytes

Abstract Introduction Brief episodes of non-lethal myocardial ischemia/reperfusion preceding more sustained ischemia and reperfusion reduce lethal ischemia/reperfusion injury. The underlying protective signaling mechanisms of such ischemic preconditioning are unclear in their details. Among other protective mediators, neuronal acetylcholine (ACh) and its signaling through cholinergic receptors is involved in protection by ischemic preconditioning. Recently, a non-neuronal cardiac cholinergic system (NNCCS) has been identified, suggesting that a nervous system-independent, cardiomyocyte-derived acetylcholine signaling with signal amplification by auto- and paracrine cholinergic receptor activation could also participate in ischemic preconditioning. Aim We aimed to examine the role of NNCCS-derived ACh as a mediator of ischemic preconditioning. We therefore, in a reductionist approach, used isolated adult cardiomyocytes with hypoxic preconditioning (HPC). Methods Adult ventricular rat cardiomyocytes were isolated (from n=9 hearts), and HPC was induced by one cycle of 10 min hypoxia / 20 min reoxygenation. Thirty min normoxia served as control. Cardiomyocytes were then exposed to 30 min sustained hypoxia / 5 min reoxygenation (H/R), or a 35 min time control (TC). Cardiomyocyte viability was quantified before and after H/R or TC, respectively, by trypan blue staining. In a subgroup of experiments (cardiomyocytes from n=6 hearts) in the presence of physostigmine (0.2 mmol/L), intracellular ACh was quantified before and after HPC or normoxia and after sustained hypoxia and reoxygenation or TC, respectively, via liquid chromatography-coupled tandem mass spectrometry. Experiments on cardiomyocyte viability (from n=9 hearts) were repeated with the addition of cholinergic receptor antagonists (nicotinic: hexamethonium [1 µmol/L], muscarinic: atropine [0.1 µmol/L]) a) during the first 30 min with HPC or normoxia, b) during H/R or TC (30-65 min), or c) throughout the total 65 min protocol. Results The viability of cardiomyocytes subjected to H/R was better preserved with HPC than without (Figure A), and intracellular ACh was increased during hypoxia after prior HPC (Figure B). Cholinergic receptor antagonists during HPC did not change HPC´s protection (Figure A). However, the combination of both antagonists during sustained hypoxia abrogated HPC´s protection (Figure A), whereas nicotinic or muscarinic cholinergic receptor antagonists alone did not. Cholinergic receptor antagonists throughout the total 65 min also abrogated HPC´s protection (Figure A). Conclusion: The NNCCS is activated by HPC and causal for HPC´s protection. HPC increases intracellular acetylcholine, and cardiomyocyte protection is mediated via both nicotinic and muscarinic cholinergic receptors, suggesting an auto- and paracrine signal amplification on the cardiomyocyte level.Figure

Effects of acute intermittent hypoxia on corticospinal excitability within the primary motor cortex

PurposeAcute intermittent hypoxia (AIH) is a safe and non-invasive treatment approach that uses brief, repetitive periods of breathing reduced oxygen air alternated with normoxia. While AIH is known to affect spinal circuit excitability, the effects of AIH on cortical excitability remain largely unknown. We investigated the effects of AIH on cortical excitability within the primary motor cortex.MethodsEleven healthy, right-handed participants completed two testing sessions: (1) AIH (comprising 3 min in hypoxia [fraction of inspired oxygen ~ 10%] and 2 min in normoxia repeated over five cycles) and (2) normoxia (NOR) (equivalent duration to AIH). Single- and paired-pulse transcranial magnetic stimulations were delivered to the primary motor cortex, before and 0, 25, and 50 min after AIH and normoxia.ResultsThe mean nadir in arterial oxygen saturation was lower (p < 0.001) during the cycles of AIH (82.5 ± 4.9%) than NOR (97.8 ± 0.6%). There was no significant difference in corticospinal excitability, intracortical facilitation, or intracortical inhibition between AIH and normoxia conditions at any time point (all p > 0.05). There was no association between arterial oxygen saturation and changes in corticospinal excitability after AIH (r = 0.05, p = 0.87).ConclusionOverall, AIH did not modify either corticospinal excitability or excitability of intracortical facilitatory and inhibitory circuits within the primary motor cortex. Future research should explore whether a more severe or individualised AIH dose would induce consistent, measurable changes in corticospinal excitability.

1317-P: Intermittent Hypoxia Increased the Expression of DBH and PNMT in Neuronal Cells via MicroRNA-Mediated Mechanism

Sleep apnea syndrome (SAS) , characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) , is a risk factor for hypertension and insulin resistance. We have reported correlation between IH and insulin resistance. However, why hypertension is induced by IH has been elusive. Here, we investigated the effect of IH on the expression of catecholamine metabolizing enzymes using in vitro IH system. Human and mouse neuronal cells, NB-1 and Neuro-2a, were exposed to IH (70 cycles of 5 min hypoxia [1% O2] and min normoxia [21% O2]) or normoxia for 24 hours. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of dopamine β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in both NB-1 (P=0.0271 and P&amp;lt;0.0001, respectively) and Neuro-2a (P=0.0135 and P=0.0341, respectively) . Western blot showed that the expression of DBH and PNMT in the NB-1 cells significantly increased by IH (P=0.0296 and P=0.0007, respectively) . We next prepared reporter plasmids containing human DBH (-1018˜+10) and PNMT (-600˜+67) upstream of luciferase reporter gene and transfected them into NB-1 cells. The promoter activities of these genes were not increased by IH. Target mRNA search of microRNA (miR) s using MicroRNA.org program revealed that the mRNAs have a potential target sequence for miR-375. The miR-375 level of IH-treated cells was significantly decreased than that of normoxia-treated cells (P=0.0222) . To clarify the role of miR-375, miR-375 mimic and non-specific control RNA (miR-375 mimic NC) were introduced into NB-1 cells. The IH-induced up-regulation of mRNAs of DBH and PNMT was abolished by introduction of miR-375 mimic but not by miR-375 mimic NC. Theses results strongly suggest that IH stress down-regulates the miR-375 in neuronal cells, resulting in the increased levels of DBH and PNMT via the inhibition of the miR-375-mediated mRNA degradation, leading SAS patients to hypertension. Disclosure S.Takasawa: None. R.Shobatake: None. Y.Takeda: None. T.Uchiyama: None. A.Yamauchi: None. M.Makino: None. K.Asai: None. H.Ota: None.

Knockdown of a Cyclic Nucleotide-Gated Ion Channel Impairs Locomotor Activity and Recovery From Hypoxia in Adult Drosophila melanogaster.

Cyclic guanosine monophosphate (cGMP) modulates the speed of recovery from anoxia in adult Drosophila and mediates hypoxia-related behaviors in larvae. Cyclic nucleotide-gated channels (CNG) and cGMP-activated protein kinase (PKG) are two cGMP downstream targets. PKG is involved in behavioral tolerance to hypoxia and anoxia in adults, however little is known about a role for CNG channels. We used a CNGL (CNG-like) mutant with reduced CNGL transcripts to investigate the contribution of CNGL to the hypoxia response. CNGL mutants had reduced locomotor activity under normoxia. A shorter distance travelled in a standard locomotor assay was due to a slower walking speed and more frequent stops. In control flies, hypoxia immediately reduced path length per minute. Flies took 30–40 min in normoxia for >90% recovery of path length per minute from 15 min hypoxia. CNGL mutants had impaired recovery from hypoxia; 40 min for ∼10% recovery of walking speed. The effects of CNGL mutation on locomotor activity and recovery from hypoxia were recapitulated by pan-neuronal CNGL knockdown. Genetic manipulation to increase cGMP in the CNGL mutants increased locomotor activity under normoxia and eliminated the impairment of recovery from hypoxia. We conclude that CNGL channels and cGMP signaling are involved in the control of locomotor activity and the hypoxic response of adult Drosophila.

Effect of Normobaric Hypoxia on Alterations in Redox Homeostasis, Nitrosative Stress, Inflammation, and Lysosomal Function following Acute Physical Exercise.

Hypoxia is a recognized inducer of oxidative stress during prolonged physical activity. Nevertheless, previous studies have not systematically examined the effects of normoxia and hypoxia during acute physical exercise. The study is aimed at evaluating the relationship between enzymatic and nonenzymatic antioxidant barrier, total antioxidant/oxidant status, oxidative and nitrosative damage, inflammation, and lysosomal function in different acute exercise protocols under normoxia and hypoxia. Fifteen competitive athletes were recruited for the study. They were subjected to two types of acute cycling exercise with different intensities and durations: graded exercise until exhaustion (GE) and simulated 30 km individual time trial (TT). Both exercise protocols were performed under normoxic and hypoxic (FiO2 = 16.5%) conditions. The number of subjects was determined based on our previous experiment, assuming the test power = 0.8 and α = 0.05. We demonstrated enhanced enzymatic antioxidant systems during hypoxic exercise (GE: ↑ catalase (CAT), ↑ superoxide dismutase; TT: ↑ CAT) with a concomitant decrease in plasma reduced glutathione. In athletes exercising in hypoxia, redox status was shifted in favor of oxidation reactions (GE: ↑ total oxidant status, ↓ redox ratio), leading to increased oxidation/nitration of proteins (GE: ↑ advanced oxidation protein products (AOPP), ↑ ischemia-modified albumin, ↑ 3-nitrotyrosine, ↑ S-nitrosothiols; TT: ↑ AOPP) and lipids (GE: ↑ malondialdehyde). Concentrations of nitric oxide and its metabolites (peroxynitrite) were significantly higher in the plasma of hypoxic exercisers with an associated increase in inflammatory mediators (GE: ↑ myeloperoxidase, ↑ tumor necrosis factor-alpha) and lysosomal exoglycosidase activity (GE: ↑ N-acetyl-β-hexosaminidase, ↑ β-glucuronidase). Our study indicates that even a single intensive exercise session disrupts the antioxidant barrier and leads to increased oxidative and nitrosative damage at the systemic level. High-intensity exercise until exhaustion (GE) alters redox homeostasis more than the less intense exercise (TT, near the anaerobic threshold) of longer duration (20.2 ± 1.9 min vs. 61.1 ± 5.4 min—normoxia; 18.0 ± 1.9 min vs. 63.7 ± 3.0 min—hypoxia), while hypoxia significantly exacerbates oxidative stress, inflammation, and lysosomal dysfunction in athletic subjects.

Extracellular vesicles derived from lung cancer cells exposed to intermittent hypoxia upregulate programmed death ligand 1 expression in macrophages

PurposeIntermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), compromises immune surveillance through the upregulation of programmed cell death-1 ligand (PD-L1). Tumor-released extracellular vesicles (EVs) have been reported to modulate immunosuppressive activities. We investigated whether or not EVs derived from intermittent hypoxic lung cancer cells can alter the expression of PD-L1 in macrophages.MethodsThe expression of PD-L1+monocytes from 40 patients with newly diagnosed non-small-cell lung cancer (NSCLC) and with (n=21) or without (n=19) OSA were detected. Plasma EVs isolated from NSCLC patients with moderate–severe OSA (n=4) and without OSA (n=4) were co-cultured with macrophages. A549 cells were exposed to normoxia or IH (48 cycles of 5 min of 1% O2 hypoxia, followed by 5 min of normoxia). EVs were isolated from cell supernatant and were co-cultured with macrophages differentiated from THP-1. PD-L1 and hypoxia-inducible factor-1 α (HIF-1α) expressions were measured by flow cytometry, immunofluorescence, and Western blot analysis.ResultsPD-L1+monocytes were elevated in NSCLC patients with OSA and increased with the severity of OSA and nocturnal desaturation. PD-L1+ macrophages were induced by EVs from NSCLC patients with OSA and positively correlated with HIF-1α expressions. EVs from IH-treated A549 can promote PD-L1 and HIF-1α expression in macrophages and the upregulation of PD-L1 expression was reversed by specific HIF-1α inhibitor.ConclusionIH can enhance the function of EVs derived from lung cancer cells to aggravate immunosuppressive status in macrophages. HIF-1α may play an important role in this process.

378-P: Upregulation of Regenerating Gene IV and Hepatocyte Growth Factor in Cardiomyocytes by Intermittent Hypoxia and Its MicroRNA-Mediated Mechanism

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for cardiovascular disease (CVD) and insulin resistance. However, the mechanisms linking IH stress and CVD remain elusive. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes either to sustained hypoxia (1% O2 [SH]), 70 cycles/24 h of IH (5 min SH/10 min normoxia [21% O2]), mimicking SAS patients, or normoxia for 24 h, analyzed mRNA expression and found that IH significantly increased the mRNA levels of regenerating gene (Reg) IV and hepatocyte growth factor (Hgf). Reg IV and Hgf in IH-treated cell medium were also increased. In order to analyze the IH-induced expression mechanisms of Reg IV and Hgf genes, we prepared reporter plasmids containing Reg IV and Hgf upstream of luciferase reporter gene, and transfected them into P19.CL6 cardiomyocytes. The promoter activities of the genes were not increased by IH. Target mRNA search of microRNA (miR)s revealed that both the mRNAs have a potential target sequence for miR-499. The miR-499 level of IH-treated cells was significantly decreased than that of normoxia-treated cells. To clarify the role of miR-499, miR-499 mimic and non-specific control RNA (miR-499 mimic NC) were introduced into P19.CL6 cells, and the IH-induced up-regulation of Reg IV and Hgf was abolished by introduction of miR-499 mimic. We also measured apoptosis of IH/SH-stimulated P19.CL6 cells by TUNEL method and found that SH but not IH increased apoptosis and that Reg IV and Hgf in cultured medium significantly reduced. The addition of Reg IV and/or Hgf in the culture medium attenuated the SH-induced apoptosis. These results indicate that IH stress increased levels of Reg IV and Hgf mRNA via the inhibition of the miR-499-mediated mRNA degradation, leading to protect cardiomyocytes against hypoxia -induced apoptosis. Disclosure S. Takasawa: None. A. Itaya-hironaka: None. A. Yamauchi: None. M. Makino: None. S. Sakuramoto-tsuchida: None. T. Uchiyama: None. Y. Takeda: None. Y. Kyotani: None. H. Ota: None.

Long-Term Effects of Late Gestation in Utero Hypoxic Stress on Mood Disorders: Sex and Age Differences

Abstract Introduction: In utero insults have been linked with increased fear and anxiety in progeny. In utero hypoxic stress is associated with a multitude of gestational complications such as pregnancy-associated hypertensive disorders and intrauterine growth restriction. Maternal hypertension during pregnancy is also associated with increased mood and anxiety disorders in progeny. However, it is unknown if these associations are due to in utero hypoxic stress. We hypothesized that exposure to late gestational hypoxia will have a long-term impact on anxiety in progeny. Methods: Timed pregnant female Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia - 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2) for a total of 10 CIH cycles/hour. At weaning (PND 28), progeny was pair-housed with a conspecific of same sex and similar weight. To examine mood and anxiety disorders, we quantified anxiety-related behaviors (time spent in the center of open field arena, marble burying test, social and anti-social behaviors with conspecifics) along with quantifying food intake and circulating sex hormone levels during puberty (postnatal day, PND 40-45) and young adulthood (PND 60-65) in male and female progeny. Results: Gestational CIH did not impact circulating sex hormones or food intake, regardless of sex or age of progeny. However, gestational CIH increased anxiety related behaviors in pubertal females. These effects of gestational CIH on anxiety in pubertal females were not maintained, as these behaviors resolved in young adulthood. Gestational CIH did not impact male progeny, regardless of age. Conclusion: Exposure to CIH during gestation resulted in increased anxiety related behaviors in pubertal female progeny. In utero hypoxia during late gestation may temporarily increase the risk for mood and anxiety disorders in pubertal females.

Long-Term Effects of Late Gestation in Utero Hypoxic Stress on Neurodegeneration: Sex and Age Differences

Abstract Introduction: In utero insults have been proposed to lead to the onset of neurodegenerative diseases later in life, such as Parkinson’s disease (PD). In utero hypoxia is associated with a multitude of conditions, such as maternal sleep apnea, preeclampsia, gestational diabetes, and maternal hypertension. Exposure to in utero hypoxia may impact male progeny more than female progeny, which may underlie the male biased sex differences in PD. It is currently unknown whether late gestational hypoxic stress has a long-term effect on brain regions associated with PD, such as the nigrostriatal pathway. We hypothesized that exposure to late gestational hypoxia will result in nigrostriatal impairment in adult male progeny compared to adult female progeny. Methods: Timed pregnant female Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia - 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2) for a total of 10 CIH cycles/hour. Gestational age at delivery was recorded and neonate’s body weights were measured within 12-16 hours from birth. At weaning (postnatal day, PND 28), progeny was pair-housed with a conspecific of the same sex and similar weight. To examine PD, we focused on PD associated characteristics of oxidative stress in the nigrostriatal pathway and behavioral impairments of motor (open field activity and ultrasonic vocalizations) and cognitive (spatial memory) function during puberty (PND 40-45) and young adulthood (PND 60-65). Results: Gestational CIH had no effect on the duration of gestation, litter size, and neonatal weight at birth. Gestational CIH did not impact circulating oxidative stress, regardless of sex or age of progeny. Offspring gross motor function (open field activity) and cognitive (Morris Water maze) function were unaffected by gestational CIH. In contrast, gestational CIH impaired ultrasonic vocalizations in adult male progeny. Gestational CIH increased the latency to vocalize and decreased the loudness of the vocalizations in adult male progeny. Conclusion: Exposure to CIH during gestation resulted in nigrostriatal impairment in adult male progeny, as evidenced by impaired ultrasonic vocalizations that require a functional nigrostriatal pathway. In utero hypoxia during late gestation may increase the risk for PD in males.

The Non‐Neuronal Cholinergic System is Causal for Cardioprotection by Hypoxic Preconditioning in Isolated Adult Rat Cardiomyocytes

Introduction Brief episodes of non-lethal myocardial ischemia/reperfusion preceding more sustained ischemia reduce lethal ischemia/reperfusion injury in all species tested so far, including humans. Although this phenomenon is known for more than 3 decades, the underlying signaling mechanisms are unclear in their details. Among various cardioprotective mediators, neuronal acetylcholine (ACh) and its signaling through cholinergic receptors is certainly involved. However, a non-neuronal cardiac cholinergic system (NNCCS) has been recently identified, suggesting a nervous system-independent, cardiomyocyte-derived ACh signaling with signal amplification by autocrine cholinergic receptor activation. Aim We aimed to examine the role of NNCCS-derived ACh as a mediator of cardioprotection. We therefore in a reductionist approach used isolated adult cardiomyocytes with hypoxic preconditioning (HPC). Methods Hearts from male Lewis rats were harvested, and adult ventricular cardiomyocytes were isolated. In suspended cardiomyocytes HPC was induced by one cycle of 10 min brief hypoxia and 20 min reoxygenation. Thirty min normoxia served as control. Afterwards, cardiomyocytes were exposed to 30 min sustained hypoxia and 5 min reoxygenation (H/R) or to 35 min normoxia as time control (TC). Cardiomyocyte viability was quantified before and after H/R or TC, respectively, as the percent fraction of rod-shaped, unstained (0.5% trypan blue) cells over all cells. In parallel experiments, physostigmine (0.2 mmol/L) was added to cardiomyocyte suspensions to block the ubiquitous ACh esterase. Intracellular ACh was quantified with and without HPC via liquid chromatography-coupled tandem mass spectrometry. In subsets, the nicotinic cholinergic receptor antagonists hexamethonium (1.0 µmol/L) and α-bungarotoxine (0.1 µmol/L) or the muscarinic cholinergic receptor antagonist atropine (0.1 µmol/L) were added to cardiomyocyte suspensions with and without HPC, respectively, and exposure to H/R or TC, respectively, afterwards. Again, cardiomyocyte viability was quantified. Results With HPC, the viability of cardiomyocytes subjected to H/R was better preserved than without (24±1% vs. 7±1%, figure A). Also, intracellular ACh was slightly (n.s.) increased after sustained hypoxia (6±2 pmol/mg protein), but significantly with HPC (13±3 pmol/mg protein) in comparison to baseline (3±1 pmol/mg protein) or TC (3±1 pmol/mg protein, figure B). However, this increase was only detectable in the presence of physostigmine (figure B). Addition of nicotinic and muscarinic cholinergic receptor antagonists attenuated the protection by HPC to the same extent (hexamethonium: 12±2%, α-bungarotoxine: 11±1%, atropine: 10±1%, figure A). Conclusion The NNCCS is activated by HPC and causal for HPC´s protection. HPC increases intracellular ACh, and cardiomyocyte protection is mediated via both nicotinic and muscarinic cholinergic receptors, suggesting an autocrine signal amplification on the cardiomyocyte level.

Hydrogen peroxide treatment of Atlantic salmon temporarily decreases oxygen consumption but has negligible effects on hypoxia tolerance and aerobic performance

Hydrogen peroxide is widely used as a treatment of ectoparasite (e.g. salmon lice, amoebic gill disease) affected Atlantic salmon (Salmo salar) in aquaculture but has frequently caused unacceptable levels of mortality. Hydrogen peroxide toxicity increases with dose, exposure duration and temperature, and can cause an acute stress response, oxidative stress, gill damage, respiratory acidosis and altered gas and ion exchange. The combined stress effects have the potential to cause sub-lethal impacts on aerobic capacity that affects hypoxia tolerance and swimming performance. Here, we tested the sub-lethal effects of hydrogen peroxide treatment on the aerobic capacity, hypoxia tolerance and gill histopathology of healthy Atlantic salmon. Hydrogen peroxide treatment (1250 ppm at 12 °C for 20 min) caused a significant but negligible reduction in acute hypoxia tolerance (lost equilibrium at slightly higher levels of dissolved oxygen) immediately following treatment, but not in the 14 days afterwards. Immediately following hydrogen peroxide treatment (1250 ppm at 16 °C for 20 min) oxygen consumption was reduced for up to 20 min in normoxia and 40 min in hypoxia. However, following transient reductions in oxygen consumption the maximum metabolic rate and Ucrit measured in a swim flume were unaffected by hydrogen peroxide exposure (1250 ppm at 16 °C). Resting metabolic rate was unaffected by hydrogen peroxide treatment in all experiments. Hydrogen peroxide exposure caused non-homogenous lamellar epithelium lifting with subepithelial oedema and multi-focal lamellar fusion. The reduction in oxygen consumption following hydrogen peroxide treatment was likely caused by an increase in blood oxygen due to the breakdown of hydrogen peroxide by catalase and other oxidative enzymes in the fish tissues. Ultimately, hydrogen peroxide did not affect aerobic performance and had negligible impacts on hypoxia tolerance, suggesting that hydrogen peroxide has minimal sub-lethal impacts on aerobic performance of healthy Atlantic salmon at the doses used here.

Cardiovascular and metabolic responses to passive hypoxic conditioning in overweight and mildly obese individuals.

Although severe intermittent hypoxia (IH) is well known to induce deleterious cardiometabolic consequences, moderate IH may induce positive effects in obese individuals. The present study aimed to evaluate the effect of two hypoxic conditioning programs on cardiovascular and metabolic health status of overweight or obese individuals. In this randomized single-blind controlled study, 35 subjects (54 ± 9.3 yr, 31.7 ± 3.5 kg/m2) were randomized into three 8-wk interventions (three 1-h sessions per week): sustained hypoxia (SH), arterial oxygen saturation ([Formula: see text]) = 75%; IH, 5 min [Formula: see text] = 75% - 3 min normoxia; normoxia. Ventilation, heart rate, blood pressure, and tissue oxygenation were measured during the first and last hypoxic conditioning sessions. Vascular function, blood glucose and insulin, lipid profile, nitric oxide metabolites, and oxidative stress were evaluated before and after the interventions. Both SH and IH increased ventilation in hypoxia (+1.8 ± 2.1 and +2.3 ± 3.6 L/min, respectively; P < 0.05) and reduced normoxic diastolic blood pressure (-12 ± 15 and -13 ± 10 mmHg, respectively; P < 0.05), whereas changes in normoxic systolic blood pressure were not significant (+3 ± 9 and -6 ± 13 mmHg, respectively; P > 0.05). IH only reduced heart rate variability (e.g., root-mean-square difference of successive normal R-R intervals in normoxia -21 ± 35%; P < 0.05). Both SH and IH induced no significant change in body mass index, vascular function, blood glucose, insulin and lipid profile, nitric oxide metabolites, or oxidative stress, except for an increase in superoxide dismutase activity following SH. This study indicates that passive hypoxic conditioning in obese individuals induces some positive cardiovascular and respiratory improvements despite no change in anthropometric data and even a reduction in heart rate variability during IH exposure.

1887-P: Intermittent Hypoxia Increased the Expressions of Interleukin (IL)-8, Osteonectin, and Myonectin via OCT1 and Nrf2 Binding to the Promoters in Muscle Cells

Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human RD and mouse C2C12 muscle cells either to 70 cycles/24 h of IH (5 min hypoxia [1% O2]/10 min normoxia [21% O2]), mimicking SAS patients, or normoxia for 24 h, measured mRNA levels by real-time reverse transcription polymerase chain reaction, and found that IH significantly increased the mRNA levels of interleukin-8 (IL-8), osteonectin (ON), and myonectin (MN). IL-8, ON, and MN in IH-treated RD cell medium were also increased (P&amp;lt;0.0001, P&amp;lt;0.0001, and P=0.0079, respectively). We further analyzed the IH-induced expression mechanisms of IL-8, ON, and MN genes in RD muscle cells. Deletion analyses revealed that -152 to -151 region in IL-8, -105∼-99 region in ON, and -3,741∼-3,738 region in MN promoter(s) were responsible for the activation by IH. The promoters contain consensus transcription factor binding sequences for octamer binding transcription factor 1 (OCT1) in IL-8 and MN, and for nuclear factor erythroid 2-related factor 2 (NRF2) in ON, respectively. The introduction of siRNA for OCT1, and NRF2 abolished the increase of mRNAs for IL-8 and MN, and ON, respectively. As IL-8, ON, and MN are reported to be myokines and associated with glucose intolerance, insulin resistance, and diabetes, the results could well explain a mechanism of glucose intolerance, insulin resistance, and diabetes in SAS patients. Disclosure S. Takasawa: None. R. Shobatake: None. A. Itaya-Hironaka: None. M. Makino: None. S. Sakuramoto-Tsuchida: None. T. Uchiyama: None. H. Ota: None. A. Yamauchi: None.

Postpartum Maternal Vascular Function in a Rat Model of Gestational Obstructive Sleep Apnea

IntroductionDe novo obstructive sleep apnea (OSA) in pregnancy, is associated with adverse gestational outcomes such as preeclampsia, gestational diabetes, and fetoplacental hypoxia. In addition, exposure to chronic intermittent hypoxia (CIH), a model of OSA, induces endothelial dysfunction and enhanced vasoconstriction in pregnant mice. It is currently unknown whether gestational OSA has a long‐term effect on maternal vascular function. We hypothesized that exposure to gestational CIH during late pregnancy will result in postpartum maternal vascular dysfunction.MethodsTimed pregnant female Long‐Evans rats were randomly assigned to two experimental groups: Normoxia (n=6) and CIH (n=6). The CIH group was exposed to five days (gestational days: 15–20) of intermittent hypoxia [6 min cycles of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2)]. Gestational age at delivery was recorded and neonate’s crown‐rump length, abdominal girth, and body weights were measured within 12–16 hours from birth. At weaning (postnatal day 28), dams were euthanized and third‐order mesenteric arteries, and the main uterine artery, and uterine perivascular adipose tissue (PVAT) were excised. To assess endothelium‐dependent relaxation, we performed concentration‐response curves to acetylcholine (ACh: 10−9 – 3×10−5 M) using wire myography. In uterine arteries, we measured responses to ACh in the presence and absence of PVAT (0.1 g) based on our previous findings that uterine PVAT has vasoactive effects on these arteries.ResultsGestational CIH had no effect on the duration of gestation (Normoxia: 22.3 ± 0.21 days vs. CIH: 22.8 ± 1.16 days, p=0.24), litter size (Normoxia: 12.2 ± 1.64 vs. CIH: 10.3 ± 1.40, p=0.34), and neonatal weight at birth (Normoxia: 6.4 ± 0.24 g vs. CIH: 6.7 ± 0.19 g, p=0.16), while it increased neonatal crown‐rump length (Normoxia: 3.98 ± 0.07 cm vs. CIH: 4.19 ± 0.07 cm, p=0.05) and abdominal girth (Normoxia: 4.72 ± 0.10 cm vs. 4.94 ± 0.03 cm, p=0.07). At weaning, dams exposed to gestational CIH were lighter compared to dams exposed to normoxia (Normoxia: 319 ± 6.5 g vs. CIH: 298 ± 8.7 g, p=0.08). Exposure to gestational CIH had no effect on ACh‐induced relaxation in mesenteric resistance arteries (pEC50, Normoxia: 7.80 ± 0.03 vs. CIH: 7.89 ± 0.06, p=0.19). In the absence of PVAT, uterine arteries from dams exposed to gestational CIH had exaggerated responses to ACh compared to dams exposed to normoxia [(−)PVAT/pEC50, Normoxia: 6.77 ± 0.08 vs. CIH: 7.13 ± 0.09, p&lt;0.01], while PVAT normalized this difference [(+)PVAT/pEC50, Normoxia: 6.67 ± 0.08 vs. CIH: 6.70 ± 0.07, p=0.99]. The effects of PVAT were due to its anti‐dilatory influences on uterine arteries from CIH‐treated rats [CIH (−PVAT) vs. CIH (+PVAT), p = 0.003], whereas it had no effect on arteries from control rats [Normoxia (−PVAT) vs. Normoxia (+PVAT), p = 0.77].ConclusionExposure to CIH during gestation resulted in neonatal macrosomia, reduced maternal weight at weaning, and exaggerated postpartum uterine vascular smooth muscle relaxation responses, while it did not affect small resistance artery reactivity. Gestational OSA may impair maternal vascular recovery after birth.Support or Funding InformationUNTHSC Basic Seed Grant

Abstract 192: Sex Dependent Differences in Cardiac Myocyte Excitation-Contraction Coupling After Chronic Stimulation of the Delta or Kappa Opioid Receptors During Normoxic and Hypoxic Conditions

Introduction: In 2016 the US government declared opioid misuse a public emergency. While most opioid related deaths can be attributed to overdose, recent data suggest a link between opioid use and cardiovascular disease. Further, epidemiological data suggest it may be sex dependent, with females (♀) being at a somewhat higher risk than males (♂). This is paradoxical because until menopause, ♀ have a lower risk of heart disease when compared to ♂. Healthy adult hearts express the δ and κ ORs, but not the μOR. Therefore, the overall goals of this study using a reductionist model were to: 1). To determine if there are sex specific differences in cardiac myocyte excitation-contraction coupling between ♂ and ♀ myocytes exposed to δ or κ OR agonists during normoxia/hypoxia. 2). Examine if 17β-Estradiol enhances or augments the effects of δ or κ OR stimulation. Methods: Myocytes were isolated from adult ♂ and ♀ Sprague-Dawley rats and cultured overnight in M199 HEPES media, or M199 HEPES media containing either 1 μM DADLE (δ agonist), 20 μM U50-488 (κ agonist), 100 nm 17β-Estradiol, 1μM DADLE + 100 nm 17β-Estradiol, or 20 μM U50-488 + 100 nm 17β-Estradiol. To simulate an infarct, a subset of the myocytes was incubated at 1%O 2 , 5% CO 2 for 4 hours + 10 nM isoproterenol followed by 60 min normoxia (21%O 2 , 5% CO 2 ) before recordings. Excitation-Contraction was measured using an IonOptix rig (IonOptix LLC, Milton MA) at 36±1° at 1 Hz pacing. Results: During normoxia, ♂ myocytes compared to ♀ myocytes had an increased peak contraction and peak Ca 2+ transient. The addition of 100 nM 17β-Estradiol did not affect peak contraction in either sex but decreased the Ca 2+ transient in ♂ myocytes while increasing it in ♀ myocytes. DADLE and U50-488 had no effect on ♀ myocyte contractility but decreased ♂ myocyte contractility by a minimum of 13% and a maximum of 26% with/without 100 nM 17β-Estradiol. ♀ myocytes were immune to the effects of hypoxia, even exhibiting a slight increase in their peak calcium compared to control conditions, arguably because of the addition of isoproterenol. ♂ myocytes on the other hand, had an overall decrease in excitation-contraction coupling during hypoxia which was made worse by OR stimulation. Conclusion: ♀ myocytes are immune to the detrimental effects of δ and κ OR stimulation.

1959-P: Upregulation of PYY, GLP-1, and NTS by Intermittent Hypoxia in Enteroendocrine Cells

The patients with sleep apnea syndrome (SAS) are exposed to intermittent hypoxia (IH) during sleep. Although accumulating evidence indicates a strong relationship between SAS and obesity, we previously demonstrated that IH up-regulates the mRNA levels of anorexigenic peptides POMC and CART via GATA transcription factors in human neuronal cells (Int. J. Biochem. Cell Biol. 95, 100, 2018). Appetite is regulated not only by the central nervous system but also by gastrointestinal peptides. Here, we investigated the effect of IH on the appetite-inhibiting gut hormone expression using in vitro IH system. Human and mouse enteroendocrine cells, Caco-2 and STC-1, were exposed to IH [64 cycles of 5 min hypoxia (1% O2) and 10 min normoxia (21% O2)] or normoxia for 24 hours. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of PYY, GLP-1, and neurotensin (NTS) in both Caco-2 and STC-1. ELISA showed that the concentrations of PYY, GLP-1, and NTS in the culture medium were significantly increased by IH. To determine whether the IH-induced increases in PYY, GLP-1, and NTS in enteroendocrine cells were caused by transcriptional activation, reporter plasmids for PYY, GCG, which encodes a preprotein, part of which is cleaved into GLP-1, and NTS were transfected into Caco-2, and the luciferase activity was measured after the IH treatment. The promoter activities of PYY, GCG and NTS were not increased by IH. We then searched targeted miRNA and found that PYY, GLP-1 and NTS mRNAs have potential target sequences for miR-96, -527, and -2116. We measured the levels of the miRNAs and found that the levels of the miRNAs were not decreased by IH. Furthermore, the mRNA levels of PYY, GLP-1, and NTS were significantly up-regulated even in normoxia by trichostatin A and were significantly decreased even in IH by 5-azacytidine, suggesting that IH increases PYY, GLP-1, and NTS mRNAs via alterations in the chromatin structure in enteroendocrine cells. IH might have an anorexigenic influence on the enteric nervous system. Disclosure R. Shobatake: None. H. Ota: None. A. Itaya-Hironaka: None. A. Yamauchi: None. M. Makino: None. S. Sakuramoto-Tsuchida: None. T. Uchiyama: None. N. Takahashi: None. S. Ueno: None. K. Sugie: None. S. Takasawa: None.

Intermittent Hypoxia Up-Regulates Gene Expressions of Peptide YY (PYY), Glucagon-like Peptide-1 (GLP-1), and Neurotensin (NTS) in Enteroendocrine Cells.

The patients with sleep apnea syndrome are exposed to intermittent hypoxia (IH) during sleep. We previously demonstrated the IH-induced up-regulation of the mRNA levels of anorexigenic peptides proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART) in human neuronal cells. Appetite is regulated not only by the central nervous system but also by the peptides from gastrointestinal tract. Here, we investigated the effects of IH on the gene expression(s) of appetite-inhibiting gut hormones. Human enteroendocrine Caco-2 and mouse STC-1 cells were exposed to IH [64 cycles of 5 min hypoxia (1% O2) and 10 min normoxia (21% O2)] or normoxia for 24 h. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of peptide YY (PYY), glucagon-like peptide-1 (GLP-1), and neurotensin (NTS) in Caco-2 and STC-1 cells. ELISA showed that the concentrations of PYY, GLP-1, and NTS in the culture medium were significantly increased by IH. The mRNA levels of PYY, GLP-1, and NTS were significantly up-regulated even in normoxia by Trichostatin A (TSA) and were significantly decreased even in IH by 5-azacytidine (5AZC), suggesting that IH increases PYY, GLP-1, and NTS mRNAs via alterations in the chromatin structure in enteroendocrine cells. IH might have an anorexigenic influence on the enteric nervous system.

Sex Differences in Autonomic Cardiac Control and Oxygen Saturation Response to Short-Term Normobaric Hypoxia and Following Recovery: Effect of Aerobic Fitness.

Introduction: The main aims of this study were to investigate autonomic nervous system (ANS) and arterial oxygen saturation (SpO2) responses to simulated altitude in males and females, and to determine the association between maximal oxygen uptake (VO2max) and these responses.Materials and Methods: Heart rate variability (HRV) and SpO2 were monitored in a resting supine position during Preliminary (6 min normoxia), Hypoxia (10 min, fraction of inspired oxygen (FiO2) of 9.6%, simulated altitude ~6,200 m) and Recovery (6 min normoxia) phases in 28 males (age 23.7 ± 1.7 years, normoxic VO2max 59.0 ± 7.8 ml.kg−1.min−1, body mass index (BMI) 24.2 ± 2.1 kg.m−2) and 30 females (age 23.8 ± 1.8 years, VO2max 45.1 ± 8.7 ml.kg−1.min−1, BMI 21.8 ± 3.0 kg.m−2). Spectral analysis of HRV quantified the ANS activity by means of low frequency (LF, 0.05–0.15 Hz) and high frequency (HF, 0.15–0.50 Hz) power, transformed by natural logarithm (Ln). Time domain analysis incorporated the square root of the mean of the squares of the successive differences (rMSSD).Results: There were no significant differences in SpO2 level during hypoxia between the males (71.9 ± 7.5%) and females (70.8 ± 7.1%). Vagally-related HRV variables (Ln HF and Ln rMSSD) exhibited no significant differences between sexes across each phase. However, while the sexes demonstrated similar Ln LF/HF values during the Preliminary phase, the males (0.5 ± 1.3) had a relatively higher (p = 0.001) sympathetic activity compared to females (−0.6 ± 1.4) during the Hypoxia phase. Oxygen desaturation during resting hypoxia was significantly correlated with VO2max in males (r = −0.45, p = 0.017) but not in females (r = 0.01, p = 0.952) and difference between regression lines were significant (p = 0.024).Conclusions: Despite similar oxygen desaturation levels, males exhibited a relatively higher sympathetic responses to hypoxia exposure compared with females. In addition, the SpO2 response to resting hypoxia exposure was related to maximal aerobic capacity in males but not females.

Hypoxic preconditioning reduces propofol-induced neuroapoptosis via regulation of Bcl-2 and Bax and downregulation of activated caspase-3 in the hippocampus of neonatal rats

ABSTRACTObjective: Evidence has shown that propofol may cause widespread apoptotic neurodegeneration. Hypoxic preconditioning (HPC) was previously demonstrated to provide neuroprotection and brain recovery from either acute or chronic neurodegeneration in several cellular and animal models. Therefore, the present study was designed to investigate the protective effects of hypoxic preconditioning on apoptosis caused by propofol in neonatal rats. Methods: Propofol (100 mg/kg) was given to 7-day-old (P7) Sprague Dawley pups. Before the propofol injection, hypoxic preconditioning was administered by subjecting rats to five cycles of 10 min of hypoxia (8% O2) and 10 min of normoxia (21% O2), then 2 h of room air. We detected neuronal structure changes and apoptosis by hematoxylin and eosin (HE) staining and TUNEL assay, respectively. Bcl-2, Bax and cleaved-caspase-3 levels were quantified using Western blotting and immunohistochemistry. Result: After treatment with propofol, Bcl-2 levels decreased and Bax and cleaved-caspase-3 levels increased. However, our results suggest that hypoxic preconditioning could reverse this change. Conclusion: Our results indicate that pretreatment with hypoxic preconditioning prevents propofol-induced neuroapoptosis by increasing the levels of Bcl-2 and decreasing the levels of Bax and cleaved-caspase-3.