Animal Model Of Sleep Apnea Research Articles

Spontaneous and evoked angiotensin II sniffer cell activity in the lamina terminalis in vitro.

Angiotensin II (ANG II) has been shown to have central nervous system effects. Although tissue renin-angiotensin systems (RAS) have been demonstrated in multiple tissues, the existence of a brain RAS is still a matter of debate. These studies test for angiotensin release from brain slices prepared from adult male Sprague-Dawley rats and male and female renin knock-out rats using Chinese hamster ovary cells modified to express both the angiotensin II type 1 receptor and a fluorescent calcium indicator. Sniffer cells were placed on the slices and calcium transients were measured from those located on or adjacent to the median preoptic nucleus with and without stimulation of the subfornical organ. Bath application of tetrodotoxin (1 µM) significantly attenuated spontaneous events while abolishing evoked sniffer cell activity. Bath application of dl-AP4 (10 µM, glutamatergic antagonist) did not affect either spontaneous or evoked release. Incubating the slices with fluorocitrate to inactive astrocytes did not influence sniffer cell activity in the MnPO. Pharmacological experiments indicate that ANG II release is largely both renin (aliskiren 10 µM) and ACE-1 (captopril 100 µM) dependent. However, experiments with brain slices prepared from male and female Renin knock-out rats suggest that alternative synthetic pathways may exist. Finally, these studies demonstrate that increases in ANG II release are observed following 7 days of chronic intermittent hypoxia. These studies suggest the existence of a tissue-specific RAS in the brain that involves canonical and alternative ANG II synthetic pathways and is upregulated in an animal model of sleep apnea.NEW & NOTEWORTHY These studies used Chinese hamster ovary cells that were cloned to express an angiotensin receptor (At1ra) and a calcium indicator (R-GECO) to detect the release of angiotensin from brain slices containing the lamina terminalis of rats. Some of the experiments use tissue from renin knockout rats. The results support the existence of an angiotensin system in the brain that may involve alternative synthetic pathways and is upregulated by intermittent hypoxia.

Hypothalamic Oxytocin Neuron Activation Attenuates Intermittent Hypoxia-Induced Hypertension and Cardiac Dysfunction in an Animal Model of Sleep Apnea.

Obstructive sleep apnea is a prevalent and poorly treated cardiovascular disease that leads to hypertension and autonomic imbalance. Recent studies that restore cardiac parasympathetic tone using selective activation of hypothalamic oxytocin neurons have shown beneficial cardiovascular outcomes in animal models of cardiovascular disease. This study aimed to determine if chemogenetic activation of hypothalamic oxytocin neurons in animals with existing obstructive sleep apnea-induced hypertension would reverse or blunt the progression of autonomic and cardiovascular dysfunction. Two groups of rats were exposed to chronic intermittent hypoxia (CIH), a model of obstructive sleep apnea, for 4 weeks to induce hypertension. During an additional 4 weeks of exposure to CIH, 1 group was treated with selective activation of hypothalamic oxytocin neurons while the other group was untreated. Hypertensive animals exposed to CIH and treated with daily hypothalamic oxytocin neuron activation had lower blood pressure, faster heart rate recovery times after exercise, and improved indices of cardiac function compared with untreated hypertensive animals. Microarray analysis suggested that, compared with treated animals, untreated animals had gene expression profiles associated with cellular stress response activation, hypoxia-inducible factor stabilization, and myocardial extracellular matrix remodeling and fibrosis. In animals already presenting with CIH-induced hypertension, chronic activation of hypothalamic oxytocin neurons blunted the progression of hypertension and conferred cardioprotection after an additional 4 weeks of CIH exposure. These results have significant clinical translation for the treatment of cardiovascular disease in patients with obstructive sleep apnea.

Intermittent Hypoxia Augments Pulmonary Vasoconstrictor Reactivity through PKCβ/Mitochondrial Oxidant Signaling.

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCβ and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCβ mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoKATP), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCβ inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O2- or mitoROS prevented enhanced PKCβ-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCβ/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoKATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCβ acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCβ/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCβ mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoKATP, and subsequent mitoROS generation.

Commentary: Intermittent Hypoxia Severity in Animal Models of Sleep Apnea

Commentary: Intermittent Hypoxia Severity in Animal Models of Sleep Apnea

Endogenous Opioid Tone in the Hypothalamic Paraventricular Nucleus in an Animal Model of Sleep Apnea: Possible Role in Enhanced Sensitivity to Opioid‐Induced Respiratory Depression

Central opioid‐induced respiratory depression (OIRD) can cause fatal respiratory arrest. Clinically, patients with sleep apnea (SA) have a higher incidence of fatal OIRD and rescue from OIRD frequently requires a higher dose of the opioid antagonist naloxone. Mechanisms conferring increased susceptibility to OIRD in SA patients have yet to be investigated. We recently established a preclinical model of SA‐related OIRD hypersensitivity in which adult Sprague‐Dawley rats are exposed for 8 h/day to 10 cycles/h of hypoxia (21% to 8% O2) with hypercapnia (0% to 8% CO2) for 1 week. We found that this chronic intermittent hypoxia with hypercapnia (CIHHC) model significantly enhances inhibition of PNA burst frequency caused by graded doses of the most lethal clinically‐relevant opioid, fentanyl (CIHHC IC50 = 2.0 mg/kg vs. Control IC50 = 11.0 mg/kg). We also discovered an endogenous opioid tonus in CIHHC exposed rats that tonically suppresses their resting respiratory drive. The latter was revealed as a significant increase of PNA burst amplitude following administration of naloxone (1 mg/kg, iv) that was largely absent in normoxic controls (CIHHC: +40.4 ± 10.6% vs. Control: +3.8 ± 2.0%; n=3/group). However, sources of endogenous opioid tonus was not revealed by systemic naloxone. One brain region implicated in SA‐related cardiorespiratory dysfunction is the hypothalamic paraventricular nucleus (PVN). Importantly, PVN neurons have potential both to produce and respond to opioids. To determine whether endogenous opioids within the PVN contribute to respiratory drive in CIHHC rats, naloxone (1 nmol/side) was administered bilaterally and effects on PNA bursting were analyzed. Similar to systemic administration, PVN naloxone increased PNA burst amplitude to a significantly greater extent in CIHHC rats (+20.1 ± 6.8%, n=3) compared to normoxic controls (+4.0 ± 1.9%, n=5) (P<0.05), indicating greater endogenous opioid tonus in the PVN of our rat model of SA. In CIHHC rats, PVN pre‐treatment with naloxone also attenuated OIRD to systemic fentanyl (20 mg/kg, iv), raising the possibility that increased endogenous opioid tone in the PVN could contribute to their increased sensitivity to OIRD. In control rats, PVN naloxone also attenuated, to a lesser extent, systemic fentanyl‐induced OIRD, which was surprising given that it failed to alter the frequency and amplitude of ongoing PNA bursts. The latter suggests that PVN naloxone stabilizes, without overtly driving, the respiratory network, thereby reducing its vulnerability to OIRD. Taken together, our findings indicate that targeting opioid receptors in the PVN has potential to attenuate OIRD and may be especially effective in patients with SA, who are at greater risk.Support or Funding InformationSupport: NIH T32‐HL007446 (ADB), HL088052 (GMT)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intermittent Hypoxia Severity in Animal Models of Sleep Apnea.

Intermittent Hypoxia Severity in Animal Models of Sleep Apnea.

Intermittent hypoxia induces disturbances in craniofacial growth and defects in craniofacial morphology

ObjectivesTo investigate intermittent hypoxia (IH) induced changes in craniofacial morphology and bone mineral density (BMD) in the mandible of growing rats. DesignSeven-week-old male Sprague-Dawley rats were exposed to IH for 4 days or 3 weeks. Sham-operated rats simultaneously breathed room air. Lateral and transverse cephalometric radiographs of the craniofacial region were obtained, and the linear distances between cephalometric landmarks were statistically analyzed. BMD and bone microstructure of the mandible were evaluated using micro-computed tomography (micro-CT). ResultsCephalometric analyses demonstrated that exposure to IH only in the two groups for 3 weeks decreased the size of the mandibular and viscerocranial bones, but not that of the neurocranial bones, in early adolescent rats. These findings are consistent with upper airway narrowing and obstructive sleep apnea (OSA). Micro-CT showed that IH increased the BMD in the cancellous bone of the mandibular condyle and the inter-radicular alveolar bone in the mandibular first molar (M1) region. ConclusionsThis study is the first to identify growth retardation of the craniofacial bones in an animal model of sleep apnea. Notably, 3 weeks of IH can induce multiple changes in the bones around the upper airway in pubertal rats, which can enhance upper airway narrowing and the development of OSA. The reproducibility of these results supports the validity and usefulness of this model. These findings also emphasize the critical importance of morphometric evaluation of patients with OSA.

Chronic intermittent hypoxia increases encoding pigment epithelium-derived factor gene expression, although not that of the protein itself, in the temporal cortex of rats.

Objective: Obstructive sleep apnea syndrome is mainly characterized by intermittent hypoxia (IH) during sleep, being associated with several complications. Exposure to IH is the most widely used animal model of sleep apnea, short-term IH exposure resulting in cognitive and neuronal impairment. Pigment epithelium-derived factor (PEDF) is a hypoxia-sensitive factor acting as a neurotrophic, neuroprotective, and antiangiogenic agent. Our study analyzed performance on learning and cognitive tasks, as well as PEDF gene expression and PEDF protein expression in specific brain structures, in rats exposed to long-term IH. Methods: Male Wistar rats were exposed to IH (oxygen concentrations of 21-5%) for 6 weeks-the chronic IH (CIH) group-or normoxia for 6 weeks-the control group. After CIH exposure, a group of rats were allowed to recover under normoxic conditions for 2 weeks (the CIH+N group). All rats underwent the Morris water maze test for learning and memory, PEDF gene expression and PEDF protein expression in the hippocampus, frontal cortex, and temporal cortex being subsequently assessed. Results: The CIH and CIH+N groups showed increased PEDF gene expression in the temporal cortex, PEDF protein expression remaining unaltered. PEDF gene expression and PEDF protein expression remained unaltered in the frontal cortex and hippocampus. Long-term exposure to IH did not affect cognitive function. Conclusions: Long-term exposure to IH selectively increases PEDF gene expression at the transcriptional level, although only in the temporal cortex. This increase is probably a protective mechanism against IH-induced injury.

Melatonin prevents hyperglycemia in a model of sleep apnea.

Obstructive sleep apnea is a common disorder associated with aging and obesity. Apneas cause repeated arousals, intermittent hypoxia, and oxidative stress. Changes in glucolipidic profile occur in apnea patients, independently of obesity. Animal models of sleep apnea induce hyperglycemia. This study aims to evaluate the effect of the antioxidants melatonin and N-acetylcysteine on glucose, triglyceride, and cholesterol levels in animals exposed to intermittent hypoxia. Two groups of Balb/c mice were exposed to intermittent hypoxia (n = 36) or sham intermittent hypoxia (n = 36) for 35 days. The intermittent hypoxia group underwent a total of 480 cycles of 30 seconds reducing the inspired oxygen fraction from 21% to 7 ± 1% followed by 30 seconds of normoxia, during 8 hours daily. Melatonin or N-acetylcysteine were injected intraperitonially daily from day 21 on. At day 35, glucose levels were significantly higher in the intermittent hypoxia group than in the control group. The intermittent hypoxia groups receiving N-acetylcysteine and vehicle showed higher glucose levels than the group receiving melatonin. The lipid profile was not affected by intermittent hypoxia or antioxidant administration. The present results suggest that melatonin prevents the well-recognized increase in glucose levels that usually follows exposure to intermittent hypoxia. Further exploration of the role of melatonin in sleep apnea is warranted.

Making Sense of Oxidative Stress in Obstructive Sleep Apnea: Mediator or Distracter?

Obstructive sleep apnea is increasingly recognized as an important contributor to cognitive impairment, metabolic derangements, and cardiovascular disease and mortality. Identifying the mechanisms by which this prevalent disorder influences health outcomes is now of utmost importance. As the prevalence of this disorder steadily increases, therapies are needed to prevent or reverse sleep apnea morbidities now more than ever before. Oxidative stress is implicated in cardiovascular morbidities of sleep apnea. What role oxidative stress plays in neural injury and cognitive impairments has been difficult to understand without readily accessible tissue to biopsy in persons with and without sleep apnea. An improved understanding of the role oxidative stress plays in neural injury in sleep apnea may be developed by integrating information gained examining neural tissue in animal models of sleep apnea with key features of redox biochemistry and clinical sleep apnea studies where extra-neuronal oxidative stress characterizations have been performed. Collectively, this information sets the stage for developing and testing novel therapeutic approaches to treat and prevent, not only central nervous system injury and dysfunction in sleep apnea, but also the cardiovascular and potentially metabolic conditions associated with this prevalent, disabling disorder.

Apnea produces neuronal degeneration in the pons and medulla of guinea pigs

Obstructive sleep apnea and other sleep-related breathing disorders result in recurrent periods of oxygen deprivation (hypoxia), hypercapnia and an increase in the cellular production of reactive oxygen species (oxidative stress-related injury). Individuals with these disorders suffer from a variety of cellular abnormalities that result in cardiopulmonary dysfunctions, disturbances in sleep and other pathologies. In the present experiment, using an animal model of sleep apnea, we determined that the degeneration of neurons and glia, due to apoptosis, occurs in specific regions of the pons and medulla. Adult guinea pigs, which were divided into control (normoxic) and experimental (hypoxic) groups, were anesthetized with α-chloralose and immobilized with Flaxedil. Apnea (hypoxia) was induced by ventilatory arrest in order to desaturate the oxyhemoglobin to 75% SpO 2. A sequence of apnea, followed by ventilation with recovery to > 95% SpO 2, was repeated for a period of 3 h. At the end of the period of recurrent apnea, the animals were perfused and brain sections were immunostained with a mouse monoclonal antibody raised against single-stranded DNA (ssDNA). Apoptotic neurons and glia, which were not found in the control group of animals, were present in brainstem regions in hypoxic group of animals; these regions involved in the control of respiration (e.g., the parafacial respiratory group and the ventral respiratory group), cardiovascular functions (e.g., the nucleus ambiguus, the nucleus tractus solitarius and the dorsal motor nucleus of the vagus) as well as REM sleep (the nucleus pontis oralis) and wakefulness (e.g., the dorsal raphe and locus ceruleus). We suggest apoptotic neurons and glia in critical areas of the pons and medulla results in many of the comorbidities experienced by patients with sleep-disordered breathing pathologies.

Intermittent hypoxia: keeping it real

in this issue of the Journal of Applied Physiology , Tamisier and colleagues ([20][1]) describe a novel method for studying the effects of chronic intermittent hypoxia (CIH) in healthy human subjects. To investigate the effects of CIH on sleep, ventilatory control, and blood pressure, the authors

Arterial Chemoreceptors and Sympathetic Nerve Activity

Chronic elevation in sympathetic nerve activity (SNA) is associated with the development and maintenance of certain types of hypertension1 and contributes to the progression of chronic heart failure (CHF).2 The mechanisms involved in sympathetic dysfunction in these disorders appear to be complex and multifactorial. A unified hypothesis is likely to encompass alterations in multiple autonomic reflex pathways, central integratory sites, and chemical mediators that control sympathetic outflow. For example, tonic restraint of sympathetic outflow by arterial and cardiopulmonary baroreflexes is depressed in CHF2 and depressed or reset in hypertension.3 Moreover, maladaptive changes also occur in the central nervous system at integrative sites for autonomic control in both disease processes.4,5 It is also clear that sympathoexcitatory cardiac,6 somatic,7 and central/peripheral chemoreceptor reflexes8 are enhanced in CHF and hypertension. Arterial chemoreceptors serve an important regulatory role in the control of alveolar ventilation, but they also exert a powerful influence on cardiovascular function.9 Activation of arterial chemoreceptors by hypoxemia increases sympathetic outflow to systemic vascular beds to compensate for the direct vasodilating effects of hypoxia on these vessels and to redistribute blood flow to essential organs. In this review, we highlight relevant information that implicates the arterial chemoreflex as a contributory mechanism for the sympathetic hyperactivity in CHF and hypertension and illustrate proposed mechanisms for this altered function. Arterial chemoreceptors located in the aortic and carotid bodies (CBs) respond to hypoxemia and hypercapnia. Because central chemoreceptors also respond to hypercapnia, hypoxia is typically used as a specific stimulus to arterial chemoreceptors. In some mammals, such as rats and rabbits, reflex responses to hypoxemia arise solely from the CB, whereas in other species, the aortic chemoreceptor contribution can be significant. However, it is not possible to experimentally separate the relative contribution of the aortic and …

Sleep apnea in mice: a useful animal model for study of SIDS?

Although the incidence of sudden infant death syndrome (SIDS) has been decreased by education programs to avoid sleeping in prone position, the pathological mechanisms of SIDS have not fully been understood. Basic research on sleep apnea using experimental animals may help further understanding and prevention of SIDS because the syndrome is thought as inability to wake up from respiratory arrest (apnea) during sleep. Although several animal models of sleep apnea have been described previously, mice would be useful experimental animals in that these animals are frequently used in genetic engineering. Those considerations prompted us to establish a method for measuring ventilation of mice concomitantly with electroencephalography and electromyography for assessing sleep–wake states. Normal wild-type mice developed two types of central sleep apneas (CSA), that is, post-sigh and spontaneous apneas, as normal humans do. Moreover, post-sigh apneas in mice were observed exclusively during slow-wave sleep (SWS) while spontaneous apneas were seen in both SWS and rapid eye movement (REM) sleep. These characteristics are very similar to those of sleep apneas in healthy human infants and children. Therefore, mice seem to be a promising experimental animal model for studying the genetic and molecular basis of respiratory regulation and dysregulation during sleep in humans, especially infants and children. However, we should keep in mind limitations in studying mice as an animal model of SIDS, since they are nocturnal rodents and they sleep in the prone position.

Sleep apnea in mice: a useful animal model for study of SIDS?

Although the incidence of sudden infant death syndrome (SIDS) has been decreased by education programs to avoid sleeping in prone position, the pathological mechanisms of SIDS have not fully been understood. Basic research on sleep apnea using experimental animals may help further understanding and prevention of SIDS because the syndrome is thought as inability to wake up from respiratory arrest (apnea) during sleep. Although several animal models of sleep apnea have been described previously, mice would be useful experimental animals in that these animals are frequently used in genetic engineering. Those considerations prompted us to establish a method for measuring ventilation of mice concomitantly with electroencephalography and electromyography for assessing sleep–wake states. Normal wild-type mice developed two types of central sleep apneas (CSA), that is, post-sigh and spontaneous apneas, as normal humans do. Moreover, post-sigh apneas in mice were observed exclusively during slow-wave sleep (SWS) while spontaneous apneas were seen in both SWS and rapid eye movement (REM) sleep. These characteristics are very similar to those of sleep apneas in healthy human infants and children. Therefore, mice seem to be a promising experimental animal model for studying the genetic and molecular basis of respiratory regulation and dysregulation during sleep in humans, especially infants and children. However, we should keep in mind limitations in studying mice as an animal model of SIDS, since they are nocturnal rodents and they sleep in the prone position.

Quantitative magnetic resonance imaging of upper airways musculature in an animal model of sleep apnea.

Electromyographic studies of patients with sleep apnea and of the English bulldog, an animal model of sleep apnea, indicate that there is increase activity of the airways dilator muscles. The muscles, when biopsied, show both adaptation and muscle injury. In this study we have utilized quantitative magnetic resonance imaging to characterize changes in the upper airway musculature of the bulldog in vivo. The imaging procedure utilized provided a quantitative measurement of the T2 relaxation times of airway muscle (geniohyoid, sternohyoid, sternothyroid, thyropharyngeus, and hyopharyngeus) and nonairway muscles spatially localized to submillimeter-resolution levels. Quantitative differences between the medians and distributions of T2 relaxation times of airway vs. nonairway muscles were demonstrated. These differences were related to the degree of sleep-disordered breathing. The changes observed are compatible with the hypothesis that there is both increased edema and fibrosis in upper airway muscle in sleep apnea.