Serotnin as a possible biomarker in obstructive sleep apnea

Accepted Manuscript Biomarkers in Obstructive Sleep Apnea Melissa C. Lipford, MD, Kannan Ramar, MBBS, MD, Yao-Jen Liang, Chii-Wann Lin, Yun-Ting Chao, Jen An, Chih-Hsien Chiu, Yi-Ju Tsai, Chih-Hung Shu, Fei-Peng Lee, Rayleigh Ping-Ying Chiang PII:

S1087-0792(15)00101-X

DOI:

10.1016/j.smrv.2015.08.003

Reference:

YSMRV 905

To appear in:

Sleep Medicine Reviews

Received Date: 12 December 2014 Revised Date:

7 August 2015

Accepted Date: 7 August 2015

Please cite this article as: Lipford MC, Ramar K, Liang Y-J, Lin C-W, Chao Y-T, An J, Chiu C-H, Tsai YJ, Shu C-H, Lee F-P, Chiang RP-Y, Biomarkers in Obstructive Sleep Apnea, Sleep Medicine Reviews (2015), doi: 10.1016/j.smrv.2015.08.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Biomarkers in Obstructive Sleep Apnea

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Melissa C. Lipford, MDa, Kannan Ramar, MBBS, MDa, Yao-Jen Liangb,c, Chii-Wann Linb,d,e, Yun-Ting Chaof, Jen And, Chih-Hsien Chiug, Yi-Ju Tsaih, Chih-Hung Shuf,i, Fei-Peng Leej , Rayleigh Ping-Ying Chiangb,f,i,j,k,l

a

Center for Sleep Medicine, Mayo Clinic, Rochester, MN, USA

International Sleep Science & Technology Association, Berlin, Germany

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b c

Department of Life Science, Fu Jen Catholic University, New Taipei City,

d

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Taiwan

Graduate Institute of Bioelectronics and Bioinformatics, National Taiwan

University e

Graduate Institute of Biomedical Engineering, National Taiwan University,

Taipei, Taiwan f

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Department of Otolaryngology Head and Neck Surgery, Taipei Veterans

General Hospital, Taipei, Taiwan g

Department of Animal Science and Technology, National Taiwan University,

Taipei, Taiwan

School of Medicine, College of Medicine, Fu Jen Catholic University, New

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Taipei City, Taiwan i

School of Medicine, National Yang-Ming University, Taipei, Taiwan

j

Department of Otolaryngology, School of Medicine, Taipei Medical University,

Taipei, Taiwan k

Sleep Technology Special Interest Group, INSIGHT Center, National Taiwan

University, Taipei, Taiwan l

Center of Sleep Medicine, Taipei Veterans General Hospital, Taipei, Taiwan

Corresponding author: Rayleigh Ping-Ying Chiang, Department of Otolaryngology Head and Neck Surgery & Center of Sleep Medicine, Taipei

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Veterans General Hospital, Taipei, Taiwan. No.201, Sec. 2, Shipai Rd, Beitou District, Taipei City, Taiwan 11217, R.O.C.

Running title: Serotonin as Biomarker for Sleep Apnea

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E-mail: [email protected]

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Conflicts of interest: No authors have indicated conflicts of interest.

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Summary Obstructive sleep apnea (OSA) is a highly prevalent disease which carries substantial public health burden. Polysomnography is the standard procedure used to diagnose OSA. However cost, accessibility, technical

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requirements, and skilled interpretation needs constrain its widespread use and have a role in the under-diagnosis of sleep disordered breathing. There is a

clinical need to develop expedient and widely accessible tools to detect this disorder., Several biochemical markers have recently been proposed as

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diagnostic tools in OSA. Numerous neurochemicals directly influence the activity of upper airway dilator motor neurons, which subsequently influence

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respiration during sleep. Serotonin (5-HT) is one such neurochemical that has a key role in ventilatory stimulation. Herein, we review the current evidence demonstrating relationships between multiple biomarkers and sleep disordered breathing and focus on relationships between OSA and 5-HT. We discuss the

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possibility of biomarker-driven detection technology in the future as a means of diagnosing and monitoring OSA. Finally, we explore the specific role 5-HT may have in the future in both the diagnosis and treatment of OSA.

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Keywords: biomarkers; obstructive sleep apnea (OSA); polysomnography;

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serotonin (5-HT); sleep-disordered breathing; alternative OSA treatments

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Abbreviations AHI, apnea-hypopnea index Au/ZnO, gold/zinc oxide

CA15-3, carbohydrate antigen 15-3 CNS, central nervous system CPAP, continuous positive airway pressure DOI, 1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane

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EBC, exhaled breath condensate

eNO, exhaled nitric oxide 5-HT, serotonin 5-HTT, serotonin transporter

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GABA, γ-amino butyric acid

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eCO, exhaled carbon monoxide EEG, electroencephalography

GG, genioglossus muscle HBV, hepatitis B virus INF-γ, interferon-γ

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LAMP, loop-mediated isothermal amplification LTF, long-term facilitation

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NMDA, N-methyl-D-aspartate nNO, nasal nitric oxide

NREM, non–rapid eye movement OSA, obstructive sleep apnea PPT, pedunculopontine tegmental PSG, polysomnography REM, rapid eye movement SDB, sleep-disordered breathing SPR, surface plasmon resonance

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CA1, cornu ammonis region 1

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SSRI, selective serotonin reuptake inhibitor

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Section I. Introduction Biochemical markers are commonly used in diagnostic testing and assessment of disease severity in persons with myocardial infarction, congestive heart failure, and renal disease. Laboratory biomarker testing allows clinicians

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rapid access to important data regarding a patient’s clinical status. There is a vital need to expand the role of biomarker assays in other disease processes, including obstructive sleep apnea (OSA).

OSA is a common condition affecting 2% to 4% of the US adult

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population [1]. OSA carries significant public health burden; it is independently associated with increased risk of cardiovascular and cerebrovascular disease

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and, due to associated daytime sleepiness, increased risk of automobile accidents [2-5]. However, for the majority of persons who have OSA, the disorder remains undiagnosed [6, 7]. In-lab polysomnography continues to be the primary modality in diagnosis of OSA [8]. Cost, accessibility, technical requirements,

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and skilled interpretation needs constrain its widespread use, and serve as factors in the under-diagnosis of OSA [9].

Home-based sleep testing is increasing in popularity. These studies do not require a sleep technologist to remain present overnight, and testing is

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performed in the patient’s own bed, resulting in less sleep disruption. However, this modality of testing is not recommended for patients with certain medical

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comorbidities, such as congestive heart failure or chronic obstructive pulmonary disease. Due to the unattended nature of this testing, signal artifact or lost electrode leads can degrade data and potentially limit test interpretation. Homebased testing may also underestimate the severity of OSA [10]. Because of the high prevalence of OSA and its associated health risks, a need continues for development of expedient and widely accessible testing. Use of biomarkers may aid in this clinical arena. Complex neuronal circuitry in the midbrain and brainstem areas provides central control of breathing. A cascade of neurotransmitter and

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neuromodulator signaling between these neurons controls ventilatory drive. Via direct and indirect pathways, these neurochemicals can stimulate or inhibit respiratory muscle tone. Serotonin (5-hydroxytryptamine, 5-HT) serves a vital role in multiple respiratory pathways and is implicated directly in the

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pathogenesis of sleep disordered breathing[11]. Chemosensitive 5-HT neurons stimulate respiration in response to elevations in carbon dioxide levels through activation of motoneurons which control upper airway activity [12]. 5-HT

systems also have a role in the autonomic response and arousal from sleep seen

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in sleep apnea [12-15]. We will review current data on how 5-HT and associated biomarkers have a role in the pathogenesis of OSA and may

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eventually aid in the diagnosis and treatment of the disease.

Section II. Overview of OSA and Neurotransmitter Physiology Hypoglossal motoneurons, innervate the intrinsic and extrinsic

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muscles of the tongue, maintain patency of the upper airway during respiration, and have a central role in the pathophysiology of OSA [16-18]. Several neurochemicals directly influence the activity of these upper airway dilator motoneurons—in particular, the hypoglossal neurons that innervate the

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genioglossus muscle (GG) of the tongue. Glycine, γ-amino butyric acid (GABA), and glutamate are major neurotransmitters that act on the upper airway

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motoneurons. Numerous neuromodulators, primarily 5-HT, as well as others including noradrenaline, and acetylcholine are also actively involved upstream in facilitating stimulation of upper airway dilator motoneurons [19, 20]. A. γ-Amino Butyric Acid Cycles of hypoxemia and arousals associated with sleep apnea lead to alterations in the synthesis and release of multiple neurotransmitters in the brainstem [21]. GABA has an inhibitory role at the hypoglossal motor nucleus [22]. In an in vitro study, exposure of pheochromocytoma cell cultures to

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intermittent hypoxia decreased glutamic acid decarboxylase-67 activity and GABA levels. GABA synthesis is primarily catalyzed by glutamic acid decarboxylase [21]. A study involving guinea pigs demonstrated recurrent periods of apnea induced extensive apoptosis in central nervous system (CNS)

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nuclei that control both non–rapid eye movement (NREM) and rapid eye

movement (REM) sleep, and eszopiclone (a GABA agonist) is capable of

preventing neuronal degeneration at these sites [23]. Human studies have shown that the Ala20Val polymorphism of the GABA (B) R1 gene may be associated

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with OSA, whereas the Gly489Ser polymorphism of the gene does not share the association. The C/C variant of the Phe658Phe polymorphism GABA (B) R1

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gene also appears to be associated with OSA (OSA) [24]. These studies suggest that GABA has a potentially important role in sleep apnea. However, further work is required to explore this hypothesis and establish the specific

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relationships in human subjects.

B. Glutamate and N-methyl-D-aspartate Hypoxemia is a powerful stimulus for the release of glutamate in the central nervous system. Glutamate is a potent neurotoxin and is involved in

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neuronal death in the setting of ischemic and hypoxic CNS injuries [25]. This phenomenon may be extrapolated to the cycles of hypoxemia associated with

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sleep-disordered breathing (SDB). Glutamate and its interactions with the Nmethyl-D-aspartate (NMDA) receptor appear to have bidirectional relations with sleep apnea.

Glutamate and NMDA also appear to be involved specifically in the

degeneration of critical memory pathways. This link may explain the evolving associations between OSA and neurodegenerative disease. Recurrent apneic episodes result in high levels of glutamate, which may lead to excitotoxicitydriven cellular damage and eventually to apoptosis of cornu ammonis region 1 (CA1) neurons within the hippocampus. This area of neuronal circuitry is

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critical in memory formation and aberrations lead to deficits in the organization and storage of new memories [26]. Animal models have replicated this type of neuronal damage after glutamate microinjections and demonstrated the

postjunctional NMDA receptors [27].

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downstream cellular effects on memory centers are in part mediated through

Macey et al studied high-resolution brain MRI in patients with OSA and controls [28]. Patients with OSA had multi-focal gray matter loss compared to controls. These degenerative-type changes were observed within memory

between OSA and cognitive dysfunction.

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centers of the hippocampus. These studies highlight the potential relationships

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Guinea pig models indicate that apnea-induced potentiation of the hippocampal CA1 field excitatory postsynaptic potential is mediated by an NMDA receptor mechanism [26]. Repetitive apneic episodes result in excess presynaptic release of glutamate in the CA1 area of the hippocampus. Glutamate

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binds to the many NMDA receptors in this area, leading to increased calcium entry, and eventually to excitotoxic driven apoptosis of CA1 area neurons [29]. Following recurrent apneic episodes, downstream post-synaptic communications between the CA1 and CA3 hippocampal areas are reduced. Use of an NMDA

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receptor antagonist, applied locally or systemically in apneic animals, blocked these downstream reductions in synaptic communications between the CA1 and

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CA3 [26]. These results suggest that recurrent apnea induced excitotoxicity may damage hippocampal circuitry, thus leading to memory deficits in the OSA patient. In fact, a double-blind randomized cross-over study, studied use of the glutamate antagonist (sabeluzole) in OSA patients. A dose-dependent reduction in oxygen desaturation index was observed with use of the drug, suggesting reduced apneic episodes [30]. However, a double-blind, randomized, placebocontrolled single-dose crossover study of the low affinity NMDA receptor antagonist AR-R15896AR in 15 male patients with moderate to severe SDB

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found that AR-R15896AR administration did not significantly change the overall apnea-hypopnea index (AHI) or oxygen saturation [27]. Cui et al [31] studied injection of L-glutamate into the insular cortex of rats and subsequent EMG activity in genioglossus and diaphragm

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musculature were measured. L-glutamate stimulation led to reduced

genioglossus and diaphragm electrical activity and induced apneic episodes in rats. The stimulation also led to significant downstream reductions in 5-HT levels in plasma and the brainstem.

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Cardiovascular research using animal models has shown that sleep apnea leads to degeneration of the nucleus ambiguous neurons expressing the

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glutamate receptor. This degeneration contributes to impairment of baroreflexmediated reductions in heart rate in Fischer 344 inbred rats exposed to intermittent hypoxia over a period of 35 to 50 days [32]. Localized neurodegeneration may explain how OSA can lead to autonomic dysregulation,

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and sympathetic overactivity. This may be one route by which OSA predisposes patients to cardiac disease [33].

The above discussion demonstrates that multiple neurotransmitters and cascades of neurochemical reactions are involved in the pathophysiology of

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sleep apnea.

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obstructive sleep apnea. We will now focus specifically on the role of 5-HT in

Section III.

Role of Serotonin in Respiratory Function

5-HT acts both as a neurotransmitter and a neuromodulator in the

human CNS. The 5-HT neurons located near the brainstem have global projections throughout the brain. These serotonergic connections have effects on numerous CNS processes, and dysfunction in this system may be implicated in respiratory pathology, such as sleep apnea and sudden infant death syndrome (SIDS)[12]. The effects of 5-HT depend upon the specific cellular receptors it acts upon (5-HT1-2 and 5-HT4-7), influences from downstream second messenger

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systems, central versus peripheral application, and interactions with other neuromodulatory chemicals [15, 34].

A. Serotonin and Respiratory Function

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The serotonergic neurons (primarily housed in the medullary raphe and ventrolateral medulla) project to multiple respiratory nuclei in the pons and medulla [35, 36]. These neurons release 5-HT, substance P, and thyrotropinreleasing hormone (TRH) [36]. Each of these neural factors participate in a

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cascade of intracellular signaling reactions, ultimately modulating neuronal excitability and playing a vital role in ventilatory control through multiple

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mechanisms.

5-HT has a predominantly excitatory central effect on respiratory function. . Electrical stimulation of the raphe (leading to 5-HT release) causes excitation of phrenic motor neurons and leads to diaphragmatic contraction[37].

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5-HT also leads to excitatory stimulation to the hypoglossal and trigeminal motoneurons, which serve upper airway dilator musculature [14, 38]. In rats with respiratory insufficiency due to spinal cord contusion, systemic application of 5-HT agonist therapy led to restoration of a normal ventilatory response [39].

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Similar studies have demonstrated that 5-HT agonists can reverse effects of morphine driven respiratory depression [40]. Similarly, substance P and TRH

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also have stimulatory effects on the rhythmicity of breathing by increasing phrenic nerve activity [41-43]. However serotonergic effects on respiration are complicated.

Inhibitory respiratory effects were observed when peripheral receptors (5-HT2A, 5-HT2C, and 5-HT3) were stimulated via intravenous injection of 5-HT. These effects are postulated to be through serotonergic effects on the nodose ganglion [44]. The potentially varying peripheral and central respiratory responses to 5HT may present a challenge in finding serotonergic-based pharmacologic agents to promote respiratory stability. However, the available data supports that the

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summative effect of 5-HT leads to stimulation of respiration and maintenance of upper airway patency. The 5-HT system also serves a role in modulating the ventilatory response to hypercapnia [45, 46]. Medullary serotonergic neurons act as

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chemoreceptors which are highly sensitive to changes in carbon dioxide levels [47-49]. Increased arterial carbon dioxide levels lead to a proportionally

increased respiratory rate. This process is driven by ventral raphe serotonergic neurons which project to breathing centers located throughout the medulla and

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spinal cord, and stimulate a respiratory response. 5-HT neuronal systems also project diffusely throughout other regions of the brain and are involved in non-

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respiratory responses to hypercapnia including sympathetic activation and precipitating arousal from sleep [12, 50, 51]. 5-HT systems provide multifaceted protective effects on upper airway stabilization and ventilation. Based on these mechanisms, it is certainly possible that decreased stimulation of the respiratory

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network by reduced activity of 5-HT neurons may lead to sleep-related hypoventilation and hypercapnia.

Serotonergic systems also likely play a role in SIDS. Autopsy studies have demonstrated 5-HT abnormalities in infants who died of SIDS, possibly

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implicating the effects of 5-HT on respiratory musculature and central

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respiratory chemoreceptor driven control [52].

Section IV. Serotonin and Sleep Disordered Breathing A. Serotonin and Sleep Architecture Serotonergic neurons have a significant influence on sleep/wake

cycles due to their multiple connections throughout the cortex, basal forebrain, limbic system, and brainstem areas. The 5-HT systems function predominantly to promote wakefulness and inhibit REM sleep [53, 54]. However, 5-HT mediated effects vary depending upon the administration route and specific binding pattern to any of the seven known classes of receptors. For example,

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microinjection of 5-HT1A agonists into the dorsal raphe nucleus increases REM sleep [55]; whereas, local administration of 5-HT1B, 5-HT2A/2C, 5-HT3, and 5HT7 agonists decrease REM sleep. In mouse models, animals engineered to lack expression of 5-HT1A or 5-HT1B receptors have greater amounts of REM sleep in

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comparison to normal mice [56, 57]. These alterations were observed without changes in wake or slow wave sleep patterns. Whereas, mice without 5-HT2A or 5-HT2C receptors have increased wakefulness and decreased slow wave sleep [58, 59]. Systemic administration of nonselective 5-HT2A/2C antagonists,

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selective 5-HT2A antagonists, or 5-HT2A inverse agonists increase slow-wave sleep and reduce wakefulness in animal models, humans with normal sleep, and

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in patients with primary or comorbid insomnia [54, 60]. When full agonists are administered (with binding to 5-HT1A, 5-HT2A/2C, 5-HT3, and 5-HT7 receptors) the net serotonergic effect on sleep is to increase wakefulness and reduce both slow wave sleep and REM [54].

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While the effects of 5-HT on sleep architecture are complicated, the overall effects may serve as protective factors in OSA. The wake promoting effects of 5-HT may be involved in provoking arousals during sleep apnea episodes. Additionally, the REM suppressant activity of 5-HT may be involved

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in lowering the severity of sleep apnea. The majority of OSA patients experience significant worsening of disease during REM [61]. Buchanan et al

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[62] demonstrated in mice genetically modified to have selective loss of 5-HT neurons in the brain, there was loss of the arousal response to hypercapnia. In another study, when wild-type mice were treated with 5-HT2A receptor antagonist therapy, there was a dose-dependent reduction in hypercapniainduced arousals [63]. These important findings suggest that arousals mediated through 5-HT systems may play a critical role in reducing the length of apneic episodes and protecting patients against severe hypercapnia.

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B. Serotonin and Sleep Disordered Breathing Serotonergic drive is diminished during sleep compared to wakefulness, leading to a relative reduction in 5-HT driven ventilatory stimulation. This reduced 5-HT activity may contribute to upper airway

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collapse in patients with OSA [15, 64]. During the process of normal aging, there is a significant decrease in available 5-HT receptors. This has been consistently documented in multiple studies through use of functional

neuroimaging techniques [65, 66]. The enhanced reduction in 5-HT activity with

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age may serve as an explanation of the increased OSA frequency observed in older individuals. In those over age 65, the prevalence of OSA is thought to be

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as high as 62% [67]. Another association between serotonergic deficiency and the development of OSA lies in the numerous studies which have demonstrated relationships between OSA and depression [68-70]. This relationship exists despite controlling for factors such as obesity and hypertension [71].

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Certain neurodevelopmental disorders such as Prader-Willi Syndrome are associated with extremely high rates of OSA. This disease process is associated with marked reductions in central 5-HT activity[72]. OSA manifests prior to the development of obesity in Prader-Willi patients [73]. This suggests a

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central serotonergic mediated role in the development of sleep apnea. Animal based studies on sleep disordered breathing frequently utilize

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the English bulldog. The natural upper airway anatomy of this breed results in nearly ubiquitous OSA [74]. 5-HT systems play a protective role in improving upper airway patency in this animal [64, 75]. In fact, when 5-HT antagonists (methysergide or ritanserin) were administered, these animals were noted to begin snoring even during wakefulness and blood oxygen saturations correspondingly decreased. In parallel to these findings, the activity of upper airway dilator muscle activity was reduced in the setting of 5-HT antagonists [64].

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Within the respiratory rhythm generator, the 5-HT1A receptor is the most extensively expressed 5-HT receptor. Besnard et al [76] investigated timeand dose-related effects of central and systemic injections of 8-OHDPAT (5HT1A agonist), SB224289 (5-HT1B antagonist), and DOI (5-HT2A/2C agonist) on

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GG activity in anesthetized and conscious rats. This study suggests that

serotonergic modulation of the respiratory component of the GG muscle

activities remains complex but is highly sensitive to 5-HT1A receptors after

central injection in rats under anesthesia. In research involving 5-HT2 receptor

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homozygous knockout mice, NREM apneas were not modified, and bradypnea following sighs were more pronounced in 5-HT2A-/- mutant mice [59]. The

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effects of [+/-]-2,5-dimethoxy-4-iodoaminophentamine, a 5-HT2A/2C receptor agonist, on pharyngeal airflow mechanics were examined in both anesthetized lean and obese rats. The net effect was reduced potential for upper airway collapse via serotonergic stimulation of hypoglossal nerves as well as

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stimulation of upstream upper airway motoneurons [77].

C. Serotonin-Based Pharmacotherapies in Sleep Disordered Breathing Based on animal model responses to 5-HT antagonists as precipitators

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of sleep disordered breathing, 5-HT agonists (such as L-tryptophan, fluoxetine and paroxetine), have been hypothesized as being potential pharmacologic

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treatments of OSA. In human trials, reduced apneas were noted with use of these medications, however this improvement has been restricted to NREM sleep [7880].

Mirtazapine, an antidepressant with 5-HT1 agonist, as well as 5-HT2

and 5-HT3 antagonist properties has also been investigated using a rat model of central apnea. Carley and Radulovacki [81] showed that mirtazapine significantly reduced central apnea expression during NREM and REM sleep in the rat. The investigators determined that this effect is most likely due to the mixed agonist/antagonist profile of mirtazapine at 5-HT receptors [81].

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However, a previous small, randomized controlled trial found an approximate halving in the severity of OSA with daily mirtazapine doses of 4.5 and 15 mg [82]. However the treatment caused weight gain, which could eventually lead to worsening of OSA.

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Administration of ondansetron, a 5-HT3 antagonist, to rats led to

reduction in central sleep apnea events. This effect appears to be peripherally driven, through activation of the nodose ganglion [83, 84]. Prospective clinical trials addressing the role of 5-HT in sleep apnea have indicated that the selective

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serotonin reuptake inhibitor (SSRI) fluoxetine is beneficial to some patients,

models of sleep apnea[85].

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whereas ondansetron seems of little value despite its efficacy in rat and dog

Both ondansetron and fluoxetine reduced the frequency of apneic episodes attributable to increased monoamine levels in a mouse model of monoamine oxidase A deficiency [86]. A combination of ondansetron and

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fluoxetine resulted in an AHI reduction of approximately 40% from baseline AHI at day 14 and day 28 and improved the trend of oximetry results in patients with OSA [85]. Trazodone is a weak SSRI and a 5-HT2C antagonist, however, its metabolite is a powerful 5-HT2C agonist. Treating 9 OSA patients with 100 mg

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of trazodone increased the arousal threshold in response to hypercapnia and promoted improved ventilatory stability [87].

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The limited clinical trials involving patients with OSA demonstrate

only modest effects of serotoninergic drugs in treating sleep apnea. However numerous animal model studies, as discussed above, suggest substantial promise in this area. Further work involving human subjects is needed to explore the potential role of serotonergic pharmacotherapies in the treatment of OSA. The varied response to serotonergic drugs in human OSA may be attributed to differences in the genetic expression of serotonergic receptors. 5HT ligands significantly modulate GG activity. Genetic study of polymorphisms in the 5-HTR2A gene in Brazilian patients with and without OSA showed that -

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1438G-A polymorphism, but not 102T-C polymorphism, is related to OSA. The prevalence of the 1438G-A polymorphism in the 5-HTR2A gene was more prevalent in OSA patients compared with controls (OR 2.3; CI 95%) [88]. Specific 5-HT ligand studies are necessary in human subjects to further

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elucidate these important relationships.

Serotonin transporter (5-HTT) gene polymorphism is another area of research applicable to sleep disordered breathing [89, 90]. 5-HTT controls the reuptake of serotonin and regulates the interaction between 5-HT and its

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receptors. The 5-HTT I allele was shown to be significantly associated with

during sleep (p=0.014) [89].

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severity of OSA in human subjects (p=0.019) as well as oxygen desaturation

The available evidence clearly demonstrates that 5-HT is involved in the pathophysiology of sleep disordered breathing. There are numerous factors which affect the presence and severity of OSA. The degree to which 5-HT is

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involved remains unclear. However, furthering our understanding of the the relationships between 5-HT and sleep apnea will be critical in the search for pharmacotherapies in the treatment of sleep apnea. It is possible that future studies in this area will also lead to the employment of 5-HT biomarker

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technology in the detection and monitoring of sleep apnea.

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Section V. Biomarker Detection Technology A. Diagnostic Tools for Sleep Disordered Breathing Clinical history and physical examination are of paramount

importance in the identification of patients at risk for sleep disordered breathing. However, actual determination of the presence, type, and severity of disease requires overnight polysomnography. This testing is expensive, labor intensive, and in many areas, poorly accessible. New diagnostic tools are required which could supplement clinical evaluation and provide screening or diagnostic results regarding the presence of sleep apnea. These tools must be readily accessible,

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inexpensive, and provide rapid data. This is the most important obstacle to overcome in the future of testing for sleep apnea. Similarly efficient tools are also needed to gauge efficacy of treatment in patients with known sleep disordered breathing.

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Several physical and biochemical markers have been proposed as

ancillary, or even substitutive, tools in the detection of sleep apnea. One of the best studied approaches involves analysis of heart rate variability [91]. Timefrequency analysis to examine heart rate variability has proven to be a useful

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tool to identify patients with OSA. The modulation of heart rate is a surrogate marker for the increased sympathetic and decreased parasympathetic activity

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seen in sleep apnea. Treatment of OSA using continuous positive airway pressure (CPAP) leads to normalization of heart rate variability [92]. Electroencephalography (EEG) alterations could also potentially be used in screening for OSA. The mean relative power of EEG was calculated for

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delta, theta, alpha, and beta frequency bands. According to the results, EEG power (theta and delta frequencies) increased in patients with severe OSA and normalized after 6 months of CPAP treatment [93]. Several inflammatory cytokines, such as C-reactive protein, tumor

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necrosis factor- α gene, interleukin 6, and others, increase in patients with OSA [94]. Investigators have postulated that the repetitive episodes of OSA (with

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associated cycles of hypoxia and reperfusion), result in vascular oxidative stress which increases the concentration of inflammatory mediators [95]. When these concentration levels were correlated with AHI, several studies have shown a significant reduction of inflammatory mediators after CPAP treatment [96, 97].

B. Point-of-Care Testing for Biomarkers A study by Petrosyan et al [98] used gas analysis of exhaled breath condensate (EBC) as a novel means to detect OSA. OSA is often accompanied by airway inflammation and oxidative stress, and the study used analysis of

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exhaled air to detect inflammatory marker suggestive of OSA. The investigators measured components of exhaled nitric oxide (eNO), nasal nitric oxide (nNO), and exhaled carbon monoxide (eCO). Leukotriene B4, hydrogen peroxide, pH, and 8-isoprostane were also measured. The results showed that the measured

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eNO, nNO, and eCO levels were higher in OSA patients than in healthy

subjects. Exhaled air from OSA patients had greater concentration of leukotriene B4, hydrogen peroxide, and 8-isoprostane than the healthy subjects. In contrast, the pH of OSA patients was lower than in control subjects. Overall, airway

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inflammation and oxidative stress were present in the OSA patients, and EBC markers demonstrated a correlation with the severity of OSA. EBC studies have

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also demonstrated use in the detection of pediatric OSA [99].

As a key respiratory modulating neurotransmitter in the CNS, 5-HT has the potential to also serve as a biomarker for sleep disordered breathing. Further research in the detection of 5-HT levels between individuals with and

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without sleep apnea is required.

C. Surface Plasmon Resonance Biosensor Technology Surface plasmon resonance (SPR) technology has been available for biomedical uses since the 1990s. SPR involves analysis of adsorption of

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materials onto a thin metal surface. The measured refractive index is sensitive to molecular interactions and the concentration of substances near the surface.

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SPR detection systems are utilized in the detection of biotoxins, herbicides and pesticides, cytokines, hormones, and tumor markers [100, 101]. This assay system is a powerful quantitative tool when working with

low concentrations of analytes, and it has provided several advantages, such as its simplicity of use, real-time result analysis, and detection of biomolecules directly without labeling. Micro-concentrations of various substances in human saliva have been analyzed using SPR technology. For example, carbohydrate antigen 15.3 (CA15-3) is generally present at lower levels in the saliva of healthy women

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than in women who are breast cancer patients. Liang et al [102] used SPR biosensor technology based on gold/zinc oxide (Au/ZnO) thin film and the Biacore SPR system (General Electric Co) to compare the sensitivity of the different systems. The group prepared different concentrations of CA15-3 and

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analyzed intensity responses to the samples. Their results showed that the

Au/ZnO thin-film SPR system has a linear detection range less than 20 U/mL better than the Biacore SPR system. The study group postulated that the Au/ZnO thin-film SPR system will become a powerful diagnostic tool in breast cancer

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detection.

Chang et al [103] developed a bifunctional, combined aptamer

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system for the detection of interferon-γ (INF-γ) in pure buffer and plasma dilutions. INF-γ is a factor implicated in tuberculosis. The study group also constructed a simple, low-cost SPR-based, loop-mediated isothermal amplification (LAMP) sensing system for the on-site detection of hepatitis B

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virus (HBV). LAMP can be detected directly through measuring the change of refractive index in the bulk LAMP solution with SPR sensing methods [104]. Several biomarkers have emerged as providing important information regarding respiratory status and specifically the presence of OSA [105]. 5-HT in

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particular may be harnessed as a specific biomarker for OSA diagnostic testing. Detection of 5-HT levels may be possible through SPR biosensor technology.

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Further studies are required evaluating analysis of 5-HT levels using Au/ZnObased thin-film SPR systems. This detection system has high sensitivity and could potentially be used for measuring 5-HT levels in human blood without need for concentrating samples. The potential competitive advantages of this type of system include relatively low cost of implementation and rapid result turnaround with high sensitivity and high specificity. We believe that measurement of 5-HT concentrations with the SPR system may eventually serve a role in the diagnosis and assessment of OSA severity.

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Section VI. Conclusion 5-HT has a critical role in influencing respiratory control during sleep. 5-HT acts as a potent central ventilatory stimulant and serves to maintain upper airway patency. 5-HT neurons also maintain eucapnia via their chemoreceptor

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properties. Relative reductions in 5-HT may lead to the development and

worsening of OSA. Further research is required on the role of 5-HT-enhancing drugs in the treatment of OSA. Studies thus far have shown promise; however, the pharmacological effects seemed to be restricted to NREM sleep. Further

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works involving both animal and human models are required to ascertain the future potential clinical use of 5-HT pharmacotherapies in the management of

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OSA.

5-HT may be a feasible biomarker for OSA and has tremendous potential in the ascertainment of the diagnosis and severity of OSA. Biomarker detection technologies may evolve to represent an accurate, accessible, and rapid

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mode of diagnosing sleep disordered breathing.

SPR has the capability to detect biochemicals at the molecular level. Although the application of SPR in 5-HT detection is not well established to date; further studies using SPR technology to quantify 5-HT levels would be an

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important milestone in developing future tools in the diagnosis of OSA.

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Practice Points 1. Obstructive sleep apnea (OSA) is a common condition with significant associated public health burden. OSA is an independent risk factor for cardiovascular and cerebrovascular disease. Untreated disease worsens:

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hypertension, atherosclerosis, oxidative stress, systemic inflammation, and increases risk of cardiac arrhythmias. The current standard for diagnosing OSA is polysomnography. However limitations in this

modality of testing include: high cost, restricted accessibility, and need

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for skilled interpretation.

2. Serotonin (5-HT) is a biomarker which serves as a powerful respiratory

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stimulant, directly activating motoneurons serving respiratory muscles as well as through actions via chemoreceptor pathways

Research Agenda

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1. There is a clinical need to develop new diagnostic tools for OSA. Harnessing the power of specific biomarkers such as 5-HT may provide an avenue which could improve accessibility and reduce healthcare burden associated with the diagnosis of OSA.

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2. Surface plasmon resonance (SPR) can detect biochemicals at the molecular level. This technology may represent a vehicle to quickly,

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accurately, and cost-effectively detect serotonin levels. Research is required to determine if SPR technology can be utilized in measuring 5HT levels and if this could represent a novel means in the diagnosis of OSA.

3. Further research is needed in the employment of 5-HT agonist medications in the treatment of OSA.

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