Spontaneous Central Apneas Research Articles

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

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

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

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

Neural drive to respiratory muscles in the spontaneously breathing rat pup

The neonatal rodent serves as useful and appropriate model within which to study respiratory system development. Despite an extensive literature that documents respiratory control in vitro, in vivo studies have relied upon whole body plethysmography to determine measures of respiratory frequency and tidal volume. However, plethysmography restricts access to the animal and thus, respiratory muscle electromyographic (EMG) activities have not been recorded in these studies previously. Electromyography yields accurate information about neural respiratory center output to the musculature and therefore, about the control of breathing in the intact animal. In this case, we documented neural drive to respiratory pump and upper airway muscles, electrocardiogram (ECG) and chest wall motions in rat pups up to 10 days of age noting sighs, spontaneous central apneas and hypopneas in room air and with successive increments in fractional inspired CO2 (FICO2). Our findings underscore the advantages of EMG recordings for purposes of determining the magnitude and distribution of neural drive to respiratory muscles and for characterizing the full range of breathing behaviors exhibited by rats in the early postnatal period.

8-OH-DPAT suppresses spontaneous central apneas in the C57BL/6J mouse strain

Apneas are common and prognostically relevant disorders of the central control of breathing, but pharmacological interventions are dissatisfying. The respiratory phenotype of C57BL/6J mice is characterized by the occurrence of spontaneous central apneas with laryngeal closure. In the present study we investigated the impact of the 5-HT(1A) receptor agonist 8-OH-DPAT on apneas in C57BL/6J mice, because of the important role of serotonin in the regulation of breathing and previous reports showing that serotonergic drugs can affect central apneas. Whole-body plethysmography in awake, unrestrained mice revealed that intraperitoneal application of 8-OH-DPAT (10microgkg(-1)) decreased the occurrence of spontaneous apneas from 1.91+/-0.25 to 1.05+/-0.05 apneas min(-1). The efficacy of 5-HT(1A) receptor activation was further verified in the in situ working heart-brainstem preparation. Here the apneas occurred at a frequency of 1.33+/-0.19min(-1). Intra-arterial perfusion with 1-2microM 8-OH-DPAT completely abolished spontaneous apneas. These results suggest that 5-HT(1A) receptor activation may be a potential treatment option for central apneas.

Spontaneous central apneas occur in the C57BL/6J mouse strain

Despite the clinical significance of central apneas in a wide range of disorders little is known about their pathogenesis. Research in this field has been hindered by the lack of appropriate animal models. Our goal was to determine whether the C57BL/6J mouse strain, which has an inherited predisposition for dysrhythmic breathing, exhibits spontaneous apneas. In vivo plethysmography of unanesthetized, unrestrained adult C57BL/6J mice revealed a regular occurrence of spontaneous apneas. In situ recordings from respiratory outputs (phrenic, vagal, hypoglossal nerves) in the working heart-brainstem preparation (WHBP) also showed spontaneous central apneas accompanied by laryngeal closure as indicated by tonic vagal postinspiratory activity and increase in subglottal pressure. The apneas were further characterized by a hypoglossal discharge with delayed onset compared to the tonic vagal postinspiratory activity. We conclude that spontaneous central apneas with active laryngeal closure occur in C57BL/6J mice. This mouse strain is a useful animal model to study neuronal mechanisms that underlie the generation of spontaneous central apneas.

Active glottal closure during central apneas limits oxygen desaturation in premature lambs.

Our laboratory previously reported that active glottal closure was present in 90% of spontaneous central apneas in premature lambs while maintaining a high-apneic lung volume (Renolleau S, Letourneau P, Niyonsenga T, and Praud JP. Am J Respir Crit Care Med 159: 1396-1404, 1999.) The present study aimed at testing whether this mechanism limits postapnea oxygen desaturation. Four premature lambs were instrumented for recording states of alertness, thyroarytenoid muscle and diaphragm electromyographic (EMG) activity, nasal airflow, lung volume changes, and pulse oximetry. One thousand four hundred fifty-two spontaneous central apneas (isolated or during periodic breathing) were analyzed in nonsedated lambs. Apneas, with high lung volume maintained by active glottal closure, were compared with apneas, with a tracheostomy opened at apnea onset. Oxygen desaturation slopes were lower when high-apneic lung volume was actively maintained during both wakefulness and quiet sleep. Furthermore, oxygen desaturation slopes were lower after isolated apneas with continuous thyroarytenoid EMG during wakefulness, compared with apneas with noncontinuous thyroarytenoid EMG (= glottis opened shortly after apnea onset). These results highlight the importance of maintaining high-alveolar oxygen stores during central apneas by active glottal closure to limit desaturation in newborns.

R-zacopride, a 5-HT3 antagonist/5-HT4 agonist, reduces sleep apneas in rats

The effects of R-zacopride, a benzamide with potent 5-HT3 receptor antagonist and 5-HT4 receptor agonist properties, on spontaneous apneas were studied in 10 Sprague-Dawley rats by monitoring respiration and sleep for 6 h. R-zacopride (0.5, 1.0 and 10.0 mg/kg) suppressed spontaneous central apneas during non-rapid-eye-movement (NREM) sleep by 50% ( P=.05 for 0.5 mg/kg, P=.02 for 1.0 mg/kg and P=.001 for 10.0 mg/kg dose vs. control), and during rapid-eye-movement (REM) sleep by 80% by all doses tested ( P<.0007) for at least 2 h after intraperitoneal injection. We conclude that R-zacopride, over a 20-fold dose range, significantly reduces central apnea expression during NREM and REM sleep in the rat. The efficacy of this compound to suppress central apneas most probably arises from its antagonist actions at 5-HT3 receptors or from its mixed agonist/antagonist profile at 5-HT4/5-HT3 receptors.

Role of Peripheral Adenosine A1 Receptors in the Regulation of Sleep Apneas in Rats

The effects of administration of N6-p-sulfophenyladenosine (p-SPA), a peripheral adenosine A1 receptor agonist, and 8-(p-sulfophenyl)theophylline (p-SPT), a peripheral adenosine A1 receptor blocker, on spontaneous apneas were studied in 10 adult Sprague–Dawley rats by monitoring respiration, sleep, and blood pressure for 6 h. Intraperitoneal injection of p-SPA (1 mg/kg) to rats suppressed spontaneous central apneas during non-rapid eye movement sleep by 50% in comparison to control recordings (p=0.03). This effect was blocked by pretreatment with an equimolar dose of p-SPT (0.67 mg/kg) indicating that p-SPA suppression of apneas was receptor mediated in the peripheral nervous system. Administration of p-SPA did not affect apnea expression in rapid eye movement sleep and had no effect on sleep or blood pressure at the dose tested. Administration of p-SPT (0.67, 6.7, and 30 mg/kg) to rats had no effect on apneas, sleep, or blood pressure. The lack of p-SPT effect on sleep apneas argues against a physiologic role for endogenous adenosine in the peripheral nervous system as a modulator of sleep apnea expression under baseline conditions.

Thyroarytenoid muscle electrical activity during spontaneous apneas in preterm lambs.

Laryngeal dynamics plays a major role during perinatal life, a period of respiratory control immaturity. Continuous electromyographic (EMG) activity of a laryngeal adductor muscle (thyroarytenoid [TA] muscle), was recently observed throughout provoked central apneas, either isolated or during induced periodic breathing, in full-term lambs. The aim of the present study was to test if continuous TA EMG activity was also present during spontaneous apneas in nonsedated preterm lambs. We studied 7 premature lambs (term 131 +/- 1 d of postconceptional age). Premature birth was induced after acceleration of fetal lung maturation. Electrodes for diaphragm, inferior pharyngeal constrictor (IPC), and TA electromyograms, electrocardiogram, electroencephalogram, eye movement, and airflow recordings were implanted. Radiotelemetry recordings were repeated from 135 to 149 +/- 8 d of postconceptional age. A total of 2,088 apneas (2,020 central and 68 mixed) >/= 3 s were recorded in the lambs, including 57 epochs of periodic breathing. Continuous TA EMG activity was present throughout 88.4% of all apneas and 98.4% of apneas during periodic breathing, regardless of the sleep stage. These results suggest that active glottic closure is frequent during spontaneous central apneas in this model of prematurity. This unique model will allow us to study controlling mechanisms and consequences of glottic closure during neonatal apneas.

Role of Peripheral Serotonin in the Regulation of Central Sleep Apneas in Rats

The aim of our study was to determine the effects of serotonin (5-HT), which does not penetrate the blood-brain barrier (BBB), and GR38032F, a 5-HT3 receptor antagonist that may cross the BBB, on spontaneous apneas in adult Sprague-Dawley rats. Rats were implanted with electrodes for EEG and electromyographic recording to monitor sleep, with a radiotelemetry transmitter for monitoring aortic BP and heart period (HP) and were placed inside a single chamber plethysmograph for monitoring respiration. Sleep, BP, HP, and respiration were monitored for 6 h following administration of drugs. Intraperitoneal injection of 5-HT (0.79 mg/kg) to rats increased spontaneous central apneas during rapid eye movement (REM) sleep by > 250% in comparison to control recording (p = 0.01). GR38032F (0.1 mg/kg), which produced no effect on apnea expression, completely blocked the 5-HT-induced increase in REM apneas. Administration of 5-HT did not affect apnea expression in non-REM sleep and had no effect on sleep or BP. From these observations, we conclude that binding at 5-HT3 receptors in the peripheral nervous system promotes REM-related apnea genesis in rats. These findings further suggest that endogenous 5-HT, acting at least at peripheral 5-HT3 receptors, may play a baseline physiologic role in the expression of spontaneous central apneas in rats.

Larynx and neonatal apneas

This paper summarizes current knowledge on laryngeal dynamics during neonatal apneas, including mechanisms potentially involved in mixed/obstructive apneas at the laryngeal level, and recent personal data on active glottic closure throughout spontaneous central apneas (either isolated or in periodic breathing) observed in preterm lambs. It is suggested that this is reminiscent of the basic respiratory pattern in vertebrates alternating periods of lung ventilation with periods of breath-holding with the exchanger full of gas. Moreover, this unique ovine model of spontaneous neonatal apneas and periodic breathing offers new opportunity for studying candidate treatments of neonatal apneas.

Serotonin 5-HT3-receptor antagonist GR 38032F suppresses sleep apneas in rats.

The effects of administration of GR38032F, a 5-HT3 receptor antagonist, on spontaneous sleep apneas were studied in adult Sprague-Dawley rats by monitoring sleep, respiration and blood pressure for 6 hours. Intraperitoneal injection of GR38032F (1 mg/kg) suppressed spontaneous central apneas during non-rapid-eye-movement (NREM) and especially during rapid-eye-movement (REM) sleep. This effect was associated with increased respiratory drive but did not cause cardiovascular changes at the dose tested. The suppressive action of GR38032F on spontaneous sleep apneas is analogous to findings in anesthetized rats in which 5-HT and 2-methyl-5-HT provoked central apneas that were antagonized by GR38032 (Yoshioka et al, JPET 1992; 260:917-924). Our data implicate 5-HT3 receptor systems in determining sleep-related respiratory drive and apnea expression in rats, effects which are most probably mediated by vagal afferents.

USEFULNESS OF RADIOTELEMETRY SYSTEM FOR STUDYING SLEEP APNEAS IN LAMBS 1819

Our research program on upper airway dynamics during neonatal apneas utilizes polysomnographic recordings in non-sedated newborn lamb. Standard equipment, however, consists of numerous sensors attached to the lamb and requires animal restraint that disrupts sleep and prevents development of normal, spontaneous apneas. In order to perform recordings in more natural conditions, we have recently develop a radiotelemetry equipment composed of an implantable 8 channel transmitter and a FM receiver. The transmitter (23× 35 × 8 mm) is supplied with a lithium battery connected in series with a magnetically activated switch to extend battery life. Studied biopotentials (EEG, EOG, EMG, nasal flow) can be transmitted to a maximal distance of 8 m. The aim of the present study was to validate this telemetry equipment in vivo. Five non-sedated lambs, aged 5 to 25 days, were studied. The first validation step was to record each biopotential simultaneously by the standard equipment and by telemetry. Results obtained confirmed the reliability and reproducibility of the signals transmitted by telemetry. The second validation step was to test the usefulness of the telemetry equipment for repeated and simultaneous recording of several signals in one lamb (EEG, EOG, ECG, diaphragm EMG, pharyngeal and laryngeal constrictor muscle EMG). Results obtained confirmed the possibility to record signals during more than 30 days. Moreover, we were able to record longer sleep periods (especially REM sleep) with more spontaneous central apneas. In conclusion, radiotelemetry is an exciting tool for research on neonatal apneas (including fetal lamb in utero, preterm or term newborn lamb). A Sa02 sensor is being developped and will soon be added. Finally, radiotelemetry has several potential clinical applications, including cardiorespiratory home monitoring of infants at risk for sudden infant death syndrome, and adult or children polysomnographic studies. Funded by MRC 7137.