Transgenic models to study disorders of respiratory control in newborn mice

Recent studies described the in vivo respiratory phenotype of mutant newborn mice with targeted deletions of genes involved in respiratory control development. Whole-body flow barometric plethysmography is the noninvasive method of choice for studying unrestrained newborn mice. The main characteristics of the early postnatal development of respiratory control in mice are reviewed, including available data on breathing patterns and on hypoxic and hypercapnic ventilatory responses. Mice are very immature at birth, and their instable breathing is similar to that of preterm infants. Breathing pattern abnormalities with prolonged apneas occur in newborn mice that lack genes involved in the development of rhythmogenesis. Some mutant newborn mice have blunted hypoxic and hypercapnic ventilatory responses whereas others exhibit impairments in responses to hypoxia or hypercapnia. Furthermore, combined studies in mutant newborn mice and in humans have helped to provide pathogenic information on genetically determined developmental disorders of respiratory control in humans.     

Sleep apnea and effect of chemostimulation on breathing instability in mice.

Sleep apnea occurs in humans and experimental animals. We examined whether it also arises in adult mice. Ventilation in male adult 129/Sv mice was recorded concomitantly by electroencephalograms and electromyograms for 6 h by use of body plethysmography. Apnea was defined as cessation of plethysmographic signals for longer than two respiratory cycles. While mice breathed room air, 32.3 +/- 6.9 (mean +/- SE, n = 5) apneas were observed during sleep but not in quiet awake periods. Sleep apneas were further classified into two types. Postsigh apneas occurred exclusively during slow-wave sleep (SWS), whereas spontaneous apneas arose during both SWS and rapid eye movement sleep. Compared with room air (9.1 +/- 1.4/h of SWS), postsigh apneas were more frequent in hypoxia (13.7 +/- 2.1) and less frequent in hyperoxia (3.6 +/- 1.7) and hypercapnia (2.8 +/- 2.1). Our data indicated that significant sleep apnea occurs in normal adult mice and suggested that the mouse could be a promising experimental model with which to study the genetic and molecular basis of respiratory regulation during sleep.     

Physiology of ventilatory regulation during sleep

During light slow-wave sleep, ventilation is principally regulated by automatic metabolic control system. An instability in the respiratory control may be the predominant disturbance leading to very irregular or periodic breathing. During deep sleep, ventilation is progressively more stable. During REM sleep, automatic regulation is abolished and ventilation is particularly dependent on the compartmental control system. The reduction in airways and respiratory muscles tone favors the occurrence of obstructive apneas. The elevation in arousal threshold leads prolongation of the obstructive events.     

Postnatal development of right atrial injection of capsaicin-induced apneic response in rats.

Apnea and respiratory failure often occur in infants with pulmonary disease. Bronchopulmonary C-fiber (PCF)-mediated apnea is an important component of respiratory dysfunction. This study was undertaken to define the postnatal development of PCF-mediated apnea. The experiments were conducted in five groups of anesthetized, tracheotomized, and spontaneously breathing rats with ages at postnatal days P1-3, P7-9, P14-16, P21-23, and P56-58. Right atrial bolus injection of three doses of capsaicin (Cap), equivalent to 2, 4, and 8 microg/kg used previously in 450-g rats, was applied to stimulate PCFs. We found that 1) Cap-induced apneic response [percent change from the baseline expiratory duration (Te) values (deltaTe%)] and the sensitivity of this response (deltaTe%.microg(-1)) were significantly greater in the rats P10; 2) the Cap-induced apneas were vagally dependent in all rats tested; and 3) bivagotomy-induced prolongation of Te was much greater in the rats P10. From these findings we concluded that, compared with the older rats (>P10), the newborn rats have a stronger PCF-mediated respiratory inhibition that may contribute to infants' vulnerability to respiratory failure.     

Novel whole body plethysmography system for the continuous characterization of sleep and breathing in a mouse

Sleep is associated with marked alterations in ventilatory control that lead to perturbations in respiratory timing, breathing pattern, ventilation, pharyngeal collapsibility, and sleep-related breathing disorders (SRBD). Mouse models offer powerful insight into the pathogenesis of SRBD; however, methods for obtaining the full complement of continuous, high-fidelity respiratory, electroencephalographic (EEG), and electromyographic (EMG) signals in unrestrained mice during sleep and wake have not been developed. We adapted whole body plethysmography to record EEG, EMG, and respiratory signals continuously in unrestrained, unanesthetized mice. Whole body plethysmography tidal volume and airflow signals and a novel noninvasive surrogate for respiratory effort (respiratory movement signal) were validated against simultaneously measured gold standard signals. Compared with the gold standard, we validated 1) tidal volume (correlation, R(2) = 0.87, P < 0.001; and agreement within 1%, P < 0.001); 2) inspiratory airflow (correlation, R(2) = 0.92, P < 0.001; agreement within 4%, P < 0.001); 3) expiratory airflow (correlation, R(2) = 0.83, P < 0.001); and 4) respiratory movement signal (correlation, R(2) = 0.79-0.84, P < 0.001). The expiratory airflow signal, however, demonstrated a decrease in amplitude compared with the gold standard. Integrating respiratory and EEG/EMG signals, we fully characterized sleep and breathing patterns in conscious, unrestrained mice and demonstrated inspiratory flow limitation in a New Zealand Obese mouse. Our approach will facilitate studies of SRBD mechanisms in inbred mouse strains and offer a powerful platform to investigate the effects of environmental and pharmacological exposures on breathing disturbances during sleep and wakefulness.     

Effects of high-frequency pressure waves applied to upper airway on respiration in central apnea.

We examined the effects of high-frequency (30-Hz) low-pressure oscillations on respiration in nine patients with central sleep apnea. All patients were studied during sleep and wore a nasal mask through which the oscillations were applied. All tests were performed during periods of repetitive central apneas. Respiratory efforts were monitored from the airflow and calibrated Respitrace signals. After several cycles of apnea were monitored, the oscillatory pressures were applied for brief periods (less than 5 s) at the midpoint of the central apneas. All trials in which arousal occurred were discarded, leaving a total of 106 trials in the nine patients. High-frequency oscillation of the upper airway stimulated respiratory effort(s) in 68% of all trials (72 of 106). Apnea length was significantly shortened in four of the nine patients. In one patient with a tracheostomy, the stimulus applied to his isolated upper airway evoked respiratory efforts during central apnea in 13 of 15 trials. We conclude that high-frequency oscillatory pressures applied to the upper airway can stimulate respiratory efforts during central apnea. This response may be mediated by upper airway receptors involved in nonrespiratory airway defense reflexes and may have implications in the treatment of patients with central sleep apnea.     

Effects of inhaled oxygen (up to 40%) on periodic breathing and apnea in preterm infants.

To discover whether increases in inhaled O2 fraction (FIO2; up to 40%) decrease apnea via an increase in minute ventilation (VE) or a change in respiratory pattern, 15 preterm infants (birth weight 1,300 +/- 354 g, gestational age 29 +/- 2 wk, postnatal age 20 +/- 9 days) breathed 21, 25, 30, 35, and 40% O2 for 10 min in quiet sleep. A nosepiece and a flow-through system were used to measure ventilation. Alveolar PCO2, transcutaneous PO2, and sleep states were also assessed. All infants had periodic breathing with apneas greater than or equal to 3 s. With an increase in FIO2 breathing became more regular and apneas decreased (P less than 0.001). This regularization in breathing was not associated with significant changes in VE. However, the variability of VE, tidal volume, and expiratory and inspiratory times decreased significantly. The results indicate that the more regular breathing observed with small increases in FIO2 was not associated with significant changes in ventilation. The findings suggest that the increased oxygenation decreases apnea and periodicity in preterm infants, not via an increase in ventilation, but through a decrease in breath-to-breath variability of VE.     

Periodicities in respiration and heart rate in newborns

Periodic breathing is common in normal infants, but may be associated with prolonged apnea leading to crib death. The mechanisms of periodic breathing and its relation to normal breathing patterns are unclear. We recorded respiratory and heart rate (HR) patterns of 11 healthy newborn infants during quiet sleep, in both normal and periodic breathing. Spectral analysis of the respiratory pattern revealed a low-frequency (LF) periodicity in normal breathing approximately equal to the frequency of periodic breathing when this occurs. Periodic breathing thus appears to be an exaggeration of an underlying slow amplitude variation which is present in regular breathing. LF periodicity also appeared in the HR pattern in both normal and periodic breathing, suggesting an LF modulation of cardiovascular control as well. The lack of a definite phase relation between HR and ventilation at LF may indicate dominant peripheral, rather than central, interactions between HR and respiration at these frequencies.     

Heart rate and respiratory patterns in mild hypoxia in unanaesthetized newborn mammals

The heart rate and respiratory patterns in hypoxia are not well documented in unanaesthetized intact newborn animals. We studied heart rate and respiratory patterns during quiet sleep in 17% inspired O2 in 31 unanaesthetized newborns of five species: lamb, piglet, puppy, kitten, and rabbit. There was no significant change in mean heart rate and respiratory rate with hypoxia for any species. Brief apneas greater than 5 s were frequent (5-8/h), both in 21 and 17% O2 only in lambs and puppies. No sustained periodic breathing was induced by hypoxia. Thus, mild hypoxia has little steady-state effect on heart rate and respiratory rate and pattern in these unanaesthetized newborns. These findings are compatible with depressed chemoreceptor threshold, but indicate a remarkably mature respiratory pattern in full-term newborns of these species.     

Developmental changes in respiratory, febrile, and cardiovascular responses to PGE(2) in newborn lambs

PGE(2) has centrally mediated respiratory, febrile, and cardiovascular effects that markedly differ between fetal and adult life. We hypothesized that the transition from fetal to adult responses to PGE(2) occurs in the newborn period. Thus effects of an intracarotid infusion of PGE(2) (3 microg/min for 60 min) were determined in unanesthetized newborn lambs at 5, 10, and 15 days after birth. At 5 days, PGE(2) reduced central CO(2) sensitivity, reduced lung ventilation due to a decrease in breathing frequency, and induced hypercapnia. By 15 days, these effects of PGE(2) had waned significantly. In contrast, phasic (expiratory) thyroarytenoid muscle electromyogram activity, number of short apneas, and incidence of Biot periodic breathing were similarly increased at all three ages. PGE(2) induced a sustained fever at 10 and 15 days. Heart rate and mean arterial blood pressure were unchanged in contrast to marked increases observed by others in adults. Results showed that the transition from fetal to adult respiratory and febrile responses to PGE(2) occurs in early postnatal life, whereas adult cardiovascular responses develop later in life in sheep.     

Changes in heart rate during breathing interrupted by recurrent apneas in humans

Heart rate varies with breathing patterns, especially in sleep apnea. To assess the effects on heart rate of recurrent apneas interrupting tidal breathing, we studied five normal awake male subjects. These subjects voluntarily changed their breathing pattern from regular tidal breathing to tidal breathing interrupted by breath holding at end expiration. This recurrent apneic breathing pattern did not change mean heart rate but increased its variance significantly. In addition, the variations in heart rate formed a cyclic pattern of oscillation with a mean cycle length identical to both arterial O2 saturation (SaO2) (R = 0.95; P less than 0.01) and ventilation (R = 0.92; P less than 0.01). Cyclic changes in either SaO2 or ventilation reproduced the oscillatory patterns of heart rate seen with tidal breathing interrupted by multiple apneas, but the amplitude of the variance in heart rate was smaller. Finally, preventing the cyclic declines in SaO2 with supplemental O2 did not significantly alter the heart rate changes seen in tidal breathing interrupted by apneas.     

Mechanism of the generation of various forms of respiratory rhythm

The respiratory muscles and neurons activity in the transitional process from rhythmic respiration to its cessation and reappearance of the usual rhythmic breathing after the apnea was registered in the acute experiments on the anesthetized cats and rabbits under the action of extra intrapulmonary oxygen pressure or intravenous injection of sodium cyanide. Different forms of disturbances of respiratory rhythm (apneusis, hasping, the combination of hasping with apneusis and respiratory movements of usual form - eupnea) observed in the critical states of the organism are considered to be the result of changes in the character of activity of the medulla oblongata respiratory neurons which occur at a definite stage of hypoxia. Hasping mechanism differs essentially from the generation of eupnea and apneusis.     

Differential respiratory muscle recruitment induced by clonidine in awake goats

The purpose of this study was to test thehypothesis that dysrhythmic breathing induced by the₂-agonist clonidine isaccompanied by differential recruitment of respiratory muscles. Inadult goats (n = 14) electromyographic(EMG) measurements were made from inspiratory muscles (diaphragm andparasternal intercostal) and expiratory muscles [triangularissterni (TS) and transversus abdominis (Abd)]. EMG of thethyroarytenoid (TA) muscle was used as an index of upper airway(glottal) patency. Peak EMG activities of all spinal inspiratory andexpiratory muscles were augmented by central and peripheralchemoreceptor stimuli. Phasic TA was apparent in the postinspiratoryphase of the breathing cycle under normoxic conditions. Duringdysrhythmic breathing episodes induced by clonidine, TS and Abdactivities were attenuated or abolished, whereas diaphragm andparasternal intercostal activities were unchanged. There was no tonicactivation of TS or Abd EMG during apneas; however, TA activity becametonic throughout the apnea. We conclude that1) ₂-adrenoceptor stimulationresults in differential recruitment of respiratory muscles duringrespiratory dysrhythmias and 2) apneas are accompanied by active glottic closure in the awake goat.

     

Quantitative Changes in the Sleep EEG at Moderate Altitude (1630 m and 2590 m)

Background

Previous studies have observed an altitude-dependent increase in central apneas and a shift towards lighter sleep at altitudes >4000 m. Whether altitude-dependent changes in the sleep EEG are also prevalent at moderate altitudes of 1600 m and 2600 m remains largely unknown. Furthermore, the relationship between sleep EEG variables and central apneas and oxygen saturation are of great interest to understand the impact of hypoxia at moderate altitude on sleep.

Methods

Fourty-four healthy men (mean age 25.0±5.5 years) underwent polysomnographic recordings during a baseline night at 490 m and four consecutive nights at 1630 m and 2590 m (two nights each) in a randomized cross-over design.

Results

Comparison of sleep EEG power density spectra of frontal (F3A2) and central (C3A2) derivations at altitudes compared to baseline revealed that slow-wave activity (SWA, 0.8–4.6 Hz) in non-REM sleep was reduced in an altitude-dependent manner (∼4% at 1630 m and 15% at 2590 m), while theta activity (4.6–8 Hz) was reduced only at the highest altitude (10% at 2590 m). In addition, spindle peak height and frequency showed a modest increase in the second night at 2590 m. SWA and theta activity were also reduced in REM sleep. Correlations between spectral power and central apnea/hypopnea index (AHI), oxygen desaturation index (ODI), and oxygen saturation revealed that distinct frequency bands were correlated with oxygen saturation (6.4–8 Hz and 13–14.4 Hz) and breathing variables (AHI, ODI; 0.8–4.6 Hz).

Conclusions

The correlation between SWA and AHI/ODI suggests that respiratory disturbances contribute to the reduction in SWA at altitude. Since SWA is a marker of sleep homeostasis, this might be indicative of an inability to efficiently dissipate sleep pressure.     

Mixed and obstructive apneas are related to ventilatory oscillations in premature infants

In our previous study of 14 premature infants, apnea occurred at the minimum phase of ventilatory oscillations. The apneas corresponded to cessation of airflow at the nose and mouth and were not distinguished as central, mixed, or obstructive. Changes in heart rate associated with the apneas were not identified. To determine whether ventilatory pattern characteristics might predict either the type of apnea or heart rate changes during the apnea, we analyzed measurements of chest wall movement and heart rate that were made during the earlier studies. Chest wall movement measured by magnetometers was compared with airflow measured with a face mask and pneumotachograph. Tidal volume, breath duration, and ventilation were calculated on a breath-by-breath basis, converted to time-axis data strings, and filtered with a comb of zero phase shift digital band-pass filters to detect breathing patterns. Of 182 apneas greater than or equal to 3 s duration, 55% were central, 31% were mixed, and 14% were obstructive. All three types of apnea were related to ventilatory oscillations. Multiple linear and logistic regressions showed that an apnea was more likely to be obstructive when it was long and when the underlying ventilatory oscillation was due primarily to an oscillation in breath duration. Multiple linear and logistic regressions showed that decreases in heart rate were related primarily to the duration of apnea and secondarily to the characteristics of the underlying breathing patterns.     

Active upper airway closure during induced central apneas in lambs is complete at the laryngeal level only.

We tested the hypotheses that active upper airway closure during induced central apneas in nonsedated lambs 1). is complete and occurs at the laryngeal level and 2). is not due to stimulation of the superior laryngeal nerves (SLN). Five newborn lambs were surgically instrumented to record thyroarytenoid (TA) muscle (glottal constrictor) electromyographic (EMG) activity with supra- and subglottal pressures. Hypocapnic and nonhypocapnic central apneas were induced before and after SLN sectioning in the five lambs. A total of 174 apneas were induced, 116 before and 58 after sectioning of the internal branch of the SLN (iSLN). Continuous TA EMG activity was observed in 88% of apneas before iSLN section and in 87% of apneas after iSLN section. A transglottal pressure different from zero was observed in all apneas with TA EMG activity, with a mean subglottal pressure of 4.3 +/- 0.8 cmH2O before and 4.7 +/- 0.7 cmH2O after iSLN section. Supraglottal pressure was consistently atmospheric. Sectioning of both iSLNs had no effects on the results. We conclude that upper airway closure during induced central apneas in lambs is active, complete, and occurs at the glottal level only. Consequently, a positive subglottal pressure is maintained throughout the apnea. Finally, this complete active glottal closure is independent from laryngeal afferent innervation.     

Sleep apnea syndromes

Sleep apnea syndromes have been identified only relatively recently. Their most frequent form is characterized by a sleep-related upper airway obstruction resulting in apneas which may repeat themselves up to several hundred times during a night's sleep. Their mean duration is about 30 to 40 seconds, but some apneas last over one minute. Breathing resumption requires an arousal, which may be clearly identified on the EEG but usually goes unnoticed by the patient. The most immediate consequence are hypoxemia and sleep fragmentation. There may be associated arrhythmias and hemodynamic changes, especially in the pulmonary circulation. The predominant clinical signs are snoring (during the breathing resumption between the apneas) and daytime somnolence due to sleep fragmentation. In addition to the risks of work and traffic accidents, these patients run a long-term risk of cardiovascular accidents. About 20% develop pulmonary hypertension, a contributing factor to right heart failure. About 50% are hypertensive, which combined with a frequently observed polycythemia, makes them vulnerable to ischemic accidents. The treatment is based upon the use of continuous positive airway pressure (CPAP) during sleep. In case of failure, surgical alternatives may be considered.     

Increased pulmonary vascular resistance and reduced stroke volume in association with CO2 retention and inferior vena cava dilatation.

Changes in cardiovascular parameters elicited during a maximal breath hold are well described. However, the impact of consecutive maximal breath holds on central hemodynamics in the postapneic period is unknown. Eight trained apnea divers and eight control subjects performed five successive maximal apneas, separated by a 2-min resting interval, with face immersion in cold water. Ultrasound examinations of inferior vena cava (IVC) and the heart were carried out at times 0, 10, 20, 40, and 60 min after the last apnea. The arterial oxygen saturation level and blood pressure, heart rate, and transcutaneous partial pressures of CO(2) and O(2) were monitored continuously. At 20 min after breath holds, IVC diameter increased (27.6 and 16.8% for apnea divers and controls, respectively). Subsequently, pulmonary vascular resistance increased and cardiac output decreased both in apnea divers (62.8 and 21.4%, respectively) and the control group (74.6 and 17.8%, respectively). Cardiac output decrements were due to reductions in stroke volumes in the presence of reduced end-diastolic ventricular volumes. Transcutaneous partial pressure of CO(2) increased in all participants during breath holding, returned to baseline between apneas, but remained slightly elevated during the postdive observation period (approximately 4.5%). Thus increased right ventricular afterload and decreased cardiac output were associated with CO(2) retention and signs of peripheralization of blood volume. These results indicate that repeated apneas may cause prolonged hemodynamic changes after resumption of normal breathing, which may suggest what happens in sleep apnea syndrome.     

Osteopathy may decrease obstructive apnea in infants: a pilot study

Background

Obstructive apnea is a sleep disorder characterized by pauses in breathing during sleep: breathing is interrupted by a physical block to airflow despite effort. The purpose of this study was to test if osteopathy could influence the incidence of obstructive apnea during sleep in infants.

Methods

Thirty-four healthy infants (age: 1.5–4.0 months) were recruited and randomized in two groups; six infants dropped out. The osteopathy treatment group (n = 15 infants) received 2 osteopathic treatments in a period of 2 weeks and a control group (n = 13 infants) received 2 non-specific treatments in the same period of time. The main outcome measure was the change in the number of obstructive apneas measured during an 8-hour polysomnographic recording before and after the two treatment sessions.

Results

The results of the second polysomnographic recordings showed a significant decrease in the number of obstructive apneas in the osteopathy group (p = 0.01, Wilcoxon test), in comparison to the control group showing only a trend suggesting a gradual physiologic decrease of obstructive apneas. However, the difference in the decline of obstructive apneas between the groups after treatment was not significant (p = 0.43).

Conclusion

Osteopathy may have a positive influence on the incidence of obstructive apneas during sleep in infants with a previous history of obstructive apneas as measured by polysomnography. Additional research in this area appears warranted.     

Hypoxemia alone does not explain blood pressure elevations after obstructive apneas

In patients with obstructive sleep apnea (OSA), substantial elevations of systemic blood pressure (BP) and depressions of oxyhemoglobin saturation (SaO2) accompany apnea termination. The causes of the BP elevations, which contribute significantly to nocturnal hypertension in OSA, have not been defined precisely. To assess the relative contribution of arterial hypoxemia, we observed mean arterial pressure (MAP) changes following obstructive apneas in 11 OSA patients during non-rapid-eye-movement (NREM) sleep and then under three experimental conditions: 1) apnea with O2 supplementation; 2) hypoxemia (SaO2 80%) without apnea; and 3) arousal from sleep with neither hypoxemia nor apnea. We found that apneas recorded during O2 supplementation (SaO2 nadir 93.6% +/- 2.4; mean +/- SD) in six subjects were associated with equivalent postapneic MAP elevations compared with unsupplemented apneas (SaO2 nadir 79-82%): 18.8 +/- 7.1 vs. 21.3 +/- 9.2 mmHg (mean change MAP +/- SD); in the absence of respiratory and sleep disruption in eight subjects, hypoxemia was not associated with the BP elevations observed following apneas: -5.4 +/- 19 vs. 19.1 +/- 7.8 mmHg (P less than 0.01); and in five subjects, auditory arousal alone was associated with MAP elevation similar to that observed following apneas: 24.0 +/- 8.1 vs. 22.0 +/- 6.9 mmHg. We conclude that in NREM sleep postapneic BP elevations are not primarily attributable to arterial hypoxemia. Other factors associated with apnea termination, including arousal from sleep, reinflation of the lungs, and changes of intrathoracic pressure, may be responsible for these elevations.