Published ahead of print on February 25, 2005, doi:10.1164/rccm.200405-595OC
© 2005 American Thoracic Society doi: 10.1164/rccm.200405-595OC
Habituation of Arousal Responses after Intermittent Hypercapnic Hypoxia in PigletsDepartments of Medicine and Pediatrics and Child Health, University of Sydney, Sydney Australia Correspondence and requests for reprints should be addressed to Karen A. Waters, M.B.B.S., Ph.D., David Read Laboratory, Room 206, Blackburn Building, DO6, University of Sydney, Sydney NSW 2006, Australia. E-mail: kaw{at}mail.med.usyd.edu.au
Rationale: Clinical studies have demonstrated arousal deficits in infants suffering obstructive sleep apnea, and some infant deaths have been attributed to such an arousal deficit. Objectives: To evaluate whether arousal deficits can be induced by intermittent asphyxia during normal development. Methods and Measurements: Young piglets were exposed to intermittent hypercapnic hypoxia for 4 days from age 9.55 ± 0.5 days. Arousal responses were compared between control animals and animals exposed to intermittent hypercapnic hypoxia. Outcome measures included time to arouse after onset of the respiratory stimulus and frequency of arousals during recovery. Main Results: Arousal deficits emerged after successive exposures to hypercapnic hypoxia on Day 1, and were exacerbated on Day 4, although after overnight recovery, the deficit only became evident during the second and subsequent episode of hypercapnic hypoxia. On Day 1, time to arouse increased from 16.9 ± 7.1 seconds in the first epoch to 41.7 ± 28.6 seconds in the fourth epoch (p = 0.004 between cycles, one-way analysis of variance). In the recovery periods after hypercapnic hypoxia, there were 64% fewer arousals than baseline on Day 1 and 90% fewer arousals on Day 4. Respiratory effort, measured by VT across 10 breaths before the arousal, increased from 25.7 ± 7.6 on Day 1 to 29.1 ± 6.8 ml/kg on Day 4 (p < 0.001, two-way analysis of variance, Day 4 vs. Day 1, respectively). Conclusions: These studies demonstrate that acute and chronic arousal deficits can be induced by intermittent asphyxia, on a background of otherwise normal postnatal development.
Key Words: intermittent hypoxia • postnatal • OSA • SIDS Clinical conditions such as obstructive sleep apnea (OSA) may be associated with significant sequelae in young infants. OSA is defined as complete cessation of airflow at the mouth and nose during sleep, despite ongoing diaphragmatic efforts, and is characterized by rapid and repeated onset of perturbations in O2 and CO2 levels as well as sleep disruption (1). The most common signs of OSA in infants are snoring, labored breathing, and profuse sweating (2). Although not well defined for infants, sequelae documented in older children include subtle behavioral changes and secondary phenomena, such as metabolic alkalosis, growth and developmental failure, chronic respiratory failure, and cardiovascular complications (3–7). In infants, OSA is correlated with depression of the arousal response to ventilatory compromise, but treatment to alleviate OSA can reverse this depression (8–10). Recent studies also suggest that OSA may be linked with the sudden infant death syndrome (SIDS) (11–13). The most compelling hypothesis regarding SIDS is a failure in the neuroregulation of cardiorespiratory control because of a brainstem abnormality. Neuropathologic studies and more recent genetic studies support the concept that SIDS victims possess underlying vulnerabilities that put them at risk for sudden death (14–20). It is hypothesized that victims of SIDS, who are believed to die quietly in their sleep, suffer as much from a defect in arousal as from a defect in ventilatory control. The vulnerability of these infants is also believed to be latent until other factors occur, such as a critical period of development and/or an environmental stressor (13, 14, 16, 21, 22). Such a deficit in arousal responses is considered a necessary abnormality for an infant to succumb to SIDS, and it is hypothesized that a brainstem abnormality is responsible for this dysfunction (14, 15). The underlying cause of the brainstem abnormality is not known and may be congenital, caused by a delay in normal maturation, or it may be induced by environmental factors (22). Acute exposure to repetitive hypoxia has been shown to result in habituation that is expressed as a decreased frequency of arousal in response to the same stimulus. Fewell and Konduri (23) showed that repeated exposure to rapidly developing hypoxemia produces an arousal response decrement in sleeping lambs. The stimulus required to induce arousal increased so that the time to arouse and the decrease in SaO2 before arousal were significantly greater after repeated exposure to rapidly developing hypoxemia. Another study suggested that the carotid chemoreceptors and/or carotid baroreceptors have an important role in initiating arousal from sleep during rapidly developing hypoxemia (23). In dogs, chronic exposure to episodic hypoxia via tracheal occlusion depresses arousal responses to asphyxial gases when rebreathing expired air (24). In cats and rats, it was found that there is an important role for dopamine and sympathetic innervation in the carotid body response to hypoxia, and that chronic hypoxia alters sympathetic control (25). A chronic study in dogs found that sleep fragmentation can result in delayed arousal responses and greater arterial oxygen desaturation after acute airway occlusion, leading to the conclusion that the changed responses in OSA are primarily caused by sleep fragmentation (26). To examine effects of acute and "chronic" intermittent hypercapnic hypoxia (IHH) exposure on arousal, unsedated piglets were exposed to 6-minute cycles of a hypercapnic hypoxia (HH) gas mixture (8% O2/7% CO2/balance N2), alternating with 6 minutes of air (21% O2/balance N2), for a total time of 48 minutes, daily for 4 days. Arousal responses were monitored on Day 4 and compared with those on Day 1.
This study used a modification of previously reported protocols (27, 28). Mixed-breed miniature piglets included 11 IHH-treated and 12 control animals (see Table 1). Electrodes were implanted under general anesthetic at 7.83 ± 0.7 days, when piglets weighed 1.68 ± 0.3 kg. Blood pressure and arterial blood gases were obtained from an arterial catheter, EEG and EMG from Grass gold cup electrodes (Model E6GH; Grass Instrument Division, Warwick, RI), and ECG from Teflon-coated stainless-steel wire (A-M Systems, Inc., Carlsborg, WA). All leads were protected in the pockets of jackets that piglets wore after surgery.
Studies were undertaken in a transparent temperature-regulated polymethylmethacrylate (Perspex; Lucite Solutions, Southampton, UK) box during the piglets' usual "sleep" time. A respiratory circuit, attached to a face mask that was sealed against the snout, provided fresh gas with the relevant concentrations of O2 or CO2. A gas-tight three-way tap was used to switch between gas mixtures with a mean time to stabilization of 13.2 ± 5.7 seconds, which did not differ among gases or studies. Signals were amplified on a Grass Model 8 polygraph (Grass Instruments, Berkshire, UK) and digitized at 100 Hz (Labdat; RHT-InfoDat, Montreal, PQ, Canada). Recordings lasted 53 minutes, including a 5-minute baseline in air. The inspired gases were alternated between HH (8% O2, 7% CO2, balance nitrogen) and air (recovery) at 6-minute intervals for a total of 48 minutes: 4 x 6-minute HH cycles and 4 x 6-minute recovery cycles (Figure 1). Blood samples for arterial blood gases were taken before each gas switch and corrected to body temperature (Radiometer, Model ABL 520; Copenhagen, Denmark). Control piglets were subjected to the same protocol, except that inspired gas from tap-switch changes was always medical air. Daily studies were undertaken for 4 days and physiologic data were recorded on Days 1 and 4. Piglets were killed painlessly with an overdose of pentobarbitone (200 mg/kg–1) after the final study. Ethical approval for the study was obtained from the Animal Ethics Committee of the University of Sydney.
Identification of Arousals The raw data were reviewed visually using a commercially available digital data acquisition and analysis program (Labdat and Anadat; RHT-InfoDat). The raw signals derived from EEG, EMG, airflow, O2, CO2, and blood pressure were reviewed in 60-second epochs and inspected for arousals. An arousal was scored if a change in frequency and amplitude occurred in two or more signals, including EEG. Signal disturbances associated with blood sampling or within 10 seconds of a gas changeover were excluded. All arousals adhering to these criteria were counted, regardless of lower limit in time, because an EEG disruption reflected disturbances at the cortical level. Figure 2 depicts a sample of the raw data. The arousal latency was determined from the point when inspired levels of O2 and CO2 had plateaued at the new levels. The time to the first arousal was the start time of the arousal from the start time of the cycle in seconds. If the piglet failed to arouse during the cycle, a time of 360 seconds (the total time of the cycle) was assigned as the time to arouse for that cycle.
Results included arousal latency and the number of spontaneous arousals per minute. For the IHH group, spontaneous arousal frequency was determined at baseline and in the recovery cycles, whereas the entire study period was used for the control group. Methods to analyze sleep state are included in the online supplement.
Ventilation
Sleep Electrophysiologic and blood gas data were analyzed for sleep and wake, arousal responses, and ventilatory and arterial blood gas parameters. Data are presented as mean ± SD, unless otherwise stated. A p value of 0.05 or less was considered statistically significant. Repeated-measures t tests and analyses of variance (ANOVAs) were performed with Analyze-it for Microsoft Excel (version 1.62; Analyse-it Software, Ltd., Leeds, UK). Comparison of ventilatory data across epochs, and by day, was undertaken using general linear modeling in SPSS version 12.0.0 for Windows (SPSS Inc., Chicago, IL). Electrophysiologic data were not available for one IHH-exposed piglet.
Arousal Latency Arousal latency showed both acute and chronic deficits. The time to arouse increased across successive cycles on each study day (p < 0.001). The time to arouse also increased on Day 4 (after 4 consecutive days of IHH exposure) compared with the same cycle on Day 1. Notably, the increase on Day 4 was not apparent during the first HH exposure. The time to arouse in each cycle (apart from the first) was greater on Day 4 compared with the same cycle on Day 1 (p < 0.001, two-way ANOVA; Figure 3).
Spontaneous Arousals from Sleep There were significantly more spontaneous arousals on Day 4 compared with Day 1 in the control animals (p = 0.03), whose results were taken to represent normal maturational changes. This was also observed during baseline for the IHH-treated group where there were more spontaneous arousals during the baseline period on Day 4 compared with Day 1. In the recovery periods after IHH, there were 64% fewer arousals on Day 1 compared with baseline, and 90% fewer arousals compared with baseline on Day 4 in the recovery periods after HH (p = 0.05 by study day, p < 0.001 by cycle; Figure 4).
Blood Gases The changes in PO2 and PCO2 reflect the inspired levels of O2 and CO2 (Table 2). PO2 fell during the HH exposures, and recovered to baseline levels during the intervening recovery periods, with no differences over successive HH exposures, or between Days 1 and 4. PCO2 was elevated during the HH exposures, with associated falls in pH and bicarbonate. During recovery periods, there was hyperventilation, and PCO2 fell below baseline levels, with only partial compensation of the respiratory acidosis induced during the HH exposures. The PaO2 and PaCO2 did not change across epoch or day in air or in HH. Accordingly, the A-a gradient of HH and control animals was never different during cycles of breathing air, and although the gradient increased to 34.9 ± 1.8 during HH exposures (from –8.4 ± 1.8 in air, p < 0.001), there were no changes across epochs or days in HH or in air.
Respiratory acidosis became progressively more severe across cycles on each day, although the acidosis was more marked on Day 4. The worsening of the respiratory acidosis over successive cycles only reached statistical significance on Day 4, suggesting that the incomplete compensation with recurrent exposure to HH was further impaired after chronic exposure to IHH as illustrated by changes in base excess (Figure 5 and Figure E1 in the online supplement). For details of blood gas changes across epochs (HH or recovery), see Figures E2–E4.
The A-a gradient was calculated, using 0.21 as the FIO2 (low inspired O2 gave similar trends, but less meaningful results). Although the gradient increased during HH exposures (from 8.4 ± 1.8 to 34.9 ± 1.8), the difference was completely explained on the basis of the change in PaO2, and there were no significant effects according to epoch, day, or epoch by day.
Ventilation
Sleep Sleep was assessed in a total of four recovery cycles. As a group, the IHH-exposed piglets spent an increasing proportion of their recovery time asleep from 81.1 ± 12.7 (mean ± SD), 92.0 ± 6.9, 95.5 ± 4.0, 95.9 ± 3.4, and 96.5 ± 3.1 (p = 0.004 between cycles, one-way ANOVA), although individual variability was significant. The control piglets showed a reduction in the time they spent asleep, over successive cycles, with mean values as follows: 91.8 ± 12.3, 83.7 ± 22.4, 85.8 ± 17.9, 76.9 ± 24.3, and 75.7 ± 24.8 (p = 0.0005 between cycles, with one-way ANOVA). The IHH-treated piglets spent an increasing proportion of their total sleep time in non-REM sleep as follows: 76.4 ± 19.1, 93.17 ± 13.7, 88.7 ± 13.4, 83.5 ± 24.5, and 87.90 ± 16.8% (p = 0.01 between cycles, one-way ANOVA). The control animals showed no difference in the proportion of their TST in non-REM as follows: 82.87 ± 31.5, 91.35 ± 12.0, 93.11 ± 9.2, 87.53 ± 21.3, and 92.03 ± 11.1% (not significant, one-way ANOVA).
The most important finding in this study was that a cumulative arousal deficit was induced by daily repetitions of an intermittent respiratory stimulus during early development. Deficits were generated by both acute and chronic exposure. After 4 days of exposure and after an overnight recovery period, we observed only partial recovery of the arousal deficit. The arousal deficit observed in the acute setting was apparently dormant in the chronic setting, but it was induced and exacerbated by repetition of the acute stimulus. Possible explanations include habituation or plasticity of arousal responses, or postexposure depression of arousability. Additional abnormalities affecting spontaneous arousability support the conclusion that the induced changes reflect true depression of arousability. This phenomenon may reflect plasticity of the central nervous system, which is well recognized during early development.
Habituation of the Arousal Response The second recording, undertaken on the fourth study day, showed that the arousal responses recovered during the first exposure, but habituation recurred, and cumulative depression of arousal was again illustrated. More severe depression of arousal was observed under conditions of chronic exposure, and this was illustrated by an exacerbation of the depression of spontaneous arousability on Day 4 compared with Day 1. Other studies have suggested that arousal responses may be subject to habituation. In infants, normal arousals followed a stereotypic pattern of progression from spinal to cortical responses, which is believed to be endogenously regulated, regardless of the sensory input of the stimulus (32, 33). Repeated, tactile stimulation of human infants leads to suppression of cortical arousals so that eventually only spinal responses were observed during sleep (32). Our study was not designed to evaluate whether subcortical responses persisted. The response was specific to the repeated stimulus, because timely responses to other irregular stimuli (e.g., noise and light) persisted, whereas the response decrement to HH was apparent. Although formal demonstration of cumulative long-term habituation requires eight data points to fit a negative exponential equation and failure of recovery of the native response to baseline, the trend of the response decrement was apparent in our dataset (30, 31).
Depression of Spontaneous Arousability
Plasticity of Respiratory Sensitivity
The Respiratory Threshold The combination of hypercapnia and hypoxemia is a potent stimulus to arouse (44). Parslow and colleagues (45) found that the longer arousal latency during non-REM can be explained by the rate of oxygen desaturation being faster in REM than in non-REM sleep in infants rather than differences in the chemoreceptor threshold. Hypercapnia is also a potent arousal stimulus, which acts through the brainstem reticular activating system (46). It is possible that the chemoreceptor set point was changed. Blanco and coworkers (47) showed that changes in the detection of O2 levels by the peripheral chemoreceptors requires 24 to 31 hours of continuous exposure to new O2 concentrations, but sensory adaptation after presentation of an intermittent respiratory stimulus has not been evaluated. Further evidence that the threshold of respiratory stimulation had increased was provided by the increase in VT preceding arousals on Day 4 compared with Day 1. These data fit with the model of Gleeson and colleagues (48), who proposed that the predominant respiratory stimulus for arousal is the amplitude of effort rather than chemostimulation.
Possible Implications for Infants Nonetheless, there is also evidence that HH occurs secondary to rebreathing of expired air in the prone sleeping position (41, 51–53). Studies on Day 1 could reflect noxious respiratory stimuli experienced by infants sleeping prone for the first time, which is associated with a 14- to 19-fold increased risk for SIDS (54). Studies on Day 4 would reflect a more chronic stimulus, such as may be observed in association with OSA, and which has also been associated with subsequent SIDS (13, 14, 55). Neuropathologic changes in the same infants suggest chronic tissue hypoxia, which supports the latter mechanism. Infants with diminished ventilatory responses to hypercapnia or hypoxia fail to arouse to hypercapnic or hypoxic stimuli (56). Such abnormalities of the ventilatory responses have been observed in infants with apparent life-threatening events, and they have been observed in association with OSA in infants (8, 9, 21). Because treatment of OSA apparently reverses this depression (10), the deficit in the arousal response appears to be inducible. Habituation of the arousal response after postnatal exposure to a noxious respiratory stimulus has been illustrated in several studies (23, 57–59). The hypothesized brainstem abnormality responsible for abnormal arousability may be congenital, but the results of this study suggest that environmental factors can induce arousal deficits in otherwise normal subjects (15, 18). It remains possible that genotype ultimately determines whether the response to repeated episodes of HH is favorable or unfavorable, and responses characteristic of polymorphisms relevant to SIDS may only be elicited if repetitive episodes of intermittent HH are experienced during developmentally vulnerable periods. Risk factors such as OSA (which may have another genetic basis) and prone sleep position may only place infants at high risk for fatal consequences during sleep if they interact with specific "vulnerable" genotypes.
Other Considerations The piglets in this study displayed normal early postnatal development, with the only variant to this being their study exposure to HH to simulate ventilatory compromise. Piglets were selected as the animal model for this study because their early postnatal development most closely approximates that of the human infant (35, 42, 62). The age range of 9 to 13 days over the period of study was carefully selected as a developmental stage equivalent to 2 to 6 months in the human infant, an age of peak vulnerability to SIDS (13), and an age of increased vulnerability to respiratory compromise (63). The associated changes in arterial gases with a progressive acidosis suggest that the repeated insults were associated with inadequate ventilatory compensation rather than habituation (64). The intermittent nature of the stimulus was apparently important because the adaptive strategies usually seen in neonates experiencing hypoxia, such as reduced metabolic rate and a fall in body temperature, were not sufficient to protect against the insult, and in such circumstances, those strategies may underlie, rather than protect against, hypoxic injury (64).
Limitations of the Study Although our study provides a model for clinical diseases such as OSA and environmental factors such as prone sleeping, the results can only be partly extrapolated to clinical settings. The mechanisms stimulating arousal in our study animals would have included alterations in autonomic control independent of mechanoreceptor stimulation and stimulation of the olfactory system, both of which are believed to participate in the generation of arousal (61, 66). Arousal from sleep in response to upper airway obstruction, such as occurs in OSA, almost certainly involves mechanoreceptors as well as chemoresponses (24, 48, 67). It remains possible that effector fatigue was a component of the response, but we had no measurement that would allow documentation of respiratory muscle fatigue. However, the increased ventilatory efforts preceding arousal on Day 4 suggest that this is unlikely. We excluded effects caused by habituation to the study environment by including a control group that underwent all study procedures except HH gas exposure. Although the study environment permitted auditory and visual stimulation, chronic noise stimulation does not induce any sustained changes in the cardiovascular system and is unlikely to have contributed to our results (68). Four days of exposure may be considered a relatively brief duration for chronic exposure, and the finding that this was able to induce an arousal deficit is important. We did not explore whether such an arousal deficit continues to progress with more prolonged exposure or the sequence or time course over which the deficit develops or "recovers." More rigorous testing of the habituation hypothesis would require additional studies, such as a change in study context (e.g., removal from the study environment) to show that this led to renewal of the native response to HH exposure, and that reinstatement in the study environment induced rehabituation. Dishabituation may also be demonstrable, if simultaneous delivery of another stimulus with HH would allow reassertion of the native response to HH.
Conclusions
The authors gratefully acknowledge Prof. Colin Sullivan's critical input to these studies.
Supported by the National Health and Medical Research Council grant 147100 and Financial Markets Trust for Children. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Conflict of Interest Statement: K.A.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.D.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form July 7, 2004; accepted in final form February 22, 2005
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ABSTRACT
METHODS
E were derived from the calibrated flow signal from the pneumotachograph using a commercially available digital data analysis system (Abreath; RHT-Infodat). Ventilation is expressed in milliliters corrected for weight (ml/kg–1) and time (ml/kg/minute). 
