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J Appl Physiol 90: 2490-2501, 2001;
8750-7587/01 $5.00
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Vol. 90, Issue 6, 2490-2501, June 2001

HIGHLIGHTED TOPICS
Physiological and Genomic Consequences of Intermittent Hypoxia
Selected Contribution: Regulation of sleep-wake states in response to intermittent hypoxic stimuli applied only in sleep

Hedieh Hamrahi, Richard Stephenson, Safraaz Mahamed, Kiong Sen Liao, and Richard L. Horner

Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recurrent sleep-related hypoxia occurs in common disorders such as obstructive sleep apnea (OSA). The marked changes in sleep after treatment suggest that stimuli associated with OSA (e.g., intermittent hypoxia) may significantly modulate sleep regulation. However, no studies have investigated the independent effects of intermittent sleep-related hypoxia on sleep regulation and recovery sleep after removal of intermittent hypoxia. Ten rats were implanted with telemetry units to record the electroencephalogram (EEG), neck electromyogram, and body temperature. After >7 days recovery, a computer algorithm detected sleep-wake states and triggered hypoxic stimuli (10% O2) or room air stimuli only during sleep for a 3-h period. Sleep-wake states were also recorded for a 3-h recovery period after the stimuli. Each rat received an average of 69.0 ± 6.9 hypoxic stimuli during sleep. The non-rapid eye movement (non-REM) and rapid-eye-movement (REM) sleep episodes averaged 50.1 ± 3.2 and 58.9 ± 6.6 s, respectively, with the hypoxic stimuli, with 32.3 ± 3.2 and 58.6 ± 4.8 s of these periods being spent in hypoxia. Compared with results for room air controls, hypoxic stimuli led to increased wakefulness (P < 0.005), nonsignificant changes in non-REM sleep, and reduced REM sleep (P < 0.001). With hypoxic stimuli, wakefulness episodes were longer and more frequent, non-REM periods were shorter and more frequent, and REM episodes were shorter and less frequent (P < 0.015). Hypoxic stimuli also increased faster frequencies in the EEG (P < 0.005). These effects of hypoxic stimuli were reversed on return to room air. There was a rebound increase in REM sleep, increased slower non-REM EEG frequencies, and decreased wakefulness (P < 0.001). The results show that sleep-specific hypoxia leads to significant modulation of sleep-wake regulation both during and after application of the intermittent hypoxic stimuli. This study is the first to determine the independent effects of sleep-related hypoxia on sleep regulation that approximates OSA before and after treatment.

rats; obstructive sleep apnea


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OBSTRUCTIVE SLEEP APNEA (OSA) affects ~4% of adults (47) and is a major public health problem (37). OSA is associated with periods of recurrent asphyxia and arousals from sleep that contribute to excessive daytime sleepiness, impaired cognition, and increased risk for vehicular accidents (10, 16, 25, 46). Treatment of OSA with nasal continuous positive airway pressure (CPAP) reverses the deleterious effects of repetitive apneas on overnight sleep quality and daytime function (9, 27, 43). Nasal CPAP also increases rapid-eye-movement (REM) sleep and deep non-REM sleep on the first night of treatment in humans (22), and a rebound increase in REM sleep also occurs after cessation of apneas in a canine model of OSA (20, 35). These marked changes in sleep after treatment suggest that stimuli associated with OSA, e.g., intermittent hypoxia in sleep, may disrupt sleep regulatory mechanisms.

To our knowledge, however, there have been no studies in animals or humans that investigated the independent effects of intermittent hypoxic stimuli, applied only in sleep, on sleep regulation and recovery sleep after removal of stimuli. Rather, the only studies investigating the effects of hypoxic stimuli on sleep-wake patterns have determined responses to continuous chronic hypoxia such as those that occur at altitude. Studies in humans have shown that continuous chronic hypoxia leads to increased wakefulness and light non-REM sleep (stages 1 and 2), decreased deep non-REM sleep (stages 3 and 4), and decreased REM sleep (6, 33, 34, 39). However, it is not possible to determine whether these effects of chronic hypoxia on sleep patterns are due to effects of hypoxia per se or due to the confounding effects of the repetitive arousals induced by sleep-disordered breathing that occur in humans in chronic hypoxia (5). Animal models have also been used to determine the effects of chronic continuous hypoxia on sleep-wake patterns. Rats have been one of the most studied animal models for this purpose, largely because of their particular suitability for chronic instrumentation and long-term experiments in sleep. In rats, continuous chronic hypoxia leads to reduced REM sleep, increased wakefulness, and disruption of non-REM sleep (28, 32, 36). Unlike humans, however, these changes in sleep can be attributed to the effects of chronic hypoxia per se because periodic breathing did not occur under these conditions in rats (32, 36). Moreover, the disruptions of sleep-wake states in rats did not occur after section of the carotid sinus nerve, suggesting that continuous chronic hypoxia acts as an arousing stimulus (40). However, the overall relevance to OSA of studies with continuous hypoxia is questionable, not least because OSA patients are subjected to intermittent hypoxia and only during sleep episodes. Given the prevalence of OSA (47) and the adverse clinical consequences (10, 16, 25, 46), it is important to determine the effects of intermittent hypoxic stimuli, applied only in sleep, on the regulation of sleep-wake states and recovery sleep after removal of stimuli.

The aim of the present study, therefore, was to determine the effects of intermittent hypoxic stimuli, applied only in sleep, on the regulation of sleep-wake states. Given the suggestion that chronic continuous hypoxia may act as an arousing stimulus (40), it is hypothesized that application of hypoxic stimuli only in sleep would increase wakefulness, decrease non-REM and REM sleep, and increase sleeping electroencephalogram (EEG) frequencies. A novel feature of this study is that it is the first to deliver hypoxic stimuli only during sleep and remove the hypoxia at arousal from sleep. As such, this design is more relevant to OSA compared with studies that simply apply intermittent hypoxia regardless of whether the animal is awake or asleep. In the present study, sleep-wake states were also recorded for several hours during application of hypoxic stimuli in sleep but also in the recovery phase after removal of hypoxic stimuli. This study therefore is also the first to determine the independent effects of sleep-related hypoxia on sleep regulation in a fashion that approximates OSA before and after treatment.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Surgical Preparation

Studies were performed on 10 adult male Sprague-Dawley rats (mean body wt = 275 g; range = 228-335 g; Charles River Laboratories). Each rat was housed individually, maintained on a 12:12-h light-dark cycle (lights on at 0700), and had access to food and water ad libitum. All procedures conformed to the recommendations of the Canadian Council on Animal Care, and the University of Toronto Animal Care Committee approved the experimental protocol.

Sterile surgery was performed under general anesthesia induced by intraperitoneal ketamine (85 mg/kg) and xylazine (15 mg/kg). Before surgery, the rats were also given intraperitoneal buprenorphine (0.03 mg/kg), atropine (1 mg/kg), and sterile saline (3 ml, 0.9%). An anesthesia mask (14) was placed over the snout, and halothane in oxygen-enriched air (50% O2) was administered as necessary for the remainder of the surgery (typically 0.5-2%). Effective anesthesia was judged by abolition of the pedal withdrawal and corneal blink reflexes. Body temperature was measured with a rectal probe and was maintained between 36 and 38°C with a heating pad (BAS, West Lafayette, IN).

Each rat underwent surgical implantation of a three-channel radiotransmitter (model TL10M3-F50-EET, Data Sciences International, St. Paul, MN) to record the EEG, neck muscle electromyogram (EMG), and core body temperature. First, midline incisions were made in the scalp and abdomen to expose the skull and peritoneal cavity. The radiotransmitter was then inserted into the peritoneal cavity and loosely sutured to the rectus abdominus muscle. The electrode leads from the implant were tunneled from the peritoneal cavity and led subcutaneously to the head. The muscle and skin of the abdomen were then sutured closed. The rats were placed in a stereotaxic apparatus (Kopf model 962, Tujunga, CA) to fix body position for subsequent implantation of the EEG and neck EMG electrodes. The EMG electrodes were sutured bilaterally to the dorsal cervical neck muscle. Three holes were then drilled into the skull, and two EEG electrodes and a reference electrode were attached using stainless steel screws (size 0-80X1/16, Plastics One, Roanoake, VA). The EEG electrodes were placed on the skull over the frontal-parietal cortex and were positioned ~2 mm anterior and 2 mm to the right of bregma and 3 mm posterior and 2 mm to the left of bregma, respectively (21). The reference electrode was placed ~5 mm anterior and 3 mm to the left of bregma. Dental acrylic was used to anchor the electrodes to the skull. The skin was sutured closed, and the rat recovered for at least 7 days (range = 7-21 days) before the experiments. After the animals recovered from surgery, they were handled daily and habituated to the experimental chamber for the experiments (see below). At the end of all experiments, the rats were overdosed with an intraperitoneal injection of pentobarbital sodium (100 mg/100 g), and the telemetry units were removed, cleaned, and resterilized.

Recording Procedures

The EEG, neck EMG, and body temperature signals emitted by the telemetry unit were detected by a receiver (model RPC-1, Data Sciences International) placed under the rats' cage. The outputs from the receiver were then converted to analog outputs (PhysioTel Multiplus, Data Sciences) for subsequent amplification (DC-936 buffer amplifiers, CWE, Ardmore, PA) and filtering (0.1-100 Hz for EEG, 3-100 Hz for EMG, BPF-932 filters; CWE). The radiotransmitter used to send the EEG and EMG signals had a high frequency cut-off at 100 Hz.

The EEG, neck EMG, and body temperature signals were routed to a Grass model 79D polygraph and recorded on chart at 5 mm/s. A personal computer (IBM-compatible 386, 16 MHz) also received the EEG and EMG signals after analog-to-digital conversion at a sampling rate of 300 Hz (Lab Master DMA, Arrow Electronics, Techmar, OH). The interval histogram method (26) was used to determine the relative (%) frequency content of the EEG signal. The following bandwidths were quantified: delta 2 (0.5-2 Hz), delta 1 (2-4 Hz), theta  (4-7.5 Hz), alpha  (7.5-13.5 Hz), beta 1 (13.5-20 Hz), and beta 2 (20-30 Hz). In addition, the beta 2-to-delta 1 ratio and EEG and EMG amplitudes were determined. An on-line sleep-scoring algorithm validated for use on rats (19) was used to define sleep-wake state every 6 s. The threshold levels of beta 2/delta 1 and neck EMG amplitudes were used as scoring criteria in the algorithm. These threshold values were calibrated for each rat during preliminary experiments lasting ~2 h that included all sleep-wake states. During each experiment, the computer-generated judgment of sleep-wake states was also verified by continuous visual inspection of the raw traces.

Preoperative Calibration of Telemetry Units

Calibration values for EEG, EMG, and body temperature were produced for each telemetry unit before implantation. The EEG and neck EMG signals were calibrated on chart and computer with a 100-µV peak-to-peak sine-wave signal produced by a signal generator. The temperature sensor was also calibrated by placing the transmitter in a water bath at four different temperatures (range = 36-40°C) and measuring the resulting voltages. Preliminary experiments showed that the implanted temperature sensor was accurate to 0.1°C with a response time constant of >40 s to step changes in temperature.

Protocol and Experimental Conditions

All studies were performed in a noise-attenuated Faraday cage (model EPC-010, BRS/LVE, Laurel, MD), with the rats allowed to freely behave and free from interruption. The rats were gently handled each day after surgery and were also habituated to the apparatus by being placed in the experimental chamber for 1- to 2-h periods for at least 2 consecutive days before the experiments.

On the day of each experiment, the rats were placed in the experimental chamber at ~0930. The experimental chamber contained food, water, and bedding from the rats own home cage. For condition 1, in which rats were studied in their home cages only (see below), the rats were picked up and then immediately placed back into their home cage before the experiment was started. After this initial handling at 0930, the rats were left alone for ~2 h for acclimatization. Between 1130 and 1430, the rats were then subjected to hypoxic or control stimuli according to the protocol described below. The stimuli were discontinued at 1430, and the rats were then studied in the recovery phase until 1730, after which they were returned to their home cage.

Each rat was studied in each of five experimental conditions as outlined in Fig. 1. Condition 1 was used as a control to determine normal sleep-wake patterns with the rat in its' home cage breathing room air throughout the day. Condition 2 was also a control to determine the potential effects of the experimental chamber on sleep patterns. In this condition, the rat was placed in the experimental chamber and the chamber was flushed continuously with room air throughout the stimulus and recovery phases of the experiment. Condition 3 was also used as a control, in this case to determine whether sleep-wake patterns were affected by activation of the solenoid valves used to switch between inspired gases (see below). For this control condition, the rat was placed in the experimental chamber, and, whenever it fell asleep or was aroused from sleep, the solenoid valves were activated in the same way as during the hypoxic interventions. In this condition, however, the inspired gas was switched from room air to room air rather than from room air to hypoxia (i.e., sham interventions). This condition served as the most direct control for application of hypoxic stimuli in sleep as the experimental conditions were identical except that room air stimuli were applied instead of hypoxic stimuli. Conditions 1-3 occurred in random order. In condition 4, the effect of hypoxic stimuli applied during sleep on sleep-wake patterns was determined. In this condition, the rat was placed in the experimental chamber and, whenever it fell asleep during the stimulus phase (i.e., 1130-1430), the solenoid valves were activated to switch the inspired gas from room air to hypoxia (10% O2). Room air was reinstated on each arousal from sleep. In the recovery phase during condition 4 (i.e., 1430-1730), the inspired gas was switched from room air to room air rather than from room air to hypoxia. In condition 5, performed at least 7 days after condition 4, continuous hypoxia (10% O2) was applied. In this condition, the solenoid valves were activated to switch the inspired gas from hypoxia to hypoxia whenever the rat fell asleep during the stimulus phase. In the recovery phase of condition 5, the inspired gas was switched from room air to room air.


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  		Fig. 1.   The experimental protocol in each rat consisted of studies in 5 separate conditions. Conditions 1-3 served as controls, condition 4 determined the effects of intermittent hypoxic stimuli applied only in sleep, and condition 5 determined the effects of continuous chronic hypoxia. Rats were placed in the experimental chamber at 0930 and acclimatized to the stimuli also applied in the stimulus phase (1130-1430). Stimuli were removed at 1430, and rats were then studied in the recovery phase until 1730. See text for further details of each experimental condition.

Triggering of Hypoxic Stimuli in Sleep

Figure 2 shows a schema of the method used to apply hypoxic stimuli during sleep and remove hypoxia at arousal. On detection of two consecutive epochs of sleep by the computer, a voltage pulse was generated that triggered a solenoid valve to switch from room air to 100% N2. The N2 caused O2 levels to fall rapidly until a threshold triggered a switch to terminate N2 flow and initiate flow of premixed gas containing 10% O2, which was maintained throughout the sleep episode. On detection of two consecutive epochs of wakefulness after arousal from sleep, the voltage returned to 0 V, which switched the inspired gas transiently to 100% O2, causing O2 levels to rise rapidly. When an upper threshold was reached, the O2 flow was terminated and room air flow was reinstated (Fig. 2).


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  		Fig. 2.   Schema to show how application of hypoxic stimuli was timed to sleep episodes. On detection of sleep by the computer, a logic circuit detected that O2 levels were above an upper threshold (threshold 1, ~15% O2 indicated by the top dashed line) such that a solenoid valve was triggered to switch the inspired gas to 100% N2. Once a lower threshold was reached (threshold 2, ~12.5% O2) and the rat was still asleep, the inspired gas was switched to 10% O2. On detection of wakefulness after arousal from sleep, the logic circuit coordinated switching from 100% O2 to room air based on thresholds 1 and 2. The levels of thresholds 1 and 2 were set in preliminary experiments to avoid overshoot of the desired O2 levels. REM, rapid eye movement.

To deliver the hypoxic stimuli rapidly in sleep, the rats were studied in an airtight experimental chamber (volume = 3.3 liters) continually flushed with room air at 17 l/min. This flow rate was chosen from preliminary experiments because it was the optimum to generate fast changes in O2 levels in the chamber following a switch to hypoxia. At this flow rate, the lag time from activation of the solenoid valve to the onset of the wash out of O2 from the chamber was 5.8 ± 0.3 (SD) s, and the time to achieve 90% of desired level of hypoxia was a further 8.2 ± 0.2 s. Oxygen levels in the chamber were measured with an O2 analyzer (type OA272, Taylor Servomex, Crowborough, Sussex, UK) and were recorded on charts. The relative humidity and temperature of the experimental chamber were also measured by a calibrated thermohygrometer probe (model 37950-10, Cole-Parmer Instruments, Vernon Hills, IL).

Analysis

The overall sleep architecture was determined from visual analyses of the chart records for the stimulus and recovery phases of the experiment. Wakefulness, non-REM sleep, and REM sleep were determined in 10-s epochs using standard EEG and EMG criteria in rats (21). Periodic visual observations of the animal were also performed as an aid to determination of sleep-wake states via a one-way mirror in the sound-attenuated Faraday cage. Brief arousals from sleep lasting from 3 to 10 s were also identified from the EEG and EMG signals (1). From the chart records, the percentage (%) of wakefulness, non-REM sleep, and REM sleep and the number of arousals per hour were calculated for both the stimulus (1130-1430) and recovery (1430-1730) phases in each of the five experimental conditions. From these visually scored sleep-wake episodes, the average duration and frequency of sleep-wake episodes were also determined in each experimental phase for each condition, as were sleep latency and the number of arousals. In addition, EEG frequencies and EEG and EMG amplitudes were determined from the computer algorithm for the same visually identified periods of wakefulness, non-REM sleep, and REM sleep. To determine the effects of experimental condition on body temperature, measurements of temperature were made at 10-min intervals. Because of the relatively long time constant of the sensor (>40 s, see above), the temperature measurements were not made on a state-specific basis.

Statistical Analysis

The analyses performed for each statistical test are included in the text where appropriate. For two-way ANOVA with repeated measures, the two factors were phase of experiment [i.e., stimulus (1130-1430) and recovery (1430-1730) phases] and experimental condition (e.g., room air stimuli in sleep vs. hypoxic stimuli in sleep). For all comparisons, differences were considered significant if the null hypothesis was rejected at a level of P < 0.05 using a two-tailed test. Where post hoc comparisons were performed, the Bonferroni corrected P value was used to infer statistical significance. Analyses were performed using Sigmastat (Jandel Scientific, San Rafael, CA). Values are means ± SE unless otherwise indicated.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Efficacy of Application of Hypoxic Stimuli in Sleep

An average of 69.0 ± 6.9 hypoxic stimuli were applied in each rat over the 3-h stimulus phase (1130 to 1430). Of these stimuli, 95.0 ± 1.6% were correctly applied in sleep (81.6 ± 2.0% in non-REM sleep and 13.3 ± 2.3% in REM sleep), 2.4 ± 1.0% were incorrectly applied in wakefulness, and 2.8 ± 1.1% were applied in drowsiness as judged by an EEG pattern intermediate between light sleep and quiet wakefulness. Furthermore, 94.9 ± 1.9% of these hypoxic stimuli were correctly removed at arousal from sleep, whereas 4.9 ± 1.9% were incorrectly removed in sleep (3.8 ± 2.0% in non-REM sleep and 1.0 ± 0.3% in REM sleep) and 0.3 ± 0.2% were removed in drowsiness. During application of hypoxic stimuli in sleep (i.e., condition 4), the durations of non-REM and REM sleep episodes averaged 50.1 ± 3.2 and 58.9 ± 6.6 s, respectively, with 32.3 ± 3.2 and 58.6 ± 4.8 s of these periods being spent in hypoxia. The time spent in hypoxia in REM sleep approximated the average REM durations because REM episodes typically followed non-REM; i.e., there was no delay to establish the hypoxia as it was already applied.

Effects of Hypoxic Stimuli Applied in Sleep on %Sleep-Wake States

Figure 3 shows the effects of application of hypoxic stimuli in sleep on the distribution of sleep-wake states in one rat compared with application of room air stimuli. In the stimulus phase, the hypoxic stimuli led to clear decreases in REM sleep and increases in wakefulness and multiple short-duration non-REM episodes. In contrast, in the recovery phase when room air stimuli were applied instead of hypoxic stimuli, there was a compensatory increase in REM sleep and a decrease in wakefulness and longer non-REM episodes.


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  		Fig. 3.   Example in 1 rat to show the effects of application of hypoxic stimuli in sleep on the distribution of sleep-wake states compared with room air stimuli. In the stimulus phase, the hypoxic stimuli led to decreased REM sleep, increased wakefulness, and multiple short-duration non-REM episodes. In the recovery phase after cessation of hypoxic stimuli, there were compensatory increases in REM sleep, decreases in wakefulness, and longer non-REM episodes.

%Wakefulness. Figure 4 shows group data from the 10 rats for the effects of hypoxic stimuli applied in sleep compared with room air stimuli (i.e., sham interventions). Analysis showed that the effects of stimulus and recovery phase on %wakefulness depended on whether hypoxic or room air stimuli were applied (P = 0.0007, 2-way ANOVA; Fig. 4A). Four planned post hoc comparisons were then performed, as such the critical P value for the following paired t-tests was 0.0125. These post hoc tests showed that application of hypoxia in sleep led to increased wakefulness compared with room air stimuli (P = 0.003). After removal of hypoxic stimuli, there was decreased wakefulness in the recovery phase (P = 0.0005). Although wakefulness in recovery after hypoxia was reduced compared with the recovery phase after room air, this difference was of borderline statistical significance after Bonferroni correction (P = 0.0166). There was no change in %wakefulness between the stimulus and recovery phases during application of room air stimuli (P = 0.590).


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  		Fig. 4.   Group data (means ± SE) from 10 rats showing the effects of application of hypoxic stimuli in sleep (, condition 4) on %sleep-wake states compared with room air stimuli (open circle , condition 3). Data are shown for both the stimulus and recovery phases of the experiment. In the stimulus phase, the hypoxic stimuli led to increased wakefulness (A), little change in overall non-REM sleep (B), and decreased REM sleep (C). These changes were reversed in the recovery phase. *Significant difference between experimental conditions (i.e., application of hypoxia in sleep vs. room air in sleep); +significant difference across experimental phase (i.e., stimulus phase vs. recovery phase) (both P < 0.05).

%Non-REM sleep. The effects of stimulus and recovery phase on %non-REM sleep also depended on whether hypoxic or room air stimuli were applied in sleep (P = 0.0038, 2-way ANOVA; Fig. 4B). Post hoc tests showed that application of hypoxia in sleep led to decreased non-REM sleep compared with room air stimuli (P = 0.046), but this change was not statistically significant after Bonferroni correction for four planned comparisons (critical P = 0.0125). There was increased non-REM sleep in the recovery phase after hypoxia, but, again, this was of borderline statistical significance after correction for multiple comparisons (P = 0.023) and was also not statistically different from recovery from room air stimuli in the control condition (P = 0.070). There was also no significant change in %non-REM sleep between the stimulus and recovery phases during application of room air stimuli (P = 0.472).

%REM sleep. The changes in REM sleep were the most robust in response to hypoxic stimuli in sleep. Like non-REM sleep and wakefulness, the effects of stimulus and recovery phase on %REM sleep depended on whether hypoxic or room air stimuli were applied (P = 0.0008, 2-way ANOVA; Fig. 4C). Post hoc analyses confirmed that application of hypoxia in sleep reduced REM sleep compared with room air stimuli (P = 0.0004). In the recovery phase after hypoxia, there was a rebound increase in REM sleep (P = 0.00002) to levels higher than those after room air stimuli (P = 0.009).

Duration and Frequency of Sleep-Wake Episodes in Response to Hypoxic Stimuli in Sleep

Figure 5 shows the changes in durations and frequency of wakefulness, non-REM sleep, and REM sleep episodes that are responsible for the changes in %sleep-wake states observed in Fig. 4. For each of wakefulness, non-REM sleep, and REM sleep, the effects of stimulus or recovery phase on the duration and frequencies of sleep-wake states depended on whether hypoxic or room air stimuli were applied (each P < 0.05, 2-way ANOVAs). Post hoc comparisons showed that the increased %wakefulness observed with application of hypoxia in sleep was due to an increased number of waking episodes that were of longer duration compared with room air (P = 0.0001 and 0.007, respectively; Fig. 5A). Analysis also showed that, although %non-REM sleep was not significantly affected by the hypoxic stimuli (Fig. 4B), there was an increased frequency of non-REM episodes with application of hypoxic stimuli compared with room air, with these non-REM episodes also being of shorter duration (P = 0.00008 and 0.0002, respectively; Fig. 5, C and D). The reduced %REM sleep with the hypoxic stimuli was due to a decreased number of REM episodes that were also of shorter duration compared with room air (P = 0.0127 and 0.002 respectively; Fig. 5, E and F).


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  		Fig. 5.   Group data (means ± SE) showing the effects of application of hypoxic stimuli in sleep (, condition 4) on the duration and frequency of sleep-wake episodes compared with room air stimuli (open circle , condition 3). In the stimulus phase, wakefulness episodes in the presence of hypoxic stimuli were of significantly longer duration (A) and more frequent (B) than with room air. Non-REM episodes with the hypoxic stimuli were of significantly shorter duration (C) and more frequent (D) than with room air. REM episodes with the hypoxic stimuli were of significantly shorter duration (E) and less frequent (F) than with room air. The duration and frequency of sleep-wake episodes returned to control levels in the recovery phase after removal of the hypoxic stimuli. *Significant difference between experimental conditions (i.e., hypoxia in sleep vs. room air in sleep); +significant difference across experimental phase (i.e., stimulus phase vs. recovery phase) (both P < 0.05).

EEG Frequencies in Response to Hypoxic Stimuli Applied in Sleep

There were significant effects of hypoxic stimuli applied in sleep on EEG frequencies. Figure 6 shows the changes in the ratio of high to low EEG frequencies (beta 2/delta 1) in the stimulus and recovery phases during application of hypoxic or room air stimuli in sleep. For wakefulness, non-REM sleep, and REM sleep, the effects of stimulus and recovery phase on beta 2/delta 1 depended on whether hypoxic or room air stimuli were applied (each P < 0.01, 2-way ANOVAs). Post hoc paired t-tests showed that for each sleep-wake state the EEG was associated with a significant shift toward faster frequencies (i.e., increased beta 2/delta 1) during application of hypoxic stimuli in sleep compared with room air stimuli (each P < 0.005). This effect was most apparent in non-REM sleep in which there were highly significant increases in the high-frequency components of the EEG signal in the beta 1 (13.5-20 Hz) and beta 2 (20-30 Hz) bands with application of hypoxia (P = 0.0005 and 0.00006, respectively). Likewise, in non-REM sleep, there were also corresponding decreases in the low-frequency components of the EEG signal in the delta 2 (0.5-2 Hz), delta 1 (2-4 Hz), and theta  (4-7.5 Hz) bands with application of hypoxia (P < 0.008, 0.00001, and 0.0003, respectively). This shift to faster EEG frequencies with application of hypoxia was also observed in REM sleep in which there was decreased activity in the alpha  band (7.5-13.5 Hz) and increased activity in the beta 2 band (P = 0.011 and 0.002, respectively). Note that the overall values of beta 2/delta 1 are lower in non-REM sleep compared with wakefulness and REM sleep regardless of experimental condition. This difference is indicative of the typical overall slower EEG frequencies in non-REM sleep.


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  		Fig. 6.   Group data (means ± SE) showing the effects hypoxic stimuli applied in sleep (, condition 4) on the ratio of high (beta 2: 20-30 Hz) to low (delta 1: 2-4 Hz) frequencies in the electroencephalogram (EEG) signal (beta 2/delta 1) compared with room air stimuli (open circle , condition 3). Note that application of hypoxic stimuli in sleep led to a shift toward faster EEG frequencies (i.e., increased beta 2/delta 1) in wakefulness (A), non-REM sleep (B), and REM sleep (C) compared with room air stimuli. The beta 2/delta 1 returned to control levels in the recovery phase after removal of the hypoxic stimuli. *Significant difference between experimental conditions (i.e., application of hypoxia in sleep vs. room air in sleep); +significant difference across experimental phase (i.e., stimulus phase vs. recovery phase) (both P < 0.05).

No Changes in Sleep-Wake States Across the Three Control Conditions

There was no difference in %wakefulness, non-REM sleep, and REM sleep recorded in the stimulus and recovery phases across the three controls conditions (i.e., conditions 1-3 in Fig. 1, all P > 0.250, 2-way ANOVAs).

Effects of Hypoxic Stimuli on Body Temperature

Figure 7 shows that application of hypoxic stimuli in sleep caused body temperature to decrease, with continuous hypoxia causing lower body temperatures than application of intermittent hypoxic stimuli timed only to sleep episodes. Statistical analysis confirmed that the effects of stimulus and recovery phase on body temperature depended on experimental condition, i.e., whether room air, intermittent hypoxia, or continuous hypoxia was applied (each P < 0.02, 2-way ANOVAs). Post hoc paired t-tests showed that body temperature was significantly decreased by hypoxic stimuli in sleep compared with room air stimuli (P < 0.001) and was further reduced by continuous hypoxia compared with both intermittent hypoxia and room air stimuli (both P < 0.0001). Body temperature returned to levels indistinguishable from control (i.e., room air stimuli) after removal of intermittent or continuous hypoxia (P = 0.115 and 0.08, respectively).


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  		Fig. 7.   Group data (means ± SE) showing that hypoxic stimuli lowered body temperature, with continuous chronic hypoxia (black-triangle, condition 5) causing lower body temperatures than intermittent hypoxic stimuli timed only to sleep episodes (, condition 4) or application of room air stimuli (open circle , condition 3). See text for further details.

Effects of Chronic Hypoxia vs. Hypoxia Applied Only in Sleep on Sleep-Wake States

Figure 8 shows the effects of continuous hypoxia vs. intermittent hypoxic stimuli applied in sleep on %wakefulness, non-REM sleep, and %REM sleep. Analyses showed that there were significant effects of experimental phase (i.e., stimulus vs. recovery) and experimental condition (i.e., chronic vs. intermittent hypoxia) on %wakefulness, %non-REM sleep, and %REM sleep (all P < 0.0025, 2-way ANOVAs). Nevertheless, there was no statistically significant interaction between experimental phase and the type of hypoxic intervention for wakefulness, non-REM sleep, or REM sleep (all P > 0.08, 2-way ANOVAs). This latter result showed that the direction of change in sleep-wake states across the stimulus and recovery phases was consistent between intermittent hypoxic stimuli applied only in sleep and chronic continuous hypoxia, although the absolute amounts of wakefulness, non-REM sleep, and REM sleep differed between hypoxic conditions.


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  		Fig. 8.   Group data (means ± SE) showing the effects of hypoxic stimuli applied only in sleep (, condition 4) on %sleep-wake states compared with continuous chronic hypoxia (black-triangle, condition 5). Continuous chronic hypoxia led to increased wakefulness (A) and decreased non-REM (B) and REM sleep (C). Rebound increases in non-REM and REM sleep and decreases in wakefulness were observed in the recovery phase after return to room air stimuli. See text for further details.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study show that application of hypoxic stimuli exclusively in sleep leads to increased wakefulness, faster EEG frequencies in non-REM sleep, and decreased REM sleep. These effects of hypoxic stimuli were reversed on return to room air stimuli; e.g., there were rebound increases in REM sleep, decreases in wakefulness, and increased slower frequencies in the EEG. These overall effects of hypoxic stimuli on sleep patterns and the effects on recovery sleep after removal of stimuli are similar to those observed in OSA patients before and after treatment (22) and in dogs after cessation of experimentally induced OSA (20, 35). As such, these results suggest that the disruptive effects of OSA on sleep patterns and the subsequent compensatory changes in sleep after treatment are likely attributable to the recurrent episodic sleep-related hypoxia in OSA rather than, for example, recurrent periods of transient hypercapnia that are also associated with each apnea. To our knowledge, this study is the first to document the independent effects of intermittent hypoxic stimuli, applied only in sleep, on sleep regulation and recovery sleep after removal of hypoxic stimuli.

Methodology and Relevance to OSA

A number of studies have investigated the consequences of repetitive intermittent hypoxic stimuli on physiological parameters such as blood pressure and cardiovascular responses in rats and related these responses to OSA (2, 11, 12, 18, 24). In all these previous studies, however, the intermittent hypoxic stimuli were applied for several hours during the light (i.e., resting) phase without reference to sleep-wake states. In those studies, and common to other protocols applying intermittent hypoxic stimuli with the aim of mimicking OSA, the assumption is that, by applying the episodic hypoxic stimuli to rats in the light phase, those stimuli are likely to be applied in sleep. However, the ultradian rhythm of the sleep-wake cycle in rats (e.g., Ref. 45) ensures that there are prolonged periods of wakefulness even during the light phase (Fig. 3), making it unlikely that most of the stimuli would be applied in sleep. In addition, with such protocols, it is also unlikely that the termination of hypoxia would be coincident with arousal as occurs in clinical disorders.

The present study is novel because it is the first to trigger the delivery of hypoxic stimuli to the onset of sleep episodes and to remove those stimuli at arousal from sleep. In this study, ~95% of the applied hypoxic stimuli were correctly applied during sleep and removed at arousal, with the majority of errors (3%) due to periods of EEG activity intermediate between light sleep and quiet wakefulness (i.e., drowsiness). Overall, these results show that the hypoxic stimuli were applied in sleep with a high degree of accuracy, such that this design is relevant and appropriate to determine the independent effects of sleep-related hypoxia in a fashion that approximates sleep-related hypoxia in OSA. The longer durations of REM episodes, compared with non-REM durations, in the presence of the hypoxic stimuli also agree with the reduced arousal responses to hypoxia in REM sleep observed in animals and humans (7, 38). For example, in a previous study in humans, decreases in arterial O2 saturation to ~70% produced arousals from non-REM sleep much more consistently than hypoxia in REM sleep (7).

Although the hypoxic stimuli in this study were applied in sleep to approximate OSA, it is important to mention that there are major differences between our protocol and natural OSA. For example, during apneic episodes, progressive hypoxia, hypercapnia, and increasing inspiratory efforts against an obstructed airway may all contribute to arousal responses and sleep disturbance. The reason we chose to perform our studies in the absence of other stimuli, e.g., hypoxia plus hypercapnia, was to allow determination of the independent effects of hypoxia on sleep patterns without the complication of determining which effects were due to hypoxia, hypercapnia, or both. Another difference between our protocol and OSA is that arterial hypoxemia progressively worsens in OSA, whereas we simply switched from room air to 10% O2 at sleep onset to produce the hypoxic stimuli. We chose to deliver our stimuli in this fashion to have reproducible interventions within and between animals, as well as for practical reasons; e.g., it was difficult to reproducibly and safely control a progressive withdrawal of O2 in the experimental chamber by progressively adding N2.

Another important caveat is that we chose to restrict our sleep-related hypoxic interventions to a 3-h period in the light phase of the rest-activity cycle and determined the effects of stimulus removal on recovery sleep for a subsequent 3-h period also in the light phase. Because rats sleep predominantly in the light, we chose this approach to maximize the opportunity of observing any potential changes in sleep-wake patterns resulting from the hypoxic stimuli and any potential compensatory changes after stimulus removal. However, this approach means that the changes we observed with application of sleep-related hypoxia in this study can only be ascribed to the acute, short-term effects of such stimuli. It remains to be determined whether these acute effects persist with long-term application of hypoxic stimuli, e.g., over 24-h periods or longer. Such chronic application of intermittent hypoxic stimuli may be more relevant to the clinical condition in which OSA patients will have typically experienced recurrent sleep-related hypoxia for many years before coming to medical attention. Another caveat resulting from studying only a limited period of the day in our study is that a longer period (e.g., 24 h) would have been better to fully characterize both the normal and disrupted sleep patterns in the different experimental conditions. Although we acknowledge this limitation, our results showed that sleep patterns were not different between each of the three control conditions, especially condition 1, in which the rat was in its most natural environment, i.e., within its home cage and breathing normal ambient air (Fig. 1). This result supports the notion that sleep was normal in the control experiments. Furthermore, we also do not think that the changes in non-REM and REM sleep observed between the stimulus and recovery phases with the hypoxic stimuli were due to time-of-day effects because responses in this study were compared with application of room air stimuli at the same times of day.

Approximately 70 hypoxic stimuli were applied in sleep in each rat over the 3-h stimulus phase, i.e., ~23 episodes per hour. This frequency of sleep-related hypoxic episodes approximates the apnea index in moderate OSA. One of the reasons we chose to deliver 10% O2 as our hypoxic stimulus was because it is likely that the level of arterial hypoxemia resulting from the transient hypoxia would approximate the levels observed in moderate to severe OSA. Exposure of unanesthetized rats to chronic, continuous 10% O2 reduces arterial PO2 to ~37 Torr when measured after ~10 min and also produces a respiratory alkalosis (pH ~7.55) due to hyperventilation induced hypocapnia (29). This level of arterial PO2 at this pH in rats approximates an arterial oxygen saturation of 65-70% (8). In practice, however, it is not likely that the arterial oxygen saturation would reach this level with the transient 10% O2 applied in our study. Indeed, because the sleep durations averaged between 50 and 60 s in the presence of hypoxic stimuli, the minimum arterial oxygen saturation in the rat will be affected by the finite time taken for O2 in the chamber to decrease to 10% (Fig. 2) and then equilibrate in the animal. As such, it is likely that the arterial oxygen saturation after a transient stimulus of 10% O2 would be greater than the minimum level of 65-70% recorded in rats exposed to continuous chronic hypoxia (29). We chose not to measure blood gases in this study to avoid potential confounding effects of tether restraint on sleep architecture.

Another reason we chose to deliver 10% O2 in this study was because it allowed for direct comparisons of sleep-wake patterns in continuous hypoxia (condition 5) to previous studies also using continuous hypoxia in rats (28, 32, 36). Our results with continuous hypoxia are in agreement with these previous studies, although these latter groups did not investigate recovery sleep after hypoxia as in our study. However, an important outcome of comparing sleep-wake patterns in continuous hypoxia vs. hypoxic stimuli only in sleep (Fig. 8) was that it showed that the effects of both hypoxic conditions on sleep were qualitatively similar but that the effects of continuous hypoxia were of larger magnitude. This result implies that mechanisms modulating sleep-wake patterns in continuous hypoxia may also be operative with intermittent hypoxia, with the reduced effects on sleep in the latter case simply being due to less overall time being spent in the presence of the hypoxic stimuli. This suggestion may be relevant in explaining the observation that continuous hypoxia caused significant reductions in non-REM sleep, whereas with intermittent hypoxia the overall amounts of non-REM were unaffected but there was evidence of disruption because of more non-REM episodes of shorter duration. The larger effects on non-REM sleep in the former condition may reflect an effect of continuous hypoxia acting to promote wakefulness and reduce the tendency to fall asleep. With intermittent hypoxic stimuli, however, non-REM sleep was entered on a background of room air breathing; therefore, sleep onset may not have been impaired. That sleep-related hypoxic stimuli in this study led to increased wakefulness, a shift to increased faster EEG frequencies in non-REM sleep, and decreased REM sleep are consistent with the hypoxia acting as an arousal stimulus (40).

As mentioned previously, hypoxia in conscious freely behaving rats produces hyperventilation-induced hypocapnia (29). In the present study, we did not prevent this hypocapnia by adding CO2 to the inspired air during hypoxic stimuli. Nevertheless, we do not think that this significantly detracts from the results of the present study, nor their interpretation, because compensation for respiratory alkalosis by adding CO2 to rats exposed to chronic 10% O2 did not significantly affect sleep compared with sleep with 10% O2 alone (36).

Temperature

It has been demonstrated previously that application of hypoxic stimuli decreases body temperature and metabolism in rats (e.g., Refs. 13, 36), and a similar decrease in body temperature was also observed in this study, especially in the presence of continuous hypoxia (Fig. 7). With intermittent hypoxic stimuli applied only in sleep, however, body temperature decreased only slightly (<0.5°C) compared with room air stimuli.

It has previously been demonstrated in rats that sleep efficiency is maximal under thermoneutral conditions (41). The quantity of sleep, especially REM sleep, is decreased when ambient temperature is raised or lowered outside the thermoneutral range (44). However, these studies on the effects of altered ambient temperatures are not directly comparable with the present study in which ambient temperature was constant. The effects of altered ambient temperatures on sleep in the previous studies could be mediated, at least in part, by changes in skin temperature, but the latter was not measured in the present study.

In general, thermal effects on sleep-wake state appear to be more closely related to thermoregulatory control mechanisms, notably the difference between body temperature and set point, rather than absolute temperature (31, 41). Hypoxia has been shown to cause a reduction in behavioral thermoregulatory set point in rats (17), and, at subthermoneutral ambient temperatures, this is accompanied by reductions in both metabolic heat production and body temperature (13). However, because we did not assess the thermoregulatory set point in this study, we cannot determine whether sleep-specific intermittent hypoxia caused hypothermia or a regulated reduction in body temperature. Hence, further studies are needed to determine the relative roles of hypoxia and decreased body temperature in the observed changes in sleep architecture. In this context, we note that the effects of continuous hypoxia on sleep-wake state are dependent on functional carotid body chemoreceptors (40), whereas the hypoxic hypometabolism and reduced body temperature are not (15, 30). This suggests that the changes in sleep-wake pattern observed in this study are not likely mediated by changes in body temperature.

Results and Overall Schema

The changes in sleep patterns before and after treatment of OSA, especially the rebound increase in REM sleep, have been well described in animals and humans (20, 22, 35, 43), but the mechanisms behind this effect have not been determined. However, the similar rebound increase in REM sleep observed in the present study after removal of intermittent hypoxic stimuli suggests that sleep-related hypoxia may be involved. It has been suggested that the propensity for REM sleep accumulates as a function of time spent in non-REM sleep, and, when this drive exceeds a certain threshold, it then triggers REM sleep (3, 4). A feature of this model, however, is that the REM sleep episode must not be disrupted for full discharge of the REM sleep drive. Premature shortening of a REM episode, e.g., in this study by the presence of hypoxic stimuli, would therefore lead to inadequate discharge of the REM propensity. In this scenario, repeated REM sleep fragmentation would therefore lead to a chronically increased REM sleep drive that would manifest itself as a rebound increase in REM sleep during uninterrupted recovery sleep (3, 4). Because integrity of REM sleep is important for functions related to memory and cognitive processes (23, 42), the impact of repetitive hypoxic stimuli on REM sleep mechanisms may also contribute to the impaired daytime function of OSA patients (9, 16, 27).

In summary, this study shows that application of hypoxic stimuli only in sleep leads to significant modulation of sleep-wake regulation, as evidenced by sleep during application of the hypoxic stimuli and recovery sleep after removal of the hypoxic stimuli. This study is the first to determine the independent effects of sleep-related hypoxic stimuli on sleep-regulation in a fashion that approximates sleep-related hypoxia in OSA before and after treatment.


    ACKNOWLEDGEMENTS

We thank Dr. Dina Brooks for help in modifying the sleep detection software in this study for delivery hypoxic stimuli in sleep and removal of stimuli at arousal.


    FOOTNOTES

This work was supported by the Medical Research Council (MRC) of Canada (MT-15563), Ontario Thoracic Society, and Canada Foundation for Innovation. R. L. Horner is a recipient of a MRC of Canada Scholarship.

Address for reprint requests and other correspondence: R. L. Horner, Rm. 6368 Medical Sciences Bldg., 1 Kings College Circle, Toronto, Ontario, Canada, M5S 1A8 (E-mail: richard.horner{at}utoronto.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 January 2001; accepted in final form 5 March 2001.


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