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J Appl Physiol 94: 525-532, 2003. First published October 25, 2002; doi:10.1152/japplphysiol.00226.2002
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Vol. 94, Issue 2, 525-532, February 2003

Sleep apnea and effect of chemostimulation on breathing instability in mice

Akira Nakamura1, Yasuichiro Fukuda1, and Tomoyuki Kuwaki1,2

Departments of 1 Autonomic Physiology and 2 Molecular and Integrative Physiology, Graduate School of Medicine, Chiba University, Chiba-city, Chiba 260-8670, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

sleep apnea syndromes; respiration; control of breathing; whole body plethysmography


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SLEEP APNEA SYNDROME, defined as an apnea-hypopnea index of >= 5 associated with daytime hypersomnolence, is estimated to affect 4% of men and 2% of women (38). Sleep apnea is classified into obstructive, central, and mixed types. Obstructive sleep apnea is characterized by repetitive obstruction of the upper airway during sleep, whereas central sleep apnea is characterized by recurrent apneic episodes in the absence of upper airway obstruction (1). Although some patients with obstructive sleep apnea may be treated with continuous positive airway pressure (5), the exact cause(s) of both obstructive sleep apnea and central sleep apnea has not been fully elucidated.

Several animal models of sleep apnea have been described over the past two decades. These have included sleep-triggered mechanical airway occlusion in dogs (16), exposure to hypoxic gas in mice (34), and artificially induced central apnea in lambs (20). In addition, English bulldogs (13) and rats (6, 25, 33) develop sleep apnea without artificial maneuvers. Among these models, the rat is of particular interest because sleep apnea occurs not only in sleep apnea syndrome patients but also in normal humans (2, 18, 38). Whether normal mice suffer from sleep apnea without artificial maneuvers remains unknown.

We studied apnea in mice because these animals are frequently used in genetic engineering. The use of transgenic mice has already allowed the effects of specific genes on physiological functions to be studied. In fact, the sleep-wake state and ventilation in mice have been simultaneously recorded, for example, to characterize the roles of leptin in respiratory depression with obesity (28). However, sleep apnea in mice has not been documented to our knowledge, and the identification of sleep apnea in normal mice might help to understand the molecular basis of sleep apnea.

The aims of the present study were 1) to examine whether normal mice develop apnea during sleep and, if so, 2) to examine the effect of respiratory chemostimulation on sleep apnea. The second aim was based on studies indicating that an elevated CO2 threshold (lowest CO2 concentration that triggers respiration) during sleep could trigger sleep apnea (7).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mice and surgical procedure. Male 129/Sv mice (23-33 wk old at the time of surgery, weighing 27-33 g, average 31 g, n = 5) were anesthetized with 2-3% isoflurane. Electrodes for electroencephalography (EEG) and electromyography (EMG) were implanted at a body temperature of 36-38°C (Physitemp Instruments, Clifton, NJ). For EEG recording, stainless steel screws were implanted bilaterally into the parietal bones of the skull at 2 mm lateral and 2 mm posterior to the bregma while the head was immobilized with a stereotaxic frame (Stoelting, Wood Dale, IL). Two electrodes for EMG recording were also implanted into the nuchal muscle. In one additional animal, an EMG of the external intercostal muscle in the eighth intercostal space was recorded instead of the EEG and the neck EMG to examine whether the absence of a plethysmographic signal (i.e., apnea, see Definition of apnea) was associated with the disappearance of an EMG signal from the respiratory muscles. These electrodes were soldered to a four-channel connector plug (MT Giken, Tokyo, Japan) that was firmly fixed to the skull with dental cement. An antibiotic (cephazolin, 100 mg/kg) was subcutaneously administered before and after the surgery. The mice were housed individually after surgery in a temperature-controlled room (23-25°C) under a 12:12-h light-dark cycle (lights on at 0600). Food and water were available ad libitum. All experimental procedures proceeded in accordance with the "Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences" recommended by the Physiological Society of Japan.

Measurement of EEG and definition of sleep stages. Mice were allowed to recover from surgery for at least 7 days before experiments. This period was selected on the basis of preliminary studies of four mice showing that apnea initially appeared 3-4 days after surgery, increased toward 7 days, and remained constant thereafter. Breathing was measured by body plethysmography (see Measurement of ventilation) in a free-running state, together with the EEG and EMG. To allow freedom of movement during recording, a slip-ring (MSR35, MT Giken) was placed at the connection of the electrodes to lines outside the plethysmograph. The EEG and EMG signals were amplified (with band-pass filters for EEG, 0.25-100 Hz; for neck EMG, 15-100 Hz; for intercostal EMG, 200-1,000 Hz) (AB-651J, Nihon Kohden, Tokyo, Japan) and fed into a personal computer (Macintosh, Apple Computer) after analog-to-digital conversion (MacLab, ADInstruments, Castle Hill, NSW, Australia) at a sampling frequency of 200 Hz (for EEG and neck EMG) or 4,000 Hz (for intercostal EMG). Neck EMG and intercostal EMG were digitally filtered (FIR filter, band pass 15-100 Hz and 200-1,000 Hz, respectively) and used for later analysis. Sleep architecture was determined by visual inspection of the neck EMG and digitally filtered EEG (0.25-4, 4-8, and 8-30 Hz). Waveforms were displayed on a wide screen at a resolution of ~5 mm/s, and transitions among vigilance states were judged by relative changes in amplitude. For every 5-s epoch, vigilance was scored as a predominant state by use of the following criteria. Quiet wakefulness (QW) was defined by a high-frequency (8-30 Hz) low-amplitude EEG with a relatively high EMG tone. Active wakefulness (AW) was defined by very-high-amplitude EMG volleys. EMG-based discrimination between QW and AW was confirmed by visual inspection of videotaped behavior of the mouse. Slow wave sleep (SWS) was defined by a low-frequency (0.25-4 Hz) high-amplitude EEG. Non-SWS sleep or rapid eye movement (REM) sleep was defined by a mixed-frequency (4-8 and 8-30 Hz) low-amplitude EEG associated with weak or absent EMG activity. Two investigators (A. Nakawana and T. Kuwaki) individually scored all recordings. The initial agreement was over 90%, and disagreements were resolved by classifying the segments with reference to the average amplitudes of the EEG and EMG.

Measurement of ventilation by whole body plethysmography. We used double-chamber whole body plethysmography (10, 27, 29) with few modifications, as described by Jacky (15). One chamber (750 ml) was used as a barometric chamber where the mouse was placed, and reference pressure was measured in the other. The outlet ports of both chambers were connected by a long tube so that the time constant for gas leakage from the chamber was large compared with the duration of the respiratory cycle. Such modification allowed both respiratory frequency (fR) and tidal volume (VT) to be measured by the flow-through system (15). Plethysmographic signals were recorded as changes in the pressure difference between the two chambers by use of a differential pressure transducer (TP-602T, Nihon Kohden). Amplified (AR-601G and AB-621G, Nihon Kohden) signals were fed into a computer (sampling frequency of 100 Hz) together with EEG and EMG signals as described above. The fR and amplitude of the plethysmographic signals (representing VT) were calculated by use of the signal analysis software, Chart (ADInstruments). Ambient temperature and pressure and the chamber temperature were measured intermittently (about every 2 h) during the 6 h of recording, and the average values were used for later calculation of VT. Chamber temperature in the thermoneutral range was maintained between 22 and 25°C by controlling the room temperature to minimize possible metabolic effects on respiration. Animal core temperature was not measured continuously. Instead, the rectal temperature of each mouse was recorded immediately after the plethysmographic recording. Individual measurements of rectal temperature revealed a mean value (±SE) of 35.8 ± 0.7°C, which did not significantly differ among four gas conditions (see below). On the basis of these values, the VT value was calculated according to the formula used by Epstein et al. (10). Minute volume (MV) was defined as the product of inspiratory VT and fR. In addition, the coefficients of variance (CV = SD/average) of VT and fR were calculated to evaluate the regularity of respiration at each sleep stage. Respiratory parameters including apneas were calculated for QW, SWS, and REM but not for AW because breathing varied widely during various behaviors (e.g., walking, grooming) in AW and hence values in AW would not be suitable as controls for those in sleep stages.

All recordings were obtained over 6 h between 1000 and 1600, which is the resting period for nocturnal mice (26). During the entire recording period, mice were allowed to move freely. Each chamber was continuously flushed with a gas mixture at a rate of 500 ml/min, either room air or hypoxic (15% O2 balance with N2), hyperoxic (100% O2), or normoxic hypercapnic (5% CO2-21% O2-N2 balance) gas mixtures. Chamber PCO2 and PO2 values were continuously monitored (Respina IH26, NEC-San-Ei-Instruments, Tokyo, Japan) at the outlet of the chamber, and we confirmed that the flushing rate in this study was sufficient to avoid both CO2 accumulation and an O2 decrease in the chamber while animals breathed room air. Each animal was tested under all four air conditions in a random order over 4-7 days.

Definition of apnea. Apnea was defined as a cessation of plethysmographic signals for at least two respiratory cycles, similar to the criterion used for sleep apnea in humans (cessation of breathing for >10 s, which is one or two missed breaths). The duration of the basal respiratory cycle was calculated as the mean value of stable breaths during the 10-20 s just before the apnea. Similar to previous reports (4, 6) of periodic respiration and sleep apneas in rats, apneas were classified as postsigh if the preceding breath was at least 25% above the average amplitude during the preceding 10 s (see Fig. 2, B and D). Apnea without a preceding sigh was defined as spontaneous. The duration of apnea was evaluated not only by an absolute length of time but also as the ratio of each apnea period to the respiratory cycle period adjacent to the apnea (termed "apnea duration index"), because the length of cycle time significantly differed among the gas conditions (see RESULTS). The "apnea occurrence index," defined as the number of apneic episodes per hour, was separately calculated for each type of apnea during each stage of sleep.

Statistical analyses. All data are expressed as means ± SE. The effects of the inspired gas composition on the appearance of apnea, sleep state, and basal respiratory parameters (VT, fR, and MV) were assessed by ANOVA with a repeated measures design, in which room air was treated as the control. Differences in respiratory parameters among vigilance states (QW, SWS, and REM sleep) were also analyzed by use of ANOVA with repeated measures. A P value below 0.05 denoted a statistically significant difference.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Basic parameters of ventilation and sleep-wake state. To examine whether normal mice develop sleep apnea, we continuously and concomitantly measured ventilation and the vigilance state for 6 h when the animals were at rest. When breathing normal room air, the mice spent 54.4 ± 3.9% of the recording period sleeping (both SWS and REM sleep, Table 1). The inhalation of hypoxic, hyperoxic, or hypercapnic gas mixtures did not affect the total amount of sleep, compared with room air (Table 1). Unlike humans but very similar to rats, SWS and wake episodes were highly fragmented, and the duration of SWS varied widely within each animal even under a specific gas condition. Consequently, sleep architecture did not significantly differ among the four gas conditions (Fig. 1).

                              
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  		Table 1.   Effects of inspired gas composition on duration of vigilance state



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  		Fig. 1.   Vigilance state in mice under different gas conditions. Values are means ± SE (n = 5). Episode duration of slow wave sleep (SWS) widely varies even under specific gas conditions. Mean values of episode duration were not significantly different among 4 gas conditions. AW, active wakefulness; QW, quiet wakefulness; REM, rapid eye movement sleep.

On the other hand, basic respiratory parameters (fR, VT, and MV) varied according to gas composition at both wakefulness and sleep stages (Table 2). Specifically, fR, VT, and MV were significantly higher while animals breathed a high CO2 gas mixture (5% CO2-21% O2) than when they breathed normal room air during every vigilance state (QW, SWS, and REM sleep). When mice breathed a low O2 gas mixture (15% O2), fR, VT, and MV similarly increased, although with a lower magnitude. Comparisons among vigilance states showed that the fR, VT, and MV values in SWS were lower than those during QW under all inspired gas mixtures. The VT value was smaller in REM than in SWS. However, the MV value in REM did not significantly differ from that in SWS because the fR value in REM was higher than or equal to that in SWS.

                              
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  		Table 2.   Respiratory parameters in each vigilance state

We also assessed the breathing rhythm during each vigilance state by calculating the CV of fR and VT (Table 3). Hypercapnia induced a significant reduction of CV in VT during all vigilance states and in fR during QW compared with room air. Comparisons among vigilance states showed that the CV of fR in SWS was the lowest under all gas conditions, indicating that respiratory rhythmogenesis was regular and stable in SWS, irrespective of chemostimulation. On the other hand, the CV of VT in REM was the smallest, indicating that different mechanisms control rhythm and amplitude during SWS and REM sleep.

                              
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  		Table 3.   Stability of respiratory parameters in each vigilance state

Parameters of apnea while breathing room air. In mice breathing room air, 32.3 ± 6.9 (n = 5) instances of apnea were recorded during the 6-h observation period (Table 4). Apneas were detected during sleep and never during QW periods. Sleep apneas were classified as postsigh and spontaneous types (Fig. 2). Most instances of postsigh apnea (>99.5%) did not begin immediately after sighs but after a lag period of several normal (or diminished) breaths (Fig. 2, B and D). The cessation of respiratory signals in whole body plethysmography was accompanied by the disappearance of intercostal EMG traces (Fig. 2, C and D), indicating that these types of apnea were central rather than obstructive. Intercostal EMG recordings during both types of apnea similarly disappeared when the animal breathed hypoxic, hyperoxic, and hypercapnic gas mixtures (data not shown).

                              
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  		Table 4.   Occurrence of sighs and apneas during 6 h of recording period



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  		Fig. 2.   Typical examples of spontaneous (A, C) and postsigh (B, D) apneas. Recordings were obtained from 2 mice (A and B were from one mouse, whereas C and D were from another) under room air. A: spontaneous apnea during REM sleep. B: postsigh apnea during SWS. C and D: absence of intercostal electromyogram (EMG) during both types of apneas, indicating central sleep apnea. No evidence indicates obstructive sleep apnea in 16 postsigh and 10 spontaneous apneas in this animal. Middle traces in each section represent positive-rectified and integrated (time constant 0.1 s) EMG. Small, sharp deflections present during apneic periods are electrocardiographic artifacts. Arrowheads in B and D indicate normal or diminished breaths interspaced between a sigh and apnea. EEG, electroencephalogram; Resp, respiration recorded by whole body plethysmography.

The type of apnea was related to sleep stages. Under room air, only postsigh apneas were observed during SWS, whereas the spontaneous type appeared only during REM sleep (Table 4). Most of the recorded apneas were of the postsigh type, presumably because SWS occupied over 90% of the entire duration of sleep (Table 1). To standardize such differences in sleep stages, we calculated the apnea occurrence index, defined as number of apneic episodes per hour of each sleep stage (Fig. 3). The occurrence index of spontaneous apnea during REM sleep (14.8 ± 9.6/h) was higher than that of postsigh apnea during SWS (9.1 ± 1.4/h). The duration of both spontaneous and postsigh apneas varied from 1.2 to 6.2 s, which corresponded to 2.3 to 8.2 respiratory cycle times adjacent to the apnea (Fig. 4). Apparent arousal immediately after apnea was infrequent (3 of 565 apneas in total; 0.5%) on the basis of visual analysis of the EEG and EMG signals.


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  		Fig. 3.   Effect of inspired gas composition on sigh and apnea occurrence index. A: during SWS, postsigh apnea occurrence index was significantly higher under hypoxia and significantly lower under hyperoxia and hypercapnia than in room air. B: during REM sleep, occurrence index of spontaneous apnea was not significantly affected by gas condition, although the tendency was similar. During both SWS and REM sleep, occurrence index of total sigh did not differ among 4 gas conditions. Values are means ± SE values of 5 mice. ND, not detected. *P < 0.05 and **P < 0.01, compared with room air.



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  		Fig. 4.   Effct of inspired gas composition on apnea duration. Left: apnea duration in absolute time. Right: apnea duration index (ratio of apnea duration to stable respiratory cycle time around apnea). Values are means ± SE values of 5 mice. Neither variable significantly differed among 4 gas conditions. Mean values of apnea duration index were ~3-5, meaning that 2-4 respirations were absent in each apnea irrespective of gas condition. (Spontaneous apnea in SWS and postsigh apnea in REM sleep were seldom or never observed, so they are not shown.)

Effect of inspired gas on apnea. A comparison of breathing patterns under the four gas conditions showed that the relationship between the type of apnea and sleep stage was generally conserved at least for postsigh apneas. Specifically, postsigh apneas occurred exclusively during SWS under every gas condition (Table 4). However, the apnea occurrence index was significantly dependent on the gas condition (Fig. 3). Hypoxia induced about a 50% increase in postsigh apneas, whereas hyperoxia and hypercapnia resulted in a 60-70% decrease compared with postsigh apneas in room air. The tendency of the spontaneous apnea occurrence index in REM sleep was similar, although the difference was not statistically significant because of a large variability (Fig. 3). The difference in the postsigh apnea occurrence index in SWS was not tightly associated with the occurrence of sigh per se (Fig. 3) but was somewhat correlated with the ratio of sighs followed by apnea to an entire sigh (Table 4).

Apnea duration and fR were reciprocally related. Thus the apnea duration indexes (for both postsigh and spontaneous types of apnea) did not significantly differ among the four gas conditions (Fig. 4). The mean apnea duration indexes were ~3-5, representing an absence of 2-4 expected breaths in each apnea irrespective of the gas condition or type of apnea.

Finally, the total period in which the mice underwent postsigh apnea per hour tended to be longer in hypoxia, shorter in hyperoxia, and significantly shorter in hypercapnia compared with the corresponding values recorded under room air (Fig. 5). The tendency for spontaneous apnea was similar during REM sleep.


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  		Fig. 5.   Effect of inspired gas composition on total apnea period. Changes were similar to those of apnea occurrence index (see also Fig. 3). Namely, total apnea period was longer under hypoxia and shorter under hyperoxia and hypercapnia than in room air. Only postsigh apnea in SWS significantly differed from that in room air, presumably because of a variation of the duration of each apnea (see Fig. 4). Values are means ± SE values from 5 mice. *P < 0.05, compared with room air.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated breathing instability during sleep in normal mice to examine whether this species is suitable for studies on sleep apnea. The major findings of the present study were as follows. 1) Episodes of apnea are frequent in normal mice during daytime sleep. 2) Sleep apneas could be classified into two types, postsigh and spontaneous apneas. Postsigh apneas occurred exclusively in SWS, although sighs without subsequent apnea arose during both SWS and REM sleep. 3) Chemostimulation by hypoxia increased the occurrence of the postsigh apnea index during SWS. In contrast, chemostimulation by hypercapnia decreased the index. The tendency of the spontaneous apnea occurrence index during REM sleep was similar. 4) The duration of apnea was equivalent to three to five respiratory cycle periods, irrespective of the basal fR or type of apnea.

Effects of inspired gas composition on sleep-wake state. Under room air breathing, the amount of total sleep and the duration of each vigilance state recorded in our study (Table 1, Fig. 1) were within the range of those identified in mice by recording only the EEG (11, 24, 36, 37), in which the REM fraction varied among the studies as 1-10%. The low REM fraction in our study (1.7%) might have resulted from the fact that the recording chamber was not the home cage. However, repeated measurements did not appear to change the REM fraction in our preliminary experiment, indicating that acclimatization to the recording chamber did not affect the REM fraction. Rather, variations in the REM fraction among the reports may have resulted from variations in the techniques used to measure EEG and to judge vigilance states in mice. For example, the position of electrodes, classification of EEG band frequencies, algorithm of the judgment, minimal duration of a vigilance state, and their combination varied among the studies. These should be considered when comparing results from various laboratories because EEG recording in mice has not been standardized. Nevertheless, our data indicated that being placed in the whole body plethysmography chamber over a long period seemed not to seriously affect the vigilance state. Moreover, the sleep-wake state was not significantly affected by changes in the inspired gas composition. This result was in concert with those in rats (21, 30), although more severe hypoxia (10% O2) distorts sleep and lengthens wakefulness (12). The degrees of both hypoxia (15% O2) and hypercapnia (5% CO2) in the present study were thought to be mild enough not to affect the sleep-wake state. This might be also interpreted to mean that the observed change in apnea according to the inspired gas composition (see below) was not likely to be due to a change in sleep architecture.

Effects of inspired gas composition on respiratory variables. Although hypoxic and hypercapnic stimuli did not result in any apparent effect on sleep architecture, they clearly stimulated respiration, as evident by changes in the fR, VT, and MV values (Table 2). Moreover, these values depended not only on the composition of the inspired gas but also on the vigilance states. The smaller fR, VT, and MV during SWS compared with QW in our mice were consistent with the findings in humans (17). In our study, the VT value during REM further decreased compared with SWS, whereas the MV value during REM was not significantly different from that during SWS. This finding was also in agreement with those of human studies (9, 17). In both humans (8) and mice (28), the ventilatory responses to acute hypoxia and hypercapnia were the largest during wakefulness and the lowest during REM sleep. Our results extended these findings to long-term hypoxic/hypercapnic stimulation (Table 2).

Similar to previous findings in humans (17), breathing was most regular during SWS among three vigilance states (Table 3). The rank order of CV in fR was hyperoxia > hypoxia > = room air > hypercapnia during SWS, whereas that of the apnea occurrence index (Fig. 3) was hypoxia > room air > hyperoxia > = hypercapnia. This means that the basic and overall regularity of breathing does not predict the occurrence of apneas. During QW, the CVs of fR and VT under hypercapnia were smaller than those under room air, indicating that chemoreflex activation overcame other sources of irregularity such as emotion under hypercapnia.

Characteristics of apnea in mice. The numbers of sighs and episodes of apnea during the entire recording period (Table 4) closely matched those found in rats (6). The 129/Sv mouse strain might be susceptible to sleep apnea because basic respiratory parameters and ventilatory responses to chemoreceptor stimulation vary among strains of mice (29, 35). However, our preliminary results found no apparent differences between 129/Sv and C57BL6/J mice, which are two major strains that are frequently used in studies of gene targeting.

Both the postsigh and spontaneous types of apnea identified in this study were probably central sleep apnea because no apparent intercostal EMG (Fig. 2) indicated absence of the paradoxical thoracic movement that arises during obstructive sleep apnea. This type of apnea is unlikely to occur in four-footed animals because of the straighter arrangement of the nasal cavity and pharynx than in upright animals such as humans. In fact, the only four-footed animal that is known to develop obstructive sleep apnea is the English bulldog (13), but this is related to the nature of the facial and upper airway structures. The absence of free-floating hyoid arches may also contribute to the absence of obstructive sleep apnea in rodents.

Postsigh apneas exclusively arose during SWS irrespective of gas conditions, although sighs without subsequent apnea developed during both SWS and REM sleep (Table 4, Fig. 3). On the other hand, spontaneous apnea was not clearly related to sleep stages. Therefore, a sigh-apnea coupling mechanism(s) in mice might be dependent on the sleep state.

The present study simply classified apneas into two types according to the presence or absence of a preceding sigh. However, postsigh apneas in humans have been further classified into those that follow several normal breaths after an augmented breath (type 1) and those immediately after a sigh (type 2) (14, 32). Moreover, spontaneous apneas have also been classified into those with neither a preceding nor subsequent sigh (type 3) and those followed by an augmented breath(s) (type 4). Although the rationale for such classification is not clear, we also observed these types of apneas in mice (type 1 accounted for 99.5% of total of postsigh apneas and type 3 was over 99% of that of spontaneous apneas; Fig. 2).

Relationship between apneas and inspired gas composition. Figure 3 shows that hypoxic stimulation increased the postsigh apnea occurrence index during SWS. Conversely, chemoreceptor activation during hypercapnia decreased the postsigh apnea occurrence index during SWS. The tendency for spontaneous apnea to occur during REM sleep was similar, although the data were inconclusive because of the low number of recorded episodes of spontaneous apnea. Thus respiratory stimulation induced by different chemoreceptor inputs results in opposite effects with regard to the occurrence of apnea. The increase in the occurrence of apnea induced by hypoxia was in agreement with that in normal humans (3, 19) but not in rats (6). Hyperoxia reduced the occurrence of apnea in the present study, which was also in agreement with the findings in patients with central apneas (22) but not in those with obstructive apnea in whom supplemental O2 rather increased the occurrence or prolonged the duration of apnea (22, 23).

In general, apneas after an overshoot are thought to result from hypocapnia (7). In our study, however, postsigh apneas developed during SWS even under hypercapnia. This finding suggests that postsigh apneas in mice did not result solely from hypocapnia induced by hyperventilation. Other possible causes of postsigh apneas include mechanoreceptor activation by augmented breaths, which may actively suppress the subsequent respiratory drive within the neural circuitry as with the persistent inhibition in the Hering-Breuer reflex (7). In fact, our preliminary experiments found that anesthesia significantly suppressed the ratio of apneas to sighs but did not affect the occurrence or numbers of sighs, emphasizing the importance of neural factors in sigh-apnea coupling.

Our observation of a hypoxia-induced increase in postsigh apneas could also be explained by hyperventilation-induced hypocapnia under this condition. However, a decrease in arterial PCO2 (PaCO2) becomes apparent when inspired PO2 is <13% in rats (31). Because we used an inspired PO2 of 15% in our study, the decrease in PaCO2 would be too moderate to trigger sleep apnea by itself. Rather, a combination of a small decrease in PaCO2, a small increase in the apnea threshold (PaCO2 below that required to induce apnea; Ref. 7), and a mild strengthening of sigh-apnea coupling (see Table 4) seemed to increase the incidence of postsigh apnea induced by hypoxia. The decrease in the incidence of apnea under hyperoxia in the present study also supports our view that not only PaCO2 but also neural mechanisms controlling sigh-apnea coupling might play a key role in the development of sleep apnea. PaCO2 theory alone cannot explain why the duration of apnea was equal to three to five respiratory cycle periods irrespective of basal fR or type of apnea (Fig. 4). This is because PaCO2 accumulation during the apneic period should depend on the duration of apnea and not on the index of the apnea duration. Nevertheless, it would be necessary to evaluate breath-by-breath PaCO2 before making any conclusions on this issue.

In conclusion, the present study demonstrated that a significant amount of sleep apnea occurs in normal mice as it does in rats and humans. We therefore believe that mice constitute a suitable animal model with which to study the genetic and molecular mechanisms involved in the regulation and dysregulation of breathing.


    ACKNOWLEDGEMENTS

We thank Chikako Kumagai and Wataru Nakamura for excellent technical assistance. We also thank Dr. Faiq G. Issa and Dr. Megumi Shimoyama for help in editing the manuscript.


    FOOTNOTES

Part of this work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by grants from the Shimadzu Science Foundation and the Yamanouchi Foundation for Research on Metabolic Disorders.

Address for reprint requests and other correspondence: T. Kuwaki, Dept. of Molecular and Integrative Physiology, Graduate School of Medicine, Chiba Univ., 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan (E-mail: kuwaki{at}faculty.chiba-u.jp).

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.

First published October 25, 2002;10.1152/japplphysiol.00226.2002

Received 15 March 2002; accepted in final form 11 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 94(2):525-532
8750-7587/03 $5.00 Copyright © 2003 the American Physiological Society


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