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. Author manuscript; available in PMC: 2014 Aug 15.
Published in final edited form as: Respir Physiol Neurobiol. 2013 Jun 14;188(2):143–151. doi: 10.1016/j.resp.2013.06.002

Development of autonomic dysfunction with intermittent hypoxia in a lean murine model

P Chalacheva a, J Thum a, T Yokoe b, CP O’Donnell b, MCK Khoo a
PMCID: PMC3729633  NIHMSID: NIHMS494313  PMID: 23774144

Abstract

Intermittent hypoxia (IH) has been previously shown in a lean murine model to produce sustained hypertension and reverse the diurnal variation of blood glucose (BG). Concomitant glucose infusion attenuated the hypertension but exacerbated the BG fluctuations. In this study, cardiovascular variability analysis was employed to track the development of autonomic dysfunction in mice exposed to room air (IA) or IH, in combination with saline or glucose infusion. Baroreflex sensitivity was found to decrease in all animals, except in the control group. Low-frequency power of pulse interval spectrum, reflecting vagal activity, decreased more rapidly in glucose relative to saline while low-frequency power of blood pressure, reflecting sympathetic activity, decreased more slowly in IH relative to IA. Ultradian (~12 h) rhythmicity was substantially suppressed in IH groups. These findings suggest that IH acted to increase sympathetic activity while glucose infusion led to reduced parasympathetic activity. The combination of IH and hyperglycemia leads to progressively adverse effects on autonomic control independent of obesity.

1. Introduction

In sleep disordered breathing (SDB), periods of apnea or hypopnea alternate with periods of transient ventilatory overshoot that are accompanied or initiated by arousal from sleep, subjecting the patient to nocturnal intermittent hypoxia (IH). The evidence to date suggests that chronic exposure to IH, resulting from SDB, constitutes an independent causative factor for the development of systemic hypertension (Dempsey et al., 2010). Epidemiological studies in humans with SDB are supported by prospective animal studies, in which chronic exposure to nocturnal episodic hypoxia produced sustained daytime hypertension after several weeks (Brooks et al., 1997; Fletcher et al., 1992).

Although knowledge about the causal pathways linking SDB to hypertension remains incomplete, many studies suggest that abnormal autonomic control plays an important role (Carlson et al., 1993; Somers et al., 1995). Recent studies have also found an association between SDB and abnormal glucose metabolism (Ip et al., 2002; Punjabi et al., 2004; Punjabi et al., 2002). Taken together, the cumulative evidence suggests that chronic exposure to IH resulting from SDB can lead to both autonomic and metabolic dysfunction. Although a variety of underlying mechanisms could be responsible for these associations, abnormal autonomic nervous system control is likely to play an important role in facilitating or even initiating metabolic dysfunction. Activation of the sympatho-adrenal axis is known to reduce insulin sensitivity and increase blood glucose, which could then lead to hyperinsulinemia and further sympathetic over-activity (Chasens et al., 2003).

To examine the effects of IH on blood pressure and glucose regulation, a series of studies has been conducted in recent years using a chronically instrumented murine model (Iiyori et al., 2007; Li et al., 2005; Polotsky et al., 2003; Polotsky et al., 2006; Yokoe et al., 2008). Lean animals were used in order to circumvent the confounding influence of obesity. In this model, each animal was placed in a customized chamber and subjected to repetitive acute hypoxic exposures in which the inspired oxygen level was reduced from room air levels to 5–6% over a 30 s duration and changed back to room air over the subsequent 30 s (Polotsky et al., 2006). These cycles of IH were maintained for the 12 hours of the light, or sleeping, phase of the mouse. During the intervening dark, or active, phase, the mouse breathed room air over the entire 12-hour duration. The total length of IH exposure varied between studies from nine hours to 3 months. In the study of Yokoe et al. (2008), this protocol was repeated over a total duration of 4 consecutive days. Controls were subjected to the same protocol, except that air (IA) instead of hypoxia was introduced on an intermittent basis during sleep. Some of the mice were made mildly hyperglycemic through continuous infusion of dextrose, and the rest were kept in euglycemic conditions with continuous saline infusion. Blood pressure was monitored continuously from the femoral artery, while blood samples were withdrawn twice a day (during the sleep and active periods) for analysis of glucose, insulin and corticosteroid levels. Exposure to IH produced sustained hypertension as well as hyperglycemia in the light/sleeping phase of the mice, and reversed the diurnal variation of blood glucose. Concomitant glucose infusion attenuated the hypertension but exacerbated the blood glucose fluctuations.

In this study, we sought to examine in greater detail the time course of changes in autonomic activity in the Yokoe murine model following exposure to IH with or without concomitant hyperglycemia (Yokoe et al., 2008). Autonomic activity was monitored continuously using indices of cardiovascular variability derived from the continuous arterial blood pressure (ABP) recordings in the mice. We hypothesized that there would be a detectable and progressive increase in sympathetic activity and decrease in parasympathetic activity over the first few days of exposure to IH, and that these changes would be exacerbated by the presence of hyperglycemia.

2. Methods

2.1. Protocols

Details regarding surgery, catheterization, and post-operative maintenance are provided in the study previously published by (Yokoe et al., 2008). In brief, male mice, aged 10–12 weeks, were chronically implanted with femoral arterial and venous catheters to allow continuous arterial pressure measurements as well as infusion of saline (0.9% sodium chloride) or glucose (50% dextrose) at a constant rate of 100 µlh−1. Three days after the surgery, the mice were kept on a 12 hour light-dark cycle beginning at 8am for 4 days with free access to food and water where they were either exposed to IA or IH during light period (sleep period) and stable room air condition during dark period (active period). Each IH cycle consisted of 30 seconds of hypoxia (5.0–6.0% inspired oxygen) followed by 30 seconds of room air (20.9% inspired oxygen), while IA cycle only consisted of switches in room air. Each mouse was subjected to one of four exposures: IA + saline (n = 5), IH + saline (n = 11), IA + glucose (n = 6), and IH + glucose (n = 9). Animal handling and experimentation were carried out in accordance with approved institutional animal care and use committee protocols at the University of Pittsburgh.

2.2. Data Processing

Continuous ABP data were sampled at 200 Hz. The pulse interval (PI) and beat-averaged ABP were extracted from the continuous ABP data. One PI was defined as the time interval between the point of maximum slope on the ABP waveform of the current beat and the point of maximum slope of the next beat. The beat-averaged ABP was determined by calculating the average blood pressure within the PI. For each analysis, if there was more than 25% of “excessively noisy” data within the data segment of interest, that segment would be excluded from the analysis. A data segment was deemed “excessively noisy” if the estimated signal amplitude to noise amplitude was less than 2 and/or there was an abrupt increase in amplitude in a beat that was ≥ 5 times the average amplitude of the signal.

2.3. Power spectral analyses of ABP and PI

Spectral analysis has been shown to be a useful tool in investigating the cardiovascular regulation in both humans and animals by allowing the quantification of the magnitude of sympathetic and parasympathetic activities (Just et al., 2000). Beat-to-beat ABP and PI were divided into 1-minute segments. For each segment, the beat-to-beat signals were resampled at 20 Hz and subjected to trend removal. Low- and high-frequency power (LFP: 0.15 – 1.5 Hz, HFP: 1.5 – 5 Hz respectively) of ABP and PI were calculated by integrating the power spectrum between the aforementioned frequency ranges (Just et al., 2000). The low-frequency power of ABP, denoted as LFPBP, reflects sympathetic activity while the low-frequency power of PI, denoted as LFPPI, in mice reflects parasympathetic activity (Janssen and Smits, 2002).

2.4. Baroreflex Sensitivity

Baroreflex sensitivity (BRS) was determined using both the sequence and spectral methods. In the sequence method, the changes in ABP and PI together in the same direction, either increasing or decreasing, for at least 3 consecutive beats were identified (Parati et al., 2000). The slope of the regression line between ABP and PI was taken to represent the estimate of the baroreflex sensitivity (BRSSEQ). The change in ABP or PI from the current beat to the next beat was not required to be greater than a minimum threshold in order to qualify as an increasing or decreasing sequence (Laude et al., 2009). PI was allowed to lag ABP by 0 to 3 beats. This follows from Laude et al. (2008) whose previous study showed that the cross-spectral analysis resulted in a phase lag in PI of 0.5 sec. Therefore, we allowed the PI to lag ABP by at most 3 beats as the delay of 3 beats was approximately close to 0.5 sec. Only the sequences with the goodness of fit (R2) of the regression line equal to or greater than 0.9 were considered in the analysis. In the spectral method, BRS was calculated as the square root of the ratio of PI and ABP power (Parati et al., 2000) in both low- and high-frequency regions and would be denoted as BRSLF and BRSHF, respectively.

2.5. Assessment of the Changes in Autonomic Responses over Time

2.5.1. Slopes of the linear trends

Linear trends were fitted to the time-series ABP, BRSSEQ, BRSLF, BRSHF, LFPBP and LFPPI of each mouse. The slope of the linear trend, reflecting the overall change in the signal over time, was tested for any difference among the exposure groups.

2.5.2. Diurnal amplitude

The time-series ABP, BRSSEQ, BRSLF, BRSHF, LFPBP and LFPPI were first smoothed out using a moving average filter with a span of 9 points (4.5 hours). The smoothed time-series of the IH groups were segmented into one-day periods. For each of these segments, the Cosinor fitting with a fixed frequency of 1 cycle/day was applied (Tong, 1976). The absolute amplitude of the fitted sinusoid, half of the peak-to-peak value, was then derived from each one-day segment. The reason why this analysis was not applied to the IA + saline and IA + glucose groups was due to the apparently pronounced ultradian rhythms (~12 hour cycle) in those groups while the diurnal rhythms of the IH + saline and IH + glucose groups were more evident.

2.5.3. Diurnal and ultradian power

After smoothing and linear trend removal, the time-series ABP, BRSSEQ, BRSLF, BRSHF, LFPBP and LFPPI were subjected to power spectral analysis. The diurnal power was the average power within the frequency range of 1 ± 0.3 cycles/day (average periodicity = 24 hours) and the ultradian power was the average power within the frequency range of 2 ± 0.3 cycles/day (average periodicity = 12 hours). To determine the relative strength of ultradian to diurnal rhythmicity, the ratio of the ultradian power to the diurnal power of each of the above time-series was computed.

2.6. Statistical Analysis

2.6.1. Two-way ANOVA

To statistically assess the effect of IH and hyperglycemia, two-way ANOVA was used to compare the slopes of the fitted linear trends and the ratios of the ultradian power to the diurnal power of ABP, BRSSEQ, BRSLF, BRSHF, LFPBP and LFPPI. The Holm-Sidak test was employed for post-hoc pairwise comparisons. If the normality or equal variance test failed, the data were first logarithm-transformed prior to performing the ANOVA calculations.

2.6.2. One-way Repeated Measures ANOVA

One-way repeated measures ANOVA was used to analyze the diurnal amplitude of ABP, BRSSEQ, BRSLF, BRSHF, LFPBP and LFPPI of the mice in the combined IH groups (IH + saline and IH + glucose) with duration of IH exposure (in days) being the repeated factor. Holm-Sidak post-hoc comparisons versus control, with the day-1 amplitude being treated as control, were conducted. If the normality or equal variance tests failed, one-way repeated measures ANOVA on ranks with Dunnett’s post-hoc test versus control was used instead.

3. Results

3.1. Changes in Blood Pressure

As the mice were becoming acclimated to the experimental routine, the ABP showed an overall decreasing trend over four days in all exposure groups, but the decrease was slower in the IH + saline and IH + glucose groups (Figure 1). ABP in the IA + glucose group decreased the most rapidly relative to the other exposure groups, with the rate of decrease being approximately three times larger relative to the IH + saline group. The difference in the mean ABP slopes between saline and glucose infusion was significant (p = 0.003). Post-hoc comparisons showed that the mean ABP slope in IA + saline was significantly less steep than from IA + glucose (p = 0.020), and ABP in IH+saline decreased less rapidly than IH+glucose (p = 0.035).

Figure 1. Beat-averaged blood pressure (ABP) of all exposure groups.

Figure 1

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Group median beat-averaged ABP time courses over the duration (4 days) of the experiment. (B) Each bar shows the mean of the slopes of the linear trends fitted to ABP. The error bar represents the standard error. * indicates pairwise difference, p < 0.05 (Holm-Sidak method).

3.2. Changes in Baroreflex Sensitivity

In contrast to the controls (IA + saline), the BRSLF of the mice in the other exposure groups decreased over the four days of exposure (Figure 2). Although the BRSLF in the control group showed a tendency to increase over time, this was not significantly different from zero (one-sample t-test, p = 0.23). The differences in mean BRSLF slopes between hypoxia levels (IA vs. IH) as well as between infusion levels (saline vs. glucose) were significant (p = 0.002 and p = 0.010, respectively). Pairwise comparisons showed that the BRSLF decreased faster in IA + glucose (p = 0.020) and IH + saline (p = 0.004) compared to IA + saline. The findings for BRSSEQ paralleled those for BRSLF.

Figure 2. Baroreflex sensitivity at low-frequency range (BRSLF) of all exposure groups.

Figure 2

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Group median BRSLF timecourses over the duration (4 days) of the experiment. (B) Each bar shows the mean of the slopes of the linear trends fitted to BRSLF. The error bar represents the standard error. (C) Each bar shows the mean of the slopes of the linear trends fitted to baroreflex sensitivity computed from sequence method (BRSSEQ). The error bar represents the standard error. * indicates pairwise difference, p < 0.05 (Holm-Sidak method).

3.3 Changes in Low-Frequency Blood Pressure oscillation amplitude

Exposure to IH acted to sustain sympathetic over-activity, as reflected by the much slower decrease in LFPBP in both IH groups compared to the IA groups (Figure 3). Two-way ANOVA confirmed this observation (p < 0.001). Pairwise comparisons showed that among the mice that were infused with saline, those that were exposed to IH had a slower decrease in LFPBP (p = 0.001). The same conclusion could be drawn from the glucose-infusion group (p = 0.008).

Figure 3. Low-frequency power of blood pressure (LFPBP) of all exposure groups.

Figure 3

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(A) Group median LFPBP time courses over the duration (4 days) of the experiment. (B) Each bar shows the mean of the slopes of the linear trends fitted to LFPBP. The error bar represents the standard error. * indicates pairwise difference, p < 0.05 (Holm-Sidak method).

3.4. Effect of Glucose Infusion

Hyperglycemia had a strong effect on reduction of parasympathetic activity, as reflected by LFPPI (Figure 4). LFPPI decreased faster over the 4 days in the IA + glucose group compared to IA + saline (p = 0.024). LFPPI in the IH + glucose group also decreased faster than the IH + saline group (p = 0.038).

Figure 4. Low-frequency power of pulse interval (LFPPI) of all exposure groups.

Figure 4

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(A) Group median LFPPI time courses over the duration (4 days) of the experiment. (B) Each bar shows the mean of the slopes of the linear trends fitted to LFPPI. The error bar represents the standard error. * indicates pairwise difference, p < 0.05 (Holm-Sidak method).

3.5. Changes in Amplitude of Diurnal and Ultradian rhythms

The IA + saline and IA + glucose groups displayed prominent ultradian rhythms (12-hour cycle oscillations) in the autonomic indices, whereas these oscillations were substantially less evident in the IH + saline and IH + glucose groups. In the IH groups, we focused our attention on how the diurnal amplitude changed over the experimental duration. We observed not only an overall decreasing trend in ABP over four days of exposure, but also a tendency for the diurnal amplitude to decrease over time. The LFPBP, which also showed the overall decreasing trend, had a significant reduction in the diurnal amplitudes over time (Figure 5). One-way repeated measures ANOVA showed that there was a significant difference among the mean diurnal amplitude of LFPBP in the combined IH group over the four days (p < 0.001). Post-hoc comparisons versus day-1 amplitude showed that the diurnal amplitudes of LFPBP in the IH + saline and IH + glucose groups from day-2 to day-4 were significantly lower than day-1 (p < 0.05).

Figure 5. Diurnal amplitude of low-frequency power of blood pressure (LFPBP) during exposure to intermittent hypoxia.

Figure 5

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(A) Group median LFPBP time courses over the duration (4 days) of the experiment. (B) Each bar shows the mean of the diurnal amplitudes of LFPBP. The error bar represents the standard error. * indicates pairwise difference, p < 0.05 (Holm-Sidak method).

In all the autonomic parameters that were analyzed in this study except for ABP, the ratio of ultradian power to diurnal power (Pultr/Pdiur) was higher in the IA + saline and IA + glucose groups compared to the IH + saline and IH + glucose groups. Thus, IH acted to suppress ultradian rhythmicity. Figure 6 illustrates this effect on all BRS indices. Two-way ANOVA showed that Pultr/Pdiur of BRSLF in IH groups was lower than that of IA groups (p < 0.001) and in glucose groups was lower than that of saline groups (p = 0.036). Pairwise comparisons showed that both IA + glucose and IH + saline had a lower ratio (Pultr/Pdiur) compared to IA + saline (p = 0.038 and p = 0.001, respectively). As was the case for BRSLF, Pultr/Pdiur of BRSHF in the IH groups was lower than that of IA groups (p < 0.001). As well, Pultr/Pdiur of BRSHF in the glucose groups was lower than that of saline groups (p = 0.041). Pairwise comparisons for BRSHF also showed similar results to BRSLF: both IA + glucose and IH + saline had Pultr/Pdiur compared to IA + saline (p = 0.015 and p < 0.001, respectively). In addition, IH + glucose had significantly lower Pultr/Pdiur compared to IA + glucose (p = 0.048). BRSSEQ, however, showed that there was a significant interaction between the hypoxia and infusion factors (p = 0.015). Pairwise comparisons showed that IH + saline had significantly lower Pultr/Pdiur compared to both IA + saline and IH + glucose (p < 0.001 and p = 0.029, respectively). The analysis of Pultr/Pdiur of LFPPI yielded findings similar to those for Pultr/Pdiur of BRS (Figure 7). The IA + glucose and IH + saline groups had lower Pultr/Pdiur of LFPPI compared to the IA + saline group (p = 0.016 and p = 0.002, respectively). The suppression of ultradian rhythmicity by IH was already apparent on the first day of IH exposure; there were no systematic changes in Pultr/Pdiur over the subsequent days of exposure.

Figure 6. Ratio of ultradian power (12-hour cycle) to diurnal power (24-hour cycle) of baroreflex sensitivity (BRS).

Figure 6

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(A) BRS by sequence method (BRSSEQ). (B) BRS at low-frequency range (BRSLF). (C) BRS at high-frequency range (BRSHF). Each bar shows the mean of the ratios of the ultradian powers to the diurnal powers of the BRS. The error bar represents the standard error. * indicates pairwise difference, p < 0.05 (Holm-Sidak method).

Figure 7. Ratio of ultradian power (12-hour cycle) to diurnal power (24-hour cycle) of low-frequency power of pulse interval (LFPPI).

Figure 7

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Each bar shows the mean of the ratios of the ultradian powers to the diurnal powers of the LFPPI. The error bar represents the standard error. * indicates pairwise difference, p < 0.05 (Holm-Sidak method).

4. Discussion

4.1. Issues related to the animal preparation

As alluded to in the Results section, there was a slow downward drift in ABP in all mice over the four day study that replicates our previous study using an identical protocol but without the IH exposure (Alonso et al., 2007). This downward drift in overall ABP was likely due to the continuing adaptation of the mice to the tethering system as well as acclimation to the daily routine of the experiment which involved blood sampling twice per day requiring opening of the environmental box (controlling for light and sound), which housed the customized cages containing the tethered mice. The faster drift downwards of the glucose-infused mice was potentially related to the vasodilating properties of glucose-mediated insulin release, which was higher in the glucose infused mice than saline-infused mice. Overall, any impact on baseline blood pressure from acclimation to tethering or the sampling schedule should be comparable among all four experimental groups.

Multiple behavioral and physiologic measures demonstrate that the mice remained healthy over the course of the four day interventional component of the study. Food intake increased over each of the three recovery days from surgery and was at normal levels throughout the subsequent four day study. Body weight at the beginning of the four day experiment was 23.8 ± 0.9 gm and was unchanged at 23.9 ± 0.8 gm at the completion of the study. Blood glucose and plasma insulin levels in the IA + saline control group were all in the normal range and did not change over time. The plasma corticosterone levels during the dark/active period for days 2, 3, and 4 during the interventional study were 34 ± 11, 14 ± 5, and 25 ± 10 ng/ml for the IA + saline control and 55 ± 16, 31 ± 10, and 27 ± 9 ng/ml in the IA + glucose group. Mean heart rate also remained relatively unchanged over the 4 days. IH exposure during the light/sleep period increased plasma corticosterone to ~150–200 ng/ml, but always returned to basal levels during room air exposure in the dark/active period. By way of comparison, a study by Jacobson et al. (2006) reported basal plasma corticosterone levels in catheterized mice of ~160 ng/ml at 5–7 days after surgery that increased to ~300 ng/ml in response to hypoglycemia. Another study in chronically instrumented mice reported a basal corticosterone level of ~80 ng/ml that increased to ~180 ng/ml in response to a restraint stress (Tjurmina et al., 2002). Thus, not only do our basal plasma corticosterone levels show a complete absence of stress and no change over the course of the four day intervention, they are considerably lower than previously reported levels in healthy chronically instrumented mice.

4.2 Cardiovascular indices of autonomic function

Cardiovascular variability indices such as blood pressure variability and HRV have been valuable markers of sympathetic and parasympathetic activities in both humans and animals. Similar to humans, the ABP and its variability in mice are predominantly controlled by the sympathetic branch of the autonomic nervous system and are reflective of sympathetic tone (Janssen et al., 2000). In humans, the LFP of HRV is under the influence of both the sympathetic and parasympathetic systems (Gehrmann et al., 2000; Taskforce, 1996), but in mice, this component is largely parasympathetic (Janssen and Smits, 2002). Also, in humans, the HFP of HRV is known to be largely modulated by the parasympathetic nervous system. However, in mice, the extent to which vagal cardiac modulation contributes to HFP is more controversial; for instance, Janssen et al. (2002) suggested that this component primarily reflects mechanical coupling of heart rate with respiration. Thus, unlike in humans (Just et al., 2000), the ratio of LFP to HFP of HRV in mice may not necessarily reflect sympathovagal balance.

We employed the sequence method as one of the ways to assess BRS. This method requires the identification of short time-segments in which beat-to-beat blood pressure and PI have to increase or decrease together. Due to the relatively low sampling rate of 200 Hz, the minimum change in the PI was automatically fixed by the sampling rate, making it more difficult to identify one sequence since the PI has to consecutively increase or decrease by 5 milliseconds at a time for at least three beats consecutively while the parallel change in blood pressure has to also occur simultaneously. This is in contrast to the suggestion by Laude et al. (2009) that the threshold for the minimum change in either the blood pressure or PI should not be imposed in order to optimize the sequence method in mice. As well, comparison of our results to other studies may be confounded by the fact that different frequency bands have been employed in the spectral analysis of both blood pressure and heart rate in mice (Baudrie et al., 2007).

4.3. Autonomic effects of intermittent hypoxia and hyperglycemia

So far, most studies have focused on the end result of the long-term exposure to IH, but few studies have looked at the early stage of development of autonomic dysfunction during chronic exposure to IH. Lin et al. (2007) showed that exposure to chronic IH resulted in a sustained impairment of baroreflex control of heart rate. As well, Dematteis et al. (2008) reported increased chemoreflex and depressed baroreflex activity as a result of exposure to chronic IH. Consistently, we found that IH suppressed the BRS and this suppression begins on the same day following the start of exposure to IH in mice. On each of the 4 days of the experiment, the mice exposed to IH had higher mean blood pressures than their corresponding counterparts exposed to IA. This is consistent with the findings in other studies on both mice (Campen et al., 2005; Kaufman et al., 2007), and humans (Cooper et al., 2005; Peppard et al., 2000) in which IH exposure, whether acute or chronic, resulted in systemic hypertension.

IH, the main characteristic of SDB, can also cause increases in sympathetic activity as reported by previous studies in both humans and animals. Direct measurements of sympathetic nervous activity from the peroneal nerve showed increased sympathetic nervous activity in SDB subjects (Narkiewicz et al., 1999) while other studies in dogs with simulated SDB (Brooks et al., 1997) and rats exposed to IH (Fletcher et al., 1999) showed elevated blood pressure but once the airway obstruction or IH was abolished, the blood pressure declined. Lai et al. (2006) investigated blood pressure variability in rats and found that LFPBP, reflecting sympathetic activity, increased after the exposure to IH for 5 days. In the current study, although all mice displayed a decreasing trend in LFPBP, the groups exposed to IH decreased less rapidly than those exposed to IA. We attribute this result in the IH-exposed mice to the additive effect of two opposing trends: a strong downward drift in sympathetic tone as all mice became acclimated to the daily routine of the experiment, and a countervailing upward trend in sympathetic activity produced by IH.

We found that parasympathetic activity decreased in mice exposed to hyperglycemia. While IH also seemed to cause reduction in vagal activity, this change did not achieve statistical significance. Rey el al. (2004) reported that chronic exposure to IH in cats resulted in reduction of the HF component of HRV, which reflects parasympathetic activity, similar to SDB patients. A population-based study investigating the association between multiple metabolic syndromes and HRV showed that insulin resistance, marked by increased fasting insulin, is associated with lower HF and LF components of HRV (Liao et al., 1998). Another study by Licht et al. (2010) showed similar result that decreased in parasympathetic activity, indicated by reduction in respiratory sinus arrhythmia, and increased sympathetic activity, indicated by reduction in pre-ejection period, are associated with increased likelihood of metabolic syndrome. Besides the fact that these studies were not performed in mice, their results are consistent with our findings.

Another interesting finding in our study is the disappearance of ultradian fluctuations of BRS and the low-frequency component of heart rate in mice exposed to IH. Previous studies showed that many ultradian rhythms are related to metabolic processes such as feeding cycle (Li et al., 1999; Wollnik, 1989). The abolished high-frequency 12-hour cycle in mice exposed to IH could in part be explained by the suppressed gustatory sensory perception by hypoxia.

4.4 Other factors

There were a number of factors in the study that were not controlled, which might have contributed independently to some of the autonomic and cardiovascular differences observed between IH and IA groups. Although we did control the light dark cycle in a 12hr:12hr format, the data have not been partitioned according to sleep state or activity as we did not have the animals instrumented for polysomnography. Body temperature was not recorded and to our knowledge there is no way to assess metabolic rate during periods of intermittent hypoxia using indirect calorimetry. However, we do know from a study in chronically instrumented rats by Hamrahi et al. (2001) that exposure to intermittent hypoxia has no effect on lowering body temperature over a period of several hours.

4.5. Implications of the findings

The findings arrived at in this study provide a useful picture of the early development of autonomic dysfunction following chronic “nocturnal” exposure to IH and how these autonomic changes are influenced also by concomitant hyperglycemia. In the murine model, the time courses for early development of insulin resistance and autonomic dysfunction appear to be quite similar. Thus, our findings do not resolve the question of whether changes in autonomic function occur first following IH exposure and that these lead to a reduction in insulin sensitivity. Extrapolation of our results to humans poses another challenge. Several prospective studies in humans have shown that the effect of IH on sympathetic over-activity is cumulative over time and that the cumulative effect occurs over a time course of several minutes. For example, Xie et al. (2000) found that exposing healthy young subjects to intermittent asphyxia over a period of 20 min led to sympathetic activation that continued even after the stimulus was removed. In another study (Cutler et al., 2004), exposure to intermittent hypoxic apnea over a 20 min duration led to prolonged sympathetic activation. The same results occurred regardless of whether these exposures occurred against a background of hypercapnia or isocapnia, thus confirming that the primary mediator for the increase in sympathetic activity was the IH. In two other studies (Leuenberger et al., 2005; Leuenberger et al., 2007), healthy young subjects exposed to repetitive hypoxic apneas for a total duration of 30 mins displayed, in the post-recovery period, a small and short-lasting increase in mean arterial blood pressure, accompanied by a more sustained and substantial increase in muscle sympathetic nerve activity. The aforementioned human studies did not include the determination of insulin sensitivity before and after IH exposure. However, it has been observed that insulin sensitivity can improve as rapidly as 48 hours after the start of CPAP treatment in patients with SDB (Harsch et al., 2004).

Taken together, the current evidence suggests that, in humans, changes in autonomic activity occur relatively rapidly with exposure to IH, whereas changes in insulin sensitivity take place at a somewhat slower pace. Thus, it is possible that some of the reduction in insulin sensitivity may have taken place as a consequence of the cumulative sympathetic over-activity. The findings from this study are consistent with this view; however, they do not exclude the possibility that exposure to IH could also directly lead to alterations in glucose metabolism, independent of preceding changes in autonomic control.

5. Conclusion

In summary, measurements of continuous ABP from a chronically instrumented murine model were used to investigate the effects of IH, occurring under conditions of euglycemia and hyperglycemia, on the early development of autonomic nervous system dysfunction. Lean animals were used in order to eliminate the confounding influence of obesity. On the basis of the indices of cardiovascular variability derived from these measurements, we conclude that IH acted to increase sympathetic activity, whereas hyperglycemia decreased parasympathetic activity. Exposure to both IH and hyperglycemia led to the largest reductions in baroreflex sensitivity. Our findings suggest that IH, combined with hyperglycemia, exerts progressively adverse effects on autonomic control independent of obesity.

Highlights.

  • A lean murine model was employed to track the development of autonomic dysfunction independent of obesity.

  • Exposure to intermittent hypoxia and continuous infusion of glucose or saline over 4 days reduced baroreflex sensitivity.

  • Intermittent hypoxia promoted sympathetic activity whereas hyperglycemia suppressed parasympathetic activity.

  • Ultradian (12-hour) rhythmicity was substantially suppressed in mice exposed to intermittent hypoxia.

  • Intermittent hypoxia combined with hyperglycemia progressively adversely affected autonomic control independent of obesity.

Acknowledgements

This work was supported in part by National Institutes of Health grants HL090451, EB001978 and HL077785.

Footnotes

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References

  1. Alonso LC, Yokoe T, Zhang P, Scott DK, Kim SK, O'Donnell CP, Garcia-Ocana A. Glucose infusion in mice: a new model to induce beta-cell replication. Diabetes. 2007;56:1792–1801. doi: 10.2337/db06-1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 2.Baudrie V, Laude D, Elghozi JL. Optimal frequency ranges for extracting information on cardiovascular autonomic control from the blood pressure and pulse interval spectrograms in mice. American journal of physiology. 2007;292:R904–R912. doi: 10.1152/ajpregu.00488.2006. [DOI] [PubMed] [Google Scholar]
  3. 3.Brooks D, Horner RL, Kozar LF, Render-Teixeira CL, Phillipson EA. Obstructive sleep apnea as a cause of systemic hypertension. Evidence from a canine model. J Clin Invest. 1997;99:106–109. doi: 10.1172/JCI119120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 4.Campen MJ, Shimoda LA, O'Donnell CP. Acute and chronic cardiovascular effects of intermittent hypoxia in C57BL/6J mice. Journal of applied physiology. 2005;99:2028–2035. doi: 10.1152/japplphysiol.00411.2005. [DOI] [PubMed] [Google Scholar]
  5. 5.Carlson JT, Hedner J, Elam M, Ejnell H, Sellgren J, Wallin BG. Augmented resting sympathetic activity in awake patients with obstructive sleep apnea. Chest. 1993;103:1763–1768. doi: 10.1378/chest.103.6.1763. [DOI] [PubMed] [Google Scholar]
  6. 6.Chasens ER, Weaver TE, Umlauf MG. Insulin resistance and obstructive sleep apnea: is increased sympathetic stimulation the link? Biol Res Nurs. 2003;5:87–96. doi: 10.1177/1099800403257088. [DOI] [PubMed] [Google Scholar]
  7. 7.Cooper VL, Pearson SB, Bowker CM, Elliott MW, Hainsworth R. Interaction of chemoreceptor and baroreceptor reflexes by hypoxia and hypercapnia - a mechanism for promoting hypertension in obstructive sleep apnoea. The Journal of physiology. 2005;568:677–687. doi: 10.1113/jphysiol.2005.094151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 8.Cutler MJ, Swift NM, Keller DM, Wasmund WL, Smith ML. Hypoxia-mediated prolonged elevation of sympathetic nerve activity after periods of intermittent hypoxic apnea. J Appl Physiol. 2004;96:754–761. doi: 10.1152/japplphysiol.00506.2003. [DOI] [PubMed] [Google Scholar]
  9. 9.Dematteis M, Julien C, Guillermet C, Sturm N, Lantuejoul S, Mallaret M, Levy P, Gozal E. Intermittent hypoxia induces early functional cardiovascular remodeling in mice. American journal of respiratory and critical care medicine. 2008;177:227–235. doi: 10.1164/rccm.200702-238OC. [DOI] [PubMed] [Google Scholar]
  10. 10.Dempsey JA, Veasey SC, Morgan BJ, O'Donnell CP. Pathophysiology of sleep apnea. Physiological reviews. 2010;90:47–112. doi: 10.1152/physrev.00043.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 11.Fletcher EC, Bao G, Li R. Renin activity and blood pressure in response to chronic episodic hypoxia. Hypertension. 1999;34:309–314. doi: 10.1161/01.hyp.34.2.309. [DOI] [PubMed] [Google Scholar]
  12. 12.Fletcher EC, Lesske J, Behm R, Miller CC, 3rd, Stauss H, Unger T. Carotid chemoreceptors, systemic blood pressure, and chronic episodic hypoxia mimicking sleep apnea. J Appl Physiol. 1992;72:1978–1984. doi: 10.1152/jappl.1992.72.5.1978. [DOI] [PubMed] [Google Scholar]
  13. 13.Gehrmann J, Hammer PE, Maguire CT, Wakimoto H, Triedman JK, Berul CI. Phenotypic screening for heart rate variability in the mouse. Am J Physiol Heart Circ Physiol. 2000;279:H733–H740. doi: 10.1152/ajpheart.2000.279.2.H733. [DOI] [PubMed] [Google Scholar]
  14. 14.Hamrahi H, Stephenson R, Mahamed S, Liao KS, Horner RL. Selected Contribution: Regulation of sleep-wake states in response to intermittent hypoxic stimuli applied only in sleep. J Appl Physiol. 2001;90:2490–2501. doi: 10.1152/jappl.2001.90.6.2490. [DOI] [PubMed] [Google Scholar]
  15. 15.Harsch IA, Schahin SP, Radespiel-Troger M, Weintz O, Jahreiss H, Fuchs FS, Wiest GH, Hahn EG, Lohmann T, Konturek PC, Ficker JH. Continuous positive airway pressure treatment rapidly improves insulin sensitivity in patients with obstructive sleep apnea syndrome. American journal of respiratory and critical care medicine. 2004;169:156–162. doi: 10.1164/rccm.200302-206OC. [DOI] [PubMed] [Google Scholar]
  16. 16.Iiyori N, Alonso LC, Li J, Sanders MH, Garcia-Ocana A, O'Doherty RM, Polotsky VY, O'Donnell CP. Intermittent hypoxia causes insulin resistance in lean mice independent of autonomic activity. American journal of respiratory and critical care medicine. 2007;175:851–857. doi: 10.1164/rccm.200610-1527OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 17.Ip MS, Lam B, Ng MM, Lam WK, Tsang KW, Lam KS. Obstructive sleep apnea is independently associated with insulin resistance. American journal of respiratory and critical care medicine. 2002;165:670–676. doi: 10.1164/ajrccm.165.5.2103001. [DOI] [PubMed] [Google Scholar]
  18. 18.Jacobson L, Ansari T, McGuinness OP. Counterregulatory deficits occur within 24 h of a single hypoglycemic episode in conscious, unrestrained, chronically cannulated mice. American journal of physiology. Endocrinology and metabolism. 2006;290:E678–E684. doi: 10.1152/ajpendo.00383.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 19.Janssen BJ, Leenders PJ, Smits JF. Short-term and long-term blood pressure and heart rate variability in the mouse. American journal of physiology. 2000;278:R215–R225. doi: 10.1152/ajpregu.2000.278.1.R215. [DOI] [PubMed] [Google Scholar]
  20. 20.Janssen BJ, Smits JF. Autonomic control of blood pressure in mice: basic physiology and effects of genetic modification. American journal of physiology. 2002;282:R1545–R1564. doi: 10.1152/ajpregu.00714.2001. [DOI] [PubMed] [Google Scholar]
  21. 21.Johansson C, Thoren P. The effects of triiodothyronine (T3) on heart rate, temperature and ECG measured with telemetry in freely moving mice. Acta Physiol Scand. 1997;160:133–138. doi: 10.1046/j.1365-201X.1997.00134.x. [DOI] [PubMed] [Google Scholar]
  22. 22.Just A, Faulhaber J, Ehmke H. Autonomic cardiovascular control in conscious mice. American journal of physiology. 2000;279:R2214–R2221. doi: 10.1152/ajpregu.2000.279.6.R2214. [DOI] [PubMed] [Google Scholar]
  23. 23.Kaufman CL, Kaiser DR, Steinberger J, Kelly AS, Dengel DR. Relationships of cardiac autonomic function with metabolic abnormalities in childhood obesity. Obesity (Silver Spring) 2007;15:1164–1171. doi: 10.1038/oby.2007.619. [DOI] [PubMed] [Google Scholar]
  24. 24.Lai CJ, Yang CC, Hsu YY, Lin YN, Kuo TB. Enhanced sympathetic outflow and decreased baroreflex sensitivity are associated with intermittent hypoxia-induced systemic hypertension in conscious rats. Journal of applied physiology. 2006;100:1974–1982. doi: 10.1152/japplphysiol.01051.2005. [DOI] [PubMed] [Google Scholar]
  25. 25.Laude D, Baudrie V, Elghozi JL. Applicability of recent methods used to estimate spontaneous baroreflex sensitivity to resting mice. American journal of physiology. 2008;294:R142–R150. doi: 10.1152/ajpregu.00319.2007. [DOI] [PubMed] [Google Scholar]
  26. 26.Laude D, Baudrie V, Elghozi JL. Tuning of the sequence technique. IEEE Eng Med Biol Mag. 2009;28:30–34. doi: 10.1109/MEMB.2009.934630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 27.Leuenberger UA, Brubaker D, Quraishi S, Hogeman CS, Imadojemu VA, Gray KS. Effects of intermittent hypoxia on sympathetic activity and blood pressure in humans. Auton Neurosci. 2005;121:87–93. doi: 10.1016/j.autneu.2005.06.003. [DOI] [PubMed] [Google Scholar]
  28. 28.Leuenberger UA, Hogeman CS, Quraishi S, Linton-Frazier L, Gray KS. Short-term intermittent hypoxia enhances sympathetic responses to continuous hypoxia in humans. J Appl Physiol. 2007;103:835–842. doi: 10.1152/japplphysiol.00036.2007. [DOI] [PubMed] [Google Scholar]
  29. 29.Li J, Thorne LN, Punjabi NM, Sun CK, Schwartz AR, Smith PL, Marino RL, Rodriguez A, Hubbard WC, O'Donnell CP, Polotsky VY. Intermittent hypoxia induces hyperlipidemia in lean mice. Circ Res. 2005;97:698–706. doi: 10.1161/01.RES.0000183879.60089.a9. [DOI] [PubMed] [Google Scholar]
  30. 30.Li P, Sur SH, Mistlberger RE, Morris M. Circadian blood pressure and heart rate rhythms in mice. Am J Physiol. 1999;276:R500–R504. doi: 10.1152/ajpregu.1999.276.2.R500. [DOI] [PubMed] [Google Scholar]
  31. 31.Liao D, Sloan RP, Cascio WE, Folsom AR, Liese AD, Evans GW, Cai J, Sharrett AR. Multiple metabolic syndrome is associated with lower heart rate variability. The Atherosclerosis Risk in Communities Study. Diabetes Care. 1998;21:2116–2122. doi: 10.2337/diacare.21.12.2116. [DOI] [PubMed] [Google Scholar]
  32. 32.Licht CM, Vreeburg SA, van Reedt Dortland AK, Giltay EJ, Hoogendijk WJ, DeRijk RH, Vogelzangs N, Zitman FG, de Geus EJ, Penninx BW. Increased sympathetic and decreased parasympathetic activity rather than changes in hypothalamic-pituitary-adrenal axis activity is associated with metabolic abnormalities. J Clin Endocrinol Metab. 2010;95:2458–2466. doi: 10.1210/jc.2009-2801. [DOI] [PubMed] [Google Scholar]
  33. 33.Lin M, Liu R, Gozal D, Wead WB, Chapleau MW, Wurster R, Cheng ZJ. Chronic intermittent hypoxia impairs baroreflex control of heart rate but enhances heart rate responses to vagal efferent stimulation in anesthetized mice. Am J Physiol Heart Circ Physiol. 2007;293:H997–H1006. doi: 10.1152/ajpheart.01124.2006. [DOI] [PubMed] [Google Scholar]
  34. 34.Narkiewicz K, Kato M, Phillips BG, Pesek CA, Davison DE, Somers VK. Nocturnal continuous positive airway pressure decreases daytime sympathetic traffic in obstructive sleep apnea. Circulation. 1999;100:2332–2335. doi: 10.1161/01.cir.100.23.2332. [DOI] [PubMed] [Google Scholar]
  35. 35.Parati G, Di Rienzo M, Mancia G. How to measure baroreflex sensitivity: from the cardiovascular laboratory to daily life. Journal of hypertension. 2000;18:7–19. [PubMed] [Google Scholar]
  36. 36.Peppard PE, Young T, Palta M, Skatrud J. Prospective study of the association between sleep-disordered breathing and hypertension. The New England journal of medicine. 2000;342:1378–1384. doi: 10.1056/NEJM200005113421901. [DOI] [PubMed] [Google Scholar]
  37. 37.Polotsky VY, Li J, Punjabi NM, Rubin AE, Smith PL, Schwartz AR, O'Donnell CP. Intermittent hypoxia increases insulin resistance in genetically obese mice. The Journal of physiology. 2003;552:253–264. doi: 10.1113/jphysiol.2003.048173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 38.Polotsky VY, Rubin AE, Balbir A, Dean T, Smith PL, Schwartz AR, O'Donnell CP. Intermittent hypoxia causes REM sleep deficits and decreases EEG delta power in NREM sleep in the C57BL/6J mouse. Sleep Med. 2006;7:7–16. doi: 10.1016/j.sleep.2005.06.006. [DOI] [PubMed] [Google Scholar]
  39. 39.Punjabi NM, Shahar E, Redline S, Gottlieb DJ, Givelber R, Resnick HE. Sleep-disordered breathing, glucose intolerance, and insulin resistance: the Sleep Heart Health Study. American journal of epidemiology. 2004;160:521–530. doi: 10.1093/aje/kwh261. [DOI] [PubMed] [Google Scholar]
  40. 40.Punjabi NM, Sorkin JD, Katzel LI, Goldberg AP, Schwartz AR, Smith PL. Sleep-disordered breathing and insulin resistance in middle-aged and overweight men. American journal of respiratory and critical care medicine. 2002;165:677–682. doi: 10.1164/ajrccm.165.5.2104087. [DOI] [PubMed] [Google Scholar]
  41. 41.Rey S, Del Rio R, Alcayaga J, Iturriaga R. Chronic intermittent hypoxia enhances cat chemosensory and ventilatory responses to hypoxia. The Journal of physiology. 2004;560:577–586. doi: 10.1113/jphysiol.2004.072033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 42.Somers VK, Dyken ME, Clary MP, Abboud FM. Sympathetic neural mechanisms in obstructive sleep apnea. J Clin Invest. 1995;96:1897–1904. doi: 10.1172/JCI118235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 43.Taskforce. Heart rate variability. Standards of measurement, physiological interpretation, and clinical use. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. European heart journal. 1996;17:354–381. [PubMed] [Google Scholar]
  44. 44.Tjurmina OA, Armando I, Saavedra JM, Goldstein DS, Murphy DL. Exaggerated adrenomedullary response to immobilization in mice with targeted disruption of the serotonin transporter gene. Endocrinology. 2002;143:4520–4526. doi: 10.1210/en.2002-220416. [DOI] [PubMed] [Google Scholar]
  45. 45.Tong YL. Parameter estimation in studying circadian rhythms. Biometrics. 1976;32:85–94. [PubMed] [Google Scholar]
  46. 46.Wollnik F. Physiology and regulation of biological rhythms in laboratory animals: an overview. Laboratory animals. 1989;23:107–125. doi: 10.1258/002367789780863538. [DOI] [PubMed] [Google Scholar]
  47. 47.Xie A, Skatrud JB, Crabtree DC, Puleo DS, Goodman BM, Morgan BJ. Neurocirculatory consequences of intermittent asphyxia in humans. J Appl Physiol. 2000;89:1333–1339. doi: 10.1152/jappl.2000.89.4.1333. [DOI] [PubMed] [Google Scholar]
  48. 48.Yokoe T, Alonso LC, Romano LC, Rosa TC, O'Doherty RM, Garcia-Ocana A, Minoguchi K, O'Donnell CP. Intermittent hypoxia reverses the diurnal glucose rhythm and causes pancreatic beta-cell replication in mice. The Journal of physiology. 2008;586:899–911. doi: 10.1113/jphysiol.2007.143586. [DOI] [PMC free article] [PubMed] [Google Scholar]

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