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1Pulmonary and Sleep Research Laboratory, Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts; 2Laboratoire du Sommeil, Laboratoire HP2 (Hypoxie PathoPhysiologie), Centre Hospitalier Universitaire de Grenoble, France
Submitted 10 May 2004 ; accepted in final form 17 September 2004
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ABSTRACT |
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chemosensitivity; vascular resistance; pathophysiology
Increasing evidence suggests that exposure to hypoxia may also influence sympathetic activity and arterial pressure after termination of the exposure. Morgan et al. (29) and others (33) documented increased muscle sympathetic nerve activity (MSNA) that persists up to an hour after termination of a 20-min hypoxic exposure. The mechanism for this persistent sympathoexcitation remains obscure. Morgan et al. (29) postulated that central potentiation of sympathetic activity might occur similar to long-term facilitation of ventilation after episodic hypoxia. Other authors demonstrated that acute intermittent hypoxia produced sympathoexcitation (9, 36). The influence of the pattern of hypoxic exposure on this sustained sympathoexcitation has not been directly compared, however.
To investigate whether the pattern of hypoxia influences sympathetic activity and hemodynamics in the postexposure period, we measured sympathetic activity (peroneal microneurography) and hemodynamics [forearm blood flow (FBF), arterial pressure, heart rate] in normal volunteers on two occasions during and after 2 h of either sustained or cyclic intermittent hypoxia.
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METHODS |
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Ten healthy, nonsmoking, normotensive subjects, free of vasoactive medications, completed the study. All subjects underwent a routine history and physical examination to exclude cardiopulmonary or neurological disease before giving written informed consent. To eliminate possible confounding hormonal effects on cardiovascular function, female subjects were studied during the week after menses and all tested negative for pregnancy (
-human chorionic gonadotrophin hormone urinary test). This protocol was reviewed and approved by the Institutional Review Board at the Beth Israel Deaconess Medical Center.
General Procedures
Subjects were studied in the supine position at the same time of day while the room temperature was maintained at 24°C. Respiratory and cardiovascular variables and sympathetic nerve activity were recorded continuously, digitized at 250 Hz and stored on a computer. The data were analyzed offline with signal-processing software (Windaq, Dataq Instruments, Akron, OH). We performed all measurements, except the progressive isocapnic hypoxic ventilatory response, in two 10-min periods before and after exposure. Exposures were for 2 h. Each exposure started when subjects decreased their arterial oxygen saturation (SaO2) below 85% and stopped 2 h later. The return to room air at the end of exposure required no more than 1 min to reach an SaO2 of 98%.
Experimental Protocols
Subjects were studied on 2 days, 
1 mo apart. On the day of the study, the subjects were exposed to 2 h of either intermittent or continuous hypoxia (Fig. 1) with the order determined randomly. Only one exposure occurred each day. Four of 10 subjects received intermittent hypoxia first. After setup each day, subjects rested for 15–30 min, until all measurements were stable for at least 5 min, after which continuous baseline recordings were made of sympathetic activity, arterial pressure, and cardiac electrical activity on room air for 10 min. Five to six plethysmographic measures of FBF were performed on two occasions during this baseline period. After this 10-min period of baseline recording, we measured the ventilatory response to hypoxia using a modification of the Rebuck and Campbell (32) rebreathing method. We then exposed the subjects to continuous or intermittent hypoxia. Subjects breathed through a leak-free face mask (8940 Series; Hans Rudolph, Kansas City, MO) to which a two-way valve was connected (2600 Series; Hans Rudolph). A three-way valve was attached to the inspiratory tubing to connect the subject to either a 30-liter respiratory bag or room air. For continuous hypoxia, the respiratory bag was filled with a gas mixture containing 9% oxygen balanced with nitrogen, so that SaO2 fell to 85% during the continuous hypoxia exposures. Nitrogen was added to the inspired gas as necessary to maintain SaO2 near 85%. For intermittent hypoxia, the respiratory bag was filled with 100% nitrogen and the subject breathed alternately from the bag and from the room. The number of breaths of pure nitrogen and the number of room air breaths were determined individually for each subject, so as to allow a 10% fluctuation in SaO2 with a nadir at 85% (range: 6–10 pure nitrogen respiratory breaths alternating with 3–4 room air breaths). This produced 30–40 drops in SaO2 per hour. Because no carbon dioxide was added during exposure, both hypoxic exposures may be considered poikilocapnic hypoxia. After a 2-h exposure, subjects were returned to room air breathing, and all measurements, cardiovascular and FBF as well as MSNA and peripheral hypoxic chemosensitivity, were continued for 10 min after SaO2 returned to baseline. We then again performed the rebreathing isocapnic hypoxic ventilatory response testing.
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Respiratory variables.
Subjects breathed through a leak-free face mask (8940 Series; Hans Rudolph) to which a two-way valve was connected (2600 Series; Hans Rudolph). To measure the ventilatory response to hypoxia, subjects breathed from a closed circuit connected to a 7-liter bag-in-box. The box was connected to a 10-liter Wedge Spirometer (Med Science, St. Louis, MO). Linear displacement of the spirometer was recorded continuously and is proportional to volume. Oxygen saturation was monitored with a pulse oximeter (Biox model 3740; Ohmeda, Louisville, KY). End-tidal carbon dioxide was measured using an infrared gas analyzer (model 17630; Vacu-med, Ventura, CA). Subjects breathed from a mouthpiece wearing noseclips. The circuit was filled with calibrated gas made up of 24% O2-7% CO2 balanced with N2 such that the bag volume is 60% of the subject's vital capacity (VC) plus 1 liter. Carbon dioxide was removed as necessary from the circuit by directing a variable amount of the flow through a scrubber to maintain isocapnia. After the subject breathed on the circuit for 1 min, N2 was added to increase the bag volume to 1 liter above VC to hasten the decrease in oxygen saturation. When SaO2 decreased to 92%, oxygen was added to the circuit at 0.1–0.2 l/min through a pediatric flowmeter to allow precise control of the rate of fall of saturation. Oxygen flow was adjusted so that 2 min of data could be collected at three SaO2: 90%, 85%, and 80%. Expiratory tidal volume was obtained by integration of the flow signal. Breath-by-breath breath frequency was obtained by the ratio 1/Ttot, where Ttot is the duration of each respiratory cycle. Breath-by-breath exhaled minute ventilation (
E) was calculated by multiplying tidal volume and breath frequency. A linear correlation was used to obtain the slope of the SaO2 and E relationship.
Cardiovascular variables. Heart rate was taken from the electrocardiogram. Right arm arterial pressure was measured at 5-min intervals using an automated arm-cuff sphygmomanometer (Dinamap Model, Critikon, Tampa, FL) as well as continuous beat-by-beat measurements by digital photoplethysmography using the Portapress device (TNO-Institute of Applied Physics Biomedical Instrumentation, Amsterdam, The Netherlands).
FBF. Blood flow was measured in the left forearm by venous occlusion plethysmography (EC6 plethysmography, Hokanson, Bellevue, WA) using mercury-in-Silastic strain gauges. The arm was placed in a passive position above the level of the left atrium. The strain gauge was placed at the midpoint of the forearm with a distally placed occlusion cuff and a proximal venous occlusion cuff. Before data collection, a series of occlusions was performed to determine the venous occlusion pressure that resulted in the steepest slope of the arterial inflow curve. This typically yielded venous pressures of 45–50 mmHg. The wrist arterial occlusion cuff was inflated to 200 mmHg. After 1 min, the collecting cuff positioned above the elbow was rapidly inflated above venous pressures for 8 s every 16 s. An average of four to six flow measurements was used in the computation of the results at each time point. FBF is expressed in milliliters per 100 milliliters of limb tissue per minute. Forearm vascular resistance (FVR) was obtained by dividing mean arterial pressure (MAP) by FBF.
Muscle sympathetic nerve activity. We obtained nerve recordings using standard tungsten microelectrodes inserted into the peroneal nerve posterior into the popliteal area after localization by surface stimulation. Signals were filtered, amplified, and full-wave rectified. The rectified signal was integrated for display on an oscilloscope and for recording (Nerve Traffic Analyzer, model 662c-3, University of Iowa, Bioengineering Dept., Iowa City, IA). Electrode position in muscle fibers was confirmed by pulse synchronous bursts of activity occurring 1.2–1.4 s after the QRS complex, reproducible activation during the second phase of the Valsalva maneuver, elicitation of afferent nerve activity by mild muscle stretching, and the absence of response to startle. Sympathetic bursts were identified using a specific algorithm described by Hamner and Taylor (19) using Matlab software (The Mathworks, Natick, MA). For purposes of quantification, MSNA was reported in 5-min periods and expressed as burst frequency (bursts/min) and average activity per minute (AUI/min).
Data Analysis
We averaged nerve activity parameters over 5-min windows of data collection at baseline, during the hypoxic exposure, and during recovery from the exposure. Heart rate and MAP were averaged over the corresponding time intervals during which plethysmographic forearm flow measurements were made.
Baseline values were compared from the two different trials and pre- to postexposure with a two-tail distribution paired t-test. Except where otherwise noted, data are reported as means ± SE in the text, tables, and figures. P values <0.05 were considered statistically significant. When changes approached significance, the sample size calculation necessary to accept the null hypothesis with 80% confidence was reported as an illustration of the trend.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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No changes occurred after either exposure in peripheral chemosensitivity as assessed by the slopes of the linear relationship between e and SaO2 during progressive isocapnic hypoxia (Table 2). Nor was there any change in room air ventilation after either exposure.
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We found no difference in baseline MAP and heart rate between exposures. Both exposures led to an increase in MAP that was evident on return to room air (Fig. 2A); however, this increase only reached significance after the continuous hypoxic exposure (P < 0.01 for continuous; P = 0.194 for cyclic hypoxia, sample size for significance: 28). MAP was lower after the cyclic compared with the continuous hypoxic exposure; however, this difference did not reach significance (P = 0.07, sample size for significance: 24). No significant change occurred in heart rate after either exposure (Fig. 2B). Heart rates were 64.9 ± 7.2 heart beats/min and 68.0 ± 5.2 heart beats/min before and 69.3 ± 9.6 heart beats/min and 70.0 ± 9.1 heart beats/min after continuous and cyclic hypoxic exposures, respectively, and these were not different between themselves.
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MSNA exhibited a significant increase after continuous hypoxic exposure (Fig. 4). This reached significance both in bursts per minute and in average activity per minute (Table 3); however, MSNA did not exhibit a significant increase after cyclic hypoxia. During recovery, MSNA activity was lower after cyclic hypoxia compared with sustained hypoxia; however, this did not reach significance (burst frequency P = 0.129, total activity per minute P = 0.357).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The cardiovascular response during an exposure to hypoxia has been studied extensively (11, 12, 17, 18, 21, 26, 27, 29, 31, 35). Sympathetic and cardiovascular changes after termination of a hypoxic exposure remain less well defined, however. Morgan and colleagues (29, 36, 37) were among the first to describe sustained sympathoexcitation during room air breathing after a 20-min exposure to moderate (SaO2 80%) sustained hypoxia. Exposing normal volunteers to both continuous (37) and intermittent (36) hypoxia, these authors documented increases in MSNA up to 180% of baseline for up to 1 h after termination of the exposure. In these studies, the increase in sympathetic activity occurred with no change in arterial pressure (36, 37). Morgan and colleagues used brief exposures of human volunteers to hypoxia, but others have shown that exposure of rats to cyclic intermittent hypoxia for 8 h/day for 14–35 days also results in sustained sympathoexcitation and increased arterial pressure even after restoration of normoxia (15). These studies, designed to mimic the cyclic changes in oxygen saturation experienced during sleep in patients with sleep apnea (4), indicate that intermittent hypoxia may have substantial effects on sympathetic activity and arterial pressure. Although not studied as extensively, several studies suggest that continuous exposures to hypoxia of days to weeks also result in sustained sympathoexcitation and arterial hypertension (6, 20). Thus, although we are not aware of any prior direct comparisons of sympathetic activity and hemodynamics after continuous and cyclic intermittent hypoxia, several studies suggest that these different patterns of hypoxic exposure result in qualitatively similar cardiovascular responses.
In contrast, a number of studies indicate that ventilatory responses to cyclic and continuous hypoxia are different. Continuous hypoxic exposure in humans produces a respiratory plasticity that varies in the time domain (30). After an initial abrupt ventilatory increase to 159% of baseline during an isocapnic hypoxic exposure, ventilation decreases so that after a 20-min exposure, ventilation is only 126% above the baseline value, a phenomenon termed "roll off" (13). Ventilation then remains stably elevated during continued hypoxic exposure for a variable period. During this time, if the hypoxic exposure is stopped, ventilation returns almost immediately to the prior room air baseline and the ventilatory response to hypoxia is not altered. However, longer hypoxic exposures, lasting hours (16, 22) or days (10), result in a further increase in ventilation, which is termed acclimatization. In contrast to the acute hypoxic ventilation increase, acclimatization outlasts the exposure, as room air breathing and hypoxic ventilatory response are augmented compared with preexposure (10). This leads to hyperventilation with PaCO2 below the baseline normoxic level. Elegant studies, primarily by Bisgard (5), indicate that acclimatization is dependent on the peripheral chemoreceptor and is associated with an increase in peripheral chemoreflex gain. The mechanism for acclimatization is thought to be altered neuromodulation of the carotid chemoreceptor with endothelin (8), angiotensin (24), and other substances (5) believed to be contributors. Although most studies have employed continuous exposures to hypoxia or altitude to induce acclimatization, recently evidence has emerged that intermittent exposure of as little as 4 h/day for 5 days/wk will also induce acclimatization in normal volunteers (23).
A totally distinct mechanism is thought to be involved in LTF of ventilation. First, the stimulus that is required to produce LTF involves episodic stimulation of respiratory centers through hypoxic exposure or direct electric stimulation of the carotid sinus nerve; classically, a sequence of 5 min of exposure followed by 5 min recovery, repeated three times (30). As the stimuli are different, the neuropathways and mediators are also different, with LTF depending on serotonergic facilitation of phrenic premotor neurons (28), which elicit brain-derivative neurotrophic factor in the spinal cord neurons (3).
In this study, we proposed to compare sympathetic and hemodynamic responses to continuous and cyclic intermittent hypoxia. We employed two 10-min periods before and after exposure to perform all measurements except for the progressive isocapnic hypoxic ventilatory response (HVR). Because this was performed after the 10-min period of measurement, the HVR had no effect on MSNA measurement. Concerning the relation between the first HVR and the postexposure measurement, we cannot totally rule out that the HVR might affect MSNA during recovery; however, the short exposure to isocapnic hypoxia (<5 min) and the temporal position of this exposure (before 2 h of sustained or cyclic hypoxia) mitigates against a significant modulation of postexposure MSNA activity. Moreover, in a recent study Ainslie and Poulin (1) reported no effect of successive acute hypoxic exposure challenges on ventilatory response and hemodynamic parameters including cerebral flow. One issue in any such study is how to match exposures to the two different patterns of hypoxia. Alternatives include matching the duration of the exposure, matching the nadir SaO2, matching the mean SaO2, and matching the time at nadir SaO2 in the cyclic exposure to the duration of the continuous exposure. All of these paradigms have advantages and disadvantages. We chose in our protocol to match total duration of the exposure and to attempt to match the nadir SaO2. Despite our attempts, however, we created lower nadir values during cyclic hypoxia. In our protocol, mean oxygen saturation was different between the two exposures with sustained hypoxia exhibiting a lower mean SaO2 than cyclic hypoxia. By design, the range of variation was greater during cyclic hypoxic exposure compared with sustained hypoxia. Possibly as a consequence of the lower mean SaO2, cyclic exposure appeared to produce smaller changes in sympathetic activity and hemodynamics compared with sustained continuous hypoxia. However, it is not known if the sympathetic/hemodynamic response after hypoxia depends more on the mean SaO2 value, the total duration, or the pattern of hypoxic exposure.
One argument that might be relevant in quantifying the severity of an hypoxic stimulus is the work done by Edmunds and Marshall (14). These authors demonstrated that during the onset of hypoxia (5-min periods) oxygen delivery was not altered until a hypoxic threshold was reached, because flow regularly increased with the severity of the exposure. Thus alterations in blood flow and oxygen delivery may decrease the variability in peripheral oxygen tissue concentration during cyclic hypoxia and minimize the effects of small differences in hypoxic exposure levels.
Another factor that might be relevant to the differences we observed between cyclic and sustained hypoxic exposures is the level of ventilation during the exposures. Because sustained hypoxia was a more profound ventilatory stimulus, this exposure is likely to produce lower carbon dioxide levels compared with cyclic hypoxia; these lower CO2 levels might, in turn, decrease sympathetic activity. In addition, the effect of greater ventilation, through volume feedback, might be further expected to reduce SNA during and after sustained hypoxia. Because we observed greater increases in MSNA after sustained, as opposed to cyclic, hypoxia, the effect of CO2 and lung volume would be expected to reduce rather than enhance the differences we observed.
In our study, neither of the two exposures induced changes in peripheral ventilatory control as assessed by the isocapnic hypoxic ventilatory response performed before and after exposure. This was expected as, to our knowledge, no study had demonstrated acclimatization before 8 h of hypoxia in humans (22), but was crucial to investigate, as an increase in carotid chemosensitivity would be likely to play a role in any sympathetic increase occurring after chronic hypoxic exposure (15). Despite the absence of evidence for increased peripheral chemosensitivity, MSNA increased after continuous hypoxia but not cyclic hypoxia. This remained true whether MSNA was expressed as burst frequency or as average activity per minute. We cannot rule out a change in MSNA after cyclic hypoxia, as there was a slight increase, although the difference did not reach significance. A change of this amplitude would require a sample size of 16 subjects to reach significance with 80% confidence. Possibly a longer exposure to cyclic hypoxia or a nadir to lower SaO2 levels would have produced a significant response with the sample we studied. Interestingly, despite a lower SaO2 nadir in our study, our results appear to contradict those of Cutler et al. (9), who showed that a cyclic hypoxic stimulus induced persistent MSNA increase after a 20-min exposure.
Several prior works reported increases in arterial blood pressure after hypoxic stimuli of different durations, such as one night (2) and 5 wk (20). In our protocol, the increase in arterial blood pressure reached significance after 2 h of sustained hypoxia but not after 2 h of cyclic hypoxia. Taking into account that the increase in MSNA was only significant for sustained hypoxia makes us believe that the increase in arterial blood pressure is likely to be driven by the increase in sympathetic tone. The increase in arterial pressure makes the sustained sympathoexcitation even more significant, however, because baroreflex mechanisms might be expected to ameliorate any sympathetic response. Interestingly, neither continuous nor cyclic hypoxia administered for 20 min produced any change in arterial pressure (9, 29, 37).
As our laboratory previously reported after 20 min of a sustained hypoxic exposure (34), 2 h of continuous hypoxia were followed by an increase in FBF. Despite a similar increase after cyclic hypoxia, this change did not achieve significance (P = 0.118) with the sample we studied. Resistance did not change significantly after either cyclic or continuous hypoxia. We cannot account for the lack of change in resistance despite the increase in sympathetic activity. The findings are consistent, however, with persistent release of an endogenous vasodilator similar to the situation after a 20-min exposure (34).
Although the increase in MSNA during an exposure to hypoxia is thought to be chemoreceptor dependent (25), the mechanisms that account for sympathoexcitation after a sustained hypoxic exposure are not well understood. It has been well established that prolonged hypoxia produces an increase in peripheral chemosensitivity that outlasts the exposure and might count, in part, for the sympathetic increase; however, previous studies on short hypoxic exposure (9, 29, 34, 37) as well as our study failed to identify a change in hypoxic ventilatory response that would suggest an increase in peripheral chemosensitivity. Impairment of the central integration of afferent information has been proposed, but this remains difficult to explore (29, 37). Hansen and Sander (20) reported increases in MSNA occurring after a 4-wk altitude exposure. Looking further into the mechanism by which MSNA was increased, they demonstrated that neither saline bolus nor hyperoxia were able to normalize MSNA. In fact, when volume expansion was combined with hyperoxia, MSNA still remained elevated. These findings were interpreted as suggesting that neither peripheral baroreflex unloading nor peripheral chemoreflex stimulation were sufficient or explain the elevation in sympathetic activity. Of course, the increase in MSNA may be due to redundant factors with elevated central sympathetic gain also contributing to the elevation observed. As noted above, our laboratory recently provided evidence that 20 min of continuous hypoxia produces a persistent release of an endogenous vasodilator after the exposure, which might engage the arterial baroreflex, and through this, increase sympathetic outflow (34). Arguing against a baroreflex-mediated increase in sympathetic activity, however, is the increase in arterial pressure. One possible way to reconcile these findings is baroreflex resetting. During a short hypoxic exposure baroreflex resetting occurs, permitting a higher heart rate and MSNA for the same blood pressure (18).
In conclusion, our study compared the hemodynamic responses to cyclic intermittent and continuous hypoxic exposures of 2-h duration. We found that continuous, but not cyclic, exposures produced increased sympathetic activity and arterial pressure. We speculate that the hemodynamic and sympathetic responses to hypoxia depend more on the magnitude of the hypoxic exposure than on the pattern of the exposure.
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FOOTNOTES |
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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.
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, Data from the cyclic intermittent hypoxic exposure; 
, values obtained from the sustained hypoxic exposure. Although this did not reach significance, lower resistances occurred after both exposures. This is particularly interesting, as an increase in muscle sympathetic activity suggests that this increase may compensate for hypoxia-induced vasodilation.