This study was approved by the human ethics committee of the University of Toronto and conforms to the Declaration of Helsinki. A group of seven healthy volunteers (5 male, 2 female) gave their informed consent after the experimental protocols and possible risks had been explained. The volunteers were non-smokers taking no medication, and with no history of cardiorespiratory diseases. Their mean ±s.d. age, height and body mass were 23.7 ± 3.1 years, 174.2 ± 4.7 cm and 65.0 ± 3.7 kg, respectively.
Each volunteer was familiarised with all procedures by performing trial runs on each set of apparatus during an introductory session. Approximately 1 week later, they began 14 consecutive days of exposures to air or hypoxia (assigned pseudorandomly). Volunteers were seated during the ≈1 h testing sessions which began with a 5 min rest, followed by the first modified rebreathing test. After a 10 min rest, volunteers were exposed to hypoxia or air for 20 min, and immediately afterwards undertook the second modified rebreathing test. The same testing schedule was repeated after a minimum of 1 month with the alternate exposure to hypoxia or air in a pseudorandom crossover design. During the quiet periods of these experiments volunteers usually spent their time reading, sitting quietly or listening to the radio.
The modification of Read's original rebreathing test (Read, 1967), first described by Casey et al. (1987) and Duffin & McAvoy (1988), has recently been updated (Mohan & Duffin, 1997), as well as the interpretation of its results (Duffin et al. 2000). In its hyperoxic form the modified rebreathing test measures central chemoreflex sensitivity equivalent to those measured using Read's technique and the end-tidal forcing technique (Mohan et al. 1999).
Two modifications are made to Read's rebreathing technique. First, rebreathing is preceded by 5 min of hyperventilation to lower stores of carbon dioxide, thereby allowing the chemoreflex threshold to be discerned as carbon dioxide rises through it. This modification also permits measurement of the subthreshold ventilation independent of the chemoreflexes, ‘basal’ ventilation estimating the ‘wakefulness drive’ (Fink, 1961).
In the second modification, iso-oxia is maintained during rebreathing by supplying oxygen to the rebreathing bag, so that both hyperoxic (150 mmHg) and hypoxic (50 mmHg) ventilatory responses to carbon dioxide can be examined. Comparison of hypoxic and hyperoxic measurements determine if changes occurred in peripheral or central chemoreflexes or both, making assumptions discussed in Duffin et al. (2000). The daily pairs of modified rebreathing tests before and after the exposures were either both hypoxic (50 mmHg) or both hyperoxic (150 mmHg); the choice was alternated between days so that over the 14 day period each volunteer undertook seven pairs of each type.
Each rebreathing test consisted of 5 min of hyperventilation, during which volunteers were coached to maintain an end-tidal partial pressure of carbon dioxide between 19 and 25 mmHg. The rebreathing bag initially contained oxygen at the desired iso-oxic partial pressure, and carbon dioxide at a partial pressure of about 42 mmHg. Rebreathing began at the end of expiration and was followed by three deep breaths, producing rapid equilibration of carbon dioxide partial pressures in the bag, lungs and arterial blood to that of mixed venous blood. The equilibration was verified by observation of a plateau in end-tidal carbon dioxide partial pressure, and was a prerequisite for continuing the test. Rebreathing was terminated either when ventilation exceeded 100 l min−1 or the end-tidal partial pressure of carbon dioxide exceeded 60 mmHg, although some volunteers, females and small males, had tests terminated at lower levels of ventilation because of discomfort.
During rebreathing, the volunteers wore nose-clips and breathed through a mouthpiece connected to one side of a wide-bore Y-valve (Collins P-319, 80 ml dead space) that permitted switching from room air to the rebreathing bag. The 5 l rebreathing bag was enclosed in a rigid container with a 50 mm diameter tube connected to a dry rolling-seal spirometer (Spiroflow model 130, Morgan) to monitor breath-by-breath changes in ventilation. A sample flow of 90 ml min−1 was drawn from the mouthpiece side of the Y-valve (gas sample tube UD 5037, Brüel and Kjær) for end-tidal monitoring (anaesthetic gas monitor type 1304, Brüel and Kjær,) of carbon dioxide partial pressure (resolution of 1 mmHg) and oxygen partial pressure (resolution of 4 mmHg), and returned to the bag during rebreathing. Iso-oxia was maintained by a flow of oxygen to the rebreathing bag side of the Y-valve under computer control.
Hypoxic or air exposures
During the 20 min exposure to hypoxia or air, isocapnia at eucapnic partial pressures was maintained using a method described by Sommer et al. (1998). Volunteers wore a facemask (8930 series, Hans Rudolph Inc.) attached to a non-rebreathing valve (8932 T-shape 2WNRBV, Hans Rudolph Inc.). Expired volume was measured from the outlet of the non-rebreathing valve with a dry gas meter (Parkinson-Cowan), and respired gases were sampled at the mouthpiece as for the rebreathing test.
At first, the inspired gas was supplied from a 3 l reservoir bag filled continuously from a compressed gas cylinder containing room air; with the flow manually set to match each volunteer's average resting ventilation during the first 5 min. This average flow was supplied from a compressed gas cylinder containing room air for the air exposures, but containing 10 % oxygen for the hypoxic exposures. If inspired flow exceeded that supplied, the reservoir bag emptied and a demand valve (Mares, Beta, modified to reduce its opening pressure) supplied flow from another compressed gas cylinder containing 6 % carbon dioxide (approximately mixed venous carbon dioxide partial pressure) and 10 % oxygen. This arrangement maintained isocapnia at eucapnic partial pressures despite increases in ventilation produced by hypoxia.
A 16-bit analog to digital converter (National Instruments, AT-MIO-16XE-50) digitised the analog signals for on-line computer analysis using specially written software (LabVIEW, National Instruments). The software calculated tidal volumes, inspiratory and expiratory times, ventilation, and end-tidal partial pressures of carbon dioxide and oxygen on a breath-by-breath basis. The volume changes and partial pressures of oxygen and carbon dioxide were also written on a chart recorder (Lineacorder mark VII WR 3101, Graphtec) to monitor the initial rebreathing equilibration. This measurement system was calibrated before each experimental session using gases of known concentrations (room air and a primary standard mixture of 5 % carbon dioxide, 10 % oxygen), and a calibrated 1 l volume syringe (model 5540, Hans Rudolph Inc.).
The data accumulated during the hypoxic and air exposures were analysed using a specially designed spreadsheet (Microsoft Excel). For display purposes, the breath-by-breath ventilation and end-tidal partial pressures of carbon dioxide and oxygen for each exposure were first averaged over 60 s to allow mean responses for all volunteers to be calculated for any day of exposure. For statistical comparisons, these 60 s averages were then combined to calculate means for all volunteers over 5 min epochs corresponding to the 5 min prior to hypoxic or air exposure (T1) and the first (T2) and last (T3) 5 min of hypoxic or air exposure for any day. A repeated measures analysis of variance with the significance level set at 5 % was used to detect significant differences, and individual parameters were contrasted using Student-Newman-Keuls comparisons (SigmaStat 2.0, SPSS).
The data accumulated from the rebreathing tests were analysed using a specially designed spreadsheet (Microsoft Excel). First, breaths from the initial three breath equilibration, as well as sighs, swallows and breaths incorrectly detected by the software were excluded from further analysis. Next, the breath-by-breath end-tidal partial pressures of carbon dioxide were plotted against time and fitted with a least-squares regression line. The equation for this line provided a predicted value of end-tidal partial pressure of carbon dioxide vs. time, thereby minimising interbreath variability due to measurement. Subsequently, tidal volume (ml body temperature and pressure, saturated, BTPS), respiratory rate (breaths min−1) and ventilation (l min−1 BTPS) were plotted against the predicted end-tidal partial pressure of carbon dioxide (mmHg). Figure 1B and C shows examples.
Figure 1. Comparison of respiratory responses
A, mean (±s.e.m.) end-tidal partial pressures of oxygen and carbon dioxide and ventilatory responses to isocapnic (eucapnic) hypoxia are greater on day 14 (filled symbols) than on day 1 (open symbols). Variables were binned (1 min) and averaged for all volunteers. B, the ventilatory response to hypoxic iso-oxic rebreathing of a representative volunteer before (open symbols) and after (filled symbols) a single 20 min exposure to isocapnic (eucapnic) hypoxia on day 8, showing a post-hypoxic increase in threshold. C, the ventilatory response to iso-oxic (hypoxic) rebreathing of a representative volunteer before a single 20 min exposure to isocapnic (eucapnic) hypoxia on day 1 (open symbols) and day 14 (filled symbols), showing a decrease in chemoreflex threshold (V̇E,T1) with daily hypoxic exposures.
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Each of these plots was fitted with a model made up of the sum of two or three segments separated by one or two breakpoints, respectively (Duffin et al. 2000). Model fitting was based on minimising the sum of least squares for non-linear regressions using commercial software (SigmaPlot 5.0, SPSS). The first segment was an exponential decline to a final value, the latter taken as a measure of the basal ventilation, basal tidal volume and basal breathing frequency (V̇E,B, VT,B and fB characteristics, respectively). The exponential decline was chosen to fit any waning of short-term potentiation of ventilation produced by hyperventilation that might have occurred; it was observed in only 17 of 384 (4.4 %) of tests. In those tests without such a trend the decay constant of the exponential decline reverted to a value less than 1, so that the basal values were equivalent to the mean values below the first breakpoint.
The second and possible third segments were straight lines from the first to the second breakpoint, and above the second breakpoint, respectively. The first breakpoint was taken as a measure of the chemoreflex threshold for the ventilation, tidal volume and breathing frequency responses to carbon dioxide (V̇E,T1, VT,T1 and fT1 characteristics, respectively). The second breakpoint was taken as the point at which the ventilatory response pattern changed, in terms of tidal volume and frequency, but was observed in so few tests that it is not reported here. The slopes of the first straight-line portions were taken as the chemoreflex sensitivity for the ventilation, tidal volume, and breathing frequency responses (V̇E,S1, VT,S1 and fS1 characteristics, respectively).
The three chemoreflex characteristics, measured 7 times over the course of 14 days, were first classified into groups, according to: type of exposure series (air or hypoxia), iso-oxia of rebreathing test (hyperoxic or hypoxic) and time of measurement (before or after exposure). To detect significant changes, hyperoxic and hypoxic rebreathing test results were compared separately using two-way repeated measures analyses of variance on 2 × 7 matrices, with the significance level set at 5 % (SigmaStat 2.0). To test for changes over the 14 days the two factors were air vs. hypoxic exposure and the seven factors were days. To test for changes due to a single exposure the two factors were before vs. after and the seven factors were days. When significance was observed, individual parameters were contrasted using Student-Newman-Keuls comparisons, and trends were specified using regression (SigmaStat 2.0).
In addition to these comparisons we also examined the variability of the parameters from all the air exposure sessions by calculating their coefficient of variation over the 14 day period, and then their correlation using the Pearson product moment correlation coefficient (SigmaStat 2.0).