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J Appl Physiol 90: 1065-1073, 2001;
8750-7587/01 $5.00
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Vol. 90, Issue 3, 1065-1073, March 2001

Depression of ventilatory responses after daily, cyclic hypercapnic hypoxia in piglets

Karen A. Waters and Kellie D. Tinworth

Departments of Medicine and Paediatrics and Child Health, University of Sydney, Sydney, New South Wales 2006, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ventilatory responses (VRs) were measured via a sealed face mask and pneumotachograph in 30 unsedated, mixed-breed miniature piglets at 12.6 ± 2.3 days of age (day 1) and then repeated after seven daily 24-min exposures to 10% O2-6% CO2 [hypercapnic hypoxia (HH)]. Arterial blood was sampled at baseline, after 10 min of exposure, and after 10 min of recovery. VRs included hypoxia (10% O2 in N2), hypercapnia (6% CO2 in air), and HH (10% O2-6% CO2-balance N2). Treatment groups (n = 10 each) were exposed to 24 min of HH from day 2 to 8 as sustained HH (24 min of HH and then 24 min of air) or cyclic HH (4 min of HH alternating with 4 min of air). Day 1 and 9 data were compared in treatment and control groups. After cyclic HH, respiratory responses to CO2 were reduced during hypercapnia and during HH (P < 0.001 vs. control for minute ventilation in both). In both treatment groups, time to peak minute ventilation was delayed in hypoxia (P = 0.02, ANOVA), and response amplitude was increased (P < 0.001 and P = 0.003, sustained and cyclic HH, respectively, vs. control). Respiratory pattern was also altered during the VRs and among treatment groups. Stimulus presentation characteristics exert effects on VRs that are independent of those elicited by daily HH.

hypercapnia; repetitive; development; ventilatory response


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DEPRESSION OF VENTILATION and/or arousal responses to blood-gas perturbations have been proposed as a mechanism underlying increased risk for sudden infant death (SID). During infancy, clinical conditions that increase an infant's risk for SID are associated with repeated exposure to hypercapnic hypoxia (HH) rather than hypoxia alone. Such conditions include facial entrapment in the prone position or in soft bedding (23, 30). Recent studies suggest that obstructive sleep apnea (OSA) may also be associated with increased risk for SID (17, 27). Older children with OSA fail to increase minute ventilation (VE) during cyclic hypercapnia and have depressed arousal responses to CO2 (11, 12, 16).

The piglet is an established model for the study of early postnatal cardiorespiratory development. Maturation of respiratory control is equivalent to that of human infants at birth, with more rapid postnatal maturation, so that the period up to 30 days of age equates to the first 6 mo in a human infant (6, 24). Previous studies in piglets demonstrated that repeated exposure to hypoxia, with uncontrolled and, therefore, low-CO2 levels, caused depression of responses to hypoxia compared with controls (31). In the acute setting, a cyclic pattern of the stimulus caused greater depression of ventilatory responses (VRs) to hypoxia compared with that of a sustained stimulus of the same total duration (29).

VR to hypoxia and hypercapnia represents stimulation of the peripheral and central chemoreceptors, respectively (26). The combination of hypoxia and hypercapnia tends to cause a synergistic increase in ventilatory and sympathetic responses in adults (26), whereas the two stimuli are thought to be simply additive in the neonatal period (21). Early postnatal maturation occurs predominantly at the peripheral chemoreceptors, and the VR to hypoxia would be expected to be more vulnerable to environmental influences than that to hypercapnia.

We hypothesized that repeated exposure to HH would induce depression of the later hypoxic responses in developing piglets, as observed in response to daily hypoxia with uncontrolled (low) CO2 levels. Because VRs to CO2 are thought to be mature at this age, we hypothesized that there would be minimal change in the VRs to CO2.

VRs were measured in piglets before and after seven daily exposures to 24 min of HH, and the results were compared with those of control piglets. To examine whether the pattern of stimulus delivery affected the changes induced in VRs, one group was exposed to 24 min of continuous HH and another group to a cyclic stimulus with the same total duration. VRs to CO2 were depressed after seven daily exposures to HH, whether or not there was accompanying hypoxia, but only if the daily stimulus had been presented in a cyclic pattern.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mixed-breed miniature piglets were transported from a commercial piggery on day 4.3 ± 4.1 (SD) after birth and then housed in an animal facility with light exposure between 12 midday and 12 midnight. Aseptic surgery was undertaken under general anesthesia on day 10.4 ± 2.8, when piglets weighed 2.5 ± 0.7 kg. The piglets were ventilated throughout the anesthetic, and heart rate was monitored continuously using surface electrodes. Anesthesia was induced using a face mask delivering 1-3% halothane with 30-50% nitrous oxide in O2 and continued throughout surgery via an endotracheal tube, adjusted according to the level of spontaneous respiratory efforts and heart rate. An arterial catheter was placed in the descending aorta via the right femoral artery, tunneled subcutaneously to exit on the ipsilateral flank, and protected in the pocket of jackets that were worn from the time of surgery. Analgesia commenced intraoperatively with paracetamol rectal suppository, to a total dose of 200 mg/kg. Studies commenced a minimum of 48 h after surgery to permit full recovery from anesthetic. The piglets were unsedated at the time of study and had returned to normal feeding and activity. Average weight gain was 135 ± 50 (SE) g/day during the period of the study. Three piglets (8%) were unable to complete the protocol: one with intractable diarrhea was euthanized, arterial line failure in another piglet left incomplete data, and one died suddenly at the end of exposure to the 4-min protocol on day 2. After the final study, all animals were killed painlessly with an overdose of pentobarbitone.

Ethical approval for the study was obtained from the Animal Ethics Committee of the University of Sydney.

VRs

VRs to hypoxia (10% O2-balance N2), hypercapnia (6% CO2-balance air), and HH (10% O2-6% CO2-balance N2) were measured on days 1 and 9. Responses were measured after a step change in the inspired gas mixture. Respiratory variables were recorded for 25 min, including a 5-min baseline, 10 min of exposure to the relevant gas mixture, and 10 min of recovery in air. Arterial blood samples were taken for gas analysis at baseline, after 10 min of exposure, and after 10 min of recovery in air. Blood-gas tensions, pH, base excess, and Hb were measured in an automated blood-gas analyzer (Nova Stat Profile 4, Waltham, MA). All values were corrected to the rectal temperature of the animal, which was recorded along with box temperature at the time each blood sample was taken (ESO-1 and Thermalert TH-8, Physitemp Instruments, Clifton, NJ). A minimum of 45 min of recovery was permitted between each VR, and the sequence of VRs was randomly assigned. For individual piglets, the same sequence of VRs was used on day 9 as on day 1, and the studies were performed at the same time of day.

The study environment comprised a temperature-regulated perspex box. Box temperature was maintained by using a servo-controlled incubator, modified to suit the experimental setup (Thermoline, RI 250, Smithfield, NSW, Australia). Piglets were placed in a vinyl hammock within the box to maintain their head position relative to the respiratory monitoring devices, while still permitting movement of the limbs. Flow was recorded via a calibrated, heated pneumotachograph (Hans Rudolph, 4500A, Kansas City, MO) attached to a full face mask. The mask was sealed against the snout by a layer of thixotropic gel under soft rubber (from a party balloon) inside the firm rubber seal of an anesthetic mask designed for animals (small 1582 or medium 1583, Lyppard, NSW, Australia). The inspiratory limb provided fresh gas flow and incorporated a gastight three-way tap to permit rapid switching between reservoir bags containing air or the required gas mix. The mean time for stabilization at the new gas level was 16.1 ± 1.7 s and was not different among the gas types. A one-way valve was incorporated into the expiratory limb of the circuit to prevent side streaming of air into the gas mix, and O2 and CO2 concentrations were measured on the distal side of the pneumotachograph.

Signals were amplified on a Grass model 8 polygraph and then digitized by using a commercially available eight-channel data-acquisition program (LABDAT, RHT-InfoDat, Montreal). The sampling frequency was 100 Hz. Recordings included continuous measurement of O2 and CO2 and calibrated flow from the pneumotachograph. VE, tidal volume (VT), and respiratory frequency (f) were derived from these raw signals. Ventilation is expressed in milliliters corrected for weight (ml/kg) and time (ml · kg-1 · min-1), whereas f is expressed in hertz.

Daily HH (Sustained HH or Cyclic HH on Study Days 2-8)

On each of the 7 days between VR measurements (days 2-8), all piglets spent a total of 48 min in the study environment during a time that coincided with a normally dark (sleep) period. Each piglet was randomly assigned to one of three treatment groups and received the assigned protocol from days 2 to 8 (see Fig. 1). Two treatment groups were exposed to HH. The three groups were control, cyclic HH, or sustained HH. Piglets in the two HH groups (cyclic and sustained) were exposed to a total of 24 min of HH (10% O2-6% CO2-balance N2) on 7 consecutive days. The control group breathed fresh air for 48 min in the same study environment and via the nasal mask. Sustained HH comprised 24 min of HH followed by 24 min in air. Cyclic HH comprised 4 min of HH alternating with 4 min in air. The cyclic study design permits maximal VR to the stimulus during each 4-min exposure, in a pattern that mimics brief, repeated exposure to HH, such as might occur in the clinical setting.


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  		Fig. 1.   Graphical representation of the study design. Blocks show the pattern of gas exposure for the 3 treatment groups during the treatment period (days 2-8). HH, hypercapnic hypoxia. n, No. of piglets.

Analysis

Outcome measurements included VE (ml · kg-1 · min-1), VT (ml/kg), f (Hz), and blood gases (pH, PCO2, PO2, and base excess). After the raw data were reviewed and signal artifact was excluded, ventilation was averaged over consecutive 1-min intervals by using the analysis software associated with the data-acquisition program (ANADAT and ABREATH, RHT-InfoDat). A total of 0.3% of data epochs was excluded, and, in 0.1% of epochs, 30 s of data were analyzed rather than a full minute. The ABREATH program calculates ventilatory parameters, including VE, VT, and f from the calibrated flow signal.

Control and baseline data were averaged for each piglet and then for the group. Results are presented for the mean of each group. To determine the effects of daily HH exposure on VRs, while accounting for differences that may be attributable to age, day 1 and 9 values were calculated for each piglet before comparisons were made among groups. Data are presented as means ± SD, unless otherwise stated. Comparisons among groups at baseline or on day 1 were performed by one-way ANOVA. Analyses for differences among groups for VRs were performed by using general linear modeling for repeated measures in SPSS for Windows (version 8.0, UK). A P value of <= 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A total of 30 piglets (12 female) completed the study. The treatment and control groups each included 10 piglets. Inspired O2 fraction during hypoxia was 8.9 ± 0.7%, and inspired CO2 fraction during hypercapnia was 6.3 ± 0.4%, and these were not different between the 2 study days or between the control and treatment groups. The first recording was at age 12.6 ± 0.5 days, with a range of 10-26 days for the total study period. Piglet characteristics with means ± SD for each group, are shown in Table 1, and baseline physiological data are shown in Table 2.

                              
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  		Table 1.   Physical characteristics of the piglets measured at baseline (day 1)


                              
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  		Table 2.   Physiological parameters of the piglets at baseline (day 1)

Effect of Daily Exposure to HH on VRs

See Figs. 2-4.


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  		Fig. 2.   Changes in raw data between days 1 () and 9 (). A: raw data for minute ventilation (VE; means ± SE) across time for baseline, 10 min of exposure to hypercapnia (6% CO-balance air), and 10 min of recovery in air on days 1 and 9 in the cyclic-HH group. The level of ventilation achieved during early exposure is reduced on day 9 compared with day 1. B: raw data for VE (means ± SE) across time for baseline, 10 min of exposure to hypoxia (10% O2-balance nitrogen), and 10 min of recovery in air on days 1 and 9 in the sustained-HH group. The level of ventilation is higher on day 9 than day 1, with continued increase in VE to the 4th min of exposure on day 9.

Control and baseline data. The increases in VE, VT, and f were significantly higher than baseline values for each gas mixture, regardless of the study group (P < 0.05). The VE achieved was greatest in HH, followed by hypercapnia, with hypoxia stimulating the lowest response. In hypoxia, all groups reached maximum VE by the second minute on day 1. In the control piglets, mean values during the exposure on day 1 were 1,845 ± 148, 1,585 ± 151, and 1,095 ± 128 ml · kg-1 · min-1 for HH, hypercapnia, and hypoxia, respectively. On day 9, mean values for the control group were 2,027 ± 194, 1,692 ± 129, and 964 ± 77 ml · kg-1 · min-1 for HH, hypercapnia, and hypoxia, respectively (P < 0.001 among gases, and P < 0.05 between days for hypoxia). On both day 9 and day 1, the control piglets showed a steady fall in VE and VT over time in hypoxia (P = 0.03 for VE and VT, F = 7.8 and 7.3, respectively). The magnitude of this fall over time [day 1 = 312 (range 1,233-921) ml · kg-1 · min-1, and day 9 = 214 (range 1,055-841) ml · kg-1 · min-1] exceeded the mean difference between days (131 ml · kg-1 · min-1; P = 0.05, F = 4.8). The slope of this fall did not change over the period of the study.

Effects of daily HH exposure on days 1 and 9. Regardless of the gas mixture, the changes in VE, VT, and f between days 1 and 9 were significantly different between the control and HH-exposed groups. Control subjects showed reduced VE (ml · kg-1 · min-1) compared with piglets with HH exposure (P = 0.001 compared with sustained HH, and P = 0.03 compared with cyclic HH). Control subjects showed reduced VT compared with the sustained-HH group (P < 0.001) but not compared with the cyclic-HH group. Changes in f were higher in the control than in the sustained-HH (P = 0.05) and the cyclic-HH groups (P = 0.04). The sustained-HH group had higher VE (P < 0.001), higher VT (P < 0.001), and overall higher f (P = 0.04). The time to reach peak VE levels during hypoxia on day 9 was delayed in the animals exposed to daily HH compared with the control group (8 and 7.9 vs. 4.3 min for sustained HH and cyclic HH vs. control group, respectively; ANOVA, P = 0.02).

VE. Differences in VE were significant among the groups during the period of exposure to all VR gases (Figs. 2 and 3). During hypoxia, the increase in VE was enhanced in the piglets exposed to HH, with the greatest effect in the sustained-HH group (Fig. 2B; P < 0.001 and P = 0.003 for control compared with sustained-HH and cyclic-HH groups, respectively). There was a reduction in the early response to hypercapnia in piglets from the cyclic-HH group (Fig. 2A). This meant that, during the HH exposure, the VE change was lower in the cyclic-HH group than in the control group (P < 0.001) but was not different for the sustained-HH group. Compared with control subjects during hypercapnic exposure, the VE change was lower in the cyclic-HH group (P < 0.001) and higher in the sustained-HH group (P = 0.05).


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  		Fig. 3.   Changes (Delta ) in day 1 and 9 data for VE in the 3 treatment groups (open circle , control; black-triangle, sustained HH; , cyclic HH). Hypoxia, 10% O2 in N2; hypercapnia, 6% CO2 in air; and HH, 10% O2-6% CO2-balance N2. For clarity, error bars have been removed. See text for results of statistical analyses.

The change in VE was higher for the sustained-HH group than the cyclic-HH group for all gas mixtures (in hypoxia, P = 0.02; in HH, P < 0.001; and in hypercapnia, P < 0.001).

During recovery from all gas exposures, VE was higher in the sustained-HH group compared with the control and cyclic-HH groups (compared with control: after hypoxia, P = 0.003; after HH, P = 0.01; and after hypercapnia, P < 0.001, and compared with the cyclic-HH group: after hypoxia, P = 0.008; after HH, P = 0.03; and after hypercapnia, P < 0.001).

Effects on VT and f

VT. During exposure to all VR gas mixes, differences in VT were significant among the groups (Fig. 4A). During hypoxia, VT increased more in the HH-exposed groups than in the control group (P < 0.001, for both the sustained-HH and cyclic-HH groups). During HH, the VT change was higher in the sustained-HH compared with the control group (P < 0.001) but was not different between the cyclic-HH and control groups. During hypercapnia, the VT change was higher in the sustained-HH than in the control group (P < 0.001) but was not different between the cyclic-HH and control groups.


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  		Fig. 4.   Changes in day 1 and 9 data for tidal volume (VT; A) and respiratory frequency (f; B) in the 3 treatment groups (open circle , control; black-triangle, sustained HH; , cyclic HH). Gases are as defined in Fig. 3 legend. For clarity, error bars have been removed. See text for results of statistical analyses.

The sustained-HH group had a higher VT change in all gas mixtures compared with the cyclic-HH group (in hypoxia, P = 0.02; in HH, P < 0.001; and in hypercapnia, P < 0.001).

During recovery from gas exposures with hypoxia, the VT change was lower in the control group than the sustained-HH group (hypoxia, P = 0.003; HH, P < 0.001), but there was no difference after hypercapnia. The control group had lower VT than the cyclic-HH group in recovery from HH (P = 0.001) but not after other gas exposures. During recovery from hypoxia and hypercapnia, VT was higher in the sustained-HH group than the cyclic-HH group (hypoxia, P = 0.001; hypercapnia, P < 0.001), but there was no difference after HH.

f. During exposure to VR gas mixtures, differences in f were only significant between the groups in HH and in hypercapnia (Fig. 4B). During HH, f increased more in the control group than either HH group (P < 0.001, compared with sustained-HH and cyclic-HH groups). During hypercapnia, f increased more in the control group than the sustained-HH group (P = 0.004) but was not different from the cyclic-HH group values.

The change in f was not different between the sustained-HH and cyclic-HH groups for any of the gas mixtures.

During the recovery periods after VRs, the changes in f were only significant in HH and hypercapnia. After HH, the control and sustained-HH piglets had higher f compared with the cyclic-HH group (P < 0.001 and P = 0.03, respectively). After hypercapnia, the control group had lower f compared with the sustained-HH (P < 0.001) and cyclic-HH groups (P = 0.02).

Respiratory Cycle Times

Inspiratory time. There were no significant differences in inspiratory time (TI) among groups during gas exposure for day 1 and 9 data across time or among gases (Fig. 5A).


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  		Fig. 5.   Changes in day 1 and 9 data for respiratory times for the 3 treatment groups (open circle , control; black-triangle, sustained HH; , cyclic HH) during ventilatory response to HH. A: change in inspiratory time (Delta TI). B: change in expiratory time (Delta TE). C: change in total breath time (Delta TT). Gases are as defined in Fig. 3 legend. For clarity, error bars have been removed. See text for results of statistical analyses.

During recovery periods, TI increased in the control group, whereas it fell in the sustained-HH group (P = 0.02). Although TI fell slightly in the cyclic-HH group, the change was not different compared with that seen in the control or sustained-HH group.

Expiratory time. There was no significant change in expiratory time (TE) among gas mixtures or across time during exposure. During exposure to the gas mixtures between day 1 and day 9, TE was longer on day 9 than day 1 for the sustained-HH group only. Control subjects showed a fall in TE that was significantly greater than that of the sustained-HH group (P < 0.001) and compared with the cyclic-HH group (P < 0.001).

The sustained-HH group showed an increase in TE that was significant compared with the fall in TE of the cyclic-HH group (P = 0.03).

During recovery periods, TE was shorter on day 9 than day 1 for all groups. TE fell more in the control group than in the sustained-HH group (P = 0.001) and the cyclic-HH group (P < 0.001). The fall in TE was not different between piglets exposed to sustained HH compared with cyclic HH. The reduction in TE on day 9 compared with day 1 was greatest after hypercapnia and significantly greater than that seen after hypoxia (P < 0.001) and after HH (P < 0.001).

Total breath time. The changes in TE were reflected in those seen for total breath time (TT) (Fig. 5C). No significant differences were found among the gas mixtures or across time during the exposure. The TT was longer on day 9 than day 1 for the sustained-HH group during exposure to all gas mixtures. The fall in TT in control subjects was significant compared with the sustained-HH (P < 0.001) and cyclic-HH groups (P < 0.001).

The sustained-HH group showed increased TE compared with the cyclic-HH group during gas exposures (P = 0.03).

During recovery periods, TT was shorter on day 9 than day 1 for all groups. TT fell more in the control than in the sustained-HH (P = 0.01) and cyclic-HH groups (P < 0.001). The change in TT during recovery was not different between the sustained-HH and the cyclic-HH groups. The fall in TT during recovery on day 9 was less after hypercapnia than after hypoxia or HH (P < 0.001 for hypercapnia compared with the other gases).

Arterial Gases

There were no significant differences in pH, arterial PO2, arterial PCO2 (PaCO2), or base excess among the groups at baseline, during the exposure, or during the recovery period for any of the VRs or on either study day. The levels of arterial PO2, PaCO2, pH, and base excess during exposures are summarized in Table 3 (means ± SE) for all piglets. Small changes in data from day 1 to day 9 were not different among the groups and were not attributable to differences in Hb or, therefore, to HH exposure. Thus the changes in blood gases were similar on days 1 and 9 for the control group and for the HH-exposed piglets.

                              
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  		Table 3.   Blood-gas values, corrected to rectal temperature, during ventilatory response exposure periods

Subgroup Analyses

Subgroup analysis showed that, compared with males, females had lower VE during HH (P < 0.01), but the effect of sex was not seen in VT or f. No effect of sex was found on the respiratory responses during exposure to hypercapnia or to hypoxia. No effect of sex was seen on VE during the recovery periods after hypercapnia, HH, or hypoxia. Effects among litters could not be examined, because of the high number of litters represented within each study group (see Table 1). Temperature responses were significant during the VR tests (P > 0.001) but not among the different gases. Temperature decreased during recovery compared with baseline (P < 0.1) and exposure (P < 0.001). Mean baseline, exposure, and recovery temperatures were 39.7, 39.7, and 39.6°C, respectively. The temperature differences between days 1 and 9 were different among groups (ANOVA, P = 0.03), being smaller in the sustained-HH group (e.g., 0.32 vs. 0.17 and 0.31°C for control, sustained-HH, and cyclic-HH groups, respectively).

Summary

Piglets exposed to cyclic HH showed depression of the early respiratory response (VE) to CO2, whether this was measured in hypercapnia or HH. Piglets exposed to sustained HH had lower f (whether measured in hypercapnia or HH) that did not translate into reduced VE because of a nonsignificant but compensatory increase in VT. The changes observed during HH most closely reflected those seen during hypercapnia in both groups. All piglets exposed to daily HH showed enhancement of VE during hypoxia compared with day 1 values, and this change was significant compared with control subjects. Piglets exposed to 24 min of HH had prolonged TE in all gases on day 9 compared with day 1. This shift in respiratory pattern did not depend on the gas type (hypoxia, HH, or hypercapnia), and the difference was significant compared with the control group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that brief exposure to HH, daily for 1 wk, causes depression of subsequent respiratory responses to CO2 and enhancement of respiratory responses to hypoxia in young piglets. If the daily stimulus is presented in a cyclic manner, the changes induced in response to CO2 are greater compared with a sustained stimulus of equal duration.

The depression of VRs to CO2 was expressed during the early component of the tests and was present whether or not the hypercapnia was associated with hypoxia. The alterations in CO2 response 1) persist for at least 24 h after the last stimulus, 2) are consistent with enhancement of the predominantly inhibitory influences of the central nervous system (CNS) during early development (18), and 3) occur despite the reported maturity of CO2 responses at this stage of development.

The amplitude of the VR to hypoxia increased beyond the level seen in age-matched controls, although there was a delay to reach peak ventilation in hypoxia. A daily, sustained stimulus caused maximal effect on the VR to hypoxia and lengthening of TE during all VRs.

Three important issues are raised by these results. As far as we are aware, this is the first study to induce sustained depression of the VRs to CO2. The second important point is that the hypoxic responses were enhanced after the same daily stimulus. Finally, the change in VRs was dependent on the pattern in which the acute stimulus was delivered.

Postnatal Development of VRs

Any changes in the control group would be attributable to normal development. That is, the VE (adjusted for weight) did not change significantly in the control piglets in response to HH or hypercapnia, and VE was maintained over the period of the study (in day 9 relative to day 1). On both days 1 and 9, the control piglets showed a steady fall in VE and VT over time in hypoxia, but at the lowest point this was still 160 and 153% of the baseline values (day 1 and day 9, respectively). On day 9, the mean VE during hypoxia was 88% of the day 1 value, with a corresponding decrease in VT but no change in f.

Depression of VRs to CO2

The changes were measurable 24 h after the last episode of HH, despite the fact that the daily HH exposure lasted only 1.7% of the day. The magnitude of change was 200-300 ml · kg-1 · min-1, which is equivalent to 50% of the baseline level of ventilation, or a 10-15% reduction in the maximum respiratory response (during HH).

Immaturity of the CNS may make the VRs of the piglets more susceptible to depression in response to environmental influences. The CNS of piglets is still undergoing maturation at the time that these modifications to cardiorespiratory responses were induced (7). The TE was prolonged in piglets with depressed VRs, consistent with enhancement of centrally mediated GABAergic mechanisms (8). Previous studies by that group show that the prolongation of TE is dependent on the dose of CO2 and almost certainly due to activation of neurons in the ventrolateral medulla (3), where GABA predominates during early development but not in adults. It is also feasible that changes in c-Fos translate into altered expression of other neuroproteins over the course of this 9-day study (3), e.g., RET, which has been specifically implicated in the functional CO2 response (4).

The changes occurred in the respiratory responses to CO2, despite reports that the VR to CO2 is thought to be mature at this stage of development. Studies using steady-state techniques for analyzing respiratory responses to CO2, equivalent to those used in this study, suggest that these are fully functional soon after birth (22) and that postnatal responses to CO2 in piglets are stable (25, 33). When taking the fast (peripheral) and slower (central) components of the CO2 response into account, Wolsink et al. (33) were able to demonstrate, in piglets, that the peripheral component of the responses to CO2 changed over the first 48 h of life. Our study methods would not identify changes in the response to CO2 that are attributable to the fast peripheral chemoreceptor responses (33); these responses are postulated to underlie the fall in PaCO2 levels and the fall in apnea threshold (9, 32) and to occur very early in the postnatal period. No ongoing maturation would be expected in our study group, because studies in piglets have found no ongoing maturation between 2 and 11 days of age (32).

Enhanced Response to Hypoxia

Enhanced ventilation has been demonstrated immediately after episodic exposure to hypoxia in adult animals (5, 13, 28), persisting for >40 min after the exposure. The serotonergic mechanisms responsible for that prolonged enhancement of ventilation originate in the raphe and may be responsible for the enhanced response during hypoxia in our study (28). Similar enhancement of ventilation in neonatal rats was attributed to the expression of nitric oxide synthase 6 h after exposure to episodic hypoxia. Eight cycles of eucapnic hypoxia (5% CO2 and 10% O2) over 2 h resulted in an increase in ventilation and increased expression of neuronal nitric oxide synthase in the brain stem (13). However, the timing of the effects described above follows hypoxic exposure and is, therefore, different from the enhanced responses we observed during the period of exposure and persisting for at least 24 h after the last exposure. Responses to hypoxia would undergo significant postnatal development, including maturation and resetting of the sensitivity of the peripheral chemoreceptors during the time that these piglets were studied (10); thus this maturational effect may have impacted the observed changes in hypoxic, as well as hypercapnic, VRs.

Stimulus Pattern Affects Outcome

Depression of the early VR to CO2 only occurred in the cyclic-HH group and was not seen after daily, sustained HH of the same total duration. The hypoxic VR was enhanced in both the cyclic-HH and sustained-HH groups, but the changes were exacerbated in the sustained-HH group.

The finding that an acute stimulus presented in a cyclic pattern can alter the long-term outcomes of repeated (chronic) exposure is completely new. Repeated hypoxia in a similar acute setting also causes state-specific failure of arousal and cardiorespiratory responses to hypoxia in lambs (14). However, we found no literature pertaining to the effect of such a cyclic stimulus over several days.

The changes seen in the ventilatory parameters were not translated into blood-gas responses, suggesting that additional changes accompanied the respiratory response, e.g., reduced metabolic response to the stimulus. The changes in response to CO2 predominated early in the exposure period and may have been associated with short-term blood-gas changes. However, the sustained respiratory response to hypoxia was also not associated with blood-gas shifts. This suggests that metabolic responses may be altered (particularly during hypoxia) along with the shift in respiratory strategy (during hypercapnia). The sustained-HH group had a smaller temperature adaptation during all VRs, and this may account for the greater respiratory response during hypoxia. Prolongation of expiratory duration may serve several purposes, including greater VT during expiration, and changes in lung mechanics serving to maximize gas exchange by regulating functional residual capacity, including elimination of CO2 and adjustment of pH (8).

The demonstration of enhanced respiratory responses to hypoxia is consistent with the studies cited above but contrasts with a previous study by our laboratory (31) examining acute responses of piglets exposed to hypocapnic hypoxia. In the presence of lowered CO2, respiratory responses were depressed after acute exposure to cyclic hypoxia compared with an equivalent continuous stimulus. This and the altered respiratory pattern observed in the present study suggest that the influence of CO2 was significant. The changes in respiratory pattern that are induced by CO2 exposure persist in piglets up to 21 days of age (1, 8); thus it is effects attributable to differences in the CO2 conditions that would account for the different results (19). Alternatively, daily repetition of the stimulus permitted compensatory strategies, such as changes in metabolism, to develop in response to the acute respiratory depression.

Other Potential Influences

The present study was designed to elucidate which chemoresponses were affected by daily HH. All VRs were tested on the same day, but we randomized the sequence of VRs to eliminate any effect of the testing sequence. We also permitted a minimum recovery period of 45 min between tests; current literature suggests that this time would have permitted full recovery of the VR to CO2. The blood gases of the piglets returned to baseline before the next test was commenced, and baseline ventilation was not different among the groups or among the tests on either day. Any influence of maturation was accounted for by the inclusion of a control group and by analyzing responses for days 1 and 9. Fitting a mask snugly around the snout may have induced inhibitory vagal responses, but these would have been consistent across groups and should not have influenced comparisons made among groups. The choice of study in the unsedated state was important in avoiding potential confounding effects of anesthesia or sedation on the hypoxic component of the response (2, 15). The respiratory controller is sensitive to classic conditioning (20), but the greatest depression of respiratory responses was found in the group exposed to the most frequent stimulus, where a conditioning effect would be expected to enhance the response. The effects were also observed among groups that were studied in equivalent test conditions and do not support this as being a relevant influence on the final testing day. Subgroup analyses resulted in very small group sizes; thus further studies should be undertaken to clarify the role of sex.

Conclusion

The present study is the first to demonstrate that episodic exposure to CO2 can induce long-term changes in VRs to hypercapnia. Cyclic exposure to HH caused depression of the early respiratory response to CO2 (whether this was presented as hypercapnia or HH) during early development. Exposure to daily HH delayed the time to reach peak ventilation during hypoxia. The amplitude of the respiratory response to hypoxia was enhanced by daily exposure to sustained HH. These changes to the VRs persisted for at least 24 h after the last stimulus and appeared to be adaptive, as there was no accompanying change in blood gases. In infants with clinical conditions such as OSA, such adaptive strategies may enable them to tolerate more severe gas disturbances with less respiratory stimulation. Thus respiratory responses to hypercapnia retain plasticity during early life, and changes induced in respiratory responses to hypercapnia or hypoxia differ according to the manner in which the stimulus is presented.


    ACKNOWLEDGEMENTS

This study was supported by The Australian Lung Foundation/Astra Career Development Award, Australian Lung Foundation Ludwig Engel Grant-in-aid for Physiology, National Health and Medical Research Council, The Financial Markets Trust for Children, The Institute of Respiratory Medicine (University of Sydney), and The Children's Hospital Fund (Royal Alexandra Hospital for Children).


    FOOTNOTES

Address for reprint requests and other correspondence: K. A. Waters, David Read Laboratory, Rm. 206, Blackburn Bldg., DO6, The Univ. of Sydney, Sydney, NSW 2006 Australia (E-mail: kaw{at}mail.med.usyd.edu.au).

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

Received 17 July 2000; accepted in final form 5 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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J APPL PHYSIOL 90(3):1065-1073
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society


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