Journal of Applied Physiology
Vol. 81, No. 4, pp. 1555-1561, October 1996
CONTROL OF BREATHING, CIRCULATION, AND TEMPERATURE

Development of ventilatory responsiveness to progressive hypoxia and hypercapnia in low-birth-weight lambs

T. J. Moss, M. G. Davey, G. J. McCrabb, and R. Harding

Fetal and Neonatal Research Unit, Department of Physiology, Monash University, Clayton, Victoria 3168, Australia

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Moss, T. J., M. G. Davey, G. J. McCrabb, and R. Harding. Development of ventilatory responsiveness to progressive hypoxia and hypercapnia in low-birth-weight lambs. J. Appl. Physiol. 81(4): 1555-1561, 1996.Our aim was to determine the effects of low birth weight on ventilatory responses to progressive hypoxia and hypercapnia during early postnatal life. Seven low-birth-weight (2.7 ± 0.3 kg) and five normal-birth-weight (4.8 ± 0.2 kg) lambs, all born at term, underwent weekly rebreathing tests during wakefulness while arterial PO₂, PCO₂, and pH were measured. Hypoxic ventilatory responsiveness (HOVR; percent increase in ventilation when arterial PO₂ fell to 60% of resting values) increased in normal lambs from 86.6 ± 7.1% at week 1 to 227.4 ± 24.9% at week 6. In low-birth-weight lambs, HOVR was not significantly different at week 1 (60.1 ± 18.7%) from that of normal lambs but did not increase with postnatal age (56.6 ± 19.3% at week 6). HOVR of all lambs at 6 wk was significantly correlated with birth weight (r² = 0.8). Hypercapnic ventilatory responsiveness (gradient of ventilation vs. arterial PCO₂) did not change with age and was not significantly different between groups [84.7 ± 7.5 (low-birth-weight lambs) vs. 89.4 ± 6.6 ml ^· min^{1 ·} kg^{1 ·} mmHg¹ (normal lambs)]. We conclude that intrauterine conditions that impair fetal growth lead to the failure of HOVR to increase with age.

newborn; growth restriction; small for gestational age; rebreathing

INTRODUCTION

THE RISK OF SUDDEN INFANT DEATH SYNDROME (SIDS) is increased in low-birth-weight infants (3, 20, 28), suggesting that respiratory control mechanisms may be impaired in such infants. During studies into the postnatal development of ventilatory responsiveness to progressive hypoxia and hypercapnia in normal lambs (5, 19), we were provided with the opportunity to examine the hypoxic and hypercapnic ventilatory responses of full-term low-birth-weight [small for gestational age (SGA)] newborn lambs.

Moss et al. (19) have previously demonstrated that normally grown lambs born at term significantly increase their ventilatory responsiveness to progressive hypoxia over the first 3-4 postnatal wk, whereas the magnitude of their ventilatory response to hypercapnia does not change. Other mammalian species (4, 8) also display increasing ventilatory responsiveness to hypoxia during early postnatal life. This is believed to be due to resetting of the sensitivity of the peripheral chemoreceptors from fetal to adult arterial PO₂ (Pa_O2) levels (14). We are unaware of any published accounts of development of ventilatory responsiveness in full-term low-birth-weight neonates. An effect of low birth weight on the ventilatory response of preterm infants to steady-state hypoxia has been demonstrated (1). However, that study may be confounded by the prematurity of its subjects (1) because we have recently shown that preterm birth affects ventilatory responsiveness to progressive hypoxia and hypercapnia (5). Our aim was to determine the time course of postnatal development of ventilatory responsiveness to progressive hypoxia and hypercapnia in SGA lambs born at full term.

MATERIALS AND METHODS

Animals and preparation. Seven SGA lambs and five lambs of normal birth weight [appropriate weight for gestational age (AGA)] were studied. Lambs were assigned to the AGA group if their birth weight (range 4.4-5.4 kg) was greater than or equal to the mean birth weight of our previous singleton or twin lambs born from ewes of the same breed (4.1 ± 0.1 kg; n = 30). Birth weights of SGA lambs (range 1.8-3.7 kg) were all below those of our normal population and were two or more SDs below the mean birth weight of AGA lambs. All lambs were born vaginally at term [147 ± 0.8 (SGA) and 146 ± 0.2 days (AGA) of gestation] from ewes of the same breed. Four SGA lambs were twin pairs from two pregnancies, one was a quadruplet, and two were from a quintuplet pregnancy. AGA lambs were from twin or singleton pregnancies. None of the SGA lambs was subjected to any prenatal treatment, and in each case, low birth weight arose naturally. All experimental procedures received ethical approval from relevant institutional bodies.

One to three days after birth, the lambs were anesthetized (2-3% halothane in O₂) and underwent aseptic surgery for the implantation of an aortic catheter inserted via a femoral artery. They were then returned to their mothers and allowed to recover for at least 24 h before the first study was conducted. Lambs were studied while awake at ~1-4 and 6 wk postnatally, after which time they were painlessly killed by an intravenous overdose of pentobarbital sodium.

To study ventilatory responsiveness, the lambs were separated from their mothers for 1-2 h and placed in a sling in ventral recumbency. A thermistor (Mon-a-therm, Malinckrodt) was used to measure rectal temperature. A blood sample (~0.3 ml) was taken for measurement of baseline blood gas tensions. A low-dead-space rubber face mask (no. 271670, Kruuse) was then sealed to the lamb's shaved snout with a vinyl polysiloxane dental impression material (Reprosil, Dentsply International) and was secured with tapes tied behind the lamb's head. The mask and sealant were easily and painlessly removed at the end of each study. Inspired O₂ and end-tidal CO₂ levels were monitored by an O₂/CO₂ analyzer (Eliza Duo, Engström).

On each study day, two progressive hypoxic and two progressive hypercapnic rebreathing tests were performed during quiet wakefulness in random order, allowing 15-20 min between tests. Before each test, a pneumotachograph (no. 0, Fleisch) was inserted into the outlet of the face mask to measure ventilation. The pneumotachograph was attached to a differential pressure transducer (model PT5A, Grass Instruments), the output signal from which was integrated (model 7P10CD, Grass Instruments) to provide a record of inspired tidal volume (VT). This signal was displayed and recorded with an analog-to-digital converter (MacLab/8, Analog Digital Instruments) connected to a computer (Power Macintosh 6100/60) running a data-logging program (Chart C3.3.5, Analog Digital Instruments). The program calculated and recorded VT and instantaneous breathing frequency (f). A short period of air breathing (~2 min) was recorded to determine baseline ventilation at the beginning of the study, and another blood sample was taken at this time to determine the effect of the face mask and pneumotachograph on blood gas tensions. Baseline ventilation was recorded, and a blood sample taken before each rebreathing test.

Hypoxic rebreathing tests. Progressive hypoxia was induced by attaching a rubber bag containing ~3 liters of room air to the pneumotachograph. The inspired O₂ fraction (FI_O2) was allowed to fall as the lamb rebreathed while the end-tidal PCO₂ was maintained at resting values by controlling the speed of a fan that passed the rebreathed gas through soda lime in a parallel circuit. When the FI_O2 fell to 14%, we began removing blood samples (0.3 ml) at 30-s intervals until the end of the test (when the FI_O2 = 10%). Hypoxic rebreathing tests lasted 3-4 min.

Hypercapnic rebreathing tests. To induce progressive hypercapnia, lambs rebreathed from a rubber bag containing ~3 liters of 5% CO₂ in air. The FI_O2 was maintained at 21% by controlling the flow of 100% O₂ into the bag. Blood samples were removed at 45-s intervals during the test that ended when end-tidal PCO₂ exceeded 60 Torr, typically after 3-4 min.

Blood sample analysis. Immediately after the completion of each test, blood samples were analyzed at 37°C with a blood gas analyzer (ABL510, Radiometer) to determine the arterial pH (pH_a), arterial PCO₂ (Pa_CO2), and Pa_O2 All measurements were adjusted to the lamb's rectal temperature as measured at the beginning of the study.

Data analysis. VT adjusted for body weight (in ml/kg) and f were determined for the periods of baseline ventilation and for 10 s spanning the periods during the rebreathing tests when blood samples were removed. Inspired minute ventilation adjusted for body weight (I; in ml ^· min^{1 ·} kg¹) was determined by calculating the product of VT and f.

Due to variations in baseline Pa_O2 within and between lambs, we quantified hypoxic ventilatory responsiveness (HOVR) as the percent increase in I when Pa_O2 fell to 60% of the value observed immediately before the test. Lambs tolerate this level of hypoxia without displaying discomfort. To estimate I at the required Pa_O2 , we interpolated between I measurements with Pa_O2 values immediately above and below the 60% level (Fig. 1). VT and f responsiveness to hypoxia were similarly determined. I, VT, and f when Pa_O2 had fallen to 60 Torr were obtained in the same manner.

Hypercapnic ventilatory responsiveness (HCVR) was calculated by determining the gradient of the relationship between I and the Pa_CO2 of coincident blood samples. VT and f responsiveness to hypercapnia were determined by calculating the gradients of the relationships between Pa_CO2 and VT and f, respectively.

Statistical analysis. Comparisons were made between body weights; baseline ventilatory parameters (I, VT, and f); pH_a, Pa_O2, and Pa_CO2; ventilatory responsiveness measurements; and induction rates of hypoxia and hypercapnia with a one between-group (AGA vs. SGA), one repeated-measures (age) analysis of variance with Statistical Analysis System software (SAS). Ventilatory parameters under baseline conditions and when Pa_O2 = 60 Torr were compared with a one between-group (AGA vs. SGA), one repeated-measures (baseline vs. Pa_O2 = 60 Torr) analysis of variance with SAS. When significant differences were detected, a least significant difference test was employed to determine the significant differences between group means. Blood gas values before and after application of the face mask were compared by paired t-test. The rates of weight gain of SGA and AGA lambs between weeks 1 and 6 were compared between groups by unpaired t-test.

All data are presented as means ± SE. Statistical significance is accepted when P < 0.05, and only significant differences are reported.

RESULTS

Body weights, postnatal growth and blood gases. At birth, SGA lambs weighed 2.7 ± 0.3 kg and AGA lambs weighed 4.8 ± 0.2 kg. Mean body weights of SGA and AGA lambs are plotted with respect to postnatal age in Fig. 2. The growth rate (161 ± 25 g/day) of SGA lambs was not different from that (193 ± 6 g/day) of AGA lambs.

Baseline blood gas values did not change with age in AGA lambs. Values for AGA lambs were Pa_O2 of 98.8 ± 1.8 Torr (range 82.5-118.6 Torr), Pa_CO2 of 37.6 ± 1.0 Torr (29.9-44.8 Torr), and pH_a of 7.413 ± 0.004 (7.377-7.465). In SGA lambs, Pa_O2 and Pa_CO2 values did not change with age and were not different from values for AGA lambs. Values for SGA lambs were Pa_O2 of 104.2 ± 2.5 Torr (80.0-154.6 Torr) and Pa_CO2 of 39.5 ± 0.6 Torr (32.1-45.8 Torr). The pH_a of SGA lambs was lower at weeks 1 (7.380 ± 0.010) and 2 (7.383 ± 0.013) than at weeks 3 (7.418 ± 0.013) and 6 (7.425 ± 0.014), but the value at week 4 (7.406 ± 0.013) was not different from that at any other age. At week 2, the pH_a was lower in SGA lambs (7.382 ± 0.013) than in AGA lambs (7.421 ± 0.007). Application of the face mask reduced Pa_O2 in SGA lambs (104.4 ± 2.5 vs. 99.5 ± 1.9 Torr) and caused a reduction in pH_a in both SGA and AGA lambs (7.402 ± 0.006 vs. 7.385 ± 0.006 and 7.414 ± 0.005 vs. 7.399 ± 0.005, respectively).

Baseline ventilatory data. Baseline I was not different between SGA and AGA lambs at any age (Fig. 3). I decreased in both SGA and AGA lambs between week 1 (774 ± 107 and 634 ± 87 ml ^· min^{1 ·} kg¹, respectively) and week 3 (398 ± 34 and 398 ± 23 ml ^· min^{1 ·} kg¹, respectively), after which time I did not change with age in either group. VT was not different between SGA (11.1 ± 0.6 ml/kg) and AGA (10.9 ± 0.3 ml/kg) lambs at any age and did not change with age for either group (Fig. 3). The f was not different between AGA and SGA lambs at any age (Fig. 3). In AGA lambs, f decreased from 60 ± 7 breaths/min at week 1 to 44 ± 4 breaths/min at week 2. In SGA lambs, f decreased from 62 ± 13.4 breaths/min at week 1 to 41.3 ± 6.1 breaths/min at week 3, after which it did not change with age (Fig. 3). Baseline ventilatory data of SGA and AGA lambs at week 6 are shown in Table 1.

Table 1. I, VT, and f under baseline (normoxic) conditions and when PaO2 fell to 60 Torr for SGA and AGA lambs at 6 wk of age SGA AGA  I, ml · min1 · kg1   Baseline 321 ± 32* 375 ± 30*   60 Torr 612 ± 99 1,353 ± 159 VT, ml/kg   Baseline 10.6 ± 1.5* 10.1 ± 0.5*   60 Torr 14.8 ± 1.1* 20.9 ± 1.9 f, breaths/min   Baseline 32 ± 4* 37 ± 3*   60 Torr 40 ± 5 64 ± 7 Values are means ± SE. I, inspired minute ventilation; VT, tidal volume; f, breathing frequency; PaO2, arterial PO2; SGA, small for gestational age; AGA, appropriate weight for gestational age. For each variable, significant differences between mean values are indicated by absence of common symbols.

Ventilatory response to hypoxia. Hypoxic induction rates did not change with age for AGA lambs (0.23 ± 0.02 mmHg/s). The value for SGA lambs was lower at week 2 (0.16 ± 0.02 mmHg/s) than at weeks 1, 3, and 6 (combined ages, 0.24 ± 0.1 mmHg/s) but not at week 4 (0.21 ± 0.02 mmHg/s). The rates of induction of hypoxia were the same for SGA and AGA lambs except at week 2 (0.16 ± 0.02 and 0.24 ± 0.01 mmHg/s, respectively).

HOVR was not different between SGA and AGA lambs at 1 wk of age (60.1 ± 18.7 and 86.6 ± 7.1%, respectively) but was lower in SGA than in AGA lambs from week 2 onward. In AGA lambs, HOVR increased from 86.6 ± 7.1% at week 1 to 138.8 ± 10.2% at week 2 and further to 191.2 ± 23.2% at week 4. HOVR did not change with age in SGA lambs (Fig. 4A ). HOVR of all lambs at 6 wk was significantly correlated with birth weight (Fig. 5A). Ventilatory data when Pa_O2 had fallen to 60 Torr are shown in Table 1.

VT responsiveness to hypoxia was not different between SGA and AGA lambs 1 wk after birth but was lower in SGA lambs from week 2 onward. VT responsiveness to hypoxia increased between weeks 4 and 6 in AGA lambs but did not change with postnatal age in SGA lambs (Fig. 4B ).

SGA lambs had a lower f responsiveness to hypoxia than AGA lambs from week 2 onward. The f responsiveness to hypoxia did not change with postnatal age in SGA lambs but increased between weeks 1 and 3 in AGA lambs, after which time it did not change further (Fig. 4C ).

Ventilatory response to hypercapnia. Hypercapnic induction rates were different between SGA and AGA lambs at weeks 1 (0.13 ± 0.03 and 0.06 ± 0.01 mmHg/s, respectively) and 3 (0.10 ± 0.02 and 0.06 ± 0.01 mmHg/s, respectively). The rate of induction of hypercapnia did not change with postnatal age in AGA lambs (0.07 ± 0.01 mmHg/s). In SGA lambs, the induction rate of hypercapnia was faster at week 1 (0.13 ± 0.01 mmHg/s) than at weeks 4 (0.07 ± 0.01 mmHg/s) and 6 (0.08 ± 0.01 mmHg/s).

HCVR of SGA lambs (70.5 ± 10.0 ml ^· min^{1 ·} kg^{1 ·} mmHg¹) was not different from that of AGA lambs (88.8 ± 12.8 ml ^· min^{1 ·} kg^{1 ·} mmHg¹) and did not change with age in either group (Fig. 6A ). HCVR was not correlated with birth weight (Fig. 5B). VT responsiveness to hypercapnia was not different between groups and did not change with postnatal age (Fig. 6B ). Hypercapnic f responsiveness was higher in AGA lambs than in SGA lambs at week 1 (2.3 ± 0.3 vs. 1.4 ± 0.1 breaths ^· min^{1 ·} mmHg¹; Fig. 6C ). Hypercapnic f responsiveness was higher in AGA lambs at week 1 than at week 6 (2.3 ± 0.3 vs. 1.4 ± 0.2 breaths ^· min^{1 ·} mmHg¹). In SGA lambs, f responsiveness was higher at week 4 (2.1 ± 0.5 breaths ^· min^{1 ·} mmHg¹) than at week 3 (1.3 ± 0.2 breaths ^· min^{1 ·} mmHg¹).

DISCUSSION

This study has shown that, in lambs born at term, factors associated with low birth weight suppress the early postnatal development of HOVR that is seen in lambs of normal birth weight. The significant correlation observed between birth weight and HOVR at 6 wk demonstrates that those lambs with the greatest impairment in development of HOVR are those that have experienced the greatest degree of intrauterine compromise (as indicated by their low birth weights). In contrast, HCVR was not affected by low birth weight. To our knowledge, ours is the first description of ventilatory responsiveness to hypoxia and hypercapnia in full-term low-birth-weight subjects during early postnatal development.

We have quantified HOVR as the increase in ventilation for a given percent reduction in Pa_O2, a method we consider to be the most appropriate for developing neonates. This method of quantifying HOVR is very similar to that used by Moss et al. and Davey et al. previously to determine the postnatal changes in ventilatory responsiveness to progressive hypoxia in normal (19) and preterm (5) lambs, respectively. By using a 40% decrease in Pa_O2 as the index by which HOVR is measured, we have ensured that the relative degree of hypoxia reached during the tests is the same for animals with different baseline Pa_O2 levels. Although our statistical analysis showed no significant differences in Pa_O2 between age groups or between SGA and AGA lambs, we observed variability in Pa_O2 even between measurements made in the same animal on the same day. We considered the relationship that exists between arterial O₂ saturation (Sa_O2) and ventilation, which has been used to measure HOVR (7), to be an inappropriate index of HOVR in our study. Pa_O2 (not Sa_O2 or arterial O₂ content) is sensed by the peripheral chemoreceptors (6), and the relationship between Pa_O2 and Sa_O2 changes during early postnatal life due to the changing ratio of fetal to adult hemoglobin (2).

Mechanical limitations of the respiratory system do not appear to be responsible for preventing the postnatal increase in HOVR in SGA lambs. Although previous studies have demonstrated impaired respiratory system mechanics in low-birth-weight infants (13, 17), these studies used subjects delivered before term and may not, therefore, provide information relevant to the present study. We are not aware of any previous studies that have examined pulmonary mechanics in full-term low-birth-weight neonates, although some indexes of lung function later in childhood have been shown to be related to birth weight independent of prematurity (27). Structural development of the lung parenchyma of late-gestation fetal sheep is not affected by experimental growth retardation, although development of the large airways in these animals is impaired (23). Because all of our SGA lambs were delivered at term, it would be expected that the normal endocrine events associated with labor that are responsible for pulmonary maturation (16) were experienced by these animals.

At no time during the first 6 postnatal wk was baseline ventilation or its components (VT and f) affected by low birth weight. During the rebreathing tests, the maximal level of ventilation reached in response to hypercapnia was always greater than that reached in response to hypoxia. These findings, together with the lack of a difference in HCVR between SGA and AGA lambs, demonstrate that mechanical limitations of the respiratory system are unlikely to have limited HOVR in SGA lambs.

An increase in HOVR in the early postnatal period has been observed in a number of mammalian species including rats (8), sheep (19), and cats (4). The increasing responsiveness has been attributed to an increase in carotid body sensitivity to hypoxia due to resetting of the peripheral chemoreceptors from fetal to adult Pa_O2 values (14). Accordingly, it has been shown that the development of HOVR in newborn rats can be postponed by delaying the postnatal increase in Pa_O2 that occurs at birth (9). However, this results in the absence of a ventilatory response to acute episodes of hypoxia (9). Our SGA lambs are clearly different in that they do increase their ventilation in response to hypoxia. For this reason, it seems unlikely that failure of peripheral chemoreceptor resetting is solely responsible for the failure of development in HOVR in our SGA lambs.

A depressed ventilatory response to hypoxia (as opposed to a complete lack of response) has been described in rabbit pups exposed to cocaine in utero and may be attributed to the associated chronic fetal hypoxemia (30). It is possible that chronic fetal hypoxemia is responsible for depressing the HOVR of our SGA lambs. In utero growth restriction has been shown to be associated with chronic fetal hypoxemia, as observed clinically and in experimental models of chronic placental insufficiency (26). The placental exchange area available to fetuses in multiple pregnancies, such as those in this study, may have been restricted because placental weight per fetus diminishes as the number of fetuses increases (25). We consider it likely that the low birth weight of our SGA lambs was due to chronic placental insufficiency resulting in a lack of O₂ (and/or other substrates). We do not believe that the impaired intrauterine growth of SGA lambs was the result of maternal undernutrition or genetic differences because all ewes were of the same breed, received the same diet, and appeared in good health throughout pregnancy.

Factors responsible for low birth weight at term may have altered the development of neurons involved in respiratory control in SGA lambs. Experimental intrauterine growth restriction has been shown to cause a significant reduction in brain growth in fetal sheep (22) and to affect areas of the brain stem involved with cardiorespiratory control in guinea pigs (24). These studies of neural development in experimentally growth-retarded fetuses suggest that compromised development of central respiratory neurons in SGA lambs could have been responsible for the impaired HOVR. Interestingly, HCVR was normal in SGA lambs, suggesting that central chemosensitivity had not been affected. It remains possible that development of the carotid bodies is affected in SGA lambs, but we know of no evidence to support this.

We have recently shown that the postnatal increase in ventilatory responsiveness to progressive hypoxia is followed by a decline in responsiveness that occurs some time between 6 wk and 6 mo of age in sheep (5). As a result of this decline, ventilatory responsiveness of adult ewes to progressive hypoxia is not different from that of 2- to 3-day-old lambs (19). Such an attenuation of HOVR into adulthood has been described for rats (11), opossums (10), dogs (21), and humans (18, 29). A relative hypersensitivity to hypoxia during early postnatal life may provide protection against hypoxic episodes that can occur as a result of apnea that is commonly experienced soon after birth (12). If humans are affected by intrauterine growth retardation in a similar way to lambs, low-birth-weight neonates may be at increased risk for developing life-threatening hypoxia because the hypoxic chemoreflex is necessary for the termination of apnea (15).

It has been reported that when one of twins dies of SIDS, it is usually the twin of lower birth weight (3), and two other studies (20, 28) have shown that SIDS victims were smaller at birth than their living siblings. If the control of breathing in human infants is, like that of lambs, affected by low birth weight, ventilatory responsiveness to hypoxic episodes (e.g., during rebreathing or apnea) may be impaired in low-birth-weight infants, contributing to increased vulnerability to SIDS. In view of our findings of a profound difference in HOVR of SGA and AGA lambs, priorities of future research should be the determination of the underlying mechanisms and investigation of ventilatory responsiveness in SGA infants.

ACKNOWLEDGEMENTS

The technical assistance of Alexandra Jakubowska and Kerryn Westcott is greatly appreciated.

FOOTNOTES

Address reprint requests to T. J. Moss (E-mail: t.moss{at}med.monash.edu.au).

Received 11 December 1995; accepted in final form 20 May 1996.

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