INTRODUCTION

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ACCUMULATING EVIDENCE SUGGESTS that many victims of sudden infant death syndrome have experienced prenatal compromise, resulting in intrauterine growth restriction (16, 21). However, few investigations of ventilatory control in newborns after exposure to adverse intrauterine conditions have been conducted.

We have recently shown that low-birth-weight lambs, unlike normal lambs, do not increase their ventilatory responsiveness to progressive hypoxia during wakefulness as they age (18). Because sudden infant death syndrome is normally associated with sleep (31), we thought it important to extend these observations and examine ventilatory responses to respiratory stimuli during sleep in lambs that had experienced prenatal compromise.

We chose to induce chronic anemia in pregnant ewes as a means of causing prenatal compromise in fetuses that could be subsequently studied after birth. Maternal anemia during human pregnancy is associated with increased risks of prematurity and intrauterine growth restriction (1). Chronic maternal anemia in a sheep model results in fetal growth restriction (20). It was our aim to determine whether ventilatory and arousal responses of newborn lambs to respiratory stimuli experienced during sleep are affected by prenatal exposure to chronic maternal anemia.

	METHODS

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Seven control lambs and six lambs from chronically anemic ewes were studied repeatedly during the first 2-3 postnatal wk. Six date-mated sheep underwent aseptic surgery (halothane anesthesia) at 91 ± 1 days postmating for the implantation of femoral artery and vein catheters to allow the induction, maintenance, and monitoring of chronic anemia. After surgery, arterial blood was sampled daily for measurement of arterial pH, PCO₂ (Pa_CO2), PO₂ (Pa_O2), and O₂ saturation (Sa_O2; ABL510, Radiometer). Hematocrit was measured in triplicate by using microcapillary tubes. Beginning on day 96 of pregnancy, "exchange transfusions" (during which 500 ml of blood were replaced with plasma) were commenced in six ewes to lower the maternal hematocrit to below 14% (20). On day 143 ± 1 of pregnancy, catheters were cut short and obstructed, and ewes were allowed to spontaneously deliver lambs at term. Control lambs were obtained from ewes that did not experience any intervention during pregnancy.

After delivery, lambs were weighed and body dimensions and ponderal index [body wt/(crown-rump length)³] were measured. Postnatal lambs (1-3 days of age) underwent aseptic surgery (halothane anesthesia) for the implantation of a femoral artery catheter (for blood sampling), an intrapleural balloon-tipped catheter, and pairs of stainless steel electrodes (AS632, Cooner) to monitor electrocorticogram (ECoG), electrooculogram (EOG), and nuchal electromyogram (EMG). Signals from these electrodes were used in the identification of sleep states and arousal.

Studies of ventilation during sleep were performed daily beginning on the day after surgery until 13 ± 2 days of age; after this point, lambs were unlikely to sleep spontaneously in the laboratory. During studies, lambs were placed prone in a sling within a soundproof room. Rectal temperature was continuously monitored. While lambs rested quietly awake, an arterial blood sample was collected for determination of pH, Pa_CO2, Pa_O2, and Sa_O2 (ABL510, Radiometer). A pulse oximeter (N-200, Nellcor) was attached to the lamb's shaved tail to continuously measure transcutaneous O₂ saturation (SO₂).

Lambs were fitted with a flexible face mask (Kruus) to allow measurement of ventilation. Before the face mask was fitted, the snout was shaved and dental impression material (President, Coltène, Switzerland) was applied to the inside rim of the mask to ensure an airtight seal. The mask was easily and painlessly removed at the end of each experiment. Gas was continually drawn from the face mask to record the inspired O₂ (FI_O2) and end-tidal CO₂ fractions (FET_CO2; Eliza Duo, Engström, Sweden). A pneumotachometer (model 4500A, Hans Rudolph) was inserted into the outlet of the face mask to measure ventilation; it was attached to a differential pressure transducer (model PT5A, Grass Instruments), and the amplified signal was integrated (model PT10, Grass Instruments) to yield a measurement of volume.

Physiological data were sampled at 200 Hz by an analog-to-digital converter (MacLab, AD Instruments) and recorded on a computer (PowerMac 7200, Apple). "Chart" software (AD Instruments) was used to record the integrated pneumotachometer signal and to calculate the volume of each breath (tidal volume; VT). Breathing frequency (f) was determined from the integrated pneumotachometer record. Electrode signals were conditioned and amplified (Grass Instruments) before being sampled.

During each study, one of three different ventilatory tests was performed (hypercapnia, combined hypoxia-hypercapnia, or hypoxia), or lambs were allowed to sleep without interruption. Manipulation of respired gases was performed remotely from outside the soundproof room. When hypercapnic and combined hypoxic-hypercapnic tests were performed, a Y piece that allowed switching of airflow was attached to the pneumotachometer; one outlet of the Y piece was attached to a rubber bag, whereas the other port was open to room air. Between tests, lambs breathed room air via the open port. To perform hypercapnic and combined hypoxic-hypercapnic tests, the direction of airflow through the Y piece was switched so that the lamb breathed to and from the rubber bag. The bag contained 200 ml/kg body weight of gas consisting of either 5% CO₂-50% O₂-balance N₂ (for hypercapnic tests) or 5% CO₂-21% O₂-balance N₂ (for combined hypoxic-hypercapnic tests).

During studies when hypoxic tests were performed, the pneumotachometer was attached to the side port in a tube through which gas mixtures flowed. Between hypoxic tests, 21% O₂ in N₂ flowed through the tube at 5-6 l/min. To perform hypoxic tests, the O₂ content of the inspired gas was rapidly reduced to, and maintained at, 10%.

Ventilatory tests were performed during identified episodes of active sleep (AS; low-voltage ECoG, active EOG, inactive EMG) and quiet sleep (QS; high-voltage ECoG, inactive EOG, tonically active EMG). Ventilatory tests were discontinued when lambs aroused from sleep. Arousal from AS was defined as the appearance of nuchal EMG activity, and arousal from QS was defined as the switching from high- to low-voltage ECoG activity. Observation of the lambs by using a video camera aided in the identification of sleep states and arousal. Up to seven ventilatory tests or episodes of uninterrupted sleep were recorded in each sleep state during each study.

Data analysis. Minute ventilation (E; ml · min¹ · kg¹; the product of VT and f), FET_CO2, and SO₂ during uninterrupted sleep were determined from averages of VT, f, FET_CO2, and SO₂ measured during entire episodes of uninterrupted AS and QS.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Minute ventilation (top), tidal volume (middle), and breathing frequency (bottom) plotted against end-tidal CO2 for combined hypoxia-hypercapnia (A) and hypercapnia (B) in one control lamb during active and quiet sleep. Data points are average values obtained from 5-s segments of ventilatory recording and were collected from single ventilatory tests.

Statistical analyses. Statistical analyses were performed by using SAS software (SAS). Initial analysis indicated that no variables changed with postnatal age (2-17 days) over the study period. Therefore, resting ventilation, arousal, and ventilatory responsiveness measurements and measurements of Pa_O2, Pa_CO2, Sa_O2, and pH were averaged for each day of study for each animal. Values were averaged to provide a single value for each animal.

	RESULTS

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Hematocrits of ewes that underwent exchange transfusions are shown in Fig. 2. Exchange transfusions initially were performed twice daily (over 3-5 days) until the ewe's hematocrit fell below 14%. After this time it was necessary to perform exchange transfusions at least twice every 3 days to maintain hematocrit at this reduced level. Maternal hematocrit was reduced from 33.5 ± 3.2% (before exchange transfusions were commenced) to 13.7 ± 0.2% throughout the anemic period.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Hematocrits of 6 ewes that underwent exchange transfusions () and a group of 6 unbled ewes (). Arrow indicates point at which exchange transfusions commenced. Timing of birth of lambs from anemic ewes (means ± SE) is indicated.

Control and treated lambs were born spontaneously at full term (control, 147 ± 1 days; treated, 147 ± 1 days). Birth weights of treated lambs (3.9 ± 0.4 kg) were lower than those of control animals (5.2 ± 0.2 kg; Fig. 3). Treated lambs had lower ponderal indexes (2.53 ± 0.16 × 10⁵ kg/cm³) than did control animals (3.15 ± 0.18 × 10⁵ kg/cm³), resulting in treated lambs appearing thin and "wasted." Parity was not different between the two groups of lambs (control, 1.4 ± 0.2; treated, 1.2 ± 0.2). At all ages, treated lambs weighed less than control animals (Fig. 3). The mean daily growth rate for control lambs (290 ± 30 g/day) was not different from that of treated lambs (220 ± 30 g/day).

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Body weights of lambs from anemic ewes () and control lambs () at birth and during first 2 postnatal wk. Values are means of individual measurements made on days when studies of ventilation during sleep were conducted.

Arterial pH (control, 7.41 ± 0.01; treated, 7.40 ± 0.01), Pa_O2 (control, 97.0 ± 2.1 Torr; treated, 94.3 ± 3.4 Torr), and Sa_O2 (control, 96.5 ± 0.6%; treated, 96.1 ± 1.6%) when lambs rested quietly awake breathing room air were not different between control and treated lambs. Mean Pa_CO2 of treated lambs (42.4 ± 0.7 Torr) was greater than in control animals (39.9 ± 0.6 Torr). Pa_CO2, pH, Pa_O2, and Sa_O2 did not change with age in either group. Rectal temperatures of control lambs (39.8 ± 0.01°C) were slightly higher than those of treated lambs (39.6 ± 0.03°C).

Measurements during sleep. E during uninterrupted sleep was not different between groups but was lower in both groups during AS than during QS (Fig. 4). VT and f were not different between control and treated lambs but were both greater during QS than during AS (data not shown). In both groups, FET_CO2 was higher during AS than during QS and was greater in treated than control lambs during both sleep states (Fig. 4). SO₂ was not different between groups but was lower during AS than during QS for both groups (Fig. 4).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Minute ventilation, end-tidal CO2, and O2 saturation (SO2) averaged in control lambs (solid bars) and lambs from anemic ewes (open bars) throughout entire episodes of uninterrupred active and quiet sleep. All 3 variables were significantly different during quiet sleep than during active sleep for both groups of lambs. End-tidal CO2 fraction (FETCO2) was significantly higher in treated than in control lambs during both sleep states. Minute ventilation and SO2 were not different between control and treated lambs.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Minute ventilation (top), tidal volume (middle), and breathing frequency (bottom) responses to combined hypoxia-hypercapnia (A), hypercapnia (B), and hypoxia (C) in control lambs (solid bars) and lambs from anemic ewes (open bars) during active and quiet sleep. Ventilatory responses to hypercapnia, combined hypoxia-hypercapnia, and hypoxia were greater during quiet sleep than during active sleep for both groups of lambs. During quiet sleep, in both groups of lambs, ventilatory responses to combined hypoxia-hypercapnia were significantly greater than responses to hypercapnia. Tidal volume and breathing frequency responses to each of the ventilatory tests were affected similarly to overall ventilatory responses.

Measurements at arousal. Measurements made at arousal from uninterrupted sleep, hypercapnic, combined hypoxic-hypercapnic, and hypoxic tests are presented in Table 1. Episodes of uninterrupted AS were significantly longer than episodes of uninterrupted QS for both groups of lambs and were not different between treatment groups for either sleep state. Similarly, lambs spent more time in AS than QS when ventilatory tests (hypercapnia, combined hypoxia-hypercapnia, or hypoxia) were performed. The duration of AS episodes was shortened by hypercapnic, combined hypoxic-hypercapnic, and hypoxic tests. QS episodes were shorter when hypercapnic and combined hypoxic-hypercapnic tests were performed but not when hypoxic tests were performed.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Exposure to maternal anemia throughout late gestation resulted in restricted fetal growth and chronic elevations in CO₂ levels throughout early postnatal life. Despite this relative hypercapnia, arterial pH, Pa_O2, Sa_O2, and ventilation were not significantly affected during sleep or wakefulness. Ventilatory responses to hypercapnia and combined hypoxia-hypercapnia tended to be reduced in treated lambs, whereas responses to hypoxia appeared to be unaffected.

We have extended the findings of Mostello et al. (20) by demonstrating that fetuses from chronically anemic ewes can survive this prenatal insult and pregnancies proceed to full-term. We have shown that lambs from anemic ewes are growth restricted at birth; their birth weights are low, but measurements of body dimensions are not affected, resulting in a thin, wasted appearance. In contrast, human neonates born to anemic mothers have shorter body lengths as well as low birth weights (27). The type of growth restriction that we observed is typical of that caused by intrauterine compromise during late gestation (15), whereas the overall retardation of growth observed in humans may reflect an effect of intrauterine compromise from earlier in gestation. Like the low-birth-weight lambs that we have studied previously (18), lambs from anemic ewes grew at a rate similar to control lambs during the first few postnatal weeks, resulting in their body weights remaining below those of control animals throughout the study period. This observation is consistent with studies of postnatal growth in infants small for their gestational age who were born at or before full term (11, 29).

The intrauterine growth restriction we observed was presumably due to a reduced nutrient supply to fetuses of anemic ewes because chronic maternal anemia throughout the final one-third of gestation in this species has been shown to cause a failure of uterine artery blood flow to increase with gestational age (20). Despite this reduced uterine blood flow, fetuses from anemic ewes studed by Mostello et al. (20) were not chronically hypoglycemic or hypoxemic, and hematocrit was not elevated. However, in our study, the presence of fetal hypoxemia and/or hypoglycemia cannot be excluded because the period of exposure to maternal anemia was ~2 wk greater than that of the fetuses studied by Mostello et al. Thus our lambs may have been more affected because of the increased duration of exposure to maternal anemia.

Mostello et al. (20) reported a tendency for Pa_CO2 to be higher in fetuses from anemic ewes than in control animals (39.5 ± 0.5 vs. 36.2 ± 0.8 Torr, P = 0.08), and this intrauterine hypercapnia (which may have been greater in our fetuses owing to the longer exposure to maternal anemia) may have altered the CO₂ ventilatory set point during prenatal development. We have attempted to determine CO₂ set points by extrapolation of the linear regression relationship between ventilation and FET_CO2. These estimates were not different between control and treated lambs; however, they may be unreliable because we have not been able to demonstrate an effect of oxygenation on these estimates (4). Lambs from anemic ewes had higher FET_CO2 levels at spontaneous arousal from AS and QS. Similarly, FET_CO2 at arousal was higher in treated lambs when hypercapnic and combined hypoxic-hypercapnic ventilatory responses were performed, confirming a tolerance of these lambs for higher levels of CO₂.

The elevated PCO₂ levels in lambs from anemic ewes could be due to impaired pulmonary gas exchange or airway function. We did not collect data relating to lung function in our lambs, but previous studies suggest that altered pulmonary development could be responsible for our observations. In humans, low birth weight is associated with impaired lung function in childhood (25) and adult life (2, 28), and this is believed to be due to constrained airway growth during gestation. Experimental growth restriction in utero, because of placental insufficiency, affects the development of the trachea in sheep, and it is possible that more distal airways are similarly affected (23). In addition, recent experiments performed in our laboratory have demonstrated abnormalities in pulmonary structure (thickened air-blood barrier) and function (increased chest wall and decreased lung compliance), persisting for at least 8 wk, in lambs that were growth restricted in utero; these lambs had elevated Pa_CO2 and reduced Pa_O2 levels (30). Thus it is possible that lambs from anemic ewes possess pulmonary abnormalities that restrict alveolar ventilation and hence elevate Pa_CO2. Impaired gas diffusion across the pulmonary membrane seems an unlikely explanation for our present observations because CO₂ levels were affected without an effect on PO₂ or SO₂. A further possible explanation for the elevated CO₂ levels observed in treated lambs is that CO₂ production may have been increased. However, we collected no data relating to O₂ consumption or CO₂ production and are unaware of studies specifically examining the effects of intrauterine growth restriction on these variables.

Lambs from anemic ewes did not have ventilatory responses to hypercapnia or combined hypoxia-hypercapnia that were significantly different from those of control lambs. There was, however, a tendency for ventilatory responsiveness to hypercapnia and combined hypoxia-hypercapnia to be lower in treated lambs than in controls. Variability of ventilatory responses was observed during AS and QS in both groups of lambs, and thus, although the mean ventilatory response of lambs from anemic ewes to hypercapnia appears low, values in these lambs were within the range observed in control lambs. Variability of ventilatory responses to hypercapnia has been demonstrated in lambs during wakefulness (19) and in human infants during sleep (5). Unlike human infants, who display greater variability in ventilatory responses during AS than during QS (5), in our lambs variability in responses appeared similar during AS and QS.

Although we found no significant differences in ventilatory responses to hypoxia, hypercapnia, or combined hypoxia-hypercapnia between lambs from anemic ewes and control lambs, responses of both groups were affected by sleep state, in concurrence with previous studies (5, 8, 10, 13, 24). This effect of sleep state was also present for arousal responses. When hypoxic, hypercapnic, and combined hypoxic-hypercapnic ventilatory tests were performed, arousal from AS was delayed when compared with QS. During hypercapnic and combined hypoxic-hypercapnic tests, greater degrees of hypercapnia were experienced during AS, and, when combined hypoxic-hypercapnic and hypoxic tests were performed in AS, greater degrees of hypoxia were experienced. These findings demonstrate that, in lambs, AS is associated with reduced ventilatory and arousal responsiveness and hence increased vulnerability to respiratory challenges.

As in previous studies of developing animals (17, 26, 33), we found a positive interaction between hypoxia and hypercapnia in stimulating ventilation. The absence of this interaction during AS may be due to the weak association between respiratory stimuli and ventilation in this state (22). A positive interaction between hypoxia and hypercapnia in stimulating arousal from QS and AS was also present in both groups of lambs.

E immediately before arousal from combined hypercapnic-hypoxic and hypercapnic tests was not different and was not affected by sleep state despite the differences in ventilatory responses between these tests and between sleep states. Our data suggest that there is a ventilatory threshold for the initiation of arousal and add support to other studies demonstrating that arousal in response to respiratory stimuli occurs because of increased respiratory drive (3, 6, 7, 9, 12, 14). Reduced ventilatory responses during AS resulted in lambs rebreathing the hypercapnic or combined hypoxic-hypercapnic gas mixtures for longer periods of time; this resulted in FET_CO2 levels increasing to a greater degree during AS before a level of ventilatory stimulation sufficient to cause arousal was attained. Our observation that arousal from QS did not occur significantly earlier when lambs were challenged with hypoxia than when ventilatory tests were not performed suggests that the level of hypoxia used did not cause a sufficient increase in respiratory drive to reach the threshold for arousal achieved by hypercapnic and combined hypoxic-hypercapnic stimulation. This is supported by the finding that ventilation at arousal from hypoxic tests was not different than at arousal from uninterrupted sleep.

In conclusion, exposure to chronic maternal anemia during late gestation results in fetal growth restriction and chronic hypercapnia during early postnatal life. The elevation in baseline CO₂ levels in postnatal lambs was not associated with hypo- or hyperventilation, but ventilatory responses to CO₂ tended to be reduced in lambs from anemic ewes. However, it appears likely that the chronic hypercapnia experienced by lambs from anemic mothers was due to altered pulmonary function.