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The transition from episodic fetal breathing to continuous neonatal breathing occurs at a time when many other changes occur, such as exclusion of the placenta, lung expansion with air, changes in O2 and CO2 in contact with the airway, a decrease in pulmonary vascular resistance, and an increase in pulmonary blood flow. Furthermore, the newborn is exposed to new afferent inputs such as light, sound, touch, and changes in temperature. Although not all of these changes have the same importance, it is difficult to isolate any single factor as being responsible for the initiation of continuous breathing activity at birth.

It has been reported previously that hypercapnia stimulates fetal breathing activity and that an increase in Paco2 is involved in the initiation of continuous breathing at birth(1). Furthermore, fast peripheral cooling results in continuous breathing activity in utero and after birth(2, 3). Therefore a decrease in peripheral temperature at birth might be involved in the initiation of continuous breathing activity. However, it has recently been proposed that, after cord occlusion at birth, breathing activity is initiated due to the disappearance of respiratory inhibitors produced by the placenta(1, 4, 5). Although the candidate substance has not been identified, this idea conflicts with our previous observation that initiation on breathing during the first minutes of life was much more dependent on blood gas changes than the presence or absence of the umbilical circulation(6).

To clarify this issue, we have used an ECMO system in the chronically instrumented fetal lamb to isolate the role of changes in Paco2 and temperature on the initiation and maintenance of continuous breathing after birth from that of umbilical cord occlusion per se.

METHODS

This project was reviewed and approved by the animal care committee of University of Limburg. Experiments were commenced on unanesthetized chronically instrumented fetal sheep in utero and continued in a water bath after umbilical cord occlusion and delivery. Five pregnant sheep were operated at 128-132-d gestational age after 48-h fasting, using a sterile technique and under general anesthesia (thiopentone 1 g/70 kg i.v. for induction, 0.8% halothane in 50% nitrous oxide and 50% oxygen for maintenance). A midline laparotomy was performed, and the uterus was partly exteriorized to allow access to the fetal head. The uterus was opened, and the fetus was exteriorized down to the umbilical cord. An inflatable cuff occluder(VO-4, 2 cm inside diameter, Rhodes Medical Instruments, Woodland Hills, CA) was placed around the umbilical cord and anchored to the abdominal skin. The volume required for total occlusion was checked by inflating the occluder briefly with saline until the lumen was closed. A fetal thoracotomy was done at the level of the 10th intercostal space. A pair of wire electrodes (AS 632 Cooner Wire, Chatsworth, CA) were sewn into the diaphragm for recording EMG activity. Thermistors (PT 100 sensors, Murata, Nijkerk Electronics, The Netherlands) were placed in the pleural cavity to measure fetal core temperature. Catheters were placed in the right axillary artery and in the right carotid artery (directed toward the head) to measure blood pressure and heart rate, and for fetal blood sampling (Radiometer ABL3, Copenhagen), and a catheter was placed in the right axillary vein to infuse antibiotics and fluids. Wire electrodes for recording ECoG activity were implanted bilaterally on the fetal parietal dura through holes drilled in the skull. A common electrode was sewn in adjacent tissue. Wire electrodes were sewn into a nuchal extensor muscle to record nuchal EMG and into periorbital muscles to record eye movements (four fetuses). A catheter was placed in the fetal trachea to measure intratracheal pressure. For the ECMO system, two catheters(Biomedicus, Eden Prairie, MN) were placed: one for drainage to the ECMO system (12-14 French) was advanced 10 cm into the right external jugular vein to the right atrium, and one for return of oxygenated blood to the fetus (8-10 French) was advanced 3 cm into the right carotid artery toward the aortic arch. These catheters were connected to 60-cm tubes of 0.65-cm inside diameter capped at the end and filled with heparinized saline (50 U/mL). These tubes permitted later connection to the ECMO system. All catheters, tubing, and electrode cables were exteriorized through a small incision in the flank of the ewe. Catheters were placed in the ewe's carotid artery and jugular vein for blood sampling or infusing antibiotics. The ECMO catheters were flushed twice/d with 5 mL of heparinized saline (50 U/mL), and both catheters were connected to a continuous infusion of 1 mL/h heparinized saline (50 U/mL). Antibiotics were given daily to the ewe (ampicillin 1 g/d) and to the fetus(ampicillin 150 mg/kg/d and gentamicin 5 mg/kg/d) until the end of experimentation.

ECMO. The ECMO system used was as previously described by Kuipers et al.(68). In brief, it consisted of 0.65-cm inside diameter silastic tubing connected to a venous reservoir, a peristaltic pump, and a membrane lung (Scimed, 0.8 m2, Plymouth, MN). The circuit was enclosed in a thermostatically controlled box that maintained fetal blood temperature at 39.5 °C. Blood was drained from the right atrium via the jugular vein and returned to the aorta through the carotid catheter. The blood gases were set by altering the gases supplied to the membrane lung. The ECMO circuit was primed with 350 mL of freshly citrated adult sheep blood. The blood in the circuit was kept at 39.5 °C, and blood gases and pH were held within the fetal physiologic range. The membrane lung was supplied with a gas composed of 0.4 L/min O2, 1.5 L/min N2, and 0.1 L/min CO2 at a total flow rate of 2.0 L/min. Heparin was infused to maintain activated coagulation time at 350-450 s. After connecting the ECMO circuit to the fetus, the pump flow was started at 50 mL/min and then increased slowly to approximately 150-200 mL/min within 15 min. At this flow the ECMO system takes over 15-20% of the combined ventricular output(9).

Experimental protocol. Baseline recordings (Hewlett Packard; 7758 A recorder, paper speed of 1 cm/min) of ECoG activity (filtered between 4 and 40 Hz), integrated diaphragmatic EMG (time constant 0.3 s, filtered between 100 and 1000 Hz), integrated nuchal EMG (time constant 1 s, filtered between 100 and 1000 Hz), eye movements (not filtered), tracheal and blood pressure, heart rate, and core temperature were started on the 2nd d postsurgery before connection to the ECMO system. On the 3rd d postsurgery, when the fetal blood gases were within the normal range and the incidence of fetal breathing movements (defined as activity of the diaphragm and/or negative deflections of tracheal pressure which occurred for > 1 min) was normal(10), the fetuses were connected to the ECMO system, maintaining fetal temperature, blood gases, and pH. Recordings were obtained before ECMO, on ECMO (during fetal normocapnia/fetal normoxia), and after cord occlusion after delivering the fetuses into a warm saline bath. Recordings on ECMO were taken at least 1 h after starting ECMO perfusion.

Labor was identified as the presence of contractions, seen as positive pressure deflections on tracheal pressure recordings (reflecting an increase in amniotic pressure) occurring with a frequency >5 per 10 min and amplitude of 12-30 mm Hg.

To study the initiation and maintenance of continuous breathing activity after birth the fetuses were delivered while on ECMO into a warm saline bath(temperature 39.5 °C), using the following procedure. The pump flow was increased to 350-500 mL/min, then the umbilical cord was occluded. The ewe was killed by injecting 20 mL of pentobarbitone sodium (Euthesate) i.v. Because the umbilical circulation was totally interrupted (confirmed after the fetus was born), the fetus was not affected. The membrane lung was supplied with a gas composed of 1.5-1.9 L/min O2, and 0.1 L/min CO2 at a flow rate of 2.0 L/min. Postnatal Po2 levels were achieved by increasing the O2 flow to the membrane lung. The abdomen of the ewe was opened, and the uterus was partly visualized and opened very carefully at the place were the fetal catheters, tubing, and electrodes were exteriorized. A glove filled with warm saline was placed over the fetal head until it was under water in the bath, to avoid expansion of the fetal lung with air. The fetus was exteriorized down to the umbilical cord, and the cord was cut distal to the occluder. The fetus was delivered and placed in a warm saline bath (39.5°C), with careful handling of the catheters, tubing, and electrodes cables. The temperature of the bath was allowed to decrease slowly, therefore the first peripheral and then central temperature decreased. The Paco2 level was held constant by adjustment of CO2 flow to the membrane lung. Neonatal blood gases and pH were measured every 15 min. After establishing continuous breathing activity, neonatal Paco2 was decreased by decreasing the concentration of CO2 flow to the membrane lung. Mild hypocapnia was defined as a fetal Paco2 1-2 kPa less than baseline.

Blood gases and pH were analyzed from preductal arterial blood samples, values being corrected for body core temperature. Blood gases and pH, the incidence per h, and the length of periods of LV ECoG, the incidence per h and length of periods of breathing movements, and the incidence of breathing movements during LV ECoG were analyzed. Results are expressed as mean ± SEM.

RESULTS

Experiments were performed on 5 fetal lambs, at gestational age of 133-135 d.

Baseline recordings before ECMO. Baseline recordings of 10-19-h duration were obtained 55-70 h after surgery in all fetuses (n = 5). Nuchal EMG was associated with HV ECoG Eye movements (n = 4) were associated with LV ECoG and fetal breathing movements. Fetal breathing activity was present for 31.7 ± 3.16% of the time, the length of periods being 8.23 ± 1.13 min. The incidence of breathing activity during LV ECoG was 65.8 ± 3.67% (Table 1, Fig. 1).

Table 1 pH, blood gases, incidence and duration of LV ECoG of the fetus before and after connection to the ECMO system after delivering into a warm saline bath and after the establishment of continuous breathing
Figure 1
figure 1

The incidence of fetal breathing movements and LV ECoG activity during baseline before ECMO, baseline on ECMO (no labor, n= 3), baseline on ECMO (labor, n = 2), periodic breathing activity after cord occlusion in a warm saline bath, and continuous breathing activity. The four horizontal bars represent, from the top, the umbilical cord intact/cut, the decrease in fetal central temperature, the maintenance in Paco2 and the increase in Pao2. BM, breathing movements; LV, LV ECoG.

On the 3rd d postsurgery the fetuses were connected to the ECMO system. Periods of fetal normocapnia/normoxia were studied to control for possible effects of ECMO on fetal behavior.

Baseline recordings on ECMO. Nuchal EMG activity was associated with HV ECoG activity. Rapid eye movements were associated with LV ECoG activity and fetal breathing movements. Two ewes went into labor before connection to the ECMO system was made. There were no breathing movements present while these two fetuses remained in utero on ECMO during these hours of labor. In these two fetuses we could not analyze fetal breathing activity during a control period on ECMO. In the other three fetuses labor was not present until near cord occlusion time (6-12 h on ECMO). In these fetuses, breathing activity was present for 36.0 ± 4.0% of the time, the length of fetal breathing periods being 9.2 ± 1.8 min. The incidence of breathing activity during LV ECoG was 75.3 ± 3.2% (see also Table 1 and Fig. 1).

Later (after 6-12 h), just before cord occlusion, all fetuses were exposed to spontaneous labor, therefore there were no breathing movements present. Nuchal EMG activity remained associated with HV ECoG activity. Rapid eye movements were associated with LV ECoG activity. Fetal temperature was kept constant by perfusing the fetus with blood maintained at 39.5 °C by the ECMO system (see “Methods”).

Breathing activity after cord occlusion in a warm saline bath. After delivering the fetuses into the saline bath at 39.5 °C all lambs showed periodic breathing activity, these breathing movements being associated only with LV ECoG. The temperature of the bath was allowed to decrease slowly, therefore first peripheral and then central temperature decreased. Central temperature decreased by a mean of 0.02 ± 0.01 °C/min. Breathing movements were present for 17.7 ± 4.57% of the total time (Fig. 1) and for 34.1 ± 9.71% of the time during LV ECoG. Nuchal EMG activity was associated with HV ECoG activity in four experiments; in one experiment nuchal EMG was also present during LV ECoG (Fig. 2). Rapid eye movements (n = 3, in one experiment electrodes were during delivery accidentally cut) were associated with LV ECoG activity and breathing movements. pH and blood gases are given every 20 min in Table 2.

Figure 2
figure 2

Recording of a neonatal lamb in the warm saline bath at 133 d of gestation, 6 d after surgery, and connected to the ECMO system. Tracings are, from the top: electrocortical activity, integrated nuchal EMG, integrated diaphragm EMG (two traces), blood pressure, heart rate, and tracheal pressure. Blood gas and pH samples were taken at the times indicated. Note that breathing movements were still present periodically for 36 min after delivering the neonate into the saline bath. Continuous breathing activity became present after 36 min. Nuchal muscle activity was still modulated by electrocortical activity

Table 2 Mean ± SEM of the pH and blood gases of the lambs delivered in the saline bath

Continuous breathing activity. After 36-192 min, breathing activity became present continuously both during LV and HV ECoG in all animals. At this time the central temperature had fallen by a mean of 1.2± 0.5 °C. At the time continuous breathing activity was initiated, blood gases and pH were: pH 7.20 ± 0.04, Paco2 7.35 ± 0.16 kPa, and Pao2 12.78 ± 2.51 kPa (Table 1). At the time breathing was continuous, nuchal muscle activity was associated with LV ECoG, which implies arousal at the moment of the initiation of continuous breathing. Eye movements remained associated with LV ECoG.

This continuous breathing activity was very dependent on the level of Paco2 because in all instances it could be stopped by decreasing Paco2 (pH 7.44 ± 0.02, Paco2 3.97 ± 0.46 kPa, and Pao2 25.79 ± 6.78 kPa). The Δ change of Paco2 in the five experiments ranged from 33 to 48%.

DISCUSSION

Our results show that 1) continuous breathing activity was not initiated after cord occlusion when an increase in fetal Paco2 and an immediate fall in core temperature were prevented and 2) that all fetuses delivered into a warm saline bath after cord occlusion and supported on ECMO still showed periodic breathing for 36-192 min when Paco2 was unchanged, but Pao2 was held at neonatal levels. Furthermore, we showed that breathing activity became continuous when central temperature was decreased by 1.2 °C by allowing the temperature of the bath to decrease while Paco2 was maintained. This continuous breathing was dependent on the level of Paco2, because it stopped by decreasing neonatal Paco2. The characteristic of the continuous breathing after delivering the lamb in the warm saline bath were similar to neonatal breathing (high amplitude, high frequency). Furthermore, all fetuses were able to breathe air after they were taken out of the bath.

The establishment of continuous breathing at birth has been the subject of several studies on the influence of different stimuli, examined by means of acute experiments(2, 1113). In those experiments fetal and environmental temperatures, fetal blood gases, and pH were not always well controlled. In previous experiments with ECMO we found that it was possible to maintain fetal lambs with good control of Paco2, skin, and core temperature(68). Therefore, the use of ECMO seemed an appropriate method for studying further the mechanisms involved in the initiation of postnatal breathing activity.

The results of our experiments pose four questions. 1) Why did continuous breathing activity not start after cord occlusion? 2) Why did breathing revert to an episodic pattern after delivery into a saline bath, whereas core temperature decreased slowly, Paco2 remained constant, and Pao2 was set at neonatal levels? 3) Why did breathing start later? 4) What mechanism maintained this continuous breathing once initiated? These questions are discussed below.

Why did continuous breathing not start after cord occlusion? The exclusion of putative respiratory modulators after cord exclusion has been suggested to be a crucial mechanism for the initiation and maintenance of breathing at birth(1, 4, 5, 14). After birth neonatal breathing differs from fetal breathing in occurring during HV ECoG as well as during LV ECoG. Therefore, it could be argued that a putative fetal respiratory modulator should inhibit breathing only during HV ECoG. To study this problem Alvaro et al.(5) infused ovine placental extracts to fetuses showing continuous breathing obtained by producing a Pao2 much higher than that which occurs naturally. The infusions produced a depression of breathing activity, and the investigators concluded that the placenta contains some substance that can inhibit breathing activity. There are, however, problems with the interpretation of this work. First, it is not clear whether the breathing produced represents spontaneous breathing activity or whether it is a response to the very high Pao2, usually associated with arousal. Furthermore, the demonstration that placental extracts depress or inhibit breathing activity does not permit the conclusion to be drawn that a substance (e.g. prostaglandin, adenosine)(15, 16) is released and is responsible for the inhibition of spontaneous breathing activity during HV ECoG. The conclusive experiments would be to show the presence of continuous breathing in utero after inhibiting release of this substance or blocking its receptors. Furthermore, other experimental manipulations, such as peripheral or central cooling(3, 30) or the placement of small lesions in the lateral pons(17), induce continuous breathing presumably through a different mechanism not related to changes in fetal plasma level of the hypothetical respiratory modulator produced by the placenta.

Our previous(6) and present work does not support the view that exclusion of respiratory modulators produced by the placenta is an essential mechanism in the initiation of continuous breathing at birth, because neonatal breathing was not established immediately after cord occlusion whenever Paco2 and temperature were maintained(6). Thus, our first conclusion is that continuous breathing was not established when Paco2 was maintained until temperature decreased, even though the umbilical cord had been occluded and Pao2 was held at neonatal levels.

Reversion to episodic breathing pattern. During labor there were no breathing movements present as previously described(18). In our experiments this inhibition was partly lifted very soon after delivering the fetuses into the warm saline bath, in that breathing returned to a periodic fetal breathing pattern. The mechanisms involved in the transition from fetal apnea during labor to periodic fetal breathing after delivery are not known, but the only factor that changed sufficiently rapidly in our experiments to explain it was the rise in Pao2 to neonatal levels. Such a rise in Pao2 will produce a cerebral vasoconstriction(19) and thus a rise in brain tissue Pco2. This may stimulate breathing, as both fetal and neonatal breathing are known to be very sensitive to the level of Paco2(1, 20, 21).

Other authors offer different explanations for the presence of breathing during hyperoxia. Because hypoxemia is inhibitory to fetal breathing(20), it has been speculated(2224) that if Pao2 is increased it will lift a tonic inhibition of breathing activity, allowing it to occur in both LV and HV ECoG. These authors propose this to be the mechanism by which fetal breathing activity is periodic. However, the large increase in Pao2 in their experiments could have caused a decrease in cerebral blood flow and local increase in tissue CO2(19)(see above) of sufficient magnitude to produce continuous breathing.

Why did continuous breathing start? The third question concerns the initiation of continuous breathing. We found that this occurred only when the temperature of the bath decreased by a mean of 1.2 °C, resulting in a decrease of fetal peripheral and central temperature. It is known that peripheral and central cooling stimulates breathing activity in utero and after birth(2, 3, 30) and although the underlying mechanisms are not known, several possibilities exist. The first is that the slow disappearance from the bloodstream of an inhibitory modulator (arising from the placenta or elsewhere) is involved. However, our observation that the onset of continuous breathing was linked to a fall in temperature leads us to propose that the increased afferent input produced by cooling produces a lifting of brainstem inhibitory processes, especially during HV ECoG. This effect can be viewed as an alteration in the behavioral control of breathing and, if so, we expect it to be accompanied by changes in the gain of spinal and autonomic reflexes. This merits further study. Another possibility is that the increase in neonatal metabolism, produced by shivering and nonshivering thermogenesis on cooling, provides an increase in the metabolic drive to breathing(25). Last, changes in sensitivity to CO2 produced by cooling may be considered as a possibility. Studies in neonatal kittens(26) support this view as in this species the gain of peripheral chemoreflex respiratory responses to hypoxia or CO2 was less in a warm than a cold environment.

Maintenance of continuous breathing. The discussion above focuses attention on the role of CO2 as a respiratory stimulus in perinatal life. Once started, we found that continuous breathing was dependent on the level of Paco2, as reducing it returned breathing to an episodic pattern or stopped it altogether. Thus, despite the relative insensitivity of the respiratory controller to hypoxia as a stimulant in early neonatal life, CO2 is clearly an important stimulus. Recent studies(27) show that the carotid chemoreceptors in the lamb have both dynamic and steady state responses to CO2 at an age when hypoxia sensitivity is weak(28).

It has been suggested that the ability to initiate continuous breathing is dependent on the maturity of the fetus (gestational age > 134 d)(23, 24). In our experiments we showed that breathing activity became continuous even in fetuses of 133 d of gestation. This is confirmed by the recent report of Tiktinsky et al.(29).

In summary, maintenance of fetal Paco2 and a slow decrease in central temperature after cord occlusion delays the establishment of continuous breathing, although we speculate that the rapid reversion to an episodic breathing pattern is a consequence of the rise in Pao2. The level of Paco2 is important in the maintenance of breathing activity in the first few hours of life. Our results do not support the hypothesis that breathing at birth is dependent on the disappearance of a respiratory inhibitor after cord occlusion. At birth several simultaneous changes occur, including changes in oxygenation, temperature, and in blood or CSF levels of respiratory modulator substances. These change the sensitivity to CO2 and permit the transition from fetal periodic breathing to neonatal continuous breathing to be effected.