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Although the disorders of the control of breathing are an important cause of mortality and morbidity during infancy, the treatment of such disorders has been symptomatic, largely due to our lack of understanding of the mechanisms responsible for the control of breathing during the perinatal period(1). An understanding of the mechanism(s) for the control of episodic breathing movements during fetal life and establishment of continuous breathing after birth may provide further insight into such disorders.

In the ovine fetus, rapid, irregular, and intermittent breathing movements occupy 30-50% of the total time and occur only during REM sleep(2). We have recently reported that an increase in fetal Pao2 achieved by distending fetal lungs with oxygen may initiate arousal and stimulate breathing during NREM sleep state in mature sheep fetuses(3, 4). Mechanisms responsible for these new and interesting behavioral and breathing responses remain unclear. Adamsonet al.(5) and Baier et al.(6) have suggested that such responses might be mediated via decreases in PGE2 or some other respiratory inhibitory factor of placental origin, because in their studies, umbilical cord occlusion in the presence of even modest increases in fetal Pao2 lead to arousal and onset of continuous breathing. Several other studies have also shown PGE2 to be a strong inhibitor of fetal and neonatal breathing(7–10). Studies by Blanco et al.(11) showed a decrease in placental blood flow during oxygenation which was proportional to the level of Pao2. Finally, in vitro studies on freshly delivered placentas also showed a constricting effect of O2 on placental vessels(12, 13).

Consequently, it is possible that, in our studies of lung distension, an increase in fetal Pao2, caused 1) a direct umbilical vascular constriction and/or 2) a decreased plasma concentraction of PGE2 leading to arousal and stimulation of breathing. The decreased plasma PGE2 could result from decreased delivery from the placenta due to decreased UBF and/or increased metabolism of PGE2 in the lungs(5, 14) during lung distension and oxygenation. Therefore, to determine the mechanisms for the stimulation of fetal breathing with increased fetal Pao2, we designed a study to test the hypotheses that an increase in fetal Pao2 will 1) cause a cessation of or a severe decrease in UBF or 2) decrease plasma PGE2 concentration.

METHODS

Animal preparation. Eight fetal sheep were studied between 137 and 143 d of gestation (140 ± 2; mean ± SD). Time-dated, pregnant ewes of mixed breed underwent surgery under general anesthesia(ketamine HCl, 10 mg/kg, for induction and 4% halothane and 1-2% halothane in O2 mixture for maintenance) between 129 and 133 d of gestation (term,≈147 d). Observing sterile conditions, the pregnant horn of the uterus was exposed through a midline abdominal incision, and fetal head and rump landmarks were identified. Longitudinal uterine and skin incisions (5-6 cm) were made just cranial to the left sacral crest and anterior to the spinal muscles as described by Rudolph and Heymann(15). Access to the common umbilical artery was obtained through a left flank incision in the fetus. A left approach was used to avoid the inferior vena cava. Retroperitoneal dissection was continued until the junction of the aorta and external iliac artery was identified. Once the common umbilical artery was identified as a continuation of the aorta, a perivascular flow probe (6SS transit time; Transonic Systems Inc., Ithaca, NY) was placed on the common umbilical artery proximal to its bifurcation. Before securing the cable of the flow probe to the epaxial muscle, we ensured that the probe reflector did not occlude the common umbilical artery. Through the same incision, a temperature probe (Physitemp Instrument Inc., Clifton, NJ) was also secured retroperitoneally. All of the fetal and maternal incisions were sutured in layers, and both flow and temperature probes were secured to the fetal skin as well as to the uterus. Before implantation, each flow probe was testedin vitro for its integrity and optimum signal reception capability. The transit time perivascular flow probe (Transonic Inc.) measures instantaneous and average volume flow using ultrasonic energy. Such measurements are independent of vessel size, vessel to flow probe alignment, flow velocity profile, turbulence, hematocrit, or electrical interference. Similarly, the temperature probe was also checked in vitro before implantation, and the values were used only if found to be comparable to a clinical thermometer. Before securing the probes, at least 10-12 cm of extra cable length were made available within the uterine cavity to facilitate the free fetal movements.

Through a separate uterine incision, the fetal head and neck were exteriorized and a carotid arterial and a jugular venous catheters (2.0 mm ID and 3.0 mm OD; Portex, Hythe, Kent, UK) were placed to obtain blood samples for pH, blood gas tensions, and PGE2 and to infuse fluids and antibiotics, respectively. The fetal trachea was cannulated with a polyvinyl Y-endotracheal tube (4.8 mm ID and 7.9 mm OD; Tygon) to apply CPAP to the fetal lungs. Three sets of electrodes were implanted to record ECoG, electro-oculogram, and EMGnk and were used to define the fetal behavioral states. Thereafter, the fetus was further exteriorized to implant a EMGdi electrode and an umbilical cord occluder. The inflatable umbilical balloon (occluder) was snugly tied around the umbilical cord, in an unobliterating fashion, as proximal as possible to the cord insertion and sutured to the fetal skin to avoid cord accidents. The technique for these surgical procedures has been given in detail elsewhere(3). Before returning the fetus to the uterus, two amniotic catheters were sutured to the back of the fetal neck. The larger (4.8 mm ID and 7.9 mm OD) catheter was connected to one of the Y-ends of the endotracheal tube to facilitate the tracheoamniotic flow, whereas the second amniotic catheter (2.0 mm ID and 3.0 mm OD) was used to monitor labor and substract the fetal blood pressure. All of the fetal and maternal incisions were sewn in layers, and the fetal arterial UBF probe cable, fetal temperature probe, umbilical cord occluder catheter, all four sets of fetal electrode wires, arterial and venous catheters, two endotracheal tube extensions, and both amniotic catheters were exteriorized through the left maternal flank and stored in a cloth pouch. To administer fluids intraoperatively and antibiotics postoperatively, the maternal jugular vein was cannulated with a polyvinyl catheter (2.0 mm ID and 3.0 mm OD; Portex). The postoperative care included provision of water and food ad libitum, flushing of all vascular catheters twice a day with heparinized saline, and administration of antibiotics for 4 postoperative days as described previously(3).

Data acquisition and analysis. Electrophysiologic signals from ECoG, electro-oculogram, EMGnk, and EMGdi were amplified and filtered through Neurolog System (NL 100 AK, 102G 125/6; Digitimer Ltd., Herts, England) to record the frequency ranges of 0.5-40 Hz, 5-40 Hz, and 50 Hz to 1 kHz, respectively. The UBF signal was amplified (Gould Electronics, Valley View, OH) and displayed to record the flow range between 0 and 1000 mL/min. The calibration and zero flow of the UBF meter were frequently checked on the chart paper and found to be steady during the entire experiment. Furthermore, to ensure the placement of the UBF probe on the umbilical artery, the zero flow was duly checked by occluding the umbilical cord 1 d before and immediately after the completion of the experiment. The fetal temperature probe was connected to a temperature monitor (Thermalert Model TH-8, Physitemp Instruments Inc., Clifton, NJ). During experiments, all signals except fetal temperature were displayed on an eight-channel chart recorder (Gould 2800S, Cleveland, OH), digitized via an eight-channel Neuro-corder (Neurodata Instruments Corp., New York, NY), and recorded on a videocassette recorder(model FVHC 4000, Fisher, Toronto, ON, Canada) for off-line analyses.

Electrographic criteria were used to define the fetal behavioral states. REM sleep was defined by the simultaneous presence of low voltage ECoG and eye movements and absence of nuchal muscle tone, whereas NREM sleep was defined by the simultaneous presence of high voltage ECoG and nuchal muscle tone and the absence of eye movements. Arousal was defined by the simultaneous presence of low voltage ECoG, nuchal tone, and eye movements(7). Low voltage ECoG was defined as the ECoG amplitude being less than 40 μV and high voltage being more than 40 μV(16). Cardiorespiratory and behavioral patterns during control and experiments included the following: 1) breathing time as a percentage of total time; 2) total durations of i) REM sleep, ii) NREM sleep, and iii) arousal as percentages of total time;3) percentage of time breathing during REM sleep/total REM sleep time; 4) percentage of time breathing in NREM sleep/total NREM sleep time; 5) percentage of time breathing in arousal/total arousal time;6) latency between the initiation of lung distension with oxygen and the onset of arousal and/or stimulation of breathing; 7) latency between the decrease in the UBF and the onset of arousal and stimulation of breathing; and 8) recovery of the UBF to the control values. The EMGdi signal was integrated and from the integrated signal, the amplitude of (∫EMGdi) breathing was measured. The frequency of breathing was defined as the number of diaphragmatic contractions per min of breathing time, whereas the amplitude was measured in arbitrary units from the total area from the integrated EMGdi. The UBF, fetal arterial blood pressure, and heart rate were continuously displayed and recorded on the chart recorder and videocassette during both the control and experimental conditions. During each sleep state, analyses of the blood flow, heart rate, and blood pressure were done every 10 min in 4-min epochs (24 min/h). The observer for the analyses of sleep state was blinded to the arterial pH and blood gas tensions; however, the observer could not be blinded to the UBF, heart rate, and blood pressure, because these measurements were done in a state-specific manner.

Experimental design. Each ewe was transferred into a metabolic cart at least 24 h before the experiments. Experiments were conducted no sooner than 4 d after surgery and only after the fetal pH and blood gas tensions were within normal range (arterial pH more than 7.30 and Pao2 more than 20 torr). Also, no experiments were carried out during labor. Control physiologic variables were recorded for 3 h before each experiment consisting of at least two cycles of REM and NREM sleep each. The morning trough period (0700-0900 h) was avoided because the incidence of FBM is the lowest during this time(17). An equal number of REM and NREM periods were included in the control. The existing tracheal fluid was suctioned through one of the endotracheal tube loops, and fetal lungs were distended at a CPAP of 30 cm H2O either with 100% nitrogen or oxygen in a randomized order. Distension with nitrogen served as the control for gaseous distension. The CPAP was attained in increments of 10 cm H2O using a Baby Bird ventilator (Bird Corp., Palm Springs, CA). Lung distension with nitrogen and oxygen was maintained for at least 2 h, and the change in gas mixture was made during NREM sleep and only after it was established for at least 5-10 min.

Arterial blood samples were collected in ice-chilled heparinized syringes for the analyses of pH, blood gas tensions, Hbo2 saturation, hematocrit, and PGE2 assays during both REM and NREM sleep states under control conditions and during lung distension with nitrogen, whereas the blood samples were drawn during both sleep states and arousal during lung distension with oxygen. The blood samples were drawn once a given state was well established (>5 min). Therefore, we were unable to withdraw blood samples during the arousal periods under control (2.9 ± 3.7 min) and during lung distension with nitrogen (1.2 ± 3.3 min), because such periods were shorter than 5 min. This step was taken to minimize blood sampling during the transitional behavioral states. Similarly, the frequency and amplitude of breathing could not be analyzed during these brief periods. The arterial pH, blood gas tensions (IL 1301; Instrumentation Laboratory System, Lexington, MA) and Hbo2 saturations (IL 2802) were measured within 15 min of collection, and the temperature of the blood gas analyzer was corrected to the fetal body temperature. The blood samples for PGE2 assays were collected in microcentrifuge tubes (Microtainer, Becton Dickinson, Rutherford, NJ) containing EDTA and 0.1 mL of indomethacin (10-4 M) and were immediately centrifuged at 1500 × g; 4 °C for 10 min. The plasma was thereafter frozen in microtest tubes (Brinkman, Westbury, NJ) at-70 °C until analysis. All of the PGE2 assays were done in one of the author's (D.M.O.) laboratories in a blinded fashion using RIA techniques as described previously by Olson et al.(18).

Statistical methods. Analysis of variance for repeated measures was done to test the differences between experimental conditions and sleep states and if a significant difference was observed, Tukey's post hoc test was done to determine the significance. Correlation coefficient analysis was undertaken to determine the relationship between the changes in UBF and onset of arousal and breathing. Similar analyses were performed to investigate the relationship between frequency and incidence of breathing during various sleep states, experimental conditions and UBF, and PGE2 concentrations. All values are given as mean ± SD with ranges if appropriate, and a p value of ≤0.05 was considered as significant.

RESULTS

Effect on arterial pH, blood gas tensions and Hbo2 saturation. Arterial pH, blood gas tensions, and Hbo2 saturation during control and lung distension with N2 and O2 in various sleep states are given inFigure 1. The pH decreased significantly from the control value of 7.33 ± 0.02 to 7.29 ± 0.03 and 7.28 ± 0.05 during lung distension with nitrogen in NREM and REM sleep states, respectively. During lung distension with oxygen, pH further decreased to 7.23± 0.02 and 7.24 ± 0.04 in NREM and REM sleep states, respectively (p < 0.05). The decrease in pH during lung distension with O2 was also significant when compared with the N2 values in both sleep states (p < 0.05;Fig. 1). Compared with the control values, Paco2 increased during oxygenation, whereas it remained unchanged during nitrogen administration. The increase in Paco2 during oxygenation was also significant when compared with values obtained during lung distension with nitrogen in both NREM and REM sleep states (p < 0.05). Lung distension with N2 had no effect either on Pao2 or Hbo2 saturation. Both Pao2 and Hbo2 saturation increased significantly during lung distension with O2 when compared with both the control and the N2 values. Sleep state had no independent effect on these variables (pH, blood gas tension, and Hbo2 saturation) either during control or lung distension with oxygen and nitrogen(Fig. 1).

Figure 1
figure 1

Arterial pH, Paco2, Pao2, and Hbo2 saturation during NREM sleep (▪), REM sleep (□), and arousal (□) under control conditions and during lung distension with 100% N2 and O2 (*p < 0.05 compared with control;†p < 0.05 compared with N2).

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Effect on sleep states. The durations of NREM sleep, REM sleep, and arousal as percentages of total time during control and lung distension are given in Figure 2. NREM sleep time remained unchanged during lung distension with N2 (49 ± 16%) or oxygen (39 ± 6%) when compared with the control values (41 ± 12%). Likewise, the decrease in NREM sleep during lung distension with oxygen was also not significant when compared with the N2 value. The percentage of REM sleep significantly decreased during lung distension with O2 (22± 6%) when compared with the control (56 ± 11) and N2 (50± 13) values (p < 0.05). The duration of arousal significantly increased during oxygenation as compared with the control and lung distension with nitrogen (Fig. 2).

Figure 2
figure 2

Duration of NREM sleep(▪), REM sleep (□), and arousal (□) as a percentage of total time under control conditions and during lung distension with 100% nitrogen and oxygen. Compared with the control period and lung distension with nitrogen, the duration of REM sleep decreased, whereas the duration of arousal increased during an increase in fetal Pao2. No effect on the duration of NREM sleep was observed(*p < 0.05 compared with control; †p < 0.05 compared with N2).

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Effect on FBM. Breathing time as a percentage of total time significantly increased during lung distension with O2 (73 ± 15) when compared with both control (22 ± 18) and N2 (10 ± 17) values (p < 0.05). Breathing times as a percentage of total NREM sleep, REM sleep, and arousal times are given in Figure 3. Breathing during REM sleep decreased significantly during lung distension with N2 administration when compared with O2. However, no significant differences were observed in the incidence of breathing in REM sleep during N2 and O2 administration as compared with the control values. Breathing was not observed during NREM sleep, either under control conditions or during lung distension with nitrogen, whereas it was consistently present during oxygenation (p< 0.05; Fig. 3). Breathing during arousal also significantly increased from 37 ± 24% during control to 93 ± 9% during lung distension with O2 (p = 0.05).

Figure 3
figure 3

Percent time breathing during NREM sleep, REM sleep, and arousal under control conditions and during lung distension with 100% N2 and O2. No breathing was observed in NREM sleep either during control or lung distension with nitrogen. Breathing during NREM sleep and arousal significantly increased during lung distension with O2 as compared with control and lung distension with nitrogen. Breathing during REM sleep increased during O2 administration compared with lung distension with nitrogen (*p < 0.05 compared with control and†p < 0.05 compared with N2). Pecent breathing during NREM/total NREM time (▪), percent breathing during REM/total REM time (□), and percent breathing during arousal/total arousal time(□).

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Lung distension with nitrogen or oxygen had no significant effects either on the amplitude (∫EMGdi) or the frequency (f) of breathing during REM sleep as compared with the control period(Fig. 4). Further correlation analyses also did not show any significant association between the PGE2 concentration (r= -0.14-0.50; p = 0.09-0.72), UBF (-0.43-0.11; p = 0.17-0.78), and the frequency of breathing.

Figure 4
figure 4

Frequency of breathing and diaphragmatic amplitude(∫EMGdi) during various sleep states (▪, NREM; □, REM; and□, arousal) under control and experimental conditions. No significant effects on amplitude or the frequency of breathing were observed during REM sleep.

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Representative tracings showing the effects of lung distension with N2 and O2 on cardiorespiratory and behavioral variables are shown in Figures 5 and6.

Figure 5
figure 5

Representative tracings from one sheep fetus at 141 d of gestation during control period (A) and lung distension with nitrogen (B). During control periods, breathing movements are present only during the REM sleep, whereas they are absent during NREM sleep. During lung distension with 100% nitrogen, no significant effects were observed on any of the recorded variables.

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Figure 6
figure 6

Representative tracings from the same fetus showing the onset of arousal (A), and continuation of breathing in both REM and NREM sleep states (B) during lung distension with 100% O2. Breathing becomes regular during the first 2 to 3 min of the onset of arousal. The UBF decreased during arousal, whereas no further decrease in UBF was observed during REM or NREM sleep states.

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UBF and plasma PGE2concentrations. In response to lung distension with oxygen, the UBF decreased from the control values (323 ± 99 and 300 ± 82 mL/min during NREM and REM states, respectively) to 235 ± 84 mL/min(-27 ± 19%) at the onset of arousal and remained decreased during both NREM (248 ± 93 mL/min) and REM (235 ± 87 mL/min) sleep states, regardless of the presence or absence of FBM (p < 0.05). Lung distension with nitrogen had no significant effect on UBF as compared with the control period (Fig. 7).

Figure 7
figure 7

Mean arterial UBF as absolute values (mL/min) and as percentage of control in various sleep states (â–ª, NREM; â–¡, REM; andâ–¡, arousal) during control and lung distension with nitrogen and oxygen. UBF decreased during oxygenation in both NREM and REM sleep states as compared with the control period (*p < 0.05 compared with control).

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Plasma PGE2 concentration significantly increased by 170 ± 67 and 130 ± 49% in NREM and REM sleep states, respectively, during lung distension with N2 when compared with the control values. No significant changes were observed during lung distension with O2. Sleep states had no independent effect on the UBF or plasma PGE2 concentration either under control conditions or during lung distension with nitrogen and oxygen (Figs. 7 and8).

Figure 8
figure 8

Plasma PGE2 as absolute values (pg/mL) and as percentage of control in various sleep states (â–ª, NREM; â–¡, REM; andâ–¡, arousal) during control and lung distension with nitrogen and oxygen. PGE2 increased during lung distension with N2, whereas no significant differences were observed either between control and O2 or N2 and O2 values (*p < 0.05 compared with control).

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Relationship between UBF and breathing and behavioral responses. Six of the eight fetuses manifested a 25 ± 2.1% decrease in UBF before the onset of arousal. In these six fetuses, the latency between lung distension with oxygen and the decrease in UBF was 33 ± 22 min. For the onset of breathing, the latency was 56 ± 24 min. The lag period between the decrease in UBF and stimulation of breathing was 23 ± 26 min and varied widely between 5 and 75 min. In the remaining two fetuses, arousal and breathing preceded the decrease in UBF by 8 and 13 min. After discontinuation of O2 administration, breathing stopped in 12 ± 6 min, whereas the UBF returned to the control values after 19 ± 14 min. The correlation coefficient between the time to the onset of arousal and the decrease in UBF was 0.3311, and the p value = 0.423.

Fetal body temperature and cardiovascular responses. The fetal body temperature, heart rate, and the blood pressure are given inTable 1. No significant changes were observed under control and during lung distension with N2 or O2 under a given sleep condition.

Table 1 Fetal temperature and cordiovascular responses during control and lung distension
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DISCUSSION

The current study was undertaken to investigate the relationship among UBF, PGE2 concentrations, arousal, and stimulation of breathing in NREM sleep during lung distension and oxygenation in fetal sheep. We provide evidence that increasing fetal Pao2 by lung distension with oxygen decreases UBF and stimulates fetal breathing without altering fetal concentrations of PGE2. Because breathing stimulation was not significantly correlated to the UBF, the onset of continuous breathing does not appear to depend on a decrease in UBF. Furthermore, fetal arousal and stimulation of breathing are not dependent on a decrease in plasma PGE2 levels.

In our present study, a significant decrease in the arterial UBF was observed during fetal oxygenation, whereas no significant change was observed during lung distension with N2. The effects of Pao2 and various drugs on umbilical/placental blood flow have been studied by several investigators(12, 13). In freshly delivered placentas, using constant perfusion pressure, Nyberg and Westin(13) found a decrease in blood flow in all experiments and an increase in pressure in the majority of experiments in response to an increase in Pao2 of 222 torr. In isolated placental vessels, hyperoxia also caused contraction of the vessels. But in studies by Lewis(12), vasoconstrictor response in both human and sheep was noted in only one-fourth of the arteries studied. An equal number of umbilical arteries constricted when the temperature was lowered to 25 °C(12). In more recent in vivo studies, Iwamotoet al.(19) did not observe a significant decrease in umbilical/placental blood flow in response to intermittent lung distension either with or without an increase in fetal Pao2, whereas the studies by Blanco et al.(11) showed a marked decrease in placental blood flow. The discrepancy in these studies may be methodologic; in vivo versus in vitro experiments, gestational age of the fetuses, the time lag between the separation of placenta and the experiments, and the effects of different ambient temperatures. In our studies, the decrease in the UBF may be either due to the direct contractile effects of oxygen or due to an increase in the pulmonary blood flow due to lung distension and an increase in Pao2; current knowledge supports the latter possibility(19, 20). Our results are in agreement with those of Blanco et al.(11), who were unable to observe a change in the placental blood flow during expansion without oxygenation, whereas a significant decrease in placental blood flow was observed during oxygenation.

Among the various prostanoids, PGE2 has been most extensively studied during the perinatal life(5, 7–10, 18, 21–23). Several studies have shown that infusion of PGE2 suppresses, whereas inhibition of PGE2 synthesis via indomethacin and meclofenamate stimulates, FBM(9, 10). Kittermanet al.(10) showed that FBM could be stimulated by prostaglandin synthase inhibitors. The observation that levels of PGE2 rise before parturition and decrease after birth corresponding to the suppression and stimulation of breathing, respectively, has led investigators to believe that PGE2 may be a primary modulator of the establishment of breathing at birth. Adamson et al.(5) were able to induce continuous FBM by occluding the umbilical cord while keeping the fetal Pao2 between 25 and 50 torr. The stimulation of breathing occurred during cord occlusion, which coincided with a decrease in the plasma PGE2 concentrations. Release of the umbilical cord resulted in an increase in the PGE2 concentrations, thereby suggesting that onset of continuous breathing may be mediated by a respiratory inhibitor produced by the placenta such as PGE2. In view of the evidence provided by Olson et al.(22) that the fetal membranes (amnion and chorioallantois) and fetal cotyledons are a major source of fetal PGE2, it is not surprising that Admson et al.(5) observed a decrease in PGE2 concentrations during cord occlusion. Kuipers et al.(24) have suggested that fetal breathing is neither initiated nor stimulated during cord occlusion. Studies by Alvarez et al.(25) and Alvaro et al.(26) have shown that FBM do not increase with umbilical cord occlusion if the fetal Pao2 remains at the preocclusion levels, confirming our previous(3, 4, 6, 7) observations that an increase in fetal Pao2 is a prerequisite for the onset of arousal and stimulation of FBM in a gestation-dependent manner(3).

The increase in PGE2 concentration during lung distension with N2 was unexpected and has not been reported previously in fetal sheep. The likely mechanism for this increase includes an increased production due to the mechanical stretch of the lung. Such an increase in response to lung distension has been documented by both in vivo and in vitro studies(27, 28). Berry et al.(27) have shown that lung distension causes release of PGE2 in both guinea pigs and rats. Leffler et al.(28) have shown that, during ventilation, the metabolism of prostaglandins (prostacyclin and PGE2) changes from net catabolism(-0.39 ng/mL) to net production (0.31 ng/mL). However, the question remains why the increase in PGE2 did not persist during oxygenation in the continuing presence of lung distension. One possibility is that pulmonary blood flow further increased during oxygenation as compared with N2 administration as shown by Iwamoto et al.(19) and Teitel et al.(20). Consequently, the increased pulmonary blood flow might have led to an increased PGE2 catabolism, because several previous studies in adult dogs, cats, rabbits, and guinea pigs have shown a 77-100% clearance of prostaglandins in one pass through the pulmonary circulation(14, 29). Olleyet al.(21) have shown that the clearance increases with the gestational age, reaching the neonatal and postneonatal values by 126 d of gestation. Furthermore, in our studies, the arterial UBF decreased during oxygenation which might have caused a decrease in the circulating PGE2 concentrations. Therefore, we suggest that, during oxygenation, an increased production of PGE2 from the lungs was counterbalanced by the decrease in placental blood flow and an increased pulmonary catabolism, thereby maintaining the PGE2 concentrations near control values.

The precise mechanisms whereby the increase in fetal Pao2 causes arousal and stimulation of breathing during NREM sleep remain unclear. However, several possibilities exist. In all previous studies an increase in CO2 and a decrease in pH was observed. Hypercarbia and metabolic acidemia are known to stimulate FBM(30). Furthermore, the increase in fetal Pao2 might have resulted in a fall in cerebral blood flow, which in turn will cause a widening of arteriovenous CO2 difference. Thus sagittal CO2 will rise, stimulating breathing. Studies by Rurak et al.(31) have shown that, during hypercapnia, oxygen consumption increases mostly via O2 extraction due to the limited oxygen availability during fetal life. However, in our lung distension studies, O2 availability was not restricted and might have led to an increase in fetal metabolic rate causing stimulation of FBM. Rurak and Gruber(32, 33) reported a 30% increase in oxygen consumption and increased Pco2 in both umbilical vessels during normal FBM, and oxygen consumption was diminished when the FBM were absent. Therefore, it is possible that, in response to increased Pao2, CO2 production increased, resulting in stimulation of breathing regardless of the sleep state.

Several adult, neonatal, and fetal studies have shown that hypoxic inhibition of breathing might be mediated via adenosine, because administration of adenosine analogs depress breathing or augment the hypoxia-induced respiratory depression, whereas adenosine antagonists prevented such depression(34, 35). Therefore, because fetal Pao2 normally ranges between 20 and 30 torr, it is conceivable that an increase in fetal Pao2 in our experiments decreased the baseline adenosine concentrations, thereby resulting in stimulation of breathing in oxygenated fetuses.

In summary, the results of this study indicate that the increase in fetal Pao2 achieved via lung distension with oxygen causes a significant decrease in the UBF. However, no causal relationship between the decrease in UBF and stimulation of breathing was observed. Furthermore, our observations do not support the role of PGE2 as a primary modulator of fetal breathing and behavior in our fetal lung distension studies. Further studies are warranted to investigate the fetal cerebral blood flow, metabolic rate, and the role of various neurotransmitters in the manifestation of these interesting breathing and behavioral responses.