Changes in lung liquid dynamics by prolonged fetal hypoxemia
induced
STUART B. HOOPER AND RICHARD HARDING Department of Physiology, Monash University, Clayton, Victoria 3 168, Australia
HOOPER, STUART B., AND RICHARD HARDING. Changes in lung liquid dynamics induced by prolonged fetal hypoxemia. J. Appl. Physiol. 69( 1): 127-135,1990.-Our aim was to determine the effect of prolonged fetal hypoxemia, induced by reduced maternal uterine blood flow (RUBF), on fetal lung liquid secretion, flow, and volume. In chronically catheterized fetal sheep, lung liquid volume (Vi,) and the secretion rate of lung liquid (Vs) were measured before and after a 24-h period of either RUBF or normoxemia. Tracheal fluid flow and the incidence of fetal breathing movements (FBM) were measured before, during, and after the 24-h period. In normoxic control fetuses Vs was not significantly altered. After 24 h of RUBF, Vs was significantly (P < 0.005) reduced compared with pre-RUBF values. During 24 h of RUBF the incidence of FBM declined initially but returned to control values after 12-16 h. In seven of eight fetuses, over the l2- to 24-h period of RUBF, large amounts of liquid (22.7-62.6 ml) were drawn into the lungs during FBM, resulting in a net movement of amniotic fluid into the lungs. During the 1% to 24-h period of RUBF, changes in the incidence of FBM were found to be significantly and positively correlated (r = 0.86, P < 0.005) with the changes in VI, that occurred over the 24-h period. Thus, prolonged RUBF can result in the inhalation of large volumes of amniotic fluid by the fetus, which could be a cause of in utero meconium aspiration.
reduced uterine blood flow; lung liquid flow; fetal breathing movements
secretion;
tracheal
fluid
oflunggrowthinthe fetus is poorly understood, it is clear that expansion of the fetal lungs by liquid is a very important factor (25, 34). Sustained alterations in lung liquid volume (V,) have major effects on lung growth and pulmonary development (1). However, the mechanisms by which changes in lung expansion result in altered rates of tissue growth are not known, although they are likely to be exerted locally rather than systemically (26). Fetal lung liquid is secreted across the pulmonary epithelium into the future air spaces (28) and leaves the lungs via the trachea. Fetal VL is therefore determined by the balance between the rate of lung liquid secretion (Vs) and the net rate at which it flows out of the trachea (VJ. The production of fetal lung liquid is oxygen dependent (28) and is inhibited by arginine vasopressin (AVP) (29, 32) and epinephrine (21, 33), both of which are elevated in fetal plasma during fetal hypoxemia (20, 23, 30). In contrast, V,, is influenced by the resistance of the upper airway and is augmented during episodes of fetal breathing movements (FBM) when this
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resistance is low (15, 16, 18). Thus, Vs and V,, are able to change independently, resulting in changes in VL. We have recently shown that 4- to 6-h periods of moderate asphyxia in fetal sheep, secondary to controlled reduction .s in maternal uterine blood flow, significantly decrease both Vs and Tit, (19). VL did not change, presumably owing to simultaneous and equal reductions in Vs and V,,. It was not known, however, wh.ether VL would remain unchanged after longer periods of fetal hypoxemia. It seemed possible that changes in VL might occur because the incidence of FBM, which is depressed by acute hypoxemia (5, 7), eventually returns to control values after prolonged hypoxemia (4, 24). Thus, if Vs remains inhibited during periods of prolonged fetal hypoxemia and V,, is augmented by the return of FBM, we hypothesized that an imbalance between the two may result, leading to a change in VL. The aim of this study was to determine the effects of 24 h of fetal hypqxemia on Vs and VL and to characterize any changes in Vt, and the incidence of FBM over this period. METHODS
A nimal preparation. Surgery was performed on 14 Border Leicester x Merino ewes, between 105 and 115 days of pregnancy, under aseptic conditions. Anesthesia was induced by 5% sodium thiopental intravenously and was maintained, after tracheal intubation, with 1.5% halothane in oxygen and nitrous oxide (50:50 vol/vol). In all ewes, polyvinyl catheters (SV116, Dural Plastics, Australia) were implanted into the maternal jugular vein and amniotic cavity. An adjustable cl.amp was placed around the maternal common internal iliac artery to produce controlled reductions in uterine blood flow (5, 6, 10, 11, 19) In each fetus, two silicone rubber cannulas (no. 601365, Dow Corning) were implanted 2-4 cm into the trachea; one was directed toward the lungs, and the other was directed toward but did not enter the larynx (l&19). Fine stainless steel wire electrodes (no. AS632, Cooner) were implanted into the fetal diaphragm muscle to record its electromyogram. Polyvinyl catheters (SV65, Dural Plastics) were implanted into the carotid artery and jugular vein of each fetus. The fetal catheters, electrode wires, and control cable for the adjustable clamp were exteriorized through an incision in the right flank of the ewe. After surgery, benzyl-penicillin (400,000 units) and streptomycin (500 mg) were injected into the amniotic fluid. The exposed tracheal cannulas were j oined to form
0 1990 the American
Physiological
Society
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an exteriorized tracheal loop, and the ewes were allowed to recover for at least 5 days after surgery. Lung liquid flow into and out of the fetal lungs was quantitated using a flowmeter connected in series with the tracheal loop (16, 18, 19). V,, was calculated by measuring the net volume of fluid that either entered or left the lungs per hour via the trachea. Vt, was given a positive value when it was directed away from the lungs. Experimental protocol. Uterine blood flow was reduced by constricting the maternal common internal iliac artery to produce a 50-60% reduction in the degree of oxygen saturation of hemoglobin (Sao,) in fetal carotid arterial blood. Measurements of fetal blood gases (Sao, and arterial Pop and PCO~ (Pao, and Pa+, pH, and hemoglobin content were made at frequent intervals (0, 1-2 h, 4-6 h, 9-11 h, 18-20 h, and 24 h) with a Radiometer ABL30 blood gas analyzer and a Radiometer OSM2 oximeter. The volume of fetal blood removed over 24 h was lo-15 ml. Fetal tracheal pressure, arterial pressure, and heart rate were also measured. Tracheal and arterial pressures were displayed on a polygraph (Grass model 7D) after subtraction of amniotic fluid pressure. The incidence and amplitude of FBM were determined from tracheal pressure recordings. The amplitude of FBM was measured at 5-min intervals during periods of FBM, and a mean value was calculated each hour. Each experiment on the effect of reduced uterine blood flow (RUBF) consisted of three consecutive periods: a control period, a period of RUBF, and a recovery period. Each control period lasted for 15 h; during the first 12 h we measured V,, and the incidence of FBM, and during the remaining 3 h we measured Vs and VL. During the period of RUBF (27 h) we measured V,, and the incidence of FBM in the first 24 h; then for 3 h we measured Vs and VL. The recovery period lasted for 12 h, during which we again measured Vtr and the incidence of FBM. In a separate series of experiments, L Iwe measured the normal daily variations in VL, Vs, Vtr, and FBM in normoxic fetuses. These experiments were the same as those described above except that RUBF was not induced. Thus, measurements of VL and Vs were made 24 h apart, and Vt,, and FBM were measured before, during, and after the 24-h study period. Vs and VL were measured using an established method of indicator dilution (12,19,28,29). We isolated the fetal lung lumen from the upper airway by connecting the descending tracheal cannula to a sterile loo-ml reservoir (open to atmosphere via bacterial filter) throughout the 3-h procedure. Lung liquid was drained into the reservoir, and an impermeant indicator (Dextran Blue 2000, Pharmacia, Uppsala, Sweden; 200 mg) was added. The indicator was thoroughly mixed by repeatedly draining the lung liquid into the reservoir and then returning it to the lungs over a period of 45 min. After this initial mixing period, lung liquid samples (0.75-3.0 ml) were collected at 15-min intervals for the rest of the 3-h period. The volume of liquid removed at each sample time was calculated to approximately equal the volume of liquid secreted over that time interval so that VL was not significantly altered. Vs was calculated from the rate of dilution of the indicator. The concentration of indicator
AND
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in lung liquid was measured using a multichannel absorbance meter (Titertek Multiskan, Flow Laboratories, UK) set a wavelength of 620 nm. StatisticaL an&&s. The results presented in the text are means t SE. In both control and RUBF fetuses, the incidence and amplitude of FBM, V,, and blood gas, pH, and hemoglobin data were analyzed using a three-way analysis of variance (ANOVA) with treatment (normoxia vs. RUBF), time, and animals as factors. The two treatments were then analyzed separately using a two-way ANOVA followed by a Newman-Keuls multiple range test to determine significant differences between time periods. The level of significance was P < 0.05. vs was analyzed using a Student’s paired t test. V,, was assigned a negative value if liquid entered the lungs and a positive value if liquid left them. The direction of Vt, was therefore determined by comparison of the value for V,, with zero, by use of a Student’s paired t test. RESULTS
All fetuses survived until near term (140.3 t 1.2 days, term = 145 days), when the ewes and fetuses were killed by an overdose of pentobarbital sodium. At the start and end of each experiment, all fetuses were considered healthy, as judged by blood gas and pH measurements. Uterine blood flow was reduced for 24 h in 11 experiments on nine fetuses. However, the fetuses in three of these experiments were found to be normoxemic at the end of the 24-h period, despite the clamp being at its maximum adjustment. The mechanism by which normoxemia was achieved in these experiments is not known but could have been increased collateral blood supply and/or a greatly reduced fetal oxygen consumption. Because the mechanism is not known, these three experiments have been eliminated from the study. Fetal blood gas and pH measurements. In each RUBF experiment (n = 8 on 8 fetuses; mean gestational age 126.8 t 21.1 days), the fetal Pao, and Sao, were initially decreased from mean control values of 22.9 t 1.0 Torr and 58.3 t 2.0% to 13.8 t 0.5 Torr and 20.9 t 1.6%, respectively, and remained significantly reduced for the remainder of the 24-h period (Fig. 1). The fetal Sao, was significantly higher 18-20 h and 24 h after the induction of RUBF than at the beginning of the RUBF period (Fig. 1). During RUBF, the Pa co, was significantly increased initially but returned to control values after 4-6 h. Similarly, arterial pH was significantly decreased initially but then returned to control values by 12-18 h after induction of RUBF (Fig. 1). Fetal hemoglobin content also increased significantly initially but returned to control levels after 4-6 h of RUBF. At the end of the 24-h period it did not differ from the control period (9.1 t 0.6 vs. 9.1 t 0.7 g/dl). In control experiments (n = 5 fetuses), Sao,, Pao,, Pa and pH did not change significantly throughout the?&-h period of normoxia. They were similar at the beginning (Sao,, 56.8 t 2.2%; Pao,, 23.0 t 1.0 Torr; Pace,, 49.6 t 1.0 Torr; pH 7.342 t 0.009) and at the end of the 24-h period (57.2 t 1.9%, 22.0 t 1.0 Torr, 49.9 t 0.9 Torr, and 7.345 t 0.009, respectively). Lung liquid secretion rate. During the 3-h control
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0, I -
E E
N 0 a, u 0:
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129
. 60
So02 po2
(v
20-
-40 * *--,,a
* 102
1
1
1
St
St
1
1
o-o
pH
e- -0
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0 VI
* z 0
- 20 ii 0 St % LL
15 -
5 Q) LL
0
1 -0
FIG. 1. Fetal blood oxygen saturation (Sao,), arterial oxygen and carbon dioxide pressures (Pao, and Pa&, and pH values during control and RUBF periods. Values are means t SE. * Significantly different from control (P < 0.025).
m 74 .
- 7.3
--I
J
0 D
-72.
-5
I
n
D 7ii L
-ii LI
45 control
I 1-2
1 4-6
1 Q-11
Period of hypoxia
(h)
period, Vs values ranged between 3.4 and 20.8 ml/h in all fetuses (Fig. 2). After 24 h of RUBF, vs was significantly reduced from 9.1 t 1.5 to 3.0 t 0.5 ml/h (P c 0.005). In contrast, vs was not significantly different at the end of the 24-h period of normoxia (10.9 t 2.6 ml/h)
15
\1 24h 1I h-.24h =
1 -71. 24
I 18-20
from that measured at the start of this period (8.2 t 1.0 ml/h). Incidence of FBIM. A significant effect of treatment (i.e., normoxemia vs. RUBF) was found for the incidence of FBM but not for the amplitude of FBM. In both
hypoxia normoxia
FIG. 2. Fetal lung liquid secretion rates before and after a 24-h period of RUBF or normoxia. Values are means t SE. * Significantly different from control (P < 0.025).
Control
After
24h
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130
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normoxic control and RUBF fetuses, the incidence of FBM measured during 12-h control periods ranged between 24.0 min/h and 44.7 min/h in different animals (Fig. 3). The incidence of FBM was decreased (P < 0.05) initially by RUBF, but it progressively increased with time and had returned to control values 12-16 h after the start of RUBF. This return in FBM occurred despite the persistence of hypoxemia in these fetuses. The mean hourly amplitude of FBM measured during the period of RUBF (range 7.1-10.2 Torr) was not different from that measured during both the control and recovery periods (range 7.1-11.9 Torr). Tracheal fluid flow. The net volume of liquid flowing through the fetal trachea of RUBF-treated fetuses and the net volume flowing during periods of FBM are displayed in Fig. 4 and Table 1. These volumes were not significantly altered at any stage throughout the 24-h study period in normoxemic control fetuses. A significant effect of treatment (normoxia vs. RUBF) was found for the net volume of liquid flowing through the fetal trachea over consecutive 6-h periods. During the 12-h period preceding RUBF, the mean net volumes of liquid leaving the lungs via the trachea in each of the two 6-h periods were 34.8 t 8.1 and 41.2 t 10.7 ml (range 3.6-87.5 ml; Fig. 4). The mean net volumes of liquid leaving the lungs during episodes of FBM were 24.4 t 7.4 and 26.8 t 7.4 ml, respectively, during these 6-h control periods. The mean net volume of liquid leaving the lungs during periods of apnea, in the same 6-h periods, were 9.7 t 1.4 and 14.4 t 4.2 ml, respectively. These data indicate that under normal conditions -70% of liquid leaving the lungs does so during periods of FBM. The mean net volume of liquid leaving the lungs during apnea did not significantly differ from control values at any stage during the 24-h period of RUBF. During the first 6-h period of RUBF, the net volume of liquid leaving the lungs and the volume leaving the lungs during FBM were significantly reduced (to 16.8 t O-
50
f .-c E
40
22 1E!!
30
z Q) 0
1
l -
-0 24h 0 24h
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3.7 and -0.4 t 3.7 ml, respectively) compared with values recorded during the control period. A negative value indicates that a net movement of liquid into the lungs was measured. During the final two 6-h periods of RUBF, the direction of net liquid movement along the fetal trachea during periods of FBM was significantly reversed (Figs. 4 and 5); that is, V,, during FBM was significantly less than zero (P < 0.025). On average, FBM were associated with a mean net influx of liquid into the fetal lungs that ranged from 60.1 ml influx to 9.4 ml efflux over 6 h. Over the second 12 h of the 24-h RUBF period, when the incidence of FBM had returned to control values (Fig. 3), the reversal in the direction of fluid movement along the trachea during FBM caused a large net movement of liquid into the lungs (range 44.6 ml influx to 21.6 ml efflux over 6 h; Table 1). In some fetuses, when periods of FBM coincided with nonlabor uterine contractions, liquid moved out of the fetal lungs during the contractions and afterwards moved back in (Fig. 5). The total amount of liquid entering the fetal lungs over the 12- to 24-h period of RUBF varied considerably between animals and ranged from 69.0 ml into to 25.2 ml out of the fetal lungs (Table 1). In the first 6 h of the recovery period, the direction and magnitude of net liquid movement along the trachea in total and during periods of FBM returned to control values (Fig. 4, Table 1). Lung liquid uolume. In the control period, VL ranged between 43.9 and 199.1 ml in different fetuses. When expressed as a percentage of control VL, VL after 24 h of RUBF ranged from 74.4 to 189.8% of the control value (range 78.7-169.8 ml). Thus, although the mean VL measured after 24 h of RUBF (117.4 t 14.1 ml) was not different from the control value of VL (117.9 t 12.2 ml), both large increases and decreases in VL were observed in different fetuses. When expressed as a percentage of control values, the change in VL over the 24-h RUBF period was significantly correlated (r = 0.86, P < 0.005,
normoxia hypoxia
IK :
t) Q)-
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60
>
-
--3
FIG. 4. Total net volume of liquid entering or leaving lungs and volume entering or leaving lungs during periods of FBM over consecutive 6-h periods: 12 h of control, 24 h of hypoxia, and 12 h of recovery. Filled circles, mean volume of liquid; bars, range of volumes measured. Positive values indicate volumes leaving lungs; negative values indicate volumes entering lungs. During 24-h RUBF period, large volumes of liquid (up to 70 ml) entered lungs during periods of FBM in some fetuses.
60
80
control
24h hypoxia
Recovery
1. Net volumes of lung liquid leaving or entering the fetal lungs
TABLE
Net Volume Fetal Age, days
Ewe No.
Control*
Negative
values
133 134 129 118 130 119 127 125 126.8k2.1 indicate
volumes
94.7
124.6 95.9 86.9 32.6 77.8 22.5 34.7 141.5 77.1t15.5 of liquid
entering
75.0 50.5 15.7 57.8 17.7 17.6 92.5 52.7t11.7 lungs.
or Leaving
Lungs,
ml Recoveryt
Hypoxia? During FBM
Total
7001 7031 7062 7098 7107 7134 7149 8041 Means t SE
Entering
* 12-h control
n = 8) with the change in the incidence of FBM over the last 6 h of the RUBF period (Fig. 6). In normoxic control fetuses, mean VL was not different at the start of the 24h period (101.5 A 22.9 ml) from the value measured at the end of this period (116.2 t 27.8 ml). When expressed as a percentage of control, the change in VL over-the 24h period of normoxia was not correlated (r = -0.02) with
During FBM
Total
-11.7 21.1 4.6 -69.0 25.2 -35.1 -25.7 -22.7 - 14.7t11.0 period;
t second
-28.7 -22.7 -25.4 -62.6 20.1 -36.6 -25.5 -72.8 -31.8t9.9
97.7
109.0 84.6 33.9 87.2 40.5
8.2 9.4 80.7 10.8 100.1 7.6 44.9k17.1
111.1
71.7 76.9t11.5
12 h of a 24-h RUBF
the change in the incidence last 6 h of this period.
During FBM
Total
period;
of FBM
$12-h
recovery
period.
recorded over the
DISCUSSION Our aim was to examine the effects of 24 h of fetal hypoxemia, secondary to controlled RUBF, on fetal VL,
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1
1
INTEGRATED EMG DIAPHRAGM loop
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1 -I
I
l3 INTEGRATED TRACHEAL FLOW (ml)
o
FIG. 5. Polygraph recordings from a sheep fetus (125 days gestation) during a control period (top 4 tracings) and after 18 h of RUBF (bottom 4 tracings). Tracings from top to bottom: EMG activity of uterus, EMG activity of diaphragm shown as a time average, integrated tracheal fluid flow (integrator resets after net movement of 3 ml in either direction; efflux +, influx -), and fetal tracheal pressure (from which amniotic fluid pressure has been subtracted). During control period, FBM are associated with a net efflux of tracheal fluid. After 18 h of RUBF, a net influx of tracheal fluid occurs during FBM, except when they coincide with nonlabor uterine contractions, in which case efflux occurs. EMG calibration shows 100 PV square-wave signal at 500 Hz.
EMG UTERUS 1mV
INTEGRATED EMG DIAPHRAGM 1oopv
INTEGRATED TRACHEAL FLOW ( m 1)
I
/l/I
0
VI/
/I
-3 ITRACHEAL PRESSURE +” -AMNIOTIC 0 PRESSURE-j0 (mmHg)
w
10 mln
Vs, Vt,, and FBM. Our findings demonstrate that during prolonged periods of fetal hypoxemia, Vs remains inhibited, the incidence of FBM returns to control values after an initial inhibition, and the pattern of fluid movement along the fetal trachea is significantly altered. As a result of these changes, the maintenance of VL, and thus lung expansion, appears to be related to the incidence of FBM after they return. The fetuses that were subjected to RUBF remained hypoxemic throughout the 24-h period, although their Pace., and pH returned to control levels by the end of the 24-h period. The recovery in the Pace,, however, occurred at least 6 h before the recovery in pH. This recovery in Pace, and pH must have coincided with a change in placental function or with a change in the metabolic state of the fetus. An increased lactate metabolism or
clearance or decreased placental/fetal lactate production resulting from a reduction in fetal plasma glucose concentrations or fetal glucose metabolism (22) could have been responsible. It is unlikely that the buffering capacity of fetal blood increased because fetal hemoglobin concentrations were not different after 24 h of RUBF. However, an alteration in the ability of the fetal kidney to excrete acid is a possibility. Whatever the cause, this correction in blood pH is likely to be a very important adaptive response to maintain fetal viability. For example, the significant increase in fetal Sao, toward the end of the RUBF period occurred without an accompanying change in Pao,. This was most likely due to the increase in fetal blood pH, over this time period, producing an increase in the affinity of fetal hemoglobin for oxygen. At the end of a 24-h period of RUBF, vs was inhib-
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133
FIG. 6. Correlation (r = 0.86, n = 8, P < 0.005) between mean incidence of FBM over last 6 h of 24-h RUBF period and lung liquid volume after 24-h RUBF period.
0
40
80 FBM
(‘10
120 of Control)
ited to a similar degree to that observed during shorter periods of fetal asphyxia (19). In our previous report on the inhibition of Vs during acute fetal asphyxia, we suggested a number of possible mechanisms, including increased plasma AVP and/or epinephrine concentrations and decreased pulmonary oxygen delivery and/or blood flow (19). In this study, as in our previous study, the reduction in Vs during RUBF was not dependent on gestational age, whereas the inhibitory effects of both AVP and catecholamines clearly are. Epinephrine has very little effect on Vs before 125 days of gestation, but the degree of inhibition then progressively increases with gestational age and immediately before and during labor epinephrine causes reabsorption (8,21,27,33). Similarly, AVP has little effect on Vs before 134 days of gestation, but again the degree of inhibition increases with gestational age (29,32). Thus it is unlikely that either of these two hormones could be responsible for the inhibition of Vs during periods of RUBF until at least very late in gestation (i.e., X35 days). It is possible that a reduction in pulmonary blood flow or oxygen delivery could be responsible. The finding that the incidence of FBM returns to control values during prolonged fetal hypoxemia has been reported previously (4, 24). It is difficult to define precisely when the incidence of FBM starts to increase again in individual fetuses because their return was usually progressive and often variable. However, we estimate that increased incidence of FBM began 7-15 h (mean 12.0 t 0.9 h) after the start of RUBF. The return in FBM was found to be significantly correlated (r = 0.51, P < 0.001) with fetal arterial pH. However, because the fetus can utilize lactate as an energy source (2, 31), it is conceivable that a reduction in fetal plasma lactate concentrations, and thus an elevation in pH, is a consequence rather than a cause of the recovery of FBM.
160
200
During acute fetal hypoxemia (1 h) (7), without associated acidemia or hypercapnia, the incidence of FBM is inhibited, indicating that the detection of a lowered fetal oxygen content is involved. During prolonged fetal hypoxemia, the recovery of the incidence of FBM may therefore result from a change in the ability of the fetus to detect a lowered oxygen content, which may or may not be associated with a changing blood pH. Recently it has been demonstrated that the inhibition of FBM during hypoxemia in the fetus is associated with a distinct group of cells located within the upper lateral pons (14). The recovery of FBM during prolonged fetal hypoxemia may therefore result from an alteration in, or a selective inhibition of, these cells. With the reestablishment of FBM during prolonged RUBF, the net movement of fluid along the fetal trachea during periods of FBM was reversed. Normally there is a net efflux of tracheal fluid associated with periods of both FBM and apnea (13,16,18), as was clearly displayed during the control periods in this study (Fig. 5). Normally, V,, is greater during periods of FBM (-2-3 times) than during periods of apnea, despite occasional infuxes of fluid occurring during periods of augmented FBM (increased frequency and/or inspiratory effort). During prolonged RUBF, however, the direction of net fluid movement along the fetal trachea during periods of FBM was reversed. We also found in some fetuses that the occurrence of nonlabor uterine contractions during these periods of tracheal fluid influx either stopped this influx or caused efflux (Fig. 5). When the fetal upper airway is bypassed, thus creating a low-resistance pathway between the fetal lung and amniotic cavity, a pattern of fluid movement similar to that observed during prolonged RUBF occurs (15). During prolonged RUBF, a low-resistance pathway may also have been created. Normally there is a high resistance to fluid flow from the
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amniotic sac to the pharynx in the fetus, but this resistance may be lowered by mouth opening accompanied by tongue depression and protrusion (15, 17). Whatever the explanation, the influxes of fluid recorded during FBM were often large enough to result in the visible inhalation of amniotic fluid, as indicated by a color change in the lumen of the externalized tracheal loop catheters. The findings of this study may be of relevance to an understanding of aspiration of meconium by the fetus in utero. Meconium aspiration syndrome is often associated with respiratory failure in newborn infants and is thought to result from the displacement of surfactant within terminal sacs by the free fatty acids contained in meconium (9). Meconium aspiration has usually been attributed to fetal gasping during severe bouts of asphyxia (3). However, if gasping is induced in fetal sheep, a net movement of fluid into the fetal lungs does not occur; the volume of liquid inhaled during the gasp is usually exhaled at the end of the gasp (unpublished observations). It would appear from our study that FBM during prolonged hypoxemia could result in the entry of meconium into the fetal lungs, if it is present in amniotic fluid. It is not known how much amniotic fluid reached the terminal sacs in this study or how much mixing with lung liquid occurred at this level. Nevertheless, because some fetuses inhaled substantial volumes of liquid (up to 60 ml), it is highly probable that amniotic fluid and some meconium (if it were present) could have reached the terminal sacs of the lungs. After 24 h of RUBF, VL varied widely between fetuses but was closely and positively correlated with the incidence of FBM over the 1% to 24-h RUBF period. VL is determined by a balance between the net movement of fluid across the pulmonary epithelium and the net movement of fluid along the trachea. Normally liquid is secreted across the epithelium and leaves the lung via the trachea; over long periods (24 h) Vt, approximates Vs and VL is relatively constant. That is, for VL to remain constant, liquid must leave the lungs at the same rate at which it enters. Although FBM may cause transient increases or decreases in VL by augmenting and/or reversing the direction of Vt,, a change in the incidence of FBM is not normally associated with a change in VL. This was clearly displayed in the normoxic control fetuses where the change in FBM over the last 6 h of the 24-h period was unrelated to the change in VL (r = -0.02). Likewise, in normoxic fetuses, stimulation of FBM with indomethacin does not reverse the direction or increase the magnitude of V,, (unpublished observations). However, after 24 h of RUBF, the incidence of FBM was closely correlated with changes in VL, probably because the production of lung liquid was greatly reduced and because liquid moved into the lungs during FBM. Thus we have found evidence to suggest that, during prolonged RUBF, lung expansion is, in part, regulated by the incidence of FBM due to the inhalation of amniotic fluid. We are indebted surgical preparation grateful to Professor
to K. Billings and J. Norman for assistance in the and postsurgical care of the animals. We are also G. D. Thorburn for his interest in this project.
AND
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This work was supported by the National Research Council of Australia. Address reprint requests to S. B. Hooper. Received
21 November
1988; accepted
in final
Health
form
and
13 February
Medical
1990.
REFERENCES 1. ALCORN, D., T. M. ADAMSON, T. F. LAMBERT, J. E. MALONEY, B. C. RITCHIE, AND P. M. ROBINSON. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J. Anat. 123: 649-660, 1977. 2. BATTAGLIA, F. C., AND G. MESCHIA. Principal substrates of fetal metabolism. Physiol. Rev. 58: 499-527, 1978. 3. BLOCK, M. F., D. A. KALLENBERGER, J. D. KERN, AND D. NEPVEUX. In utero meconium aspiration by the baboon fetus. Obstet. Gynecol. 57: 37-40, 1981. 4. BOCKING, A. D., R. GAGNON, K. M. MILNE, AND S. E. WHITE. Behavioral activity during prolonged hypoxemia in fetal sheep. J. Appl. Physiol. 65: 2420-2426, 1988. 5. BOCKING, A. D., AND R. HARDING. Effects of reduced uterine blood flow on electrocortical activity, breathing and skeletal muscle activity in fetal sheep. Am. J. Obstet. Gynecol. 154: 655-662, 1986. 6. BOCKING, A. D., I. C. MCMILLEN, R. HARDING, AND G. D. THORBURN. Effects of reduced uterine blood flow on fetal and maternal cortisol. J. Dev. Physiol. Oxf. 8: 237-245, 1986. 7. BODDY, K., G. S. DAWES, R. L. FISHER, J. PINTER, AND J. S. ROBINSON. Foetal respiratory movements, electrocortical and cardiovascular responses to hypoxemia and hypercapnia in sheep. J. Physiol. Lond. 243: 500-518, 1974. M. J., R. E. OLVER, C. A. RAMSDEN, L. B. STRANG, AND 8. BROWN, D. V. WALTERS. Effects of adrenaline and of spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb. J. Physiol. Lond. 344: 137-152, 1983. D. A., G. F. NIEMAN, J. E. THOMPSON, A. M. PASKANIK, 9. CLARK, J. E. ROKHAR, AND C. E. BREDENBERG. Surfactant displacement by meconium free fatty acids: an alternative explanation for atelectasis in meconium aspiration syndrome. J. Pediatr. 110: 765770,1987.
K. E., M. DURNWALD, AND ?J. E. AUSTIN. A model for 10. CLARK, studying chronic reduction in uterine blood flow in pregnant sheep. Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H297-H301, 1982. 11. CLARK, K. E., AND C. A. SKILMAN. Methods of acute and chronic reduction of uteroplacental blood flow. In: Animal Models in Fetal Medicine, edited by P. W. Nathanielsz. Ithaca, NY: Perinatology, 1984,p. 37-57. 12. DICKSON, K. A., AND R. HARDING. Restoration of lung liquid volume following its acute alteration in fetal sheep. J. Physiol. Lond. 385: 531-544,1987. 13. DICKSON, K. A., J. E. MALONEY, AND P. J. BERGER. Stateregulated changes in lung liquid secretion and tracheal flow rate in fetal lambs. J. Appl. Physiol. 62: 34-38, 1987. 14. GLUCKMAN, P. D., ANII B. M. JOHNSTON. Lesions in the upper lateral pons abolish the hypoxic depression of breathing in unanesthetised fetal lambs in utero. J. Physiol. Lond. 382: 373-383, 1987. 15. HARDING, R., A. D. BOCKING, AND J. N. SIGGER. Upper airway resistances in fetal sheep: influence of breathing activity. J. Appl. Physiol. 60: 160-165, 1986. 16. HARDING, R., A. D. HOCKING, AND J. N. SIGGER. Influence of upper respiratory tract on lung liquid flow to and from fetal lungs. J. Appl. Physiol. 61: 68-74, 1986. 17. HARDING, R., P. JOHNSON, AND M. E. MCCLELLAND. Respiratory function of the larynx in developing sheep and the influence of sleep state. Respir. Physiol. 40: 165-179, 1980. 18. HARDING, R., J. N. SIGGER, P. J. D. WICKHAM, AND A. D. BOCKING. The regulation of flow of pulmonary fluid in fetal sheep. Respir. Physiol. 57: 47-59, 1984. 19. HOOPER, S. B., K. A. DICKSON, AND R. HARDING. Fetal lung liquid secretion, flow and volume in response to reduced uterine blood flow in fetal sheep. J. Dev. Physiol. Oxf. 10: 473-485, 1988. S. B., AND R. HARDING. Arginine vasopressin concentra20. HOOPER, tions increase in the fetus during periods of reduced uterine blood flow (Abstract). Proc. Aust. Physiol. Pharmacol. Sot. 18: 66P, 1987. 21. HOOPER, S. B., AND R. HARDING. Effects of ,+adrenergic blockade on lung liquid secretion during fetal asphyxia. Am. J. Physiol. 257
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(Regulatory Integrative Comp. Physiol. 26): R705-R710, 1989. 22. JONES, C. T., J. W. K. RITCHIE, AND D. WALKER. The effects of hypoxia on glucose turnover in the fetal sheep. J. Dev. Physiol. Oxf. 5: 223-235, 1983. 23. JONES, C. T., AND R. 0. ROBINSON. Plasma catecholamines in foetal and adult plasma. J. Physiol. Lond. 248: 15-33, 1975. 24. Koos, B. J., T. KITANAKA, K. MATSUDA, R. D. GILBERT, AND L. D. LONGO. Fetal breathing adaptation to prolonged hypoxaemia in sheep. J. Dev. Physiol. Oxf. 10: 161-166, 1988. 25. LIGGINS, G. C., AND J. A. KITTERMAN. Development of the fetal lung. In: The Fetus and Independent Life. London: Pitman, 1981, p. 308-330. (Ciba Found. Symp. 86). 26. MOESSINGER, A. C., R. HARDING, T. M. ADAMSON, M. SINGH, AND G. KIT. Local versus systemic factors at play in fetal lung growth and maturation (Abstract). Pediatr. Res. 25: 56A, 1989. 27. OLVER, R. E., C. A. RAMSDEN, L. B. STRANG, AND D. V. WALTERS. The role of amiloride-blockade of sodium transport in adrenalineinduced lung liquid reabsorption in the fetal lamb. J. Physiol. Lond. 376:321-340,1986. 28. OLVER, R. E., AND L. B. STRANG. Ion fluxes across the pulmonary epithelium and the secretion of the lung liquid in the foetal lamb.
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J. Physiol. Lond. 241: 327-357, 1974. 29. PERKS, A. M., AND S. CASSIN. The effects of arginine vasopressin on lung liquid secretion in fetal sheep. In: The Physiological Development of the Fetus and Newborn, edited by C. T. Jones and P. W. Nathanielsz. London: Academic, 1985, p. 253-257. 30. RURAK, D. W. Plasma vasopressin levels during hypoxaemia and the cardiovascular effects of exogenous vasopressin in foetal and adult sheep. J. Physiol. Lond. 277: 341-357, 1978. 31. SHELLY, H. J. The use of chronically catheterized fetal lambs for the study of fetal metabolism. In: Foetal and Neonatal Physiology. Cambridge, UK: Cambridge University Press, 1972, p. 360-381. 32. WALLACE, M. J., S. B. HOOPER, AND R. HARDING. Regulation of lung liquid secretion by arginine vasopressin in fetal sheep. Am. J. Physiol. 258 (Regulatory Integrative Comp. Physiol. 27): R104Rlll, 1990. 33. WALTERS, D. V., AND R. E. OLVER. The role of catecholamines in lung liquid absorption at birth. Pediatr. Res. 12: 239-242, 1978. 34. WIGGLESWORTH, J. S. Factors affecting fetal lung growth. In: Physiology of the Fetal and Neonatal Lung, edited by D. V. Walters, L. B. Strang, and F. Geubelle. Lancaster, UK: MTP, 1987, p. 2536.
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induced
STUART B. HOOPER AND RICHARD HARDING Department of Physiology, Monash University, Clayton, Victoria 3 168, Australia
HOOPER, STUART B., AND RICHARD HARDING. Changes in lung liquid dynamics induced by prolonged fetal hypoxemia. J. Appl. Physiol. 69( 1): 127-135,1990.-Our aim was to determine the effect of prolonged fetal hypoxemia, induced by reduced maternal uterine blood flow (RUBF), on fetal lung liquid secretion, flow, and volume. In chronically catheterized fetal sheep, lung liquid volume (Vi,) and the secretion rate of lung liquid (Vs) were measured before and after a 24-h period of either RUBF or normoxemia. Tracheal fluid flow and the incidence of fetal breathing movements (FBM) were measured before, during, and after the 24-h period. In normoxic control fetuses Vs was not significantly altered. After 24 h of RUBF, Vs was significantly (P < 0.005) reduced compared with pre-RUBF values. During 24 h of RUBF the incidence of FBM declined initially but returned to control values after 12-16 h. In seven of eight fetuses, over the l2- to 24-h period of RUBF, large amounts of liquid (22.7-62.6 ml) were drawn into the lungs during FBM, resulting in a net movement of amniotic fluid into the lungs. During the 1% to 24-h period of RUBF, changes in the incidence of FBM were found to be significantly and positively correlated (r = 0.86, P < 0.005) with the changes in VI, that occurred over the 24-h period. Thus, prolonged RUBF can result in the inhalation of large volumes of amniotic fluid by the fetus, which could be a cause of in utero meconium aspiration.
reduced uterine blood flow; lung liquid flow; fetal breathing movements
secretion;
tracheal
fluid
oflunggrowthinthe fetus is poorly understood, it is clear that expansion of the fetal lungs by liquid is a very important factor (25, 34). Sustained alterations in lung liquid volume (V,) have major effects on lung growth and pulmonary development (1). However, the mechanisms by which changes in lung expansion result in altered rates of tissue growth are not known, although they are likely to be exerted locally rather than systemically (26). Fetal lung liquid is secreted across the pulmonary epithelium into the future air spaces (28) and leaves the lungs via the trachea. Fetal VL is therefore determined by the balance between the rate of lung liquid secretion (Vs) and the net rate at which it flows out of the trachea (VJ. The production of fetal lung liquid is oxygen dependent (28) and is inhibited by arginine vasopressin (AVP) (29, 32) and epinephrine (21, 33), both of which are elevated in fetal plasma during fetal hypoxemia (20, 23, 30). In contrast, V,, is influenced by the resistance of the upper airway and is augmented during episodes of fetal breathing movements (FBM) when this
ALTHOUGHTHEREGULATION
0161-7567/90
$1.50
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resistance is low (15, 16, 18). Thus, Vs and V,, are able to change independently, resulting in changes in VL. We have recently shown that 4- to 6-h periods of moderate asphyxia in fetal sheep, secondary to controlled reduction .s in maternal uterine blood flow, significantly decrease both Vs and Tit, (19). VL did not change, presumably owing to simultaneous and equal reductions in Vs and V,,. It was not known, however, wh.ether VL would remain unchanged after longer periods of fetal hypoxemia. It seemed possible that changes in VL might occur because the incidence of FBM, which is depressed by acute hypoxemia (5, 7), eventually returns to control values after prolonged hypoxemia (4, 24). Thus, if Vs remains inhibited during periods of prolonged fetal hypoxemia and V,, is augmented by the return of FBM, we hypothesized that an imbalance between the two may result, leading to a change in VL. The aim of this study was to determine the effects of 24 h of fetal hypqxemia on Vs and VL and to characterize any changes in Vt, and the incidence of FBM over this period. METHODS
A nimal preparation. Surgery was performed on 14 Border Leicester x Merino ewes, between 105 and 115 days of pregnancy, under aseptic conditions. Anesthesia was induced by 5% sodium thiopental intravenously and was maintained, after tracheal intubation, with 1.5% halothane in oxygen and nitrous oxide (50:50 vol/vol). In all ewes, polyvinyl catheters (SV116, Dural Plastics, Australia) were implanted into the maternal jugular vein and amniotic cavity. An adjustable cl.amp was placed around the maternal common internal iliac artery to produce controlled reductions in uterine blood flow (5, 6, 10, 11, 19) In each fetus, two silicone rubber cannulas (no. 601365, Dow Corning) were implanted 2-4 cm into the trachea; one was directed toward the lungs, and the other was directed toward but did not enter the larynx (l&19). Fine stainless steel wire electrodes (no. AS632, Cooner) were implanted into the fetal diaphragm muscle to record its electromyogram. Polyvinyl catheters (SV65, Dural Plastics) were implanted into the carotid artery and jugular vein of each fetus. The fetal catheters, electrode wires, and control cable for the adjustable clamp were exteriorized through an incision in the right flank of the ewe. After surgery, benzyl-penicillin (400,000 units) and streptomycin (500 mg) were injected into the amniotic fluid. The exposed tracheal cannulas were j oined to form
0 1990 the American
Physiological
Society
127
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128
PROLONGED
HYPOXEMIA
an exteriorized tracheal loop, and the ewes were allowed to recover for at least 5 days after surgery. Lung liquid flow into and out of the fetal lungs was quantitated using a flowmeter connected in series with the tracheal loop (16, 18, 19). V,, was calculated by measuring the net volume of fluid that either entered or left the lungs per hour via the trachea. Vt, was given a positive value when it was directed away from the lungs. Experimental protocol. Uterine blood flow was reduced by constricting the maternal common internal iliac artery to produce a 50-60% reduction in the degree of oxygen saturation of hemoglobin (Sao,) in fetal carotid arterial blood. Measurements of fetal blood gases (Sao, and arterial Pop and PCO~ (Pao, and Pa+, pH, and hemoglobin content were made at frequent intervals (0, 1-2 h, 4-6 h, 9-11 h, 18-20 h, and 24 h) with a Radiometer ABL30 blood gas analyzer and a Radiometer OSM2 oximeter. The volume of fetal blood removed over 24 h was lo-15 ml. Fetal tracheal pressure, arterial pressure, and heart rate were also measured. Tracheal and arterial pressures were displayed on a polygraph (Grass model 7D) after subtraction of amniotic fluid pressure. The incidence and amplitude of FBM were determined from tracheal pressure recordings. The amplitude of FBM was measured at 5-min intervals during periods of FBM, and a mean value was calculated each hour. Each experiment on the effect of reduced uterine blood flow (RUBF) consisted of three consecutive periods: a control period, a period of RUBF, and a recovery period. Each control period lasted for 15 h; during the first 12 h we measured V,, and the incidence of FBM, and during the remaining 3 h we measured Vs and VL. During the period of RUBF (27 h) we measured V,, and the incidence of FBM in the first 24 h; then for 3 h we measured Vs and VL. The recovery period lasted for 12 h, during which we again measured Vtr and the incidence of FBM. In a separate series of experiments, L Iwe measured the normal daily variations in VL, Vs, Vtr, and FBM in normoxic fetuses. These experiments were the same as those described above except that RUBF was not induced. Thus, measurements of VL and Vs were made 24 h apart, and Vt,, and FBM were measured before, during, and after the 24-h study period. Vs and VL were measured using an established method of indicator dilution (12,19,28,29). We isolated the fetal lung lumen from the upper airway by connecting the descending tracheal cannula to a sterile loo-ml reservoir (open to atmosphere via bacterial filter) throughout the 3-h procedure. Lung liquid was drained into the reservoir, and an impermeant indicator (Dextran Blue 2000, Pharmacia, Uppsala, Sweden; 200 mg) was added. The indicator was thoroughly mixed by repeatedly draining the lung liquid into the reservoir and then returning it to the lungs over a period of 45 min. After this initial mixing period, lung liquid samples (0.75-3.0 ml) were collected at 15-min intervals for the rest of the 3-h period. The volume of liquid removed at each sample time was calculated to approximately equal the volume of liquid secreted over that time interval so that VL was not significantly altered. Vs was calculated from the rate of dilution of the indicator. The concentration of indicator
AND
FETAL
LUNG
LIQUID
in lung liquid was measured using a multichannel absorbance meter (Titertek Multiskan, Flow Laboratories, UK) set a wavelength of 620 nm. StatisticaL an&&s. The results presented in the text are means t SE. In both control and RUBF fetuses, the incidence and amplitude of FBM, V,, and blood gas, pH, and hemoglobin data were analyzed using a three-way analysis of variance (ANOVA) with treatment (normoxia vs. RUBF), time, and animals as factors. The two treatments were then analyzed separately using a two-way ANOVA followed by a Newman-Keuls multiple range test to determine significant differences between time periods. The level of significance was P < 0.05. vs was analyzed using a Student’s paired t test. V,, was assigned a negative value if liquid entered the lungs and a positive value if liquid left them. The direction of Vt, was therefore determined by comparison of the value for V,, with zero, by use of a Student’s paired t test. RESULTS
All fetuses survived until near term (140.3 t 1.2 days, term = 145 days), when the ewes and fetuses were killed by an overdose of pentobarbital sodium. At the start and end of each experiment, all fetuses were considered healthy, as judged by blood gas and pH measurements. Uterine blood flow was reduced for 24 h in 11 experiments on nine fetuses. However, the fetuses in three of these experiments were found to be normoxemic at the end of the 24-h period, despite the clamp being at its maximum adjustment. The mechanism by which normoxemia was achieved in these experiments is not known but could have been increased collateral blood supply and/or a greatly reduced fetal oxygen consumption. Because the mechanism is not known, these three experiments have been eliminated from the study. Fetal blood gas and pH measurements. In each RUBF experiment (n = 8 on 8 fetuses; mean gestational age 126.8 t 21.1 days), the fetal Pao, and Sao, were initially decreased from mean control values of 22.9 t 1.0 Torr and 58.3 t 2.0% to 13.8 t 0.5 Torr and 20.9 t 1.6%, respectively, and remained significantly reduced for the remainder of the 24-h period (Fig. 1). The fetal Sao, was significantly higher 18-20 h and 24 h after the induction of RUBF than at the beginning of the RUBF period (Fig. 1). During RUBF, the Pa co, was significantly increased initially but returned to control values after 4-6 h. Similarly, arterial pH was significantly decreased initially but then returned to control values by 12-18 h after induction of RUBF (Fig. 1). Fetal hemoglobin content also increased significantly initially but returned to control levels after 4-6 h of RUBF. At the end of the 24-h period it did not differ from the control period (9.1 t 0.6 vs. 9.1 t 0.7 g/dl). In control experiments (n = 5 fetuses), Sao,, Pao,, Pa and pH did not change significantly throughout the?&-h period of normoxia. They were similar at the beginning (Sao,, 56.8 t 2.2%; Pao,, 23.0 t 1.0 Torr; Pace,, 49.6 t 1.0 Torr; pH 7.342 t 0.009) and at the end of the 24-h period (57.2 t 1.9%, 22.0 t 1.0 Torr, 49.9 t 0.9 Torr, and 7.345 t 0.009, respectively). Lung liquid secretion rate. During the 3-h control
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PROLONGED
0, I -
E E
N 0 a, u 0:
25 -
HYPOXEMIA
0-0 -0 l -
0
AND
FETAL
LUNG
LIQUID
129
. 60
So02 po2
(v
20-
-40 * *--,,a
* 102
1
1
1
St
St
1
1
o-o
pH
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* z 0
- 20 ii 0 St % LL
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5 Q) LL
0
1 -0
FIG. 1. Fetal blood oxygen saturation (Sao,), arterial oxygen and carbon dioxide pressures (Pao, and Pa&, and pH values during control and RUBF periods. Values are means t SE. * Significantly different from control (P < 0.025).
m 74 .
- 7.3
--I
J
0 D
-72.
-5
I
n
D 7ii L
-ii LI
45 control
I 1-2
1 4-6
1 Q-11
Period of hypoxia
(h)
period, Vs values ranged between 3.4 and 20.8 ml/h in all fetuses (Fig. 2). After 24 h of RUBF, vs was significantly reduced from 9.1 t 1.5 to 3.0 t 0.5 ml/h (P c 0.005). In contrast, vs was not significantly different at the end of the 24-h period of normoxia (10.9 t 2.6 ml/h)
15
\1 24h 1I h-.24h =
1 -71. 24
I 18-20
from that measured at the start of this period (8.2 t 1.0 ml/h). Incidence of FBIM. A significant effect of treatment (i.e., normoxemia vs. RUBF) was found for the incidence of FBM but not for the amplitude of FBM. In both
hypoxia normoxia
FIG. 2. Fetal lung liquid secretion rates before and after a 24-h period of RUBF or normoxia. Values are means t SE. * Significantly different from control (P < 0.025).
Control
After
24h
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130
PROLONGED
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AND
normoxic control and RUBF fetuses, the incidence of FBM measured during 12-h control periods ranged between 24.0 min/h and 44.7 min/h in different animals (Fig. 3). The incidence of FBM was decreased (P < 0.05) initially by RUBF, but it progressively increased with time and had returned to control values 12-16 h after the start of RUBF. This return in FBM occurred despite the persistence of hypoxemia in these fetuses. The mean hourly amplitude of FBM measured during the period of RUBF (range 7.1-10.2 Torr) was not different from that measured during both the control and recovery periods (range 7.1-11.9 Torr). Tracheal fluid flow. The net volume of liquid flowing through the fetal trachea of RUBF-treated fetuses and the net volume flowing during periods of FBM are displayed in Fig. 4 and Table 1. These volumes were not significantly altered at any stage throughout the 24-h study period in normoxemic control fetuses. A significant effect of treatment (normoxia vs. RUBF) was found for the net volume of liquid flowing through the fetal trachea over consecutive 6-h periods. During the 12-h period preceding RUBF, the mean net volumes of liquid leaving the lungs via the trachea in each of the two 6-h periods were 34.8 t 8.1 and 41.2 t 10.7 ml (range 3.6-87.5 ml; Fig. 4). The mean net volumes of liquid leaving the lungs during episodes of FBM were 24.4 t 7.4 and 26.8 t 7.4 ml, respectively, during these 6-h control periods. The mean net volume of liquid leaving the lungs during periods of apnea, in the same 6-h periods, were 9.7 t 1.4 and 14.4 t 4.2 ml, respectively. These data indicate that under normal conditions -70% of liquid leaving the lungs does so during periods of FBM. The mean net volume of liquid leaving the lungs during apnea did not significantly differ from control values at any stage during the 24-h period of RUBF. During the first 6-h period of RUBF, the net volume of liquid leaving the lungs and the volume leaving the lungs during FBM were significantly reduced (to 16.8 t O-
50
f .-c E
40
22 1E!!
30
z Q) 0
1
l -
-0 24h 0 24h
FETAL
LUNG
LIQUID
3.7 and -0.4 t 3.7 ml, respectively) compared with values recorded during the control period. A negative value indicates that a net movement of liquid into the lungs was measured. During the final two 6-h periods of RUBF, the direction of net liquid movement along the fetal trachea during periods of FBM was significantly reversed (Figs. 4 and 5); that is, V,, during FBM was significantly less than zero (P < 0.025). On average, FBM were associated with a mean net influx of liquid into the fetal lungs that ranged from 60.1 ml influx to 9.4 ml efflux over 6 h. Over the second 12 h of the 24-h RUBF period, when the incidence of FBM had returned to control values (Fig. 3), the reversal in the direction of fluid movement along the trachea during FBM caused a large net movement of liquid into the lungs (range 44.6 ml influx to 21.6 ml efflux over 6 h; Table 1). In some fetuses, when periods of FBM coincided with nonlabor uterine contractions, liquid moved out of the fetal lungs during the contractions and afterwards moved back in (Fig. 5). The total amount of liquid entering the fetal lungs over the 12- to 24-h period of RUBF varied considerably between animals and ranged from 69.0 ml into to 25.2 ml out of the fetal lungs (Table 1). In the first 6 h of the recovery period, the direction and magnitude of net liquid movement along the trachea in total and during periods of FBM returned to control values (Fig. 4, Table 1). Lung liquid uolume. In the control period, VL ranged between 43.9 and 199.1 ml in different fetuses. When expressed as a percentage of control VL, VL after 24 h of RUBF ranged from 74.4 to 189.8% of the control value (range 78.7-169.8 ml). Thus, although the mean VL measured after 24 h of RUBF (117.4 t 14.1 ml) was not different from the control value of VL (117.9 t 12.2 ml), both large increases and decreases in VL were observed in different fetuses. When expressed as a percentage of control values, the change in VL over the 24-h RUBF period was significantly correlated (r = 0.86, P < 0.005,
normoxia hypoxia
IK :
t) Q)-
HYPOXEMIA
AND
FETAL
LUNG
131
LIQUID
60
>
-
--3
FIG. 4. Total net volume of liquid entering or leaving lungs and volume entering or leaving lungs during periods of FBM over consecutive 6-h periods: 12 h of control, 24 h of hypoxia, and 12 h of recovery. Filled circles, mean volume of liquid; bars, range of volumes measured. Positive values indicate volumes leaving lungs; negative values indicate volumes entering lungs. During 24-h RUBF period, large volumes of liquid (up to 70 ml) entered lungs during periods of FBM in some fetuses.
60
80
control
24h hypoxia
Recovery
1. Net volumes of lung liquid leaving or entering the fetal lungs
TABLE
Net Volume Fetal Age, days
Ewe No.
Control*
Negative
values
133 134 129 118 130 119 127 125 126.8k2.1 indicate
volumes
94.7
124.6 95.9 86.9 32.6 77.8 22.5 34.7 141.5 77.1t15.5 of liquid
entering
75.0 50.5 15.7 57.8 17.7 17.6 92.5 52.7t11.7 lungs.
or Leaving
Lungs,
ml Recoveryt
Hypoxia? During FBM
Total
7001 7031 7062 7098 7107 7134 7149 8041 Means t SE
Entering
* 12-h control
n = 8) with the change in the incidence of FBM over the last 6 h of the RUBF period (Fig. 6). In normoxic control fetuses, mean VL was not different at the start of the 24h period (101.5 A 22.9 ml) from the value measured at the end of this period (116.2 t 27.8 ml). When expressed as a percentage of control, the change in VL over-the 24h period of normoxia was not correlated (r = -0.02) with
During FBM
Total
-11.7 21.1 4.6 -69.0 25.2 -35.1 -25.7 -22.7 - 14.7t11.0 period;
t second
-28.7 -22.7 -25.4 -62.6 20.1 -36.6 -25.5 -72.8 -31.8t9.9
97.7
109.0 84.6 33.9 87.2 40.5
8.2 9.4 80.7 10.8 100.1 7.6 44.9k17.1
111.1
71.7 76.9t11.5
12 h of a 24-h RUBF
the change in the incidence last 6 h of this period.
During FBM
Total
period;
of FBM
$12-h
recovery
period.
recorded over the
DISCUSSION Our aim was to examine the effects of 24 h of fetal hypoxemia, secondary to controlled RUBF, on fetal VL,
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132
PROLONGED EMG UTERUS 1mV
1
1
INTEGRATED EMG DIAPHRAGM loop
HYPOXEMIA
AND
FETAL
LUNG
LIQUID
1 -I
I
l3 INTEGRATED TRACHEAL FLOW (ml)
o
FIG. 5. Polygraph recordings from a sheep fetus (125 days gestation) during a control period (top 4 tracings) and after 18 h of RUBF (bottom 4 tracings). Tracings from top to bottom: EMG activity of uterus, EMG activity of diaphragm shown as a time average, integrated tracheal fluid flow (integrator resets after net movement of 3 ml in either direction; efflux +, influx -), and fetal tracheal pressure (from which amniotic fluid pressure has been subtracted). During control period, FBM are associated with a net efflux of tracheal fluid. After 18 h of RUBF, a net influx of tracheal fluid occurs during FBM, except when they coincide with nonlabor uterine contractions, in which case efflux occurs. EMG calibration shows 100 PV square-wave signal at 500 Hz.
EMG UTERUS 1mV
INTEGRATED EMG DIAPHRAGM 1oopv
INTEGRATED TRACHEAL FLOW ( m 1)
I
/l/I
0
VI/
/I
-3 ITRACHEAL PRESSURE +” -AMNIOTIC 0 PRESSURE-j0 (mmHg)
w
10 mln
Vs, Vt,, and FBM. Our findings demonstrate that during prolonged periods of fetal hypoxemia, Vs remains inhibited, the incidence of FBM returns to control values after an initial inhibition, and the pattern of fluid movement along the fetal trachea is significantly altered. As a result of these changes, the maintenance of VL, and thus lung expansion, appears to be related to the incidence of FBM after they return. The fetuses that were subjected to RUBF remained hypoxemic throughout the 24-h period, although their Pace., and pH returned to control levels by the end of the 24-h period. The recovery in the Pace,, however, occurred at least 6 h before the recovery in pH. This recovery in Pace, and pH must have coincided with a change in placental function or with a change in the metabolic state of the fetus. An increased lactate metabolism or
clearance or decreased placental/fetal lactate production resulting from a reduction in fetal plasma glucose concentrations or fetal glucose metabolism (22) could have been responsible. It is unlikely that the buffering capacity of fetal blood increased because fetal hemoglobin concentrations were not different after 24 h of RUBF. However, an alteration in the ability of the fetal kidney to excrete acid is a possibility. Whatever the cause, this correction in blood pH is likely to be a very important adaptive response to maintain fetal viability. For example, the significant increase in fetal Sao, toward the end of the RUBF period occurred without an accompanying change in Pao,. This was most likely due to the increase in fetal blood pH, over this time period, producing an increase in the affinity of fetal hemoglobin for oxygen. At the end of a 24-h period of RUBF, vs was inhib-
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FIG. 6. Correlation (r = 0.86, n = 8, P < 0.005) between mean incidence of FBM over last 6 h of 24-h RUBF period and lung liquid volume after 24-h RUBF period.
0
40
80 FBM
(‘10
120 of Control)
ited to a similar degree to that observed during shorter periods of fetal asphyxia (19). In our previous report on the inhibition of Vs during acute fetal asphyxia, we suggested a number of possible mechanisms, including increased plasma AVP and/or epinephrine concentrations and decreased pulmonary oxygen delivery and/or blood flow (19). In this study, as in our previous study, the reduction in Vs during RUBF was not dependent on gestational age, whereas the inhibitory effects of both AVP and catecholamines clearly are. Epinephrine has very little effect on Vs before 125 days of gestation, but the degree of inhibition then progressively increases with gestational age and immediately before and during labor epinephrine causes reabsorption (8,21,27,33). Similarly, AVP has little effect on Vs before 134 days of gestation, but again the degree of inhibition increases with gestational age (29,32). Thus it is unlikely that either of these two hormones could be responsible for the inhibition of Vs during periods of RUBF until at least very late in gestation (i.e., X35 days). It is possible that a reduction in pulmonary blood flow or oxygen delivery could be responsible. The finding that the incidence of FBM returns to control values during prolonged fetal hypoxemia has been reported previously (4, 24). It is difficult to define precisely when the incidence of FBM starts to increase again in individual fetuses because their return was usually progressive and often variable. However, we estimate that increased incidence of FBM began 7-15 h (mean 12.0 t 0.9 h) after the start of RUBF. The return in FBM was found to be significantly correlated (r = 0.51, P < 0.001) with fetal arterial pH. However, because the fetus can utilize lactate as an energy source (2, 31), it is conceivable that a reduction in fetal plasma lactate concentrations, and thus an elevation in pH, is a consequence rather than a cause of the recovery of FBM.
160
200
During acute fetal hypoxemia (1 h) (7), without associated acidemia or hypercapnia, the incidence of FBM is inhibited, indicating that the detection of a lowered fetal oxygen content is involved. During prolonged fetal hypoxemia, the recovery of the incidence of FBM may therefore result from a change in the ability of the fetus to detect a lowered oxygen content, which may or may not be associated with a changing blood pH. Recently it has been demonstrated that the inhibition of FBM during hypoxemia in the fetus is associated with a distinct group of cells located within the upper lateral pons (14). The recovery of FBM during prolonged fetal hypoxemia may therefore result from an alteration in, or a selective inhibition of, these cells. With the reestablishment of FBM during prolonged RUBF, the net movement of fluid along the fetal trachea during periods of FBM was reversed. Normally there is a net efflux of tracheal fluid associated with periods of both FBM and apnea (13,16,18), as was clearly displayed during the control periods in this study (Fig. 5). Normally, V,, is greater during periods of FBM (-2-3 times) than during periods of apnea, despite occasional infuxes of fluid occurring during periods of augmented FBM (increased frequency and/or inspiratory effort). During prolonged RUBF, however, the direction of net fluid movement along the fetal trachea during periods of FBM was reversed. We also found in some fetuses that the occurrence of nonlabor uterine contractions during these periods of tracheal fluid influx either stopped this influx or caused efflux (Fig. 5). When the fetal upper airway is bypassed, thus creating a low-resistance pathway between the fetal lung and amniotic cavity, a pattern of fluid movement similar to that observed during prolonged RUBF occurs (15). During prolonged RUBF, a low-resistance pathway may also have been created. Normally there is a high resistance to fluid flow from the
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amniotic sac to the pharynx in the fetus, but this resistance may be lowered by mouth opening accompanied by tongue depression and protrusion (15, 17). Whatever the explanation, the influxes of fluid recorded during FBM were often large enough to result in the visible inhalation of amniotic fluid, as indicated by a color change in the lumen of the externalized tracheal loop catheters. The findings of this study may be of relevance to an understanding of aspiration of meconium by the fetus in utero. Meconium aspiration syndrome is often associated with respiratory failure in newborn infants and is thought to result from the displacement of surfactant within terminal sacs by the free fatty acids contained in meconium (9). Meconium aspiration has usually been attributed to fetal gasping during severe bouts of asphyxia (3). However, if gasping is induced in fetal sheep, a net movement of fluid into the fetal lungs does not occur; the volume of liquid inhaled during the gasp is usually exhaled at the end of the gasp (unpublished observations). It would appear from our study that FBM during prolonged hypoxemia could result in the entry of meconium into the fetal lungs, if it is present in amniotic fluid. It is not known how much amniotic fluid reached the terminal sacs in this study or how much mixing with lung liquid occurred at this level. Nevertheless, because some fetuses inhaled substantial volumes of liquid (up to 60 ml), it is highly probable that amniotic fluid and some meconium (if it were present) could have reached the terminal sacs of the lungs. After 24 h of RUBF, VL varied widely between fetuses but was closely and positively correlated with the incidence of FBM over the 1% to 24-h RUBF period. VL is determined by a balance between the net movement of fluid across the pulmonary epithelium and the net movement of fluid along the trachea. Normally liquid is secreted across the epithelium and leaves the lung via the trachea; over long periods (24 h) Vt, approximates Vs and VL is relatively constant. That is, for VL to remain constant, liquid must leave the lungs at the same rate at which it enters. Although FBM may cause transient increases or decreases in VL by augmenting and/or reversing the direction of Vt,, a change in the incidence of FBM is not normally associated with a change in VL. This was clearly displayed in the normoxic control fetuses where the change in FBM over the last 6 h of the 24-h period was unrelated to the change in VL (r = -0.02). Likewise, in normoxic fetuses, stimulation of FBM with indomethacin does not reverse the direction or increase the magnitude of V,, (unpublished observations). However, after 24 h of RUBF, the incidence of FBM was closely correlated with changes in VL, probably because the production of lung liquid was greatly reduced and because liquid moved into the lungs during FBM. Thus we have found evidence to suggest that, during prolonged RUBF, lung expansion is, in part, regulated by the incidence of FBM due to the inhalation of amniotic fluid. We are indebted surgical preparation grateful to Professor
to K. Billings and J. Norman for assistance in the and postsurgical care of the animals. We are also G. D. Thorburn for his interest in this project.
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This work was supported by the National Research Council of Australia. Address reprint requests to S. B. Hooper. Received
21 November
1988; accepted
in final
Health
form
and
13 February
Medical
1990.
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