Pulmonary feedback and gestational age-dependent regulation of fetal breathing movements

Prenatal lung development requires fetal breathing movements (FBM). To investigate the dependence of FBM on feedback originating from the lung, we hypothesized that pneumonectomy stimulates FBM. Time-dated fetal sheep underwent bilateral pneumonectomy, unilateral pneumonectomy, or sham surgery at 125-130 days gestation. The incidence of FBM decreased in sham-operated fetuses at 142 days versus 130 days (p = 0.013), but was unchanged across all gestational ages in bilaterally pneumonectomized fetuses (p > or = 0.52). In unilaterally pneumonectomized fetuses, the incidence of FBM remained unchanged until 139 days and was higher than that of the bilaterally pneumonectomized fetuses at 130-136 days gestation (p < or = 0.03). The amplitude of integrated diaphragmatic electromyographic activity (integralEMG(di)) and total respiratory output (frequency of breathing x integralEMG(di)) were lower in pneumonectomized fetuses versus sham-operated fetuses at later gestational ages (p < 0.05). These decreases in integralEMG(di) and total respiratory output were most pronounced at 142 days in bilaterally pneumonectomized fetuses versus sham-operated fetuses (p = 0.006 and 0.016, respectively). Low-voltage electrocortical activity (ECoG) increased, and high-voltage ECoG decreased, in unilaterally pneumonectomized fetuses compared with sham-operated fetuses (p = 0.04). In conclusion, we provide new evidence that feedback from the fetal lung modulates the incidence and various components of phrenic nerve output, suggesting a positive feedback mechanism between FBM and lung development.     

Changes in fetal organ flow during intrauterine mechanical ventilation with or without oxygen

To investigate whether the changes in circulation at birth are due to lung ventilation, changes in PaO2 or both we mechanically ventilated in utero the lungs of 10 fetal sheep (120-127 days of gestational age) five days after instrumentation under general anaesthesia. Electrocortical activity (ECoG), eye movements (EOG), electromyographic activity from diaphragm and posterior neck activity (EMG) and electrocardiogram (ECG) were recorded. Fetal catheters (artery and vein of the hindlimb, arteries of both forelimbs which in three occasions were advanced into the left ventricle, fetal trachea and amniotic cavity), and an endotracheal tube were placed. After recovery radioactive 15 mu microspheres (I125, Ce141, Sr85 and Sc46) were injected into the inferior vena cava or left ventricle during high voltage electrocortical activity before and after lung expansion with N2 and after expansion with O2 for two levels of PaO2. PaCO2 did not change. The percentage of spheres trapped in the lungs increased from 9.6% to 44% after expanding the lungs with N2 and to 90% when fetal PaO2 increased (P less than 0.001). Blood flow to different organs did not change during normoxic expansion but it decreased significantly to the brain (91 +/- 25 to 27 +/- 8 ml/min per 100g, [mean +/- SD]) placenta (160 +/- 57 to 54 +/- 33 ml/min/100g) and coronaries (239 +/- 91 to 117 +/- 60 ml/min per 100g) when PaO2 was increased. In conclusion fetal circulation responds to raised levels of PaO2 well before birth probably by a direct action of oxygen on the vessels.     

Changes in intra-abdominal pressure during postural and respiratory activation of the human diaphragm.

In humans, when the stability of the trunk is challenged in a controlled manner by repetitive movement of a limb, activity of the diaphragm becomes tonic but is also modulated at the frequency of limb movement. In addition, the tonic activity is modulated by respiration. This study investigated the mechanical output of these components of diaphragm activity. Recordings were made of costal diaphragm, abdominal, and erector spinae muscle electromyographic activity; intra-abdominal, intrathoracic, and transdiaphragmatic pressures; and motion of the rib cage, abdomen, and arm. During limb movement the diaphragm and transversus abdominis were tonically active with added phasic modulation at the frequencies of both respiration and limb movement. Activity of the other trunk muscles was not modulated by respiration. Intra-abdominal pressure was increased during the period of limb movement in proportion to the reactive forces from the movement. These results show that coactivation of the diaphragm and abdominal muscles causes a sustained increase in intra-abdominal pressure, whereas inspiration and expiration are controlled by opposing activity of the diaphragm and abdominal muscles to vary the shape of the pressurized abdominal cavity.     

Lack of correlation of severity of lung disease with the phosphatidylcholine concentration in fetal lung fluid from premature lambs at 133-136 days gestational age

Fetal lung fluid was collected following tracheotomy at the time of delivery of 40 premature lambs at 133-136 days gestational age. The concentration of phosphatidylcholine and saturated photophatidylcholine in fetal lung fluid was compared with the severity of lung disease of the lambs as assessed after 3 to 10 h of controlled mechanical ventilation with only peak inspiratory pressures varied to control the PCO2 values. Phosphatidylcholine concentration in fetal lung fluid did not correlate with the peak inspiratory pressures needed to ventilate the lambs, total lung compliance values, or the surfactant phosphatidylcholine pool sizes measured by alveolar wash after sacrifice. The ratio of saturated to total phosphatidylcholine was constant (0.55 +/- 0.02) and independent of concentration of phosphatidylcholine in the fetal lung fluid. The fetal lung fluid contained only about 0.7% of the final surfactant phosphatidylcholine pool released by the lambs to the alveoli after birth. Within a narrow gestational age range characterized by lung disease of widely varying severity, the phosphatidylcholine concentrations in fetal lung fluid were not predictive of the severity of lung disease.     

Increased pulmonary vascular filtration pressure does not alter lung liquid secretion in fetal sheep.

The purpose of this study was to determine whether an increase in pulmonary vascular filtration pressure affects net production of liquid within the lumen of the fetal lung. We studied 14 chronically catheterized fetal lambs [130 +/- 3 (SD) days gestation] before, during, and after a 4-h rapid (500 ml/h) intravenous infusion of isotonic saline. In seven fetuses we measured pulmonary arterial and left atrial pressures, lung lymph flow, and protein osmotic pressures in plasma and lymph. In eight lambs with a chronically implanted tracheal loop cannula, we measured the change in luminal lung liquid volume over time by progressive dilution of tracheally instilled 125I-albumin, which stays within the lung lumen. Saline infusion increased pulmonary vascular pressures by 2-3 mmHg and decreased the plasma-lymph difference in protein osmotic pressure by 1 mmHg. Lung lymph flow increased from 1.9 +/- 0.6 to 3.9 +/- 1.2 (SD) ml/h; net production of luminal lung liquid did not change (12 +/- 5 to 12 +/- 6 ml/h). Thus an increase in net fluid filtration pressure in the pulmonary circulation, which was sufficient to double lung lymph flow, had no significant effect on luminal lung liquid secretion in fetal sheep.     

Human diaphragmatic EMG: changes with lung volume and posture during supramaximal phrenic stimulation

If esophageal and chest wall recordings of diaphragmatic electromyographic activity (EMG) accurately reflect neural drive to this muscle, then compound muscle action potentials (CMAPs) produced by supramaximal stimulation of the phrenic nerve should not alter with changes in diaphragmatic position. Maximal CMAPs were therefore recorded 1) during changes in lung volume from near residual volume to near total lung capacity, 2) during isovolume maneuvers at different lung volumes, and 3) while subjects were lying, sitting, and standing. The areas of maximal CMAPs recorded with the gastroesophageal catheter increased 5.1 +/- 3.6 times (mean +/- SD) between these volumes, increased 2.4 +/- 1.3 times as the diaphragm descended during an isovolume maneuver (at functional residual capacity), and increased 4.4 +/- 2.4 times between the lying and standing positions. Because the stimuli were supramaximal, these changes in EMG reflect changes in the relationship between the esophageal electrodes and the diaphragmatic muscle fibers. Artifactual changes were also documented for surface electrodes on the chest wall. Because of these positional changes in maximal CMAPs, previous studies, which used integrated diaphragmatic EMG to document "reflex" changes in neural drive, should be reevaluated.     

Differential costal and crural diaphragm compensation for posture changes

The electromyographic (EMG) activities of the costal and crural diaphragm were recorded from bipolar fine-wire electrodes placed in the costal fibers adjacent to the central tendon and in the anterior portions of the crural fibers in 12 anesthetized cats. The EMG activities of costal and crural recordings were compared during posture changes from supine to head up and during progressive hyperoxic hypercapnia in both positions. The activity of both portions of the diaphragm was greater in the head up compared with supine posture at all levels of CO2; and increases in crural activity were greater than those in costal activity both as a result of changes in posture and with increasing CO2 stimuli. These results are consistent with the concept that diaphragm activation is modulated in response to changes in resting muscle length, and further, that neural control mechanisms allow separate regulation of costal and crural diaphragm activation.     

Fetal baboons convert 18:3n-3 to 22:6n-3 in vivo. A stable isotope tracer study

Using [13C]-tracers and direct fetal doses, we show for the first time that the fetal primate converts alpha-linolenic acid (18:3) to docosahexaenoic acid (22:6) in vivo, and we estimate the relative bioefficacy of the two substrates for brain 22:6 accretion. Pregnant female baboons consumed a diet free of long chain polyunsaturates (LCP), with n-6/n-3 ratio of 10/1. In the third trimester of pregnancy (normal gestation = 182 days), they were instrumented with chronic indwelling catheters in the maternal femoral artery and the fetal jugular artery. Doses of either [U-13C]-18:3 (18:3*, n = 3) or [U-13C]-22:6 (22:6*, n = 2) were administered directly to the fetus. Blood was collected from fetus and mother, and the fetus was taken by cesarean section when electromyographic activity indicated that parturition was imminent. Fetal liver, brain, retina, and retinal pigment epithelium (RPE) were collected, and (13)C fatty acids determined. In 18:3*- dosed animals, labeled n-3 LCP were detected in fetal plasma at 1 day post-dose and peaked at 2;-3 days; brain 22:6* was constant at 3, 5, and 9 days post-dose, at 0.57 +/- 0.03 percent of dose (%Dose). In 22:6*- dosed animals, brain 22:6* was similar at 3 and 9 days post-dose (4.64 +/- 0.43%Dose). From these data, we estimate that preformed 22:6 in the fetal bloodstream is 8-fold more efficacious for brain 22:6 accretion than is 18:3. Retina 22:6* was stable at about 0.0008%Dose from 3 to 9 days in 18:3-dosed animals, but RPE 22:6* dropped over the period; brain results were consistent with these observations. Liver showed about 0.5%Dose in 22:6* and in intermediary n-3 fatty acid metabolites 20:5* and 22:5* at 3 days post-dose, and declined afterward. Back-transfer of labeled fatty acids to the maternal bloodstream was measurable but not sufficient to compromise the quantitative conversion data in fetuses. We conclude 1) primate fetuses have the capacity to convert 18:3 to 22:6 in vivo; 2) fetal brain 22:6* as %Dose plateaus by 3 days post-dose; 3) fetal plasma 22:6 is about 8-fold more effective as a substrate for brain 22:6 accretion compared with 18:3; and 4) the fetal liver is likely to be an important site of 18:3 to 22:6 conversion.     

Alterations in respiratory muscle activation in the ischemic fatigued canine diaphragm

The purpose of the present study was to examine the respiratory motor response to diaphragm fatigue. Studies were performed using in situ diaphragm muscle strips dissected from the left costal diaphragm in anesthetized dogs. The left inferior phrenic artery was isolated, and diaphragmatic strip fatigue was elicited by occluding this vessel. Strip tension, strip electromyographic activity, parasternal electromyographic activity, and the electromyogram of the right hemidiaphragm were recorded during spontaneous breathing efforts before, during, and after periods of phrenic arterial occlusion. In separate trials, we examined the neuromuscular responses to phrenic arterial occlusion at arterial PCO2 (PaCO2) of 40, 55, and 75 Torr. No fatigue and no alteration in electromyographic activities were observed in trials at PaCO2 of 40 Torr. During trials at PaCO2 of 55 and 75 Torr, however, diaphragm tension fell, the peak height of the diaphragm strip electromyogram decreased, and the peak heights of the parasternal and right hemidiaphragm electromyograms increased. Relief of phrenic arterial occlusion resulted in a return of strip tension and all electromyograms toward base-line values. In additional experiments, the left phrenic nerve was sectioned in the chest after producing fatigue. Phrenic section was followed by an increase in the peak height of the left phrenic neurogram (recorded above the site of section). This latter finding suggests that diaphragm strip motor drive may be reflexly inhibited during the development of fatigue by neural traffic carried along phrenic afferents.     

The ratio of plasma bioactive to immunoreactive ACTH-like activity increases with gestational age in the fetal lamb.

The fetal ovine pituitary-adrenal axis plays an important role in the timing of parturition, in fetal lung maturation, and in fetal and neonatal responses to stress. While the ovine pituitary during the last third of gestation (term = 145 days) is capable of secreting immunoreactive ACTH (iACTH) in response to various stimuli, plasma cortisol levels frequently do not reflect the rise in plasma ACTH. Therefore, we examined the relationship between plasma iACTH and steroidogenic ACTH-like activity (bACTH) in a group of immature fetal lambs (Group I: gestational age = 97 +/- 2 days, mean +/- SEM, n = 16) and a group of near-term fetuses (Group II: gestational age = 136 +/- 1 days, n = 13) following acute exteriorization. Plasma iACTH was determined by RIA. Plasma bACTH was determined by the ability of glass-extracted material to stimulate corticosterone (B) production in an acutely dispersed rat adrenal bioassay. Plasma iACTH and bACTH levels varied among animals within age groups, with iACTH tending to be higher in immature fetal lambs (Group I) than near-term lambs (Group II) and bACTH being higher (P < 0.05) near term than earlier (Group I: iACTH = 807 +/- 273 pg/ml, bACTH = 173 +/- 44 pg/ml; Group II: iACTH = 405 +/- 85 pg/ml, bACTH = 371 +/- 96 pg/ml). The proportion of iACTH that had biologic activity (e.g. B/I ratio) was significantly greater in the older than in the younger fetuses (Group II: B/I = 0.862 +/- 0.109; Group I: B/I = 0.462 +/- 0.105 P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex-specific differences in rabbit fetal lung maturation in response to epidermal growth factor.

Males and females exhibit different stages of lung development at the same gestation with males lagging behind. We hypothesized that one of the mechanisms responsible for the sex-specific difference in fetal lung maturation is a delay in the onset of epidermal growth factor (EGF) activity in the male fetal lung. EGF influences growth and differentiation during development. We studied the effects of EGF on the incorporation of glycerol into lamellar body disaturated phosphatidylcholine (DSPC) in sex-specific fetal rabbit lung explants prepared at 21 and 24 days gestation (term 31 days). The explants were maintained in Waymouth's media + 10% stripped fetal calf serum with or without EGF (10 ng/ml). The incorporation of [1,3-14C]glycerol into lamellar body DSPC was assessed after 3, 5, or 7 days of culture. Female lung explants prepared at 21 days of gestation had increased incorporation of glycerol into DSPC over time in response to EGF treatment. Male lung explants prepared at 21 days did not respond to EGF treatment. In explants prepared at 24 days gestation, baseline glycerol incorporation into DSPC was higher in female as compared to male fetal lung explants. EGF-responsiveness was also sex-specific in these more mature explants, with the male explants now responding to EGF with a consistent increase in the incorporation of glycerol into lamellar body DSPC. We conclude that one of the mechanisms responsible for the lag in male fetal lung development is a delay in the onset of EGF activity.     

Effect of increased arterial CO2 on fetal breathing and behavior in sheep

We studied breathing and behavioral response to increased arterial CO2 (PaCO2) in 12 fetal sheep between 130 and 145 days of gestation. Of these 12 fetuses, 10 had an increase in PaCO2 through maternal rebreathing of CO2; in the other 2 fetuses CO2 was increased via an endotracheal tube and application of continuous distending airway pressure. We used our window technique to observe and videotape fetal behavior. The experiments consisted of recording breathing activity and behavior during resting conditions (1 low- and high-voltage ECoG cycle) and during administration of CO2. We measured electrocortical activity (ECoG), eye movements (EOG), electromyography of the diaphragm (EMGdi) and neck muscles, tracheal (Ptr), amniotic, and carotid arterial pressures. Administration of CO2 by the rebreathing technique produced an increase in the amplitude of breathing activity as reflected by an increase in Ptr from 5.0 +/- 0.6 to 12 +/- 1.9 mmHg (P less than 0.01) and an increase in SEMGdi from 32 +/- 4 to 77 +/- 8% max (P less than 0.001). Frequency increased due to a decrease in inspiratory (TI) and expiratory duration. Ptr/TI increased from 11.0 +/- 2.0 to 37.4 +/- 9.0 mmHg/s (P less than 0.05) and SEMGdi/TI increased from 67 +/- 7 to 221 +/- 28% max/s (P less than 0.001). Although the response was at times prolonged into the transitional high-voltage zone, it did not persist during established high-voltage ECoG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coordination between glottic adductor muscle and diaphragm EMG activity in fetal lambs in utero

It haspreviously been reported that active glottic adduction is presentduring prolonged apneas but absent during periods of breathingmovements in fetal lambs in utero. The present study was aimed atexamining the precise coordination between fetal breathing movements[diaphragm electromyographic (EMG) activity (Di EMG)] andglottic adduction [thyroarytenoid muscle EMG activity (TAEMG)]. Electrodes for electroencephalogram, eye movements, TAEMG, and Di EMG and an arterial catheter were surgically implanted infetal lambs 123-142 days postconception. Polygraphic recordings were performed without sedation while the ewe breathed room air (n = 11) or various gas mixtures(hypoxia, n = 5; hyperoxia,n = 4; hypercapnia,n = 5; hypercapnia+hyperoxia,n = 5). Tonic TA EMG was observedthroughout >90% of apneas (>6 s) in both non-rapid-eye-movement and rapid-eye-movement sleep, and when Di EMG frequency decreased inrapid-eye-movement sleep. In all but two fetuses, TA EMG was immediately inhibited when Di EMG appeared. Altering blood gases did not modify these results. In conclusion, Di EMG and TAEMG are well coordinated in late gestation in fetal lambs,except in a few cases. These findings may have consequencesfor understanding the pathogenesis of mixed/obstructiveapneas of prematurity.

     

Chemical and postural influence on scalene and diaphragmatic activation in humans

The electromyographic activity of the diaphragm (EMGdi) and scalene muscle (EMGsc) was studied in the supine and upright positions, respectively, during hyperoxic progressive hypercapnic rebreathing (HCVR) in five healthy males. End-expiratory esophageal pressure (EEPes) was quantified on a breath-to-breath basis as a reflection of altered end-expiratory lung volume. There was no significant difference in the slopes of EMGdi, expressed as a percentage of maximum at total lung capacity vs. minute volume of ventilation (VI), between the supine and upright positions [0.79 +/- 0.05 (SE) vs. 0.92 +/- 0.17, respectively]. In contrast, the slope of the regression line relating EMGsc to VI was steeper in the upright than in the supine position (0.69 +/- 0.05 vs. 0.35 +/- 0.04, respectively; P less than 0.005). Positive EEPes at comparable VI at the ends of HCVRs were of greater magnitude upright than supine (3.27 +/- 0.68 vs. 4.35 +/- 0.60 cmH2O, respectively, P less than 0.001). We conclude that altering posture has a greater effect on scalene and expiratory muscle activity than on diaphragmatic activity during hypercapnic stimulation.     

The influence of oligohydramnios on thoracic dimensions of fetal sheep.

Oligohydramnios frequently leads to lung hypoplasia in the fetus, but the underlying mechanisms are incompletely understood. Our aim was to determine the effects of oligohydramnios on the dimensions of the fetal thorax. Using pairs of implanted ultrasound transducers in 6 fetal sheep, we measured 4 thoracic dimensions (transverse, anterior-posterior, manubrium to left and right hand sides of the diaphragmatic dome) for 2 control days, 3 days of amniotic and allantoic fluid drainage (oligohydramnios), and 2 days after the return of drained fluids. The effect of oligohydramnios, which began at 121-2 days of gestation (term being c.145 days), on each dimension was quantified daily as the difference between the measured value and the value predicted from the growth of that dimension over the study period. Oligohydramnios led, within 48 hours, to significant reductions in the transverse dimension (5.9-6.1%) and in the distance between the manubrium and the dome of the diaphragm (1.7-2.2%). There was no change in the anterior-posterior dimension. We conclude that oligohydramnios causes alterations, within 48 hours, in the dimensions of the fetal thorax which can be reversed, at least partially, by re-expansion of the fluid sacs. These changes, which are expected to produce reductions in thoracic volume, may, if prolonged, lead to lung hypoplasia.     

Influence of genioglossus tonic activity on upper airway dynamics assessed by phrenic nerve stimulation.

Upper airway (UA) dynamics can be evaluated during wakefulness by using electrical phrenic nerve stimulation (EPNS) applied at end-expiration during exclusive nasal breathing by dissociating twitch flow and phasic activation of UA muscles. This technique can be used to quantify the influence of nonphasic electromyographic (EMG) activity on UA dynamics. UA dynamics was characterized by using EPNS when increasing tonic EMG activity with CO(2) stimulation in six normal awake subjects. Instantaneous flow, esophageal and nasopharyngeal pressures, and genioglossal EMG activity were recorded during EPNS at baseline and during CO(2) ventilatory stimulation. The proportion of twitches presenting an inspiratory-flow limitation pattern decreased from 100% at baseline to 78.7 +/- 21.4% (P = 10(-4)) during CO(2) rebreathing. During CO(2) stimuli, maximal inspiratory twitch flow (VI(max)) of flow-limited twitches significantly rose, with the driving pressure at which flow limitation occurred being more negative. For the group as a whole, the increase in VI(max) and the decrease in pressure were significantly correlated with the rise in end-expiratory EMG activity. UA stability assessed by EPNS is dramatically modified during CO(2) ventilatory stimulation. Changes in tonic genioglossus EMG activity significantly contribute to the improvement in UA stability.     

Validation of improved recording site to measure phrenic conduction from surface electrodes in humans.

Phrenic nerve stimulation, electrical (ES) or from cervical magnetic stimulation (CMS), allows one to assess the diaphragm contractile properties and the conduction time of the phrenic nerve (PNCT) through recording of an electromyographic response, traditionally by using surface electrodes. Because of the coactivation of extradiaphragmatic muscles, signal contamination can jeopardize the determination of surface PNCTs. To address this, we compared PNCTs with ES and CMS from surface and needle diaphragm electrodes in five subjects (10 phrenic nerves). At a modified recording site, lower and more anterior than usual (lowest accessible intercostal space, costochondral junction) with electrodes 2 cm apart, surface and needle PNCTs were similar (CMS: 6.0 +/- 0.25 ms surface vs. 6.2 +/- 0.13 ms needle, not significant). Electrodes recording the activity of the most likely sources of signal contamination, i.e., the serratus anterior and pectoralis major, showed distinct responses from that of the diaphragm, their earlier occurrence strongly arguing against contamination. With ES and CMS, apparently uncontaminated signals could be consistently recorded from surface electrodes.     

Blood circulation and oxygen transport in the fetal guinea pig

Anaesthetized fetal guinea pigs near term were studied under conditions, where maternal placental flow of haemoglobin was maintained within the normal range. The rate of maternal fetal equilibration of intravenously injected 3H2O was found to be similar as in unanaesthetized animals (half time 4 min) indicating that fetal circulation was undisturbed under the present experimental conditions. Umbilical blood flow as determined by a modified 3H2O method was 0.13 ml . min-1 . g-1 of fetal body mass. Radioactive microspheres, injected into the fetal saphenous (jugular) vein, were distributed to the placenta, the lower body, the upper body and the lungs at a ratio of 31(47):27(39):30(6):12(8). From these data, cardiac output was calculated (0.38 ml . min-1 . g-1) and found to be almost equally distributed between the placenta, the lower body and the upper body. There was preferential streaming of the inferior vena caval blood to the upper body. There was no evidence for flow through a ductus venosus. The O2-saturation in the fetal carotid arterial blood was 59 +/- 4%. The O2-supply to the fetal tissues was estimated to be 3 times the oxygen consumption.