Development of tolerance to ethanol-induced suppression of breathing movements and brain activity in the near-term fetal sheep during short-term maternal administration of ethanol

The effect of short-term maternal ethanol administration on the ethanol-induced suppression of fetal breathing movements, electrocortical (ECoG) activity, and electroocular (EOG) activity was determined in the near-term fetal sheep. Twelve conscious instrumented pregnant ewes (between 125 and 139 days of gestation; term, 147 days) received 1-h intravenous infusion of 1 g ethanol/kg total body weight daily for six days (n = 6) or an equivalent volume of normal saline daily for six days (n = 6). On the seventh day, the ethanol- and saline-pretreated animals were administered 1 g ethanol/kg total body weight. A further six ewes received 1-h intravenous infusion of 1 g ethanol/kg total body weight (n = 3) or an equivalent volume of normal saline (n = 3) daily for thirteen days with both groups receiving 1 g ethanol/kg total body weight on day fourteen. Fetal ECoG and EOG activities, and fetal breathing movements were monitored continuously over the post- operative and experimental periods. Saline infusion had no significant effect on the parameters studied. Fetal breathing movements were suppressed for 8 h after the first ethanol dose, and were not significantly suppressed after fourteen days of once-daily, maternal ethanol administration. Low-voltage ECoG and EOG activities were suppressed for 3 h after the first ethanol dose, and were not significantly suppressed after seven days of repeated ethanol administration. Maternal and fetal blood gases and acid-base balance were not significantly affected by maternal ethanol administration. These data demonstrate that short-term maternal administration of ethanol results in the development of tolerance to ethanol in the mature fetus.     

Indomethacin reversal of ethanol-induced suppression of ovine fetal breathing movements and relationship to prostaglandin E2

The effects of indomethacin on the ethanol-induced suppression of fetal breathing movements and fetal arterial plasma and cerebrospinal fluid (CSF) PGE2 concentrations and maternal arterial plasma PGE2 concentration were determined in the near-term fetal lamb. Eight conscious instrumented pregnant ewes (between 130 and 133 days of gestation; term, 147 days) received 1-h maternal intravenous infusion of 1 g ethanol/kg total body weight, and the fetus received 6-h intravenous infusion of indomethacin (1 mg/h per kg fetal body weight) commencing 30 min later. Serial fetal and maternal arterial blood samples (n = 8) and fetal CSF samples (n = 5) were collected at selected times throughout the 12-h study for the determination of PGE2 concentration. Fetal breathing movements were monitored continuously throughout the experimental period. Maternal ethanol infusion resulted in initial suppression (P less than 0.05) of fetal breathing movements for 2 h below pretreatment value, followed by a rapid increase in the incidence of fetal breathing movements after the onset of fetal indomethacin treatment. Fetal and maternal plasma PGE2 concentrations and fetal CSF PGE2 concentration were increased (P less than 0.05) above the pre-infusion value during the administration of ethanol and 1 h thereafter. Fetal indomethacin treatment suppressed (P less than 0.05) to undetectable levels fetal plasma and CSF PGE2 concentrations, which then became similar (P greater than 0.05) to pretreatment by 12 h. There was a positive correlation between fetal plasma and CSF PGE2 concentrations. There was an inverse correlation between the incidence of fetal breathing movements and fetal CSF PGE2 concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine stimulates breathing in fetal sheep with brain stem section.

Breathing responses to adenosine were determined in 12 chronically catheterized fetal sheep (greater than 0.8 term) in which hypoxic inhibition of breathing had been eliminated by brain stem section. The caudal extent of transection varied from the rostral midbrain to the pontomedullary junction. Isocapnic hypoxia [delta arterial PO2 (PaO2) of -12 Torr] doubled the incidence and depth of breathing activity and increased the incidence of eye movements. Intra-arterial infusion of adenosine (0.30 +/- 0.03 mg.min-1.kg fetal wt-1) increased the incidence and amplitude of breathing without affecting blood gases. Adenosine did not significantly alter the incidence of eye activity. Intra-arterial injection of oligomycin (120 +/- 26 micrograms/kg fetal wt), an inhibitor of mitochondrial oxidative phosphorylation, also stimulated breathing activity. In four fetuses with brain stem section, peripheral arterial chemodenervation blunted the stimulatory effects of hypoxia on breathing activity and abolished altogether the excitatory effects of adenosine. It is concluded that 1) hypoxia and adenosine likely inhibit breathing in normal fetuses by affecting similar areas of the brain stem and 2) in fetuses with brain section, hypoxic hyperpnea depends on peripheral and central mechanisms, whereas adenosine stimulates breathing via the peripheral arterial chemoreceptors.     

Upper airway resistances in fetal sheep: the influence of breathing activity

Fetal breathing movements (FBM) and lung liquid volume are known to affect lung development, but little is known about mechanisms controlling movement of liquid through the upper respiratory tract (URT). Therefore we measured resistances of the URT in 8 unanesthetized fetal sheep during late gestation while FBM were monitored from pressures in the lower trachea or from electromyogram of respiratory muscles. URT resistance to liquid flow toward the amniotic sac increased from 3.5 +/- 1.9 Torr X ml-1 X min during episodes of FBM to 21.1 +/- 5.7 Torr X ml-1 X min during apnea. Laryngeal resistance during apnea was greater (P less than 0.001) than supralaryngeal resistance in each of six fetuses in which URT resistance was partitioned. Fetal paralysis abolished the increase in laryngeal resistance to efflux that was previously related to the high-voltage electrocortical state and apnea. We were unable to quantify URT resistance to fluid movement toward the lungs because the larynx acted as a valve, permitting flow toward the lungs only in the presence of FBM. The supralaryngeal portion of the URT also apparently acts as a valve, normally preventing the entry of amniotic fluid into the pharynx. These findings help to explain our earlier observations that efflux of liquid from the fetal lungs is greater during episodes of FBM than during apnea.     

The effects of indomethacin and prostaglandin E2 on the ethanol-induced suppression of ovine fetal breathing movements.

A study was performed to examine the role of prostaglandins (PGs) in the mechanism of the ethanol-induced suppression of FBM, in which the objective was to test the hypothesis that fetal administration of PGE2 can suppress the incidence of FBM following reversal of ethanol-induced suppression of FBM by indomethacin, a fatty acid cyclooxygenase inhibitor. Instrumented near-term pregnant ewes received 1-h maternal infusion of ethanol (1 g/kg maternal body weight) followed 0.5 h later by a 3-h fetal infusion of indomethacin (1 mg/kg fetal body weight/h), and then a 2-h fetal infusion of PGE2 (400 ng/kg fetal body weight/min). Prior to drug administration, FBM occurred approximately 36.1 +/- 2.6% of the time. FBM were suppressed during the period of ethanol infusion (9.6 +/- 1.7%); the ethanol-induced suppression of FBM was reversed by fetal indomethacin treatment (77.5 +/- 14.1%); shortly after the onset of fetal PGE2 infusion, the incidence of FBM decreased to a 2-h mean incidence of 14.1 +/- 4.2%, which was similar in magnitude to that observed after maternal ethanol infusion. After the completion of PGE2 infusion, the incidence of FBM rapidly increased to a peak incidence of 83.4 +/- 19.2%, which was indicative of a prolonged effect of indomethacin on FBM. The data indicate that PGs mediate the ethanol-induced suppression of ovine FBM and that the action of indomethacin to antagonize ethanol-induced suppression of FBM is primarily due to its inhibition of PG synthesis.     

Indomethacin inhibits thrombin-, but not thyroxin-stimulated resorption of fetal rat limb bones

The bone-resorbing effects of thrombin and thyroxin, two agents that stimulate resorption in neonatal mouse calvaria by prostaglandin-dependent mechanisms, were examined in cultures of fetal rat limb bones. Thrombin produced maximal resorption in the limb bone cultures at a concentration of 100 U/ml when bones were cultured in BGJ supplemented with 1 mg/ml bovine serum albumin. The effects of thrombin were partially inhibited by 0.5 and 10 uM indomethacin. Thrombin failed to elicit resorption when the limb bones were cultured in DMEM with 15% horse serum. Thyroxin stimulated the resorption of limb bones in both BGJ-albumin and DMEM-serum media. Resorption was elicited by thyroxin concentrations of 10 nM-10 uM. 30 uM thyroxin failed to stimulate resorption. The dose-response curve to thyroxin was shallow, and the agent did not produce maximal resorption. The bone-resorbing effects of thyroxin were not affected by 0.5 or 10 uM indomethacin.     

Indomethacin inhibits thrombin-, but not thyroxin- stimulated resorption of fetal rat limb bones

The bone-resorbing effects of thrombin and thyroxin, two agents that stimulate resorption in neonatal mouse calvaria by prostaglandin-dependent mechanisms, were examined in cultures of fetal rat limb bones. Thrombin produced maximal resorption in the limb bone cultures at a concentration of 100 U/ml when bones were cultured in BGJ supplemented with 1 mg/ml bovine serum albumin. The effects of thrombin were partially inhibited by 0.5 and 10 uM indomethacin. Thrombin failed to elicit resorption when the limb bones were cultured in DMEM with 15% horse serum.Thyroxin stimulated the resorption of limb bones in both BGJ-albumin and DMEM-serum media. Resorption was elicited by thyroxin concentrations of 10 nM − 10 uM. 30 uM thyroxin failed to stimulate resorption. The dose-response curve to thyroxin was shallow, and the agent did not produce maximal resorption. The bone-resorbing effects of thyroxin were not affected by 0.5 or 10uM indomethacin.     

Supraspinal FMRFamide antagonizes morphine-induced horizontal, but not vertical, locomotor activity

Morphine and the molluscan neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) were administered to mice alone or in combination intracerebroventricularly (ICV) and the effect on locomotor activity was measured. Morphine given alone (0.5 micrograms) significantly increased horizontal locomotor activity compared to vehicle-treated controls. FMRFamide at low doses (0.01-10 micrograms) had no effect of its own, but blocked the morphine-induced increase in horizontal locomotor activity. Unlike the opiate antagonist naloxone (1.0 micrograms), FMRFamide (up to 10 micrograms) had no effect on morphine-induced decrease in vertical activity. These data further support a role for FMRFamide as a modulator of opiate action, but comparison to naloxone suggests that FMRFamide might not act as a pure competitive antagonist of this opiate effect.     

The central effects of morphine on fetal breathing movements in the fetal sheep

The fetal respiratory and electrocortical effects of 0.6 microgram to 600 micrograms of morphine, administered into the lateral cerebral ventricle, have been studied in chronically catheterised, unanaesthetized fetal sheep at 115-135 days gestation. Morphine at 0.6 microgram had no effect on breathing movements or electrocorticographic activity, and at 6 micrograms induced a period of apnoea (43-122 min) but had no effect on electrocortical activity. Intravenous naloxone (2 mg bolus and infusion of 2 mg/kg/h for 2 h) to the fetus had no effect on this apnoea. Morphine at 60 micrograms induced an initial period of apnoea (30-65 min) followed by episodic but significantly deep breathing movements with no effect on electrocortical activity and at 600 micrograms induced an initial period of apnoea (22-95 min) which was followed by deep, irregular and continuous (126-302 min) breathing movements. During the apnoea electrocortical activity initially remained cyclic, but as apnoea progressed there was a gradual reduction in the voltage of the electrocorticogram to a low voltage state. Intravenous naloxone (2 mg bolus and infusion of 2 mg/kg/h for 2 h) reversed both the respiratory and electrocortical effects. The hyperventilation was also inhibited by hypoxia. Naloxone alone had no effect on fetal breathing activity.     

Effects of increased core temperature on breathing movements and electrocortical activity in fetal sheep

Core temperature of fetal sheep was raised by perfusing warm water through a loop implanted into the abdomen, or into the stomach via the oesophagus. Raising fetal temperature by 0.8-2 degrees C was associated with an increase in amplitude and incidence of breathing movements, and an increase in the proportion of breathing movements that occurred during high voltage electrocortical (ECoG) activity. Fetal hyperthermia was maintained for 8 h, but the augmentation of breathing movements did not last for more than 2-3 h. The results indicate that changes of maternal temperature caused by hot weather, exercise, fever, and possibly diurnal changes of body temperature could alter the amplitude and pattern of fetal breathing movements.     

Effect of beta-endorphin on fetal breathing movements in sheep

It has been suggested that endogenous opioids, such as beta-endorphin (beta-EP), act to depress respiration in the fetus and newborn. We have investigated the effect of infusing beta-EP either intravenously or into a lateral cerebral ventricle on breathing movements and electrocortical activity in eight fetal lambs between 116 and 133 days gestation. Intravenous infusion of beta-EP (200 or 500 micrograms over 1 h) increased plasma beta-EP concentrations 2- to 230-fold and was associated with a small decrease in the percent time spent breathing, from 57.8 +/- 9.1 to 51.3 +/- 8.2%/h (n = 6 exp). There was no change in the amount of high- or low-voltage electrocortical activity. Intracerebroventricular beta-EP infusion (1 or 2 micrograms beta-EP/min for 120 min) was not associated with any change of breathing movements (n = 5 exp) during the period of the infusion. However, in four experiments, in the 6-h period after the end of the beta-EP infusion there were episodes of 2-4 h when the percent time per hour spent breathing exceeded 70%. Electrocortical activity increased in amplitude and distinct episodes of high- and low-voltage activity were sometimes lost in these experiments. We conclude that high concentrations of beta-EP in plasma or cerebrospinal fluid do not totally suppress fetal breathing directly in the fetal lamb.     

Almitrine inhibits breathing movements in fetal sheep in utero

Whilst hypoxia stimulates fetal peripheral chemoreceptors, fetal breathing movements do not increase as hypoxia also has central effects. We wondered whether specific stimulation of the arterial chemoreceptors by almitrine would produce a stimulation of fetal breathing movements. When almitrine was given to 5 intact and 3 peripherally-chemodenervated fetal sheep in utero, fetal breathing movements rapidly ceased for 1-12 h. There was also a decrease in the amount of time spent in low voltage electrocortical activity. The effects of almitrine are therefore similar to those of hypoxia, and are independent of the peripheral chemoreceptors. Thus it may be a valuable tool in the study of the control of fetal breathing.     

Effects of moderate hypoxia on fetal electrocortical activity, eye movements, and breathing activity in sheep

Isocapnic hypoxaemia (delta PaO2 = -8.0 +/- 0.5 mmHg; delta CaO2 = -2.86 +/- 0.20 ml/dl) was produced in fetal sheep by having the ewe breathe for one hour a gas mixture (v/v) of 10.5% O2 and 1.5% CO2 in N2. Mean fetal heart rate, blood pressure, and incidence of low voltage electrocortical activity were not affected. However, the incidence of rapid-eye-movements and breathing activity was reduced by about 40%. Breathing movements during hypoxaemia had a mean inspiratory time, breath interval, and tracheal pressure amplitude which did not differ significantly from those during control experiments in which the ewe breathed air from the plastic bag. These observations suggest that hypoxia decreases the incidence of breathing movements but does not affect the amplitude or pattern of breathing activity and that it may reduce the incidence of eye movements and breathing activity through a common mechanism.     

Relationship between fetal electrocorticographic changes and umbilical blood flow in the near-term sheep fetus

Local blood flow was measured with radioactive microspheres in 9 near-term ewes 2 min into successive high and low voltage electrocortical activity states. In an additional 8 animals the umbilical blood flow was measured using an electromagnetic flow-probe on the common umbilical vein. The microsphere data indicated that the blood flow during low and high voltage electrocortical activity was 185 +/- 22 ml/min per kg of fetus (SEM) and 165 +/- 22 ml/min per kg of fetus (P less than 0.01) respectively. Using the electromagnetic flowprobe the average flow during low and high voltage electrocortical activity was 203 +/- 14 ml/min per kg of fetus and 196 +/- 13 ml/min per kg of fetus (P less than 0.05) respectively. We observed that the decrease in the umbilical blood flow preceded the change from low to high voltage electrocortical activity by approximately 1 min. In that time the flow is significantly lower than it was during the preceding measurements taken during the low voltage electrocortical activity periods. This depression was still significantly lower at 3 min into the high voltage electrocortical activity whereas at 5 min into the high voltage state it was elevated to near average values. We conclude that the umbilical blood flow, on the average, is lower in high voltage states than it is in low voltage states and that this change precedes the switch from low to high voltage electrocortical activity.     

Denervation of peripheral chemoreceptors decreases breathing movements in fetal sheep

The role of the peripheral chemoreceptors in the control of fetal breathing movements has not been fully defined. To determine whether denervation of the peripheral chemoreceptors affects fetal breathing movements, we studied 14 chronically catheterized fetal sheep from 120 to 138 days of gestation. In seven fetuses the chemoreceptors were denervated by bilateral section of the vagus and carotid sinus nerves; in seven others, sham operations were performed. We compared several variables during two study periods: 0-5 and 6-13 days after operation. In the denervated fetuses there were significant decreases in the incidence and amplitude of fetal breathing movements during both study periods. There were no differences between the two groups in incidence of low-voltage electrocortical activity, arterial pH and blood gas tensions, fetal heart rate, mean arterial blood pressure, or duration of survival after operation or birth weight. We conclude that denervation of the peripheral chemoreceptors decreases fetal breathing movements. These results indicate that the peripheral chemoreceptors are active during fetal life and participate in the control of fetal breathing movements.     

Effect of morphine on breathing and behavior in fetal sheep

To define the dose response of apnea and breathing to morphine we studied 12 fetuses at 116-141 days of gestation using our window technique. We instrumented the fetus to record electrocortical activity (ECoG), eye movements (EOG), diaphragmatic activity (integral of EMGdi), heart rate, carotid blood pressure, and amniotic pressure. Saline and morphine in doses of 0.03, 0.1, 0.5, 1, and 3 mg/kg were injected in random order in the jugular vein of the fetus during low-voltage ECoG. Fetuses were videotaped for evaluation of fetal behavior. We found 1) that saline did not elicit a response; 2) apnea, associated with a change from low- to high-voltage ECoG, increased from 2.2 +/- 1.5 (SE) min in two fetuses at a dose of 0.03 mg to 20 +/- 6.3 min in seven fetuses at 3 mg/kg (P less than 0.005); 3) the length of the breathing responses, associated with a change from high- to low-voltage ECoG, were 15 +/- 1.8 and 135.9 +/- 18.1 min (P less than 0.0005); 4) integral of EMGdi X frequency, an index equivalent to minute ventilation, increased from 1,763 +/- 317 arbitrary units to 10,658 +/- 1,843 at 1.0 mg/kg and then decreased to 7,997 +/- 1,335 at 3.0 mg/kg. These changes were related to a steady increase in integral of EMGdi, whereas frequency decreased at 3 mg/kg. There was an increase in breathing response to morphine plasma concentrations or morphine doses.(ABSTRACT TRUNCATED AT 250 WORDS)