Corticotropin and cortisol responses to corticotropin-releasing factor in the chronically hypoxemic ovine fetus DeWana R. Kerr, PhD: Maria I. Castro, PhD:' b Nancy K. Valego, PhD: Nayel M. Rawashdeh, MD, PhD, and James C. Rose, PhD"' b Winston-Salem, North Carolina OBJECTIVE: The purpose of this study was to determine if mild hypoxemia (- 25% below normal) of at least 5 days' duration alters corticotropin and cortisol responses to corticotropin-releasing factor. STUDY DESIGN: We studied 14 (hypoxemic, n = 5; normoxemic, n = 9) fetuses of 135 ± 1 (mean ± SEM) days' gestational age. Fetuses were placed in the experimental group if arterial P02 was :5 16 mm Hg for 5 days. In normoxemic animals arterial P02 was ~ 17 mm Hg. Plasma hormone responses were compared by analysis of variance. RESULTS: Resting corticotropin levels were not different (hypoxemic 26 ± 5 pg/ml, normoxemic 29 ± 12 pg/ml), and corticotropin-releasing factor (530 ± 30 ng/kg) increased (p = 0.01) corticotropin levels similarly in both groups. Basal plasma cortisol levels (hypoxemic 20 ± 10 ng/ml, normoxemic, 30 ± 7 ng/ml) were not significantly different. Both groups had similarly increased (p < 0.01) plasma cortisol levels after corticotropin-releasing factor administration. CONCLUSION: Mild hypoxemia lasting 5 days does not significantly alter corticotropin and cortisol responses to corticotropin-releasing factor in the late-gestation ovine fetus. (AM J OBSTET GYNECOL 1992;167:1686-90.)
Key words: Hypoxemia, fetus, sheep, corticotropin, cortisol
The stress of a reduction in oxygen supply leading to hypoxemia is a potentially serious danger faced by the fetus during gestation and labor. J Most fetal studies of hypoxemia have involved reducing fetal arterial P0 2 30% to 45% below normal. We refer to such conditions as a certain percent hypoxemia (e.g., 30% to 45% hypoxemia). Induction of such levels of hypoxemia and their maintenance for up to 7 hours increase plasma corticotropin concentrations in ovine fetuses between gestational ages of 96 and 145 days (term - 145 days),"·6 Corticosteroid responses to such hypoxemia are undetectable or low at the younger ages and greater later in gestation. 2 . 6 It has also been shown that adrenal secretion rates of corticosteroids are increased by profound hypoxemia. 7 The previously mentioned studies involve the acute induction of fetal hypoxemia late in gestation. The effects of long-term naturally occurring From the Departments of PhysiologylPharmacology" and ObstetricslGynecologyb and the Perinatal Research Laboratories, Bowman Gray School of Medicine, Wake Forest University. Supported by National Institutes of Health grant HD 11210. Presented in part at the Thirty-Seventh Annual Meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990. Reprint requests: James c. Rose, PhD, Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1083. 616/41932
1686
fetal hypoxemia on circulating corticotropin and cortisol levels and their responses to corticotropin-releasing factor (CRF) are unknown. In this report we describe results from a group of fetuses that manifested mild hypoxemia ( < 30% hypoxemia) in the absence of hypercapnia, acidosis, or elevations in hematocrit. We studied these animals to determine if mild hypoxemia, possibly naturally occurring, of ;;:: 5 days' duration altered resting plasma corticotropin and cortisol levels and the responses to synthetic ovine CRF.
Material and methods All surgical and experimental protocols were approved by the Animal Care and Use Committee of the Bowman Gray School of Medicine. We studied 14 chronically cannulated ovine fetuses at 135 ± 1 (mean ± SEM) days of gestation. Term in the sheep is approximately 145 days. Fetuses were placed in an experimental group (hypoxemic, n = 5) if the arterial P0 2 was ::;; 16 mm Hg for ;;:: 5 days. The arterial P0 2 levels were also lower at the time of surgery during maternal ventilation with 100% oxygen, which suggests that the condition probably existed before our receiving the animals in the laboratory. The control group (normoxemic, n = 9) consisted of fetuses with an arterial P0 2 of ;;:: 17 mm Hg. In an attempt to avoid the
Volume 167 Number 6
complicating effects of acidosis and hypercapnia on plasma corticotropin and cortisol levels, only fetuses with normal arterial pH and Pc0 2 values were included in this report. Surgery. Ewes with known single insemination dates in which pregnancy was confirmed at 50 days of gestation by ultrasonography were brought to the vivarium to acclimate them to the environment approximately 5 days before surgery. Food was withheld for 48 hours and water for 24 hours before surgery. Gentamicin (80 mg) (Elkins-Sinn, Cherry Hill, N.J.) was administered intravenously to the ewe before surgery. Anesthesia was induced with intravenous ketamine hydrochloride (Butler Veterinary Supply, Kernersville, N.C.) and pentobarbital sodium (Barber Veterinary Supply, Richmond, Va.) and maintained with 0.75% to 1.00% halothane delivered in 100% oxygen. Strict aseptic precautions were observed. Mter anesthesia, an intravneous infusion of Ringer's solution was started and continued throughout the course of surgery. The uterus was exposed through a midline abdominal incision, delivered, and covered in warm saline solution-moistened towels. The fetal hind limbs were located and exposed through a small uterine incision. Polyvinyl catheters were inserted into the right and left fetal tibial arteries and right saphenous vein and advanced to the descending aorta and inferior vena cava, respectively. A 0.5 ml sample of arterial blood was taken for determination of pH, Pco 2 , P0 2 , and hematocrit to assess animal well-being. Catheters were filled with 1000 D/ml heparin sodium. The exterioized portion of the fetus was then returned to the uterus, after which a polyvinyl catheter was placed in the amniotic cavity. Mter the injection of 80 mg of gentamicin into the amniotic fluid, the uterus was closed with a double row of sutures. Mter completion of the abdominal surgery, all fetal catheters were led to the left flank of the ewe and exteriorized. Polyvinyl chloride catheters were placed into the left maternal femoral artery and vein and advanced to the abdominal aorta and inferior vena cava, respectively. These catheters were filled with heparin, plugged, and exited through a flank incision. Daily the fetal and maternal vascular catheters were drained of heparin, flushed with sterile saline solution, filled with heparin, and plugged to maintain patency. Postoperatively the ewes were housed in metabolic carts in air-conditioned, light-cycled rooms. Experiment. Mter a 5-day recovery period from surgery, the animals were studied with the ewe in a portable metabolic cart. Food and water were withdrawn 1 to 2 hours before the experiment. Each experiment was preceded by 80 mg of gentamicin administered intravenously to the ewe. All experiments were initiated between 11 AM and 1 PM hours. Care was taken to conduct the experiments in a quiet environment. An aliquot of lyophilized ovine CRF (BaChem, Phil-
Plasma corticotropin and cortisol in chronic fetal hypoxemia
1687
adelphia) was diluted with a solution of 0.1 % bovine serum albumin in normal saline solution immediately before each experiment. Fetal weight was estimated on the basis of gestational age and later determined by back calculation from birth or postmortem weight, by assuming a 2% weight gain per day.s Control arterial blood samples were taken. A I-minute intravenous injection of ovine CRF (530 ± 30 ng/kg) in 2 ml of 0.1 % bovine serum albumin in normal saline solution was administered and followed by additional arterial sampling from the ewe and fetus at 30, 60, 120, 240, and 360 minutes after the injection. Arterial blood samples were taken at all sample times from the ewe and fetus for cortisol, blood gas, and hematocrit measurements. Fetal blood samples were also taken for corticotropin at these times. The total volume removed at each sample time was 2.0 m!. Hormone assays. Plasma immunoreactive corticotropin and cortisol concentrations were measured by radioimmunoassays, the characteristics of which have been described in detail elsewhere. g • 10 Blood gas determination. All blood gases and pH were measured at 39% C with a Radiometer ABL30 (Radiometer, Copenhagen) instrument. Statistical procedures. Weight, basal plasma hormone levels, blood gases, and hematocrit were compared with independent group t tests. Two-way analysis of variance for repeated measures was used to assess differences between hypoxemic and normoxemic groups in hormone changes across the length of the experiment. A significance level of p < 0.05 was used. Results
Weight, age, ovine CRF dose, and basal values of blood gases, pH, and hematocrit are shown in Table I. Weight was not different between the two groups. Resting pH, Pc0 2 , and hematocrit were not significantly different and did not change across the experiment. All were within normal limits. Arterial P0 2 levels during surgery (5 days before the experiment), 1 and 2 days before the experiment (hypoxemic group only), and at the beginning of the experiment are shown in Table II. Arterial P0 2 levels were lower in the hypoxemic group during catheterization in the presence of maternal ventilation. Values on days 1 and 2 before the study were not different from those on the day of the experiment in the hypoxemic group. At the beginning of the experiment arterial P0 2 levels remained significantly lower in the hypoxemic group than in the normoxemic group (p < 0.02). During the experiment arterial P0 2 levels did not change in either group. Fig. 1 shows fetal plasma corticotropin responses to the ovine CRF injection. Resting plasma corticotropin levels were not different. The increase in plasma corticotropin levels after ovine CRF administration was sig-
1688 Kerr et al.
December 1992 Am J Obstet Gyneco1
900
•
700
..--. ......
600
"'tlO
S
500
0..
400
'-"
..c: ...., CJ -
Key words: Hypoxemia, fetus, sheep, corticotropin, cortisol
The stress of a reduction in oxygen supply leading to hypoxemia is a potentially serious danger faced by the fetus during gestation and labor. J Most fetal studies of hypoxemia have involved reducing fetal arterial P0 2 30% to 45% below normal. We refer to such conditions as a certain percent hypoxemia (e.g., 30% to 45% hypoxemia). Induction of such levels of hypoxemia and their maintenance for up to 7 hours increase plasma corticotropin concentrations in ovine fetuses between gestational ages of 96 and 145 days (term - 145 days),"·6 Corticosteroid responses to such hypoxemia are undetectable or low at the younger ages and greater later in gestation. 2 . 6 It has also been shown that adrenal secretion rates of corticosteroids are increased by profound hypoxemia. 7 The previously mentioned studies involve the acute induction of fetal hypoxemia late in gestation. The effects of long-term naturally occurring From the Departments of PhysiologylPharmacology" and ObstetricslGynecologyb and the Perinatal Research Laboratories, Bowman Gray School of Medicine, Wake Forest University. Supported by National Institutes of Health grant HD 11210. Presented in part at the Thirty-Seventh Annual Meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990. Reprint requests: James c. Rose, PhD, Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1083. 616/41932
1686
fetal hypoxemia on circulating corticotropin and cortisol levels and their responses to corticotropin-releasing factor (CRF) are unknown. In this report we describe results from a group of fetuses that manifested mild hypoxemia ( < 30% hypoxemia) in the absence of hypercapnia, acidosis, or elevations in hematocrit. We studied these animals to determine if mild hypoxemia, possibly naturally occurring, of ;;:: 5 days' duration altered resting plasma corticotropin and cortisol levels and the responses to synthetic ovine CRF.
Material and methods All surgical and experimental protocols were approved by the Animal Care and Use Committee of the Bowman Gray School of Medicine. We studied 14 chronically cannulated ovine fetuses at 135 ± 1 (mean ± SEM) days of gestation. Term in the sheep is approximately 145 days. Fetuses were placed in an experimental group (hypoxemic, n = 5) if the arterial P0 2 was ::;; 16 mm Hg for ;;:: 5 days. The arterial P0 2 levels were also lower at the time of surgery during maternal ventilation with 100% oxygen, which suggests that the condition probably existed before our receiving the animals in the laboratory. The control group (normoxemic, n = 9) consisted of fetuses with an arterial P0 2 of ;;:: 17 mm Hg. In an attempt to avoid the
Volume 167 Number 6
complicating effects of acidosis and hypercapnia on plasma corticotropin and cortisol levels, only fetuses with normal arterial pH and Pc0 2 values were included in this report. Surgery. Ewes with known single insemination dates in which pregnancy was confirmed at 50 days of gestation by ultrasonography were brought to the vivarium to acclimate them to the environment approximately 5 days before surgery. Food was withheld for 48 hours and water for 24 hours before surgery. Gentamicin (80 mg) (Elkins-Sinn, Cherry Hill, N.J.) was administered intravenously to the ewe before surgery. Anesthesia was induced with intravenous ketamine hydrochloride (Butler Veterinary Supply, Kernersville, N.C.) and pentobarbital sodium (Barber Veterinary Supply, Richmond, Va.) and maintained with 0.75% to 1.00% halothane delivered in 100% oxygen. Strict aseptic precautions were observed. Mter anesthesia, an intravneous infusion of Ringer's solution was started and continued throughout the course of surgery. The uterus was exposed through a midline abdominal incision, delivered, and covered in warm saline solution-moistened towels. The fetal hind limbs were located and exposed through a small uterine incision. Polyvinyl catheters were inserted into the right and left fetal tibial arteries and right saphenous vein and advanced to the descending aorta and inferior vena cava, respectively. A 0.5 ml sample of arterial blood was taken for determination of pH, Pco 2 , P0 2 , and hematocrit to assess animal well-being. Catheters were filled with 1000 D/ml heparin sodium. The exterioized portion of the fetus was then returned to the uterus, after which a polyvinyl catheter was placed in the amniotic cavity. Mter the injection of 80 mg of gentamicin into the amniotic fluid, the uterus was closed with a double row of sutures. Mter completion of the abdominal surgery, all fetal catheters were led to the left flank of the ewe and exteriorized. Polyvinyl chloride catheters were placed into the left maternal femoral artery and vein and advanced to the abdominal aorta and inferior vena cava, respectively. These catheters were filled with heparin, plugged, and exited through a flank incision. Daily the fetal and maternal vascular catheters were drained of heparin, flushed with sterile saline solution, filled with heparin, and plugged to maintain patency. Postoperatively the ewes were housed in metabolic carts in air-conditioned, light-cycled rooms. Experiment. Mter a 5-day recovery period from surgery, the animals were studied with the ewe in a portable metabolic cart. Food and water were withdrawn 1 to 2 hours before the experiment. Each experiment was preceded by 80 mg of gentamicin administered intravenously to the ewe. All experiments were initiated between 11 AM and 1 PM hours. Care was taken to conduct the experiments in a quiet environment. An aliquot of lyophilized ovine CRF (BaChem, Phil-
Plasma corticotropin and cortisol in chronic fetal hypoxemia
1687
adelphia) was diluted with a solution of 0.1 % bovine serum albumin in normal saline solution immediately before each experiment. Fetal weight was estimated on the basis of gestational age and later determined by back calculation from birth or postmortem weight, by assuming a 2% weight gain per day.s Control arterial blood samples were taken. A I-minute intravenous injection of ovine CRF (530 ± 30 ng/kg) in 2 ml of 0.1 % bovine serum albumin in normal saline solution was administered and followed by additional arterial sampling from the ewe and fetus at 30, 60, 120, 240, and 360 minutes after the injection. Arterial blood samples were taken at all sample times from the ewe and fetus for cortisol, blood gas, and hematocrit measurements. Fetal blood samples were also taken for corticotropin at these times. The total volume removed at each sample time was 2.0 m!. Hormone assays. Plasma immunoreactive corticotropin and cortisol concentrations were measured by radioimmunoassays, the characteristics of which have been described in detail elsewhere. g • 10 Blood gas determination. All blood gases and pH were measured at 39% C with a Radiometer ABL30 (Radiometer, Copenhagen) instrument. Statistical procedures. Weight, basal plasma hormone levels, blood gases, and hematocrit were compared with independent group t tests. Two-way analysis of variance for repeated measures was used to assess differences between hypoxemic and normoxemic groups in hormone changes across the length of the experiment. A significance level of p < 0.05 was used. Results
Weight, age, ovine CRF dose, and basal values of blood gases, pH, and hematocrit are shown in Table I. Weight was not different between the two groups. Resting pH, Pc0 2 , and hematocrit were not significantly different and did not change across the experiment. All were within normal limits. Arterial P0 2 levels during surgery (5 days before the experiment), 1 and 2 days before the experiment (hypoxemic group only), and at the beginning of the experiment are shown in Table II. Arterial P0 2 levels were lower in the hypoxemic group during catheterization in the presence of maternal ventilation. Values on days 1 and 2 before the study were not different from those on the day of the experiment in the hypoxemic group. At the beginning of the experiment arterial P0 2 levels remained significantly lower in the hypoxemic group than in the normoxemic group (p < 0.02). During the experiment arterial P0 2 levels did not change in either group. Fig. 1 shows fetal plasma corticotropin responses to the ovine CRF injection. Resting plasma corticotropin levels were not different. The increase in plasma corticotropin levels after ovine CRF administration was sig-
1688 Kerr et al.
December 1992 Am J Obstet Gyneco1
900
•
700
..--. ......
600
"'tlO
S
500
0..
400
'-"
..c: ...., CJ -
