INTRODUCTION

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ANTENATAL ADMINISTRATION of glucocorticoids is widely used to improve lung maturity and function in premature infants and has resulted in improved clinical outcomes in this group of patients. In addition to promoting lung development, antenatal glucocorticoids have significant effects on postnatal cardiovascular, renal, and endocrine functions (5, 9, 10). The mechanisms by which glucocorticoids facilitate postnatal circulatory function are uncertain but may be related to augmented vascular, cardiac, humoral, and autonomic functions.

Glucocorticoids result in significant abnormalities in vascular function, enhancing responsiveness to vasoconstrictors while reducing the activity of depressor systems (25, 41). However, in addition to these peripheral mechanisms, there are reasons to believe that neural mechanisms may also be involved in the blood pressure response to glucocorticoids. Central administration of hydrocortisone increases blood pressure and sympathetic activity in adult rats (36). Glucocorticoids also modulate baroreflex control of heart rate (HR) and renal sympathetic nerve activity (RSNA) and enhance the pressor effect of central administration of ANG II, likely through augmenting activation of the sympathetic nervous system (26-28). Our laboratory previously reported that antenatal administration of glucocorticoids increases the RSNA response at birth in prematurely delivered lambs and decreases the sensitivity of the HR baroreflex (30).

Glucocorticoids stimulate cytodifferentiation and induce expression of developmentally regulated proteins in a large number of tissues (3). Concerns have recently been expressed regarding repetitive courses of corticosteroids and the potential for long-term detrimental effects related to antenatal glucocorticoid exposure. In particular, data from a variety of sources suggest that prenatal glucocorticoid exposure may predispose an individual to hypertension later in life (4, 20). Understanding the mechanisms by which glucocorticoids alter cardiovascular function in the fetus and newborn is critically important. Studies of the effects of glucocorticoids on central autonomic function early in life may provide an important contribution to the etiology of hypertension in the adult. The present study was therefore designed to test the hypothesis that fetal adrenal cortisol production modulates developmental changes in arterial baroreflex function and sympathetic drive after birth. More specifically, we examined the effects of fetal adrenalectomy without (ADX) and with physiological cortisol replacement (ADX + F) on birth-related changes in systemic hemodynamics, RSNA, and baroreflex control of HR and RSNA in lambs delivered near term by cesarean section.

	METHODS

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Studies were performed on conscious, chronically instrumented fetal sheep. Pregnant ewes of Dorset and Suffolk mixed breeding were obtained from a local source; gestational ages were based on the induced ovulation technique as previously described (13). All surgical and experimental procedures were performed within the regulation of the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Guiding Principles in the Care and Use of Animals approved by the Council of the American Physiological Society and governed by the Animal Care and Use Committee of the University of Iowa were strictly adhered to.

Surgical preparations. The initial surgery, consisting of vascular catheter placement and bilateral adrenalectomy, was performed at 130-131 days gestation (term 145 days). This timing of surgery was chosen as it is before the beginning of the normal increase in plasma cortisol levels that occurs in sheep beginning ~2 wk before parturition (16). Briefly, after induction with 12 mg/kg of thiopental sodium (Abbott Laboratories, North Chicago, IL), anesthesia was maintained using a mixture of halothane (1%), oxygen (33%), and nitrous oxide (66%). After a maternal abdominal flank incision was performed, the uterus was partially externalized and opened over the fetal hindlimbs. Polyethylene catheters were placed into the fetal femoral arteries and veins bilaterally. A catheter for recording amniotic pressure was also secured to the fetal skin. With the use of a retroperitoneal approach, the adrenal was isolated, the vascular supply was ligated with suture, and the organ was removed. The flank incision was closed, the fetus was gently rotated, and the process was repeated on the other side. The fetus was returned to the uterus and after closure of all incisions, catheters were exteriorized through subcutaneous tunnels and placed in a cloth pouch on the ewe's flank. Ampicillin sodium was administered to the ewe intramuscularly before surgery (2 g) and infused into the amniotic cavity following surgery (2 g). After surgery, pregnant ewes were returned to individual pens and allowed free access to food and water. During the 3 days after surgery, the ewe received ampicillin sodium (1 g/day im).

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Physiological studies. Before the start of the experiments, the ewe was transferred to the laboratory in a small cart that was placed in a Faraday cage. The pregnant ewe was then sedated with diazepam (0.3 mg/kg), given an intravenous bolus infusion of vecuronium bromide (0.1 mg/kg), intubated, and ventilated to maintain venous blood gas values similar to those obtained during spontaneous respiration. Muscle paralysis was necessary to eliminate movements that interfere with nerve recording. Maternal sedation with diazepam and paralysis had no effect on fetal HR, arterial pressure, or plasma catecholamine concentrations (unpublished data). Diazepam was administered every 2 h while additional doses of vecuronium (0.1 mg/kg) were administered when movement was detected. During the experiments, a constant infusion of a solution of 5% dextrose and 0.2% NaCl was administered to the ewe at a rate of 125 ml/h and to the fetus at 100 ml · kg¹ · day¹. After intubation of the ewe, a 1-h stabilization period was allowed before the start of the experiment.

Experimental protocol. Fetal baseline values for HR, MABP, and RSNA were continuously recorded and averaged over 30 min. Fetal arterial blood for determination of blood gases and pH as well as plasma norepinephrine, epinephrine, cortisol, ANG II, and arginine vasopressin (AVP) levels were obtained at the completion of the baseline period. The volume of blood sampled from the fetus was replaced immediately with an equivalent volume of maternal blood to avoid any hemodynamic effects of sampling. Baroreflex function in the fetus was then determined by producing ramp changes in MABP with a continuous intravenous infusion of progressive doses of phenylephrine or nitroprusside (1-30 µg · kg¹ · min¹ over a 5- to 7-min period using a Harvard infusion pump) while simultaneously recording HR and RSNA. A 40- to 60-min recovery period was allowed for MABP, HR, and RSNA to return to baseline values before the alternative drug was administered. At the completion of the baroreflex studies, the amount of background noise in the fetal nerve signal was assessed by inhibiting nerve activity using an intravenous infusion of the ganglionic blocking agent tetraethylammonium bromide (10 mg/kg).

Analytic procedures. Arterial blood for pH, PCO₂, and PO₂ was collected anaerobically in heparinized syringes, and measurements were immediately determined using a BGM 1302 pH/blood gas analyzer (Instrumentation Laboratory, Lexington, MA). All blood gas values were corrected for fetal temperature. Measurements of plasma AVP and ANG II were determined by radioimmunoassay (University of Wisconsin School of Veterinary Medicine Radioimmunoassay Laboratory, Dr. M. Brownfield, Director). Plasma norepinephrine, epinephrine, and cortisol concentrations were determined by radioimmunoassay in our laboratory using commercially available kits according to the manufacturer's specifications (Katcombi RIA RE29291, KMI Diagnostics, Minneapolis, MN and Cortisol RIA kit, Diagnostic Products, Los Angeles, CA).

Computation and data analysis. RSNA was integrated and corrected by subtracting the background noise level obtained in the presence of ganglionic blockade. RSNA was normalized for each animal and expressed as the percentage of activity observed in the fetus; the amount of activity measured during the initial fetal baseline period was defined as 100%. Baroreflex function, expressed as the relationship between MABP and HR or integrated RSNA, was analyzed using a logistic sigmoid function (Sigma Plot, SPSS Science, Chicago, IL) according to the equation RSNA or HR = P₄ + P₁/{1 + exp[P₂(MABP  P₃)]}, where P₁ is the range between the upper and lower plateaus, P₂ is a coefficient to calculate the gain as a function of pressure, P₃ is the MABP at midrange of the curve, and P₄ is the lower plateau (14). The gain was calculated as the first derivative of the above equation. Statistical analyses of differences in HR, MABP, RSNA, baroreflex parameters, plasma hormone concentrations, and arterial blood gas values during the described study periods were performed using a two-way, repeated-measures analysis of variance, factoring for treatment group and time. If the F statistic was found to be significant (P < 0.05), comparison among means was performed by the Bonferroni t-test for multiple comparisons. Data exhibiting a lack of homogeneity of variances among groups were analyzed nonparametrically using the Kruskal-Wallis test. Differences were considered significant when P < 0.05. All results are expressed as means ± SE.

	RESULTS

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Arterial pH and blood gas values. Fetal arterial pH, PO₂, and PCO₂ were similar between the two groups (Table 1). Similar increases in PO₂ occurred in both groups after birth. PCO₂ significantly decreased after birth in both groups, although newborn values did not differ between ADX and ADX + F lambs. Fetal arterial pH was also similar in both groups. After delivery, pH remained similar to fetal values in the ADX group. However, arterial pH was significantly greater after birth in the ADX + F compared with ADX lambs.

Effect of adenalectomy and cortisol replacement on baseline hemodynamics and RSNA. The changes in HR, MABP, and RSNA occurring with delivery in ADX and ADX + F sheep are shown in Table 2. HR was similar in both fetal groups and increased after birth in ADX + F but not ADX lambs (P < 0.05). MABP was significantly lower in ADX compared with ADX + F fetuses. After delivery, MABP did not increase in ADX animals (44 ± 2 vs. 44 ± 3 mmHg for fetus and 1-h newborn, respectively), whereas MABP significantly increased after birth in ADX + F lambs and remained elevated for the duration of the study. Large differences in the sympathetic response at birth, as determined by RSNA, were also seen between groups, being significantly greater in ADX + F animals than in ADX lambs at 1 and 3 h. More specifically, RSNA almost doubled in ADX + F lambs 1 h after delivery and remained at this level for the duration of the study. In contrast, RSNA was similar before and after birth in ADX animals.

Effect of adenalectomy and cortisol replacement on baroreflex control of HR and RSNA. Cortisol replacement had no effect on the upper or lower plateaus or the gain (slope) of the fetal HR baroreflex (Table 3 and Fig. 1). The HR baroreflex curve was shifted to the right in ADX + F compared with ADX fetuses, as indicated by the significant increase in the curve midpoint pressure (54 ± 3 vs. 45 ± 2 mmHg for ADX + F and ADX, respectively). A similar effect of cortisol was seen for the fetal RSNA baroreflex curves with a significant shift to the right for the curve midpoint pressure in the ADX + F (56 ± 4 mmHg) compared with the ADX fetus (46 ± 2 mmHg) but no change in the gain or curve plateau values (Table 4 and Fig. 2).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of adrenalectomy (ADX) and ADX with cortisol replacement (ADX + F) on baroreflex control of heart rate before (fetus; A) and after birth (newborn; B). Curves are generated from mean values for baroreflex function parameters. Standard error for upper and lower plateaus and curve midpoint are depicted. Individual values for baroreflex function parameters are presented in Table 3. , Resting mean arterial blood pressure (MABP) and heart rate. bpm, Beats/min.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of ADX and ADX + F on baroreflex control of renal sympathetic nerve activity (RSNA) before (fetus; A) and after birth (newborn; B). RSNA is expressed as a percentage of activity seen in the fetus at rest, defined as 100%. Curves are generated from mean values for baroreflex function parameters. Standard error for upper and lower plateaus and curve midpoint are depicted. Individual values for baroreflex function parameters are presented in Table 4. , Resting MABP and RSNA.

Effect of adenalectomy and cortisol replacement on circulating hormone concentrations. In both groups of animals, fetal and newborn plasma epinephrine concentrations were undetectable. No differences were detected between fetal values for norepinephrine, ANG II, or AVP (Table 5). After birth, norepinephrine, ANG II, and AVP significantly increased above fetal values in ADX lambs, whereas only norepinephrine increased in ADX + F animals. The postnatal increases in plasma norepinephrine concentrations were similar in both groups. Plasma cortisol concentrations in ADX animals were below the level of sensitivity of the assay (~5 ng/ml). In ADX + F fetuses, plasma cortisol concentration was 58 ± 9 ng/ml at the time of study, and it decreased to 19 ± 8 ng/ml 1 h after delivery and ~90 min after stopping the cortisol infusion.

	DISCUSSION

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The results from this study demonstrate that physiological replacement of cortisol in adrenalectomized fetal lambs results in higher fetal blood pressure and greater postnatal increases in HR, MABP, and RSNA. In addition, cortisol replacement results in a resetting or shift of the efferent limb of the HR and RSNA baroreflex toward higher arterial pressure. The immediate postnatal increases in resting HR, MABP, and RSNA and the resetting of the HR and RSNA baroreflex curves are similar to those previously described by this laboratory in intact lambs (31), suggesting that the natural prepartum increase in fetal plasma cortisol concentration is vital for normal autonomic and sympathetic function at birth.

Studies of the ovine fetus demonstrate a progressive increase in fetal plasma cortisol levels during the last 2 wk of gestation that likely prepares the fetus for the transition to extrauterine life (16, 19). The increasing availability of endogenous glucocorticoids late in fetal life modulates the development and rate of differentiation of numerous tissues, the most well studied being the lung. However, fetal adrenal cortisol production also plays an important role in cardiovascular development. Unno et al. (39) reported that bilateral fetal adrenalectomy attenuates the normal gestational age-dependent increase in fetal blood pressure occurring in late gestation, whereas physiological replacement of cortisol produced a sustained increase in blood pressure. Although the rise in fetal blood pressure associated with administration of glucocorticoids appears related to increased peripheral vascular resistance, the mechanisms for this are unclear. In vivo glucocorticoids increase smooth reactivity and enhance pressor responsiveness (2, 6). Cortisol infusion increases the pressor response to intravenous ANG II in intact ovine fetuses, although several studies suggest the peripheral renin-angiotensin system does not mediate the fetal blood pressure response to glucocorticoids (12, 29, 37). Glucocorticoids also reduce the activity of depressor systems, including vasodilator prostaglandins and nitric oxide (41). Clearly, more studies investigating the effects of glucocorticoids and fetal hemodynamic regulation are needed.

Padbury et al. (21) demonstrated that HR, blood pressure, and cardiac output fail to increase after birth in adrenalectomized near-term ovine fetuses. Because replacement doses of hydrocortisone were administered, the authors attributed the attenuated physiological responses to the lack of adrenal epinephrine secretion. Our findings of increased postnatal HR and MABP in ADX + F animals contrast with those of Padbury and suggest that the normal increase in circulating epinephrine is not vital for these responses. The primary difference between the studies is the amount of cortisol replacement. In the Padbury study, plasma cortisol levels were significantly lower in adrenalactomized compared with control animals and below the normal prepartum levels. On the other hand, we achieved plasma cortisol levels well within the physiological range present immediately before delivery. Because newborn systemic hemodynamic measurements in ADX + F animals were similar to those reported by others and us in intact animals, we suggest that adequate levels of circulating glucocorticoids are of primary importance in cardiovascular adaptation at birth (23, 31, 32). Antenatal administration of glucocorticoids augments cardiovascular performance in prematurely delivered lambs, despite these animals' having lower plasma epinephrine levels than controls (22, 30). Taken together, these findings support an important role for glucocorticoids in postnatal hemodynamic adaptation.

The lack of increase in RSNA at birth in ADX animals is similar to that which we previously reported in prematurely delivered lambs (30). Exogenous administration of glucocorticoids, either via physiological replacement of cortisol, as in the present study, or antenatal maternal administration of betamethasone, as performed in our study of prematurely delivered lambs, impacted significantly on the changes in RSNA after birth. Thus corticosteroids appear to have a maturational effect on the sympathetic response at birth inasmuch as steroid-treated animals had increases in RSNA after birth, similar to that seen in intact near-term animals (31). We speculate the augmented sympathetic outflow and enhanced noradrenergic function contribute to the increased postnatal blood pressure seen in these animals.

Relatively little is known about the physiological impact of glucocorticoids on sympathetic function, and no consensus exists as to the effects. The majority of studies in adult animals and humans demonstrate that resting or stimulated sympathetic nerve activity is decreased or unchanged by glucocorticoids (7, 15, 40). Central administration of hydrocortisone acutely increases blood pressure and sympathetic activity in adult rats (36). Our finding of an attenuated RSNA response at birth in ADX relative to ADX + F lambs and intact lambs (31) suggests the effects of glucocorticoids on the sympathetic nervous system may differ depending on the stage of development. Central autonomic function may be influenced by glucocorticoids by the integration of multiple mechanisms, including the regulation of genes encoding neuropeptides and ion channels, neuronal excitability, and neurosecretion (17). Studies in a number of species show that glucocorticoid receptors in the brain are preferentially concentrated within the limbic system, the nucleus of the solitary tract, and the paraventricular nucleus of the hypothalamus, these latter two regions being strongly implicated as cardiovascular control centers (1, 18). In fact, we recently demonstrated that the paraventricular nucleus participates in regulating the sympathoexcitatory response at birth (8). In young rats, glucocorticoids increase phenylethanolamine N-methyltransferase within the hypothalamus, accelerate development of central noradrenergic function, and induce expression of the norepinephrine transporter (33, 34, 38). Alterations in serotonin concentrations, an important central neurotransmitter, are also present in the hypothalamus and brain stem of rat pups exposed to prenatal dexamethasone (17). In addition to influencing the expression of target genes (genomic effects), glucocorticoids may have rapid, nongenomic effects by interacting with specific membrane receptors and involving traditional second messengers. Iontophoretic application of glucocorticoids acutely increases activity of barosensitive cardiovascular neurons in the rostral ventral lateral medulla and has rapid effects on neural activity in the hypothalamus (11, 24). Further studies are required to increase our understanding of these potential effects of glucocorticoids on centrally mediated sympathetic outflow.

The in vivo effects of exogenous administration of glucocorticoids on function of the arterial baroreflex in adult rats have been recently examined. Scheuer and colleagues (26, 28) reported that elevation of corticosterone levels reset baroreflex control of HR and RSNA toward higher pressure and reduced the gain of the responses. These effects were reversed within hours of administering the glucocorticoid type II receptor antagonist mifepristone. We similarly demonstrated in premature fetuses and newborn lambs that antenatal administration of betamethasone decreased the sensitivity of baroreflex-mediated changes in HR to increases in MABP (30). Unno et al. (39) found in adrenalectomized fetuses that cortisol infusion increased blood pressure and basal HR. Although a direct stimulatory effect of cortisol on the heart is possible, this finding is also consistent with a resetting of the baroreflex. In the present study, restoring circulating cortisol levels to the physiological range in ADX animals shifted the baroreflex curves for HR and RSNA to the right without altering the slope of the curves. Resetting of the baroreflex curves immediately after birth was seen in the ADX + F animals, similar to that which we previously described in intact newborn lambs (31). In contrast, no postnatal resetting was seen in the ADX animals. Thus, there are consistent data demonstrating that glucocorticoids influence baroreflex function; this may be one mechanism by which corticosteroids modulate blood pressure.

We did not investigate the mechanism(s) by which glucocorticoid replacement alters baroreflex function. In the present studies, we found no effect of hydrocortisone on the sensitivity of the fetal baroreflex responses, whereas previous studies showed an attenuation of the slope of the reflex curve for HR and RSNA (26, 28, 30). Differences in animal species, experimental conditions, dose of cortisol replacement, and use of anesthetics may contribute to this discrepancy in findings. Resetting of the arterial baroreflex may occur at the level of the baroreceptors (i.e., peripheral resetting) or centrally, via alterations in neuronal activity, activation of other reflex pathways, or changes in the level of circulating hormones that influence autonomic activity. Determining whether the increased resting blood pressure seen in the ADX + F fetuses and newborns relative to the ADX animals contributed to or resulted from resetting of the baroreflex will require additional studies.

Perspectives