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Am J Physiol Regul Integr Comp Physiol 287: R209-R217, 2004. First published March 11, 2004; doi:10.1152/ajpregu.00701.2003
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DEVELOPMENT AND TISSUE PLASTICITY

Long-term hypoxia alters ovine fetal endocrine and physiological responses to hypotension

Keiichi Adachi,1 Hikaru Umezaki,1 Kanchan M. Kaushal,1 and Charles A. Ducsay1,2

1Departments of Physiology/Pharmacology and 2Pediatrics, School of Medicine, Center for Perinatal Biology, Loma Linda University, Loma Linda, California 92350

Submitted 8 December 2003 ; accepted in final form 8 March 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Exposure to long-term hypoxia (LTH) results in altered cortisol responses in the ovine fetus. The present study was designed to test the hypothesis that LTH alters adrenal responsiveness to fetal hypotension. Pregnant ewes were maintained at high altitude (3,820 meters) from day 30 of gestation. Normoxic control and LTH fetuses were catheterized on day 132 of gestation. In the LTH group, maternal PO2 was maintained comparable to that observed at altitude (~60 mmHg) by nitrogen infusion through a tracheal catheter. On day 137, fetuses received a 5-h saline infusion followed by infusion of sodium nitroprusside to reduce fetal arterial pressure by 30–35% for 10 min. The study was repeated on day 139 of gestation with a continuous cortisol infusion (10 µg/min). Hypothalamic and pituitary tissues were collected from additional fetuses for assessment of glucocorticoid receptors. During the saline infusion in response to hypotension, plasma ACTH increased over preinfusion mean values in both groups (P < 0.05). Plasma cortisol concentrations increased in both groups concomitant with increased ACTH secretion. However, peak values in the LTH fetuses were significantly higher compared with controls (P < 0.05). During the cortisol infusion, the ACTH response was eliminated in both groups, with ACTH levels significantly lower in the LTH group (P < 0.05). Glucocorticoid receptor binding was not different between groups. These results demonstrate an enhanced cortisol response to hypotension in LTH fetuses that does not appear to be the result of an increase in negative feedback sensitivity of the hypothalamic-pituitary-adrenal axis.

adrenocorticotropin hormone; cortisol; nitroprusside

THE OVINE FETUS RESPONDS TO an acute stressor with a dramatic elevation in plasma ACTH and cortisol. Acute hypoxia (1, 4, 16), hypotension (26, 32), and hemorrhage (25, 34) all have profound stimulatory effects on the fetal hypothalamic-pituitary-adrenal axis (HPA). Short-term reductions in uterine blood flow also enhance cortisol secretion in the ovine fetus (3, 27). Placental embolization to induce fetal hypoxia for up to 3 wk (20) has also been shown to result in elevated fetal ACTH and cortisol concentrations. The response of the fetal HPA to stressors lasting more than a few weeks, however, is far less clear. Phillips et al. (23) used surgical removal of caruncles to subject the fetus to sustained hypoxia throughout gestation and found that plasma cortisol concentrations were elevated despite ACTH levels similar to controls. The ability of the fetus to respond to a secondary stressor, after exposure to chronic hypoxic or hypotensive stress, is less clearly defined. Although a few studies have examined the response of the fetal HPA to a superimposed secondary stressor after hypoxia lasting up to a few weeks (9, 22), there are no data on the effects of such a challenge after fetal hypoxia for more than 3–4 wk.

Our laboratory has focused on the effects of chronic, long-term hypoxia (LTH) with a model in which the ewe is maintained at 3,800 meters from day 30 of gestation to near term (14, 15). During this time, the maternal arterial PO2 was ~60 mmHg, and fetal arterial PO2 was ~17–19 mmHg. Basal fetal ACTH and cortisol concentrations were similar to values in the normoxic fetus, whereas the cortisol response to exogenously administered ACTH was blunted (14). However, in response to an episode of severe asphyxia induced by 5 min of complete cord occlusion, the cortisol response was actually enhanced in the LTH ovine fetus compared with normoxic controls (15).

We designed the present study to test the hypothesis that a less severe secondary stressor like fetal hypotension also results in enhanced cortisol secretion in LTH fetuses compared with normoxic controls. By infusing cortisol before hypotension and measuring ACTH and cortisol responses, we also tested the hypothesis that LTH alters cortisol negative feedback sensitivity.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Presurgical Procedures

Time-dated pregnant ewes of mixed Western breed (Nebeker Farms, Lancaster, CA) were housed in outdoor sheltered pens at the Barcroft Laboratory White Mountain Research Station (elevation 3,820 meters) from day 30 to 123–125 of gestation. The ewes were allowed alfalfa pellets, mineral supplements, and water ad libitum.

Surgical Procedures

Between days 123 and 125 of gestation, the LTH ewes were transported from the White Mountain Research Station to Loma Linda University Medical Center Animal Research Facility (elevation: 346 meters) where they were implanted with a nonocclusive tracheal catheter (4.0 mm OD) and an arterial catheter. Maternal PO2 for the LTH group was maintained at ~60 mmHg (mean PO2 measured in animals at altitude) throughout the studies by adjusting humidified nitrogen gas flow through the tracheal catheter. Normoxic, age-matched pregnant ewes were maintained near sea level (~300 meters) throughout the study.

On day 132 of gestation, surgeries were performed on normoxic control and LTH fetuses (n = 6 for each group) under halothane general anesthesia, induced with intravenous thiopental as previously described (14, 15). The uterus was exposed through a midline vertical laparotomy, and the fetal head was delivered. We placed Tygon catheters (2.28 mm OD) in the fetal carotid artery and jugular vein and advanced them to the ascending aorta and superior vena cava (SVC), respectively. A third catheter (1.78 mm OD) was placed in the tibial vein and advanced to the inferior vena cava (IVC). An amniotic fluid catheter was anchored to the fetal hindlimb to measure amniotic fluid pressure by methodology previously described in detail (14, 15). All catheters were tunneled under the ewe's skin, exteriorized through the left flank, and stored in a nylon pouch sutured to the skin. All procedures were approved by the Institutional Animal Care and Use Committee, and the animals were maintained in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.

Postoperative Care

After surgery, the ewes were maintained in a metabolic cart with food and water provided ad libitum. The ewes received antibiotics (3 ml Crystiben im; 450,000 units penicillin G-procaine and 450,000 units penicillin G-bensathine; Solvay Animal Health, Mendota Heights, MN) one time a day for the first 3 days after surgery. The fetus received antibiotics [40 mg Tobramycin iv (Bristol-Myers Squibb, Princeton, NJ), 150 mg Clindamycin iv (Abbott Laboratories), and 1 g Mezlocillin in the amniotic fluid (Miles, West Haven, CT)] two times daily for the first 3 days after surgery. Afterward, 500 mg Mezlocillin was administered in the amniotic fluid two times daily until the start of the experiment. All vascular catheters were flushed two times daily with heparinized saline.

Hemodynamic Measurements

The ewes were monitored while standing undisturbed. Fetal mean arterial pressure, heart rate, and amniotic fluid pressure were monitored using an eight-channel recorder (Gould 2800S; Gould, Cleveland, OH). Analog signals were converted to digital input by a microcomputer (IBM PC-AT; IBM, Armonk, NY) and processed with real-time data acquisition software developed in our laboratory (5). Fetal mean arterial pressure was corrected for changes in amniotic fluid pressure.

Experimental Protocol

On day 137, we administered 5% ethanol saline (vehicle) in the SVC at the rate of 2.5 ml/h for 7 h by an infusion pump (model 944; Harvard Apparatus, South Natick, MA). Fetal arterial blood samples were collected at –30 and –15 min before the start of the saline infusion and then 1, 2, 3, 4, and 5 h after the infusion was started. Blood was collected in chilled potassium-EDTA syringes (Monovette; Sarstedt, Nümbrecht, Germany) and centrifuged at 4,000 rpm for 10 min at 4°C. Plasma was then separated and stored at –70°C until analyzed. Fetal erythrocytes were reconstituted with sterile saline and returned to the fetal circulation after withdrawal of the next sample. After the start of saline infusion (5 h), sodium nitroprusside (Nitropress; Abbott Laboratories) in 5% dextrose was infused in the IVC for 10 min at a rate (ranging from 17 to 50 µg/min) sufficient to reduce fetal arterial pressure by 30–35% of baseline. Additional blood samples for ACTH and cortisol were collected during and after the start of the nitroprusside infusion (0, 5, 10, 15, 30, 60, and 120 min). Fetal arterial blood samples were also drawn at selected time intervals for determination of fetal blood gas using an automated blood gas analyzer (ABL300; Radiometer, Copenhagen, Denmark).

To examine the potential effects of cortisol negative feedback on pituitary ACTH secretion, the study was repeated on day 139 with a continuous cortisol infusion (Hydrocortisone; Sigma, St. Louis, MO; 10 µg/min, in 5% ethanol saline). A 10 µg/min dose of cortisol was chosen, since doses in this range have been shown to effectively inhibit ACTH secretion in the near-term ovine fetus (33).

Hormonal Assays

Plasma immunoreactive ACTH concentrations were determined by RIA (DiaSorin, Stillwater, MN), which has been previously described and validated in our laboratory for use in the ovine fetus (2, 14). The intra-assay and interassay coefficients of variation were 7 and 11%, respectively. Assay sensitivity was 1 pg/ml.

Plasma cortisol concentrations were measured by RIA, as previously described and validated. The intra-assay and interassay coefficients of variation were 8 and 11%, respectively, whereas the assay sensitivity was 0.2 ng/ml (2, 14).

Glucocorticoid Receptor Quantification

To examine a potential mechanism of differences in glucocorticoid negative feedback sensitivity, we collected hypothalamic and pituitary tissue from additional control and LTH fetuses (n = 10 for each group). Between days 139 and 141 of gestation, ewes were c-sectioned, and the fetuses were delivered and killed. The fetal brains were removed immediately, the hypothalamus was dissected out, and the pituitary was removed from the sella turcica. The tissues were snap-frozen in liquid nitrogen and stored at –70°C until analyzed.

The methodology for the binding assay was similar to that previously described (24). Briefly, tissues were homogenized in cold 20 mM Tris, 10 mM thioglycerol, 1 mM EDTA, and 10% glycerol, at pH 7.5 (TEGT). This was followed by centrifugation at 100,000 g for 30 min at 4°C. The protein concentration was determined by the Lowry method, and the cytosolic prep was diluted with cold buffer for a final protein concentration of 2 mg/ml. The binding assays were performed using 100 µl cytosol with 20 nM [3H]triamcinolone acetonide (26 Ci/mmol; Amersham) with and without a 100-fold excess of cortisol and incubated overnight. After the incubation, the reaction was stopped with the addition of 200 µl of dextran-coated charcoal slurry (1% charcoal and 0.5% dextran in TEGT buffer). After centrifugation for 15 min at 2,220 rpm (800 g), 150-µl aliquots in duplicate were added to 4 ml of scintillation cocktail and counted in a Packard 1900CA analytic scintillation counter. Scatchard analysis for determination of maximal binding and dissociation constant (Kd) was performed using GraphPad Prism analytical software (GraphPad Software, San Diego, CA).

Statistical Analyses

Differences over time and between control and LTH fetuses were analyzed using two-way ANOVA with repeated measures and Bonferroni's post hoc test where appropriate. All values were expressed as means ± SE, and P < 0.05 was considered statistically significant. Statistical comparisons of glucocorticoid receptor binding parameters were performed using a Students t-test.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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Maternal Blood Gases

Nitrogen flow was maintained through the maternal tracheal catheter at a rate sufficient to maintain maternal arterial blood gas values at a level similar to that observed at 3,820 meters at the Barcroft Laboratory (14). As expected, mean arterial PO2 in the LTH ewes was significantly lower than in the normoxic controls (57.5 ± 1.5 vs. 102.5 ± 2.0 mmHg, P < 0.01, LTH vs. control). Mean maternal pH values (7.43 ± 0.01 LTH and 7.42 ± 0.01 control) and arterial PCO2 (31.6 ± 0.5 LTH and 32.2 ± 0.8 control) did not differ between the groups. Maternal blood gas values were unaffected by fetal nitroprusside or cortisol infusion (data not shown).

Effects of Hypotension During Vehicle Infusion

Fetal arterial pressure and heart rate. Saline vehicle infusion had no effect on blood pressure or heart rate in either control or LTH fetuses (Fig. 1). The sodium nitroprusside infusion reduced mean arterial pressure in both groups of fetuses to a similar extent, a decrease of ~30–35% from baseline. After the termination of the infusion, mean arterial pressure returned to prehypotension levels within ~10 min. Fetal heart rate was unaffected by hypotension and remained unchanged throughout the study in both control and LTH fetuses (Fig. 1). Mean arterial pressure in the LTH group was higher than control over the course of the study (P < 0.01).



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  		Fig. 1. Fetal mean arterial pressure and heart rate in response to 10 min of nitroprusside-induced hypotension during saline infusion. Overall mean arterial pressure was significantly different between control and long-term hypoxia (LTH) fetuses during the course of the experiment (P < 0.01), although heart rates did not differ. The 10-min period of hypotension is represented by the shaded area, and all values represent means ± SE in this and Figs. 2–7.

 
Fetal blood gases. Mean basal arterial PO2 in the LTH fetuses during the saline infusion was significantly lower than in the normoxic controls (18.6 ± 0.4 vs. 22.6 ± 0.4 mmHg, P < 0.01; Fig. 2, top). Mean arterial PCO2 was also significantly lower in the LTH group compared with the normoxic controls (43.8 ± 0.6 vs. 49.9 ± 0.5 mmHg, P < 0.01; Fig. 2, middle), whereas arterial pH was higher in the LTH fetuses compared with control (7.35 ± 0.01 vs. 7.31 ± 0.01, P < 0.01; Fig. 2, bottom). However, PO2, PCO2, and pH values did not change significantly in response to hypotension in either treatment group.



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  		Fig. 2. Fetal blood gas parameters in control and LTH fetuses during saline infusion. All three parameters were different between control and LTH fetuses (P < 0.01) but were unaffected by saline infusion or hypotension.

 
ACTH and cortisol responses to hypotension. During the saline infusion, basal ACTH concentrations were similar in both groups (24.9 ± 4.2 pg/ml control vs. 24.4 ± 3.2 pg/ml LTH), and levels were unaffected by the saline infusion (Fig. 3, top). Hypotension significantly increased plasma ACTH concentrations in both groups, and the time course of the plasma ACTH rise in response to hypotension was similar in both groups. Peak plasma ACTH concentrations were attained at the end of the nitroprusside infusion and were similar in both groups (92.9 ± 17.0 pg/ml control vs. 119 ± 22.1 pg/ml LTH; Fig. 3, top). After termination of the nitroprusside infusion, plasma ACTH gradually returned to near-preinfusion values in both groups. No differences in ACTH concentrations were observed between LTH and control fetuses.



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  		Fig. 3. Fetal plasma ACTH and cortisol concentrations in control and LTH fetuses in response to 10 min nitroprusside-induced hypotension during saline infusion. *P < 0.05 compared with control.

 
Basal cortisol values were also similar in both groups (10.3 ± 1.4 ng/ml control vs. 12.7 ± 2.8 ng/ml LTH), and saline infusion had no effect on basal plasma cortisol concentrations in either group (Fig. 3, bottom). After hypotension, cortisol concentrations increased significantly over baseline values (P < 0.05) in both control and LTH fetuses. However, cortisol concentrations were significantly higher in the LTH fetuses (P < 0.05 compared with control) at 15, 30, and 60 min after the start of hypotension. By the end of the study period, mean plasma cortisol concentrations returned to preinfusion values in both groups.

Effects of Hypotension During Cortisol Infusion

Fetal arterial pressure and heart rate. During the cortisol infusion, there was a gradual increase in mean arterial pressure in the LTH fetuses. Immediately before the start of the nitroprusside treatment, blood pressure was significantly higher in the LTH fetuses compared with control (P < 0.05, Fig. 4, top). Because the nitroprusside infusion rate was adjusted to reduce mean arterial pressure by 30–35%, the nadir in mean arterial pressure did not reach the same level in the LTH and control groups. After the end of the hypotensive episode, mean arterial pressure remained higher in the LTH fetuses (P < 0.01 compared with control). Fetal heart rate was significantly higher in the LTH fetuses compared with control after hypotension (P < 0.01; Fig. 4, bottom).



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  		Fig. 4. Fetal mean arterial pressure and heart rate in response to 10 min of nitroprusside-induced hypotension during cortisol infusion. Mean arterial pressure and heart rate were significantly different between control and LTH fetuses during the course of the experiment (P < 0.01).

 
Fetal blood gasses. Cortisol infusion had no effect on any of the parameters measured (Fig. 5). As observed in the fetuses during the saline infusion, mean basal arterial PO2 values were lower in the LTH group compared with control (18.1 ± 1.0 vs. 23.8 ± 0.8 mmHg, P < 0.01). After hypotension, there was a trend toward a decrease in arterial PO2 in the LTH, but the change did not reach statistical significance. Mean arterial PCO2 values were consistently lower in the LTH fetuses (44.4 ± 1.1 vs. 50.1 ± 0.7 mmHg, P < 0.01 compared with control) and were unchanged by hypotension. Mean basal arterial pH values were higher in the LTH group compared with control (7.33 ± 0.01 vs. 7.30 ± 0.01, P < 0.01) but were unaffected by hypotension.



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  		Fig. 5. Fetal blood gas parameters in control and LTH fetuses during cortisol infusion. All 3 parameters were different between control and LTH fetuses (P < 0.01) but were unaffected by cortisol infusion or hypotension.

 
ACTH and cortisol responses to hypotension. Before the start of the cortisol infusion, basal levels of ACTH were similar in both groups (27.0 ± 5.8 pg/ml control vs. 29.0 ± 5.3 pg/ml LTH; Fig. 6, top). After the start of the cortisol infusion, there was a significant effect of time, with a decline in plasma ACTH concentrations in both control and LTH fetuses during the course of the study (P < 0.01). Hypotension had no effect on ACTH, however, after the end of the hypotensive period; ACTH levels in the LTH group remained lower than values for the control group for the duration of the experiment (P < 0.01, Fig. 6, top).



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  		Fig. 6. Fetal plasma ACTH and cortisol concentrations in control and LTH fetuses in response to 10 min of nitroprusside-induced hypotension during cortisol infusion. Both ACTH and cortisol values were significantly different between control and LTH fetuses during the course of the experiment (P < 0.01). Note scale changes compared with Fig. 3.

 
As expected, during the cortisol infusion, plasma cortisol concentrations were significantly higher than preinfusion values in both groups (P < 0.01; Fig. 6, bottom). Hypotension had no further effect on plasma cortisol levels in either group. However, despite the same rate of cortisol infusion and no difference in fetal weight (3.56 ± 0.19 vs. 3.77 ± 0.29 kg, control vs. LTH), cortisol concentrations were significantly higher in the LTH group compared with control (P < 0.01) during the course of the cortisol infusion. Plasma clearance of cortisol was estimated based on the equilibrium values of cortisol attained during the cortisol infusion. Although there was a trend toward reduced cortisol clearance in the LTH fetuses compared with controls, the difference did not reach statistical significance (2.99 ± 0.40 vs. 2.31 ± 0.15 l·kg–1·h–1 in control vs. LTH, respectively, P > 0.05).

Glucocorticoid receptor binding. Chronic hypoxia had no effect on glucocorticoid receptor density in the fetal hypothalamus or pituitary (Fig. 7). Likewise, the Kd did not differ between the two treatment groups in either region.



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  		Fig. 7. Hypothalamic (A) and pituitary (B) glucocorticoid receptor values in near-term control and LTH fetuses. Maximal binding (Bmax) values are fmol/mg protein, and dissociation constant values are listed inside each histogram.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There is a wealth of information regarding responsiveness of the ovine fetal HPA to a wide range of acute physiological stressors ranging from hypoxia (1, 4, 16) to hypotension (26, 32). Efforts to study more LTH have centered on the use of placental embolization (9) or oxytocin-induced uterine contractures (22) and also showed that these forms of stress stimulated ACTH and cortisol levels in the ovine fetus. Phillips et al. (23) used surgical removal of caruncles to subject the fetus to sustained hypoxia throughout gestation and found that plasma cortisol concentrations were elevated despite ACTH levels similar to controls. The ability of the fetus to respond to a secondary stressor, after exposure to a long-term, chronic stress, is not clearly defined. A small number of studies have examined the response of the fetal HPA to a superimposed secondary stressor after hypoxia lasting up to a few weeks (9, 11, 22). However, there are no data on the effects of such a challenge after fetal hypoxia lasting through the course of gestation. This underscores the fact that the magnitude of the HPA response may not only depend on the stage of development but also on the duration and the degree of the hypoxic stress. Furthermore, such factors may alter the ability of the fetus to respond to a secondary stressor (10).

In our model of hypoxia lasting the course of gestation, we previously showed that LTH fetuses had an attenuated cortisol response to exogenously administered ACTH (14). In marked contrast, umbilical cord occlusion resulted in an enhanced cortisol response in LTH fetuses compared with normoxic controls, despite a similar ACTH response (15). The present study was designed to determine if a less severe secondary stressor like hypotension also resulted in a differential cortisol response in LTH fetuses. In this study, we demonstrated that the ACTH response of LTH ovine fetuses was similar to normoxic controls. However, the cortisol response was enhanced in the LTH group in response to the secondary stressor.

The issue of what adaptive value enhanced cortisol secretion may have for the LTH fetus is unclear at present. In addition to the role of cortisol in the initiation of labor in the sheep, the stimulation of glucocorticoid secretion in response to stress is a central adaptive mechanism among mammals (18). Acute elevations in cortisol result in a number of catabolic effects, including glycogenolysis, increased lipolysis, and protein catabolism, with a resultant elevation of blood glucose levels (19). We have not yet determined potential differences in energy substrate between control and LTH fetuses in response to a secondary stressor. Enhanced glucose availability could be viewed as an adaptive advantage during an acute stressor.

It is interesting to note that the cortisol response was greater in LTH fetuses compared with control during saline infusion despite similar ACTH responses. A number of factors may contribute to the enhanced circulating cortisol levels in the LTH fetuses. ACTH clearly stimulates short-term cortisol output. However, neuronal input to the adrenal may also play an important role in the regulation of cortisol secretion. Splanchnic nerve section (21) in the near-term ovine fetus attenuated the cortisol response to hypotension, whereas peak ACTH levels were unaffected. Carotid denervation had a similar effect on the fetal response to acute hypoxemia (12). An additional study found that sinoaortic denervation attenuates reflex responses to hypotension (32). In the LTH animals in the present study, one could therefore hypothesize an enhanced neuroendocrine reflex arc.

A difference in cortisol clearance could also potentially explain the differential cortisol response. Despite similar fetal weights between groups and the same cortisol infusion rate, the overall levels of cortisol attained during the infusion were significantly higher in the LTH fetuses. This potential difference in cortisol clearance could result in higher cortisol levels observed in response to hypotension. This possibility is further strengthened by the fact that cortisol levels remained elevated for a longer period of time in the LTH group during the saline infusion. Although not significant, estimates of plasma clearance from the present study suggest that a reduction in cortisol clearance may indeed play a role in the observed higher cortisol concentrations in the LTH fetuses. Metabolic clearance studies using dual-labeled cortisol may be helpful to more accurately assess the potential effects of LTH on cortisol clearance. One could also suggest that perhaps adrenal blood flow is enhanced in the LTH fetuses. However, previous studies from our group demonstrated no differences in total adrenal blood flow in response to a superimposed hypoxia between LTH and normoxic fetuses (17).

It is also of interest that the enhanced cortisol response to hypotension in the LTH fetuses compared with normoxic controls is similar to the change observed after a 5-min, complete umbilical cord occlusion (15). It is interesting to note, however, that the maximal ACTH response after cord occlusion was 530 ± 90 pg/ml, whereas, in the present study, peak values only reached 119 ± 22 pg/ml. These data suggest that a much milder stressor like hypotension is sufficient to elicit a maximal cortisol response and that the baroreceptor, like chemoreceptor stimulation of the cortisol response, is enhanced in the LTH fetus.

Unlike the observed baroreceptor stimulation of the cortisol response to hypotension, the lack of baroreflex heart rate response to hypotension in both the control and LTH fetuses in the present study is puzzling. Pervious studies using a similar protocol indeed showed the typical bradycardia associated with hypotension in the fetus. There were, however, two key differences between these studies and the protocol in the present study. First, the percent ethanol in the vehicle solution was less than our study. Second and more important, the vehicle infusion was stopped for 1 h before the start of the hypotension. One could speculate that ethanol may have played a role in blunting the heart rate response to hypotension. Although to our knowledge there are no studies in the literature examining the effect of ethanol on fetal baroreceptor function, there are abundant data in adult animals and humans to suggest that ethanol can attenuate baroreflex-mediated heart rate function (7, 29). The apparent dissociation between baroreflex mediation of endocrine and cardiovascular responses in the fetus observed in the present study will serve as an area of future study.

The principal focus of the present study was to determine the effects of LTH on ACTH and cortisol responses to hypotension. However, we secondarily studied potential differences in cortisol negative feedback after LTH. Previous studies demonstrated that the fetal sheep between 117 and 131 days of gestation are very sensitive to the negative feedback effects of cortisol, and the ACTH response to nitroprusside-induced hypotension was completely inhibited after cortisol infusion (6, 30). In near-term fetuses, however, although basal ACTH levels are suppressed by cortisol (33), the ACTH response to hypotension was unaffected by cortisol infusion (31). In the present study, the ACTH response to hypotension was blunted in both control and LTH fetuses (Fig. 6). Although the exact reason for the difference between the two studies is unknown, there are a number of potential explanations. In the present study, the cortisol infusion was maintained during the nitroprusside infusion, whereas, in the study by Wood (31), the cortisol infusion was terminated 1 h before the start of nitroprusside. Also, all of the fetuses in the present study underwent a previous hypotensive episode at day 137, which could have potential effects on cortisol negative feedback. Previous studies have clearly shown that exposure to a repetitive stressor can alter HPA function (13, 22).

Regardless of the causes, in our experimental design, there was no increase in fetal ACTH concentrations in response to hypotension in either control or LTH fetuses. However, after hypotension, the degree of suppression of ACTH was greater in the LTH fetuses compared with the control group. Alone, these data suggest that there may be enhanced negative feedback sensitivity to cortisol in the LTH fetuses. However, the overall cortisol levels attained during the cortisol infusion were also higher in the LTH fetuses. A higher level of cortisol would be expected to produce a greater suppression of ACTH. Furthermore, there was no difference in either hypothalamic or pituitary glucocorticoid receptor density in the LTH fetuses compared with normoxic controls. Together, these data suggest that there is no change in cortisol negative feedback sensitivity after LTH.

It is interesting to note the effects of cortisol on fetal mean arterial pressure. Although saline infusion had no effect on blood pressure, cortisol infusion caused a significant increase in mean arterial pressure in the LTH group. There was a trend toward elevated pressure in the control animals over the first 3 h of the infusion, but the change did not reach statistical significance. Previous studies using different age fetuses and different cortisol infusion protocols have demonstrated an increase in blood pressure after cortisol infusion (8, 28, 35) and suggested that the hypertensive effect of cortisol is through activation of the renin-angiotensin system, increasing vascular sensitivity to ANG II (8, 28). These data lead to the speculation that the apparent increased sensitivity of the LTH fetuses to the hypertensive effects of cortisol may be the result of LTH on the renin-angiotensin system. Future studies utilizing ANG II and ANG type 1-specific antagonists will be necessary to elucidate the effects of LTH.

Fetal PO2 values are typical of what we have previously reported for chronically catheterized LTH animals (14, 15). PCO2 and pH are also in the range of values previously observed in these animals. Although all three blood gas parameters were different between control and LTH fetuses, neither hypotension nor cortisol infusion had an effect on these values. Previous studies also failed to demonstrate an effect of hypotension on fetal blood gasses (26).

To our knowledge, this is the first study to describe the effects of exposure to a long-term (longer than a few weeks) stressor on the response to hypotension in the ovine fetus. Taken together, the findings from the present study indicate that LTH enhances the fetal cortisol response to hypotension compared with normoxic controls without differences in ACTH output. Furthermore, the LTH fetus appears to be more sensitive to the effects of cortisol infusion at the level of the cardiovascular system. These findings suggest an alteration or resetting of HPA to maintain hormone levels within a normal physiological range, despite LTH, and respond to a secondary stressor in a robust manner. Such information may have important clinical implications.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Child Health and Human Development Grant HD-31226.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. A. Ducsay, Center for Perinatal Biology, School of Medicine, Loma Linda Univ., Loma Linda, California 92350 (E-mail: cducsay{at}som.llu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akagi K and Challis G Jr. Hormonal and biophysical responses to acute hypoxemia in fetal sheep at 0.7–0.8 gestation. Can J Physiol Pharmacol 68: 1527–1532, 1990.[Web of Science][Medline]
  2. Apostalakis EM, Longo LD, and Yellon SM. Regulation of basal adrenocorticotropin and cortisol secretion by arginine vasopressin in the fetal sheep during late gestation. Endocrinology 129: 295–300, 1991.[Abstract/Free Full Text]
  3. Bocking AD, McMillen IC, Harding R, and Thorburn GD. Effect of reduced uterine blood flow on fetal and maternal cortisol. J Dev Physiol 8: 237–245, 1986.[Web of Science][Medline]
  4. Boddy K, Jones CT, Mantnell C, Ratcliffe JG, and Robinson JS. Changes in plasma ACTH and corticosteroid of the maternal and fetal sheep during hypoxia. Endocrinology 94: 588–591, 1974.[Abstract/Free Full Text]
  5. Dale PS, Ducsay CA, Gilbert RD, Koos BJ, Longo LD, and Power GG. A microcomputer program for real time data acquisition in the perinatal physiology laboratory. J Dev Physiol 11: 56–61, 1989.
  6. Dix PM, Rose JC, Morris M, Hargrave BY, and Meis PJ. Cortisol infusion blocks adrenocorticotropic hormone but not vasopressin responses to hypotension in fetal lambs. Am J Obstet Gynecol 148: 317–321, 1984.[Web of Science][Medline]
  7. Fazio M, Bardelli M, Macaluso L, Fiammengo F, Mattei PL, Bossi M, Fabris B, Fischetti F, Pascazio L, Candido R, and Carretta R. Mechanics of the carotid artery wall and baroreflex sensitivity after acute ethanol administration in young healthy volunteers. Clin Sci (Lond) 101: 253–260, 2001.[Medline]
  8. Forhead AJ, Broughton PF, and Fowden AL. Effect of cortisol on blood pressure and the renin-angiotensin system in fetal sheep during late gestation. J Physiol 526: 167–176, 2000.[Abstract/Free Full Text]
  9. Gagnon R, Murotsuki J, Challis JR, Fraher L, and Richardson BS. Fetal sheep endocrine responses to sustained hypoxemic stress after chronic fetal placental embolization. Am J Physiol Endocrinol Metab 272: E817–E823, 1997.[Abstract/Free Full Text]
  10. Gardner DS, Fletcher AJ, Bloomfield MR, Fowden AL, and Giussani DA. Effects of prevailing hypoxaemia, acidaemia or hypoglycaemia upon the cardiovascular, endocrine and metabolic responses to acute hypoxaemia in the ovine fetus. J Physiol 540: 351–366, 2002.[Abstract/Free Full Text]
  11. Gardner DS, Fletcher AJ, Fowden AL, and Giussani DA. Plasma adrenocorticotropin and cortisol concentrations during acute hypoxemia after a reversible period of adverse intrauterine conditions in the ovine fetus during late gestation. Endocrinology 142: 589–598, 2001.[Abstract/Free Full Text]
  12. Giussani DA, McGarrigle HH, Moore PJ, Bennet L, Spencer JAD, and Hanson MA. Carotid sinus nerve section and the increse in plasma cortisol during acute hypoxia in fetal sheep. J Physiol 477: 75–80, 1994.[Abstract/Free Full Text]
  13. Green LR, Kawagoe Y, Fraser M, Challis JR, and Richardson BS. Activation of the hypothalamic-pituitary-adrenal axis with repetitive umbilical cord occlusion in the preterm ovine fetus. J Soc Gynecol Investig 7: 224–232, 2000.[CrossRef][Web of Science][Medline]
  14. Harvey LM, Gilbert RD, Longo LD, and Ducsay CA. Changes in ovine fetal adrenocortical responsiveness after long-term hypoxemia. Am J Physiol Endocrinol Metab 264: E741–E747, 1993.[Abstract/Free Full Text]
  15. Imamura T, Umezaki H, Kaushal K, and Ducsay CA. Long-term hypoxia alters endocrine and physiolocic responses to umbilical cord occlusion in the ovine fetus. J Soc Gynecol Investig 11: 131–140, 2004.[Abstract/Free Full Text]
  16. Jones CT, Boddy K, Robinson JS, and Ratcliffe JG. Developmental changes in the response of the adrenal glands of the fetal sheep to endogenous corticotropin, as indicated by hormone responses to hypoxaemia. Endocrinology 72: 279–292, 1977.
  17. Kamitomo M, Alonso JG, Okai T, Longo LD, and Gilbert RD. Effects of long-term, high-altitude hypoxemia on ovine fetal cardiac output and blood flow distribution. Am J Obstet Gynecol 169: 701–707, 1993.[Web of Science][Medline]
  18. Meaney MJ, Viau V, Bhatnagar S, Betito K, Iny LJ, O'Donnell D, and Mitchell JB. Cellular mechanisms underlying the development and expression of individual differences in the hypothalamic-pituitary-adrenal stress response. J Steroid Biochem Mol Biol 39: 265–274, 1991.[CrossRef][Web of Science][Medline]
  19. Munck A, Guyre PM, and Holbrook NJ. Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr Rev 5: 25–44, 1984.[Abstract/Free Full Text]
  20. Murotsuki J, Gagnon R, Matthews SG, and Challis JR. Effects of long-term hypoxemia on pituitary-adrenal function in fetal sheep. Am J Physiol Endocrinol Metab 271: E678–E685, 1996.[Abstract/Free Full Text]
  21. Myers DA, Robertshaw D, and Nathanielsz PW. Effect of bilateral splanchnic nerve section on adrenal function in the ovine fetus. Endocrinology 127: 2328–2335, 1990.[Abstract/Free Full Text]
  22. Owiny JR, Jenkins SL, Sadowsky DW, and Nathanielsz PW. Effect of pulsatile oxytocin administration to the pregnant ewe in the last third of gestation on fetal ACTH and cortisol response to acute hypoxemia. J Soc Gynecol Investig 2: 673–677, 1995.[CrossRef][Web of Science][Medline]
  23. Phillips ID, Simonetta G, Owens JA, Robinson JS, Clarke IJ, and McMillen C. Placental restriction alters the functional development of the pituitary-adrenal axis in the sheep fetus during late gestation. Pediatr Res 40: 861–866, 1996.[Web of Science][Medline]
  24. Rose JC, Kute TE, and Winkler L. Glucocorticoid receptors in sheep brain tissues during development. Am J Physiol Endocrinol Metab 249: E345–E349, 1985.[Abstract/Free Full Text]
  25. Rose JC, Macdonald AA, Heymann MA, and Rudolph AM. Developmental aspects of the pituitary-adrenal axis response to hemorrhagic stress in lamb fetuses in utero. J Clin Invest 61: 4224–4232, 1978.
  26. Rose JC, Meis PJ, and Morris M. Ontogeny of endocrine (ACTH, vasopressin, cortisol) responses to hypotension in lamb fetuses. Am J Physiol Endocrinol Metab 240: E656–E661, 1981.[Abstract/Free Full Text]
  27. Stein PE, White SE, Homan J, Fraher L, McGarrigle HH, Hanson MA, and Bocking AD. Fetal endocrine responses to prolonged reduced uterine blood flow are altered following bilateral sectioning of the carotid sinus and vagus nerves. J Endocrinol 157: 149–155, 1998.[Abstract]
  28. Tangalakis K, Lumbers ER, Moritz KM, Towstoless MK, and Wintour EM. Effect of cortisol on blood pressure and vascular reactivity in the ovine fetus. Exp Physiol 77: 709–717, 1992.[Abstract]
  29. Varga K and Kunos G. Inhibition of baroreflex bradycardia by ethanol involves both GABAA and GABAB receptors in the brainstem of the rat. Eur J Pharmacol 214: 223–232, 1992.[CrossRef][Web of Science][Medline]
  30. Wood CE. Sensitivity of cortisol-induced inhibition of ACTH and renin in fetal sheep. Am J Physiol Regul Integr Comp Physiol 250: R795–R802, 1986.[Abstract/Free Full Text]
  31. Wood CE. Does a decrease in cortisol negative feedback efficacy precede ovine parturition? Am J Physiol Regul Integr Comp Physiol 252: R624–R627, 1987.[Abstract/Free Full Text]
  32. Wood CE. Sinoaortic denervation attenuates the reflex responses to hypotension in fetal sheep. Am J Physiol Regul Integr Comp Physiol 256: R1103–R1110, 1989.[Abstract/Free Full Text]
  33. Wood CE. Cortisol inhibits ACTH secretion in late-gestation fetal sheep. Am J Physiol Regul Integr Comp Physiol 260: R385–R388, 1991.[Abstract/Free Full Text]
  34. Wood CE, Chen HG, and Bell ME. Role of vagosympathetic fibers in the control of adrenocorticotropic hormone, vasopressin, and renin responses to hemorrhage in fetal sheep. Circ Res 64: 515–523, 1989.[Abstract/Free Full Text]
  35. Wood CE, Cheung CY, and Brace RA. Fetal heart rate, arterial pressure, and blood volume responses to cortisol infusion. Am J Physiol Regul Integr Comp Physiol 253: R904–R909, 1987.[Abstract/Free Full Text]



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