Restriction of placental growth results in greater hypotensive response to -adrenergic blockade in fetal sheep during late gestation
1 Centre for the Early Origins of Adult Health, Discipline of Physiology, School of Molecular and Biomedical Sciences, University of Adelaide, Adelaide, SA 5005, Australia
Abstract
Placental insufficiency resulting in restriction of fetal substrate supply and fetal hypoxaemia is a major cause of restricted fetal growth and increased neonatal morbidity. Fetal adaptations to placental restriction (PR) include increases in circulating catecholamines and cortisol and decreased fetal body growth, with relative sparing of brain growth. The mechanisms underlying the redistribution of fetal cardiac output in PR fetuses are not known and the aim of this study was to determine whether maintenance of fetal blood pressure (BP) in the PR fetus is dependent on -adrenergic stimulation. PR was induced by removing the majority of uterine caruncles in the ewe before conception. Sterile vascular surgery was performed on seven PR and six control fetuses at 113–120 days' gestation (term = 150 ± 3 days). Fetuses with a mean arterial PO2 < 17 mmHg between 123 and 127 days' gestation were defined as hypoxic. There was a greater fall (P < 0.05) in fetal BP during phentolamine infusion (I.V: 5 mg bolus, 0.2 mg kg–1 min–1 for 2 h) in the hypoxic PR group (–15 ± 2 mmHg) compared with normoxic controls (–5 ± 1 mmHg). The fall in fetal BP during phentolamine infusion was directly related to the level of fetal PO2. Fetal BP and HR responses to phenylephrine (I.V.: 40 μg kg–1) were not different between PR and control fetuses. The maintenance of BP in the chronically hypoxic fetus is therefore dependent on -adrenergic activation, and this fetal adaptation to a suboptimal intrauterine environment pre-dates the development of significant growth restriction. While this adaptation may play a critical role in the redistribution of fetal cardiac output to ensure the sparing of brain growth, it may have adverse consequences for peripheral vascular function in the neonatal period and in adult life.
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Introduction
Placental insufficiency resulting in a restriction of fetal substrate supply and associated chronic fetal hypoxaemia is a major cause of reduced fetal growth and is associated with increased neonatal mortality and morbidity (Bernstein et al. 2000). A range of clinical studies have demonstrated cardiovascular adaptations to chronic hypoxia which include a decrease in fetal growth and a relative sparing of brain growth due to a redistribution of cardiac output (Baschat et al. 1997; Bernstein et al. 2000; Dubiel et al. 2002). The mechanisms that underlie this redistribution of cardiac output in the chronically hypoxic fetus are not well understood.
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Fetal cardiovascular responses to acute hypoxia have been well defined and include bradycardia, an increase in arterial blood pressure (BP) and peripheral vasoconstriction (Boddy et al. 1974; Cohn et al. 1974; Bartelds et al. 1993; Giussani et al. 1993). The mechanisms mediating these cardiovascular responses include chemoreflex, endocrine and local components (Giussani et al. 1994; Green et al. 2001) which act in combination to ensure a redistribution of fetal cardiac output towards the brain, heart and adrenals and away from the periphery (Rudolph et al. 1981; Jensen et al. 1987a,b; Yaffe et al. 1987; Jansen et al. 1989; Giussani et al. 1993). During acute hypoxaemia there is an increase in fetal plasma noradrenaline (NA) concentrations (Jones & Robinson, 1975) and hypoxia-mediated peripheral vasoconstriction is dependent on -adrenergic activation in the sheep fetus (Giussani et al. 1993) and chick embryo (Mulder et al. 2002). Chronic fetal hypoxia has been induced following placental embolization in late gestation (Murotsuki et al. 1997a) or exposure of the pregnant ewe to either high altitude (Longo & Pearce, 1998) or low inspired oxygen (Kitanaka et al. 1989). These models of chronic fetal hypoxia result in a decrease in fetal arterial PO2 for varying periods during late gestation. Placental embolization in late gestation, results in low fluctuating fetal PO2 and an increase in fetal BP and a decrease in fetal heart rate (HR) (Murotsuki et al. 1997b). An alternative experimental model is one in which the majority of endometrial caruncles are removed from the uterus of the non-pregnant ewe resulting in placental restriction (PR), a maintained decrease in fetal arterial PO2 and decreased fetal growth (Robinson et al. 1979). In hypoxic PR fetuses, arterial pressure is maintained at the same level as in normoxic control fetuses (Robinson et al. 1983; Walker et al. 1990; Edwards et al. 1999) while HR is lower (Walker et al. 1990). Independently of whether hypoxaemia is induced over a period of days or months, however, there is an increase in the plasma concentrations of NA in hypoxic fetuses (Jones & Robinson, 1983; Murotsuki et al. 1997b; Simonetta et al. 1997; Gardner et al. 2002). Furthermore, in chronically hypoxic fetuses, there is an inverse relationship between the prevailing fetal arterial PO2 and plasma NA concentrations (Simonetta et al. 1997).
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Studies of total body catecholamine kinetics before and after birth in spontaneously hypoxaemic fetal lambs indicate that increases in circulating catecholamine levels in these fetuses are a consequence of increased sympathoadrenal activity, rather than a decreased clearance of the catecholamines (Smolich & Esler, 1999). While an increase in circulating catecholamines appears to be an important fetal adaptation to hypoxia, no studies have investigated the contribution of sympathetic activation to the maintenance of BP in the hypoxic PR fetus. The aim of the present study therefore was to determine the BP and HR responses to the -adrenergic antagonist, phentolamine and to the -adrenergic agonist, phenylephrine in chronically hypoxic PR and normoxic control fetal sheep during late gestation.
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Methods
Animals and surgery
All experiments were performed according to guidelines of the University of Adelaide Animal Ethics Committee.
The majority of uterine caruncles were removed in seven ewes prior to conception as previously described (Robinson et al. 1979). Ewes were monitored for 4–7 days after surgery and, after 10 weeks, entered a mating programme. Pregnancy was confirmed in both PR ewes (singleton, n = 5; twin, n = 2) and control ewes (singleton, n = 2; twin, n = 4) at around 50 days' gestation.
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At 110–120 days' gestation, surgery was performed in six control and seven PR ewes under aseptic conditions with general anaesthesia induced by sodium thiopentone (1.25 g; Pentothal, Rhone Merieux, Pinkenba, Queensland, Australia) and maintained by inhalation of halothane (2.5–4%) in oxygen. Vascular catheters were inserted in the maternal jugular vein and carotid artery (Critchley Electrical Products, Silverwater, Australia), the fetal femoral and carotid arteries and jugular vein and in the amniotic cavity as previously described (Edwards et al. 1999; Morrison et al. 2001). Fetal catheters were exteriorized through a small incision in the ewe's flank. At surgery, antibiotics were administered to the ewe (3.5 ml; 150 mg ml–1 procaine penicillin, 112.5 mg ml–1 benzathine penicillin, 2 ml; 250 g ml–1 dihydrostreptomycin, Lyppards, Adelaide, Australia) and fetus (1 ml; 150 mg ml–1 procaine penicillin, 112.5 mg ml–1 benzathine penicillin 1 ml; 250 g ml–1 dihydrostreptomycin, Lyppards, Adelaide, Australia). Antibiotics were administered intramuscularly to each ewe for 3 days after surgery and intra-amniotically (5 ml; 100 mg ml–1 ampicillin, Lyppards, Adelaide, Australia) for 4 days after surgery. Animals were allowed to recover from surgery for at least 4 days prior to experimentation.
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Arterial blood gas measurements
Fetal arterial blood gas samples (0.5 ml) were collected daily for the measurement of PO2, PCO2, pH, oxygen saturation (SO2) and haemoglobin (Hb) (ABL 520 analyser, Radiometer, Copenhagen, Denmark).
Physiological measurements
Fetal femoral artery and amniotic catheters were connected to displacement transducers and a quad-bridge amplifier (ADInstruments, Castle Hill, Australia) to record fetal blood pressure (BP) and amniotic pressure. All data were digitized and recorded using Chart (ADInstruments, Castle Hill, Australia).
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Experimental protocol
Basal data. Fetal BP was recorded for 3 h prior to infusion of drugs. The number of animals in each study is shown in Table 1.
Phentolamine infusion. Each fetus received either saline or phentolamine (bolus, 5 mg; infusion, 0.2 mg kg–1 min–1 (Giussani et al. 1993; Giussani et al. 1999); Sigma Aldrich, Castle Hill, Australia) I.V. for 2 h on two consecutive days between 123 and 127 days' gestation. We used the following fetal growth curves previously established in this laboratory to make an initial estimate of fetal weight in the control and PR groups at 123–127 days' gestation (Edwards et al. 1999):
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Based on these estimates of fetal weights, a total of 72 mg phentolamine was infused in each control fetus and a total of 48 mg phentolamine was infused in each PR fetus during the 2 h infusion period. At post mortem, all fetuses were weighed and, based on this weight and the fetal growth curves, we were able to estimate the weight of each fetus on the day of infusion. Using these estimates the dose of phentolamine infused was not different between the control group (0.21 ± 0.02 mg kg–1 min–1) and the PR group (0.17 ± 0.01 mg kg–1 min–1). Fetal arterial blood samples (0.5 ml) were collected at 3 h and at 5 min before, and 5, 60, 115, 125 min and 4 h after the start of the infusion for the measurement of fetal arterial blood gases and pH. Plasma from the –3 h sample was stored at –80°C for NA assay. Fetal BP and HR were recorded throughout this period.
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Phenylephrine infusion. Completeness of the -adrenergic receptor block was confirmed for all fetuses by I.V. administration of the -adrenergic agonist, phenylephrine (100 μg, Sigma Aldrich, Castle Hill, Australia), at the end of saline and phentolamine infusion. There was no difference in the phenylephrine dose administered in the control (36 ± 3 μg kg–1) and PR (42 ± 2 μg kg–1) groups. The mean change in mean arterial pressure (MAP) after phenylephrine administration in the saline-treated fetuses was 15.3 ± 1.9 mmHg and 1.9 ± 1.3 mmHg in the phentolamine treated group (P < 0.01) when data from the control and PR fetuses were combined.
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Post mortem
At 138–141 days, or after fetal death, ewes were humanely killed with an overdose of sodium pentobarbitone (Lyppards; 25 ml at 325 mg ml–1; Virbac Aus, Peakhurst, Australia).
Analysis of fetal arterial BP and HR data
Fetal arterial BP was sampled at a rate of 400 Hz using Chart 5 (ADIstruments, Castle Hill, Australia) and corrected by subtracting intra-amniotic pressure. Fetal systolic (SBP) and diastolic (DBP) BP were calculated as the mean maximum and minimum pressure, respectively. MAP was calculated using the formula:
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HR was derived from the BP signal.
Noradrenaline assay
Fetal plasma NA concentrations were measured using a radioimmunoassay kit (BioSource Labelled Europe S.A., Nivelles, Belgium). Samples were measured in duplicate using 150–300 μl of plasma, and all samples were measured in one assay.
Statistical analysis
All data are presented as means ± S.E.M.
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Blood gas data. Mean arterial PO2, SO2, Hb, PCO2 and pH were calculated as the mean of the values measured on each infusion day. Fetuses were classified as hypoxic if the mean PO2 was <17 mmHg (Edwards et al. 1999). All fetuses in the control group were normoxic, and all fetuses in the PR group were hypoxic. Mean fetal blood gases, pH and arterial oxygen content per 100 ml (oxygen content (CO2) = (PO2 x 0.003) + [Hb] x (SO2/100) x 1.39) (Edwards et al. 1999) were compared between the control and PR groups using analysis of variance (ANOVA) with repeated measures.
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Basal data. Basal values for SBP, DBP, MAP and HR were calculated for each fetus by averaging the first minute of each 5 min period during a 1 h baseline recording prior to infusion on both days. The effects of group (control versus PR) and treatment (saline versus phentolamine) were compared using multifactorial ANOVA with repeated measures. Basal plasma NA concentrations were compared between the two groups using an unpaired t test.
Phentolamine infusion. Fetal BP and HR were calculated by averaging the first minute of each 5 min period for 1 h before and 2 h during infusion. The effects of group (control versus PR), treatment (saline versus phentolamine) and time (time points before and during the infusion) were compared using multifactorial ANOVA with repeated measures. When a significant interaction between the effects of treatment group and time were found, the data were split and re-analysed based on the interacting factor. Duncan's post hoc test was performed to identify significant differences between mean values. The relationship between mean fetal PO2 on the experimental days and the maximum change in fetal MAP after the start of the phentolamine infusion were determined using linear regression analysis.
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Phenylephrine injection. Fetal BP and HR were calculated by averaging the first minute of each 5 min period for 1 h after saline infusion. The change in fetal BP and HR from the mean baseline period (20 min prior to phenylephrine injection), and the time course of the responses to phenylephrine in the control and PR fetuses (group) were compared using ANOVA. Duncan's post hoc test was performed to identify significant differences between mean values.
A probability value of 5% (P < 0.05) was considered significant.
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Results
Fetal outcomes
The estimated weight of fetuses on the day of phentolamine infusion (124–127 days) was not significantly different between the PR (2.4 ± 0.1 kg) and control (2.9 ± 0.2 kg; P = 0.09) groups. At post mortem (138–141 days), fetal weight was lower in the PR group (3.5 ± 0.3 kg) compared to controls (4.4 ± 0.4 kg; P = 0.030).
Fetal arterial blood gas status
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Fetal PO2, SO2 and CO2 were significantly lower (F1,11 > 11.48, P < 0.01) and Hb was higher (F1,11 = 8.02, P = 0.02) in PR compared to control fetuses between surgery and 127 days' gestation (Fig. 1A–D). Fetal PCO2 was also higher in PR compared to control fetuses (F1,11 = 5.33, P = 0.04; Fig. 1E). There was no significant difference in fetal pH, however, between the groups (Fig. 1F).
Fetal arterial blood gases (A–E) and pH (F) between the first day after surgery to completion of the protocol (open bars, control; filled bars, placentally restricted (PR)). SO2, oxygen saturation; CO2, oxygen content. *P < 0.05 difference between groups. Error bars show means ± S.E.M.
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Basal cardiovascular status and plasma NA concentrations
There was no difference in basal SBP, DBP, MAP or HR between PR and control fetuses at 123–127 days' gestation. On the day of the saline or phentolamine infusion, the fetal plasma NA concentrations were higher in PR fetuses (1.4 ± 0.2 pmol ml–1) compared to control fetuses (0.8 ± 0.1 pmol ml–1; P < 0.05). An inverse relationship between plasma NA concentrations and arterial PO2 was observed (y = 2.29 – 0.06x, F1,8 = 5.59, P = 0.05).
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Fetal arterial blood gas responses to phentolamine
SO2 was lower in PR fetuses during the saline and phentolamine infusion period compared to control fetuses (F1,9 > 20.48, P < 0.002). SO2 was also lower (F1,8 = 7.28, P < 0.03) in both the control and PR groups during infusion of phentolamine compared to saline. While fetal pH did not change during the saline infusion in either the control or PR group, the pH decreased (F6,29 = 6.36, P = 0.001) after phentolamine infusion in the PR, but not the control group (Table 2). Fetal PO2 and CO2 were lower and PCO2 and Hb were higher in PR fetuses compared with control fetuses throughout the phentolamine and saline infusions. These differences were independent of whether the fetuses were infused with saline or phentolamine (F1,9 > 5.86, P < 0.001; Table 2).
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Fetal cardiovascular responses to phentolamine
Fetal BP. Fetal SBP was higher at only one time point during the saline infusion when compared to baseline values in the control group. There was no effect of saline infusion on fetal DBP or MAP in control fetuses or on SBP, DBP or MAP in the PR group.
The fetal SBP, DBP and MAP responses to phentolamine infusion were significantly different (F34,306 > 2.94, P < 0.001) in the PR group compared to the control group (Fig. 2B, D and F). In the control group, SBP, DBP and MAP were only lower (F34,135 > 2.35, P < 0.001) at 10 min after the start of the phentolamine infusion when compared with any point at 5–15 min before the infusion. In the PR group, SBP, DBP and MAP were lower (F34,170 > 9.69, P < 0.001) than baseline values at 10 min after the start of the phentolamine infusion, and the decrease in SBP, DBP and MAP was sustained for 120 min (Figs 2B, D and F). The maximum fall in MAP after the start of phentolamine infusion was also greater in the PR group (+10min; –15.2 ± 1.6 mmHg) compared with controls (+10min; –6.2 ± 1.3 mmHg; P < 0.005).
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A and B, systolic blood pressure (SBP); C and D, diastolic blood pressure (DBP); E and F, mean arterial blood pressure (MAP); and G and H, change in heart rate (delta HR) responses to intravenous infusion of saline (left panel) or phentolamine (right panel) in control () and placentally restricted (PR; ) fetuses between 123 and 127 days' gestation. *Differences (P < 0.05) across time in the control or PR group. Error bars show means ± S.E.M.
Fetal HR. Fetal HR was higher at the end of the saline infusion when compared to baseline values in control fetuses (F34,135 = 1.95, P < 0.004) and there was no change in fetal HR throughout the saline infusion period in the PR group (Fig. 2G). There was an increase in fetal HR during the first 35 min after the start of the phentolamine infusion in the control fetuses (F34,136 = 3.80, P < 0.001), with a return to baseline before a smaller rise which occurred during the last 20 min of the infusion period (Fig. 2H). In contrast, the rise in fetal HR in PR fetuses was not observed until after 40 min of phentolamine infusion (F34,135 = 1.95, P = 0.004) (Fig. 2H).
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Relationship between the maximum fall in fetal MAP in response to phentolamine and the prevailing fetal PO2. There was a significant relationship between the maximum fall in MAP in response to phentolamine (y) and the fetal PO2 (x) on the day of the infusion (y = 0.87x – 27.01, r2 = 0.78, P = 0.003) when data from the control and PR fetuses were combined (Fig. 3). In addition, there was a trend toward a relationship (P = 0.06) between the fall in MAP in response to phentolamine and the prevailing plasma NA concentrations.
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Relationship between the fall in mean arterial blood pressure (MAP) in response to phentolamine and fetal PO2 measured on the day of phentolamine infusion.
Fetal cardiovascular response to phenylephrine
Following the saline infusion period, the increases in fetal SBP, DBP and MAP and the fall in fetal HR in response to a phenylephrine bolus were not different between the control and PR fetuses (Fig. 4). In both groups, the increase in SBP and MAP was significant by 5 min after phenylephrine injection, and these increases were sustained for 25 min (F16,127 > 21.15, P < 0.001). The increase in fetal DBP, however, was sustained for 45 min in both control and PR groups (F16,127 = 17.69, P = 0.001). Fetal HR fell to a minimum at 10 min after phenylephrine injection and then increased to a maximum value by 35 min after the injection (F16,127 = 6.52, P = 0.001).
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Fetal cardiovascular responses to phenylephrine injection in control () and placentally restricted (PR; ) fetuses between 123 and 127 days' gestation. SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial blood pressure; HR, heart rate. *Differences (P < 0.05) between mean values during the infusion period and baseline values in the control and PR groups.
Discussion
This study is the first to determine the extent to which fetal BP is actively maintained by -adrenergic stimulation in a model of placental insufficiency and chronic fetal hypoxaemia. There was a significantly greater hypotensive response to -adrenergic blockade in hypoxic PR fetuses compared to normoxic controls, and there was a direct relationship between the amplitude of this hypotensive response and the prevailing PO2 in the cohort of hypoxic and normoxic animals.
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As we have previously shown, restriction of placental growth from conception in the sheep results in a sustained decrease in fetal PO2, a mild hypercapnia and no change in pH (Edwards et al. 1999).
In normoxic control fetuses, phentolamine infusion resulted in a transient fall in MAP of around 6 mmHg. Previous studies report that -adrenergic blockade resulted in either no fall (Giussani et al. 1993) or up to an 8 mmHg decrease in MAP in normoxic fetal sheep during late gestation (Kimura et al. 1996). While there were no differences in basal SBP, DBP or MAP between hypoxic PR and normoxic control fetuses, the fetal hypotensive response to phentolamine was greater (11–21 mmHg) and sustained for a longer period in the hypoxic PR group. These data indicate therefore that the maintenance of basal MAP in the hypoxic PR fetus is dependent to a greater extent on activation of peripheral vascular -adrenergic receptors than in normoxic control animals. The lack of a difference between the PR and control group in the fetal BP responses to phenylephrine injection, suggests that postreceptor events are unlikely to contribute to increased -adrenergic activation in the peripheral vasculature of the PR fetus. Our results in the chronically hypoxic sheep fetus are consistent with those obtained when an -adrenergic blocker is administered to the fetal llama (Giussani et al. 1999), a species adapted to the chronic hypobaric hypoxaemia of pregnancy at altitude. Administration of phentolamine to a llama fetus results in a marked fall (20%) in MAP during basal conditions. Circulating NA concentrations are high in both the fetal llama (Giussani et al. 1999) and the hypoxic PR fetus (Simonetta et al. 1997), and we have previously reported that there was a significant inverse relationship between plasma NA concentrations and PO2 in a cohort of PR and control fetuses during late gestation. In this latter study, there was a 0.4 pmol ml–1 increase in plasma NA concentrations for every 1 mmHg decrease in PO2 (Simonetta et al. 1997). In the present study, plasma NA concentrations were also higher in the chronically hypoxic PR fetuses, and one possibility is that NA acts in turn to increase peripheral vascular tone, which would explain the direct relationship between the hypotensive response to -adrenergic blockade and the degree of fetal hypoxia. This possibility is supported by the finding of a trend (P = 0.06) toward a relationship between the hypotensive response to phentolamine and the prevailing plasma NA concentrations.
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The source of the increase in circulating NA concentrations in the hypoxic PR fetus is unclear. Intrafetal infusion of tyramine, which acts to displace NA from vesicles within postganglionic sympathetic neurones, results in a significantly greater increase in plasma NA in PR than control fetal sheep (Simonetta et al. 1997). One possibility is that low PO2 acts via chemoreflex mechanisms to stimulate catecholamine secretion from sympathetic neurones which innervate the peripheral vasculature in the PR fetus. There is evidence that the peripheral chemoreceptors mediate fetal cardiovascular responses to a 24 h period of reduced uterine blood flow (Stein et al. 1999) but it is also the case, that carotid sinus denervation does not affect the intense peripheral vasoconstriction induced by acute hypoxaemia in the fetal llama (Giussani et al. 1996). Thus the contribution of the carotid chemoreflex to the cardiovascular and catecholamine responses to periods of hypoxia which are sustained for days or weeks during late gestation are not well understood in the chronically hypoxic PR fetus. In this context it is interesting that exposure of the chick embryo to chronic moderate hypoxia leads to sympathetic hyperinnervation of the arterial system (Ruijtenbeek et al. 2000).
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Independently of the mechanisms underlying the increase in circulating NA, it is clear that the maintenance of basal BP in the hypoxic PR fetus is dependent on activation of the sympathetic nervous system (SNS) from as early as 123 days' gestation. In a previous study we reported that infusion of an angiotensin converting enzyme (ACE) inhibitor, captopril, had no effect on basal BP in hypoxic PR or normoxic fetal sheep before 125 days' gestation (Edwards et al. 1999). After 135 days' gestation, however, captopril infusion resulted in a greater hypotensive response (8 mmHg) in PR fetuses compared to controls (Edwards et al. 1999). It was speculated that the greater hypotensive response to ACE inhibition in the older PR fetuses was related to the greater increase in circulating cortisol which occurs in these animals from 135 days' gestation (Phillips et al. 1996; Simonetta et al. 1997). Thus there appears to be a sequential activation of the SNS and renin angiotensin system in the PR fetus that maintains basal BP throughout late gestation. Such neuroendocrine and cardiovascular adaptations may underpin the sparing of brain growth that occurs in the chronically hypoxic sheep and human fetus during late gestation (McMillen et al. 2001). While increased sympathetic tone may contribute to the sparing of brain growth and, potentially, fetal survival in response to an adverse intrauterine environment, it may have longer term negative outcomes. Ultrasound studies in the human show that growth-restricted fetuses have reduced superior mesenteric artery and coeliac axis blood flow velocity and are at an increased risk of necrotizing entercolitis (Kempley et al. 1991). Furthermore, a recent epidemiological study found a direct relationship between birth weight and adult pulse rate, and concluded that elevated SNS activity established in utero may be one mechanism contributing to the association between small size at birth and high blood pressure in adult life (Flanagan et al. 1999).
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In summary we have demonstrated that the maintenance of BP in the chronically hypoxic fetus is dependent on -adrenergic activation, and that this fetal adaptation to a suboptimal intrauterine environment pre-dates the development of significant fetal growth restriction. While this adaptation may play a critical role in the sparing of brain growth (McMillen et al. 2001), probably through the redistribution of fetal cardiac output, it may have adverse consequences for peripheral vascular function in the neonatal period and potentially in adult life.
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Abstract
Placental insufficiency resulting in restriction of fetal substrate supply and fetal hypoxaemia is a major cause of restricted fetal growth and increased neonatal morbidity. Fetal adaptations to placental restriction (PR) include increases in circulating catecholamines and cortisol and decreased fetal body growth, with relative sparing of brain growth. The mechanisms underlying the redistribution of fetal cardiac output in PR fetuses are not known and the aim of this study was to determine whether maintenance of fetal blood pressure (BP) in the PR fetus is dependent on -adrenergic stimulation. PR was induced by removing the majority of uterine caruncles in the ewe before conception. Sterile vascular surgery was performed on seven PR and six control fetuses at 113–120 days' gestation (term = 150 ± 3 days). Fetuses with a mean arterial PO2 < 17 mmHg between 123 and 127 days' gestation were defined as hypoxic. There was a greater fall (P < 0.05) in fetal BP during phentolamine infusion (I.V: 5 mg bolus, 0.2 mg kg–1 min–1 for 2 h) in the hypoxic PR group (–15 ± 2 mmHg) compared with normoxic controls (–5 ± 1 mmHg). The fall in fetal BP during phentolamine infusion was directly related to the level of fetal PO2. Fetal BP and HR responses to phenylephrine (I.V.: 40 μg kg–1) were not different between PR and control fetuses. The maintenance of BP in the chronically hypoxic fetus is therefore dependent on -adrenergic activation, and this fetal adaptation to a suboptimal intrauterine environment pre-dates the development of significant growth restriction. While this adaptation may play a critical role in the redistribution of fetal cardiac output to ensure the sparing of brain growth, it may have adverse consequences for peripheral vascular function in the neonatal period and in adult life.
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Introduction
Placental insufficiency resulting in a restriction of fetal substrate supply and associated chronic fetal hypoxaemia is a major cause of reduced fetal growth and is associated with increased neonatal mortality and morbidity (Bernstein et al. 2000). A range of clinical studies have demonstrated cardiovascular adaptations to chronic hypoxia which include a decrease in fetal growth and a relative sparing of brain growth due to a redistribution of cardiac output (Baschat et al. 1997; Bernstein et al. 2000; Dubiel et al. 2002). The mechanisms that underlie this redistribution of cardiac output in the chronically hypoxic fetus are not well understood.
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Fetal cardiovascular responses to acute hypoxia have been well defined and include bradycardia, an increase in arterial blood pressure (BP) and peripheral vasoconstriction (Boddy et al. 1974; Cohn et al. 1974; Bartelds et al. 1993; Giussani et al. 1993). The mechanisms mediating these cardiovascular responses include chemoreflex, endocrine and local components (Giussani et al. 1994; Green et al. 2001) which act in combination to ensure a redistribution of fetal cardiac output towards the brain, heart and adrenals and away from the periphery (Rudolph et al. 1981; Jensen et al. 1987a,b; Yaffe et al. 1987; Jansen et al. 1989; Giussani et al. 1993). During acute hypoxaemia there is an increase in fetal plasma noradrenaline (NA) concentrations (Jones & Robinson, 1975) and hypoxia-mediated peripheral vasoconstriction is dependent on -adrenergic activation in the sheep fetus (Giussani et al. 1993) and chick embryo (Mulder et al. 2002). Chronic fetal hypoxia has been induced following placental embolization in late gestation (Murotsuki et al. 1997a) or exposure of the pregnant ewe to either high altitude (Longo & Pearce, 1998) or low inspired oxygen (Kitanaka et al. 1989). These models of chronic fetal hypoxia result in a decrease in fetal arterial PO2 for varying periods during late gestation. Placental embolization in late gestation, results in low fluctuating fetal PO2 and an increase in fetal BP and a decrease in fetal heart rate (HR) (Murotsuki et al. 1997b). An alternative experimental model is one in which the majority of endometrial caruncles are removed from the uterus of the non-pregnant ewe resulting in placental restriction (PR), a maintained decrease in fetal arterial PO2 and decreased fetal growth (Robinson et al. 1979). In hypoxic PR fetuses, arterial pressure is maintained at the same level as in normoxic control fetuses (Robinson et al. 1983; Walker et al. 1990; Edwards et al. 1999) while HR is lower (Walker et al. 1990). Independently of whether hypoxaemia is induced over a period of days or months, however, there is an increase in the plasma concentrations of NA in hypoxic fetuses (Jones & Robinson, 1983; Murotsuki et al. 1997b; Simonetta et al. 1997; Gardner et al. 2002). Furthermore, in chronically hypoxic fetuses, there is an inverse relationship between the prevailing fetal arterial PO2 and plasma NA concentrations (Simonetta et al. 1997).
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Studies of total body catecholamine kinetics before and after birth in spontaneously hypoxaemic fetal lambs indicate that increases in circulating catecholamine levels in these fetuses are a consequence of increased sympathoadrenal activity, rather than a decreased clearance of the catecholamines (Smolich & Esler, 1999). While an increase in circulating catecholamines appears to be an important fetal adaptation to hypoxia, no studies have investigated the contribution of sympathetic activation to the maintenance of BP in the hypoxic PR fetus. The aim of the present study therefore was to determine the BP and HR responses to the -adrenergic antagonist, phentolamine and to the -adrenergic agonist, phenylephrine in chronically hypoxic PR and normoxic control fetal sheep during late gestation.
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Methods
Animals and surgery
All experiments were performed according to guidelines of the University of Adelaide Animal Ethics Committee.
The majority of uterine caruncles were removed in seven ewes prior to conception as previously described (Robinson et al. 1979). Ewes were monitored for 4–7 days after surgery and, after 10 weeks, entered a mating programme. Pregnancy was confirmed in both PR ewes (singleton, n = 5; twin, n = 2) and control ewes (singleton, n = 2; twin, n = 4) at around 50 days' gestation.
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At 110–120 days' gestation, surgery was performed in six control and seven PR ewes under aseptic conditions with general anaesthesia induced by sodium thiopentone (1.25 g; Pentothal, Rhone Merieux, Pinkenba, Queensland, Australia) and maintained by inhalation of halothane (2.5–4%) in oxygen. Vascular catheters were inserted in the maternal jugular vein and carotid artery (Critchley Electrical Products, Silverwater, Australia), the fetal femoral and carotid arteries and jugular vein and in the amniotic cavity as previously described (Edwards et al. 1999; Morrison et al. 2001). Fetal catheters were exteriorized through a small incision in the ewe's flank. At surgery, antibiotics were administered to the ewe (3.5 ml; 150 mg ml–1 procaine penicillin, 112.5 mg ml–1 benzathine penicillin, 2 ml; 250 g ml–1 dihydrostreptomycin, Lyppards, Adelaide, Australia) and fetus (1 ml; 150 mg ml–1 procaine penicillin, 112.5 mg ml–1 benzathine penicillin 1 ml; 250 g ml–1 dihydrostreptomycin, Lyppards, Adelaide, Australia). Antibiotics were administered intramuscularly to each ewe for 3 days after surgery and intra-amniotically (5 ml; 100 mg ml–1 ampicillin, Lyppards, Adelaide, Australia) for 4 days after surgery. Animals were allowed to recover from surgery for at least 4 days prior to experimentation.
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Arterial blood gas measurements
Fetal arterial blood gas samples (0.5 ml) were collected daily for the measurement of PO2, PCO2, pH, oxygen saturation (SO2) and haemoglobin (Hb) (ABL 520 analyser, Radiometer, Copenhagen, Denmark).
Physiological measurements
Fetal femoral artery and amniotic catheters were connected to displacement transducers and a quad-bridge amplifier (ADInstruments, Castle Hill, Australia) to record fetal blood pressure (BP) and amniotic pressure. All data were digitized and recorded using Chart (ADInstruments, Castle Hill, Australia).
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Experimental protocol
Basal data. Fetal BP was recorded for 3 h prior to infusion of drugs. The number of animals in each study is shown in Table 1.
Phentolamine infusion. Each fetus received either saline or phentolamine (bolus, 5 mg; infusion, 0.2 mg kg–1 min–1 (Giussani et al. 1993; Giussani et al. 1999); Sigma Aldrich, Castle Hill, Australia) I.V. for 2 h on two consecutive days between 123 and 127 days' gestation. We used the following fetal growth curves previously established in this laboratory to make an initial estimate of fetal weight in the control and PR groups at 123–127 days' gestation (Edwards et al. 1999):
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Based on these estimates of fetal weights, a total of 72 mg phentolamine was infused in each control fetus and a total of 48 mg phentolamine was infused in each PR fetus during the 2 h infusion period. At post mortem, all fetuses were weighed and, based on this weight and the fetal growth curves, we were able to estimate the weight of each fetus on the day of infusion. Using these estimates the dose of phentolamine infused was not different between the control group (0.21 ± 0.02 mg kg–1 min–1) and the PR group (0.17 ± 0.01 mg kg–1 min–1). Fetal arterial blood samples (0.5 ml) were collected at 3 h and at 5 min before, and 5, 60, 115, 125 min and 4 h after the start of the infusion for the measurement of fetal arterial blood gases and pH. Plasma from the –3 h sample was stored at –80°C for NA assay. Fetal BP and HR were recorded throughout this period.
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Phenylephrine infusion. Completeness of the -adrenergic receptor block was confirmed for all fetuses by I.V. administration of the -adrenergic agonist, phenylephrine (100 μg, Sigma Aldrich, Castle Hill, Australia), at the end of saline and phentolamine infusion. There was no difference in the phenylephrine dose administered in the control (36 ± 3 μg kg–1) and PR (42 ± 2 μg kg–1) groups. The mean change in mean arterial pressure (MAP) after phenylephrine administration in the saline-treated fetuses was 15.3 ± 1.9 mmHg and 1.9 ± 1.3 mmHg in the phentolamine treated group (P < 0.01) when data from the control and PR fetuses were combined.
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Post mortem
At 138–141 days, or after fetal death, ewes were humanely killed with an overdose of sodium pentobarbitone (Lyppards; 25 ml at 325 mg ml–1; Virbac Aus, Peakhurst, Australia).
Analysis of fetal arterial BP and HR data
Fetal arterial BP was sampled at a rate of 400 Hz using Chart 5 (ADIstruments, Castle Hill, Australia) and corrected by subtracting intra-amniotic pressure. Fetal systolic (SBP) and diastolic (DBP) BP were calculated as the mean maximum and minimum pressure, respectively. MAP was calculated using the formula:
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HR was derived from the BP signal.
Noradrenaline assay
Fetal plasma NA concentrations were measured using a radioimmunoassay kit (BioSource Labelled Europe S.A., Nivelles, Belgium). Samples were measured in duplicate using 150–300 μl of plasma, and all samples were measured in one assay.
Statistical analysis
All data are presented as means ± S.E.M.
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Blood gas data. Mean arterial PO2, SO2, Hb, PCO2 and pH were calculated as the mean of the values measured on each infusion day. Fetuses were classified as hypoxic if the mean PO2 was <17 mmHg (Edwards et al. 1999). All fetuses in the control group were normoxic, and all fetuses in the PR group were hypoxic. Mean fetal blood gases, pH and arterial oxygen content per 100 ml (oxygen content (CO2) = (PO2 x 0.003) + [Hb] x (SO2/100) x 1.39) (Edwards et al. 1999) were compared between the control and PR groups using analysis of variance (ANOVA) with repeated measures.
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Basal data. Basal values for SBP, DBP, MAP and HR were calculated for each fetus by averaging the first minute of each 5 min period during a 1 h baseline recording prior to infusion on both days. The effects of group (control versus PR) and treatment (saline versus phentolamine) were compared using multifactorial ANOVA with repeated measures. Basal plasma NA concentrations were compared between the two groups using an unpaired t test.
Phentolamine infusion. Fetal BP and HR were calculated by averaging the first minute of each 5 min period for 1 h before and 2 h during infusion. The effects of group (control versus PR), treatment (saline versus phentolamine) and time (time points before and during the infusion) were compared using multifactorial ANOVA with repeated measures. When a significant interaction between the effects of treatment group and time were found, the data were split and re-analysed based on the interacting factor. Duncan's post hoc test was performed to identify significant differences between mean values. The relationship between mean fetal PO2 on the experimental days and the maximum change in fetal MAP after the start of the phentolamine infusion were determined using linear regression analysis.
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Phenylephrine injection. Fetal BP and HR were calculated by averaging the first minute of each 5 min period for 1 h after saline infusion. The change in fetal BP and HR from the mean baseline period (20 min prior to phenylephrine injection), and the time course of the responses to phenylephrine in the control and PR fetuses (group) were compared using ANOVA. Duncan's post hoc test was performed to identify significant differences between mean values.
A probability value of 5% (P < 0.05) was considered significant.
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Results
Fetal outcomes
The estimated weight of fetuses on the day of phentolamine infusion (124–127 days) was not significantly different between the PR (2.4 ± 0.1 kg) and control (2.9 ± 0.2 kg; P = 0.09) groups. At post mortem (138–141 days), fetal weight was lower in the PR group (3.5 ± 0.3 kg) compared to controls (4.4 ± 0.4 kg; P = 0.030).
Fetal arterial blood gas status
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Fetal PO2, SO2 and CO2 were significantly lower (F1,11 > 11.48, P < 0.01) and Hb was higher (F1,11 = 8.02, P = 0.02) in PR compared to control fetuses between surgery and 127 days' gestation (Fig. 1A–D). Fetal PCO2 was also higher in PR compared to control fetuses (F1,11 = 5.33, P = 0.04; Fig. 1E). There was no significant difference in fetal pH, however, between the groups (Fig. 1F).
Fetal arterial blood gases (A–E) and pH (F) between the first day after surgery to completion of the protocol (open bars, control; filled bars, placentally restricted (PR)). SO2, oxygen saturation; CO2, oxygen content. *P < 0.05 difference between groups. Error bars show means ± S.E.M.
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Basal cardiovascular status and plasma NA concentrations
There was no difference in basal SBP, DBP, MAP or HR between PR and control fetuses at 123–127 days' gestation. On the day of the saline or phentolamine infusion, the fetal plasma NA concentrations were higher in PR fetuses (1.4 ± 0.2 pmol ml–1) compared to control fetuses (0.8 ± 0.1 pmol ml–1; P < 0.05). An inverse relationship between plasma NA concentrations and arterial PO2 was observed (y = 2.29 – 0.06x, F1,8 = 5.59, P = 0.05).
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Fetal arterial blood gas responses to phentolamine
SO2 was lower in PR fetuses during the saline and phentolamine infusion period compared to control fetuses (F1,9 > 20.48, P < 0.002). SO2 was also lower (F1,8 = 7.28, P < 0.03) in both the control and PR groups during infusion of phentolamine compared to saline. While fetal pH did not change during the saline infusion in either the control or PR group, the pH decreased (F6,29 = 6.36, P = 0.001) after phentolamine infusion in the PR, but not the control group (Table 2). Fetal PO2 and CO2 were lower and PCO2 and Hb were higher in PR fetuses compared with control fetuses throughout the phentolamine and saline infusions. These differences were independent of whether the fetuses were infused with saline or phentolamine (F1,9 > 5.86, P < 0.001; Table 2).
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Fetal cardiovascular responses to phentolamine
Fetal BP. Fetal SBP was higher at only one time point during the saline infusion when compared to baseline values in the control group. There was no effect of saline infusion on fetal DBP or MAP in control fetuses or on SBP, DBP or MAP in the PR group.
The fetal SBP, DBP and MAP responses to phentolamine infusion were significantly different (F34,306 > 2.94, P < 0.001) in the PR group compared to the control group (Fig. 2B, D and F). In the control group, SBP, DBP and MAP were only lower (F34,135 > 2.35, P < 0.001) at 10 min after the start of the phentolamine infusion when compared with any point at 5–15 min before the infusion. In the PR group, SBP, DBP and MAP were lower (F34,170 > 9.69, P < 0.001) than baseline values at 10 min after the start of the phentolamine infusion, and the decrease in SBP, DBP and MAP was sustained for 120 min (Figs 2B, D and F). The maximum fall in MAP after the start of phentolamine infusion was also greater in the PR group (+10min; –15.2 ± 1.6 mmHg) compared with controls (+10min; –6.2 ± 1.3 mmHg; P < 0.005).
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A and B, systolic blood pressure (SBP); C and D, diastolic blood pressure (DBP); E and F, mean arterial blood pressure (MAP); and G and H, change in heart rate (delta HR) responses to intravenous infusion of saline (left panel) or phentolamine (right panel) in control () and placentally restricted (PR; ) fetuses between 123 and 127 days' gestation. *Differences (P < 0.05) across time in the control or PR group. Error bars show means ± S.E.M.
Fetal HR. Fetal HR was higher at the end of the saline infusion when compared to baseline values in control fetuses (F34,135 = 1.95, P < 0.004) and there was no change in fetal HR throughout the saline infusion period in the PR group (Fig. 2G). There was an increase in fetal HR during the first 35 min after the start of the phentolamine infusion in the control fetuses (F34,136 = 3.80, P < 0.001), with a return to baseline before a smaller rise which occurred during the last 20 min of the infusion period (Fig. 2H). In contrast, the rise in fetal HR in PR fetuses was not observed until after 40 min of phentolamine infusion (F34,135 = 1.95, P = 0.004) (Fig. 2H).
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Relationship between the maximum fall in fetal MAP in response to phentolamine and the prevailing fetal PO2. There was a significant relationship between the maximum fall in MAP in response to phentolamine (y) and the fetal PO2 (x) on the day of the infusion (y = 0.87x – 27.01, r2 = 0.78, P = 0.003) when data from the control and PR fetuses were combined (Fig. 3). In addition, there was a trend toward a relationship (P = 0.06) between the fall in MAP in response to phentolamine and the prevailing plasma NA concentrations.
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Relationship between the fall in mean arterial blood pressure (MAP) in response to phentolamine and fetal PO2 measured on the day of phentolamine infusion.
Fetal cardiovascular response to phenylephrine
Following the saline infusion period, the increases in fetal SBP, DBP and MAP and the fall in fetal HR in response to a phenylephrine bolus were not different between the control and PR fetuses (Fig. 4). In both groups, the increase in SBP and MAP was significant by 5 min after phenylephrine injection, and these increases were sustained for 25 min (F16,127 > 21.15, P < 0.001). The increase in fetal DBP, however, was sustained for 45 min in both control and PR groups (F16,127 = 17.69, P = 0.001). Fetal HR fell to a minimum at 10 min after phenylephrine injection and then increased to a maximum value by 35 min after the injection (F16,127 = 6.52, P = 0.001).
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Fetal cardiovascular responses to phenylephrine injection in control () and placentally restricted (PR; ) fetuses between 123 and 127 days' gestation. SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial blood pressure; HR, heart rate. *Differences (P < 0.05) between mean values during the infusion period and baseline values in the control and PR groups.
Discussion
This study is the first to determine the extent to which fetal BP is actively maintained by -adrenergic stimulation in a model of placental insufficiency and chronic fetal hypoxaemia. There was a significantly greater hypotensive response to -adrenergic blockade in hypoxic PR fetuses compared to normoxic controls, and there was a direct relationship between the amplitude of this hypotensive response and the prevailing PO2 in the cohort of hypoxic and normoxic animals.
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As we have previously shown, restriction of placental growth from conception in the sheep results in a sustained decrease in fetal PO2, a mild hypercapnia and no change in pH (Edwards et al. 1999).
In normoxic control fetuses, phentolamine infusion resulted in a transient fall in MAP of around 6 mmHg. Previous studies report that -adrenergic blockade resulted in either no fall (Giussani et al. 1993) or up to an 8 mmHg decrease in MAP in normoxic fetal sheep during late gestation (Kimura et al. 1996). While there were no differences in basal SBP, DBP or MAP between hypoxic PR and normoxic control fetuses, the fetal hypotensive response to phentolamine was greater (11–21 mmHg) and sustained for a longer period in the hypoxic PR group. These data indicate therefore that the maintenance of basal MAP in the hypoxic PR fetus is dependent to a greater extent on activation of peripheral vascular -adrenergic receptors than in normoxic control animals. The lack of a difference between the PR and control group in the fetal BP responses to phenylephrine injection, suggests that postreceptor events are unlikely to contribute to increased -adrenergic activation in the peripheral vasculature of the PR fetus. Our results in the chronically hypoxic sheep fetus are consistent with those obtained when an -adrenergic blocker is administered to the fetal llama (Giussani et al. 1999), a species adapted to the chronic hypobaric hypoxaemia of pregnancy at altitude. Administration of phentolamine to a llama fetus results in a marked fall (20%) in MAP during basal conditions. Circulating NA concentrations are high in both the fetal llama (Giussani et al. 1999) and the hypoxic PR fetus (Simonetta et al. 1997), and we have previously reported that there was a significant inverse relationship between plasma NA concentrations and PO2 in a cohort of PR and control fetuses during late gestation. In this latter study, there was a 0.4 pmol ml–1 increase in plasma NA concentrations for every 1 mmHg decrease in PO2 (Simonetta et al. 1997). In the present study, plasma NA concentrations were also higher in the chronically hypoxic PR fetuses, and one possibility is that NA acts in turn to increase peripheral vascular tone, which would explain the direct relationship between the hypotensive response to -adrenergic blockade and the degree of fetal hypoxia. This possibility is supported by the finding of a trend (P = 0.06) toward a relationship between the hypotensive response to phentolamine and the prevailing plasma NA concentrations.
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The source of the increase in circulating NA concentrations in the hypoxic PR fetus is unclear. Intrafetal infusion of tyramine, which acts to displace NA from vesicles within postganglionic sympathetic neurones, results in a significantly greater increase in plasma NA in PR than control fetal sheep (Simonetta et al. 1997). One possibility is that low PO2 acts via chemoreflex mechanisms to stimulate catecholamine secretion from sympathetic neurones which innervate the peripheral vasculature in the PR fetus. There is evidence that the peripheral chemoreceptors mediate fetal cardiovascular responses to a 24 h period of reduced uterine blood flow (Stein et al. 1999) but it is also the case, that carotid sinus denervation does not affect the intense peripheral vasoconstriction induced by acute hypoxaemia in the fetal llama (Giussani et al. 1996). Thus the contribution of the carotid chemoreflex to the cardiovascular and catecholamine responses to periods of hypoxia which are sustained for days or weeks during late gestation are not well understood in the chronically hypoxic PR fetus. In this context it is interesting that exposure of the chick embryo to chronic moderate hypoxia leads to sympathetic hyperinnervation of the arterial system (Ruijtenbeek et al. 2000).
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Independently of the mechanisms underlying the increase in circulating NA, it is clear that the maintenance of basal BP in the hypoxic PR fetus is dependent on activation of the sympathetic nervous system (SNS) from as early as 123 days' gestation. In a previous study we reported that infusion of an angiotensin converting enzyme (ACE) inhibitor, captopril, had no effect on basal BP in hypoxic PR or normoxic fetal sheep before 125 days' gestation (Edwards et al. 1999). After 135 days' gestation, however, captopril infusion resulted in a greater hypotensive response (8 mmHg) in PR fetuses compared to controls (Edwards et al. 1999). It was speculated that the greater hypotensive response to ACE inhibition in the older PR fetuses was related to the greater increase in circulating cortisol which occurs in these animals from 135 days' gestation (Phillips et al. 1996; Simonetta et al. 1997). Thus there appears to be a sequential activation of the SNS and renin angiotensin system in the PR fetus that maintains basal BP throughout late gestation. Such neuroendocrine and cardiovascular adaptations may underpin the sparing of brain growth that occurs in the chronically hypoxic sheep and human fetus during late gestation (McMillen et al. 2001). While increased sympathetic tone may contribute to the sparing of brain growth and, potentially, fetal survival in response to an adverse intrauterine environment, it may have longer term negative outcomes. Ultrasound studies in the human show that growth-restricted fetuses have reduced superior mesenteric artery and coeliac axis blood flow velocity and are at an increased risk of necrotizing entercolitis (Kempley et al. 1991). Furthermore, a recent epidemiological study found a direct relationship between birth weight and adult pulse rate, and concluded that elevated SNS activity established in utero may be one mechanism contributing to the association between small size at birth and high blood pressure in adult life (Flanagan et al. 1999).
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In summary we have demonstrated that the maintenance of BP in the chronically hypoxic fetus is dependent on -adrenergic activation, and that this fetal adaptation to a suboptimal intrauterine environment pre-dates the development of significant fetal growth restriction. While this adaptation may play a critical role in the sparing of brain growth (McMillen et al. 2001), probably through the redistribution of fetal cardiac output, it may have adverse consequences for peripheral vascular function in the neonatal period and potentially in adult life.
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