Calcitonin gene-related peptide contributes to the umbilical haemodynamic defence response to acute hypoxaemia
1 Department of Physiology, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK
Abstract
Despite clinical advances in obstetric practice, undiagnosed fetal hypoxaemia still contributes to a high incidence of perinatal morbidity. The fetal defence to hypoxaemia involves a redistribution of blood flow away from peripheral circulations towards essential vascular beds, such as the umbilical, cerebral, myocardial and adrenal circulations. In marked contrast to other essential vascular beds, the mechanisms mediating maintained perfusion of the umbilical circulation during hypoxaemia remain unknown. This study determined the role of calcitonin gene-related peptide (CGRP) in the maintenance of umbilical blood flow during basal and hypoxaemic conditions. Under anaesthesia, five sheep fetuses were instrumented with catheters and a Transonic probe around an umbilical artery, inside the fetal abdomen, at 0.8 of gestation. Five days later, fetuses were subjected to 0.5 h hypoxaemia during either I.V. saline or a selective CGRP antagonist in randomised order. Treatment started 30 min before hypoxaemia and ran continuously until the end of the challenge. The CGRP antagonist did not alter basal blood gas or cardiovascular status in the fetus. A similar fall in Pa,O2 occurred in fetuses during either saline (21 ± 0.8 to 9 ± 0.9 mmHg) or antagonist treatment (20 ± 0.9 to 9 ± 1.2 mmHg). Hypoxaemia during saline led to significant increases in arterial blood pressure, umbilical blood flow and umbilical vascular conductance. In marked contrast, hypoxaemia during CGRP antagonist treatment led to pronounced falls in both umbilical blood flow and umbilical vascular conductance without affecting the magnitude of the hypertensive response. In conclusion, CGRP plays an important role in the umbilical haemodynamic defence response to hypoxaemia in the late gestation fetus.
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Introduction
Acute fetal hypoxaemia is one of the major challenges that the fetus may face during gestation (Huch et al. 1977) and it may occur by insufficiency of either uterine blood flow (Yaffee et al. 1987) or umbilical blood flow (Itskovitz et al. 1983), or by a decrease in maternal arterial oxygen content (Parer, 1980). Other mechanisms such as fetal anaemia or increased fetal oxygen consumption (e.g. in pyrexia) are relatively rare in clinical practice (Parer & Livingston, 1990). Risk factors which predispose to the development of fetal hypoxaemia can be classified into maternal (e.g. diabetes (Macfarlane & Tsakalakos, 1985), pregnancy induced or chronic hypertension (Galanti et al. 2000), Rhesus sensitisation (Thilaganathan et al. 1992), maternal infection (Dalitz et al. 2003), sickle cell anaemia (Manzar, 2000), chronic substance abuse (Mukherjee & Hodgen, 1982; Slotkin, 1998), asthma (Witlin, 1997), seizure disorders (Tomson et al. 1997) or smoking (Socol et al. 1982)), intrapartum (e.g. multiple pregnancy (Maier et al. 1995), pre- or post-term birth (Stubblefield & Berek, 1980; Salafia et al. 1995), prolonged labour (Leszczynska-Gorzelak et al. 2002), placental abruption (Salafia et al. 1995), placenta praevia (Kovalovszki et al. 1990), prolapsed umbilical cord (Faiz et al. 2003) or abnormal presentation of the fetus (Mukhopadhyay & Arulkumaran, 2002)) and iatrogenic (e.g. epidural anaesthesia (Preston et al. 1993)). Despite recent clinical advances in obstetric practice and perinatal care in dramatically reducing infant mortality, there is still a high incidence of perinatal morbidity. Much of this can be attributed to birth asphyxia during complicated labour (Hall, 1989) or undetected episodes of antenatal fetal hypoxaemia (Low et al. 1995). Both situations render the infant at increased risk of developing complications in the central nervous (e.g. cerebral palsy (Johnston et al. 2001), neonatal seizures (Arpino et al. 2001), hypoxic–ischaemic encephalopathy (Low et al. 1985)), cardiovascular (e.g. left ventricular myocardial dysfunction (Walther et al. 1985)) and respiratory (e.g. meconium aspiration syndrome (Klingner & Kruse, 1999)) systems.
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The fetal defence to acute hypoxaemia involves cardiovascular responses that redistribute the combined ventricular output towards essential vascular beds such as the umbilical, adrenal, myocardial and cerebral circulations (for reviews, see Rudolph, 1984 and Giussani et al. 1994a,b). Redistribution of blood flow is largely accomplished by peripheral vasoconstriction, which is triggered by a carotid chemoreflex (Giussani et al. 1993) and then maintained or subsequently modified by endocrine (Iwamoto, 1990; Giussani et al. 1994b; Fletcher et al. 2000) and local components (Vane et al. 1990; Morrison et al. 2003). The resulting increase in pressure-dependent perfusion through the cerebral, myocardial and adrenal vascular beds is further facilitated by active vasodilatation (Carter et al. 1993; Thornburg et al. 2000; Riquelme et al. 2002; Blood et al. 2003). In contrast to the adrenal, myocardial and cerebral circulations, the physiological mechanisms maintaining perfusion of the umbilical vascular bed during adverse intrauterine conditions remain comparatively less well understood.
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Calcitonin gene-related peptide (CGRP) is the most potent endogenous vasodilator peptide known (Brain et al. 1985). Recently it has been shown that CGRP has a powerful vasodilator action, which in part is mediated via nitric oxide (NO), in vivo in the ovine fetal lung (Takahashi et al. 2000) and in vitro in the human umbilical artery and vein (Dong et al. 2004). However the in vivo role of CGRP in the control of the umbilical vascular bed during basal or stressful conditions has not been investigated. The present study tested the hypothesis that CGRP plays an important role in the maintenance of umbilical blood flow during acute hypoxaemia in the fetus. Late gestation fetal sheep surgically prepared for long-term recording in the conscious state were used as an experimental model. The hypothesis was tested by investigating the effects of fetal treatment with a selective CGRP antagonist on in vivo umbilical haemodynamics via an indwelling Transonic flow probe during basal and hypoxaemic conditions.
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Methods
Surgical preparation
All procedures were performed under the UK Animals (Scientific Procedures) Act 1986 and were approved by the Ethical Review Committee of the University of Cambridge. Five Welsh Mountain sheep fetuses, at 120 ± 2 days of gestation (term is 145 days), were surgically prepared for long-term recording using strict aseptic conditions as previously described in detail (Giussani et al. 2001). In brief, food, but not water, was withheld from the pregnant ewes for 24 h prior to surgery. Following induction with 20 mg kg–1 I.V. sodium thiopentone (Intraval Sodium; Merial Animal Health Ltd; Rhone Merieux, Dublin, Ireland), general anaesthesia (1.5–2.0% halothane in 50 : 50 O2 : N2O) was maintained using positive pressure ventilation. Midline abdominal and uterine incisions were made, the fetal hind limbs were exteriorized and on one side femoral arterial (i.d., 0.86 mm; o.d., 1.52 mm; Critchly Electrical Products, NSW, Australia) and venous (i.d., 0.56 mm; o.d., 0.96 mm) catheters were inserted. The catheter tips were advanced carefully to the ascending aorta and superior vena cava, respectively. Another catheter was anchored onto the fetal hind limb for recording of the reference amniotic pressure. In addition, a transit-time flow transducer (4SB; Transonic Systems Inc., Ithaca, NY, USA) was placed around one of the umbilical arteries, close to the common umbilical artery, inside the fetal abdominal cavity, for continuous measurement of unilateral umbilical blood flow (Gardner et al. 2001). The fetus was returned to the uterine cavity, the uterine incisions were closed in layers, the dead space of the catheters was filled with heparinized saline (80 i.u. heparin ml–1 in 0.9% NaCl) and the catheters' ends were plugged with sterile brass pins. The catheters and flow probe lead were then exteriorised via a key-hole incision in the maternal flank and kept inside a plastic pouch sewn onto the maternal skin.
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Postoperative care
The ewes were housed in individual pens with free access to hay and water, in rooms with a 12 h : 12 h dark : light cycle. They were fed concentrates twice daily (100 g; sheep nuts no. 6; H & C Beart Ltd, Kings Lynn, UK). All ewes received postoperative analgesia (10–20 mg kg–1 oral Penylbutazone; Equipalozone paste, Arnolds Veterinary Products Ltd, Shropshire, UK) and antibiotics (0.20–0.25 mg kg–1 I.M. Depocillin; Mycofarm, Cambridge, UK). The patency of the fetal catheters was maintained by a slow continuous infusion of heparinised saline (80 i.u. heparin ml–1 at 0.1 ml h–1 in 0.9% NaCl) containing antibiotic (1 mg ml–1 benzylpenicillin; Crystapen, Schering-Plough, Animal Health Division, Welwyn Garden City, UK). In addition, the fetuses also received daily antibiotics (150 mg kg–1 Penbritin, SmithKline Beecham Animal Health, Welwyn Garden City, Hertfordshire, UK) I.V and into the amniotic cavity.
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Experimental protocol
Following at least 5 days of postoperative recovery, all fetuses were subjected to two experimental protocols, carried out on consecutive days in randomised order. Each protocol consisted of a 2.5 h period divided into1 h normoxia, 0.5 h hypoxaemia and 1 h recovery, during either a slow I.V. infusion of vehicle (80 i.u. heparin ml–1 in 0.9% NaCl) or during fetal treatment with a selective CGRP antagonist (50 μg kg–1 I.A. bolus followed by 20 μgmin–1 I.V. infusion; calcitonin gene related peptide fragment 8–37, CGRP8-37; C-2806; Sigma Chemicals, UK, Fig. 1) which was made in a solution of heparinised saline, identical to that of the vehicle. Acute hypoxaemia in the fetus was induced by maternal inhalational hypoxia. In brief, a large transparent respiratory hood was placed over the ewe's head into which air was passed at a rate of 50 l min–1 for the 1 h period of normoxia. Following this control period, acute fetal hypoxaemia was induced for 30 min by changing the concentrations of gases breathed by the ewe to 6% O2 in N2 with small amounts of CO2 (15 l min–1 air : 35 l min–1 N2 : 1.5–2.5 l min–1 CO2). This mixture was designed to reduce fetal Pa,O2 to 9 mmHg while maintaining Pa,CO2. Following the 0.5 h period of hypoxaemia, the ewe was returned to breathing air for the 1 h recovery period. The dose of CGRP antagonist was chosen from a previous study by Takahashi et al. (2000). In that study, treatment of late gestation fetal sheep with a similar dose of the CGRP antagonist markedly diminished the vasodilator response of the pulmonary vascular bed to exogenous treatment with CGRP. In the present study, fetal treatment with the CGRP antagonist started 30 min before the onset of hypoxaemia and ran continuously until the end of the hypoxaemic challenge. During any acute hypoxaemia protocol, descending aortic blood samples were taken from the fetus at set intervals (arrows; Fig. 1) to determine arterial blood gas and acid base status (ABL5 blood gas Analyser, Radiometer, Copenhagen, Denmark; measurements corrected to 39.5°C; Fig. 1). At the end of the experimental protocol, the ewes and fetuses were killed painlessly using a lethal dose of sodium pentobarbitone (200 mg kg–1 I.V. Pentoject; Animal Ltd, York, UK), the positions of the implanted catheters and the flow probe were confirmed and the fetuses were weighed.
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The experimental protocol consisted of a 2.5-h period divided into: 1 h normoxia, 0.5 h hypoxaemia (black box) and 1 h recovery, during saline infusion or during treatment with the CGRP antagonist (grey box). Arrows represent the time at which arterial blood samples were collected.
Measurements and statistical analysis
Calibrated mean fetal arterial blood pressure (corrected for amniotic pressure) and mean umbilical blood flow were recorded continually at 1 s intervals using a computerised Data Acquisition System (Department of Physiology, Cambridge University, UK). Umbilical vascular conductance was calculated by dividing mean umbilical blood flow by mean supra-amniotic arterial blood pressure. Values for all variables are expressed as mean ± S.E.M. Cardiovascular variables are expressed as minute averages of either absolute values or of the absolute change from mean normoxic baseline. Summary measure analysis was then applied to the serial data to focus the number of comparisons, and areas under the curve were calculated for the absolute change from baseline for statistical comparison, as previously described in detail (Matthews et al. 1990). Variables were assessed using two-way ANOVA with repeated measures (Sigma-Stat; SPSS Inc., Chicago, IL, USA), comparing the effect of time (normoxia versus hypoxaemia/recovery), group (control versus CGRP antagonist) and interactions between time and group. Where a significant effect of time or group was indicated, the post hoc Student–Newman–Keuls test was used to isolate the statistical difference. For all comparisons, statistical significance was accepted when P < 0.05.
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Results
Fetal arterial blood gas and acid base status
Basal values for arterial blood gas and acid base status were similar in fetuses during saline infusion or during treatment with the CGRP antagonist. In all fetuses, acute hypoxaemia induced significant falls in arterial pH (pHa), arterial partial pressure of oxygen (Pa,O2), percentage saturation of haemoglobin with oxygen (Sat Hb) and acid base excess (ABE) without any alteration to arterial partial pressure of carbon dioxide (Pa,CO2) (Fig. 2). The magnitude of these changes was similar during saline infusion or during treatment with the CGRP antagonist. During recovery, both Pa,O2 and Sat Hb recovered to basal values in all fetuses. In contrast fetal pHa and ABE remained significantly lower than basal values until the end of the experimental protocol.
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Values represent the mean ± S.E.M. at 0 (N0) and 45 min (N45) of normoxia, at 5 (H5), 15 (H15) and 30 min (H30) of hypoxaemia, and at 30 (R30) and 60 min (R60) of recovery for fetuses exposed to 30 min hypoxaemia (box) during saline infusion (; n = 5) or during treatment with CGRP antagonist (; n = 5). Significant differences: *P < 0.05 for normoxia versus hypoxaemia or recovery. pHa, arterial pH; Pa,CO2, arterial partial pressure of CO2; Pa,O2, arterial partial pressure of O2; Sat Hb, percentage saturation of haemoglobin with oxygen; ABE, acid base excess.
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Umbilical haemodynamic responses to acute hypoxaemia
Basal values for fetal arterial blood pressure (62.5 ± 1.7 versus 61.1 ± 1.5 mmHg), unilateral umbilical blood flow (132 ± 9 versus 140 ± 14 ml min–1) and umbilical vascular conductance (2.1 ± 0.2 versus 2.3 ± 0.3 (ml min–1 mmHg–1) were similar in fetuses during saline infusion or during treatment with the CGRP antagonist, respectively (Fig. 3). During acute hypoxaemia, a significant increase in arterial blood pressure occurred in all fetuses during either saline infusion or during treatment with the CGRP antagonist, with arterial blood pressure increasing from baseline by 16.9 ± 2.0 and 17.4 ± 1.9 mmHg, respectively (Figs 3 and 4). In contrast, while umbilical blood flow (from 132 ± 9 to 198 ± 15 ml min–1) and umbilical vascular conductance (from 2.1 ± 0.2 to 2.7 ± 0.2 (ml min–1 mmHg–1) increased during the hypoxaemic period in fetuses during saline infusion, pronounced falls in both umbilical blood flow (from 140 ± 14 to 90 ± 10 ml min–1) and umbilical vascular conductance (from 2.3 ± 0.3 to 1.3 ± 0.2 ml min–1 mmHg–1) occurred early during the hypoxaemic period in fetuses during treatment with the CGRP antagonist (Figs 3 and 4). After 10 min of acute hypoxaemia during treatment with the CGRP antagonist, umbilical flow and vascular conductance returned towards basal values. By the end of the hypoxaemic challenge during the antagonist infusion, umbilical flow had increased to values above baseline (from 140 ± 13 to 187 ± 18 ml min–1), and umbilical vascular conductance had recovered to basal values (from 2.3 ± 0.3 to 2.5 ± 0.2 ml min–1 mmHg–1, Figs 3 and 4). During recovery, values for arterial blood pressure and umbilical blood flow remained significantly elevated by the end of the experimental protocol in all fetuses, during either saline infusion or treatment with the CGRP antagonist (Figs 3 and 4).
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Values are mean ± S.E.M. calculated every minute for arterial blood pressure, unilateral umbilical blood flow and umbilical vascular conductance during 1 h of normoxia, 0.5 h of hypoxaemia (box) and 1 h of recovery for fetuses during saline infusion or during treatment with the CGRP antagonist.
Values are A, mean ± S.E.M. calculated every minute for the change from normoxic baseline for arterial blood pressure, unilateral umbilical blood flow and umbilical vascular conductance and B, the statistical summary of these changes during saline infusion (; n = 5) and during treatment with the CGRP antagonist (; n = 5). Values for the statistical summary represent mean ± S.E.M. for the area under the curve of the umbilical haemodynamic variables over every 15 min. Significant differences: *P < 0.05 for normoxia versus hypoxaemia or recovery; P < 0.05, saline versus CGRP antagonist.
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Discussion
There has been a long-standing clinical and physiological interest in the haemodynamic changes in the umbilical vascular bed during pregnancy, due to its functional importance in representing feto-placental blood flow and hence fetal wellbeing. Clinically, indirect measurements of umbilical blood flow in human pregnancy are obtained routinely by Doppler flow velocimetry. However, application of this technique in detailed experiments has shown that analysis of the umbilical vessel Doppler waveforms is complicated, particularly during fetal distress. For example, while Downing et al. (1991) and Tchirikov et al. (1998) reported an increase in the umbilical artery pulsatility index (PI), signifying an increase in umbilical vascular resistance, during episodes of hypoxaemia, Muijsers et al. (1990), Morrow et al. (1990), and van Huisseling et al. (1991), were unable to find any significant change in the umbilical artery PI. Similarly, using direct, spot measurement of umbilical blood flow with radioactive microspheres in the sheep fetus, Cohn et al. (1974), and Parer (1983), reported either an increase in umbilical vascular resistance or no change in umbilical blood flow during hypoxaemia. To avoid complications in the interpretation of either indirect indices or point measurements of umbilical blood flow, or of examination of changes in the umbilical vascular bed under the effects of anaesthesia, studies in our laboratory have used continuous measurement of umbilical blood flow by an implanted Transonic flow probe around an umbilical artery within the fetal abdomen in chronically instrumented, unanaesthetised fetal sheep preparations.
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Previous investigations from this laboratory, which have measured continuous changes in flow in the umbilical vascular bed, have reported increases in umbilical blood flow during acute hypoxaemia as a result of pressure-dependent (Gardner et al. 2001) and pressure-independent mechanisms (Gardner & Giussani, 2003). Past data show that during moderate fetal hypoxaemia (i.e. a decrease in fetal Pa,O2 to 13 mmHg) there are equivalent elevations in arterial blood pressure and umbilical blood flow with no changes in umbilical vascular conductance. This suggests that during moderate fetal hypoxaemia, maintenance of umbilical perfusion is entirely pressure dependent (van Huisseling et al. 1991). However, in fetuses which have previously suffered a sustained period of intrauterine adversity, the increase in umbilical blood flow during moderate acute hypoxaemia is greater than can be accounted for by the increase in perfusion pressure (Gardner & Giussani, 2003). Therefore, in pregnancies complicated by chronic adverse intrauterine conditions, pressure-independent mechanisms may become recruited to induce an increase in umbilical conductance, signifying active vasodilatation of the umbilico-placental vascular beds. Additional data suggest that, under these conditions, recruited pressure-independent mechanisms elicit vasodilatation via an increased production of NO (Gardner & Giussani, 2003), although their identity remains unknown.
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Increasing clinical (Franco-Cereceda et al. 1987; Gennari et al. 1990; Hasbak et al. 2002; Hasbak et al. 2003) and scientific (DiPette et al. 1987; Dhillo et al. 2003) evidence suggests a role for the novel and potent vasodilator peptide CGRP in cardiovascular regulation in the adult circulation. In addition, increased levels of CGRP during pregnancy (Saggese et al. 1990), in cord blood and human neonatal blood (Parida et al. 1998), and its distribution at the site of implantation (Tsatsaris et al. 2002) and throughout the human placenta (Graf et al. 1996; Lafond et al. 1997) suggest a vasomotor role for the peptide in the feto-placental vascular beds. In the present study, we investigated whether CGRP is a potential candidate for mediating pressure-independent increases in umbilical blood flow in the late gestation fetus during stressful conditions, and tested the hypothesis that CGRP plays an important role in the umbilical vascular bed during basal and hypoxaemic conditions. In this study, acute severe hypoxaemia (reducing the fetal Pa,O2 to 9 mmHg) during a background of saline infusion also led to greater increases in umbilical blood flow than could be accounted for by the magnitude of the fetal hypertension alone. Consequently, during acute severe hypoxaemia a significant increase in umbilical vascular conductance occurred. Fetal treatment with a selective CGRP antagonist did not modify basal umbilical haemodynamics but caused pronounced falls in both umbilical blood flow and umbilical vascular conductance during acute hypoxaemia. Therefore, these data support the hypothesis tested and suggest that during acute severe hypoxaemia, pressure-independent mechanisms mediated by CGRP are recruited to maintain or increase umbilical perfusion. Interestingly, as the hypoxaemic challenge progressed during treatment with the CGRP antagonist, umbilical vascular conductance returned to basal levels and increased above baseline during the recovery period. This suggests that, at this time, additional vasoactive mechanisms may become recruited to compensate for the loss of the potent umbilical vasodilator
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CGRP. Such agents may include prostaglandin E2 (Young & Thorburn, 1994), oestrogen (Rosenfeld et al. 1996), corticotrophin releasing hormone (Perkins & Linton, 1995) and adenosine (Read et al. 1993). They may act to modify fetal vasomotor responses to acute hypoxaemia either by nitric oxide-dependent or nitric oxide-independent mechanisms.
Combined, past and present data therefore suggest that pressure-dependent mechanisms suffice to maintain adequate perfusion of the umbilical vascular bed during fetal exposure to acute moderate hypoxaemia. However, when the fetus is exposed to acute severe hypoxaemia in normal pregnancies or to acute moderate hypoxaemia in pregnancies complicated with chronic adverse intrauterine conditions, active vasodilator mechanisms are recruited to ensure maintained adequate delivery of oxygen and nutrients to the umbilical vascular bed. This study has shown that during severe acute fetal hypoxaemia, such a mechanism is provided for by CGRP.
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Abstract
Despite clinical advances in obstetric practice, undiagnosed fetal hypoxaemia still contributes to a high incidence of perinatal morbidity. The fetal defence to hypoxaemia involves a redistribution of blood flow away from peripheral circulations towards essential vascular beds, such as the umbilical, cerebral, myocardial and adrenal circulations. In marked contrast to other essential vascular beds, the mechanisms mediating maintained perfusion of the umbilical circulation during hypoxaemia remain unknown. This study determined the role of calcitonin gene-related peptide (CGRP) in the maintenance of umbilical blood flow during basal and hypoxaemic conditions. Under anaesthesia, five sheep fetuses were instrumented with catheters and a Transonic probe around an umbilical artery, inside the fetal abdomen, at 0.8 of gestation. Five days later, fetuses were subjected to 0.5 h hypoxaemia during either I.V. saline or a selective CGRP antagonist in randomised order. Treatment started 30 min before hypoxaemia and ran continuously until the end of the challenge. The CGRP antagonist did not alter basal blood gas or cardiovascular status in the fetus. A similar fall in Pa,O2 occurred in fetuses during either saline (21 ± 0.8 to 9 ± 0.9 mmHg) or antagonist treatment (20 ± 0.9 to 9 ± 1.2 mmHg). Hypoxaemia during saline led to significant increases in arterial blood pressure, umbilical blood flow and umbilical vascular conductance. In marked contrast, hypoxaemia during CGRP antagonist treatment led to pronounced falls in both umbilical blood flow and umbilical vascular conductance without affecting the magnitude of the hypertensive response. In conclusion, CGRP plays an important role in the umbilical haemodynamic defence response to hypoxaemia in the late gestation fetus.
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Introduction
Acute fetal hypoxaemia is one of the major challenges that the fetus may face during gestation (Huch et al. 1977) and it may occur by insufficiency of either uterine blood flow (Yaffee et al. 1987) or umbilical blood flow (Itskovitz et al. 1983), or by a decrease in maternal arterial oxygen content (Parer, 1980). Other mechanisms such as fetal anaemia or increased fetal oxygen consumption (e.g. in pyrexia) are relatively rare in clinical practice (Parer & Livingston, 1990). Risk factors which predispose to the development of fetal hypoxaemia can be classified into maternal (e.g. diabetes (Macfarlane & Tsakalakos, 1985), pregnancy induced or chronic hypertension (Galanti et al. 2000), Rhesus sensitisation (Thilaganathan et al. 1992), maternal infection (Dalitz et al. 2003), sickle cell anaemia (Manzar, 2000), chronic substance abuse (Mukherjee & Hodgen, 1982; Slotkin, 1998), asthma (Witlin, 1997), seizure disorders (Tomson et al. 1997) or smoking (Socol et al. 1982)), intrapartum (e.g. multiple pregnancy (Maier et al. 1995), pre- or post-term birth (Stubblefield & Berek, 1980; Salafia et al. 1995), prolonged labour (Leszczynska-Gorzelak et al. 2002), placental abruption (Salafia et al. 1995), placenta praevia (Kovalovszki et al. 1990), prolapsed umbilical cord (Faiz et al. 2003) or abnormal presentation of the fetus (Mukhopadhyay & Arulkumaran, 2002)) and iatrogenic (e.g. epidural anaesthesia (Preston et al. 1993)). Despite recent clinical advances in obstetric practice and perinatal care in dramatically reducing infant mortality, there is still a high incidence of perinatal morbidity. Much of this can be attributed to birth asphyxia during complicated labour (Hall, 1989) or undetected episodes of antenatal fetal hypoxaemia (Low et al. 1995). Both situations render the infant at increased risk of developing complications in the central nervous (e.g. cerebral palsy (Johnston et al. 2001), neonatal seizures (Arpino et al. 2001), hypoxic–ischaemic encephalopathy (Low et al. 1985)), cardiovascular (e.g. left ventricular myocardial dysfunction (Walther et al. 1985)) and respiratory (e.g. meconium aspiration syndrome (Klingner & Kruse, 1999)) systems.
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The fetal defence to acute hypoxaemia involves cardiovascular responses that redistribute the combined ventricular output towards essential vascular beds such as the umbilical, adrenal, myocardial and cerebral circulations (for reviews, see Rudolph, 1984 and Giussani et al. 1994a,b). Redistribution of blood flow is largely accomplished by peripheral vasoconstriction, which is triggered by a carotid chemoreflex (Giussani et al. 1993) and then maintained or subsequently modified by endocrine (Iwamoto, 1990; Giussani et al. 1994b; Fletcher et al. 2000) and local components (Vane et al. 1990; Morrison et al. 2003). The resulting increase in pressure-dependent perfusion through the cerebral, myocardial and adrenal vascular beds is further facilitated by active vasodilatation (Carter et al. 1993; Thornburg et al. 2000; Riquelme et al. 2002; Blood et al. 2003). In contrast to the adrenal, myocardial and cerebral circulations, the physiological mechanisms maintaining perfusion of the umbilical vascular bed during adverse intrauterine conditions remain comparatively less well understood.
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Calcitonin gene-related peptide (CGRP) is the most potent endogenous vasodilator peptide known (Brain et al. 1985). Recently it has been shown that CGRP has a powerful vasodilator action, which in part is mediated via nitric oxide (NO), in vivo in the ovine fetal lung (Takahashi et al. 2000) and in vitro in the human umbilical artery and vein (Dong et al. 2004). However the in vivo role of CGRP in the control of the umbilical vascular bed during basal or stressful conditions has not been investigated. The present study tested the hypothesis that CGRP plays an important role in the maintenance of umbilical blood flow during acute hypoxaemia in the fetus. Late gestation fetal sheep surgically prepared for long-term recording in the conscious state were used as an experimental model. The hypothesis was tested by investigating the effects of fetal treatment with a selective CGRP antagonist on in vivo umbilical haemodynamics via an indwelling Transonic flow probe during basal and hypoxaemic conditions.
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Methods
Surgical preparation
All procedures were performed under the UK Animals (Scientific Procedures) Act 1986 and were approved by the Ethical Review Committee of the University of Cambridge. Five Welsh Mountain sheep fetuses, at 120 ± 2 days of gestation (term is 145 days), were surgically prepared for long-term recording using strict aseptic conditions as previously described in detail (Giussani et al. 2001). In brief, food, but not water, was withheld from the pregnant ewes for 24 h prior to surgery. Following induction with 20 mg kg–1 I.V. sodium thiopentone (Intraval Sodium; Merial Animal Health Ltd; Rhone Merieux, Dublin, Ireland), general anaesthesia (1.5–2.0% halothane in 50 : 50 O2 : N2O) was maintained using positive pressure ventilation. Midline abdominal and uterine incisions were made, the fetal hind limbs were exteriorized and on one side femoral arterial (i.d., 0.86 mm; o.d., 1.52 mm; Critchly Electrical Products, NSW, Australia) and venous (i.d., 0.56 mm; o.d., 0.96 mm) catheters were inserted. The catheter tips were advanced carefully to the ascending aorta and superior vena cava, respectively. Another catheter was anchored onto the fetal hind limb for recording of the reference amniotic pressure. In addition, a transit-time flow transducer (4SB; Transonic Systems Inc., Ithaca, NY, USA) was placed around one of the umbilical arteries, close to the common umbilical artery, inside the fetal abdominal cavity, for continuous measurement of unilateral umbilical blood flow (Gardner et al. 2001). The fetus was returned to the uterine cavity, the uterine incisions were closed in layers, the dead space of the catheters was filled with heparinized saline (80 i.u. heparin ml–1 in 0.9% NaCl) and the catheters' ends were plugged with sterile brass pins. The catheters and flow probe lead were then exteriorised via a key-hole incision in the maternal flank and kept inside a plastic pouch sewn onto the maternal skin.
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Postoperative care
The ewes were housed in individual pens with free access to hay and water, in rooms with a 12 h : 12 h dark : light cycle. They were fed concentrates twice daily (100 g; sheep nuts no. 6; H & C Beart Ltd, Kings Lynn, UK). All ewes received postoperative analgesia (10–20 mg kg–1 oral Penylbutazone; Equipalozone paste, Arnolds Veterinary Products Ltd, Shropshire, UK) and antibiotics (0.20–0.25 mg kg–1 I.M. Depocillin; Mycofarm, Cambridge, UK). The patency of the fetal catheters was maintained by a slow continuous infusion of heparinised saline (80 i.u. heparin ml–1 at 0.1 ml h–1 in 0.9% NaCl) containing antibiotic (1 mg ml–1 benzylpenicillin; Crystapen, Schering-Plough, Animal Health Division, Welwyn Garden City, UK). In addition, the fetuses also received daily antibiotics (150 mg kg–1 Penbritin, SmithKline Beecham Animal Health, Welwyn Garden City, Hertfordshire, UK) I.V and into the amniotic cavity.
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Experimental protocol
Following at least 5 days of postoperative recovery, all fetuses were subjected to two experimental protocols, carried out on consecutive days in randomised order. Each protocol consisted of a 2.5 h period divided into1 h normoxia, 0.5 h hypoxaemia and 1 h recovery, during either a slow I.V. infusion of vehicle (80 i.u. heparin ml–1 in 0.9% NaCl) or during fetal treatment with a selective CGRP antagonist (50 μg kg–1 I.A. bolus followed by 20 μgmin–1 I.V. infusion; calcitonin gene related peptide fragment 8–37, CGRP8-37; C-2806; Sigma Chemicals, UK, Fig. 1) which was made in a solution of heparinised saline, identical to that of the vehicle. Acute hypoxaemia in the fetus was induced by maternal inhalational hypoxia. In brief, a large transparent respiratory hood was placed over the ewe's head into which air was passed at a rate of 50 l min–1 for the 1 h period of normoxia. Following this control period, acute fetal hypoxaemia was induced for 30 min by changing the concentrations of gases breathed by the ewe to 6% O2 in N2 with small amounts of CO2 (15 l min–1 air : 35 l min–1 N2 : 1.5–2.5 l min–1 CO2). This mixture was designed to reduce fetal Pa,O2 to 9 mmHg while maintaining Pa,CO2. Following the 0.5 h period of hypoxaemia, the ewe was returned to breathing air for the 1 h recovery period. The dose of CGRP antagonist was chosen from a previous study by Takahashi et al. (2000). In that study, treatment of late gestation fetal sheep with a similar dose of the CGRP antagonist markedly diminished the vasodilator response of the pulmonary vascular bed to exogenous treatment with CGRP. In the present study, fetal treatment with the CGRP antagonist started 30 min before the onset of hypoxaemia and ran continuously until the end of the hypoxaemic challenge. During any acute hypoxaemia protocol, descending aortic blood samples were taken from the fetus at set intervals (arrows; Fig. 1) to determine arterial blood gas and acid base status (ABL5 blood gas Analyser, Radiometer, Copenhagen, Denmark; measurements corrected to 39.5°C; Fig. 1). At the end of the experimental protocol, the ewes and fetuses were killed painlessly using a lethal dose of sodium pentobarbitone (200 mg kg–1 I.V. Pentoject; Animal Ltd, York, UK), the positions of the implanted catheters and the flow probe were confirmed and the fetuses were weighed.
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The experimental protocol consisted of a 2.5-h period divided into: 1 h normoxia, 0.5 h hypoxaemia (black box) and 1 h recovery, during saline infusion or during treatment with the CGRP antagonist (grey box). Arrows represent the time at which arterial blood samples were collected.
Measurements and statistical analysis
Calibrated mean fetal arterial blood pressure (corrected for amniotic pressure) and mean umbilical blood flow were recorded continually at 1 s intervals using a computerised Data Acquisition System (Department of Physiology, Cambridge University, UK). Umbilical vascular conductance was calculated by dividing mean umbilical blood flow by mean supra-amniotic arterial blood pressure. Values for all variables are expressed as mean ± S.E.M. Cardiovascular variables are expressed as minute averages of either absolute values or of the absolute change from mean normoxic baseline. Summary measure analysis was then applied to the serial data to focus the number of comparisons, and areas under the curve were calculated for the absolute change from baseline for statistical comparison, as previously described in detail (Matthews et al. 1990). Variables were assessed using two-way ANOVA with repeated measures (Sigma-Stat; SPSS Inc., Chicago, IL, USA), comparing the effect of time (normoxia versus hypoxaemia/recovery), group (control versus CGRP antagonist) and interactions between time and group. Where a significant effect of time or group was indicated, the post hoc Student–Newman–Keuls test was used to isolate the statistical difference. For all comparisons, statistical significance was accepted when P < 0.05.
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Results
Fetal arterial blood gas and acid base status
Basal values for arterial blood gas and acid base status were similar in fetuses during saline infusion or during treatment with the CGRP antagonist. In all fetuses, acute hypoxaemia induced significant falls in arterial pH (pHa), arterial partial pressure of oxygen (Pa,O2), percentage saturation of haemoglobin with oxygen (Sat Hb) and acid base excess (ABE) without any alteration to arterial partial pressure of carbon dioxide (Pa,CO2) (Fig. 2). The magnitude of these changes was similar during saline infusion or during treatment with the CGRP antagonist. During recovery, both Pa,O2 and Sat Hb recovered to basal values in all fetuses. In contrast fetal pHa and ABE remained significantly lower than basal values until the end of the experimental protocol.
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Values represent the mean ± S.E.M. at 0 (N0) and 45 min (N45) of normoxia, at 5 (H5), 15 (H15) and 30 min (H30) of hypoxaemia, and at 30 (R30) and 60 min (R60) of recovery for fetuses exposed to 30 min hypoxaemia (box) during saline infusion (; n = 5) or during treatment with CGRP antagonist (; n = 5). Significant differences: *P < 0.05 for normoxia versus hypoxaemia or recovery. pHa, arterial pH; Pa,CO2, arterial partial pressure of CO2; Pa,O2, arterial partial pressure of O2; Sat Hb, percentage saturation of haemoglobin with oxygen; ABE, acid base excess.
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Umbilical haemodynamic responses to acute hypoxaemia
Basal values for fetal arterial blood pressure (62.5 ± 1.7 versus 61.1 ± 1.5 mmHg), unilateral umbilical blood flow (132 ± 9 versus 140 ± 14 ml min–1) and umbilical vascular conductance (2.1 ± 0.2 versus 2.3 ± 0.3 (ml min–1 mmHg–1) were similar in fetuses during saline infusion or during treatment with the CGRP antagonist, respectively (Fig. 3). During acute hypoxaemia, a significant increase in arterial blood pressure occurred in all fetuses during either saline infusion or during treatment with the CGRP antagonist, with arterial blood pressure increasing from baseline by 16.9 ± 2.0 and 17.4 ± 1.9 mmHg, respectively (Figs 3 and 4). In contrast, while umbilical blood flow (from 132 ± 9 to 198 ± 15 ml min–1) and umbilical vascular conductance (from 2.1 ± 0.2 to 2.7 ± 0.2 (ml min–1 mmHg–1) increased during the hypoxaemic period in fetuses during saline infusion, pronounced falls in both umbilical blood flow (from 140 ± 14 to 90 ± 10 ml min–1) and umbilical vascular conductance (from 2.3 ± 0.3 to 1.3 ± 0.2 ml min–1 mmHg–1) occurred early during the hypoxaemic period in fetuses during treatment with the CGRP antagonist (Figs 3 and 4). After 10 min of acute hypoxaemia during treatment with the CGRP antagonist, umbilical flow and vascular conductance returned towards basal values. By the end of the hypoxaemic challenge during the antagonist infusion, umbilical flow had increased to values above baseline (from 140 ± 13 to 187 ± 18 ml min–1), and umbilical vascular conductance had recovered to basal values (from 2.3 ± 0.3 to 2.5 ± 0.2 ml min–1 mmHg–1, Figs 3 and 4). During recovery, values for arterial blood pressure and umbilical blood flow remained significantly elevated by the end of the experimental protocol in all fetuses, during either saline infusion or treatment with the CGRP antagonist (Figs 3 and 4).
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Values are mean ± S.E.M. calculated every minute for arterial blood pressure, unilateral umbilical blood flow and umbilical vascular conductance during 1 h of normoxia, 0.5 h of hypoxaemia (box) and 1 h of recovery for fetuses during saline infusion or during treatment with the CGRP antagonist.
Values are A, mean ± S.E.M. calculated every minute for the change from normoxic baseline for arterial blood pressure, unilateral umbilical blood flow and umbilical vascular conductance and B, the statistical summary of these changes during saline infusion (; n = 5) and during treatment with the CGRP antagonist (; n = 5). Values for the statistical summary represent mean ± S.E.M. for the area under the curve of the umbilical haemodynamic variables over every 15 min. Significant differences: *P < 0.05 for normoxia versus hypoxaemia or recovery; P < 0.05, saline versus CGRP antagonist.
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Discussion
There has been a long-standing clinical and physiological interest in the haemodynamic changes in the umbilical vascular bed during pregnancy, due to its functional importance in representing feto-placental blood flow and hence fetal wellbeing. Clinically, indirect measurements of umbilical blood flow in human pregnancy are obtained routinely by Doppler flow velocimetry. However, application of this technique in detailed experiments has shown that analysis of the umbilical vessel Doppler waveforms is complicated, particularly during fetal distress. For example, while Downing et al. (1991) and Tchirikov et al. (1998) reported an increase in the umbilical artery pulsatility index (PI), signifying an increase in umbilical vascular resistance, during episodes of hypoxaemia, Muijsers et al. (1990), Morrow et al. (1990), and van Huisseling et al. (1991), were unable to find any significant change in the umbilical artery PI. Similarly, using direct, spot measurement of umbilical blood flow with radioactive microspheres in the sheep fetus, Cohn et al. (1974), and Parer (1983), reported either an increase in umbilical vascular resistance or no change in umbilical blood flow during hypoxaemia. To avoid complications in the interpretation of either indirect indices or point measurements of umbilical blood flow, or of examination of changes in the umbilical vascular bed under the effects of anaesthesia, studies in our laboratory have used continuous measurement of umbilical blood flow by an implanted Transonic flow probe around an umbilical artery within the fetal abdomen in chronically instrumented, unanaesthetised fetal sheep preparations.
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Previous investigations from this laboratory, which have measured continuous changes in flow in the umbilical vascular bed, have reported increases in umbilical blood flow during acute hypoxaemia as a result of pressure-dependent (Gardner et al. 2001) and pressure-independent mechanisms (Gardner & Giussani, 2003). Past data show that during moderate fetal hypoxaemia (i.e. a decrease in fetal Pa,O2 to 13 mmHg) there are equivalent elevations in arterial blood pressure and umbilical blood flow with no changes in umbilical vascular conductance. This suggests that during moderate fetal hypoxaemia, maintenance of umbilical perfusion is entirely pressure dependent (van Huisseling et al. 1991). However, in fetuses which have previously suffered a sustained period of intrauterine adversity, the increase in umbilical blood flow during moderate acute hypoxaemia is greater than can be accounted for by the increase in perfusion pressure (Gardner & Giussani, 2003). Therefore, in pregnancies complicated by chronic adverse intrauterine conditions, pressure-independent mechanisms may become recruited to induce an increase in umbilical conductance, signifying active vasodilatation of the umbilico-placental vascular beds. Additional data suggest that, under these conditions, recruited pressure-independent mechanisms elicit vasodilatation via an increased production of NO (Gardner & Giussani, 2003), although their identity remains unknown.
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Increasing clinical (Franco-Cereceda et al. 1987; Gennari et al. 1990; Hasbak et al. 2002; Hasbak et al. 2003) and scientific (DiPette et al. 1987; Dhillo et al. 2003) evidence suggests a role for the novel and potent vasodilator peptide CGRP in cardiovascular regulation in the adult circulation. In addition, increased levels of CGRP during pregnancy (Saggese et al. 1990), in cord blood and human neonatal blood (Parida et al. 1998), and its distribution at the site of implantation (Tsatsaris et al. 2002) and throughout the human placenta (Graf et al. 1996; Lafond et al. 1997) suggest a vasomotor role for the peptide in the feto-placental vascular beds. In the present study, we investigated whether CGRP is a potential candidate for mediating pressure-independent increases in umbilical blood flow in the late gestation fetus during stressful conditions, and tested the hypothesis that CGRP plays an important role in the umbilical vascular bed during basal and hypoxaemic conditions. In this study, acute severe hypoxaemia (reducing the fetal Pa,O2 to 9 mmHg) during a background of saline infusion also led to greater increases in umbilical blood flow than could be accounted for by the magnitude of the fetal hypertension alone. Consequently, during acute severe hypoxaemia a significant increase in umbilical vascular conductance occurred. Fetal treatment with a selective CGRP antagonist did not modify basal umbilical haemodynamics but caused pronounced falls in both umbilical blood flow and umbilical vascular conductance during acute hypoxaemia. Therefore, these data support the hypothesis tested and suggest that during acute severe hypoxaemia, pressure-independent mechanisms mediated by CGRP are recruited to maintain or increase umbilical perfusion. Interestingly, as the hypoxaemic challenge progressed during treatment with the CGRP antagonist, umbilical vascular conductance returned to basal levels and increased above baseline during the recovery period. This suggests that, at this time, additional vasoactive mechanisms may become recruited to compensate for the loss of the potent umbilical vasodilator
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CGRP. Such agents may include prostaglandin E2 (Young & Thorburn, 1994), oestrogen (Rosenfeld et al. 1996), corticotrophin releasing hormone (Perkins & Linton, 1995) and adenosine (Read et al. 1993). They may act to modify fetal vasomotor responses to acute hypoxaemia either by nitric oxide-dependent or nitric oxide-independent mechanisms.
Combined, past and present data therefore suggest that pressure-dependent mechanisms suffice to maintain adequate perfusion of the umbilical vascular bed during fetal exposure to acute moderate hypoxaemia. However, when the fetus is exposed to acute severe hypoxaemia in normal pregnancies or to acute moderate hypoxaemia in pregnancies complicated with chronic adverse intrauterine conditions, active vasodilator mechanisms are recruited to ensure maintained adequate delivery of oxygen and nutrients to the umbilical vascular bed. This study has shown that during severe acute fetal hypoxaemia, such a mechanism is provided for by CGRP.
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