Calcitonin gene-related peptide antagonism attenuates the haemodynamic and glycaemic responses to acute hypoxaemia in the late gestation she
1 Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK
2 Faculty of Medicine, Imperial College, Hammersmith Hospital, London W12 ONN, UK
Abstract
The fetal defence to acute hypoxaemia involves cardiovascular and metabolic responses, which include peripheral vasoconstriction and hyperglycaemia. Both these responses are mediated via neuroendocrine mechanisms, which require the stimulation of the sympathetic nervous system. In the adult, accumulating evidence supports a role for calcitonin gene-related peptide (CGRP) in the activation of sympathetic outflow. However, the role of CGRP in stimulated cardiovascular and metabolic functions before birth is completely unknown. This study tested the hypothesis that CGRP plays a role in the fetal cardiovascular and metabolic defence responses to acute hypoxaemia by affecting sympathetic outflow. Under anaesthesia, five sheep fetuses at 0.8 of gestation were surgically instrumented with catheters and a femoral arterial Transonic flow-probe. 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. Arterial samples were taken for blood gases, metabolic status and hormone analyses. CGRP antagonism did not alter basal arterial blood gas, metabolic, cardiovascular or endocrine status. During hypoxaemia, similar falls in Pa,O2 occurred in all fetuses. During saline infusion, hypoxaemia induced hypertension, bradycardia, femoral vasoconstriction, hyperglycaemia and an increase in haemoglobin, catecholamines and neuropeptide Y (NPY). In contrast, CGRP antagonism markedly diminished the femoral vasoconstrictor and glycaemic responses to hypoxaemia, and attenuated the increases in haemoglobin, catecholamines and NPY. Combined, these results strongly support the hypothesis that CGRP plays a role in the fetal cardiovascular and metabolic defence to hypoxaemia by affecting sympathetic outflow.
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Introduction
A common challenge that the fetus faces during late gestation is an episode of acute hypoxaemia (Huch et al. 1977). Inadequately compensated periods of acute fetal hypoxaemia may lead to a wide range of developmental abnormalities and perinatal complications in the cardiovascular, respiratory and central nervous systems. Thus, fetal hypoxaemia may promote left ventricular myocardial dysfunction (Walther et al. 1985), meconium aspiration syndrome (Klingner & Kruse, 1999), neonatal seizures (Arpino et al. 2001), hypoxic–ischaemic encephalopathy (Low et al. 1985) and cerebral palsy (Johnston et al. 2001).
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The fetal defence to acute hypoxaemia involves integrated cardiovascular and metabolic responses, which facilitate fetal survival during the period of reduced oxygen availability. The cardiovascular defence responses to acute hypoxaemia are well established, and involve a transient bradycardia, a gradual increase in arterial blood pressure and peripheral vasoconstriction (for reviews, see Rudolph, 1984; Giussani et al. 1994). The latter aids the redistribution of the fetal combined ventricular output, away from peripheral circulations and towards more essential vascular beds such as the cerebral, adrenal, and myocardial circulations (Cohn et al. 1974; Peeters et al. 1979; Jensen & Berger, 1991). The fetal metabolic responses to acute hypoxaemia involve an increase in the circulating concentrations of glucose and lactate (Jones & Ritchie, 1976; Jones, 1977). The fetal hyperglycaemia during acute hypoxaemia results from a decrease in glucose uptake and utilisation by peripheral tissues (Jones et al. 1983), and an increase in hepatic glucose production (Jones & Ashton, 1976). The fetal lactacidaemia results from anaerobic metabolism of glucose in hypoxic fetal tissues, particularly in the carcass where blood flow and oxygen delivery are markedly declined.
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The physiological mechanisms mediating both the cardiovascular and metabolic defence responses to acute hypoxaemia involve the activation of the fetal sympathetic nervous system. Acute hypoxaemia triggers a carotid chemoreflex, which promotes femoral vasoconstriction via -adrenergic efferent pathways (Giussani et al. 1993). Increased sympathetic outflow also inhibits insulin release from the fetal pancreas, thereby decreasing glucose uptake and utilisation by the fetal tissues (Jones et al. 1983). As the hypoxaemic challenge progresses, catecholamines (Jones & Robinson, 1975) and neuropeptide Y (NPY; Fletcher et al. 2000) are released into the fetal circulation, and act to maintain the peripheral vasoconstrictor response (Sanhueza et al. 2003). In addition, catecholamines are also known to mobilise and release glucose from glycogen stores in the fetal liver (Apatu & Barnes, 1991).
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In the adult, calcitonin gene-related peptide (CGRP) is a novel peptide which is rapidly gaining clinical and scientific interest for its role in cardiovascular and metabolic regulation (DiPette et al. 1987; Franco-Cereceda et al. 1987; Gennari et al. 1990; Hasbak et al. 2002,; Dhillo et al. 2003). In addition to being one of the most potent vasodilator peptides known (Brain et al. 1985), accumulating evidence suggests that it can also selectively activate sympathetic outflow (Hasegawa et al. 1993; Herbison et al. 1993). The distribution of CGRP at the site of implantation (Tsatsaris et al. 2002) and throughout the human placenta (Graf et al. 1996), increased maternal plasma levels of CGRP during pregnancy (Saggese et al. 1990), and its presence in human cord and neonatal blood (Parida et al. 1998) all suggest a functional role for CGRP in development. However, the role of CGRP in fetal cardiovascular or metabolic functions, during either basal or stimulated conditions, is completely unknown.
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The present study tested the hypothesis that CGRP plays a role in the fetal cardiovascular and metabolic defence responses to acute hypoxaemia by affecting sympathetic outflow. The hypothesis was tested using the unanaesthetised late gestation sheep fetus as our experimental model, in which cardiovascular and metabolic responses to acute hypoxaemia were investigated during either saline infusion or treatment with a selective CGRP antagonist dissolved in saline.
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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 were surgically instrumented for long-term recording at 120 ± 2 days of gestation (term is 145 days), 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 exteriorised 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 descending aorta and inferior 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 (2R or 3S; Transonic Systems Inc., Ithaca, NY, USA) was placed around the contralateral femoral artery, for continuous measurement of femoral blood flow. The uterine incisions were closed in layers, the dead space of the catheters was filled with heparinised saline (80 i.u. heparin ml–1 in 0.9% NaCl), and the catheter ends were plugged with sterile brass pins. The catheters and flow probe lead were then exteriorised via a keyhole incision in the maternal flank, and kept inside a plastic pouch sewn onto the maternal skin.
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Postoperative care
During recovery, ewes were housed in individual pens in rooms with a 12 h: 12 h light: dark cycle where they had free access to hay and water and were fed concentrates twice daily (100 g sheep nuts no. 6; H & C Beart Ltd, Kings Lynn, UK). Antibiotics were administered daily to the ewe (0.20–0.25 mg kg–1 I.M. Depocillin; Mycofarm, Cambridge, UK) and fetus I.V and into the amniotic cavity (150 mg kg–1 Penbritin; SmithKline Beecham Animal Health, Welwyn Garden City, Hertfordshire, UK). The ewes also received 2 days of postoperative analgesia if in pain (10–20 mg kg–1 oral Penylbutazone; Equipalozone paste, Arnolds Veterinary Products Ltd, Shropshire, UK), as assessed by their general demeanour and feeding patterns. Generally, normal feeding patterns were restored within 48 h of recovery. Following 72 h of postoperative recovery, ewes were transferred to metabolic crates where they were housed for the remainder of the protocol. The arterial and amniotic catheters were connected to sterile pressure transducers (COBE; Argon Division, Maxxim Medical, Athens, Texas, USA), and the flow probe lead to a flow meter (T206; Transonics Systems Inc., Ithaca, NY, USA). Calibrated mean fetal arterial blood pressure (corrected for amniotic pressure), fetal heart rate (triggered via a tachometer from the pulsatility in either the arterial blood pressure or femoral blood flow signals), and mean femoral blood flow were recorded continually at 1 s intervals using a computerised data acquisition system (DAS; Department of Physiology, Cambridge University, UK). While on the metabolic crates, 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).
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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 a randomised order (Fig. 1). Each protocol consisted of a 2.5 h period divided into: 1 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 the CGRP antagonist dissolved in heparinised saline (50 μg kg–1 I.A. bolus followed by 8 μg kg–1.min–1 I.V. infusion; calcitonin gene related peptide fragment 8–37, CGRP8—37; C-2806; Sigma Chemicals, UK). Acute hypoxaemia in the fetus was induced by maternal inhalational hypoxia. In brief, a large transparent respiratory hood was placed over the ewes' 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: 5 l min–1 N2: 1.5–2.5 l min–1 CO2). This mixture was designed to reduce fetal Pa,O2 to 10 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 our own pilot experiments based on 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. At the end of the experimental protocol, the ewes and fetuses were humanely killed 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 times at which arterial blood samples were collected.
Blood sampling regimen
During any acute hypoxaemia protocol, descending aortic blood samples (0.3 ml) were taken using sterile techniques from the fetus at set time intervals (Fig. 1, arrows) to determine arterial blood gas and acid–base status (ABL5 Blood Gas Analyser, Radiometer; Copenhagen, Denmark; measurements corrected to 39.5°C). Values for percentage saturation of haemoglobin with oxygen (Sat Hb) and the blood haemoglobin concentration ([Hb]) were determined using a haemoximeter (OSM2; Radiometer). In addition, blood glucose and lactate concentrations were measured by an automated analyser (Yellow Springs 2300 Stat Plus Glucose/Lactate Analyser; YSI Ltd, Farnborough, UK). An additional 4 ml of arterial blood was withdrawn at set intervals for hormone analyses (Fig. 1, arrows). These samples were collected under sterile conditions into chilled heparin tubes (2 ml Li+/heparin tubes; L.I.P. Ltd, Shipley, West Yorkshire, UK) containing reduced glutathione (4 nmol tube–1; G-4251; Sigma Chemicals, UK) and EGTA (5 nmol tube–1; E-4378; Sigma Chemicals, UK) for catecholamines analysis or into chilled EDTA tubes (2 ml K+/EDTA; L.I.P. Ltd) for NPY analysis. All samples were then centrifuged at 4000 r.p.m. for 4 min at 4°C. The plasma obtained was then dispensed into prelabelled tubes and the samples were stored at –80°C until analysis. All hormone measurements were performed within 2 months of sample collection.
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Hormone analyses
NPY assay. Fetal plasma NPY concentrations were measured by radioimmunoassay as previously described in detail (Allen et al. 1984). All samples were analysed in duplicate at the same time. The assay used rabbit antiserum (produced in horse) and 125I-labelled porcine peptide. Separation of the free and bound fractions was performed with dextran-coated charcoal. The assay was validated for use with ovine plasma using stripped sheep plasma. The assay could detect less than 1 pmol l–1 (95% confidence interval). The interassay coefficient of variation was 6.8%. There was no detectable cross-reactivity of the anti-NPY antiserum with the peptide YY.
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Catecholamine assay. Fetal plasma noradrenaline and adrenaline concentrations were measured by high-pressure liquid chromatography (HPLC) using electrochemical detection as previously described in detail (Fowden et al. 1998). The samples were prepared by absorption of 250 μl of plasma onto acid-washed alumina, and 20 μl aliquots of the 100 μl perchloric acid elutes were injected onto the column. Dihydroxybenzylamine was added as the internal standard to each plasma sample before absorption. The limit of sensitivity for the assay was 20 pg ml–1 for adrenaline and noradrenaline. Recovery ranged from 63% to 97%, and all catecholamine values were corrected for their respective recovery. The interassay coefficients of variation for adrenaline and noradrenaline were 7.3% and 6.2%.
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Data and statistical analyses
Femoral vascular resistance was calculated using Ohm's principle by dividing mean corrected arterial blood pressure by mean femoral blood flow. Values for all arterial blood gas, acid–base and metabolic variables are expressed as mean ± S.E.M. at 0 (N0) and 45 (N45) min of normoxia, 5 (H5), 15 (H15) and 30 (H30) min of hypoxaemia, and at 30 (R30) and 60 (R60) min of recovery. Values for all endocrine variables are expressed as mean ± S.E.M. at 0 (N0) and 45 (N45) min of normoxia, 15 (H15) and 30 (H30) min of hypoxaemia, and at 30 (R30) and 60 (R60) min of recovery. The cardiovascular variables are expressed as mean ± S.E.M. minute averages. Summary measure analysis was then applied to the serial cardiovascular data to focus the number of comparisons, as previously described in detail (Matthews et al. 1990), and area under the curve was calculated for the absolute data every 30 min. All measured variables were first analysed for normality of distribution, and then assessed statistically using two-way ANOVA with repeated measures (RM; 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 Tukey test was used to isolate the statistical differences. For all comparisons, statistical significance was accepted when P < 0.05.
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Results
Fetal arterial blood gas and acid–base status during acute hypoxaemia
Basal values for arterial blood gas and acid–base status were similar in all fetuses. Infusion with saline or treatment with the CGRP antagonist had no effect on basal arterial blood gas and acid–base status (Table 1). In all fetuses, acute hypoxaemia induced significant falls in arterial pH (pHa), acid–base excess (ABE), [HCO3–], Pa,O2 and Sat Hb, without any alteration to Pa,CO2. The magnitude of these changes was similar during saline infusion or during treatment with the CGRP antagonist (Table 1). However the maximal increment in fetal [Hb] during acute hypoxaemia was significantly diminished during treatment with the CGRP antagonist (0.58 ± 0.57 g dl–1) when compared to saline-infused controls (1.98 ± 0.16 g dl–1; P < 0.05). During recovery pHa, ABE and [HCO3–] remained significantly depressed by the end of the experimental protocol in all fetuses, whereas Pa,O2, Sat Hb and [Hb] returned to basal values.
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Fetal metabolic status during acute hypoxaemia
Basal values for fetal blood glucose and lactate concentrations were similar in all fetuses. Infusion with saline or treatment with the CGRP antagonist had no effect on basal blood glucose and lactate concentrations (Fig. 2). Acute hypoxaemia during saline infusion induced significant increases in blood glucose and lactate concentrations. Treatment with the CGRP antagonist did not affect the increment in blood lactate (3.68 ± 0.68 versus 3.00 ± 0.37 mM, P > 0.05), but it did significantly diminish the increment in blood glucose concentration (0.83 ± 0.23 versus 0.34 ± 0.10 mM, P < 0.05) during acute hypoxaemia (Fig. 2). During recovery, blood lactate concentrations remained significantly elevated by the end of the experimental protocol in all fetuses, whereas blood glucose concentrations returned to basal values.
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Values represent the mean ± S.E.M. for blood glucose and lactate concentrations at 0 (N0) and 45 (N45) min of normoxia, at 5 (H5), 15 (H15) and 30 (H30) min of hypoxaemia, and at 30 (R30) and 60 (R60) min of recovery for fetuses exposed to 0.5 h hypoxaemia (open box) during saline infusion (; n = 5) or during treatment with the CGRP antagonist (; n = 5). Significant differences: *P < 0.05, for normoxia versus hypoxaemia or recovery; P < 0.05, saline versus CGRP antagonist (Two-way RM ANOVA with post hoc Tukey test).
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Fetal cardiovascular responses during acute hypoxaemia
Basal values for fetal arterial blood pressure, heart rate, femoral blood flow (FBF) and femoral vascular resistance (FVR) were similar in all fetuses. Infusion with saline, or treatment with the CGRP antagonist had no effect on basal cardiovascular variables (Fig. 3). Acute hypoxaemia during saline infusion induced significant increases in arterial blood pressure and femoral vascular resistance, and significant falls in heart rate and femoral blood flow (Fig. 3). Treatment with the CGRP antagonist did not affect the magnitude of the mean pressor (9.4 ± 1.0 versus 9.9 ± 0.9 mmHg) or bradycardic (–36 ± 11 versus –32 ± 11 beats min–1) responses to acute hypoxaemia (P > 0.05), but significantly attenuated the mean increment in the femoral vasoconstrictor response (FBF: –30 ± 4 versus –21 ± 2 ml min–1; FVR: 6.8 ± 1.5 versus 2.9 ± 0.4 mmHg. (ml min–1)–1; P < 0.05; Fig. 3). During recovery, fetal arterial blood pressure and heart rate remained significantly elevated by the end of the experimental protocol in all fetuses, whereas femoral blood flow and vascular resistance returned to basal values.
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A, values represent the mean ± S.E.M. calculated every minute for arterial blood pressure, heart rate, femoral blood flow and femoral vascular resistance during 1 h of normoxia, 0.5 h of hypoxaemia (open box) and 1 h of recovery for fetuses during saline infusion (n = 5) or during treatment with the CGRP antagonist (n = 5). B, values for the statistical summary of these responses represent the mean ± S.E.M. for the area under the curve over every 30 min during normoxia (N1, N2), hypoxaemia (H) and recovery (R1, R2) for fetuses during saline infusion (; n = 5) and during treatment with the CGRP antagonist (; n = 5). Significant differences: *P < 0.05, normoxia versus hypoxaemia or recovery; P < 0.05, saline versus CGRP antagonist (Two-way RM ANOVA with post hoc Tukey test).
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Plasma catecholamine and NPY concentrations
Basal values for fetal plasma neuropeptide Y, adrenaline and noradrenaline concentrations were similar in all fetuses. Infusion with saline or treatment with the CGRP antagonist had no effect on basal NPY or catecholamines levels (Fig. 4). Acute hypoxaemia during saline infusion induced a significant increase in plasma NPY, adrenaline and noradrenaline concentrations. Treatment with the CGRP antagonist diminished the maximal increment in plasma NPY (52 ± 16 versus 29 ± 11 pmol l–1) and adrenaline (2376 ± 1223 versus 1010 ± 379 pg ml–1; P < 0.05) concentrations during hypoxaemia, however, the effect on noradrenaline plasma concentrations (3258 ± 1557 versus 2091 ± 799 pg ml–1; P = 0.06) fell outside significance (Fig. 4). During recovery, plasma concentrations of NPY, adrenaline and noradrenaline returned to basal values.
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Values represent the mean ± S.E.M. for plasma concentrations of neuropeptide Y, adrenaline and noradrenaline at 0 (N0) and 45 (N45) min of normoxia, at 15 (H15) and 30 (H30) min of hypoxaemia, and at 30 (R30) and 60 (R60) min of recovery for fetuses exposed to 0.5 h hypoxaemia (open box) during saline infusion (; n = 5) or during treatment with the CGRP antagonist (; n = 5). Significant differences: *P < 0.05, normoxia versus hypoxaemia or recovery; P < 0.05, saline versus CGRP antagonist (Two-way RM ANOVA with post hoc Tukey test).
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Discussion
The results of the present study show that fetal treatment with a CGRP antagonist had no effect on cardiovascular or metabolic status during basal conditions. However, CGRP antagonism during acute hypoxaemia markedly diminished the increases in fetal femoral vascular resistance, blood glucose and haemoglobin concentrations, and in plasma levels of NPY and catecholamines. These data support the hypothesis that CGRP plays an important role in the activation of the sympathetic nervous system during acute hypoxaemia in the late gestation sheep fetus.
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The fetal peripheral vasoconstrictor response to acute hypoxaemia is mediated via carotid chemoreflex activation of the sympathetic nervous system, since both bilateral section of the carotid sinus nerves (Giussani et al. 1993; Bartelds et al. 1993) and -adrenergic blockade (Reuss et al. 1982; Giussani et al. 1993) markedly attenuate the increase in peripheral vascular resistance in response to hypoxaemia in the late gestation sheep fetus. As the hypoxaemic episode continues, plasma concentrations of catecholamines (Jones & Robinson, 1975; Jones & Wei, 1985) and NPY (Fletcher et al. 2000) are increased in the fetal circulation, and act to maintain the peripheral vasoconstriction. The origin of the increase in both adrenaline and noradrenaline in fetal plasma during acute hypoxaemia has been shown to be primarily from the fetal adrenal gland, since adrenal demedullation completely abolished the rise in plasma adrenaline concentration and reduced the noradrenaline response to 10% of normal (Jones et al. 1988). Since a large component of this increase in total catecholamine output from the fetal adrenal gland during acute hypoxia is mediated via activation of the splanchnic nerves (Jones et al. 1988), elevations in plasma concentrations of noradrenaline and adrenaline in the fetal sheep circulation are good indices of increased sympathetic outflow during stimulated conditions. In contrast to noradrenaline, the hypoxaemia-induced increase in fetal plasma NPY concentration is more likely to represent increased overspill from perivascular sympathetic nerve terminals. Despite the relatively high adrenal medullary NPY content, evidence suggests a negligible role for the gland in contributing to circulating plasma NPY levels during acute stress (Mormede et al. 1990). Since NPY lacks synaptic reuptake mechanisms, elevations in plasma NPY concentrations may provide a more robust measure than noradrenaline of increased sympathetic nervous activity (Lundberg, 1996). The present study showed that fetal treatment with the CGRP antagonist diminished the magnitude of the increase in femoral vascular resistance and in the concentrations of catecholamines and NPY in the fetal circulation; effects which are all consistent with the involvement of CGRP in the activation of the sympathetic nervous system during acute hypoxaemia in the sheep fetus.
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Although CGRP antagonism affected the femoral vasoconstrictor response to acute hypoxaemia, it had no effect on the hypertensive response. This does not suggest that changes in total peripheral vascular resistance are not main determinants of changes in arterial blood pressure during periods of acute stress, but the data merely emphasise that the femoral vascular bed is not representative of the whole of the peripheral vasculature in the late gestation ovine fetus.
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CGRP is one of the most potent vasodilator peptides known (Brain et al. 1985), having a potency of 10-fold greater than prostaglandins and 100–1000-fold greater than classical vasodilators, such as acetylcholine, adenosine and 5-HT (Brain & Grant, 2004). Recent in vivo experiments in the ovine fetus have shown that CGRP has a powerful vasodilator action in the pulmonary vascular bed (Takahashi et al. 2000), which is, in part, mediated via nitric oxide. Based on this evidence, CGRP may have been expected to act as a nitric oxide-dependent vasodilator during basal conditions and during acute hypoxaemia and, thereby, to reduce femoral vascular resistance during normoxia, and partially offset the femoral vasoconstrictor response to hypoxaemia in the ovine fetus. However, the results of the present study show that CGRP antagonism did not affect basal cardiovascular function. This is consistent with the available literature which has demonstrated that intravenous injection of the CGRP antagonist had no effect on basal blood pressure in rodents (Gardiner et al. 1991), and no effect on either systemic blood pressure or regional vascular beds in the conscious dog and anaesthetised rat (Shen et al. 2001). Although receptors for CGRP are widely distributed throughout the vasculature, a lack of an effect on basal cardiovascular function by the antagonist treatment is not inconsistent with the well-known vasodilator effect of exogenous administration of the agonist. Indeed, various groups have suggested that endogenous CGRP acts as a ‘rescue’ molecule, being upregulated and released from nerve terminals during periods of stress (Geppetti et al. 1991; Franco-Cereceda et al. 1993; Parida et al. 1998; Peng et al. 2002). The results of the present study also suggest that during episodes of acute hypoxaemic stress in the ovine fetus, there is an increased release of CGRP, whose effect on sympathetic outflow outweighs any local vasodilator actions of this peptide in the femoral circulation.
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The increase in blood glucose concentrations during acute hypoxaemia in fetuses infused with saline may be due to depression of insulin-dependent glucose uptake by the fetal tissues (Jones, 1977), and/or activation of endogenous glucose production and/or stimulated glycogenolysis (Jones, 1980; Jones et al. 1983; Hay et al. 1984; Apatu & Barnes, 1991; Hooper, 1995). Since fetal treatment with the -adrenergic antagonist, phentolamine, prevented the glycaemic response but enhanced insulin secretion during hypoxaemia (Jones & Ritchie, 1983), both the reduction in insulin-dependent glucose uptake and the increase in glucose production by the fetal tissues may be mediated via sympathetic -adrenergic pathways under these circumstances. Abolition of the glycaemic response to acute hypoxemia following fetal treatment with the CGRP antagonist in the present study may therefore represent an effect on insulin release and/or on the glucogenic pathways mediated either by sympathetic fibres and/or via elevations in circulating catecholamines. Either way, an involvement of CGRP in the activation of the sympathetic nervous system during acute hypoxaemia in the late gestation sheep fetus is again supported. Concomitant attenuation of the glycaemic and adrenergic responses to acute hypoxaemia by CGRP antagonism further underlines an important role of the increased plasma catecholamines in mediating the increase in blood glucose concentrations during acute hypoxaemia in the late gestation ovine fetus (Apatu & Barnes, 1991). However, in contrast to the attenuation in the glycaemic and femoral haemodynamic responses, circulating blood lactate concentrations remained unaffected during CGRP antagonism. This suggests that additional mechanisms must contribute to the circulating lactate concentration in the ovine fetus during periods of acute stress, such as an increase in lactate output from other sources, a reduction in hepatic lactate uptake, and an increased clearance of lactate by the placenta (see Gardner et al. 2003), and that these are not affected by CGRP antagonism.
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In adult sheep and other animals, the spleen can act as a reservoir of red blood cells. Sympathetic stimulation of splenic contraction and other venous reservoirs (Hoka et al. 1989) will thus decrease venous capacitance, and shift blood into the systemic circulation. In addition, Brace (1986) suggested that an increased sympathetic tone during acute hypoxaemia was responsible for the increased arterial, venous and capillary pressures in the fetus, which in turn resulted in haemoconcentration, by increasing the filtration coefficient and thereby reducing fetal blood volume. Combined, these results demonstrate why, during acute hypoxaemia, there is an acute increase in fetal red blood cell count and, thereby, haemoglobin concentration. In the present study, while the effect of CGRP antagonism on the magnitude of the increase in haemoglobin concentration during acute hypoxaemia further supports the involvement of CGRP in the activation of the sympathetic nervous system in fetal sheep, whether the spleen and venous circulations act as important reservoirs of red blood cells during the fetal period is still under debate (Potocnik & Wintour, 1996).
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The site of action of CGRP in mediating sympathetic nervous system responses during stimulated conditions in the sheep fetus is unclear. It is possible that CGRP antagonism may have affected the fetal defence responses to acute hypoxaemia by acting at the level of the sensor, the integrator and/or the target organs along the chemoreflex pathway. For example, it has been shown that CGRP-containing nerve fibres have been detected around the blood vessels as well as close to the clusters of glomus and sustentacular cells in the carotid body (Kummer & Habeck, 1991; Kameda, 1998). These nerve fibres originate from the petrosal, jugular and nodose ganglia (Kummer, 1988; Ichikawa et al. 1993), and are thought to have effects on chemosensory activity. In addition, immunocytochemical studies have revealed that the nucleus tractus solitarius and other areas involved in the integration or modulation of cardiovascular reflex responses, such as the ventrolateral medulla and the hypothalamic paraventricular nucleus, are all innervated with CGPP-containing neurones (Torrealba, 1992; Herbison et al. 1993; Batten, 1995; Coelho et al. 2004). While some reports suggest an inhibitory role of CGRP on adrenergic sympathetic neurones in the periphery (Kawasaki, 2002), the weight of evidence suggests that CGRP acts centrally to activate the sympathetic nervous system. In this context, it has been reported that microinjections of CGRP into the hypothalamus selectively activated sympathetic outflow in rats (Hasegawa et al. 1993). Similarly, microinjections of CGRP into the trigeminal subnucleus caudalis increased adrenal secretion of adrenaline and mean arterial blood pressure in cats (Bereiter & Benetti, 1991). Furthermore, Fisher et al. (1983) showed that while I.V. injection of CGRP evoked a dose-dependent hypotension, intra cerebro ventricular (I.C.V.) administration of CGRP caused an increase in arterial blood pressure, accompanied by a pronounced elevation in plasma noradrenaline concentrations. Pan & Kastin (2004) have reported that peptides are able to cross the blood–brain barrier after delivery into the peripheral bloodstream by simple diffusion or active transport mechanisms. In addition, in sheep, the blood–brain barrier in the fetus, even near term, is not completely mature and is permeable to low-molecular weight agents, as elegantly demonstrated by Stonestreet et al. (1996). Together, the weight of the evidence supports a possible central action of the antagonist in the late gestation ovine fetus. Finally, CGRP immunoreactive fibres have also been found to innervate the chromaffin adrenergic cells within the adrenal gland (Orezzoli et al. 1995). Here, CGRP may play an important role in the release of catecholamines in response to splanchnic nerve stimulation. Accordingly, Tortorella et al. (2001) reported that exogenous CGRP enhanced basal catecholamine secretion from rat adrenomedullary fragments in a concentration-dependent manner.
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In conclusion, the results from the present study show that treatment of fetal sheep during late gestation with a selective CGRP antagonist did not alter basal cardiovascular or metabolic function. In contrast, CGRP antagonism during acute hypoxaemia markedly diminished cardiovascular, metabolic and haematological responses known to be mediated by an increased activity of the sympathetic nervous system. Combined, these data strongly support the hypothesis that CGRP plays an important role in mediating the fetal defence responses to acute hypoxaemia by affecting sympathetic outflow.
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2 Faculty of Medicine, Imperial College, Hammersmith Hospital, London W12 ONN, UK
Abstract
The fetal defence to acute hypoxaemia involves cardiovascular and metabolic responses, which include peripheral vasoconstriction and hyperglycaemia. Both these responses are mediated via neuroendocrine mechanisms, which require the stimulation of the sympathetic nervous system. In the adult, accumulating evidence supports a role for calcitonin gene-related peptide (CGRP) in the activation of sympathetic outflow. However, the role of CGRP in stimulated cardiovascular and metabolic functions before birth is completely unknown. This study tested the hypothesis that CGRP plays a role in the fetal cardiovascular and metabolic defence responses to acute hypoxaemia by affecting sympathetic outflow. Under anaesthesia, five sheep fetuses at 0.8 of gestation were surgically instrumented with catheters and a femoral arterial Transonic flow-probe. 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. Arterial samples were taken for blood gases, metabolic status and hormone analyses. CGRP antagonism did not alter basal arterial blood gas, metabolic, cardiovascular or endocrine status. During hypoxaemia, similar falls in Pa,O2 occurred in all fetuses. During saline infusion, hypoxaemia induced hypertension, bradycardia, femoral vasoconstriction, hyperglycaemia and an increase in haemoglobin, catecholamines and neuropeptide Y (NPY). In contrast, CGRP antagonism markedly diminished the femoral vasoconstrictor and glycaemic responses to hypoxaemia, and attenuated the increases in haemoglobin, catecholamines and NPY. Combined, these results strongly support the hypothesis that CGRP plays a role in the fetal cardiovascular and metabolic defence to hypoxaemia by affecting sympathetic outflow.
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Introduction
A common challenge that the fetus faces during late gestation is an episode of acute hypoxaemia (Huch et al. 1977). Inadequately compensated periods of acute fetal hypoxaemia may lead to a wide range of developmental abnormalities and perinatal complications in the cardiovascular, respiratory and central nervous systems. Thus, fetal hypoxaemia may promote left ventricular myocardial dysfunction (Walther et al. 1985), meconium aspiration syndrome (Klingner & Kruse, 1999), neonatal seizures (Arpino et al. 2001), hypoxic–ischaemic encephalopathy (Low et al. 1985) and cerebral palsy (Johnston et al. 2001).
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The fetal defence to acute hypoxaemia involves integrated cardiovascular and metabolic responses, which facilitate fetal survival during the period of reduced oxygen availability. The cardiovascular defence responses to acute hypoxaemia are well established, and involve a transient bradycardia, a gradual increase in arterial blood pressure and peripheral vasoconstriction (for reviews, see Rudolph, 1984; Giussani et al. 1994). The latter aids the redistribution of the fetal combined ventricular output, away from peripheral circulations and towards more essential vascular beds such as the cerebral, adrenal, and myocardial circulations (Cohn et al. 1974; Peeters et al. 1979; Jensen & Berger, 1991). The fetal metabolic responses to acute hypoxaemia involve an increase in the circulating concentrations of glucose and lactate (Jones & Ritchie, 1976; Jones, 1977). The fetal hyperglycaemia during acute hypoxaemia results from a decrease in glucose uptake and utilisation by peripheral tissues (Jones et al. 1983), and an increase in hepatic glucose production (Jones & Ashton, 1976). The fetal lactacidaemia results from anaerobic metabolism of glucose in hypoxic fetal tissues, particularly in the carcass where blood flow and oxygen delivery are markedly declined.
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The physiological mechanisms mediating both the cardiovascular and metabolic defence responses to acute hypoxaemia involve the activation of the fetal sympathetic nervous system. Acute hypoxaemia triggers a carotid chemoreflex, which promotes femoral vasoconstriction via -adrenergic efferent pathways (Giussani et al. 1993). Increased sympathetic outflow also inhibits insulin release from the fetal pancreas, thereby decreasing glucose uptake and utilisation by the fetal tissues (Jones et al. 1983). As the hypoxaemic challenge progresses, catecholamines (Jones & Robinson, 1975) and neuropeptide Y (NPY; Fletcher et al. 2000) are released into the fetal circulation, and act to maintain the peripheral vasoconstrictor response (Sanhueza et al. 2003). In addition, catecholamines are also known to mobilise and release glucose from glycogen stores in the fetal liver (Apatu & Barnes, 1991).
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In the adult, calcitonin gene-related peptide (CGRP) is a novel peptide which is rapidly gaining clinical and scientific interest for its role in cardiovascular and metabolic regulation (DiPette et al. 1987; Franco-Cereceda et al. 1987; Gennari et al. 1990; Hasbak et al. 2002,; Dhillo et al. 2003). In addition to being one of the most potent vasodilator peptides known (Brain et al. 1985), accumulating evidence suggests that it can also selectively activate sympathetic outflow (Hasegawa et al. 1993; Herbison et al. 1993). The distribution of CGRP at the site of implantation (Tsatsaris et al. 2002) and throughout the human placenta (Graf et al. 1996), increased maternal plasma levels of CGRP during pregnancy (Saggese et al. 1990), and its presence in human cord and neonatal blood (Parida et al. 1998) all suggest a functional role for CGRP in development. However, the role of CGRP in fetal cardiovascular or metabolic functions, during either basal or stimulated conditions, is completely unknown.
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The present study tested the hypothesis that CGRP plays a role in the fetal cardiovascular and metabolic defence responses to acute hypoxaemia by affecting sympathetic outflow. The hypothesis was tested using the unanaesthetised late gestation sheep fetus as our experimental model, in which cardiovascular and metabolic responses to acute hypoxaemia were investigated during either saline infusion or treatment with a selective CGRP antagonist dissolved in saline.
, http://www.100md.com 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 were surgically instrumented for long-term recording at 120 ± 2 days of gestation (term is 145 days), 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 exteriorised 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 descending aorta and inferior 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 (2R or 3S; Transonic Systems Inc., Ithaca, NY, USA) was placed around the contralateral femoral artery, for continuous measurement of femoral blood flow. The uterine incisions were closed in layers, the dead space of the catheters was filled with heparinised saline (80 i.u. heparin ml–1 in 0.9% NaCl), and the catheter ends were plugged with sterile brass pins. The catheters and flow probe lead were then exteriorised via a keyhole incision in the maternal flank, and kept inside a plastic pouch sewn onto the maternal skin.
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Postoperative care
During recovery, ewes were housed in individual pens in rooms with a 12 h: 12 h light: dark cycle where they had free access to hay and water and were fed concentrates twice daily (100 g sheep nuts no. 6; H & C Beart Ltd, Kings Lynn, UK). Antibiotics were administered daily to the ewe (0.20–0.25 mg kg–1 I.M. Depocillin; Mycofarm, Cambridge, UK) and fetus I.V and into the amniotic cavity (150 mg kg–1 Penbritin; SmithKline Beecham Animal Health, Welwyn Garden City, Hertfordshire, UK). The ewes also received 2 days of postoperative analgesia if in pain (10–20 mg kg–1 oral Penylbutazone; Equipalozone paste, Arnolds Veterinary Products Ltd, Shropshire, UK), as assessed by their general demeanour and feeding patterns. Generally, normal feeding patterns were restored within 48 h of recovery. Following 72 h of postoperative recovery, ewes were transferred to metabolic crates where they were housed for the remainder of the protocol. The arterial and amniotic catheters were connected to sterile pressure transducers (COBE; Argon Division, Maxxim Medical, Athens, Texas, USA), and the flow probe lead to a flow meter (T206; Transonics Systems Inc., Ithaca, NY, USA). Calibrated mean fetal arterial blood pressure (corrected for amniotic pressure), fetal heart rate (triggered via a tachometer from the pulsatility in either the arterial blood pressure or femoral blood flow signals), and mean femoral blood flow were recorded continually at 1 s intervals using a computerised data acquisition system (DAS; Department of Physiology, Cambridge University, UK). While on the metabolic crates, 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).
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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 a randomised order (Fig. 1). Each protocol consisted of a 2.5 h period divided into: 1 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 the CGRP antagonist dissolved in heparinised saline (50 μg kg–1 I.A. bolus followed by 8 μg kg–1.min–1 I.V. infusion; calcitonin gene related peptide fragment 8–37, CGRP8—37; C-2806; Sigma Chemicals, UK). Acute hypoxaemia in the fetus was induced by maternal inhalational hypoxia. In brief, a large transparent respiratory hood was placed over the ewes' 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: 5 l min–1 N2: 1.5–2.5 l min–1 CO2). This mixture was designed to reduce fetal Pa,O2 to 10 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 our own pilot experiments based on 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. At the end of the experimental protocol, the ewes and fetuses were humanely killed 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 times at which arterial blood samples were collected.
Blood sampling regimen
During any acute hypoxaemia protocol, descending aortic blood samples (0.3 ml) were taken using sterile techniques from the fetus at set time intervals (Fig. 1, arrows) to determine arterial blood gas and acid–base status (ABL5 Blood Gas Analyser, Radiometer; Copenhagen, Denmark; measurements corrected to 39.5°C). Values for percentage saturation of haemoglobin with oxygen (Sat Hb) and the blood haemoglobin concentration ([Hb]) were determined using a haemoximeter (OSM2; Radiometer). In addition, blood glucose and lactate concentrations were measured by an automated analyser (Yellow Springs 2300 Stat Plus Glucose/Lactate Analyser; YSI Ltd, Farnborough, UK). An additional 4 ml of arterial blood was withdrawn at set intervals for hormone analyses (Fig. 1, arrows). These samples were collected under sterile conditions into chilled heparin tubes (2 ml Li+/heparin tubes; L.I.P. Ltd, Shipley, West Yorkshire, UK) containing reduced glutathione (4 nmol tube–1; G-4251; Sigma Chemicals, UK) and EGTA (5 nmol tube–1; E-4378; Sigma Chemicals, UK) for catecholamines analysis or into chilled EDTA tubes (2 ml K+/EDTA; L.I.P. Ltd) for NPY analysis. All samples were then centrifuged at 4000 r.p.m. for 4 min at 4°C. The plasma obtained was then dispensed into prelabelled tubes and the samples were stored at –80°C until analysis. All hormone measurements were performed within 2 months of sample collection.
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Hormone analyses
NPY assay. Fetal plasma NPY concentrations were measured by radioimmunoassay as previously described in detail (Allen et al. 1984). All samples were analysed in duplicate at the same time. The assay used rabbit antiserum (produced in horse) and 125I-labelled porcine peptide. Separation of the free and bound fractions was performed with dextran-coated charcoal. The assay was validated for use with ovine plasma using stripped sheep plasma. The assay could detect less than 1 pmol l–1 (95% confidence interval). The interassay coefficient of variation was 6.8%. There was no detectable cross-reactivity of the anti-NPY antiserum with the peptide YY.
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Catecholamine assay. Fetal plasma noradrenaline and adrenaline concentrations were measured by high-pressure liquid chromatography (HPLC) using electrochemical detection as previously described in detail (Fowden et al. 1998). The samples were prepared by absorption of 250 μl of plasma onto acid-washed alumina, and 20 μl aliquots of the 100 μl perchloric acid elutes were injected onto the column. Dihydroxybenzylamine was added as the internal standard to each plasma sample before absorption. The limit of sensitivity for the assay was 20 pg ml–1 for adrenaline and noradrenaline. Recovery ranged from 63% to 97%, and all catecholamine values were corrected for their respective recovery. The interassay coefficients of variation for adrenaline and noradrenaline were 7.3% and 6.2%.
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Data and statistical analyses
Femoral vascular resistance was calculated using Ohm's principle by dividing mean corrected arterial blood pressure by mean femoral blood flow. Values for all arterial blood gas, acid–base and metabolic variables are expressed as mean ± S.E.M. at 0 (N0) and 45 (N45) min of normoxia, 5 (H5), 15 (H15) and 30 (H30) min of hypoxaemia, and at 30 (R30) and 60 (R60) min of recovery. Values for all endocrine variables are expressed as mean ± S.E.M. at 0 (N0) and 45 (N45) min of normoxia, 15 (H15) and 30 (H30) min of hypoxaemia, and at 30 (R30) and 60 (R60) min of recovery. The cardiovascular variables are expressed as mean ± S.E.M. minute averages. Summary measure analysis was then applied to the serial cardiovascular data to focus the number of comparisons, as previously described in detail (Matthews et al. 1990), and area under the curve was calculated for the absolute data every 30 min. All measured variables were first analysed for normality of distribution, and then assessed statistically using two-way ANOVA with repeated measures (RM; 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 Tukey test was used to isolate the statistical differences. For all comparisons, statistical significance was accepted when P < 0.05.
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Results
Fetal arterial blood gas and acid–base status during acute hypoxaemia
Basal values for arterial blood gas and acid–base status were similar in all fetuses. Infusion with saline or treatment with the CGRP antagonist had no effect on basal arterial blood gas and acid–base status (Table 1). In all fetuses, acute hypoxaemia induced significant falls in arterial pH (pHa), acid–base excess (ABE), [HCO3–], Pa,O2 and Sat Hb, without any alteration to Pa,CO2. The magnitude of these changes was similar during saline infusion or during treatment with the CGRP antagonist (Table 1). However the maximal increment in fetal [Hb] during acute hypoxaemia was significantly diminished during treatment with the CGRP antagonist (0.58 ± 0.57 g dl–1) when compared to saline-infused controls (1.98 ± 0.16 g dl–1; P < 0.05). During recovery pHa, ABE and [HCO3–] remained significantly depressed by the end of the experimental protocol in all fetuses, whereas Pa,O2, Sat Hb and [Hb] returned to basal values.
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Fetal metabolic status during acute hypoxaemia
Basal values for fetal blood glucose and lactate concentrations were similar in all fetuses. Infusion with saline or treatment with the CGRP antagonist had no effect on basal blood glucose and lactate concentrations (Fig. 2). Acute hypoxaemia during saline infusion induced significant increases in blood glucose and lactate concentrations. Treatment with the CGRP antagonist did not affect the increment in blood lactate (3.68 ± 0.68 versus 3.00 ± 0.37 mM, P > 0.05), but it did significantly diminish the increment in blood glucose concentration (0.83 ± 0.23 versus 0.34 ± 0.10 mM, P < 0.05) during acute hypoxaemia (Fig. 2). During recovery, blood lactate concentrations remained significantly elevated by the end of the experimental protocol in all fetuses, whereas blood glucose concentrations returned to basal values.
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Values represent the mean ± S.E.M. for blood glucose and lactate concentrations at 0 (N0) and 45 (N45) min of normoxia, at 5 (H5), 15 (H15) and 30 (H30) min of hypoxaemia, and at 30 (R30) and 60 (R60) min of recovery for fetuses exposed to 0.5 h hypoxaemia (open box) during saline infusion (; n = 5) or during treatment with the CGRP antagonist (; n = 5). Significant differences: *P < 0.05, for normoxia versus hypoxaemia or recovery; P < 0.05, saline versus CGRP antagonist (Two-way RM ANOVA with post hoc Tukey test).
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Fetal cardiovascular responses during acute hypoxaemia
Basal values for fetal arterial blood pressure, heart rate, femoral blood flow (FBF) and femoral vascular resistance (FVR) were similar in all fetuses. Infusion with saline, or treatment with the CGRP antagonist had no effect on basal cardiovascular variables (Fig. 3). Acute hypoxaemia during saline infusion induced significant increases in arterial blood pressure and femoral vascular resistance, and significant falls in heart rate and femoral blood flow (Fig. 3). Treatment with the CGRP antagonist did not affect the magnitude of the mean pressor (9.4 ± 1.0 versus 9.9 ± 0.9 mmHg) or bradycardic (–36 ± 11 versus –32 ± 11 beats min–1) responses to acute hypoxaemia (P > 0.05), but significantly attenuated the mean increment in the femoral vasoconstrictor response (FBF: –30 ± 4 versus –21 ± 2 ml min–1; FVR: 6.8 ± 1.5 versus 2.9 ± 0.4 mmHg. (ml min–1)–1; P < 0.05; Fig. 3). During recovery, fetal arterial blood pressure and heart rate remained significantly elevated by the end of the experimental protocol in all fetuses, whereas femoral blood flow and vascular resistance returned to basal values.
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A, values represent the mean ± S.E.M. calculated every minute for arterial blood pressure, heart rate, femoral blood flow and femoral vascular resistance during 1 h of normoxia, 0.5 h of hypoxaemia (open box) and 1 h of recovery for fetuses during saline infusion (n = 5) or during treatment with the CGRP antagonist (n = 5). B, values for the statistical summary of these responses represent the mean ± S.E.M. for the area under the curve over every 30 min during normoxia (N1, N2), hypoxaemia (H) and recovery (R1, R2) for fetuses during saline infusion (; n = 5) and during treatment with the CGRP antagonist (; n = 5). Significant differences: *P < 0.05, normoxia versus hypoxaemia or recovery; P < 0.05, saline versus CGRP antagonist (Two-way RM ANOVA with post hoc Tukey test).
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Plasma catecholamine and NPY concentrations
Basal values for fetal plasma neuropeptide Y, adrenaline and noradrenaline concentrations were similar in all fetuses. Infusion with saline or treatment with the CGRP antagonist had no effect on basal NPY or catecholamines levels (Fig. 4). Acute hypoxaemia during saline infusion induced a significant increase in plasma NPY, adrenaline and noradrenaline concentrations. Treatment with the CGRP antagonist diminished the maximal increment in plasma NPY (52 ± 16 versus 29 ± 11 pmol l–1) and adrenaline (2376 ± 1223 versus 1010 ± 379 pg ml–1; P < 0.05) concentrations during hypoxaemia, however, the effect on noradrenaline plasma concentrations (3258 ± 1557 versus 2091 ± 799 pg ml–1; P = 0.06) fell outside significance (Fig. 4). During recovery, plasma concentrations of NPY, adrenaline and noradrenaline returned to basal values.
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Values represent the mean ± S.E.M. for plasma concentrations of neuropeptide Y, adrenaline and noradrenaline at 0 (N0) and 45 (N45) min of normoxia, at 15 (H15) and 30 (H30) min of hypoxaemia, and at 30 (R30) and 60 (R60) min of recovery for fetuses exposed to 0.5 h hypoxaemia (open box) during saline infusion (; n = 5) or during treatment with the CGRP antagonist (; n = 5). Significant differences: *P < 0.05, normoxia versus hypoxaemia or recovery; P < 0.05, saline versus CGRP antagonist (Two-way RM ANOVA with post hoc Tukey test).
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Discussion
The results of the present study show that fetal treatment with a CGRP antagonist had no effect on cardiovascular or metabolic status during basal conditions. However, CGRP antagonism during acute hypoxaemia markedly diminished the increases in fetal femoral vascular resistance, blood glucose and haemoglobin concentrations, and in plasma levels of NPY and catecholamines. These data support the hypothesis that CGRP plays an important role in the activation of the sympathetic nervous system during acute hypoxaemia in the late gestation sheep fetus.
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The fetal peripheral vasoconstrictor response to acute hypoxaemia is mediated via carotid chemoreflex activation of the sympathetic nervous system, since both bilateral section of the carotid sinus nerves (Giussani et al. 1993; Bartelds et al. 1993) and -adrenergic blockade (Reuss et al. 1982; Giussani et al. 1993) markedly attenuate the increase in peripheral vascular resistance in response to hypoxaemia in the late gestation sheep fetus. As the hypoxaemic episode continues, plasma concentrations of catecholamines (Jones & Robinson, 1975; Jones & Wei, 1985) and NPY (Fletcher et al. 2000) are increased in the fetal circulation, and act to maintain the peripheral vasoconstriction. The origin of the increase in both adrenaline and noradrenaline in fetal plasma during acute hypoxaemia has been shown to be primarily from the fetal adrenal gland, since adrenal demedullation completely abolished the rise in plasma adrenaline concentration and reduced the noradrenaline response to 10% of normal (Jones et al. 1988). Since a large component of this increase in total catecholamine output from the fetal adrenal gland during acute hypoxia is mediated via activation of the splanchnic nerves (Jones et al. 1988), elevations in plasma concentrations of noradrenaline and adrenaline in the fetal sheep circulation are good indices of increased sympathetic outflow during stimulated conditions. In contrast to noradrenaline, the hypoxaemia-induced increase in fetal plasma NPY concentration is more likely to represent increased overspill from perivascular sympathetic nerve terminals. Despite the relatively high adrenal medullary NPY content, evidence suggests a negligible role for the gland in contributing to circulating plasma NPY levels during acute stress (Mormede et al. 1990). Since NPY lacks synaptic reuptake mechanisms, elevations in plasma NPY concentrations may provide a more robust measure than noradrenaline of increased sympathetic nervous activity (Lundberg, 1996). The present study showed that fetal treatment with the CGRP antagonist diminished the magnitude of the increase in femoral vascular resistance and in the concentrations of catecholamines and NPY in the fetal circulation; effects which are all consistent with the involvement of CGRP in the activation of the sympathetic nervous system during acute hypoxaemia in the sheep fetus.
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Although CGRP antagonism affected the femoral vasoconstrictor response to acute hypoxaemia, it had no effect on the hypertensive response. This does not suggest that changes in total peripheral vascular resistance are not main determinants of changes in arterial blood pressure during periods of acute stress, but the data merely emphasise that the femoral vascular bed is not representative of the whole of the peripheral vasculature in the late gestation ovine fetus.
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CGRP is one of the most potent vasodilator peptides known (Brain et al. 1985), having a potency of 10-fold greater than prostaglandins and 100–1000-fold greater than classical vasodilators, such as acetylcholine, adenosine and 5-HT (Brain & Grant, 2004). Recent in vivo experiments in the ovine fetus have shown that CGRP has a powerful vasodilator action in the pulmonary vascular bed (Takahashi et al. 2000), which is, in part, mediated via nitric oxide. Based on this evidence, CGRP may have been expected to act as a nitric oxide-dependent vasodilator during basal conditions and during acute hypoxaemia and, thereby, to reduce femoral vascular resistance during normoxia, and partially offset the femoral vasoconstrictor response to hypoxaemia in the ovine fetus. However, the results of the present study show that CGRP antagonism did not affect basal cardiovascular function. This is consistent with the available literature which has demonstrated that intravenous injection of the CGRP antagonist had no effect on basal blood pressure in rodents (Gardiner et al. 1991), and no effect on either systemic blood pressure or regional vascular beds in the conscious dog and anaesthetised rat (Shen et al. 2001). Although receptors for CGRP are widely distributed throughout the vasculature, a lack of an effect on basal cardiovascular function by the antagonist treatment is not inconsistent with the well-known vasodilator effect of exogenous administration of the agonist. Indeed, various groups have suggested that endogenous CGRP acts as a ‘rescue’ molecule, being upregulated and released from nerve terminals during periods of stress (Geppetti et al. 1991; Franco-Cereceda et al. 1993; Parida et al. 1998; Peng et al. 2002). The results of the present study also suggest that during episodes of acute hypoxaemic stress in the ovine fetus, there is an increased release of CGRP, whose effect on sympathetic outflow outweighs any local vasodilator actions of this peptide in the femoral circulation.
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The increase in blood glucose concentrations during acute hypoxaemia in fetuses infused with saline may be due to depression of insulin-dependent glucose uptake by the fetal tissues (Jones, 1977), and/or activation of endogenous glucose production and/or stimulated glycogenolysis (Jones, 1980; Jones et al. 1983; Hay et al. 1984; Apatu & Barnes, 1991; Hooper, 1995). Since fetal treatment with the -adrenergic antagonist, phentolamine, prevented the glycaemic response but enhanced insulin secretion during hypoxaemia (Jones & Ritchie, 1983), both the reduction in insulin-dependent glucose uptake and the increase in glucose production by the fetal tissues may be mediated via sympathetic -adrenergic pathways under these circumstances. Abolition of the glycaemic response to acute hypoxemia following fetal treatment with the CGRP antagonist in the present study may therefore represent an effect on insulin release and/or on the glucogenic pathways mediated either by sympathetic fibres and/or via elevations in circulating catecholamines. Either way, an involvement of CGRP in the activation of the sympathetic nervous system during acute hypoxaemia in the late gestation sheep fetus is again supported. Concomitant attenuation of the glycaemic and adrenergic responses to acute hypoxaemia by CGRP antagonism further underlines an important role of the increased plasma catecholamines in mediating the increase in blood glucose concentrations during acute hypoxaemia in the late gestation ovine fetus (Apatu & Barnes, 1991). However, in contrast to the attenuation in the glycaemic and femoral haemodynamic responses, circulating blood lactate concentrations remained unaffected during CGRP antagonism. This suggests that additional mechanisms must contribute to the circulating lactate concentration in the ovine fetus during periods of acute stress, such as an increase in lactate output from other sources, a reduction in hepatic lactate uptake, and an increased clearance of lactate by the placenta (see Gardner et al. 2003), and that these are not affected by CGRP antagonism.
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In adult sheep and other animals, the spleen can act as a reservoir of red blood cells. Sympathetic stimulation of splenic contraction and other venous reservoirs (Hoka et al. 1989) will thus decrease venous capacitance, and shift blood into the systemic circulation. In addition, Brace (1986) suggested that an increased sympathetic tone during acute hypoxaemia was responsible for the increased arterial, venous and capillary pressures in the fetus, which in turn resulted in haemoconcentration, by increasing the filtration coefficient and thereby reducing fetal blood volume. Combined, these results demonstrate why, during acute hypoxaemia, there is an acute increase in fetal red blood cell count and, thereby, haemoglobin concentration. In the present study, while the effect of CGRP antagonism on the magnitude of the increase in haemoglobin concentration during acute hypoxaemia further supports the involvement of CGRP in the activation of the sympathetic nervous system in fetal sheep, whether the spleen and venous circulations act as important reservoirs of red blood cells during the fetal period is still under debate (Potocnik & Wintour, 1996).
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The site of action of CGRP in mediating sympathetic nervous system responses during stimulated conditions in the sheep fetus is unclear. It is possible that CGRP antagonism may have affected the fetal defence responses to acute hypoxaemia by acting at the level of the sensor, the integrator and/or the target organs along the chemoreflex pathway. For example, it has been shown that CGRP-containing nerve fibres have been detected around the blood vessels as well as close to the clusters of glomus and sustentacular cells in the carotid body (Kummer & Habeck, 1991; Kameda, 1998). These nerve fibres originate from the petrosal, jugular and nodose ganglia (Kummer, 1988; Ichikawa et al. 1993), and are thought to have effects on chemosensory activity. In addition, immunocytochemical studies have revealed that the nucleus tractus solitarius and other areas involved in the integration or modulation of cardiovascular reflex responses, such as the ventrolateral medulla and the hypothalamic paraventricular nucleus, are all innervated with CGPP-containing neurones (Torrealba, 1992; Herbison et al. 1993; Batten, 1995; Coelho et al. 2004). While some reports suggest an inhibitory role of CGRP on adrenergic sympathetic neurones in the periphery (Kawasaki, 2002), the weight of evidence suggests that CGRP acts centrally to activate the sympathetic nervous system. In this context, it has been reported that microinjections of CGRP into the hypothalamus selectively activated sympathetic outflow in rats (Hasegawa et al. 1993). Similarly, microinjections of CGRP into the trigeminal subnucleus caudalis increased adrenal secretion of adrenaline and mean arterial blood pressure in cats (Bereiter & Benetti, 1991). Furthermore, Fisher et al. (1983) showed that while I.V. injection of CGRP evoked a dose-dependent hypotension, intra cerebro ventricular (I.C.V.) administration of CGRP caused an increase in arterial blood pressure, accompanied by a pronounced elevation in plasma noradrenaline concentrations. Pan & Kastin (2004) have reported that peptides are able to cross the blood–brain barrier after delivery into the peripheral bloodstream by simple diffusion or active transport mechanisms. In addition, in sheep, the blood–brain barrier in the fetus, even near term, is not completely mature and is permeable to low-molecular weight agents, as elegantly demonstrated by Stonestreet et al. (1996). Together, the weight of the evidence supports a possible central action of the antagonist in the late gestation ovine fetus. Finally, CGRP immunoreactive fibres have also been found to innervate the chromaffin adrenergic cells within the adrenal gland (Orezzoli et al. 1995). Here, CGRP may play an important role in the release of catecholamines in response to splanchnic nerve stimulation. Accordingly, Tortorella et al. (2001) reported that exogenous CGRP enhanced basal catecholamine secretion from rat adrenomedullary fragments in a concentration-dependent manner.
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In conclusion, the results from the present study show that treatment of fetal sheep during late gestation with a selective CGRP antagonist did not alter basal cardiovascular or metabolic function. In contrast, CGRP antagonism during acute hypoxaemia markedly diminished cardiovascular, metabolic and haematological responses known to be mediated by an increased activity of the sympathetic nervous system. Combined, these data strongly support the hypothesis that CGRP plays an important role in mediating the fetal defence responses to acute hypoxaemia by affecting sympathetic outflow.
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