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High altitude hypoxia and blood pressure dysregulation in adult chickens

Published online by Cambridge University Press:  19 September 2012

E. A. Herrera ,
C. E. Salinas ,
C. E. Blanco ,
M. Villena  and
D. A. Giussani
E. A. Herrera
Affiliation:
Laboratorio de Función y Reactividad Vascular, Programa de Fisiopatología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
C. E. Salinas
Affiliation:
Instituto Boliviano de Biología de Altura, Facultad de Medicina, Universidad Mayor de San Andrés, La Paz, Bolivia
C. E. Blanco
Affiliation:
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
M. Villena
Affiliation:
Instituto Boliviano de Biología de Altura, Facultad de Medicina, Universidad Mayor de San Andrés, La Paz, Bolivia
D. A. Giussani*
Affiliation:
Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
*
*Address for correspondence: Prof. D. A. Giussani, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK. Email dag26@cam.ac.uk
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Abstract

Although it is accepted that impaired placental perfusion in complicated pregnancy can slow fetal growth and programme an increased risk of cardiovascular dysfunction at adulthood, the relative contribution of reductions in fetal nutrition and in fetal oxygenation as the triggering stimulus remains unclear. By combining high altitude (HA) with the chick embryo model, we have previously isolated the direct effects of HA hypoxia on embryonic growth and cardiovascular development before hatching. This study isolated the effects of developmental hypoxia on cardiovascular function measured in vivo in conscious adult male and female chickens. Chick embryos were incubated, hatched and raised at sea level (SL, nine males and nine females) or incubated, hatched and raised at HA (seven males and seven females). At 6 months of age, vascular catheters were inserted under general anaesthesia. Five days later, basal blood gas status, basal cardiovascular function and cardiac baroreflex responses were investigated. HA chickens had significantly lower basal arterial PO2 and haemoglobin saturation, and significantly higher haematocrit than SL chickens, independent of the sex of the animal. HA chickens had significantly lower arterial blood pressure than SL chickens, independent of the sex of the animal. Although the gain of the arterial baroreflex was decreased in HA relative to SL male chickens, it was increased in HA relative to SL female chickens. We show that development at HA lowers basal arterial blood pressure and alters baroreflex sensitivity in a sex-dependent manner at adulthood.

Keywords

baroreflexcardiovascular diseasechronic hypoxiafetal programming
Type
Original Article
Information
Journal of Developmental Origins of Health and Disease , Volume 4 , Issue 1 , February 2013 , pp. 69 - 76
Copyright
Copyright © Cambridge University Press and the International Society for Developmental Origins of Health and Disease 2012 

Introduction

Overwhelming evidence obtained from studies in human links development under sub-optimal intrauterine conditions with increased risk of cardiovascular disease.Reference Barker1 Common sub-optimal intrauterine conditions include impaired fetal nutrition and oxygenation. Consequently, many studies have now accumulated to report that nutrientReference Fowden, Giussani and Forhead2–Reference Martin-Gronert and Ozanne4 and oxygenReference Fowden, Giussani and Forhead2, Reference Thompson5–Reference Giussani, Camm and Niu9 restriction during development promotes intrauterine growth restriction (IUGR) and programmes cardiovascular dysfunction in the adult offspring.

Several studies, including our own, have used exposure of the pregnant rodent to a prolonged period of hypoxia during gestation and have studied adverse effects on fetal growth and on the cardiovascular system of the offspring at adulthood. These studies have reported that prenatal chronic hypoxia with or without IUGR programmes in the adult offspring impaired cardiac performance with increased susceptibility to cardiac ischaemia/reperfusion injuryReference Hauton and Ousley6–Reference Li, Xiao and Estrella10 and pronounced endothelial dysfunction in peripheral resistance circulations.Reference Rueda-Clausen, Morton and Davidge8, Reference Giussani, Camm and Niu9, Reference Morton, Rueda-Clausen and Davidge11 However, because maternal exposure to hypoxia can lead to a significant decrease in maternal food intake,Reference de Grauw, Myers and Scott12, Reference Camm, Hansell and Kane13 the extent to which any adverse effects on the offspring are due to fetal undernutrition and/or underoxygenation remain unclear.

More than 140 million people live at altitudes higher than 3000 m, providing the largest single human group at risk for fetal exposure to hypoxia.Reference Moore, Charles and Julian14 Studies of highland populations have reported fetal growth restriction and adverse cardiovascular alterations in offspring of high altitude (HA) relative to those of sea level (SL) pregnancies.Reference Lichty, Ting, Bruns and Dyer15–Reference Huicho and Niermeyer23 However, because most of HA populations are also impoverished, the relative contributions of fetal hypoxia and of malnutrition in slowing fetal growth and programming cardiovascular dysfunction, again, remain uncertain.

The chick embryo provides a useful model to investigate the direct effects on fetal growth and on programming of cardiovascular dysfunction of developmental hypoxia independent of additional effects on the nutrition of the mother and/or on the maternal or placental physiology and/or on lactation.Reference Ruijtenbeek, De Mey and Blanco24–Reference Tintu, Rouwet and Verlohren26 By combining HA exposure with the chick embryo model, we have previously been able to isolate the direct effects of HA hypoxia on fetal growthReference Giussani, Salinas, Villena and Blanco27 and on fetal cardiovascular development.Reference Salinas, Blanco and Villena28 Incubation of fertilized eggs from SL hens at HA promoted growth restriction, cardiomegaly and cardiac and aortic wall thickening in the chick embryo by the end of the incubation period.Reference Giussani, Salinas, Villena and Blanco27, Reference Salinas, Blanco and Villena28 These effects of HA incubation could be prevented by incubating eggs from HA hens at SL or by incubating eggs from SL hens at HA with oxygen supplementation.Reference Giussani, Salinas, Villena and Blanco27, Reference Salinas, Blanco and Villena28 In the present study, we have combined HA exposure with the chick embryo model, to isolate the effects of HA hypoxia during embryonic development and postnatal life on cardiovascular function measured in vivo at adulthood. Because in this model the hypoxic environmental condition persists before and after hatching, the study also permits the investigation of the effects of a ‘double insult’ in triggering cardiovascular dysfunction. As it is established that differences in sex affect developmental origins of cardiovascular disease,Reference Gilbert and Nijland29 this study investigated both male and female adult chickens.

Methods

All procedures were approved by the local ethics committee of the Bolivian Institute for High Altitude Biology (Consejo Técnico, IBBA, Universidad Mayor de San Andrés, La Paz, Bolivia) and were performed under the UK Animals (Scientific Procedures) Act 1986.

Animals

The study was conducted in Bolivia, in the HA city of La Paz (HA, 3600 m, 494 mmHg, PO2 100 mmHg) and the SL city of Santa Cruz (SL, 420 m, 760 mmHg, PO2 160 mmHg). Eighteen (nine males and nine females) Black Leghorn chicken embryos were incubated, hatched and raised at SL and 14 (seven males and seven females) Black Leghorn chicken embryos were incubated, hatched and raised at HA. At 6 months, which in this species represents early adulthood, the animals were subjected to general anaesthesia (10 mg/kg Xylazine 2%, Millpledge Pharmaceuticals, United Kingdom and 15 mg/kg Ketamine, Ketaset, Fort Dodge Animal Health, Iowa, USA, i.m.) and the femoral artery and vein were isolated via a hind limb medial incision. Polyvinyl catheters (i.d. 0.58 mm; o.d. 0.96 mm; Critchly Electrical Products, NSW, Australia) were placed in the descending aorta and inferior vena cava. The catheters were filled with heparinized saline (100 i.u. heparin in 0.9% NaCl), plugged with a brass pin and tunnelled subcutaneously to exit between the origin of the wings at the back of the chicken. After surgery, the animals were returned to a recovery room and then to floor pens. At least 5 days of post-operative recovery were allowed before the beginning of any experiment. Catheters were maintained patent by daily flushing with heparinized saline. At this time, an arterial blood sample was taken to monitor well-being.

Experimental protocol

On the day of experiments, the chicken was placed in a sling inside a dark wooden box with appropriate ventilation. The catheters were extended. The arterial catheter was connected to a pressure transducer (COBE; Argon Division, Maxxim Medical, Athens, Texas, USA) at the level of the base of the heart. Mean (MAP), systolic (SAP) and diastolic arterial pressure (DAP) and heart rate (HR) were recorded continuously via a data acquisition system (MPAQ – Maastricht Programmable AcQuisition system, Maastricht Instruments, The Netherlands, 500 Hz sample rate).

During basal recording, before the start of any experiment, 0.5 ml of arterial blood was taken to determine pHa, PaO2 and PaCO2 (ABL 500, Radiometer, Copenhagen, Denmark, measurements corrected to 37°C), percentage saturation of haemoglobin (SaO2) and haematocrit (Ht; OSM3, Radiometer, Copenhagen, Denmark).

The chicken was then subjected to a protocol consisting of a 10-min period of baseline (B), a 10-min intravenous infusion (I) and 10-min period of recovery (R). The infusion consisted of either phenylephrine (50 μg/kg per min) or sodium nitroprusside (20 μg/kg per min) in separate experiments. The experiments were performed on the same day with at least 2 h between them to allow the cardiovascular variables to return to basal values. The phenylephrine experiment always preceded the infusion of nitroprusside.

At the end of the experiments, the chicken was humanely killed with an overdose of anaesthetic (100 mg/kg, Thiopental injection BP, Link Pharmaceuticals Ltd, UK, i.v.). Post mortem, the chicken was weighed and several body measurements were taken.

Data and statistical analyses

Baseline values for MAP, HR, pulse interval (PI; HR/60,000) and the rate pressure product [RPP; (HR × SAP)/1000] were calculated. In brief, the calculation of the baroreflex gain required two steps. The first step was the calculation of the slope of the HR–blood pressure relationship and the maximum and minimum values for HR for each animal. This was achieved by plotting the minute-by-minute HR and blood pressure responses during the beginning of the infusion period from baseline to plateau. Only the period from baseline to plateau is used to avoid confounding by resetting of the baroreflex. Arterial blood pressure and HR responses to the drugs were fitted to sigmoidal curves as follows: HR = HRmin +((HRmax − HRmin)/(1 + 10(Mid-point-MAP) × Gain coefficient). The value for gain represents the gain coefficient of the Hill slope that describes the steepness of the curve. The second step was to calculate the baroreflex sensitivity according to McDowall and DampneyReference McDowall and Dampney30 applying the values for gain, maximum and minimum HR to the following equation: Baroreflex Gain =((HRmax − HRmin) × Gain coefficient)/4. This was done for each individual animal and thereafter the mean ± s.e.m. of the baroreflex gain for each group was calculated.

All data are expressed as mean ± s.e.m. Comparisons between groups were assessed statistically using two-way ANOVA with the Student–Newman–Keuls post-hoc test, with altitude and sex as factors (Prism 5, GraphPad Software, Inc.). For all comparisons, statistical significance was accepted when P < 0.05.

Results

Arterial blood gas status

There were no differences in basal arterial pH and pCO2 between groups. However, HA chickens had lower arterial pO2, SaO2 and increased Ht compared with SL chickens (Fig. 1). There was no effect of sex on arterial blood gas status either at SL or at HA. Therefore, these data were grouped at each altitude (Fig. 1).

Fig. 1 Arterial blood gas status in sea level (SL) and high altitude (HA) adult chickens. Values are the mean ± s.e.m. for pH (a), pO2 (b), pCO2 (c), O2 haemoglobin saturation (d, SaO2) and haematocrit (e, Ht) in nine males and nine female chickens incubated, hatched and raised at SL (□) and in seven male and seven female chickens incubated, hatched and raised at HA (▪). There was no effect of sex on arterial blood gas status either at SL or at HA. Therefore, these data were grouped at each altitude. Significant differences (P < 0.05) are: *SL v. HA (Two-way ANOVA + Student–Newman–Keuls post-hoc test).

In vivo basal cardiovascular function

Values for SAP, DAP and MAP during basal conditions were significantly lower in HA compared with SL chickens, and these differences were independent of the sex of the animal (Fig. 2a–2c). In contrast, basal HR was significantly higher and basal PI significantly lower in female than in male chickens at SL. Although basal HR and PI were significantly altered in HA males compared with SL males, there was no effect of altitude on basal HR or PI in female chickens (Fig. 2d and 2e). The RPP, an index of myocardial workload and oxygen consumption,Reference Gobel, Norstrom, Nelson, Jorgensen and Wang31 was significantly elevated during basal conditions in female chickens compared with male chickens at SL. Although HA had no effect of RPP in males, values for RPP in HA females were significantly lower compared with SL females (Fig. 2f).

Fig. 2 Basal cardiovascular function in sea level (SL) and high altitude (HA) adult chickens. Values are the mean ± s.e.m. for systolic arterial pressure (a, SAP), diastolic arterial pressure (b, DAP), mean arterial pressure (c, MAP), heart rate (d, HR), pulse interval (e, PI) and the rate-pressure product (f, RPP) in nine males and nine female chickens incubated, hatched and raised at SL (white solid and white hatched bars) and in seven male and seven female chickens incubated, hatched and raised at HA (black solid and black hatched bars). Significant differences (P < 0.05) are: †male v. female, same altitude (hypoxia independent of sex) or *SL v. HA, for same sex (sex independent of hypoxia). There was no interaction between hypoxia and sex (Two-way ANOVA + Student–Newman–Keuls post-hoc test).

In vivo baroreflex function

Independent of sex and altitude, all animals responded with reciprocal changes in HR to increases and decreases in arterial blood pressure induced by the administration of phenylephrine and sodium nitroprusside, respectively. Although values representing the cardiac baroreflex gain were significantly depressed in HA males compared with SL males, baroreflex gain was markedly increased in HA females compared with SL females (Fig. 3).

Fig. 3 Baroreflex function in sea level (SL) and high altitude (HA) adult chickens. Values are the mean ± s.e.m. for cardiac baroreflex function curves (a) and the gain of the cardiac baroreflex (b) in nine males and nine female chickens incubated, hatched and raised at SL and in seven male and seven female chickens incubated, hatched and raised at HA. Groups are sea level males (white circles, white bars), HA males (black circles, black bars), SL females (white squares, white hatched bars) and HA females (black squares, black hatched bars) chickens. Significant differences (P < 0.05) are: †male v. female, same altitude (hypoxia independent of sex) or *SL v. HA, for same sex (sex independent of hypoxia). There was no interaction between hypoxia and sex (Two-way ANOVA + Student–Newman–Keuls post-hoc test). The calculation of the baroreflex gain required two steps. The first step was the calculation of the slope of the heart rate (HR)–blood pressure relationship and the maximum and minimum values for HR for each animal. This was achieved by plotting the minute-by-minute HR and blood pressure responses during the beginning of the infusion period from baseline to plateau. Only the period from baseline to plateau is used to avoid confounding by resetting of the baroreflex. Arterial blood pressure and HR responses to the drugs were fitted to sigmoidal curves as follows: HR = HRmin + ((HRmax − HRmin)/(1 + 10(Mid-point-MAP) × Gain coefficient). The value for gain represents the gain coefficient of the Hill slope that describes the steepness of the curve. The second step was to calculate the baroreflex sensitivity according to McDowall and DampneyReference McDowall and Dampney30 applying the values for gain, maximum and minimum HR to the following equation: Baroreflex Gain = ((HRmax − HRmin) × Gain coefficient)/4, The three points with s.e.m. on the curve represent the summary measures of the analysis: the gain and maximum and minimum HR values.

Biometry

Body weight was significantly lower in females than in males either at SL or HA. Body length [crown–rump length (CRL)] and other longitudinal measurements also tended to be shorter in females than in males at either SL or HA; however, only tibial and meta-tarsal length reached significant differences in SL animals (Table 1). Values for ponderal index were higher in male and female chickens at HA; however, the mean calculation was not significantly different from SL. The head length:CRL ratio was significantly elevated in HA compared with SL male but not female chickens (Table 1). The same trend was observed for the head diameter:body weight ratio, but the difference in males did not reach significance.

Table 1 Body weight and dimensions in SL and HA adult chickens

SL, sea level; HA, high altitude; CRL, crown–rump length; HD, head diameter; HL, head length; BW, body weight.

Values are mean ± s.e.m. for BW, CRL, femur, tibia and meta-tarsal lengths, HD, HL, beak length, ponderal index, the ratio of HL:CRL and of HD:BW in nine males and nine female chickens incubated, hatched and raised at SL and in seven male and seven female chickens incubated, hatched and raised at HA. Significant differences (P < 0.05) are: †male v. female, same altitude or *SL v. HA (Two-way ANOVA + Student–Newman–Keuls post-hoc test).

Discussion

By combining the chick embryo model with incubation at HA, we have previously isolated the effects of developmental hypoxia on fetal growth and on fetal cardiac and aortic wall remodelling.Reference Giussani, Salinas, Villena and Blanco27, Reference Salinas, Blanco and Villena28 Here, we isolate the effects of developmental hypoxia on cardiovascular function at adulthood measured in conscious chickens in vivo, and show that development at HA lowers basal arterial blood pressure and alters baroreflex sensitivity in a sex-dependent manner at adulthood.

Several experimental models of adverse intrauterine conditions, including maternal protein deprivation,Reference Langley and Jackson32–Reference Watkins, Wilkins and Cunningham38 maternal overnutritionReference Langley-Evans, Clamp, Grimble and Jackson39–Reference Elahi, Cagampang and Anthony43 and excess glucocorticoid exposureReference Dodic, Abouantoun, O'Connor, Wintour and Moritz44–Reference Roghair, Wemmie and Volk47 have been reported to programme an increase in basal arterial blood pressure at adulthood. By contrast, there is little information on the effects of developmental hypoxia on the regulation of arterial blood pressure at adulthood, measured in vivo, particularly in the absence of anaesthesia. This is surprising as fetal hypoxia is the most common challenge in complicated pregnancy, such as during preeclampsiaReference Soleymanlou, Jurisica and Nevo48 and placental insufficiency.Reference Baschat49 The present study shows that developmental hypoxia is associated with a fall, rather than an increase, in mean, systolic and diastolic basal arterial blood pressure in adult conscious chickens and that this effect is independent of the sex of the animal. Given that hypotension occurred in the absence of bradycardia during basal conditions, an effect on lowering peripheral vascular resistance of development at HA is favoured. A lack of a programmed hypertensive effect in adult chickens of isobaric hypoxia in ovo has also been reported by Ruijtenbeek et al.Reference Ruijtenbeek, Kessels and Janssen50 Similar findings have been reported in chickReference Lindgren, Crossley, Villamor and Altimiras51 and alligatorReference Crossley and Altimiras52 embryos incubated under chronic isobaric hypoxia, yielding basal hypotension without basal bradycardia, even before hatching. Coney and MarshallReference Coney and Marshall53 reported that adult rats of chronically hypoxic pregnancies had significantly elevated resting femoral blood flow compared with adult rats of normoxic pregnancies, supporting a programmed fall in peripheral vascular resistance contributing to the programmed hypotensive effects of developmental hypoxia. Further, Herrera et al.Reference Herrera, Pulgar and Riquelme54 reported that HA chronic hypoxia during gestation yielded newborn lambs with significantly lower systemic vascular resistance than lowland newborn lambs, although basal systemic arterial blood pressure was not different. The mechanism via which developmental hypoxia may promote a fall in peripheral vascular resistance and a fall in resting blood pressure at adulthood is unclear. However, there is an established relationship between increased plasma Ht, blood viscosity and shear stress-induced increases in nitric oxide (NO) bioavailability.Reference Martini, Carpentier, Negrete, Frangos and Intaglietta55 Interestingly, moderate elevations in blood viscosity by increasing Ht by 10% from baseline could produce reductions in basal blood pressure by 10 mmHg, an effect that could be abolished by treatment with the NO synthase blocker l-N omega-nitro-l-arginine methyl ester (l-NAME) or in NO synthase-deficient mice.Reference Martini, Carpentier and Chávez Negrete56 In the present study, chickens at HA had significantly lower arterial PO2 and haemoglobin oxygen saturation and a persistent increase in Ht of ca. 8%.

Investigation of the effects of adverse intrauterine conditions on baroreflex function in the offspring is scarce relative to reported programmed changes in basal arterial blood pressure. Within the few studies available, it is known that maternal undernutrition can increase the set point and blunt baroreflex responses in the adult offspringReference Pladys, Lahaie and Cambonie57–Reference Gopalakrishnan, Gardner and Rhind59 and that embryonic and fetal exposure to excess glucocorticoids can increase the set point and decrease the sensitivity of the baroreflex in fetal, newborn and adult life.Reference Dodic, Abouantoun, O'Connor, Wintour and Moritz44, Reference Shaltout, Rose and Figueroa46, Reference Fletcher, McGarrigle, Edwards, Fowden and Giussani60–Reference Roghair, Segar and Volk62 By contrast, as with effects on basal arterial blood pressure, there is less information on the effects of developmental hypoxia on baroreflex function. One study has reported that chronic hypoxic pregnancy decreases baroreflex sensitivity in the late-gestation ovine fetusReference Pulgar, Hong and Jessup63 and Peyronnet et al.Reference Peyronnet, Dalmaz and Ehrström64 predicted but did not measure alterations in baroreflex function in rat adult offspring of chronically hypoxic pregnancies.

Gilbert and Nijland,Reference Gilbert and Nijland65 in an elegant and useful review, brought to attention the growing body of evidence reporting sex differences in the developmental programming of alterations in arterial blood pressure and its regulation. Again, most of this evidence comes from studies involving alterations in maternal nutritionReference Fowden, Giussani and Forhead2, Reference Roghair, Segar and Volk62, Reference do Carmo Pinho Franco, Nigro and Fortes66 or from models of maternal stress and glucocorticoid excessReference Roghair, Segar and Volk62, Reference Igosheva, Taylor, Poston and Glover67 than from models of prenatal hypoxia. The weight of the evidence has generally, but not exclusively, suggested that female offspring are less sensitive in manifestation of cardiovascular disease caused by prenatal stimuli.Reference Gilbert and Nijland65 Accordingly, Davidge and colleagues have reported that peripheral vascular function and cardiac hypertrophy is more profoundly affected in male than in female rat offspring of hypoxic pregnancies, particularly as they age.Reference Hemmings, Williams and Davidge68–Reference Morton, Rueda-Clausen and Davidge70 Similarly, prenatal hypoxia caused an increase in heart susceptibility to ischaemia reperfusion injury in male but not in female adult rat offspring.Reference Xue and Zhang71 To our knowledge, only one study has reported sexually dimorphic effects in programming changes in baroreflex function induced by maternal malnutrition or fetal glucocorticoid exposure.Reference Roghair, Segar and Volk62 Certainly, there have been no reports on sex-dependent programming of baroreflex dysfunction by developmental hypoxia. In the present study, we show that while the effects of developmental hypoxia on resting arterial blood pressure at adulthood were sex independent, development at HA had reciprocal effects on the gain of the baroreflex in adult male and female chickens. Baroreflex gain was decreased in adult males, but it was increased in adult females at HA relative to SL controls. A decrease in baroreflex gain may render the male less able to respond to alterations in blood pressure homoeostasis. Further, the rate-pressure product, an index of myocardial workload and oxygen consumption,Reference Gobel, Norstrom, Nelson, Jorgensen and Wang31 although unchanged in males by altitude, was decreased in highland relative to lowland females. Finally, the head:CRL ratio, an index of blood flow redistribution, although unchanged in females by altitude, was increased in highland relative to lowland males. Collectively, therefore, the present data also suggest a greater resilience of the female offspring to development under conditions of HA hypoxia, again adding to the increasing body of evidence showing greater susceptibility to programmed cardiovascular dysfunction in male than female offspring. It is important to acknowledge that there are important differences in sexual differentiation in avian and mammalian species. For instance, in the chicken, males rather than females are homogametic and sex determination is cell autonomous. Therefore, translation of the sexually dimorphic results between species should be considered cautiously.

It has been suggested that it is the mismatch between the pre- and postnatal environment that renders the offspring susceptible to developing disease at adulthood.Reference Gluckman and Hanson72 In the present study, the environmental condition of hypoxia occurs in ovo and persists after hatching. In this context, it is of interest that significant alterations in blood pressure homoeostasis were evident in male and female adult chickens despite matching of the incubation and post-hatching environments. Cardiovascular dysfunction may therefore have developed in response to a double insult; one occurring before and one after hatching. The partial contributions of incubation v. post-hatching HA hypoxia in triggering cardiovascular dysfunction at adulthood await investigation by study of adult offspring raised at SL following HA incubation, and by study of adult offspring raised at HA following SL incubation. However, evidence of similar alterations in blood pressure homoeostasis than those reported here at adulthood in the chronically hypoxic chick embryo even before hatchingReference Lindgren, Crossley, Villamor and Altimiras51 supports a primary effect triggered during the incubation period rather than after hatching.

In conclusion, by combining the chick embryo model with incubation at HA, we have isolated the effects of developmental hypoxia on cardiovascular function at adulthood measured in conscious chickens in vivo, and show that development at HA promotes blood pressure dysregulation, altering baroreflex sensitivity in a sex-dependent manner at adulthood.

Acknowledgements

This work was supported by The British Heart Foundation, The Royal Society and the U-Apoya Program, Universidad de Chile. Emilio A. Herrera is a fellow of Beca Presidente de la Republica (CONICYT) from the Chilean Government. Dino A. Giussani is a Royal Society Wolfson Research Merit Award holder.

References

1.Barker, DJ. In utero programming of chronic disease. Clin Sci (Lond). 1998; 95, 115–128.CrossRefGoogle ScholarPubMed
2.Fowden, AL, Giussani, DA, Forhead, AJ. Intrauterine programming of physiological systems: causes and consequences. Physiology (Bethesda). 2006; 21, 29–37.Google Scholar
3.Gluckman, PD, Hanson, MA, Cooper, C, Thornburg, KL. Effect of in utero and early-life conditions on adult health and disease. N Engl J Med. 2008; 359, 61–73.Google Scholar
4.Martin-Gronert, MS, Ozanne, SE. Mechanisms underlying the developmental origins of disease. Rev Endocr Metab Disord. 2012; 13, 85–92.CrossRefGoogle ScholarPubMed
5.Thompson, LP. Effects of chronic hypoxia on fetal coronary responses. High Alt Med Biol. 2003; 4, 215–224.Google Scholar
6.Hauton, D, Ousley, V. Prenatal hypoxia induces increased cardiac contractility on a background of decreased capillary density. BMC Cardiovasc Disord. 2009; 9, 1.CrossRefGoogle ScholarPubMed
7.Patterson, AJ, Zhang, L. Hypoxia and fetal heart development. Curr Mol Med. 2010; 10, 653–666.Google Scholar
8.Rueda-Clausen, CF, Morton, JS, Davidge, ST. The early origins of cardiovascular health and disease: who, when, and how. Semin Reprod Med. 2011; 29, 197–210.Google Scholar
9.Giussani, DA, Camm, EJ, Niu, Y, et al. Developmental programming of cardiovascular dysfunction by prenatal hypoxia and oxidative stress. PLoS One. 2012; 7, e31017.Google Scholar
10.Li, G, Xiao, Y, Estrella, JL, et al. Effect of fetal hypoxia on heart susceptibility to ischemia and reperfusion injury in the adult rat. J Soc Gynecol Investig. 2003; 10, 265–274.Google Scholar
11.Morton, JS, Rueda-Clausen, CF, Davidge, ST. Mechanisms of endothelium-dependent vasodilation in male and female, young and aged offspring born growth restricted. Am J Physiol Regul Integr Comp Physiol. 2010; 298, R930–R938.CrossRefGoogle Scholar
12.de Grauw, TJ, Myers, RE, Scott, WJ. Fetal growth retardation in rats from different levels of hypoxia. Biol Neonate. 1986; 49, 85–89.Google Scholar
13.Camm, EJ, Hansell, JA, Kane, AD, et al. Partial contributions of developmental hypoxia and undernutrition to prenatal alterations in somatic growth and cardiovascular structure and function. Am J Obstet Gynecol. 2010; 203(5), 495.e24–34.Google Scholar
14.Moore, LG, Charles, SM, Julian, CG. Humans at high altitude: hypoxia and fetal growth. Respir Physiol Neurobiol. 2011; 178, 181–190.Google Scholar
15.Lichty, JA, Ting, RY, Bruns, P, Dyer, E. Studies of babies born at high altitude. 1. Relation of altitude to birth weight. Am J Dis Child. 1957; 93, 666–669.Google Scholar
16.McClung, J. Effects of High Altitude on Human Birth, 1969. Harvard University Press: Cambridge.Google Scholar
17.Haas, JD, Frongillo, EF, Stepcik, C, Beard, J, Hurtado, L. Altitude, ethnic and sex differences in birthweight and length in Bolivia. Hum Biol. 1980; 52, 459–477.Google Scholar
18.Giussani, DA, Phillips, PS, Anstee, S, Barker, DJ. Effects of altitude vs. economic status on birth weight and body shape at birth. Ped Res. 2001; 49, 490–494.Google Scholar
19.Zamudio, S, Postigo, L, Illsley, NP, et al. Maternal oxygen delivery is not related to altitude- and ancestry-associated differences in human fetal growth. J Physiol. 2007; 582(Pt 2), 883–895.Google Scholar
20.Niermeyer, S. Cardiopulmonary transition in the high altitude infant. High Alt Med Biol. 2003; 4, 225–239.Google Scholar
21.Peñaloza, D, Arias-Stella, J, Sime, F, Recavarren, S, Marticorena, E. The heart and pulmonary circulation in children at high altitudes: physiological, anatomical, and clinical observations. Pediatrics. 1964; 34, 568–582.Google Scholar
22.Peñaloza, D, Sime, F, Ruiz, L. Pulmonary hemodynamics in children living at high altitudes. High Alt Med Biol. 2008; 9, 199–207.CrossRefGoogle ScholarPubMed
23.Huicho, L, Niermeyer, S. Cardiopulmonary pathology among children resident at high altitude in Tintaya, Peru: a cross-sectional study. High Alt Med Biol. 2006; 7, 168–179.Google Scholar
24.Ruijtenbeek, K, De Mey, JG, Blanco, CE. The chicken embryo in developmental physiology of the cardiovascular system: a traditional model with new possibilities. Am J Physiol Regul Integr Comp Physiol. 2002; 283, R549–R550.Google Scholar
25.Miller, SL, Green, LR, Peebles, DM, Hanson, MA, Blanco, CE. Effects of chronic hypoxia and protein malnutrition on growth in the developing chick. Am J Obstet Gynecol. 2002; 186, 261–267.Google Scholar
26.Tintu, A, Rouwet, E, Verlohren, S, et al. Hypoxia induces dilated cardiomyopathy in the chick embryo: mechanism, intervention, and long-term consequences. PLoS One. 2009; 4, e5155.CrossRefGoogle ScholarPubMed
27.Giussani, DA, Salinas, CE, Villena, M, Blanco, CE. The role of oxygen in prenatal growth: studies in the chick embryo. J Physiol. 2007; 585(Pt 3), 911–917.Google Scholar
28.Salinas, CE, Blanco, CE, Villena, M, et al. Developmental origin of cardiac and vascular disease in chick embryos incubated at high attitude. J DOHaD. 2010; 1, 60–66.Google Scholar
29.Gilbert, JS, Nijland, MJ. Sex differences in the developmental origins of hypertension and cardiorenal disease. Am J Physiol Regul Integr Comp Physiol. 2008; 295, R1941–R1952.Google Scholar
30.McDowall, LM, Dampney, RA. Calculation of threshold and saturation points of sigmoidal baroreflex function curves. Am J Physiol Heart Circ Physiol. 2006; 291, H2003–H2007.CrossRefGoogle ScholarPubMed
31.Gobel, FL, Norstrom, LA, Nelson, RR, Jorgensen, CR, Wang, Y. The rate-pressure product as an index of myocardial oxygen consumption during exercise in patients with angina pectoris. Circulation. 1978; 57, 549–556.Google Scholar
32.Langley, SC, Jackson, AA. Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets. Clin Sci (Lond). 1994; 86, 217–222.Google Scholar
33.Woodall, SM, Johnston, BM, Breier, BH, Gluckman, PD. Chronic maternal undernutrition in the rat leads to delayed postnatal growth and elevated blood pressure of offspring. Pediatr Res. 1996; 40, 438–443.Google Scholar
34.Gardner, DS, Jackson, AA, Langley-Evans, SC. Maintenance of maternal diet-induced hypertension in the rat is dependent on glucocorticoids. Hypertension. 1997; 30, 1525–1530.CrossRefGoogle ScholarPubMed
35.Manning, J, Vehaskari, VM. Low birth weight-associated adult hypertension in the rat. Pediatr Nephrol. 2001; 16, 417–422.Google Scholar
36.Gilbert, JS, Lang, AL, Grant, AR, Nijland, MJ. Maternal nutrient restriction in sheep: hypertension and decreased nephron number in offspring at 9 months of age. J Physiol. 2005; 565(Pt 1), 137–147.Google Scholar
37.Cambonie, G, Comte, B, Yzydorczyk, C, et al. Antenatal antioxidant prevents adult hypertension, vascular dysfunction, and microvascular rarefaction associated with in utero exposure to a low-protein diet. Am J Physiol Regul Integr Comp Physiol. 2007; 292, R1236–R1245.Google Scholar
38.Watkins, AJ, Wilkins, A, Cunningham, C, et al. Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring. J Physiol. 2008; 586, 2231–2244.Google Scholar
39.Langley-Evans, SC, Clamp, AG, Grimble, RF, Jackson, AA. Influence of dietary fats upon systolic blood pressure in the rat. Int J Food Sci Nutr. 1996; 47, 417–425.Google Scholar
40.Langley-Evans, SC. Intrauterine programming of hypertension in the rat: nutrient interactions. Comp Biochem Physiol A Physiol. 1996; 114, 327–333.Google Scholar
41.Khan, IY, Taylor, PD, Dekou, V, et al. Gender-linked hypertension in offspring of lard-fed pregnant rats. Hypertension. 2003; 41, 168–175.CrossRefGoogle ScholarPubMed
42.Armitage, JA, Lakasing, L, Taylor, PD, et al. Developmental programming of aortic and renal structure in offspring of rats fed fat-rich diets in pregnancy. J Physiol. 2005; 565(Pt 1), 171–184.Google Scholar
43.Elahi, MM, Cagampang, FR, Anthony, FW, et al. Statin treatment in hypercholesterolemic pregnant mice reduces cardiovascular risk factors in their offspring. Hypertension. 2008; 51, 939–944.Google Scholar
44.Dodic, M, Abouantoun, T, O'Connor, A, Wintour, EM, Moritz, KM. Programming effects of short prenatal exposure to dexamethasone in sheep. Hypertension. 2002; 40, 729–734.Google Scholar
45.Ortiz, LA, Quan, A, Zarzar, F, Weinberg, A, Baum, M. Prenatal dexamethasone programs hypertension and renal injury in the rat. Hypertension. 2003; 41, 328–334.Google Scholar
46.Shaltout, HA, Rose, JC, Figueroa, JP, et al. Acute AT(1)-receptor blockade reverses the hemodynamic and baroreflex impairment in adult sheep exposed to antenatal betamethasone. Am J Physiol Heart Circ Physiol. 2010; 299, H541–H547.CrossRefGoogle ScholarPubMed
47.Roghair, RD, Wemmie, JA, Volk, KA, et al. Maternal antioxidant blocks programmed cardiovascular and behavioural stress responses in adult mice. Clin Sci (Lond). 2011; 121, 427–436.Google Scholar
48.Soleymanlou, N, Jurisica, I, Nevo, O, et al. Molecular evidence of placental hypoxia in preeclampsia. J Clin Endocrinol Metab. 2005; 90, 4299–4308.Google Scholar
49.Baschat, AA. Fetal responses to placental insufficiency: an update. BJOG. 2004; 111, 1031–1041.CrossRefGoogle ScholarPubMed
50.Ruijtenbeek, K, Kessels, CG, Janssen, BJ, et al. Chronic moderate hypoxia during in ovo development alters arterial reactivity in chickens. Pflugers Arch. 2003; 447, 158–167.Google Scholar
51.Lindgren, I, Crossley, D II, Villamor, E, Altimiras, J. Hypotension in the chronically hypoxic chicken embryo is related to the β-adrenergic response of chorioallantoic and femoral arteries and not to bradycardia. Am J Physiol Regul Integr Comp Physiol. 2011; 301, R1161–R1168.Google Scholar
52.Crossley, DA II, Altimiras, J. Cardiovascular development in embryos of the American alligator Alligator mississippiensis: effects of chronic and acute hypoxia. J Exp Biol. 2005; 208(Pt 1), 31–39.Google Scholar
53.Coney, AM, Marshall, JM. Effects of maternal hypoxia on muscle vasodilatation evoked by acute systemic hypoxia in adult rat offspring: changed roles of adenosine and A1 receptors. J Physiol. 2010; 588(Pt 24), 5115–5125.Google Scholar
54.Herrera, EA, Pulgar, VM, Riquelme, RA, et al. High-altitude chronic hypoxia during gestation and after birth modifies cardiovascular responses in newborn sheep. Am J Physiol Regul Integr Comp Physiol. 2007; 292, R2234–R2240.Google Scholar
55.Martini, J, Carpentier, B, Negrete, AC, Frangos, JA, Intaglietta, M. Paradoxical hypotension following increased hematocrit and blood viscosity. Am J Physiol Heart Circ Physiol. 2005; 289, H2136–H2143.Google Scholar
56.Martini, J, Carpentier, B, Chávez Negrete, A, et al. Beneficial effects due to increasing blood and plasma viscosity. Clin Hemorheol Microcirc. 2006; 35, 51–57.Google ScholarPubMed
57.Pladys, P, Lahaie, I, Cambonie, G, et al. Role of brain and peripheral angiotensin II in hypertension and altered arterial baroreflex programmed during fetal life in rat. Pediatr Res. 2004; 55, 1042–1049.Google Scholar
58.Gardner, DS, Pearce, S, Dandrea, J, et al. Peri-implantation undernutrition programs blunted angiotensin II evoked baroreflex responses in young adult sheep. Hypertension. 2004; 43, 1290–1296.Google Scholar
59.Gopalakrishnan, GS, Gardner, DS, Rhind, SM, et al. Programming of adult cardiovascular function after early maternal undernutrition in sheep. Am J Physiol Regul Integr Comp Physiol. 2004; 287, R12–R20.CrossRefGoogle ScholarPubMed
60.Fletcher, AJ, McGarrigle, HH, Edwards, CM, Fowden, AL, Giussani, DA. Effects of low dose dexamethasone treatment on basal cardiovascular and endocrine function in fetal sheep during late gestation. J Physiol. 2002; 545(Pt 2), 649–660.Google Scholar
61.Segar, JL, Roghair, RD, Segar, EM, et al. Early gestation dexamethasone alters baroreflex and vascular responses in newborn lambs before hypertension. Am J Physiol Regul Integr Comp Physiol. 2006; 291, R481–R488.CrossRefGoogle ScholarPubMed
62.Roghair, RD, Segar, JL, Volk, KA, et al. Vascular nitric oxide and superoxide anion contribute to sex-specific programmed cardiovascular physiology in mice. Am J Physiol Regul Integr Comp Physiol. 2009; 296, R651–R662.Google Scholar
63.Pulgar, VM, Hong, JK, Jessup, JA, et al. Mild chronic hypoxemia modifies expression of brain stem angiotensin peptide receptors and reflex responses in fetal sheep. Am J Physiol Regul Integr Comp Physiol. 2009; 297, R446–R452.Google Scholar
64.Peyronnet, J, Dalmaz, Y, Ehrström, M, et al. Long-lasting adverse effects of prenatal hypoxia on developing autonomic nervous system and cardiovascular parameters in rats. Pflugers Arch. 2002; 443, 858–865.Google Scholar
65.Gilbert, JS, Nijland, MJ. Sex differences in the developmental origins of hypertension and cardiorenal disease. Am J Physiol Regul Integr Comp Physiol. 2008; 295, R1941–R1952.CrossRefGoogle ScholarPubMed
66.do Carmo Pinho Franco, M, Nigro, D, Fortes, ZB, et al. Intrauterine undernutrition – renal and vascular origin of hypertension. Cardiovasc Res. 2003; 60, 228–234.Google Scholar
67.Igosheva, N, Taylor, PD, Poston, L, Glover, V. Prenatal stress in the rat results in increased blood pressure responsiveness to stress and enhanced arterial reactivity to neuropeptide Y in adulthood. J Physiol. 2007; 582(Pt 2), 665–674.Google Scholar
68.Hemmings, DG, Williams, SJ, Davidge, ST. Increased myogenic tone in 7-month-old adult male but not female offspring from rat dams exposed to hypoxia during pregnancy. Am J Physiol Heart Circ Physiol. 2005; 289, H674–H682.Google Scholar
69.Rueda-Clausen, CF, Morton, JS, Davidge, ST. Effects of hypoxia-induced intrauterine growth restriction on cardiopulmonary structure and function during adulthood. Cardiovasc Res. 2009; 81, 713–722.Google Scholar
70.Morton, JS, Rueda-Clausen, CF, Davidge, ST. Flow-mediated vasodilation is impaired in adult rat offspring exposed to prenatal hypoxia. J Appl Physiol. 2011; 110, 1073–1082.Google Scholar
71.Xue, Q, Zhang, L. Prenatal hypoxia causes a sex-dependent increase in heart susceptibility to ischemia and reperfusion injury in adult male offspring: role of protein kinase C epsilon. J Pharmacol Exp Ther. 2009; 330, 624–632.Google Scholar
72.Gluckman, PD, Hanson, MA. Mismatch: the lifestyle diseases timebomb. Oxford University Press. 2006; 6, 137–211.Google Scholar
Figure 0

Fig. 1 Arterial blood gas status in sea level (SL) and high altitude (HA) adult chickens. Values are the mean ± s.e.m. for pH (a), pO2 (b), pCO2 (c), O2 haemoglobin saturation (d, SaO2) and haematocrit (e, Ht) in nine males and nine female chickens incubated, hatched and raised at SL (□) and in seven male and seven female chickens incubated, hatched and raised at HA (▪). There was no effect of sex on arterial blood gas status either at SL or at HA. Therefore, these data were grouped at each altitude. Significant differences (P < 0.05) are: *SL v. HA (Two-way ANOVA + Student–Newman–Keuls post-hoc test).

Figure 1

Fig. 2 Basal cardiovascular function in sea level (SL) and high altitude (HA) adult chickens. Values are the mean ± s.e.m. for systolic arterial pressure (a, SAP), diastolic arterial pressure (b, DAP), mean arterial pressure (c, MAP), heart rate (d, HR), pulse interval (e, PI) and the rate-pressure product (f, RPP) in nine males and nine female chickens incubated, hatched and raised at SL (white solid and white hatched bars) and in seven male and seven female chickens incubated, hatched and raised at HA (black solid and black hatched bars). Significant differences (P < 0.05) are: †male v. female, same altitude (hypoxia independent of sex) or *SL v. HA, for same sex (sex independent of hypoxia). There was no interaction between hypoxia and sex (Two-way ANOVA + Student–Newman–Keuls post-hoc test).

Figure 2

Fig. 3 Baroreflex function in sea level (SL) and high altitude (HA) adult chickens. Values are the mean ± s.e.m. for cardiac baroreflex function curves (a) and the gain of the cardiac baroreflex (b) in nine males and nine female chickens incubated, hatched and raised at SL and in seven male and seven female chickens incubated, hatched and raised at HA. Groups are sea level males (white circles, white bars), HA males (black circles, black bars), SL females (white squares, white hatched bars) and HA females (black squares, black hatched bars) chickens. Significant differences (P < 0.05) are: †male v. female, same altitude (hypoxia independent of sex) or *SL v. HA, for same sex (sex independent of hypoxia). There was no interaction between hypoxia and sex (Two-way ANOVA + Student–Newman–Keuls post-hoc test). The calculation of the baroreflex gain required two steps. The first step was the calculation of the slope of the heart rate (HR)–blood pressure relationship and the maximum and minimum values for HR for each animal. This was achieved by plotting the minute-by-minute HR and blood pressure responses during the beginning of the infusion period from baseline to plateau. Only the period from baseline to plateau is used to avoid confounding by resetting of the baroreflex. Arterial blood pressure and HR responses to the drugs were fitted to sigmoidal curves as follows: HR = HRmin + ((HRmax − HRmin)/(1 + 10(Mid-point-MAP) × Gain coefficient). The value for gain represents the gain coefficient of the Hill slope that describes the steepness of the curve. The second step was to calculate the baroreflex sensitivity according to McDowall and Dampney30 applying the values for gain, maximum and minimum HR to the following equation: Baroreflex Gain = ((HRmax − HRmin) × Gain coefficient)/4, The three points with s.e.m. on the curve represent the summary measures of the analysis: the gain and maximum and minimum HR values.

Figure 3

Table 1 Body weight and dimensions in SL and HA adult chickens