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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY
1Laboratorio de Fisiología y Fisiopatología del Desarrollo, Programa de Fisiopatología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago; 2Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago; 3International Center for Andean Studies, Universidad de Chile, Santiago-Arica-Putre, Chile; 4Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, California; 5Facultad de Medicina, Pontificia Universidad Católica Madre y Maestra, Santiago de los Caballeros, República Dominicana; 6Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom; 7Department of Pediatrics, Academic Hospital Maastricht, Maastricht, The Netherlands; 8Centre for Developmental Origins of Health and Disease, University of Southampton, Southampton, United Kingdom; and 9Universidad de Tarapacá and Centro de Investigaciones del Hombre en el Desierto, Arica, Chile
Submitted 29 December 2006 ; accepted in final form 10 February 2007
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ABSTRACT |
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hypoxemia; pulmonary hypertension; vascular reactivity; neonatal lamb; highlands
In newborns, either at altitude or sea level, pulmonary hypertension is due to a failure to regulate PVR at birth, leading to hypoxemia and sustained pulmonary hypertension. One of the major factors associated with persistent pulmonary hypertension in the newborn is chronic hypoxia in utero (1). In humans, this syndrome occurs at a rate of 1 to 2 per 1,000 live births at low altitude, and is probably much higher at high altitude (15). As an example of how serious this condition can be, the pulmonary arterial pressure (PAP) of a group of newborns in Perú at 4,540 m above sea level was close to the systemic arterial pressure (SAP) (7).
The mechanisms resulting in pulmonary hypertension at high altitude have not yet been fully elucidated. However, it has been suggested that during chronic hypoxia in newborns, production of vasocontrictor factors within the lung is enhanced, while synthesis of vasodilators may be reduced (1, 6, 31).
We hypothesized that newborn sheep gestated and born at high altitude would have a higher basal PAP and an increased pulmonary vascular response to a superimposed episode of acute hypoxemia. In addition, these animals would show an enhanced systemic vascular response to acute hypoxia relative to lowland born species.
To test the hypothesis, we investigated basal pulmonary and systemic cardiorespiratory function and the response to a period of superimposed hypoxia in newborn lambs gestated and born either at sea level or at altitude (3,600 m). To determine the mechanisms underlying changes in cardiorespiratory physiology, we assessed the sensitivity of the pulmonary vasculature to several degrees of oxygenation and determined vasoconstrictor and vasodilator responses in isolated small pulmonary and femoral arteries.
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METHODS |
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All experimental protocols were reviewed and approved by the Faculty of Medicine Ethics Committee of the University of Chile. Animal care, maintenance, procedures, and experimentation were performed in accordance with the American Physiological Society Guiding Principles for Research Involving Animals and Human Beings (2).
Fourteen newborn sheep born and raised at the University of Chile farm, Santiago, 580 m above sea level [Lowland newborn (LLNB), mean weight of 7.0 ± 0.4 kg; 7–12 days of age] and 10 newborn sheep, born and raised at Putre Research Station, International Center for Andean Studies (INCAS), University of Chile, 3,600 m above sea level [Highland newborn (HLNB), mean weight of 5.6 ± 0.4 kg; P < 0.05, compared with LLNB; 8–12 days of age] were studied. The highland sheep had been at altitude for at least 50 generations. The newborns and ewes were housed in an open yard with access to food and water ad libitum. Similar but uninstrumented animals (LLNB: n = 6, HLNB: n = 7) were used for the collection of small arteries for ex vivo experiments.
Surgical Preparation
The lambs were premedicated with atropine (0.04 mg/kg im; Atropina Sulfato; Laboratorio Chile, Santiago, Chile). All surgical procedures were performed under general anesthesia with ketamine, 10 mg/kg im (Ketostop; Drag Pharma-Invectec, Santiago, Chile) and diazepam 0.1–0.5 mg/kg im (Laboratorio Biosano, Santiago, Chile) with additional local infiltration of 2% lidocaine (Dimecaína; Laboratorio Beta, Santiago, Chile). Polyvinyl catheters (1.2 mm ID) were placed in the descending aorta and inferior vena cava via a hindlimb artery and vein. In the contralateral femoral artery, a Transonic blood flow transducer (Transonic Systems, Ithaca, NY) was installed. The polyvinyl catheters and the flow probe cable were exteriorized percutaneously through the animal's flank and kept in a pouch sewn onto the skin. In addition, an Edwards Swan-Ganz catheter (5 French; Baxter Healthcare, Irvine, CA) was inserted into the pulmonary artery via an external jugular vein, exteriorized, and placed in a pouch around the neck of the animal. All vascular catheters were filled with a heparinized solution of 0.9% NaCl (500 IU heparin/ml 0.9% NaCl) and plugged with a copper pin. Ampicillin 10 mg/kg iv (Ampicilina, Laboratorio Best-Pharma, Santiago, Chile) and gentamicin 4 mg/kg iv (Gentamicina Sulfato, Laboratorio Biosano), were administered every 12 h while the animals were instrumented. The experiments commenced 3 days after surgery.
Experimental Protocols
In vivo experiments.
ACUTE HYPOXIA.
All experiments were based on a 3-h protocol divided into three periods: 60 min of basal (breathing room air), 60 min of hypoxemia, and 60 min of recovery. To induce hypoxemia, a transparent loosely tied polyethylene bag was placed over the animal's head into which a controlled mixture of air, N2, and CO2 (
10% O2 and 2–3% CO2 in N2) was passed at 20 l/min. The gas mixture reduced arterial PO2 to 30 mmHg. After the 60 min of hypoxemia, the animal was returned to breathing air for a further 60 min (recovery).
Arterial blood samples (0.3 ml) were taken in heparinized syringes at 15 and 45 min of normoxemia, each 15 min during the hypoxemic hour, and after 15 and 45 min of recovery. Arterial pH, PO2, PCO2 (model ABL 555 blood gas monitor; Radiometer, Copenhagen, Denmark; measurements corrected to 39°C), hemoglobin concentration, percentage saturation of hemoglobin (SaO2), and oxygen content using an animal (ovine) program option (model OSM3 hemoximeter; Radiometer) were calculated. SAP, PAP, and right atrial pressure were measured continuously using pressure transducers and recorded by a data acquisition system (Powerlab/8SP System and Chart v4.1.2 Software; ADInstruments, New South Wales, Australia) connected to a personal computer. Heart rate and mean systemic arterial blood pressure (MAP) were obtained from this record. In addition, femoral blood flow (FBF) was also measured continuously and recorded by the data acquisition system connected to a personal computer.
Cardiac output was determined just after the blood sampling by the thermodilution method as the average of three determinations after injection of 3 ml of chilled (0°C) NaCl 0.9% into the pulmonary artery (model COM-2 cardiac output computer, Baxter, Irvine, CA).
Systemic vascular resistance (SVR), PVR, total oxygen consumption (
O2), and total oxygen extraction were calculated by using the following equations
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GRADED HYPOXIA.
To evaluate the PO2 sensitivity of the pulmonary vascular bed, a graded hypoxia protocol was performed, during which the aortic PO2 (PaO2) was reduced in steps of 10 mmHg (PaO2) in lowland newborns. In highland newborns, an oxygenated mixture was given to increase the PaO2 in 10 mmHg steps, until reaching values equivalent to those at sea level. In each experimental step, pH and blood gases, PAP, and SAP were registered.
Ex vivo experiments.
SMALL ARTERY ISOLATION.
Newborn sheep were euthanized with an overdose of sodium thiopenthone 100 mg/kg iv. (Tiopental; Laboratorio Biosano, Santiago, Chile). The left lung and a hindlimb were removed by dissection and immediately immersed in cold saline. Fourth generation pulmonary arteries (counting from the pulmonary artery trunk, internal diameter: LLNB, 410 ± 20 µm; HLNB, 454 ± 20 µm) and third generation femoral arteries (counting from the main femoral artery, internal diameter: LLNB, 406 ± 31 µm; HLNB, 388 ± 15 µm) were dissected from each vascular bed.
MEASUREMENT OF ARTERIAL REACTIVITY.
Isolated arteries were mounted between an isometric force transducer (model DSC 6; Kistler Morce, Seattle, WA) and a displacement device in a myograph (dual-wire myograph; Danish Myo Technologies, Aarhus, Denmark) using two stainless steel wires (diameter: 40 µm). During mounting and experimentation, the myograph organ bath was filled with 10 ml Krebs-Henseleit buffer maintained at 39°C and aerated with 95% O25% CO2. Each artery was stretched to its individual optimal lumen diameter, i.e., the diameter at which it developed the strongest contractile response to 125 mM K+, using a diameter-tension protocol as previously described for pulmonary and femoral arteries (28, 36, 37).
Contractile agonists were evaluated under basal tone. A concentration-response curve was constructed for potassium chloride (KCl) by exposing the arteries to 11 different concentrations of KCl (4.75–125 mM) with each dose maintained for 2 min, and the segment washed with Krebs-Henseleit buffer before the next concentration was introduced. Cumulative concentration-response curve for noradrenaline (10–10–10–3 M) and phenylephrine (10–10–10–3 M) were constructed by increasing the organ chamber concentration of the drug incrementally after a steady-state response had been measured. Concentration-response curves for the nitric oxide donor sodium nitroprusside (10–8 M–10–4 M) were constructed during contraction induced by 125 mM K+.
SOLUTIONS AND DRUGS.
Krebs-Henseleit buffer contained (in mM) 118.5 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, and 5.5 glucose with a pH of 7.4. In 125 mM K+ buffer, all of the NaCl was replaced by an equimolar amount of KCl. Noradrenaline and phenylephrine were obtained from Sigma (St Louis, MO) and sodium nitroprusside from Prolabo (Paris, France).
Data Analysis
For in vivo experiments all values were expressed as means ± SE. Statistical analysis was performed using a two-way analysis of variance. Differences between means were assessed using the Newman-Keuls test. Significance was accepted when P < 0.05 (12).
For ex vivo experiments dose-response curves were analyzed in terms of sensitivity and maximal response (Emax) by fitting experimental data to a sigmoid equation (Origin version 5.0; MicroCal Software, MA). Contractile responses were expressed in terms of tension [force in miliNewton divided by length of the arterial segment (in mN/mm)] or as percentage of maximal response to KCl (%Emax). Relaxant responses were expressed as a percentage of reduction of 125 mM K+-induced contraction. Sensitivity was calculated as pD2, where pD2 = –log [EC50], EC50 being the concentration at which 50% of the maximal response was obtained. Data are shown as means ± SE. Differences between mean values were assessed by Student's t-test and were considered significant if P < 0.05.
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RESULTS |
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During the basal period PaO2, PaCO2, SaO2, and O2 content were lower in HLNB than in the LLNB, but pH was higher. Moreover, O2 and extraction were also augmented in HLNB (Table 1). During acute hypoxia, significant falls in PaO2, SaO2, and O2 content occurred in all animals. In addition, the PaCO2 was maintained at similar values in both groups (isocapnic hypoxia). O2 was also maintained, associated in both groups with increased oxygen extraction (Table 1). During recovery, the altered variables returned to basal values in both groups, except for the PaCO2 in LLNB and pH in HLNB, which remained decreased in this period (Table 1).
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Pulmonary Cardiovascular Variables
During the basal period, PAP, cardiac output, PVR, and heart rate were significantly higher in the HLNB than in the LLNB (Fig. 1). During acute hypoxemia, there was a brisk and maintained increase of PAP, reaching higher values in HLNB compared with the LLNB (Fig. 1). Cardiac output did not change significantly in hypoxemia, although PVR increased in both groups of animals, but PVR reached higher values in HLNB than LLNB (Fig. 1). Heart rate increased only significantly in LLNB during hypoxia (Fig. 1).
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The SAP was similar in LLNB and HLNB. As noted above, cardiac output was higher in the HLNB, and the SVR was also lower in HLNB (Fig. 3).
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During recovery, all the cardiovascular variables returned to normoxemic levels in both groups (Figs. 1 and 3). The SAP and SVR values did not change during the graded hypoxia (data not shown).
Contractile Responses of Small Pulmonary Arteries
Response to potassium chloride. The contractile responses of small pulmonary arteries to potassium chloride from HLNB showed a higher maximal response than LLNB (P < 0.05) with similar sensitivity between both groups (Fig. 4A, Table 2).
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Adrenergic response. The contractile responses of small pulmonary arteries to noradrenaline showed no significant differences between the groups in maximal response or sensitivity (Fig. 4C, Table 2). However, the response to phenylephrine showed a lower maximal response in HLNB with similar sensitivity compared with LLNB (Fig. 4D, Table 2).
Contractile Responses of Small Femoral Arteries
Response to potassium chloride. Isolated small femoral arteries showed a lower maximal response in HLNB than in LLNB, but a similar sensitivity (Fig. 5A, Table 2).
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Adrenergic response. The response to noradrenaline showed a significant increase in maximal response and sensitivity in HLNB compared with LLNB. In addition, the response to phenylephrine in small femoral vessels also showed a higher maximal response and sensitivity in HLNB compared with LLNB (Fig. 5, C–D, Table 2).
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DISCUSSION |
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Highland lambs are lighter compared with lowlanders at the same age, showing intrauterine growth restriction (11). This intrauterine growth reduction may also be an adaptation to the effects of chronic hypoxia and it appears to be independent of nutrition since the highland and lowland pregnant ewes were maintained with the same food intake per day.
At high altitude, the HLNB showed a higher O2, while having lower oxygen content under basal conditions. We propose that this higher O2 in HLNB may result from the higher respiratory effort of hyperventilation at high altitude. In fact, when the acute hypoxic episode was induced, the O2 reached similar levels in both groups of lambs. This shows that these lambs have a physiological adaptation, which allows the maintenance of O2 even under acute hypoxia (24).
Pulmonary Vascular Bed
A variety of investigations have found a reduced (17, 18, 25, 39), no change, or enhanced (4, 6, 31, 39) in pulmonary hypoxic response after long-term exposure to low PO2. Here, we found that the pulmonary vasoconstriction under basal and hypoxic conditions is enhanced in newborns that have developed in utero and been born in the chronic hypoxia of the Andean altiplano.
The basal higher PAP in HLNB than in LLNB may be explained by our findings that pulmonary small arteries show a greater maximal response to KCl, with the same sensitivity, suggesting a greater vascular smooth-muscle mass in HLNB (23, 33). Chronic hypoxia induces overexpression of vasoconstrictors in pulmonary tissue, as well smooth muscle cell mechanisms that could be acting in the regulation of the pulmonary vascular tone producing the basal increase in PAP in our HLNB (5, 8, 21, 33). Besides, we observed a higher relaxant effect of exogenous nitric oxide in isolated pulmonary arteries, indicating a higher expression and/or function of the nitric oxide-cGMP-PKG transduction pathway located in the arterial smooth muscle.
Acute hypoxia increases PAP, PVR, and heart rate, in LLNB and HLNB, but with a lower increment in HLNB compared with their basal period. This is explained by the different basal PO2 of each group. Furthermore, as we induce an isocapnic hypoxemia, HLNB showed a higher PAP in acute hypoxemia at a lower PCO2 and higher pH compared with LLNB. Therefore, we would expect a higher pulmonary vascular tone and arterial pressure at same PCO2 and pH at the highlands. To evaluate the pulmonary vascular response at a specific PO2, we designed a graded hypoxia/oxygenation experiment. This study revealed that in the pulmonary bed there is indeed a greater degree of contraction in HLNB. This higher PAP in HLNB at the same PO2 could be the result of vascular remodeling with more muscularization of the pulmonary vessels (32).
The lower maximal response to phenylephrine observed in the isolated pulmonary arteries in HLNB agrees with the minor role of catecholamines in the pulmonary circulation (29), particularly in hypoxic newborns (30).
Systemic Cardiovascular Responses
In the femoral vascular bed, HLNB presents a higher FVR than LLNB during hipoxia. Moreover, the initial response to hypoxia was a brisk vasoconstriction in HLNB, not seen in LLNB. This marked vasoconstriction may represent persistence of a chemoreflex in origin followed by catecholamine action on the femoral vessels, as seen in fetal lambs (9, 16). The higher sensitivity and maximal response observed for noradrenaline and phenylephrine in HLNB suggests that adrenergic responses are playing a major role in the peripheral circulation in high-altitude-exposed animals, as is also observed in animals chronically exposed to hypoxia, such as the llama (10, 16), chronically hypoxic chicken embryos (27), and rats gestated under hypoxia (38). A higher response to sodium nitroprusside may indicate a major role for nitric oxide in the systemic circulation of high-altitude-exposed animals, to counteract the intense femoral vasoconstriction during hypoxia in HLNB.
In conclusion, the chronic hypoxia during pregnancy and early postnatal life at high altitude enhances the vascular contractile responses in the pulmonary and femoral beds, during basal conditions and during a superimposed hypoxic challenge. These changes determined in intrauterine life and early after birth at altitude could be adaptive to the chronic hypoxic milieu of the Andean altiplano.
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GRANTS |
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ACKNOWLEDGMENTS |
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Current address of V. Pulgar: Department of Obstetrics and Gynecology, Wake Forest University, Winston Salem, NC 27157.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* E. A. Herrera and V. M. Pulgar contributed equally to this work. 
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P < 0.05 for sensitivity tested by two-sample t-test HLNB vs. LLNB.