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Ann Thorac Surg 1995;59:1155-1161
© 1995 The Society of Thoracic Surgeons

Endothelial Dysfunction in Venous Pulmonary Hypertension in the Neonatal Piglet

Laboratoire de Chirurgie Expérimentale and Centre National de Recherche Scientifique, Unité de Recherche Associée 1159, Marie Lannelongue Hospital, Le Plessis-Robinson, France

Accepted for publication January 25, 1995.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
In a group of neonatal piglets an increase in pulmonary arterial pressure was obtained within 2 weeks after a partial mechanical obstruction of the left atrium by a balloon catheter. Mean pulmonary artery pressure in the hypertensive animals (n = 6) was 24 ± 2 mm Hg as compared (p < 0.01) with 15 ± 1 mm Hg in controls (n = 6) or 9 ± 2 mm Hg in sham-operated piglets (n = 6). Cardiac index was reduced in hypertensive versus control and sham groups: 0.15 ± 0.01 versus 0.32 ± 0.05 and 0.29 ± 0.04 L • min^-1 • kg^-1 (p < 0.05), respectively. There was no detectable difference on histologic examination in the pulmonary arteries between the three groups. Right ventricular hypertrophy was observed in the group with pulmonary hypertension. In hypertensive piglets, isolated conduit pulmonary arteries did not relax when stimulated with acetylcholine; they always relaxed to sodium nitroprusside. These data suggest that the first stages of perturbations reported during pulmonary venous hypertension occur at the level of the pulmonary vascular endothelium. This neonatal model of pulmonary hypertension is simple to perform and might be useful for further investigations.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Pulmonary hypertension (PH) is an important source of morbidity after open heart operations for congenital heart diseases [1]. Extensive experimental [2–4] and clinical research [5, 6] have demonstrated endothelial dysfunction in these patients both preoperatively and postoperatively, even after repair. Current strategies to avoid pulmonary complications after intracardiac repair involve early repair during the first weeks of life [7, 8] and postoperative pharmacologic modulation of the pulmonary vascular tone by profound sedation and adequate ventilation [9], and more recently by the use of nitric oxide inhalation [6, 10]. Despite drastic improvements in the postoperative course when these approaches are adopted, some patients still have a stormy postoperative course with PH crises and low cardiac output [10], and eventually death. Therefore, research in modeling of such heart defects is mandatory to obtain a better understanding of the general and cellular mechanisms underlying these perturbations.

Several experimental models of PH have been described, but few studies have centered on the development of a neonatal model. Silov and associates [11] in 1972 reported a model of PH created by coarcting the pulmonary veins of neonatal calves. Labourène and co-workers [12] described a similar model of progressive pulmonary venous obstruction in the neonatal piglet. These investigators used a two-stage procedure by placing bands around the pulmonary veins first on one side and 1 week later on the other side. The bands were not tightened at the onset of the procedure, and venous constriction occurred only with the growth of the piglet. More recently, some interesting models of neonatal PH by intrauterine manipulations emerged [13, 14] that may contribute to a more thorough understanding of this pathology. In each of these studies, different topics were investigated, and each study brought new insights in the pathophysiology of this condition.

The normal transition from fetal to neonatal circulation is associated with a drastic increase in the pulmonary flow and a decrease in pulmonary vascular resistances and vascular and alveolar recruitment [15]. The mechanisms for these changes have been investigated in detail but still remain unclear [16]. Since the work of Furchgott and Zawadzki [17], particular attention has been oriented toward the endothelial function in several pathologic states. In response to stimuli, the endothelial cells release endothelial-derived relaxing factor, which has been identified as nitric oxide [18] and induces vascular smooth muscle relaxation. Endothelial-derived relaxing factor directly activates soluble guanylate cyclase of vascular smooth muscle causing an increase in guanosine 3`, 5` cyclic monophosphate and thereby causing relaxation of the vascular smooth muscle. As expected, the pulmonary vascular endothelium in models [4] or in patients [5, 6, 19] with PH presents different levels of dysfunction resulting in an impairment of the pulmonary vascular tone. Recent studies have shown that in the fetal circulation, basal release of endothelial-derived relaxing factor–nitric oxide by the pulmonary endothelial cells was present. However, the stimulated release of endothelial-derived relaxing factor–nitric oxide in response to acetylcholine was reduced markedly [20] in the fetal circulation and increased rapidly during the first week of life [21]. In presence of congenital heart defects with PH, whether endothelial-derived relaxing factor activity is or is not blunted at birth is under debate.

To investigate the interaction between high pulmonary arterial pressure and endothelial dysfunction, we developed a model of venous PH in the neonatal piglet. The aim of this study was to examine whether a mechanical perturbation that induced increased pulmonary arterial pressure would alter endothelium-dependent vascular responses in conduit pulmonary arteries before inducing direct morphologic or functional vascular smooth muscle modifications.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
All surgical procedures and experimental protocols were performed in compliance with the guidelines formulated by the Institute of Laboratory Animal Resources and published by the National Institute of Health in the ``Guide for the Care and Use of Laboratory Animals'' (NIH publication 85-23, revised 1985). The animals were fed with diluted milk and bread ad libitum, a regimen that allowed for normal dietary growth in spite of early weaning.

Surgical Preparation
Neonatal piglets (age, 5.5 ± 1.4 days; weight, 3.3 ± 0.7 kg; n = 18) were purchased from a local farmer. After arrival in the laboratory, they were divided randomly into three groups of 6 animals each: control, sham-operated, and animals that underwent an operation for provocation of pulmonary hypertension. The piglets were fasted for 12 hours before the operation, sedated with intramuscular ketamine (250 mg), and anesthetized with pentobarbital sodium (15 mg/kg intravenously). Subsequently, the animals were intubated and general anesthesia was maintained with N₂O (60%)/O₂ (40%) under intermittent positive ventilation pressure (MMS 107 Ventilator, Paris, France) at 40 breaths/min with a tidal volume of 15 mL/kg.

After sterile preparation, a left thoracotomy in the fourth intercostal space was performed. The pericardium was opened by a small incision anterior to the phrenic nerve. A pursestring suture was placed at the junction between left atrium and pulmonary veins. A pediatric Foley catheter (no. 6) was introduced through the pursestring suture into the left atrium and was secured to the left atrial wall. The distal end of the Foley catheter then was tailored and passed through the chest wall incision. The lumen of the catheter was filled with silicone wax. The thoracotomy was closed over a chest tube, which was removed after 1 hour.

During the surgical procedure the animals received oxacillin (0.25 g intravenously) and gentamycin (40 mg intramuscularly). Sham-operated and PH piglets received unfractioned heparin (300 IU/kg intravenously) at the intervention and subsequently received the long-acting low molecular weight heparin fraxiparin (0.1 mg • kg^-1 • day^-1 subcutaneously) until hemodynamic evaluation was performed. In PH animals the intraatrial balloon was inflated progressively with 3 mL (1.5 mL/day during 2 days) of physiologic saline solution, whereas in sham-operated piglets the Foley catheter was present in the left atrial cavity but the balloon was not inflated. Preliminary studies performed in 6 piglets (not included in the analysis) showed that acute inflation of the 3-mL balloon in the left atrial cavity led invariably to acute pulmonary edema and death. However, morphologic studies in the same animals revealed that the overall volume of the left atrium was greater than 3 mL. In 3 piglets not included in the analysis the progressive inflation of the left atrial balloon resulted in a 10 ± 2 mm Hg increase in pulmonary arterial occlusion pressure and pulmonary arterial pressure after 3 days.

Hemodynamic Studies
Two weeks after the initial operation (15 ± 2.8 days; mean ± standard deviation) the piglets again were anesthetized and ventilated at an inspired oxygen fraction of 0.60 in a supine position. In PH piglets the balloon was emptied immediately before anesthesia and left deflated throughout the hemodynamic study, because the animals exhibited a poor cardiovascular status. None of them received antifailure treatment. The right carotid artery and external jugular vein were exposed through a small cervical incision and catheterized. A Swan-Ganz triple-lumen thermodilution catheter (4F) was advanced into the pulmonary artery under fluoroscopic guidance. Pulmonary arterial pressure, pulmonary arterial occlusion pressure, systemic arterial pressure, and right atrial pressure were measured by use of Bentley-Trantec pressure transducers (model 800) and CGR electromanometers (model Max-31). The electrocardiographic and pressure signals were recorded continuously on a multichannel direct writing ink pen oscillograph (Gould model 2800). The piglets were in a supine position, and zero level was set at the mid-axillary line. Cardiac output was determined by thermodilution (American Edwards Labs Model 9520A) using injections of 3 mL of ice-cold saline solution (0°C), and determination was the mean of three measurements performed in approximately 2 minutes. The pH, oxygen tension, and carbon dioxide tension of arterial blood were measured using a Dow-Corning blood gas system (model 288). All animals were in a stable condition at least 20 minutes before data acquisition.

The following calculations were performed: Cardiac index was obtained by dividing cardiac output by the body weight. Pulmonary arterial resistance was calculated as (pulmonary artery pressure - pulmonary arterial occlusion pressure)/cardiac index, and systemic arterial resistance as (systemic arterial pressure - right atrial pressure)/cardiac index.

After the hemodynamic assessment, a median sternotomy was performed. Heparin (200 IU/kg intravenously) was injected and the animals were killed by exsanguination. The heart and the lungs quickly were removed en bloc. The left upper lung lobe was fixed in 10% formaldehyde while maintained in inflated state and was prepared for histologic evaluation. The left lower lung lobe was weighed immediately (wet weight) and dried in a dessicator (60°C) until constant weight (dry weight) was achieved. Pulmonary water content was obtained as the ratio of (wet weight - dry weight)/wet weight.

The ventricles were dissected free from right and left atria and from the great vessels. The right ventricle then was divided from the left ventricle and septum. Both ventricular structures were weighed and indexed to the body weight.

Isolated Tissue Studies
Intrapulmonary arteries carefully were dissected free from adjoining connective tissue and lung parenchyma. The preparations were placed in a Tyrode's solution and cut into rings 3 mm in length and 2 mm in diameter. Four rings were obtained from the right lung of each animal. In paired preparation from the same arterial segment, the endothelium was removed by rubbing the luminal surface with a moistened cotton swab (rubbed preparations). All tissues were used immediately after operation. The rings were mounted in a 10-mL organ bath under an initial load of 1.5 g.

The tissues were allowed to equilibrate for 90 minutes in Tyrode's solution containing indomethacin (10 µmol) and aerated at 37°C with 5% CO₂ in O₂. During this period the bath fluid was exchanged every 10 minutes with fresh Tyrode's solution. The composition of the Tyrode's solution was as follows (millimoles per liter): NaCl, 139.2; KCl, 2.7; CaCl₂, 1.8; NaHCO₃, 11.9; MgCl₂, 0.49; NaH₂PO₄, 0.4; and glucose, 5.5 at pH 7.4. Isometric force displacement transducers (Narco F-60) and Linseis recorders were used to monitor the changes in force.

Subsequent to the equilibration period, two different protocols were followed. In the first protocol, pulmonary arterial preparations with and without endothelium were contracted in a cumulative fashion with noradrenaline (10 nmol/L to 0.1 mmol/L). In the second protocol, the rings were precontracted with noradrenaline (10 µmol/L); when the contraction reached a plateau, preparations were relaxed with cumulative concentrations of acetylcholine or sodium nitroprusside (1 nmol/L to 0.1 mmol/L).

Histologic Examination
After formalin fixation and dissection, tissue samples from each animal left lung were embedded in paraffin, and 5-µm-thick sections were stained with hematoxylin and eosin. Slides were examined microscopically, without knowledge of treatment group.

Data Analysis
Results from the hemodynamic studies are shown as means ± standard error of the mean. Comparisons between the different groups were performed by analysis of variance. Comparisons of concentration–effect curves were performed by the nonparametric Mann-Whitney test.

In the isolated tissue studies, changes in force were measured from isometric recording and expressed in grams. The maximal response induced with an agonist (E_max value) and the pD₂ value were interpolated from individual concentration–effect curves for both relaxation and contraction. The pD₂ values were defined as the negative logarithm of the EC₅₀ value. Values are presented as means ± standard error of the mean, and statistical analysis was performed using multirange analysis of variance followed by a post-hoc test (Tukey) with a confidence limit level of 95%.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Preoperatively the control animals weighed 3.65 ± 0.26 kg; sham, 3.58 ± 0.3 kg; and PH, 2.87 ± 0.25 kg. There was no difference in the weight gain of the control group (1.7 ± 0.07 kg) versus the sham group (1.37 ± 0.39 Kg) during the 2-week interval. However, a significantly (p < 0.05) lower weight gain was observed in the PH group (0.55 ± 0.08 kg).

Hemodynamic and Blood Gas Analysis
Pulmonary artery pressure and resistance were markedly elevated in PH versus control or sham-operated animals (Fig 1), but there was no difference in right atrial pressure or pulmonary arterial occlusion pressure among the three groups of animals (Table 1). Systemic arterial pressure and cardiac index were reduced markedly in PH animals versus others (see Fig 1; Table 1). There was no difference in blood gas values among the three groups (see Table 1).

View larger version (15K): [in this window] [in a new window]   Fig 1. . Hemodynamic measurements in neonatal piglets. Values are means ± standard error of the mean (see Table 1 for number of animals). (PH = pulmonary hypertension; * Values significantly different [p < 0.05] from data obtained in control or sham animals.)

Isolated Tissue Studies
The maximal contractions induced by noradrenaline in pulmonary arterial preparations were similar for the tissues obtained from the three groups of animals; these data are presented in Figure 2 and Table 2. Acetylcholine induced relaxation in the intact arterial rings derived from control and sham-operated piglets, an effect that was not observed in rubbed preparations (Fig 3; Table 3). In vascular segments derived from PH animals, the acetylcholine relaxation was significantly attenuated when compared with other groups (see Fig 3; Table 3). The relaxation induced with sodium nitroprusside was not different in any of the three groups (Fig 4; Table 4).

View larger version (25K): [in this window] [in a new window]   Fig 2. . Noradrenaline concentration–effect curves in isolated conduit pulmonary arteries obtained from neonatal piglets. The contractions are presented as force in grams, and values are means ± standard error of the mean. The number of animals used is presented in Table 2. (PH = pulmonary hypertension.)

View larger version (24K): [in this window] [in a new window]   Fig 3. . Acetylcholine-induced relaxations in isolated conduit pulmonary arteries derived from neonatal piglets. Values (means ± standard error of the mean) are presented as percent of the contraction induced by noradrenaline (see Table 3 for number of animals and contraction data). (PH = pulmonary hypertension.)

View larger version (24K): [in this window] [in a new window]   Fig 4. . Nitroprusside-induced relaxations in isolated conduit pulmonary arteries derived from neonatal piglets. Values (means ± standard error of the mean) are presented as percent of the contraction induced by noradrenaline (see Table 4 for number of animals and contraction data). (PH = pulmonary hypertension.)

View larger version (436K): [in this window] [in a new window]   Fig 5. . Muscular pulmonary artery and adjacent parenchyma obtained from one representative animal of each group: (A) control; (B) sham-operated; (C) pulmonary hypertension. There was no detectable difference in medial thickness or alveolar structure between the three groups.

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

Pulmonary hypertension secondary to congenital heart defects may occur as a result of a left to right shunt, an obstruction to the pulmonary venous drainage, or both. What exactly causes the loss of endothelial response to a dilator drug such as acetylcholine in this condition is unknown. From our study, it is certainly possible to advance that it is not from parenchymal origin because there was no evidence of gas exchange abnormalities. Several other mechanisms have been proposed to explain the loss of endothelium-dependent relaxation. Shear stress, generally present in left to right shunts, has been demonstrated to induce the production of vasodilatory mediators in vascular beds [22] that should contribute to decrease of pulmonary artery pressure. On the other side, abnormal balance of intrapulmonary eicosanoids [23, 24] along with an immature endothelial pulmonary vascular barrier [25] might be one possible explanation. Rabinovitch and associates also have demonstrated abnormalities of platelets [26] as well as at the endothelial cell level [27] in patients with PH. Interactions between these components may interfere with the ability of the endothelial cells to induce a vasodilatation in response to endothelium dependent dilator drugs. Moreover, it is not uncommon to obtain an exacerbation of the pulmonary vasoreactivity after open heart repair [4].

All these mechanisms remain hypothetic and mandate a clear demonstration; therefore, a reliable animal model is necessary. The present model is similar to that of Silov and colleagues [11]; however, in their original work there was no allusion to the pulmonary vascular endothelial function, although there was a loss of vasodilator responses to oxygen, acetylcholine, and isoproterenol. In the model of Labourène and colleagues [12], bands were placed on pulmonary veins but constriction paralleled growth. Under these conditions, one may assume that spontaneous maturation of the pulmonary vasculature was normal until pulmonary venous constriction occurred. Finally, we believe an intrauterine model would better reproduce the condition of PH associated with congenital heart defects but requires an experienced team and environment.

Interestingly, in the present study, no histologic modifications of the pulmonary arteries were found. This finding is in contrast with several previous works that showed more or less severe vascular remodeling of the pulmonary vascular bed [2]. However, all the animals operated on received large doses of heparin at the time of intervention and subsequently were treated with daily subcutaneous heparin to prevent intraatrial thrombosis. Given the fact that heparin has potent inhibitory effects on smooth muscle cell proliferation [28], it is possible that this continuous infusion of heparin modulated or slowed the remodeling process.

In conclusion, the partial obstruction of the pulmonary venous drainage system by an inflated left atrial balloon provides a simple surgical model of PH in piglets. This model permitted a significant perturbation of the pulmonary vascular bed within 15 days. The endothelium dysfunction that was demonstrated at the level of the conduit pulmonary arteries may indicate that, more generally, a loss of relaxant factors originating from the pulmonary vascular endothelium plays an important role in sustaining PH even after correction of its cause by balloon deinflation. Further studies are necessary to determine what exactly is responsible for endothelial cell dysfunction in this condition and what are the kinetics of endothelium recovery after correction of the anomaly.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We are grateful to the laboratory technicians, Chantal Verriest and Michèle Gaillard.

This work was supported in part by a grant from the Société d'Etudes et de Soins pour les Enfants Attients de Rhumatismes Articulaire aigu et de Cardiopathies Congénitales and the Caisse Régionale d'Assurance Maladie de l'Ile de France.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Address reprint requests to Dr Serraf, Marie-Lannelongue Hospital, 133, Avenue de la Résistance, 92350 Le Plessis-Robinson, France.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Serraf, A. Articles by Brink, C. Search for Related Content PubMed PubMed Citation Articles by Serraf, A. Articles by Brink, C.