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(Circulation. 1995;92:606-613.)
© 1995 American Heart Association, Inc.

Articles

In Utero Placement of Aortopulmonary Shunts

A Model of Postnatal Pulmonary Hypertension With Increased Pulmonary Blood Flow in Lambs

V. Mohan Reddy, MD; Barbara Meyrick, PhD; Jackson Wong, MD; Andras Khoor, MD; John R. Liddicoat, MD; Frank L. Hanley, MD; Jeffrey R. Fineman, MD

From the Departments of Cardiothoracic Surgery (V.M.R., J.R.L., F.L.H.) and Pediatrics (J.W., J.R.F.), University of California San Francisco; and the Departments of Pathology and Medicine (B.M., A.K.), Vanderbilt University, Nashville, Tenn.

Correspondence to Jeffrey R. Fineman, MD, University of California, San Francisco, 505 Parnassus Ave, Box 0106, M-680, San Francisco, CA 94143-0106.

	Abstract

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Background The development of pulmonary hypertension and its associated increased vascular reactivity is a common accompaniment of congenital heart disease with increased pulmonary blood flow. Although the morphology of the pulmonary vascular changes is well described, the mechanisms of vascular remodeling and increased reactivity remain incompletely understood.

Methods and Results To elucidate these mechanisms, we established an accurate and reliable experimental model of pulmonary hypertension with increased pulmonary blood flow. An aortopulmonary shunt was created with an 8.0-mm expanded polytetrafluoroethylene vascular graft in 11 late-gestation fetal lambs. At 1 month of age, shunted lambs had a pulmonary–to–systemic blood flow ratio of 2.2±1.2. Compared with 11 age-matched control lambs, mean pulmonary arterial pressure (44.8±11.7 versus 16.2±2.9 mm Hg) and the ratio of pulmonary to systemic arterial pressure were significantly increased (P<.05). Pulmonary vascular resistance was not significantly increased. The pulmonary vasoconstricting response to the infusion of U46619 (a thromboxane A₂ mimic) or acute alveolar hypoxia also was augmented in the shunted lambs. Morphometric analysis of the barium-filled pulmonary artery bed revealed medial hypertrophy, abnormal extension of muscle distally into the walls of the intra-acinar arteries, and increased numbers of barium-filled intra-acinar arteries.

Conclusions In utero placement of aortopulmonary shunts reproduces the aberrant hemodynamic state of children with congenital heart disease with left-to-right shunts; postnatal pulmonary hypertension, increased pulmonary blood flow, and vascular remodeling. In addition, the lambs have a unique paradoxical increase in pulmonary vascular volume that attenuates an increase in pulmonary vascular resistance. This experimental preparation provides a useful and consistent model for the study of the pathogenesis of pulmonary hypertension.

Key Words: pulmonary heart disease • heart defects • congenital • hypertension • pulmonary

	Introduction

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The development of pulmonary hypertension and its associated increased vascular reactivity is a common accompaniment of congenital heart disease with increased pulmonary blood flow.¹ After birth, the presence of a systemic-to-pulmonary communication (ie, truncus arteriosus, atrioventricular canal, or ventricular septal defect) results in increased pulmonary blood flow as pulmonary vascular resistance normally decreases. This abnormal postnatal hemodynamic state results in progressive structural and functional abnormalities of the pulmonary vascular bed.^{2 3 4 5 6} If uncorrected, these vascular changes result in reduction and obliteration of the pulmonary vascular bed and death secondary to severe cyanosis and myocardial failure. Early structural changes are reversible after surgical correction. However, even in patients with minimal structural changes, there may be morbidity and mortality in the perioperative period due to increased vascular reactivity, which may be secondary, in part, to early endothelial injury and the resulting aberrant production of vasoactive substances.^{7 8 9 10 11} Severe structural changes are irreversible and progressive. Therefore, the status of the pulmonary vasculature often is the principal determinant of the clinical course and feasibility of surgical treatment.¹²

Although the vascular morphology of pulmonary hypertension is well described, the mechanisms of vascular remodeling and increased vascular reactivity remain incompletely understood. A clearer understanding of these mechanisms would provide potential new avenues for the prevention and treatment of this disorder. To obtain this information, an accurate and reliable animal model of the disease process is necessary.¹³ Previous attempts to produce animal models of increased pulmonary blood flow have involved the surgical creation of an aorta-to-pulmonary communication in postnatal animals.^{14 15 16 17 18 19 20 21 22 23 24 25 26} Such models, however, do not directly simulate congenital heart disease because pulmonary vascular resistance has previously fallen and a period of normal lung growth and remodeling has already taken place.

The purpose of the present study was to establish a chronic model of pulmonary hypertension with increased pulmonary blood flow. We hypothesized that establishment of a systemic-to-pulmonary communication in utero would delay the fall in pulmonary vascular resistance that occurs at birth and, therefore, be well tolerated and more closely simulate congenital heart disease. We placed an 8.0-mm expanded polytetrafluoroethylene vascular graft between the ascending aorta and main pulmonary artery in 11 late-gestation fetal lambs. At 1 month of age, we compared the general hemodynamics and pulmonary vascular morphology of these lambs with 11 age-matched control lambs. In addition, we compared the alterations in pulmonary vasoresponsiveness to acute alveolar hypoxia and the infusion of U46619 (a thromboxane A₂ mimic).

	Methods

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Surgical Preparation and Care
Ewes
Sixteen mixed-breed Western ewes (140±1.2 days' gestation; term, 145 days) were operated on under sterile conditions with the use of local anesthesia (2% lidocaine hydrochloride), epidural anesthesia (4 mL of 1% tetracaine hydrochloride), and intravenous sedation (50 to 100 mg ketamine hydrochloride). An 18-gauge catheter was inserted into the maternal pedal vein. A midline incision was made in the ventral abdomen, and the pregnant horn of the uterus was exposed. Through a small uterine incision, the left fetal forelimb and chest were exposed, and a left lateral thoracotomy was performed in the third intercostal space. Fetal anesthesia consisted of local anesthesia with 1% lidocaine hydrochloride and ketamine hydrochloride (20 mg IM). Succinylcholine hydrochloride (3 to 5 mg IM) was administrated to prevent fetal breathing movements. The pericardium was incised along the main pulmonary trunk, and suspended with tacking sutures. The bovine arterial trunk and the main pulmonary artery were dissected and controlled with vessel loops. The ascending aorta was side-clamped with a side-biting vascular clamp. An aortotomy was performed with a No. 11 bladed knife. The aortotomy was extended to 8 mm with fine scissors, and a strip of aortic wall was excised to create an oval opening in the ascending aorta. The anastomosis between the 8.0-mm expanded polytetrafluoroethylene vascular graft (2 mm length) (Gore-tex; W.L. Gore and Assoc) and ascending aorta was performed with 7.0-Proline using a continuous suture technique (Ethicon Inc). A large vascular clip was placed to temporarily occlude the graft, and the vascular clamp was gradually released to minimize any bleeding at the suture line. The vascular clamp then was applied to the pulmonary artery. A pulmonary arteriotomy was performed, a strip of the posterior wall was excised, and the free end of the graft was sutured to the pulmonary artery. The vascular clamp was gradually released, allowing any air in the graft to escape through the suture line and needle holes. The vascular clip then was removed, establishing the graft patency (Fig 1). The thoracotomy incision was closed in layers. Warm saline was infused to replace the lost amniotic fluid, and the uterine incision was closed. After recovery from anesthesia, the ewe was returned to the cage with free access to food and water. Antibiotics (2 million U penicillin G procaine and 100 mg gentamicin sulfate) were administered intravenously to the ewe during surgery and daily thereafter until 2 days after spontaneous delivery of the lamb. Nine of the 11 control lambs were the twin of a lamb who underwent the surgical preparation described. Two of these control lambs underwent sham thoracotamies; the procedure was identical to that described above, without placement of the vascular graft.

View larger version (57K): [in this window] [in a new window]   Figure 1. Illustrations of surgical technique. A, Exposure of fetal heart via left thoracotomy. Ao indicates ascending aorta; BT, bovine trunk; DAo, descending aorta; G, gauze sponge over the right atrium; LA, left atrial appendage; LPA, left pulmonary artery; PA, main pulmonary artery; and PDA, patent ductus arteriosus. B, bovine trunk, aorta, and main pulmonary artery are dissected. Vessel loops are passed around the bovine trunk and main pulmonary artery. C, Continuous suture technique of the aorta to vascular graft anastamosis. D, Completed anastomosis. Vascular clip is subsequently removed to establish graft patency.

Lambs
After spontaneous delivery, antibiotics (1 million U penicillin G procaine and 25 mg gentamicin sulfate IM) were administered for 2 days. The lambs were weighed daily, and the respiratory rate and heart rate were obtained. Furosemide (1 mg/kg IM) was administered once or twice daily to lambs with respiratory rates of more than 100 associated with decreased activity. Elemental iron (100 mg IM) was given weekly. Supplemental nasogastric feeds were administered intermittently to lambs with poor weight gain.

At 1 month of age, 22 lambs (11 shunted and 11 age-matched controls) had polyvinyl catheters placed in an artery and a vein of one hind leg while under local anesthesia with 1% lidocaine hydrochloride. These catheters were advanced to the descending aorta and the inferior vena cava, respectively. The lambs were then anesthetized with ketamine hydrochloride (1 mg/kg per minute), intubated with a 5.5-mm-OD endotracheal tube, and mechanically ventilated with a Healthdyne pediatric time-cycled, pressure-limited ventilator. Succinylcholine chloride (2 mg/kg per dose) was given intermittently for muscle relaxation. Ventilation with 21% oxygen was adjusted to maintain a PaCO₂ between 35 and 45 mm Hg. A midsternotomy incision was performed, and the pericardium was incised. Two single-lumen polyurethane catheters were inserted into the left and right atrium, respectively. A double-lumen polyurethane catheter was placed in the main pulmonary artery distal to the vascular graft. An ultrasonic flow probe (Transonics Systems Inc) was placed around the left pulmonary artery to measure left pulmonary blood flow. After a 30-minute recovery, blood was obtained from the left and right atria, distal pulmonary artery, right ventricle, and descending aorta for hemoglobin and oxygen saturation determinations. The thoracotomy incision then was closed in layers. An intravenous injection of 250 000 U penicillin G procaine and 25 mg gentamicin sulfate suspension was administered. All protocols were approved by the Committee on Animal Research of the University of California, San Francisco.

Measurements
Pulmonary and systemic arterial, and right and left atrial pressures were measured using Statham P23Db pressure transducers (Statham Instruments). Mean pressures were obtained by electrical integration. Heart rate was measured by a cardiotachometer triggered from the phasic systemic arterial pressure pulse wave. Left and right pulmonary blood flows were measured with an ultrasonic flowmeter (Transonic Systems). All hemodynamic variables were recorded continuously on a Gould multichannel electrostatic recorder. Systemic arterial blood gases and pH were measured with a Corning 158 pH/blood gas analyzer (Corning Medical and Scientific). Hemoglobin concentration and oxygen saturation were measured with a hemoximeter (model OSM 2, Radiometer). The ratio of pulmonary to systemic blood flow (Q_p/Q_s) was calculated with the use of the Fick equation. Systemic blood flow was calculated as the total pulmonary blood flow divided by the Q_p/Q_s. Pulmonary vascular resistance was calculated using standard formulas.

Drug Preparation
U46619 (9,11-dideoxy-9-epoxymethano-prostaglandin F₂; Sigma Chemical Co) was suspended in 95% ethanol and stored at -20°C. Immediately before the study, 100 µg was dissolved in 20 mL of 0.9% saline.

Experimental Protocol
Hemodynamic Study
Sixty minutes after chest closure, baseline measurements of the hemodynamic variables (pulmonary and systemic arterial pressure, heart rate, left pulmonary blood flow, left and right atrial pressures), and systemic arterial blood gases and pH were measured. U46619 (1 µg/kg per minute) then was infused intravenously for 15 minutes. The hemodynamic variables were measured continuously, and systemic arterial blood gases and pH were measured after a new steady state was achieved. The infusion was then stopped. This dosage of U46619 was chosen because we have previously shown that 1 µg/kg per minute approximately doubles mean pulmonary arterial pressure in healthy lambs, and is well tolerated. After a 30-minute recovery, all measurements were repeated. Acute alveolar hypoxia (10% oxygen) was induced by the addition of nitrogen to the ventilation gas mixture. After 15 minutes, all measurements were obtained, and ventilation with 21% oxygen was resumed.

After a 60-minute recovery period, the chest was re-opened in 14 of the lambs (7 controls and 7 shunted), and an ultrasonic flow probe was also placed around the right pulmonary artery to measure total pulmonary blood flow. In the shunted lambs, the vascular graft was then closed with vascular clips. After 30 minutes, measurements of the hemodynamic variables and systemic arterial blood gases and pH were obtained. At the end of the study, the lambs were given a lethal dose of pentobarbital sodium followed by a bilateral thoracotomy.

Structural Studies
The lungs, heart, and trachea were removed intact from four shunted and four control lambs, and the pulmonary arterial bed was distended with a barium gelatin suspension (563 mL micropaque powder, Nicholas Picker Co; 50 g gelatin, Bloom 8-G, Fisher Scientific Co; 387 mL distilled water; and a few crystals of phenol) at 60°C from a pressure of 70 mm Hg for 2 minutes.²⁷ After arterial injection, the lungs were inflated by way of the trachea with 10% formol-saline from a pressure of 35 cm H₂O and placed in a bath of formalin for fixation. After approximately 7 days, arteriograms were made of the barium-injected arteries and of 1-cm slices of the lungs. The arteriograms allowed a simple, overall assessment of the pulmonary arterial tree, including the smallest arterial branches, which were seen as a background haze.

Random blocks for routine light microscopic examination were taken from each slice of lung; approximately 6 blocks were taken from each lung. Two 5-µm sections were cut from each block—one was stained with hematoxylin and eosin, and the other was stained with Verhoeff's elastin stain followed by van Gieson's stain. The sections were then examined for the characteristic structural changes of chronic pulmonary hypertension by the use of well-established quantitative techniques.^{3 27} Briefly, the external diameters of at least 100 arterial profiles were measured as were medial thicknesses of the muscular and partially muscular arteries. Medial thickness was then related to arterial size with the following formula: percent medial thickness=2xmedial thickness/external diameterx100. The structure of each artery was also noted—muscular, partially muscular, or nonmuscular—as was the structure of the accompanying airway—bronchus, bronchiolus, terminal bronchiolus, respiratory bronchiolus, alveolar duct, and alveolar wall. The density of the barium-filled intra-acinar arteries also was assessed. With a x25 objective and an eyepiece reticule, the number of barium-filled arteries of <200-µm external diameter was counted and related to the number of alveolar profiles in these same fields. At least 25 consecutive microscopic fields were counted for each animal.

Right ventricular hypertrophy was assessed after fixation in five animals of each group. The right ventricle was dissected from the left ventricle plus septum, and each was weighed. Right ventricular weight was expressed as a ratio of the right ventricle to the left ventricle plus septum.

Statistical Analysis
The mean±SD was calculated for the baseline hemodynamic variables, and systemic arterial blood gases and pH. The variables of the shunted lambs were compared with those of the control lambs with the use of the unpaired t test. The variables of the shunted lambs before shunt closure were compared with those after shunt closure and with those for control lambs with the use of repeated-measures ANOVA. The effects of each pulmonary vasoconstricting stimulus (alveolar hypoxia or U46619) were compared with their previous steady-state condition with the use of the paired t test, with the Bonferroni correction for multiple comparisons. The absolute change in pulmonary arterial pressure induced by these stimuli was compared between study groups using the unpaired t test. For each animal, the mean value was calculated for each structural variable and the mean±SEM was determined for each group of animals. Structural data were compared with the use of the unpaired t test. P<.05 was considered statistically significant.

	Results

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Spontaneous delivery occurred 2 to 9 days after fetal surgery. Four control fetuses and two shunted fetuses were stillborn. Two additional shunted lambs died at 2 weeks of age with increasing respiratory distress. The overall mortality rate was 33.3%. The mortality rate of the shunted lambs after birth was 18.2%. In general, shunted lambs had higher resting respiratory rates and were less active than control lambs. Although the birth weights were similar for the two study groups, shunted lambs had decreased weight gain after the first week of life (P<.05) (Fig 2). There were no differences in the sex distribution or age between the two study groups.

View larger version (15K): [in this window] [in a new window]   Figure 2. Plot showing that after 1 week of life, shunted lambs weighed less than control lambs. Values are mean±SD. *P<.05 vs control lambs.

Hemodynamic Study
At 1 month of age, the baseline systemic arterial blood gases and pH, hemoglobin, and oxygen saturations were similar for the two groups and within the normal range for our laboratory (Table 1). All shunted lambs had an audible continuous murmur and an increase in oxygen saturation between the right ventricle and distal pulmonary artery. The Q_p/Q_s was 2.2±1.2. Pulmonary arterial pressure was dramatically elevated (44.8±11.7 versus 16.2±2.9 mm Hg, P<.05), which approximated 76% of systemic values. This was associated with an increase in pulmonary blood flow, systemic blood flow, and left and right atrial pressures (P<.05). Mean and diastolic systemic arterial pressures were decreased (P<.05). The calculated pulmonary vascular resistance in the shunted lambs was not significantly different from that of control lambs (Table 2).

View this table: [in this window] [in a new window]   Table 1. Oxygenation Indexes

View this table: [in this window] [in a new window]   Table 2. General Hemodynamics

Closure of the vascular graft resulted in a significant decrease in mean pulmonary arterial pressure (from 41.4±13.1 to 28.5±11.4 mm Hg, P<.05) and total pulmonary blood flow (from 3464.0±1132.4 to 1825.5±674.8 mL/min, P<.05) and an increase in pulmonary vascular resistance (from 10.6±6.4 to 16.7±6.6 mm Hg/L per minute, P<.05). Mean systemic arterial pressure increased (from 65.5±8.9 to 85.6±16.3 mm Hg, P<.05). Compared with control lambs, mean pulmonary arterial pressure, pulmonary blood flow, and pulmonary vascular resistance remained markedly elevated after graft closure (Table 3).

View this table: [in this window] [in a new window]   Table 3. General Hemodynamics After Shunt Closure

Before graft closure, the infusion of U46619 resulted in increased mean pulmonary arterial pressure, left pulmonary vascular resistance, and mean systemic arterial pressure, and decreased left pulmonary blood flow in both shunted and control lambs. The induction of alveolar hypoxia also increased mean pulmonary arterial pressure and left pulmonary vascular resistance in both groups of animals. Mean systemic arterial pressure was unchanged in both groups. Left pulmonary blood flow decreased only in shunted lambs (Table 4). The absolute increase in mean pulmonary arterial pressure induced by either U46619 or alveolar hypoxia was greater in shunted lambs than in control lambs (P<.05) (Fig 3).

View this table: [in this window] [in a new window]   Table 4. Hemodynamic Effects of Pulmonary Vasoconstricting Stimuli

View larger version (21K): [in this window] [in a new window]   Figure 3. Bar graph showing that the increase in mean pulmonary arterial pressure induced by the infusion of U46619 (a thromboxane A2 mimic) or acute alveolar hypoxia is greater in shunted lambs than in control lambs. Values are mean±SD. *P<.05 vs control lambs.

Structural Studies
Arteriograms of the shunted lambs showed a modest degree of dilatation of the main pulmonary artery branches, and an obvious increase in background haze compared with age-matched control lambs (Fig 4). Percent medial thickness of arteries of <200-µm external diameter was significantly increased in the shunted animals compared with age-matched controls and tended to be increased in the larger arteries (Fig 5). Analysis of the structure of the intra-acinar arteries established the appearance of muscle in the walls of smaller and more peripheral arteries than normal in the shunted animals (Table 5). Morphometric analysis also revealed an increased number of arteries per unit area in the shunted animals compared with the control animals (4.5±1.0 versus 2.1±0.5, P<.05). This finding was confirmed when barium-filled arterial number was related to the number of alveolar profiles (4.2±1.2 versus 2.1±0.3, P<.05). The number of alveolar profiles per unit area was similar between groups (110±33 versus 99±16 per unit area).

View larger version (89K): [in this window] [in a new window]   Figure 4. Pulmonary arteriogram of a 4-week-old shunted lamb (left) and its twin control lamb (right). In the shunted lamb, notice the dilation of the proximal pulmonary arteries and the increase in the degree of background haze, suggesting an increase in the number or size of the intra-acinar arteries.

View larger version (17K): [in this window] [in a new window]   Figure 5. Plot showing that shunted lambs have increased medial thickness of the arteries of <200 µm in external diameter compared with control lambs. Values are mean±SEM. *P<.05 vs control lambs.

View this table: [in this window] [in a new window]   Table 5. Percentage of Fully Muscular, Partially Muscular, and Nonmuscular Pulmonary Arteries in Shunted and Control Lambs

In general, the structure of the airways and peripheral lung tissue of the shunted animals appeared to be qualitatively similar to that of the age-matched controls. However, although not qualitatively assessed, examination of the venous system suggested that the smaller branches of this side of the circulation were increased as was noted for the peripheral arteries. In addition, supernumerary arteries arising from the parent artery appeared to be more frequently encountered and to be larger in diameter in the shunted lungs than those of the age-matched controls.

The weights of both the right ventricle and the left ventricle plus septum were increased in the shunted lambs (P<.05). The ratio of right ventricular weight to left ventricular plus septal weight was similar between the groups (Table 6).

View this table: [in this window] [in a new window]   Table 6. Heart Weights

	Discussion

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We report that in utero placement in lambs of a large vascular graft between the ascending aorta and main pulmonary artery produces postnatal pulmonary hypertension with increased pulmonary blood flow. The shunted lambs failed to thrive, had a Q_p/Q_s of 2.2, and had dramatic and clinically significant elevations of mean pulmonary arterial pressure (44.8±11.7 mm Hg, which represented 76% of systemic values). The shunted lambs also showed an augmented response to pulmonary vasoconstricting stimuli. Structurally, these lambs had early changes that were similar to those seen in children with pulmonary hypertension with increased pulmonary blood flow: dilation of the proximal pulmonary arteries, increased medial wall thickness of the small muscular arteries, abnormal extension of muscle to peripheral pulmonary arteries, and biventricular enlargement. In addition, these lambs had a unique finding—an increased number of barium-filled intra-acinar pulmonary arteries. This experimental preparation represents the first animal model of pulmonary hypertension with increased pulmonary blood flow in which the left-to-right shunt is present at birth, and thus truly mimics the characteristics of congenital heart defects.

The majority of attempts to produce animal models of increased pulmonary blood flow have involved the surgical placement of an aorta-to-pulmonary communication in adult animals.^{14 15 16 17 18 19 20} These models have had little success in producing elevations in pulmonary arterial pressure because pulmonary vascular resistance is low at surgical placement, and the large left-to-right shunts that are produced result in congestive heart failure and death. Models in young animals have had increased success in producing modest elevations in pulmonary arterial pressure and the associated vascular smooth muscle remodeling.^{21 22 23 24 25} In newborn calves, an anastomosis between the aorta and left pulmonary artery was recently shown to produce left pulmonary arterial pressure near systemic levels after 10 weeks, with associated significant vascular remodeling.²⁶ However, all of these models failed to simulate conditions of congenital heart disease; the systemic-to-pulmonary communication was placed after the dramatic fall in pulmonary vascular resistance at birth, and after a period of lung growth and development had occurred. In children with congenital heart disease and left-to-right shunts, many alterations of the pulmonary vasculature occur, including a delay in the normal fall in pulmonary vascular resistance and rise in pulmonary blood flow.²⁸ We hypothesized that placement of large aorta-to-pulmonary communications in utero would also result in a delayed fall in pulmonary vascular resistance and, therefore, produce significant elevations of pulmonary arterial pressure and flow. In addition, we hypothesized that such changes would be better tolerated. We found that in utero surgery did allow the placement of a very large, low-resistance vascular graft (8.0-mm diameter and 2- to 3-mm length), which resulted in striking elevations of pulmonary arterial pressure with an acceptable mortality of 18.2% after live birth. Although we did not measure the postnatal fall in pulmonary vascular resistance, we speculate that a delayed fall in resistance was one of the alterations resulting in the surprisingly low morbidity and mortality of this model.

This model was also associated with striking alterations in the morphology of the pulmonary vascular bed. Morphometric analysis of the lungs of children with congenital heart disease has shown a progression of disturbed growth and remodeling of the pulmonary vascular bed that correlated with the child's hemodynamic state.^{3 4 5 6 9 29} These changes are characterized by abnormal extension of muscle into small peripheral arteries and, in some cases, a mild medial hypertrophy of normally muscular arteries (grade A), more severe medial hypertrophy of normally muscular arteries (grade B), and reduced arterial concentration (grade C). In the present model, the shunted lambs had morphological changes that would correlate with grade A to early grade B: abnormal extension of muscle into small peripheral arteries with medial hypertrophy of the small muscular arteries of <200 µm. Rendas et al²⁵ produced similar morphological changes in growing pigs when an anastomosis between the aorta and pulmonary trunk was placed before 4 weeks of age, and Fasules et al²⁶ produced similar changes in calves when an anastomosis between the aorta and left pulmonary artery was placed during the newborn period.

In previous animal models of pulmonary hypertension, moderate morphological changes are associated with normal numbers of intra-acinar pulmonary arteries.²⁵ In contrast, the present study shows an increase in the number of barium-filled intra-acinar pulmonary arteries. The exact etiology of our findings is unclear and requires further investigation. Although some of the increased number of vessels may represent venous filling secondary to shunts, it is unlikely to be a significant component, since the majority of veins were not barium filled. The increased number of vessels may represent compensatory vessel recruitment to the elevation in pulmonary blood flow and/or pulmonary arterial pressure, which has been described after the increase in pulmonary blood flow at birth and after the increase in pulmonary blood flow after pneumonectomy.^{30 31} However, the pulmonary arterial circulation in our lambs was filled with barium from a hypertensive pressure of 75 mm Hg. Thus, it is likely that the arteries in control lambs as well as the hypertensive lambs were fully recruited. Therefore, the most likely explanation is that the increased number of filled arteries represents a compensatory burst of small vessel growth in response to the maintained elevation in pulmonary blood flow and/or pulmonary arterial pressure. Although no previous animal model of increased pulmonary blood has documented an increased number of pulmonary arteries, the youngest animals previously shunted were 1 to 4 weeks old.^{24 25 26} Adaptations to the hemodynamic disturbance at birth of the more premature lung may be quite different. For example, coronary artery angiogenesis has been observed in animal models of right ventricular hypertrophy when the hypertrophy occurs in fetal or newborn animals, but not in adult animals.^{32 33 34} Furthermore, the size of the shunt and the elevation of pulmonary arterial pressure achieved in the present model are much greater than previously described. In children with congenital heart disease and pulmonary hypertension, moderate morphological changes are associated with normal numbers of intra-acinar pulmonary arteries, whereas late morphological changes are associated with decreased numbers of intra-acinar pulmonary arteries.^{2 3 4 5 6} Although increased numbers of arteries have not been reported in children with pulmonary hypertension with increased pulmonary blood flow, the present finding may represent an early and transient adaptation that is not found on later biopsy or autopsy studies.

Children with early, reversible morphological changes may have significant morbidity and mortality in the perioperative and postoperative periods secondary to acute elevations in pulmonary vascular resistance.⁷ This is produced by active contraction of the structurally abnormal vessels during a period of extreme sensitivity to vasoconstricting stimuli, such as hypoxia. This typical increased reactivity of the pulmonary vasculature when exposed to increased flow and pressure was also present in our lamb model. We found that the shunted lambs had a markedly exaggerated response to both acute alveolar hypoxia and the infusion of U46619, a thromboxane A₂ mimic. The mechanisms for this exaggerated presser response are not known; it may be secondary to not only structural abnormalities of the vascular smooth muscle, as has been suggested in the adult animal models of pulmonary hypertension, but also functional abnormalities of the vascular endothelium.^{35 36} For example, substantial data suggest that nitric oxide is released in response to pulmonary vasoconstricting stimuli in an attempt to modulate the response, and it has recently been shown that children with increased pulmonary blood flow and pressure have an early impairment of nitric oxide production.^{11 37} The contribution of impaired endothelial function in the abnormal responses of the shunted lambs is unclear, but preliminary data suggests that these lambs also have an impairment of endothelium-dependent pulmonary vasodilation.³⁸

In summary, we present a novel and representative animal model of postnatal pulmonary hypertension with increased pulmonary blood flow. The model entails in utero placement of an aortopulmonary vascular graft, which most closely represents the aberrant hemodynamic state of children who have congenital heart disease with increased pulmonary blood flow. This model also produced early and characteristic morphological changes of the pulmonary vasculature, a typical exaggerated response to pulmonary vasoconstricting stimuli, and the most rapid and dramatic increases in pulmonary arterial pressure in the literature. However, a few limitations of the model should be considered. First, the hemodynamic variables were obtained in anesthetized animals after an extensive thoracotomy. Although baseline hemodynamics were obtained 60 minutes after chest closure, the effects of surgery and anesthesia must be noted. Because control lambs were studied under the same conditions, differences between the groups should be independent of these factors. Second, the Q_p/Q_s was obtained with the Fick principle, using oxygen saturation differences between the right ventricle and distal pulmonary artery. In this model, there is no chamber for complete mixing of blood between the shunt and the sampling site in the pulmonary artery. Therefore, streaming of blood may produce inaccuracies in the calculated Q_p/Q_s. In addition, the systemic blood flow was not directly measured but rather calculated from the Q_p/Q_s. Therefore, the determination of systemic blood flow is subject to similar inaccuracies. To assess this potential limitation, we placed ultrasonic flow probes on the aortic arch and bovine trunk to measure systemic blood flow (minus coronary blood flow) and on the right and left pulmonary arteries to measure total pulmonary blood flow in three additional shunted lambs. The Q_p/Q_s measured with the Fick principle (3.8±1.3) was similar to that measured with the flow probes (3.9±0.9). Third, the calculation of pulmonary vascular resistance is based on the hydraulic equivalent of Ohm's law. In the shunted lambs, a dramatic elevation in mean pulmonary arterial pressure was associated with a non-significant, modest elevation of calculated pulmonary vascular resistance compared with controls. The increase in the number of barium-filled intra-acinar pulmonary arteries and resulting increase in pulmonary blood volume may limit the increase in pulmonary vascular resistance, and thus may represent an adaptive response of the immature pulmonary vasculature to increased flow and pressure. However, when there are large differences in pulmonary blood flow between groups, the use of Ohm's law in single-point comparisons may be inaccurate.³⁹ Last, closure of the shunt resulted in an increase in the calculated pulmonary vascular resistance when determined 30 minutes later. Although this may represent true active vasoconstriction, the limitations of Ohm's law and the possibility that this was an acute transient response must be considered. Despite these limitations, this experimental preparation is the first animal model of pulmonary hypertension with increased pulmonary blood flow from birth, and may provide a useful tool with which to study the mechanisms of vascular remodeling and increased reactivity associated with this disorder.

	Acknowledgments

 
This work was supported by grant 94-212 from the American Heart Association, California Affiliate; by grant HL-43357 from the National Heart, Lung, and Blood Institute; and by the University of California San Francisco Research Evaluation and Allocation Committee. The authors thank Roger Chang and Michael Johengen for expert technical assistance and Randy Kikukawa for editorial assistance.

Received October 10, 1994; revision received January 9, 1995; accepted January 17, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

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