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Ann Thorac Surg 2007;83:631-639
© 2007 The Society of Thoracic Surgeons

Original Articles: Cardiovascular

Revisiting Animal Models of Aortic Stenosis in the Early Gestation Fetus

Pirooz Eghtesady, MD, PhDa,b,*, Erik Michelfelder, MDa, Mekibib Altaye, PhDc, Edgar Ballard, MDd, Russel Hirsh, MDa, Robert H. Beekman, III, MDa

a Divisions of Pediatric Cardiac Surgery and Pediatric Cardiology, University of Cincinnati College of Medicine and Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
b Department of Surgery, University of Cincinnati College of Medicine and Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
c Department of Biostatistics and Epidemiology, University of Cincinnati College of Medicine and Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
d Department of Pediatric Pathology, University of Cincinnati College of Medicine and Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio

Accepted for publication September 6, 2006.

* Address correspondence to Dr Eghtesady, Division of Pediatric Cardiac Surgery, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, MLC 2004, Cincinnati, OH 45229-3039 (Email: pirooz.eghtesady{at}cchmc.org).


Pediatric cardiac surgery: The Annals of Thoracic Surgery CME Program is located online at http://cme.ctsnetjournals.org. To take the CME activity related to this article, you must have either an STS member or an individual non-member subscription to the journal.

 

   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: Mechanisms leading to left ventricular hypoplasia and endocardial fibroelastosis in the fetus remain unknown. Prevailing theory is that obstruction to blood flow through the left ventricle leads to elevated end-diastolic pressures, compromised myocardial perfusion, and endocardial ischemia. Fetal interventions are now being performed, based on the presumption that they would prevent such pathogenic mechanisms.

METHODS: Forty first-trimester fetal sheep (mean gestational age, 53 days) were studied. Severe fetal left ventricular outflow obstruction was created by banding the ascending aorta in 25 fetuses; 15 control fetuses underwent "sham" surgery with thoracotomy. Serial fetal echocardiography was used to assess left ventricular growth and fetal hemodynamics. Findings were correlated to morphologic and histopathologic changes, and intracardiac pressure measurements obtained from fetal cardiac catheterization.

RESULTS: Surviving banded fetuses (n = 13) had one of two phenotypes: compensatory left ventricular hypertrophy (n = 7) or noncompensatory left ventricular dilatation (n = 6) with hydrops and severe left ventricular dysfunction. All fetuses had elevated left ventricular end-diastolic pressures (mean, 21 mm Hg; range, 14 to 28 mm Hg), which correlated to the gradient across the ascending aorta (mean, 41 mm Hg; range, 28 to 73 mm Hg). In vivo echocardiography findings were incongruous with those at autopsy, and demonstrated preservation of left ventricular growth indices in all fetuses. Endocardial fibroelastosis and myocardial fibrosis were not observed in any banded fetus.

CONCLUSIONS: While early gestational obstruction to flow can compromise left ventricular function in the fetus, it does not retard normal growth. Similarly, an elevated left ventricular end-diastolic pressure is not sufficient to cause myocardial fibrosis or endocardial fibroelastosis in the fetus.

Mechanisms leading to left ventricular hypoplasia and endocardial fibroelastosis (EFE) in the fetus remain unknown. Prevailing theory is that obstruction to blood flow through the left ventricle is a major contributor to this pathophysiology, in part because of myocardial ischemia that follows elevations in left ventricular (LV) end-diastolic pressures (LVEDP). Experimental fetal cardiac interventions are being performed based on the presumption that they would prevent the evolution of this pathophysiology [1, 2].

The sequence of events leading to pressure overload of the left ventricle as a result of LV obstruction have been well described for both the neonate and the adult. Compensatory LV hypertrophy (LVH) and associated elevated LVEDP develop as the affected heart compensates to decrease wall stress and maintain the LV ejection fraction [3]. While beneficial in supporting cardiac output, the pathologic hypertrophy leads to dropout of myocardial cells, subendocardial ischemia, and potentially fibrosis in the heart [4]. Whether similar mechanisms occur in the developing left ventricle is not known, but is often suggested in textbooks of pediatric cardiology and cardiac surgery [5].

These preceding hypotheses partly derive from animal studies that show LV obstruction in the form of aortic banding is sufficient to cause LV hypoplasia in late-gestation fetuses (third trimester) [6, 7]. It is conceivable, therefore, that earlier and more significant obstruction would lead to further LV hypoplasia and perhaps an animal model of hypoplastic left heart syndrome. With these thoughts, we set out to extend previous animal studies by carrying out aortic banding in the early gestation, first-trimester sheep fetus, equivalent to 11 to 17 weeks of human gestation. Also, we chose to study the in utero history of this model through serial fetal echocardiography, not previously reported.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Experimental Design
Forty time-dated pregnant ewes between 38 and 65 days of gestation (term, approximately 148 days) were studied. After fetal surgery (described below), the animals were followed to term using serial fetal echocardiography. Near term (135 to 140 days’ gestation), the animals underwent a final echocardiographic analysis, fetal cardiac catheterization, autopsy, and an evaluation of cardiac pathology. All experiments were terminated before birth.

Animals and Surgical Protocols
All procedures followed the principles of laboratory animal care developed by the National Institutes of Health (NIH publication 85-23, revised 1985). Pregnant ewes were anesthetized with ketamine (20 mg/kg) and diazepam (0.5 mg/kg), and received 0.9% normal saline through a central venous catheter placed in the jugular vein. The animals were maintained on inhalation anesthesia (mixture of oxygen and isoflurane; minimal alveolar concentration = 2% to 2.5%). To minimize fetal distress, supplemental intramuscular fetal analgesia (diazepam 0.1 mcg/kg and ketamine 10 mcg/kg) was given based on estimated fetal weight normograms [8].

The surgical procedure included maternal laparotomy and limited hysterotomy. After delivering the fetus, we performed a limited left thoracotomy, pericardiotomy, and dissection of fetal great vessels. In the experimental group, we placed a 2.0-mm Dacron (C. R. Bard, Haverhill, Pennsylvania) band on the ascending aorta. The band length was measured to circumference of a circle whose diameter equaled the diameter of the ascending aorta. Afterward, the fetus was returned to the amniotic cavity, lost amniotic fluid was replaced with warm saline containing 100 mg gentamicin sulfate and 2 million units penicillin G procaine, and all incisions were closed. In a control group, all procedures were carried out except placement of a band. Maternal antibiotics were continued for 3 days postoperatively. Studies described below were initiated after a 1- to 2-week recovery period.

Fetal Assessment
Baseline echocardiograms were obtained on fetuses before surgical banding. Subsequently, serial echocardiograms were obtained every 2 weeks until term. To guard against any confounders, echocardiography studies were performed on awake and alert animals using a specially modified sheep chair (Premier 1 Supplies, Washington, Iowa). The following data were obtained:

Two-dimensional and M-mode imaging
Transverse imaging of the fetal thorax was used to obtain a four-chamber view of the fetal heart. From this imaging window, the following measurements were made: (1) M-mode assessment of LV and right ventricular (RV) transverse dimensions at end-diastole (maximal chamber dimension); (2) M-mode quantitation of left and right ventricular free wall and interventricular septal thickness; (3) mitral and tricuspid annulus dimensions measured in the transverse, four-chamber view of the fetal heart; aortic and pulmonary annulus dimension assessed in modified transverse imaging planes; (4) RV and LV areas at end-diastole by digital planimetry in the four-chamber view of the heart; (5) qualitative assessments of both LV and RV systolic function (characterized as normal, mildly depressed, moderately depressed, or severely depressed).

Pulsed- and continuous-wave Doppler examination
Using pulsed wave Doppler, fetal heart rate was calculated from the Doppler tracings. Pulsed- or continuous-wave Doppler, or both, was used to assess gradients across the site of banding.

Color flow Doppler examination
Color flow Doppler imaging of the mitral and tricuspid valves was performed to assess for atrioventricular valve insufficiency. The assessment of the flow pattern across the foramen ovale and in the transverse aortic arch was performed after banding.

In addition to the echocardiographic evaluations, limited ultrasonography studies were performed every other day to confirm fetal viability and to rule out hydrops.

Fetal Cardiac Catheterization
Fetal hemodynamic measurements were obtained at term in one of two fashions. In fetuses undergoing planned angiography, measurements were obtained under single plane fluoroscopic guidance after femoral arterial and venous access was gained by surgical cut-down. In the remainder of the fetuses, and in those with hydrops, we carried out a more rapid and direct chamber pressure measurements after a median sternotomy. As in the echocardiography studies, no sedatives were given to the fetuses.

Measurements were obtained in sequence from the superior vena cava, right atrium, right ventricle, branch pulmonary artery, descending aorta, ascending aorta, left ventricle and left atrium. Angiography was performed after collecting all oximetric and hemodynamic values. Nonionic contrast (1 mL/kg) was injected by hand through the arterial catheter in the ascending aorta to define arch anatomy and delineate the area of the aortic band.

Pathology and Histology Evaluation
After euthanasia (intravenous pentobarbital at 100 mg/kg), the heart was quickly extirpated and examined. Fetal autopsy was performed after completion of cardiac-specific morphometry measurements and recovery of samples for storage at –80°C. Subcutaneous edema, effusions, ascites, or evidence of lung or liver congestion were noted. Tissue samples (1 to 1.5 cm in size) were removed from each of the heart, lung, and liver specimens for histopathology study. The tissue was incubated overnight at 4°C in 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.4, then rinsed in PBS and cryoprotected by infiltration of 30% sucrose in PBS at 4°C before sectioning on a microtome. After routine histology processing, the mean transverse diameter of 100 cardiomyocytes was measured in 10 random regions of the interventricular septum of each fetus by light microscopy. Cellular morphometric analysis was performed to determine total nuclear surface area and length on slices stained with Harris hematoxylin after treatment with 1% periodic acid. We assessed cardiac fibrosis by staining with Sirius red.

Owing to necrosis and postmortem changes related to intrauterine resorption, only limited evaluations were performed on fetuses that died intrauterine.

Statistical Analysis
Univariate analysis using the mean ± SD or median (range) was calculated for each outcome measure. For serial data, the values for each animal were first calculated over the serial time points before calculating mean and median values for each group. Group comparisons were made using independent t test and a nonparametric Mann-Whitney U test when appropriate. Statistical significance was assigned for p value less than 0.05. Because of the serial measurements taken for the echocardiography data, generalized estimating equations in a generalized linear models framework were used to compare groups. Each outcome was modeled as a function of the group while adjusting for gestational age as a confounder. Correlation between morphometric and echocardiography measurements was investigated using Pearson correlation using the mean values of echocardiography measurements taken for each animal. All statistical analyses were conducted using the statistical program SAS V9.1 (SAS Institute, Cary, North Carolina).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Outcome of Aortic Banding in Early Fetal Life
Forty fetuses underwent the experimental protocol at a median gestational age of 53 days (approximately 14 weeks’ human gestation; Fig 1). In the control group (n = 15), there were 3 perioperative deaths (20%), defined as death within the 7-day recovery period after surgery; and in the banded animals (n = 25), there were 6 perioperative losses (24%). Autopsy of these fetuses demonstrated an average fetal weight of 143 ± 11.5 g.


Figure 1
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  		Fig 1. Representative photographs of first-trimester sheep fetus (A) and fetal heart (B). The white block arrow points to the band on the ascending aorta.

 
The outcome of fetuses surviving surgery is shown in Figure 2. Ten animals had intrauterine fetal death, the majority (85%) occurring at approximately 105 to 110 days of gestation. Gestational age at banding did not correlate with these deaths. In the control group, there was a 27% intrauterine fetal death rate (4 of 15). There was a 24% intrauterine death rate (6 of 25) in banded animals. Limited autopsy of these fetuses did not show evidence of fetal compromise. Similarly, serial ultrasonographic studies of these fetuses were not suspect for fetal distress (no bradycardia or evidence of hydrops) before their death. An approximately 25% spontaneous intrauterine death or abortive loss is consistent with other fetal lamb models [9].


Figure 2
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  		Fig 2. Outcome of aortic banding in early fetal life. Upper time line corresponds to the control group, and the lower time line to the experimental group. The markings along the time lines correspond to the progressing gestational age in days (d). Compensatory left ventricular hypertrophy (COMP LVH) and left ventricular (LV) dilatation refer to the two distinct phenotypic groups described in text. (POD = perioperative death; IUD = intrauterine death.)

 
The remaining fetuses had one of two phenotypes in response to aortic banding: compensatory LVH (7 of 25, approximately 28%), and severe LV dilation and dysfunction with fetal distress as evidenced by hydrops (6 of 25; approximately 24%). Gestational age at banding did not correlate with either phenotype.

In Utero History of the Model
We performed serial fetal echocardiograms to study the in utero history of our model. While data acquisition was not feasible in all animals because of poor imaging windows or lack of cooperation by the unsedated animals, serial data were obtained in 13 experimental fetuses and 13 control fetuses. We obtained a median of 4 serial fetal echocardiograms (range, 1 to 8) in each fetus.

In comparison with control animals, significant and progressive LVH developed in a subset of the banded animals, manifested as an increase in LV free wall thickness (p = 0.01; Fig 3) and ventricular septal thickness (p = 0.01). Left ventricular systolic function was preserved, and there was no evidence of reversal of flow across either the foramen ovale or transverse aortic arch in these animals. All other LV morphometric indices measured on echocardiography were comparable to control animals, and the LV remained apex-forming throughout gestation (data not shown). Mitral and aortic valve annular dimensions were not significantly different when compared with controls.


Figure 3
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  		Fig 3. Progressive in vivo increase in left ventricle free wall (LVFW) thickness during pregnancy in response to obstruction to blood flow. The LVFW thickness is plotted on the y-axis and the gestational age is on the x-axis. The values (mm) for banded fetuses are plotted using squares, the control animals are plotted using diamonds. (d = days.)

 
In contrast, in another subset of animals, fetal hydrops developed in response to banding as manifested by pleural and pericardial effusion and ascites (Fig 4A). Hydrops developed at a mean gestational age of 94 days (range, 89 to 101), equivalent to approximately 25 weeks of human gestation. They had poorly functioning and dilated left ventricles (Fig 4B) and severe mitral regurgitation (Fig 4C). The first two hydropic fetuses died within 2 weeks from onset of hydrops, preventing us from obtaining data; therefore, we examined subsequent hydropic fetuses (n = 4) when death appeared imminent as evidenced by relative fetal bradycardia (median, 3 weeks after onset of hydrops; range, 1 to 4). On serial echocardiography, these fetuses had LV free wall thinning and chamber dilation (with significant increase in LV diastolic dimension [p = 0.01], LV diastolic area [p = 0.02], and LV length [p = 0.01]) with progressing gestation. In none, however, did LV hypoplasia develop.


Figure 4
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  		Fig 4. (A) Fetal hydrops secondary to aortic banding. Note the severe and generalized edema. (B) Transverse short-axis view of the heart from same fetus. (C) Color-Doppler long-axis view of the same heart showing severe mitral regurgitation.

 
Elevated LVEDP and the Fetal LV
Cardiac catheterization (Table 1) and angiography (Fig 5A) were performed to assess the fetal hemodynamics in 6 fetuses with compensatory LVH, 4 hydropic fetuses, and 5 controls. Blood gas values for control fetuses were within normal range (pH = 7.35 ± 0.01; pCO2 = 49.5 ± 0.5; pO2 = 21.4 ± 1.2). As expected, no pressure difference was noted across the ascending aorta of control animals, and the mean LVEDP and left atrial pressures measured 6.8 ± 0.8 mm Hg and 7 ± 0.8 mm Hg, comparable to previously reported normal values [6, 10].


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  		Table 1. Fetal Hemodynamics
 

Figure 5
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  		Fig 5. (A) Left ventricle angiogram at term from a fetus with compensatory left ventricular hypertrophy. Note the severity of supravalvar aortic stenosis. The findings correlate with the pathology findings in the same fetus seen in (B), showing severe narrowing of the ascending aorta at the level of the band (black arrows).

 
Blood gas values for fetuses with compensatory LVH were also within normal range (pH = 7.33 ± 0.05; pCO2 = 50.5 ± 1.3; pO2 = 22.8 ± 1.3). In these fetuses, a mean pressure gradient of 52.2 ± 5.5 mm Hg (range, 35 to 73) was measured from the left ventricle to the ascending aorta across the banded area, consistent with severe narrowing seen on pathology study (Fig 5B). Moreover, in this group, the mean LVEDP and left atrial pressures measured 14.2 ± 1.3 mm Hg and 9.7 ± 0.08 mm Hg, respectively (p = 0.02 versus controls). Of note, two fetuses with the highest LVEDP (15 mm Hg and 16 mm Hg, respectively) had the lowest gradients (40 mm Hg and 35 mm Hg, respectively). In addition to the significant differences in band gradients (p = 0.01), the mean values for LVEDP (p = 0.02), LV systolic (p = 0.01), and superior vena cava pressures (p = 0.02) were also significantly higher in fetuses with compensatory LVH than in control fetuses. There was a trend toward higher left atrial pressures that did not reach statistical significance (p = 0.06).

Blood gas values from hydropic fetuses demonstrated evidence of hypoxemia and acidosis, consistent with their compromised hemodynamic state (pH = 7.13 ± 0.04; pCO2 = 67.3 ± 3.6; pO2 = 11.3 ± 2.2). In 2 of these fetuses, the hemodynamic studies were terminated early owing to severe bradycardia. The other 2 fetuses (with complete hemodynamic data) demonstrated mean pressure differences of 18 and 23 mm Hg, respectively, across the banded area. At the same time, these fetuses had LVEDP values of 23 and 28 mm Hg and left atrial pressures of 19 and 23 mm Hg, respectively. All four hydropic fetuses had elevated mean superior vena cava pressure (21.8 ± 3.1 mm Hg) and right atrial pressure (25.2 ± 1.6 mm Hg).

"Relative" LV Hypoplasia
To correlate autopsy and pathology findings to fetal echocardiography observations, we measured cardiac morphometry, according to Figure 6A. The data are shown in Table 2. Similar to echocardiography findings, an increase in LV free wall thickness was seen in the pathology specimens from fetuses with compensatory LVH (Fig 6B). In contrast to echocardiography findings, however, these specimens showed a decrease in LV inner diameter, suggesting apparent LV hypoplasia. Although there was a trend toward decreased mitral valve annulus size in these animals, the difference did not reach statistical significance.


Figure 6
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  		Fig 6. (A) Cross-sectional diagram of morphometric measurements recorded at pathology. The cut surface is at the equator of the heart, corresponding to the midportion of the papillary muscles, with (a), (b), (c), (d), and (e) corresponding to the left ventricle (LV) outer diameter, LV inner diameter, LV free wall thickness, interventricular septal wall thickness, and right ventricle (RV) free wall thickness, respectively. (B) Cardiac cross-sections of pathology specimens demonstrating ventricular cavity size and wall thickness at term. The heart of a fetus with compensatory left ventricular hypertrophy (COMP LVH [left]) and a control animal (right) are shown. Note the apparent reduction in LV chamber volume in the heart from the fetus with compensatory LVH.

 

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  		Table 2. Cardiac Morphometry
 
Owing to the small sample size, both parametric and nonparametric methods were used for analysis of hydropic fetuses. For outcomes found to be statistically significant, we further used regression analysis to account for the obvious differential on estimated gestational age (hydropic fetuses being younger). When compared with controls or fetuses with LVH, after adjusting for this age differential, LV weight (p = 0.03; p = 0.04), the aortic root to LV apex distance (p = 0.002; p = 0.005), and LV inner diameter (p = 0.01; p = 0.04) and outer diameter (p = 0.005; p = 0.002) were significantly larger in the hydropic fetuses, consistent with the severe LV dilatation and dysfunction seen on echocardiography. Of note, RV weight was significantly increased (p = 0.03) in the hydropic fetuses when compared with the control cohort (but not when compared with fetuses with compensatory LVH), suggesting transference of some hemodynamic load to the right ventricle. The RV values were otherwise all within normal range.

Severe Pressure Overload Does Not Lead to EFE
Evaluation of histology demonstrated no evidence of cardiomyocyte hypertrophy in any of the fetuses with aortic stenosis by comparison with normal, age-matched controls (Fig 7). Also, although there was a trend toward increased number of binucleated cells, this was not statistically significant (data not shown). No myocardial fiber disarray was noted. These results suggest that myocyte hyperplasia is the predominant mechanism for increased LV free wall thickness. Finally, no EFE was noted in any of the heart specimens, nor was there any evidence of increased fibrosis.


Figure 7
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  		Fig 7. Representative hematoxylin and eosin stains of myocardial specimens from banded fetuses (left) and control fetuses (right) at x40 magnification taken from the left ventricular free wall. Note the absence of significant cellular hypertrophy in the banded animals.

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
Mechanisms leading to hypoplasia in the developing fetal heart remain unknown. Various etiologies, including premature closure of foramen ovale, embryonic coronary compromise, LV inflow limitation, and LV outflow limitation, have been suggested [11]. Central to these potential etiologies is the "flow theory," which states that obstruction to flow through the developing ventricle leads to its underdevelopment [5]. Great attention has been recently directed toward mechanisms of potential LV outflow limitation such a fetal aortic stenosis [6, 11–17], in part because of ongoing experimental fetal interventions that have resulted in a few, but intriguing successes [1, 18].

Despite its appeal, the flow theory does not explain some inconsistencies. For example, even with fetal aortic stenosis and hypoplastic left heart syndrome, the theory fails to account for such observations as hypoplasia in the setting of an absent aortic valve [19–21], large ventricular septal defects (that allow for unobstructed flow through the LV) [11, 22], or in hearts with otherwise normal aortic and mitral valves [11, 17, 23]. Similarly, the flow theory does not account for why some fetuses go on to develop hypoplasia despite successful in utero treatment [18], whereas still others with severe obstruction (manifested by retrograde flow in the transverse arch) do not [14, 15].

Our results relate to the preceding and raise further questions for future investigations. First, in contrast to previous studies, we have found that aortic banding is not sufficient to cause LV hypoplasia, even when performed early in gestation. Previous experimental studies had suggested that this model (aortic banding) can lead to mild LV hypoplasia [6, 7]. Although our pathology findings were consistent with these previous reports and suggested a degree of LV hypoplasia, in vivo echocardiographic findings did not demonstrate any evidence of LV growth inhibition. Previous investigators would not have recognized this discordance, likely because of postmortem changes in autopsy specimens, without real-time fetal echocardiography. Therefore, future investigators would benefit from trying an alternate model and ensuring autopsy studies are complemented with in vivo data.

Second, it has been suggested that LV hypoplasia progresses through an intermediate and requisite stage characterized by severe LV dysfunction and dilatation [18]. Because of the parallel nature of fetal circulation, classic textbook teaching has been that compromise of the fetal LV function is relatively inconsequential to the fetus, with the right ventricle being able to adequately compensate [5]. Although a few clinical case series have clearly shown progression from LV dilatation to LV hypoplasia in some babies [12, 15–17], in many other instances, the left ventricle is already hypoplastic on the early fetal echocardiogram (eg, 12 weeks’ gestation) [24]. In our model, none of the fetuses with severe LV compromise and dilatation went on to have LV hypoplasia; rather, these findings were harbingers of imminent fetal death, as confirmed by recent clinical series [24–26].

Third, elevated LVEDP in response to pressure overload of the left ventricle has been suggested as an explanation for the commonly observed EFE in hearts affected by LV hypoplasia [12, 13, 27–29]. Increased impedance to ejection and a produced pressure gradient with peak intraventricular pressures exceeding systolic pressures are believed to cause increased wall stress and then compensatory LV hypertrophy that normalizes the stress [30, 31]. This adaptive mechanism places the endocardium at risk for hypoperfusion and ischemia because of the elevated LVEDP [32]. In our experiments, none of the fetuses demonstrated evidence of EFE or fibrosis, even in the setting of severe LV dilatation or elevated LVEDP.

Our studies have an important limitation: the location of the band. An obstruction below the coronaries has a different effect on myocardial blood supply than one above (although one might anticipate some subendocardial ischemia with such high LVEDPs as seen in our fetuses). We have made several attempts at creating an aortic banding model below the coronaries without success; 1 animal survived this approach (out of 10 tries) only to succumb 2 weeks later with evidence of hydrops (but no LV hypoplasia). We have recently begun an alternate approach combining the current model with coronary artery banding, trying to differentiate the effects of pressure overload from coronary flow changes. It is also possible that the timing of banding in our model may still be too "late" to produce hypoplasia or EFE. Advanced EFE has been documented in humans at 12 to 14 weeks’ gestation [33]. Our model does not examine the effects of banding earlier in fetal development, and therefore does not exclude the possibility of a causal relationship between pressure load, elevated LVEDP, and the formation of EFE if the flow perturbation occurs earlier in gestation. In addition, our model looks at the role of obstruction in the fetal period, after formation of a four-chambered heart. Other studies have shown that obstruction to flow during embryonic period can lead to significant perturbations in the developing heart with many severe malformations, including LV hypoplasia [11]. We cannot address this "timing" question, as earlier banding of the fetal aorta has not been technically possible in our hands. Our study is also limited by analysis of data obtained from surviving fetuses. It is conceivable, had it been possible, that data gained from these other animals would have shown different hemodynamics or perhaps evidence of EFE or alternative cardiac pathology; these fetuses, however, died invariably without prior echocardiographic evidence of cardiac compromise. Finally, in our model, the rate of stenosis is dependent on gradual and progressive growth of the fetus and its great vessels. That may or may not represent the rate of progression of disease in fetal aortic stenosis.

We failed to accomplish our original goal with these studies—developing an animal model of LV hypoplasia. Nevertheless, our findings provide valuable insight to future investigators. Mechanisms leading to EFE or LV hypoplasia are likely more complex than previously proposed. It is possible that changes in cardiac flow patterns do not cause, but rather are a consequence of, developing hypoplastic left heart syndrome. Importantly, in most convincing animal models (eg, chick embryos), the harmful effects of flow perturbation have been shown during embryonic life, namely, before the formation of the four-chambered heart [11, 34]. Closer examination of these concepts is warranted, especially when proposed as justification for intervention during fetal life [2].

Some have argued that hypoplastic left heart syndrome is a predetermined program of abnormal myocardial development, and unrelated to changes in blood flow, based on histologically disorganized myocardial fibers seen in some autopsy specimens [4, 35, 36]. Our model does not address other proposed mechanisms such as premature closure of foramen ovale, a model that we are currently attempting in our laboratory. It is possible, however, that beyond flow, other mechanisms (eg, genetic or immunologic) play a part in manifestation of these pathologic changes. Finally, future gene-expression studies, for example using microarrays, may provide valuable insight into molecular changes behind the abnormal fetal heart development.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This work was supported by the TRI grant of CHRF and charitable contributions of Cincinnati Children’s Heart Association. The authors also wish to thank Ms Dawn Burman for her technical assistance on this project.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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