Introduction

Pulmonary hypertension is a syndrome characterized by elevated pulmonary arterial pressure. Pulmonary hypertension occurs in a variety of clinical situations and differs in aetiology, therapy and prognosis. Recommendations for the diagnosis, classification and treatment of pulmonary hypertension in children are regularly updated by professional societies [1]. Acute and chronic right ventricular failure contributes significantly to morbidity and mortality of children with pulmonary hypertension.

Pulmonary artery banding (PAB) has been used in prevention of pulmonary hypertension as part of surgical palliation of various congenital heart defects in the paediatric population for almost six decades now [2, 3]. PAB reduces excessive pulmonary blood flow caused by congenital heart defect and protect pulmonary circulation from irreversible changes leading to severe pulmonary hypertension. The main indications currently include complex congenital heart defects where almost 90 % of the patients require early palliative surgical treatment [4]. PAB represents usually the first step in a series of palliations in selected patients from this group. Progress in operative and perioperative management of small infants with haemodynamically significant intracardiac left-to-right shunt (ventricular septal defect, atrioventricular septal defect) led to decreased use of pulmonary artery banding in the treatment of such patients following its initial wide and successful use [5–7].

The aims of the present experimental study were based on clinical practice. Signs of pressure overload of the right ventricle are frequently found in critically ill children, and therefore, the right ventricle is called “the intensivists chamber”. Our experimental study was designed as a prospective and comparative one.

Materials and Methods

The study was conducted with the approval of a multidisciplinary institutional ethics committee in an EU-accredited experimental centre of the Faculty of Medicine of Charles University in Pilsen, Czech Republic, where all animals receive humane care in compliance with the European Convention on Animal Care. The project was approved by the Faculty Committee for the Prevention of Cruelty to Animals, respecting the principles of Declaration of Helsinki.

The black mottled breed of domestic pig was obtained from an accredited breeding station for laboratory use. The study included a total of 12 clinically healthy piglets of 42 days of age with average weight of 21.5 kg (range 20.25–22.75 kg).

General Anaesthesia and Ventilator Support

Premedication was given by intramuscular administration of atropine 0.07 mg/kg (Atropin inj. sol., Spofa, Prague, Czech Republic) and azaperone 5.0 mg/kg (Stresnil inj. sol, Janssen Pharmaceutica NV, Belgium). Marginal ear veins were cannulated. General anaesthesia was induced by intravenous application of thiopental 10.0 mg/kg (Thiopental Valeant inj. sol., VUAB Pharma, Czech Republic), and all animals were orotracheally intubated. Intubated animals were mechanically ventilated using servo-ventilator (Siemens Elema 900C, Germany) in pressure-controlled mode while maintaining the tidal volume at 7.0 ml/kg by the following settings: T insp 0.7 s, Pinsp 10 cmH20, BR 28/min., I:E 0.4, PEEP 7 cmH20, FiO2 0.4. Combined general anesthesia was maintained by intravenous fentanyl 0.5–1.0 μg/kg (Fentanyl-Janssen inj. sol., GlaxoSmithKline Manufacturing S.p.A., Italy), azaperone 3.0–4.0 mg/kg and verocuronium 0.08–0.1 mg/kg (Norcuron 4 mg inj. sol., Schering-Plough S.A., Eragny-sur-Epte, France) [8]. The heart and breath rate, blood pressures, ECG curves and pulse oximetry were monitored using bedside monitor (Nihon Kohden, Tokyo, Japan).

Animal Preparation

Pulmonary artery bands were placed on the pulmonary trunk about one centimetre above pulmonary valve annulus through median sternotomy or left parasternal thoracotomy in 12 pigs using a 0.6-mm-thick strip of Gore-Tex. The bands were fixed by a Prolene suture (6/0 or 7/0) to constrict the main pulmonary artery to about 70–80 % of its original dimension. The thoracotomy was then closed. During the operation, each animal received a prophylactic dose of aminopenicillin–clavulanate 1500 mg (Amoksiklav inj., Sandoz and Novartis, Slovenia) intravenously.

After surgery, the animals were placed in special heated stabling and were received granted post-operative care lasting an average of 40 days, including a defined nutritionally complete diet.

Study Protocol

The animals were randomized into two groups and clearly marked. Animals with PAB in group E (n = 4) were administered enoximone 1.5 ± 0.8 mg/kg three times daily orally (Perfan inj. sol., Carinopharm GmbH, Germany) for a total of 40 days. Nutritional granules were always saturated with a fresh solution of enoximone and administered to animals three times daily. In the control group C (n = 8) were included piglets with PAB without enoximone treatment.

To compare the morphological changes between groups C and E, samples of heart tissues of the remaining animals were used.

Blood Pressure Measurements and Echocardiographic Assessment

Invasive blood pressure measurements were taken by direct cannulation of the chambers of the heart and great arteries during an operation under general anaesthesia and mechanical ventilation. Of the directly measured data, we calculated the systolic pressure gradient across the PAB (PG).

Following PAB and closure of chest cavity, transthoracic echocardiography was performed still under general anaesthesia using continuous-wave Doppler interrogation of blood flow targeted by two-dimensional pictures with 3.5 MHz probe (Sonoline Siem, Siemens, Germany). Doppler pulmonary artery flow velocity was recorded within 2 cm of the valve. Systolic pressure gradient across PAB was estimated using main pulmonary artery peak systolic blood flow velocity and Bernoulli equation. Each measurement was taken three times, and the values were averaged.

To study histological myocardial changes, samples of right ventricular myocardium were taken from all animals prior to completion of the experiment.

All animals were euthanized at the end of the procedure by an intravenous bolus of cardioplegic 7.5 % potassium chlorate solution in a dose of 2.0–3.0 ml/kg (Kalium Chloratum 7.5 % sol. inj., Zentiva a.s., Czech Republic) while under general anaesthesia. The carcasses were disposed of in accordance with regulations of the Czech Republic and European Union.

Histological Processing and Quantification

All tissue samples were fixed immediately after collection in a neutral formaldehyde solution. Each sample was stained using four methods: haematoxylin and eosin stain was used for tracing morphological changes, the Van Gieson method was used for differential staining of connective tissue and cardiac muscle. Adjacent sections were processed immunohistochemically using Ventana Benchmark XT automated stain (Ventana Medical System, Inc., Tucson, AZ, USA) with diaminobenzidine visualization and counterstaining with Mayer’s haematoxylin.

For detection of apoptosis, we used immunohistochemical detection of the cleaved (i.e. activated) caspase 3 (polyclonal rabbit anti-human Signal Stain Asp175 detection kit, Cell Signalling Technology, Danvers, MA, USA). Activated caspase 3 is a critical executioner of apoptosis responsible for proteolysis of many key nuclear proteins.

For detection of proliferation, we used immunohistochemical detection of the Ki-67 antigen (monoclonal mouse anti-human, clone MIB-1, Dako, Glostrup, Denmark). Ki-67 is a nuclear protein preferentially expressed during active phases of the cell cycle (G1, S, G2 and M phases), but absent in resting cells (G0 phase). While the Ki-67 positivity was evaluated in the nuclei only, the caspase 3 positivity was both in nuclei and in the cytoplasm [9].

Subsequently, we calculated the proliferation index (PI) as a ratio between the Ki-67-positive nuclear profiles and the total number of nuclear myocyte profiles observed using the 40× objective. We counted the apoptotic index (AI) as a ratio between caspase 3-positive nucleated myocyte profiles and the total number of the nucleated myocyte profiles using the 40× objective. We used the VersaCount manual tally software [10] for counting PI and AI of the Ki-67- and Caspase 3-positive cells.

The fibrosis was assessed in sections stained with the Van Gieson method. Using a 20× objective, five fields of view were sampled from the sections in a systematic uniform random manner, but the vicinity of major coronary arteries and veins was excluded to avoid a bias caused by the connective tissue irradiating from the tunica adventitia of these vessels. We used the stereological point counting method [11] to calculate the ratio of points of a uniform stereological grid hitting the connective tissue to the cardiac myocytes (CT/CM). The point grid method was performed using the Ellipse software (ViDiTo, Košice, Slovakia).

Statistical Analysis

Parametric data were expressed as median, standard deviation (SD) and 95 % confidence interval (95 % CI). As some of the histological data sets did not pass normality test, we used the Mann–Whitney U test to test equality of population medians between the C and E groups. Correlation analysis (Spearman) and polynomial regression was used to compare the data obtained. Collinearity between variables (Pearson) was tested prior to modelling by computing the correlation of estimates, with a R 2  > 0.5 considered to be significant. For qualitative analysis of accuracy of the variables reference interval dispersion, the linearity, reproducibility agreements were used [12]. The data were processed with the Statistica Base 7 (StatSoft, Inc., Tulsa, OK, USA). All the results with p value <0.05 were considered statistically significant.

Results

Within 24 h after surgery, a total of four piglets died. These animals were not included in the study. The cause of death was acute cor pulmonale, unresponsive to appropriate treatment. Early post-operative morbidity in our study was 25 %.

Twelve piglets were clinically and haemodynamically evaluated. Among them were eight animals selected for histological assessment.

Clinical Evaluation

At termination of the study, all of the operated animals with PAB were less active and showed a clear exertional dyspnoea. After an average of 40.0 ± 1.5 days after surgery (at time-2), their weight was higher at 40.25 ± 2.16 kg (95 % CI 1.56) versus 21.5 ± 1.25 kg (at time-1; 95 % CI 0.87; p < 0.001).

Breath sounds were symmetric, without secondary phenomena. The visible mucous membranes were without cyanosis but with mild hypoxaemia (pulse oximetry SpO2 90 ± 1.52  %; 95 % CI 0.88 vs. 98 ± 1 .17  %; 95 % CI 0.92; p < 0.001) compared with time-1. An ejection systolic murmur could be heard in all animals with PAB. Heart rate was higher (p < 0.05) at HR 108 ± 7 beats/min (95 % CI 5.34) versus 86 ± 5 beats/min (95 % CI 3.67) compared with time-1. Subcutaneous oedema or other signs of fluid retention were not present. Also noticeable were the distension of peripheral veins and hepatomegaly. Clinical finding corresponded with right ventricular hypertension.

At time-2, there were ECG signs of right ventricular pressure load, i.e. the right-sided deviation of QRS complexes’ axis, peaked P wave, dominant R wave in right-sided precordial leads and inverted T waves over the right precordium with prolonged activation period of the right ventricle.

During thoracotomy at time-2, there was noticeable and obvious enlargement of the heart, affecting particularly right-sided heart chambers. When harvesting the heart tissue, we found a marked hypertrophy of the myocardium of the right ventricle without significant pericardial effusion in all animals with PAB.

Haemodynamics

A total of 648 parallel data sets including data on haemodynamics were acquired.

The differences in all directly measured haemodynamic values (n = 432) during the study period are summarized in Table 1.

Table 1 Differences in directly measured data during the study
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At time-2, there was higher (p < 0.01) systolic pressure gradient across the PAB (PG) (46.59 ± 15.87 mmHg; 95 % CI 12.46) compared to time-1 (20.29 ± 5.76 mmHg; 95 % CI 4.82). At termination of the experiment, PG correlated with RAP (Pearson’s R 2 = 0.847) and also with a weight of piglets (Pearson’s R 2 = 0.998). These correlations were not present at time-1.

A spontaneous increase in directly measured systolic pressure gradient across the PAB during the study period is shown in Fig. 1.

Fig. 1
figure 1

Increase in systolic pressure gradient across the PAB during the study

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Echocardiographic parameters measured (n = 216) also showed significant differences in haemodynamic data during the study with excellent reproducibility (p < 0.01) and acceptable diversity (p = 0.082). Differences in quality of examinations between the groups were not statistically significant (p = 0.623).

At time-2, there was higher (p < 0.01) echocardiographic systolic pressure gradient across the PAB (CW-Doppler PG) (52.93 ± 12.23 mmHg; 95 % CI 8.19) compared to time-1 (27.33 ± 7.45 mmHg; 95 % CI 5.77). Peak flow velocity (PFV) in the pulmonary artery was lower (p < 0.01) at 0.47 ± 0.28 m/s (95 % CI 0.12) compared to time-1 (0.81 ± 0.39 m/s; 95 % CI 0.18). Tricuspid valve regurgitation velocity (TVRV) was higher (p < 0.01) at time-2 (1.04 ± 0.87 m/s; 95 % CI 0.56) compared to time-1 (0.65 ± 0.24 m/s; 95 % CI 1.08).

When comparing all the haemodynamic data between groups C and E, there were no significant differences detected (p = 0.576).

Histological Assessment

For evaluation and comparison of the tissue samples, there were 28 evaluable blocks. All the samples were without signs of ischaemic damage, necrosis, interstitial oedema, haemorrhage, inflammatory infiltrates, or infarction scars. Apoptosis generally prevailed over proliferation, the latter being very rare in cardiac myocytes, but more frequent in interstitial cells. The fibrosis of the tissue had a medium positive correlation with the apoptotic index (Spearman’s R = 0.48, p < 0.05).

We found no difference between the C and E groups when comparing the values of the proliferation index, apoptotic index and the fibrosis of the samples.

Summary of original values is summarized in Table 2.

Table 2 Quantitative histology of samples under study
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The histological slides on the right ventricle myocardium of groups C and E are shown in Figs. 2, 3 and 4.

Fig. 2
figure 2

Caspase 3-positive apoptotic cells in right ventricle. The corresponding micrographs demonstrating the caspase 3-positive nuclear profiles in two animals from the control groups (a, b) and in two animals from the enoximone group (c, d). At least 1000 nuclear myocyte profiles were evaluated, and the ratio between the number of caspase 3-positive nuclear profiles of cardiac myocytes (red arrow in a) and the total number of nuclear myocyte profiles was expressed as the apoptotic index (AI). The AI was comparable in both groups. Immunohistochemical detection of the caspase 3 antigen (marker of apoptosis, positive nuclei stained dark brown), scale bar 50 µm (a–d)

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Fig. 3
figure 3

Ki-67-positive proliferating cells in right ventricle. The corresponding micrographs demonstrating the Ki-67-positive nuclear profiles in two animals from the control groups (a ,b) and in two animals from the enoximone group (c, d). At least 1000 nuclear myocyte profiles were evaluated, and the ratio between the number of Ki-67-positive nuclear profiles of cardiac myocytes (green arrow in a) and the total number of nuclear myocyte profiles was expressed as the proliferation index (PI). The PI was comparable in both groups. Immunohistochemical detection of the Ki-67 antigen (marker of cellular proliferation, positive nuclei stained dark brown), scale bar 50 µm (a–d)

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Fig. 4
figure 4

Connective tissue in right ventricle. The corresponding micrographs demonstrating the myocardium and connective tissue in two animals from the control groups (a, b) and in two animals from the enoximone group (c, d). Five fields of view were sampled from each tissue block, and the ratio between collagenous connective tissue (yellow arrow in b) and the cardiac muscle was expressed. The connective tissue-to-cardiac muscle ratio was comparable in both groups. Van Gieson’s stain (differentiating pink collagen connective tissue and light brown muscle tissue), scale bar 100 µm (a–d)

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When samples from all cardiac chambers were compared (right atrium, right ventricle, left atrium and left ventricle) between the C and E groups, there were no significant differences in the PI (p = 0.486), AI (p = 0.642) and CT/CM (p = 0.853).

Testing of samples from the right and left chambers between groups C and E did not show significant differences in PI (p = 0.156), AI (p = 0.698) and CT/CM (p = 0.519).

Likewise, samples from the right and left atrium during comparison between groups C and E do not show significant differences in PI (p = 0.698), AI (p = 0.796) and CT/CM (p = 0.897).

The number of high-quality samples was too small for detailed testing between different cardiac sections in groups and between groups. Comparison of the difference between the samples of groups C and E is shown in Fig. 5a–c.

Fig. 5
figure 5

Histological quantification of proliferation of cardiac myocytes (a), apoptosis of cardiac myocytes (b) and fibrosis of myocardium (c) in both groups under study. There was no difference between the C and E groups in any of the quantitatively assessed histological parameters: a proliferation index (Mann–Whitney U test p = 0.51), b apoptotic index (Mann–Whitney U test p = 0.66), c connective tissue-to-cardiac muscle ratio (Mann–Whitney U test p = 0.87). The data are presented as medians. The boxes span the limits of first and third quartile; the whiskers show the minimum–maximum range for each group

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Discussion

The present study created a clinically relevant experimental model of right ventricular pressure overload. Clinical findings in animals correspond with right-sided heart failure due to increased right ventricular afterload, which describes a number of clinical trials [13–15].

A number of experimental studies dealt with pulmonary hypertension and right heart pressure overload [16, 17].

The myocardial changes related to pressure-overload-induced right ventricular hypertrophy were studied in infant rabbits [18]. This study demonstrated that pressure overload increased cardiomyocyte apoptosis from 4 weeks post-operatively, and fibrosis occurred in the right ventricular myocardium at 8 weeks after operation. Apoptosis of right ventricular myocytes was progressive. Experimental work focusing on the development of left ventricular diastolic dysfunction after pulmonary artery banding in newborn rabbits showed 2–8 weeks after banding a direct correlation between the right ventricular pressure overload and right ventricular hypertrophy with diastolic dysfunction and molecular changes in left ventricular myocardium [19].

In our experimental study, we used the same methodology to achieve a pressure overload of the right ventricle by pulmonary artery banding. The work was performed on larger laboratory animals and was focused on wider range of parameters. We demonstrated the similar haemodynamic and morphologic changes of the right heart following PAB as in the preceding studies. Forty days after the banding, we demonstrated pulmonary diastolic dysfunction of the right ventricle with progressive increase in the systolic pressure gradient across PAB and also the right ventricular myocardial hypertrophy in growing animals. Our haemodynamic experimental model among other things also served to verify the potential benefit of oral treatment with a phosphodiesterase inhibitor, as alluded to by previous clinical and pharmacological studies [20, 21]. We used enoximone for this treatment [22, 23]. Our results differ from other experimental studies using similar model [24, 25]. We did not find significant differences in haemodynamic data between piglets without treatment (group C), and animals with oral treatment by enoximone (group E). Consequently, haemodynamic benefit of long-term oral treatment with enoximone was not demonstrated. In our prospective study, we quantified the apoptosis, proliferation and the fibrosis in a porcine model after 40 days of pulmonary artery banding. Unlike previous studies, we obtained evidence for induction of apoptosis, proliferation and fibrosis in the whole left heart [17–19, 21]. These morphological changes were identified in myocardium of the right-sided and left-sided cardiac chambers without significant differences. We found no differences between animals with or without administration of enoximone.

Limitations

Our work has a variety of limitations. Firstly, we were faced with high early post-operative mortality of animals. For this reason, we had a relatively small number of probands and tissue samples. We perceive our inability to determine the serum levels of enoximone due to no commercially or individually available essay as further limitation of our experiment. Finally, we were unable to study and quantify the connexins (four-pass transmembrane proteins) in our myocardial tissue samples. We took samples for this test, but did not get specialized institutional results. Despite the above-mentioned limitations, we consider results of our work of experimental and clinical importance.

Conclusions

We developed and validated experimental model of increased right ventricular afterload by pulmonary artery banding in a large growing animal. Our study did not show evidence of haemodynamic benefit of oral enoximone treatment in this setting.

We have shown that pulmonary artery banding leads to morphological myocardial changes including apoptosis, proliferation and fibrosis affecting myocardium of the right and left heart. No protective effect of oral administration of enoximone was demonstrated either.