Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

Summary

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

Abstract

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Introduction

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) is a subtype of Pulmonary Hypertension (PH) due to chronic pulmonary vascular bed obstruction by persistent and organized clots related to one or more acute pulmonary embolisms^1-3. A combination of obstructive and non-obstructive microvascular disease leads to an additional increase in pulmonary vascular resistance⁴. The right ventricle must first adapt with compensated hypertrophy to maintain the cardiac output. Without treatment, the right ventricle dilates and fails over time. In the modern era, PH remains a progressive and often fatal disease despite the use of modern targeted therapies⁵. Many studies have shown that right ventricular (RV) adaptation to pressure overload is the main determinant of survival in PH patients. For that reason, understanding the mechanisms underlying the transition from adaptive to maladaptive RV remodeling is a keystone for treatment and development of new therapies. Because PH is rare and tissue sampling is almost infeasible in these frail patients, experimental studies are required. Furthermore, preclinical studies are mandatory to ascertain that a drug with benefit in the pulmonary vasculature does not cause RV impairment.

For many years, different experimental models of PH and RV failure have been developed with advantages and limitations^6,7. In the pharmacological murine models (Monocrotaline, SU5416, Hypoxia), PH and RV failure occur secondary to a massive inflammation, ischemia or toxic stressor that might induce several “sides effects” and bias in molecular pathway analysis. Furthemore, RV endomyocardial biopsies in a murine model should be very challenging without sacrifying the animal. Surgical models in larger animals are more physiological but do not affect the pulmonary vasculature (pulmonary artery banding, systemic-to-pulmonary shunt) or induce acute PH and RVF (acute pulmonary embolism). The aim of this article is to describe an original model of CTEPH in piglet that is more representative of CTEPH pathophysiology. This large animal model enables repeated noninvasive and invasive measurements usually performed in clinical practice (right heart catheterization) to follow changes in pulmonary hemodynamics and RV function.

Protocol

This protocol was approved by the local ethics committee on animal experiments and by the Institutional Committee on Animal Welfare of our institution. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996).

General considerations: All animals must be treated with respect, according to the 3Rs rules (National Centre for Replacement Refinement and Reduction of Animals in Research). Surgical procedures must be performed with a strict sterility and in the same way that for human beings. All medical devices must be sterile.

1. Anesthesia Protocol

NOTE: Large White piglets weighing 20 Kg (3-weeks-old) were used. Pulmonary Hypertension was induced progressively. The first step involved a left pulmonary artery (PA) ligation through a left thoracotomy. Following steps consisted of performing weekly PA embolizations during 5 weeks. All procedures were performed under general anesthesia.

1. Do not feed the animals 12 hr before the procedure.
2. Do a premedication of the piglet, perform an intramuscular injection using 30 mg/kg of Ketamine Hydrochloride and aropine 0.05 mg/kg in the neck muscles, 30 min before the procedure.⁸
3. When the pig is sedated, insert a catheter into the ear vein.
4. Perform intravenous a bolus of fentanyl (0.005 mg/kg) and propofol (2mg/kg) before intubation. Inject intravenously in the ear vein Cisatracurium (0.3 mg/kg) and intubate the pig (non-selective intubation with a 7 French probe)⁹.
5. Place continuous monitoring devices on the piglet: continuous EKG, expiratory CO₂ and oxymetry^8,9. Insert an arterial fluid field catheter through the carotid artery under echography guidance to monitor the systemic arterial pressure^8-10.
6. Maintain general anesthesia with isoflurane (2%) in 100% oxygen supplement, continuous intravenous infusion of fentanyl (0.004 mg/kg) and propofol (3 mg/kg).
7. Add an antibioprophylaxy with an injection of Cefatoxine (1 g) and Gentamycine (80 mg).
8. Prevent intra operative and post operative pain with injection of nalbuphine (0.01 mg/kg) t.i.d.
9. Check every 15 min the completeness of the anesthesia: absence of movement, stable heart rate, blood pressure, oxygenation.
10. Use of vet ointment on eyes to prevent dryness while under anesthesia.

2. Ligation of the Left Pulmonary Artery

1. Install the piglet in left side-lying position, shave the operative area and disinfect the skin with an alcoholic solution. Use a local sterile field.
2. Open the chest through a small left lateral thoracotomy (5 to 10 cm) in the 4^th intercostals’ space. Do not go to behind the tip of the scapula. Carefully retract the lung towards the diaphragm.
3. Once ideal surgical window located, retract the left azygos vein and dissect the main left pulmonary artery before tying it with a non-absorbable 2/0 silk.
   NOTE: It is very important not to open the pericardium.
4. Close the chest layer after layer with absorbable sutures. Use a chest tube to remove the post operative pneumothorax. Remove the chest tube just after the piglet extubation.

3. Embolization of the Right Lower Lobe Pulmonary Artery

1. After general anesthesia, put the piglet in a supine position. Carry out a continuous monitoring of the oxymetry, expiratory CO₂, EKG, mean systemic blood pressure (mPA) and mean pulmonary artery pressure (mPAP) throughout the procedure.
2. Insert all catheters percutaneously with echographic guidance. Insert an arterial 6 French catheter in the carotid artery for blood pressure monitoring and an 8 French sheath in the superior vena cava through the jugular vein (Perform the puncture 2 cm above the suprasternal notch with a 45° angle direction).
3. Under fluoroscopic guidance, insert, through the 8 Fr sheath, a 5 French angiographic catheter in the right pulmonary artery. The tip of the catheter must be in a segmental lower lobe pulmonary artery.
4. Prepare the material for pulmonary artery embolization: 1 ml of soft tissue glue containing N-butyl-2-cyanoacrylate is added to 2 ml of a lipidic contrast dye.
   CAUTION: Avoid skin or eyes contact with the N-butyl-2-cyanoacrylate.
5. When the angiographic catheter is well positioned, inject 0.2 ml to 0.4 ml of the preparation in the pulmonary artery. Assess the tolerance of the embolization by measuring the mPAP / mPA ratio that should not exceed 0.5. Stop embolization if oxygen saturation was < 90% and/or the mPA dropped under 60 mm Hg and/or the cardiac output was under 2 L/min.
6. Remove the angiographic catheter and sheaths and perform digital compressions of the puncture site.

4. Hemodynamic Assessment

1. After general anesthesia, position the piglet in a supine position. Carry out a continuous monitoring of the oxymetry, expiratory CO₂, EKG, systemic blood pressure (mPA) and pulmonary blood pressure (mPAP) throughout the procedure.
2. Ventilate the piglets with the lowest FiO₂ as possible according to the oxygen saturation (> 95%).
3. Insert all catheter percutaneously under echographic guidance. An arterial 6 French catheter is inserted in the carotid artery and an 8 French sheath is inserted in the superior vena cava (Perform the puncture 2 cm above the suprasternal notch with a 45° angle direction).
4. Insert a 7 French Swan-Ganz catheter in the pulmonary artery trunk. Assess the cardiac output by the thermo dilution technique, with injection of 10 ml of saline solution at 4 °C.
5. Record the following parameters: systolic, diastolic and mean systemic and pulmonary arterial pressure, heart rate, oxygen saturation, right atrial pressure, cardiac output.

5. Echocardiographic Assessment of the Right Ventricle

1. After general anesthesia, install the piglet in supine position and perform a trans thoracic echocardiography according human guidelines for RV screening. Record video loops during an end-expiratory pause.

6. Pressure-Volume Loops Assessment with the Conductance Method

1. After general anesthesia, install the piglet in a supine position. Carry out a continuous monitoring of the oxymetry, expiratory CO₂, EKG, systemic blood pressure (mPA) and pulmonary blood pressure (mPAP) throughout the procedure.
2. Insert an arterial 6 French catheter in the right or left carotid artery and an angiographic catheter in the left ventricle. Insert an 9 French sheath in the superior vena cava, an 8 French in the right or left femoral vein and an arterial PiCCO catheter in the right or left femoral artery. Insert all catheters percutaneously under echographic guidance.
3. Perform the pressure and volume calibration of the conductance probe according to the manufacturer’s recommendations. Measure the blood resistivity (Rho) by sampling 5 ml of arterial blood. Harvest 5 ml of arterial blood, de-air the syringe and fill the probe for the measurement of the blood resistivity. ^10-13.
4. Insert the conductance catheter in the right ventricle through the 9Fr sheath in the superior veina cava. Place properly the end of the catheter with fluoroscopic guidance. Place the end of the catheter in the apex of the right ventricle and insert all long as possible in the ventricle.
5. Control the quality of the loops. (Figure 6)
6. Insert an ballon expendable in the inferior veina cava through the femoral vein. Place the extremity just below the right atrial. Use fluoroscopic guidance.
7. Record pressure-volume loops of the right ventricle at basal state and during inferior vena cava occlusion as previously described^10-12. (Figure 7)

7. Endomyocardial Biopsies of the Right Ventricle

1. After general anesthesia, position the piglet in a supine position. Carry out a continuous monitoring of the oxymetry, expiratory CO₂, EKG, systemic blood pressure (mPA) and pulmonary blood pressure (mPAP) throughout the procedure.
2. Insert percutaneously a 10 French sheath in the superior vena cava. Insert a 7 French Swan-Ganz (SG) probe and a long 7.5 French catheter sheath in the right atrium. When the tip of the SG probe is well placed in the right ventricle (RV), inflate the balloon of the SG probe, push the long-sheath catheter in the RV against the balloon. Deflate the balloon and remove the SG probe leaving the long-sheath catheter in the RV. The good position of the long-sheath’s tip is controlled by fluoroscopy and echocardiography.
3. Insert the biotome in the long-sheath and perform endomyocardial biopsies under echographic, fluoroscopic and EKG control.

8. General Post-surgery Care Considerations

1. After surgery, extubate the piglet only after recovery of spontaneous respiratory functions.
2. For post operative medication, perform an intramuscular injection of Cefatoxine (1 g) Die for 5 days and deliver appropieted analgesia (Buprenorphin, intramusculary injection 0.01 mg/kg Bid for 10 days.
3. Do not leave an animal that has undergone surgery to the company of other animals until fully recovered.

9. Euthanasia Method

1. Euthanize the animals in the followed situations; End of experimentation, lost of weight 15% or more in one week, unibability to adequately feed themselves, anorexia, not treatable infection, pain or other didease, desease too sévère : cyanosis, major dyspnea
2. During general anesthesia with sevoluorane 8%, inject a high dose of propofol (0.5 mg/kg) associated with a lethal dose of potassium chloride (0.2 g/kg). When the heart is stopped in diastole, harveste the heart and lung bloc in order to do a histological and molecular study.

Results

Feasability

This piglet model of chronic post-embolic pulmonary hypertension has been established in our laboratory in 2009-2010. Since 2011, we used 70 piglets and we performed 63 completed models. In our experience, the realization of this model required a learning curve. 

Regarding the mortality, we observed 5 unplanned deaths (7.1%), mainly in the first part of our experience. The two critical steps were first, the pulmonary embolization and s...

Discussion

As in human clinical practice, respect of asepsis rules is mandatory during all surgical procedures. In the original CTEPH piglet model described by O. Mercier et al., the left pulmonary artery ligation was performed after opening the pericardium, through a median sternotomy¹⁴. Because the pericardium was left opened, the interaction between the right ventricle and the pericardium was impaired and right heart failure was delayed. Adverse effect of RV enlargement on cardiac output has been demonstrated...