The authors thank the team at the Laboratory of Surgical Research, Marie Lannelongue Hospital, for technical assistance and animal care. The VividE9 cardiac ultrasound system (General Electric Medical System) was financed by a grant from the Cardio-vasculaire-Ob&#233;sit&#233; Domaine D&#39;Int&#233;r&#234;t Majeur (CODDIM cod 100158, R&#233;gionIle-de-France, France).

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 &#8220;Principles of Laboratory Animal Care&#8221; formulated by the National Society for Medical Research and the &#8220;Guide for the Care and Use of Laboratory Animals&#8221; 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.
<ol>
	<li>Do not feed the animals 12 hr before the procedure.</li>
	<li>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&#160;before the procedure.8</li>
	<li>When the pig is sedated, insert a catheter into the ear vein.</li>
	<li>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.</li>
	<li>Place continuous monitoring devices on the piglet: continuous EKG, expiratory CO2 and oxymetry8,9. Insert an arterial fluid field catheter through the carotid artery under echography guidance to monitor the systemic arterial pressure8-10.</li>
	<li>Maintain general anesthesia with isoflurane (2%) in 100% oxygen supplement, continuous intravenous infusion of fentanyl (0.004 mg/kg) and propofol (3 mg/kg).</li>
	<li>Add an antibioprophylaxy with an injection of Cefatoxine (1 g) and Gentamycine (80 mg).</li>
	<li>Prevent intra operative and post operative pain with injection of nalbuphine (0.01 mg/kg) t.i.d.</li>
	<li>Check every 15 min&#160;the completeness of the anesthesia: absence of movement, stable heart rate, blood pressure, oxygenation.</li>
	<li>Use of vet ointment on eyes to prevent dryness while under anesthesia.</li>
</ol>
2. Ligation of the Left Pulmonary Artery
<ol>
	<li>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.</li>
	<li>Open the chest through a small left lateral thoracotomy (5 to 10 cm) in the 4th intercostals&#8217; space. Do not go to behind the tip of the scapula. Carefully retract the lung towards the diaphragm.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Embolization of the Right Lower Lobe Pulmonary Artery
<ol>
	<li>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.</li>
	<li>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&#176; angle direction).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#60; 90% and/or the mPA dropped under 60 mm Hg and/or the cardiac output was under 2 L/min.</li>
	<li>Remove the angiographic catheter and sheaths and perform digital compressions of the puncture site.</li>
</ol>
4. Hemodynamic Assessment
<ol>
	<li>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.</li>
	<li>Ventilate the piglets with the lowest FiO2 as possible according to the oxygen saturation (&#62; 95%).</li>
	<li>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&#176; angle direction).</li>
	<li>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 &#176;C.</li>
	<li>Record the following parameters: systolic, diastolic and mean systemic and pulmonary arterial pressure, heart rate, oxygen saturation, right atrial pressure, cardiac output.</li>
</ol>
5. Echocardiographic Assessment of the Right Ventricle
<ol>
	<li>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.</li>
</ol>
6. Pressure-Volume Loops Assessment with the Conductance Method
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Perform the pressure and volume calibration of the conductance probe according to the manufacturer&#8217;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.</li>
	<li>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.</li>
	<li>Control the quality of the loops. (Figure 6)</li>
	<li>Insert an ballon expendable in the inferior veina cava through the femoral vein. Place the extremity just below the right atrial. Use fluoroscopic guidance.</li>
	<li>Record pressure-volume loops of the right ventricle at basal state and during inferior vena cava occlusion as previously described10-12. (Figure 7)</li>
</ol>
7. Endomyocardial Biopsies of the Right Ventricle
<ol>
	<li>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.</li>
	<li>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&#8217;s tip is controlled by fluoroscopy and echocardiography.</li>
	<li>Insert the biotome in the long-sheath and perform endomyocardial biopsies under echographic, fluoroscopic and EKG control.</li>
</ol>
8. General Post-surgery Care Considerations
<ol>
	<li>After surgery, extubate the piglet only after recovery of spontaneous respiratory functions.</li>
	<li>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.</li>
	<li>Do not leave an animal that has undergone surgery to the company of other animals until fully recovered.</li>
</ol>
9. Euthanasia Method
<ol>
	<li>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 s&#233;v&#232;re&#160;: cyanosis, major dyspnea</li>
	<li>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.</li>
</ol>

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	<li>Simonneau, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Updated+clinical+classification+of+pulmonary+hypertension.">Updated clinical classification of pulmonary hypertension.</a> J Am Coll Cardiol. 54, S43-S54 (2009).</li><li>Dartevelle, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+thromboembolic+pulmonary+hypertension.">Chronic thromboembolic pulmonary hypertension.</a> Eur Respir J. 23 (4), 637-648 (2004).</li><li>Hoeper, M. M., Mayer, E., Simonneau, G., Rubin, L. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+microvascular+disease+in+chronic+thromboembolic+pulmonary+hypertension.">Pulmonary microvascular disease in chronic thromboembolic pulmonary hypertension.</a> Proc Am Thorac Soc. 3 (7), 571-576 (2006).</li><li>Humbert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+in+patients+with+idiopathic,+familial,+and+anorexigen-associated+pulmonary+arterial+hypertension+in+the+modern+management+era.">Survival in patients with idiopathic, familial, and anorexigen-associated pulmonary arterial hypertension in the modern management era.</a> Circulation. 122 (2), 156-163 (2010).</li><li>Guihaire, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+right+heart+failure:+a+window+for+translational+research+in+pulmonary+hypertension.">Experimental models of right heart failure: a window for translational research in pulmonary hypertension.</a> Semin Respir Crit Care Med. 34 (5), 689-699 (2013).</li><li>Mercier, O., Fadel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+thromboembolic+pulmonary+hypertension:+animal+models.">Chronic thromboembolic pulmonary hypertension: animal models.</a> Eur Respir J. 41 (5), 1200-1206 (2013).</li><li>Kaiser, G. 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A., Yamazaki, T., Burkhoff, D., Maughan, W. L., Sagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+left+ventricular+end-systolic+pressure-volume+relationships+by+the+conductance+(volume)+catheter+technique.">Determination of left ventricular end-systolic pressure-volume relationships by the conductance (volume) catheter technique.</a> Circulation. 73 (3), 586-595 (1986).</li><li>Mercier, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piglet+model+of+chronic+pulmonary+hypertension.">Piglet model of chronic pulmonary hypertension.</a> Pulm Circ. 3 (4), 908-915 (2013).</li><li>Brooks, H., Kirk, E. S., Vokonas, P. S., Urschel, C. W., Sonnenblick, E. 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No conflict of interest to disclose.

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...

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 embolisms1-3. 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 limitations6,7. 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 &#8220;sides effects&#8221; 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.

chronic thromboembolic pulmonary hypertension and assessment of right ventricular function in the piglet

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.

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.&#160;
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...

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

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.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

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12.2K Views.  Marie Lannelongue Hospital. 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.

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

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. 
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

Bioengineering

Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to pulmonary hypertension. For a better understanding and treatment of this disease, precise hemodynamic monitoring of left and right ventricular parameters is important. For this reason, it is essential to establish experimental pig models of cardiac hemodynamics and measurements for research purpose. 
This article shows the induction of ARDS by using oleic acid (OA) and consequent right ventricular dysfunction, as well as the instrumentation of the pigs and the data acquisition process that is needed to assess hemodynamic parameters. To achieve right ventricular dysfunction, we used oleic acid (OA) to cause ARDS and accompanied this with pulmonary artery hypertension (PAH). With this model of PAH and consecutive right ventricular dysfunction, many hemodynamic parameters can be measured, and right ventricular volume load can be detected. 
All vital parameters, including respiratory rate (RR), heart rate (HR) and body temperature were recorded throughout the whole experiment. Hemodynamic parameters including femoral artery pressure (FAP), aortic pressure (AP), right ventricular pressure (peak systolic, end systolic and end diastolic right ventricular pressure), central venous pressure (CVP), pulmonary artery pressure (PAP) and left arterial pressure (LAP) were measured as well as perfusion parameters including ascending aortic flow (AAF) and pulmonary artery flow (PAF). Hemodynamic measurements were performed using transcardiopulmonary thermodilution to provide cardiac output (CO). Furthermore, the PiCCO2 system (Pulse Contour Cardiac Output System 2) was used to receive parameters such as stroke volume variance (SVV), pulse pressure variance (PPV), as well as extravascular lung water (EVLW) and global end-diastolic volume (GEDV). Our monitoring procedure is suitable for detecting right ventricular dysfunction and monitoring hemodynamic findings before and after volume administration.

We present a protocol of creating right ventricular dysfunction in a pig model by inducing ARDS. We demonstrate invasive monitoring of left and right ventricular cardiac output using flow probes around the aorta and the pulmonary artery, as well as blood pressure measurements in the aorta and pulmonary artery.

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

A Novel Right Ventricular Volume and Pressure Loaded Piglet Heart Model for the Study of Tricuspid Valve Function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. Mechanistic studies to improve our&#160;understanding of the underlying pathophysiology responsible for the development of premature TV failure are lacking. Due to the inability to conduct these studies in humans, an animal model is required. In this manuscript, we describe the protocols for a novel chronic recovery infant piglet heart model for the study of changes in the TV when placed under combined volume and pressure stress. In this model, volume loading of the right ventricle&#160;and the TV is achieved through the disruption of the pulmonary valve. Then pressure loading is accomplished through the placement of a pulmonary artery band. The success of this model is assessed at four weeks post intervention surgery through echocardiography, intracardiac pressure measurement, and pathologic examination of the heart specimens.

A novel recovery piglet heart model with combined pressure and volume overload on the right ventricle is described for the study of tricuspid valve function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. ...

Closed Chest Biventricular Pressure-Volume Loop Recordings with Admittance Catheters in a Porcine Model

Pressure-volume (PV) loop recording enables the state-of-the-art investigation of load-independent variables of ventricular performance. Uni-ventricular evaluation is often performed in preclinical research. However, the right and left ventricles exert functional interdependence due to their parallel and serial connections, encouraging simultaneous evaluation of both ventricles. Furthermore, various pharmacological interventions may affect the ventricles and their preloads and afterloads differently. 
We describe our closed chest approach to admittance-based bi-ventricular PV loop recordings in a porcine model of acute right ventricular (RV) overload. We utilize minimally invasive techniques with all vascular accesses guided by ultrasound. PV catheters are positioned, under fluoroscopic guidance, to avoid thoracotomy in animals, as the closed chest approach maintains the relevant cardiopulmonary physiology. The admittance technology provides real-time PV loop recordings without the need for post-hoc processing. Furthermore, we explain some essential troubleshooting steps during critical timepoints of the presented procedure. 
The presented protocol is a reproducible and physiologically relevant approach to obtain a bi-ventricular cardiac PV loop recording in a large animal model. This can be applied to a large variety of cardiovascular animal research.

Here we present a closed chest approach to admittance-based bi-ventricular pressure-volume loop recordings in pigs with acute right ventricular dysfunction.

Pressure-volume (PV) loop recording enables the state-of-the-art investigation of load-independent variables of ventricular performance. Uni-ventricular ...

Chronic Ovine Model of Right Ventricular Failure and Functional Tricuspid Regurgitation

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to suboptimal clinical results. We set out to establish a chronic ovine model of FTR and right heart failure to investigate the mechanisms of FTR. Twenty adult male sheep (6-12 months old, 62 &#177; 7 kg) underwent a left thoracotomy and baseline echocardiography. A pulmonary artery band (PAB) was placed and cinched around the main pulmonary artery (PA) to at least double the systolic pulmonary artery pressure (SPAP), inducing right ventricular (RV) pressure overload and signs of RV dilatation. PAB acutely increased the SPAP from 21 &#177; 2 mmHg to 62 &#177; 2 mmHg. The animals were followed for 8 weeks, symptoms of heart failure were treated with diuretics, and surveillance echocardiography was used to assess for pleural and abdominal fluid collection. Three animals died during the follow-up period due to stroke, hemorrhage, and acute heart failure. After 2 months, a median sternotomy and epicardial echocardiography were performed. Of the surviving 17 animals, 3 developed mild tricuspid regurgitation, 3 developed moderate tricuspid regurgitation, and 11 developed severe tricuspid regurgitation. Eight weeks of pulmonary artery banding resulted in a stable chronic ovine model of right ventricular dysfunction and significant FTR. This large animal platform can be used to further investigate the structural and molecular basis of RV failure and functional tricuspid regurgitation.

Right ventricular failure and functional tricuspid regurgitation are associated with left-sided heart disease and pulmonary hypertension, which contribute significantly to morbidity and mortality in patients. Establishing a chronic ovine model to study right ventricular failure and functional tricuspid regurgitation will help in understanding their mechanisms, progression, and possible treatments.

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to ...

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

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