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>

<ol>
	<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. J. <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> Circulation. 113 (16), 2011-2020 (2006).</li><li>Galie, N., Kim, N. 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. M., Heuer, M. M., Fruhauf, N. R., Kuhne, C. A., Broelsch, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+handling+and+anesthesia+for+experimental+surgery+in+pigs.">General handling and anesthesia for experimental surgery in pigs.</a> J Surg Res. 130 (1), 73-79 (2006).</li><li>Flegal, M. C., Kuhlman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+monitoring+equipment+for+laboratory+animals.">Anesthesia monitoring equipment for laboratory animals.</a> Lab Anim (NY). 33 (7), 31-36 (2004).</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=Right+ventricular+reserve+in+a+piglet+model+of+chronic+pulmonary+hypertension.">Right ventricular reserve in a piglet model of chronic pulmonary hypertension.</a> Eur Respir J. , (2014).</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=Right+ventricular+plasticity+in+a+porcine+model+of+chronic+pressure+overload.">Right ventricular plasticity in a porcine model of chronic pressure overload.</a> J Heart Lung Transplant. 33 (2), 194-202 (2014).</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=Non-invasive+indices+of+right+ventricular+function+are+markers+of+ventricular-arterial+coupling+rather+than+ventricular+contractility:+insights+from+a+porcine+model+of+chronic+pressure+overload.">Non-invasive indices of right ventricular function are markers of ventricular-arterial coupling rather than ventricular contractility: insights from a porcine model of chronic pressure overload.</a> Eur Heart J Cardiovasc Imaging. 14 (12), 1140-1149 (2013).</li><li>Kass, D. 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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+the+right+ventricle+under+stress:+relation+to+right+coronary+flow.">Performance of the right ventricle under stress: relation to right coronary flow.</a> J Clin Invest. 50 (10), 2176-2183 (1971).</li><li>Boulate, D., 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=Pulmonary+microvascular+lesions+regress+in+reperfused+chronic+thromboembolic+pulmonary+hypertension.">Pulmonary microvascular lesions regress in reperfused chronic thromboembolic pulmonary hypertension.</a> J Heart Lung Transplant. , (2014).</li></ol>

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...

Watch this Scientific Journal Video about Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet at JoVE.com

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. ...

chronic-thromboembolic-pulmonary-hypertension-assessment-right

Research

JoVE Journal

Medicine

12.1K 次观看.  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.

视频: 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, ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

A Pulmonary Trunk Banding Model of Pressure Overload Induced Right Ventricular Hypertrophy and Failure

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary disorders. Reliable and reproducible animal models of RV failure are therefore warranted in order to investigate disease mechanisms and effects of potential therapeutic strategies. Banding of the pulmonary trunk is a common method to induce isolated RV hypertrophy but in general, previously described models have not succeeded in creating a stable model of RV hypertrophy and failure.
We present a rat model of pressure overload induced RV hypertrophy caused by pulmonary trunk banding (PTB) that enables different phenotypes of RV hypertrophy with and without RV failure. We use a modified ligating clip applier to compress a titanium clip around the pulmonary trunk to a pre-set inner diameter. We use different clip diameters to induce different stages of disease progression from mild RV hypertrophy to decompensated RV failure.
RV hypertrophy develops consistently in rats subjected to the PTB procedure and depending on the diameter of the applied banding clip, we can accurately reproduce different disease severities ranging from compensated hypertrophy to severe decompensated RV failure with extra-cardiac manifestations.
The presented PTB model is a valid and robust model of pressure overload induced RV hypertrophy and failure that has several advantages to other banding models including high reproducibility and the possibility of inducing severe and decompensated RV failure.

We present a surgical method to induce right ventricular hypertrophy and failure in rats.

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary ...

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 ...

Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of group 2 PH, due to mitral stenosis (MS), to facilitate the investigation of disease mechanisms and potential therapeutic strategies. We present a novel rat model of pulmonary venous congestion-induced pulmonary venous arterialization and group 2 PH caused by left atrial stenosis (LAS). LAS is achieved by constricting the left atrium using a half-closed titanium clip. After the LAS surgery, a rat model with a transmitral inflow velocity greater than or equal to 2.0 m/s on echocardiography gradually develops pulmonary venous arterialization and group 2 PH over an 8- to 10-week period. In this protocol, we provide the step-by-step procedure of how to perform the LAS surgery. The presented LAS rat model mimics MS in humans and is useful for studying the underlying molecular mechanism of pulmonary venous arterialization and for the preclinical evaluation of therapies for group 2 PH.

Left atrial stenosis (LAS) is a novel surgical technique used for studying group 2 pulmonary hypertension (PH) and mechanisms underlying pulmonary venous arterialization. Here, we present a protocol to constrict the left atrium using a titanium clip to cause pulmonary venous arterialization and moderate PH in a rat.

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...

Speed

Subtitles

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