The authors acknowledge the Mitacs-Japan Society for the Promotion of Science (JSPS) Summer Program. Ping Yu Xiong was supported by funding from the Mitacs-JSPS Summer Program to visit the Jikei University School of Medicine. Dr. Minamisawa is supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (S.M.), the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (S.M.), the Vehicle Racing Commemorative Foundation (S.M.), and The Jikei University Graduate Research Fund (S.M.) with financial support for this project. Dr. Archer is supported in part by U.S. National Institutes of Health (NIH) grants NIH 1R01HL113003-01A1 (S.L.A.) and NIH 2R01HL071115-08 (S.L.A.), the Canada Foundation for Innovation, Tier 1 Canada Research Chair in Mitochondrial Dynamics and Translational Medicine (S.L.A.), the William J. Henderson Foundation, the Canadian Vascular Network, and the Queen&#39;s Cardiopulmonary Unit (QCPU).
The authors acknowledge Mr. Tadashi Kokubo, Chief of Photographic Services of the Academic Information Center at the Jikei University School of Medicine, for filming the video.

The LAS experimental protocol has been approved by the Jikei University School of Medicine Animal Care Committee and the University Research and Ethics Committee (protocol #2015-118).
1. Pre-operative Preparation
<ol>
	<li>After arriving at the animal facility, provide 5-week-old male Sprague Dawley rats between 150 to 200 g with 1 week to acclimate to their new home prior to the operation.</li>
	<li>Prepare the following equipment before the surgery by autoclaving: 1) a small animal respirator, 2) an anesthetic machine, 3) an intubation kit (composed of a pair of hemostat forceps, a tongue depressor, and an 18 G angiocatheter), 4) surgical instruments (which include a pair of curved forceps, a pair of straight forceps, a needle driver, a chest retractor, a pair of scissors, a 5-0 monofilament suture, a clip applicator, medium-large clips, and a 23 G chest tube).</li>
	<li>Have sterile Q-tips and gauze ready to deal with bleeding.</li>
	<li>Use a heating pad to maintain the animal body temperature around 37 &#176;C during the surgery.</li>
</ol>
2. Anesthesia and Endotracheal Intubation
<ol>
	<li>Anesthetize the rat in an induction chamber with 5% isoflurane mixed with 2 L/min room air.</li>
	<li>Prior to intubation, shave the rat&#39;s chest hair with a hair shaver and apply hair removal cream to remove fine hair.</li>
	<li>Check the pedal reflex to confirm successful anesthesia prior to intubation.</li>
	<li>Hook the front teeth with a string and secure the string with two pins.</li>
	<li>Open the rat&#39;s mouth with the hemostat forceps and insert the tongue depressor into the mouth.</li>
	<li>Lift the tongue depressor to visualize the vocal cord. 
	NOTE: It is helpful to shine a strong light over the head region of the rat to help visualize the vocal cord.</li>
	<li>Insert the 18 G angiocatheter as an endotracheal tube into the trachea, and then, quickly, connect the catheter to the respirator.</li>
	<li>Set the tidal volume to 10 &#181;L per gram with a respiratory rate of 100 breaths/minute.</li>
	<li>Maintain anesthesia with 2% isoflurane mixed with 2 L/min room air.</li>
</ol>
3. Preparation of the Surgical Site
<ol>
	<li>Prepare the surgical site with alternating scrubs of chlorhexidine and alcohol x3.</li>
	<li>Give buprenorphine 0.01 mg/kg subcutaneously.</li>
	<li>Cover the rat with a sterile drape.</li>
	<li>Check the pedal reflex to confirm a successful intubation and maintenance of anesthesia.</li>
</ol>
4. Left Atrial Stenosis Surgery
<ol>
	<li>Mark the incision site 2 cm below the rat&#39;s left armpit with a rule.</li>
	<li>Make a 2 cm left lateral chest wall incision with a pair of scissors.</li>
	<li>Separate the intercostal muscles between the fourth and the fifth rib, using the straight and the curved forceps, until entering the chest cavity.</li>
	<li>Insert the chest retractor into the chest cavity. Continue to use the straight and curved forceps to separate the intercostal muscle to obtain a direct visualization of the thymus and the heart.</li>
	<li>Lift the thymus with a pair of straight forceps. Remove the thymus covering the heart with a pair of scissors. Avoid cutting or poking into any major blood vessels.</li>
	<li>Carefully pass a 5-0 monofilament suture through the surface of the left ventricle, right below the left atrial appendage. Avoid passing the needle through the major coronary arteries.</li>
	<li>After the suture is in place and there is no significant bleeding, tie a loose knot.</li>
	<li>Pull the suture thread up and forward to lift the heart out of the chest.</li>
	<li>Once the heart is lifted out of the chest, quickly apply a medium-large clip to the left atrium, just above the mitral valve. 
	NOTE: The clip is half-way closed, with the tip of the clip pinching the left atrium, causing left atrial stenosis.</li>
	<li>Quickly put the heart back into the chest. Ensure the heart is not outside of the chest for longer than 30 s.</li>
	<li>Remove the stay suture used to lift the heart.</li>
	<li>Close the chest with a 5-0 monofilament suture, using a simple interrupted pattern.</li>
	<li>Insert a 23 G chest tube attached to a 10 mL syringe into the chest, and then, proceed with closing the chest wall muscle and skin with simple interrupted sutures.</li>
	<li>Draw out any air, blood, and pleural effusion via the inserted chest tube, using the attached 10 cc syringe, and then, pull the tube.</li>
	<li>Close the skin layer with a 5-0 monofilament suture, using a simple interrupted pattern.</li>
	<li>Give buprenorphine 0.01 mg/kg subcutaneously.</li>
	<li>Turn off the isoflurane.</li>
	<li>Disconnect the respirator after spontaneous respiration is observed.</li>
	<li>Keep the rat intubated and allow it to recover on the heating pad until it wakes up.</li>
	<li>Safely extubate the rat after one or more of the following signs is/are observed: the rat starts moving its four limbs, it regains its righting reflex, it regains its gag reflex, or it displays spontaneous voiding.</li>
</ol>
5. Post-operative Care
<ol>
	<li>Every 8 - 12 h, give buprenorphine 0.01 mg/kg subcutaneously. Carprofen 5 mg/kg is given subcutaneously on a daily basis for 2 days and, then, as needed, if the rat is not moving around well and looks like it is in pain.</li>
	<li>Give 5 mL of normal saline subcutaneously right after the surgery, as the rat may have difficulty drinking from the water spigot, immediately postoperative.</li>
</ol>
6. Confirmation of the Success of the Left Atrial Stenosis with Echocardiography
<ol>
	<li>Perform a transthoracic echocardiography 2 weeks after the surgery to determine the LV inflow velocity.</li>
	<li>Anesthetize the rat following the steps outlined in section 1.</li>
	<li>After the induction of anesthesia, maintain anesthesia using a nose cone with 2% isoflurane mixed with 2 L/min room air.</li>
	<li>Shave the rat&#39;s chest wall with a hair shaver and use hair removal cream to remove any remaining hair.</li>
	<li>Place the ultrasound probe at the apex of the heart, which is around the fifth intercostal space on the left side of the chest. Move the probe around in this region until a good four-chamber view is obtained.</li>
	<li>Measure the LV inflow velocity using the pulsed-wave Doppler mode just above the mitral valve annulus.</li>
	<li>An LV inflow velocity greater than 2.0 m/s is required for the development of moderate pulmonary hypertension at 8 - 10 weeks post-LAS surgery.</li>
</ol>
7. Sham Operation
<ol>
	<li>Except for applying the clip (step 4.9), perform all the steps above to create age-matched, sham-operated control (SOC) rats.</li>
</ol>

<ol><li>McGuire, L. B., Nolan, T. B., Reeve, R., Dammann Jr, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cor+Triatriatum+as+a+Problem+of+Adult+Heart+Disease%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Cor Triatriatum as a Problem of Adult Heart Disease.</a> Circulation. 31, 263-272 (1965).</li>
<li>Strange, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pulmonary+hypertension%3A+prevalence+and+mortality+in+the+Armadale+echocardiography+cohort%5BTitle%5D)+AND+%22Heart%22%5BJournal%5D)">Pulmonary hypertension: prevalence and mortality in the Armadale echocardiography cohort.</a> Heart. 98 (24), 1805-1811 (2012).</li>
<li>Moreira, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+of+Pulmonary+Hypertension+in+the+General+Population%3A+The+Rotterdam+Study%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Prevalence of Pulmonary Hypertension in the General Population: The Rotterdam Study.</a> PLoS ONE. 10 (6), e0130072(2015).</li>
<li>Wijeratne, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+Incidence+and+Prevalence+of+World+Health+Organization+Groups+1+to+4+Pulmonary+Hypertension%3A+A+Population-Based+Cohort+Study+in+Ontario%2C+Canada%5BTitle%5D)+AND+%22Circulation%3A+Cardiovascular+Quality+and+Outcomes%22%5BJournal%5D)">Increasing Incidence and Prevalence of World Health Organization Groups 1 to 4 Pulmonary Hypertension: A Population-Based Cohort Study in Ontario, Canada.</a> Circulation: Cardiovascular Quality and Outcomes. 11 (2), (2018).</li>
<li>Briongos Figuero, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predictors+of+persistent+pulmonary+hypertension+after+mitral+valve+replacement%5BTitle%5D)+AND+%22Heart+and+Vessels%22%5BJournal%5D)">Predictors of persistent pulmonary hypertension after mitral valve replacement.</a> Heart and Vessels. 31 (7), 1091-1099 (2016).</li>
<li>Jiang, B. H., Tardif, J. C., Shi, Y., Dupuis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bosentan+does+not+improve+pulmonary+hypertension+and+lung+remodeling+in+heart+failure%5BTitle%5D)+AND+%22European+Respiratory+Society%22%5BJournal%5D)">Bosentan does not improve pulmonary hypertension and lung remodeling in heart failure.</a> European Respiratory Society. 37 (3), 578-586 (2011).</li>
<li>Dayeh, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Echocardiographic+validation+of+pulmonary+hypertension+due+to+heart+failure+with+reduced+ejection+fraction+in+mice%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Echocardiographic validation of pulmonary hypertension due to heart failure with reduced ejection fraction in mice.</a> Scientific Reports. 8, (2018).</li>
<li>Yin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sildenafil+preserves+lung+endothelial+function+and+prevents+pulmonary+vascular+remodeling+in+a+rat+model+of+diastolic+heart+failure%5BTitle%5D)+AND+%22Circulation%3A+Heart+Failure%22%5BJournal%5D)">Sildenafil preserves lung endothelial function and prevents pulmonary vascular remodeling in a rat model of diastolic heart failure.</a> Circulation: Heart Failure. 4 (2), 198-206 (2011).</li>
<li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Effects+and+Mechanism+of+Atorvastatin+on+Pulmonary+Hypertension+Due+to+Left+Heart+Disease%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">The Effects and Mechanism of Atorvastatin on Pulmonary Hypertension Due to Left Heart Disease.</a> PLoS ONE. 11 (7), e0157171-e0157171 (2016).</li>
<li>Hunt, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pulmonary+veins+in+the+normal+lung+and+pulmonary+hypertension+due+to+left+heart+disease%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">Pulmonary veins in the normal lung and pulmonary hypertension due to left heart disease.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 305 (10), L725-L736 (2013).</li>
<li>Fujimoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pulmonary+hypertension+due+to+left+heart+disease+causes+intrapulmonary+venous+arterialization+in+rats%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Pulmonary hypertension due to left heart disease causes intrapulmonary venous arterialization in rats.</a> The Journal of Thoracic and Cardiovascular Surgery. 154 (5), 1742-1753 (2017).</li>
<li>Katz, M. G., Fargnoli, A. S., Hajjar, R. J., Hadri, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pulmonary+hypertension+arising+from+left+heart+disease+causes+intrapulmonary+venous+arterialization+in+rats%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Pulmonary hypertension arising from left heart disease causes intrapulmonary venous arterialization in rats.</a> The Journal of Thoracic and Cardiovascular Surgery. 155 (1), 281-282 (2018).</li>
<li>Alsoufi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Not+a+Cinderella+story%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Not a Cinderella story.</a> The Journal of Thoracic and Cardiovascular Surgery. 155 (1), 282-284 (2018).</li>
<li>Kato, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pulmonary+vein+stenosis+and+the+pathophysiology+of+%26%2334%3Bupstream%26%2334%3B+pulmonary+veins%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Pulmonary vein stenosis and the pathophysiology of &#34;upstream&#34; pulmonary veins.</a> The Journal of Thoracic and Cardiovascular Surgery. 148 (1), 245-253 (2014).</li>
</ol>

The authors have nothing to disclose.

The LAS rat is a novel group 2 PH model that has already received substantial interest from researchers in the field12,13. Comparing to the two existing group 2 models, namely the pulmonary vein stenosis (PVS) model14, using piglets, and the supracoronary aortic banding (SAB) rat model8,9,10, the LAS rat model has several advantages. Compared to t...

The purpose of this article is to demonstrate the step-by-step procedure of how to perform the LAS surgery in rats. Surgically induced LAS closely mimics MS and cor triatriatum in humans, which involve the creation of a mechanical obstruction in the left atrium1. Obstruction of the left ventricular (LV) inflow often causes a congestion of the pulmonary venous circulation, and patients gradually develop PH. The World Health Organization classifies PH due to left heart diseases as group 2, which is the most prevalent group of PH2,3,4. The diagnosis of PH in patients with left heart diseases is associated with a greater than a sevenfold increase in the 1-year standardized mortality4. Currently, there is no approved therapy for group 2 PH apart from treating the underlying left heart diseases (e.g., surgically replacing the stenotic mitral valve). However, even effective mitral valve replacement does not resolve PH fully in up to half of the patients with MS5. This persistent PH is due to adverse pulmonary vascular remodeling, which is poorly understood. Hence, animal models are important to enhancing our understanding of the underlying molecular mechanisms of adverse pulmonary vascular remodeling in group 2 PH.
There are a few animal models of group 2 PH. Coronary artery ligation6,7 and transverse aortic banding8,9,10 in rodents are the most commonly used group 2 PH animal models. The major disadvantage of these models is the involvement of LV, which makes the outcome of group 2 PH studies difficult to interpret. In contrast, the LV remains intact in the LAS model. Furthermore, the LAS model is clinically relevant because it results in the slow and progressive development of PH over a 10-week period11. In humans, MS is considered significant if the transmitral Doppler flow velocity is greater than 2.0 m/s11, and we also use this number as a cut-off to determine whether the LAS surgery has produced significant stenosis. Furthermore, although the LAS model generates mild or moderate PH, it demonstrates characteristic histologic changes, similar to those in human patients, namely the development of intrapulmonary venous arterialization11. The LAS rat model is a novel and clinically relevant group 2 PH model with preserved LV function. It is suitable for studying the pathophysiology of persistent pulmonary vascular remodeling, identifying molecular targets, and testing novel therapies for group 2 PH.

<table><tbody><tr><td>5-0 Prolene Suture</td><td>Johnson &amp;amp; Johnson - Ethicon</td><td>8725H</td><td>Polypropylene suture with HEMO-SEAL Technology</td></tr><tr><td>Anaesthesia Machine</td><td>Wakenyaku Co., Ltd.</td><td>BRTK-100A</td><td>Air pump and anaethesia machine</td></tr><tr><td>Angiocatheter guidewire</td><td>Self-made</td><td></td><td>10 cm guidewire glued to a 1 cc syringe&amp;nbsp;</td></tr><tr><td>Chest retractor</td><td>Natsume Seisakusho Co., Ltd.</td><td>F-2</td><td></td></tr><tr><td>Chest tube 23G</td><td>Self-made</td><td></td><td>10 cc syringe attached to a 23G needle plus plastic tube</td></tr><tr><td>Curved forceps</td><td>Natsume Seisakusho Co., Ltd.</td><td>A-14</td><td></td></tr><tr><td>Heating pad</td><td>Vivaria</td><td>MP-916-NV</td><td>Keep body temperature at 37 degree celsius</td></tr><tr><td>Horizon Ligating Clips</td><td>Teleflex</td><td>REF 003200</td><td>Size Medium-Large</td></tr><tr><td>Horizon Manual-Load Ligating Clip Applier For Medium-Large Size Horizon</td><td>Teleflex</td><td>REF 337085</td><td>Ligation Clips Angled Jaw, (20cm)</td></tr><tr><td>Needle holder</td><td>Natsume Seisakusho Co., Ltd.</td><td>MC-40</td><td></td></tr><tr><td>Rodent Respirator</td><td>CWE Inc</td><td>SAR-830/P</td><td>Small animal ventilator</td></tr><tr><td>Scissors</td><td>Natsume Seisakusho Co., Ltd.</td><td>B-12</td><td>Straight scissors ideally with round tips</td></tr><tr><td>Straight forceps</td><td>Natsume Seisakusho Co., Ltd.</td><td>A-7</td><td></td></tr><tr><td>Tongue depressor</td><td>Uchida Yoko Co., Ltd.</td><td>8-615-2417</td><td>Use the wide end</td></tr></tbody></table>

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.

The effectiveness of the LAS is confirmed using echocardiography, 2 weeks postoperative. Rats with an LV inflow velocity greater than 2.0 m/s, measured with a four-chamber view, are considered to have developed significant stenosis (Figure 1) and reliably develop moderate PH and pulmonary venous arterialization 8 - 10 weeks post-LAS surgery.
Ten weeks post-LAS surgery, the rats in the LAS group show...

Watch this Scientific Journal Video about Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat at JoVE.com

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

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 ...

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7.1K Views.  Queen's University. Bedankt voor het bekijken van onze video's. Ik ben Susumu Minamisawa van de Jikei University School of Medicine Tokyo, Japan. Onze kans op dierlijke pulmonale hypertensie als gevolg van ratten hart-en vaatziekten die is geclassificeerd als groep twee long hypertensie.De Wereldgezondheidsorganisatie classificeert long hypertensie in vijf groepen. De linker atrial stenose rat is een diermodel groep twee pulmonale hypertensie die de meest voorkomende vorm van pulmonale hypertensie en mome...

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

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 ...

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.

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. ...

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 ...

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 ...

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 ...

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...

A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation of signal transduction pathways, to counteract, in the short-term, for the cardiac myocyte loss and or the increase in wall stress. However, prolonged activation of these pathways becomes detrimental leading to the initiation and propagation of cardiac remodeling leading to changes in left ventricular geometry and increases in left ventricular volumes; a phenotype seen in patients with systolic heart failure (HF). Here, we describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction (MOD) by ascending aortic banding (AAB) via a vascular clip with an internal area of 2 mm2. The surgery is performed in 200 g Sprague-Dawley rats. The MOD HF phenotype develops at 8-12 weeks after AAB and is characterized noninvasively by means of echocardiography. Previous work suggests the activation of signal transduction pathways and altered gene expression and post-translational modification of proteins in the MOD HF phenotype that mimic those seen in human systolic HF; therefore, making the MOD HF phenotype a suitable model for translational research to identify and test potential therapeutic anti-remodeling targets in HF. The advantages of the MOD HF phenotype compared to the overt systolic HF phenotype is that it allows for the identification of molecular targets involved in the early remodeling process and the early application of therapeutic interventions. The limitation of the MOD HF phenotype is that it may not mimic the spectrum of diseases leading to systolic HF in human. Moreover, it is a challenging phenotype to create, as the AAB surgery is associated with high mortality and failure rates with only 20% of operated rats developing the desired HF phenotype.

We describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction where signal transduction pathways involved in the initiation of the remodeling process are activated. This animal model will aid in identifying molecular targets for applying early therapeutic anti-remodeling strategies for heart failure.

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation ...

A Model of Reverse Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease by Aortic Debanding in Rats

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary arterial hypertension (PAH). As a result, approved therapeutic interventions for the treatment or prevention of PH-LHD are missing. Medications used to treat PH in PAH patients are not recommended for treatment of PH-LHD, as reduced pulmonary vascular resistance (PVR) and increased pulmonary blood flow in the presence of increased left-sided filling pressures may cause left heart decompensation and pulmonary edema. New strategies need to be developed to reverse PH in LHD patients. In contrast to PAH, PH-LHD develops due to increased mechanical load caused by congestion of blood into the lung circulation during left heart failure. Clinically, mechanical unloading of the left ventricle (LV) by aortic valve replacement in aortic stenosis patients or by implantation of LV assist devices in end-stage heart failure patients normalizes not only pulmonary arterial and right ventricular (RV) pressures but also PVR, thus providing indirect evidence for reverse remodeling in the pulmonary vasculature. Using an established rat model of PH-LHD due to left heart failure triggered by pressure overload with subsequent development of PH, a model is developed to study the molecular and cellular mechanisms of this physiological reverse remodeling process. Specifically, an aortic debanding surgery was performed, which resulted in reverse remodeling of the LV myocardium and its unloading. In parallel, complete normalization of RV systolic pressure and significant but incomplete reversal of RV hypertrophy was detectable. This model may present a valuable tool to study the mechanisms of physiological reverse remodeling in the pulmonary circulation and the RV, aiming to develop therapeutic strategies for treating PH-LHD and other forms of PH.

The present protocol describes a surgical procedure to remove ascending-aortic banding in a rat model of pulmonary hypertension due to left heart disease. This technique studies endogenous mechanisms of reverse remodeling in the pulmonary circulation and the right heart, thus informing strategies to reverse pulmonary hypertension and/or right ventricular dysfunction.

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Biology

Optimizing Pulmonary Artery Banding for Consistent Right Heart Failure Induction

Rat Model of Right-Sided Cardiac Remodeling and Arrhythmia Using Pulmonary Artery Banding

Clinical conditions, including chronic obstructive pulmonary disease or pulmonary arterial hypertension (PAH), can lead to chronic right ventricle pressure overload and progressive right heart failure (RHF). RHF can be identified by right-sided cardiac hypertrophy and dilation associated with abnormal myocardial function affecting the RV and the right atrium (RA). We recently demonstrated that severe RHF is accompanied by an increased risk of atrial inflammation, atrial fibrosis, and atrial fibrillation (AF), the most common type of cardiac arrhythmia (CA). Recent studies have shown that RV and RA inflammation plays an important role in the arrhythmogenesis of CA, including AF. However, the impact of inflammation in the development of CA and AF in RHF is poorly described.
Experimental models of RHF are required to better understand the association between right-sided myocardial inflammation and CA. The rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH) is well-established to provoke RHF. However, MCT triggers severe pneumo-toxicity and pulmonary inflammation. Hence, MCT-induced RHF does not help to distinguish whether the subsequent myocardial inflammation originates from the RHF per se or circulating inflammatory signals secreted by the injured lung.
In this article, a mechanical method involving pulmonary artery trunk banding (PAB) was used to provoke right-sided cardiac arrhythmogenesis. The PAB consists of performing a permanent suture of the pulmonary artery trunk for 3 weeks. Such an approach generates increased right-sided pressure overload. At D21 post-PAB, the suture results in hypertrophied, dilated, and inflamed RV and RA. The PAB-induced RHF is also accompanied by vulnerability to ventricular and atrial arrhythmias, including AF.

Right heart failure (RHF) is characterized by right-sided cardiac dilation and hypertrophy, leading to ventricular and atrial malfunction. Cardiopulmonary conditions associated with RHF are accompanied by an increased risk for cardiac arrhythmias. This article describes a standardized model of pulmonary artery banding-induced RHF associated with enhanced ventricular and atrial arrhythmogenesis.

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

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