Name: left atrial stenosis induced pulmonary venous arterialization and group 2 pulmonary hypertension in rat
Uploaded: 2018-11-18T14:00:00
Description: Watch this Scientific Journal Video about Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat at JoVE.com

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 respira...

<ol>
	<li>McGuire, L. B., Nolan, T. B., Reeve, R., Dammann Jr, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cor+Triatriatum+as+a+Problem+of+Adult+Heart+Disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+hypertension:+prevalence+and+mortality+in+the+Armadale+echocardiography+cohort.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+Pulmonary+Hypertension+in+the+General+Population:+The+Rotterdam+Study.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+Incidence+and+Prevalence+of+World+Health+Organization+Groups+1+to+4+Pulmonary+Hypertension:+A+Population-Based+Cohort+Study+in+Ontario,+Canada.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+persistent+pulmonary+hypertension+after+mitral+valve+replacement.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bosentan+does+not+improve+pulmonary+hypertension+and+lung+remodeling+in+heart+failure.">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. 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J., Hadri, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+hypertension+arising+from+left+heart+disease+causes+intrapulmonary+venous+arterialization+in+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+a+Cinderella+story.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vein+stenosis+and+the+pathophysiology+of+&amp;#34;upstream&amp;#34;+pulmonary+veins.">Pulmonary vein stenosis and the pathophysiology of &amp;#34;upstream&amp;#34; pulmonary veins.</a> The Journal of Thoracic and Cardiovascular Surgery. 148 (1), 245-253 (2014).</li></ol>

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.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:159px;">5-0 Prolene Suture</td>
			<td style="width:132px;">Johnson &#38; Johnson - Ethicon</td>
			<td style="width:123px;">8725H</td>
			<td style="width:222px;">Polypropylene suture with HEMO-SEAL Technology</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:159px;">Anaesthesia Machine</td>
			<td style="width:132px;">Wakenyaku Co., Ltd.</td>
			<td style="width:123px;">BRTK-100A</td>
			<td>Air pump and anaethesia machine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:159px;">Angiocatheter guidewire</td>
			<td style="width:132px;">Self-made</td>
			<td style="width:123px;"></td>
			<td>10 cm guidewire glued to a 1 cc syringe&#160;</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:159px;">Chest retractor</td>
			<td style="width:132px;">Natsume Seisakusho Co., Ltd.</td>
			<td style="width:123px;">F-2</td>
			<td></td>
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			<td style="width:132px;">Self-made</td>
			<td style="width:123px;"></td>
			<td>10 cc syringe attached to a 23G needle plus plastic tube</td>
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			<td height="62" style="height:63px;width:159px;">Curved forceps</td>
			<td style="width:132px;">Natsume Seisakusho Co., Ltd.</td>
			<td style="width:123px;">A-14</td>
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			<td style="width:132px;">Vivaria</td>
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			<td>Keep body temperature at 37 degree celsius</td>
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			<td height="21" style="height:21px;">Horizon Ligating Clips</td>
			<td style="width:132px;">Teleflex</td>
			<td style="width:123px;">REF 003200</td>
			<td>Size Medium-Large</td>
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			<td height="21" style="height:21px;">Horizon Manual-Load Ligating Clip Applier For Medium-Large Size Horizon</td>
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			<td>Ligation Clips Angled Jaw, (20cm)</td>
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			<td height="62" style="height:63px;width:159px;">Needle holder</td>
			<td style="width:132px;">Natsume Seisakusho Co., Ltd.</td>
			<td style="width:123px;">MC-40</td>
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			<td height="21" style="height:21px;width:159px;">Rodent Respirator</td>
			<td style="width:132px;">CWE Inc</td>
			<td style="width:123px;">SAR-830/P</td>
			<td>Small animal ventilator</td>
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			<td height="62" style="height:63px;width:159px;">Scissors</td>
			<td style="width:132px;">Natsume Seisakusho Co., Ltd.</td>
			<td style="width:123px;">B-12</td>
			<td>Straight scissors ideally with round tips</td>
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			<td height="62" style="height:63px;width:159px;">Straight forceps</td>
			<td style="width:132px;">Natsume Seisakusho Co., Ltd.</td>
			<td style="width:123px;">A-7</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:159px;">Tongue depressor</td>
			<td style="width:132px;">Uchida Yoko Co., Ltd.</td>
			<td style="width:123px;">8-615-2417</td>
			<td>Use the wide end</td>
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</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 ...

Thank you for watching our videos. I am Susumu Minamisawa of the Jikei University School of Medicine Tokyo, Japan. Our opportunity on animal pulmonary hypertension due to rat heart disease which is classified as Group Two pulmonary hypertension.The World Health Organization classifies pulmonary hypertension into five groups. The left atrial stenosis rat is an animal model group two pulmonary hypertension which is the most prevalent form of pulmonary hypertension and currently there is no approved therapies for this group of pulmonary hypertension. In this videos we are demonstrating the step by step procedure of how to perform left atrial stenosis surgery in rat.Surgically created right atrial stenosis mimic mitral stenosis in human. When we use it to study the possibility and under of group two pulmonary hypertension. Let's get started.Prepare the following equipment before the surgery, a small animal respirator and an anesthetic machine. An incubation kit composes of a pair of hemostat forceps, a tongue depressor and a 18 gauge angio-catheter. Surgical instruments 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, a medium large clip and a 23 gauge chest tube.Anesthetize the rat in an induction chamber with five percent isoflurane mixed with two meter per minute room air. Prior to intubation shave the chest hair. Apply hair removal cream to remove fine hair not removed by the shaver.Hook the front teeth with a string and secure the strings to pins. After the string is secured open the mouth with the hemostat forceps and insert the tongue depressor into the mouth. Lift the tongue depressor to visualize the vocal chord.Insert the 18 gauge angio-catheter as an endotracheal tube into the trachea and then quickly connect the catheter to the respirator. Set the tidal volume to 10 microliter per gram with a respiratory rate of 100 breaths per minute. Maintain anesthesia with two percent isoflurane mixed with two meter per minute room air.Sterilize the surgical site with alternating scrubs of chlorhexidine and alcohol. Give Buprenorphine 01 milligram per kilogram subcutaneously. Cover the rat with a sterile drape.Check pedal reflex to confirm successful anesthesia prior to surgery. Mark the incision site two centimeters below the armpit. Make a two centimeter left lateral chest wall incision with a pair of scissors.Carefully separate the intercostal muscle between the fourth and the fifth rib using a straight and curved forceps until entering the chest cavity. Insert the chest retractor into the chest cavity. Use some force to open up the chest retractor to split the fourth and fifth rib apart by about one centimeter.Lift the thymus with a pair of straight forceps. Carefully remove the thymus covering the heart with a pair of scissors. Carefully pass a 5-0 monofilament suture through the surface of the left ventricle right below the left atrial appendage.Care should be taken to avoid passing the needle through the major coronary arteries. After the suture is in place and there is no significant bleeding tie a loose knot. Pull the suture thread up and forward to lift the heart out of the chest.Once the heart is lifted outside of the chest quickly apply a medium large clip to the left atrium just above the mitral valve. Carefully remove the stay suture used to lift the heart. Begin close the chest with a 5-0 monofilament suture using simple interrupted pattern.Insert a 23 gauge chest tube attached to a 10cc syringe into the chest cavity. And then proceed with closing the chest. Draw out any air, blood and pleurae effusion near the inserted chest tube using the attached 10cc syringe.And then pull the tube when nothing is coming out. Close the skin area with 5-0 monofilament suture using simple interrupted pattern. Remove the surgical drape.Give Buprenorphine 01 milligram per kilogram subcutaneously. Turn off the isoflurane. Excavate the rat when it starts to wake up.Two weeks post op use echo cardiography to confirm the success of left atrial stenosis surgery. The effectiveness of the left atrial stenosis is confirmed using echo cardiography two weeks post op. Rats with left ventricle inflow velocity greater than two meters per second measured on four chamber view are considered to have developed significant stenosis.10 weeks post left atrial stenosis surgery the rats in the LAS group show left atrial enlargement, pulmonary congestion, right ventricle pressure overload and increased pulmonary venous flow compared to the rats in the sham operated group. In addition there is significant increase of right ventricular systolic pressure in the left atrial stenosis group versus the sham operated group. However, the left ventricle systolic pressure remains the same in both groups.Histological examination of long cross section stained with elastic Van Gieson shows increased pulmonary arterial and pulmonary venous medium sickness. An increased pulmonary venous dimension the left atrial stenosis group versus the sham operated group was a more of a smooth muscle acting in the staining shows increased number of smooth muscle cells in the pulmonary artery and the pulmonary vein of the left atrial stenosis group versus the control group. I hope you enjoyed watching this video.Although the surgery is relatively simple care should be taken when applying the suture to the left ventricle to avoid hitting the major coronary arteries. When lifting the heart pull the suture up toward the head of the rat so the left atrium its running structure can be clearly visualized. By applying the clip to the left atrium the tightness of the clip should be sufficient to cause significant left atrial stenosis, but not too tight to induce acute pulmonary edema and right heart failure.Thank you for watching this video. And good luck with your experiment.

left-atrial-stenosis-induced-pulmonary-venous-arterialization-group-2

Research

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Medicine

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

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 ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

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 ...

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

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. ...

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 ...