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. R., 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=Echocardiographic+validation+of+pulmonary+hypertension+due+to+heart+failure+with+reduced+ejection+fraction+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sildenafil+preserves+lung+endothelial+function+and+prevents+pulmonary+vascular+remodeling+in+a+rat+model+of+diastolic+heart+failure.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+and+Mechanism+of+Atorvastatin+on+Pulmonary+Hypertension+Due+to+Left+Heart+Disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+veins+in+the+normal+lung+and+pulmonary+hypertension+due+to+left+heart+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+hypertension+due+to+left+heart+disease+causes+intrapulmonary+venous+arterialization+in+rats.">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/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.

<table border="0" cellpadding="0" cellspacing="0" style="width:635px;" width="634">
	<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:159px;">Chest tube 23G</td>
			<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>
		</tr>
		<tr height="62">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:159px;">Heating pad</td>
			<td style="width:132px;">Vivaria</td>
			<td style="width:123px;">MP-916-NV</td>
			<td>Keep body temperature at 37 degree celsius</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horizon Manual-Load Ligating Clip Applier For Medium-Large Size Horizon</td>
			<td style="width:132px;">Teleflex</td>
			<td>REF 337085</td>
			<td>Ligation Clips Angled Jaw, (20cm)</td>
		</tr>
		<tr height="62">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="62">
			<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>
		</tr>
		<tr height="62">
			<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>
		</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 ...

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.

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7.1K visualizações.  Queen's University. Obrigado por assistir nossos vídeos. Sou Susumu Minamisawa, da Escola de Medicina da Universidade Jikei, em Tóquio, Japão. Nossa oportunidade de hipertensão pulmonar animal devido a doença cardíaca de rato que é classificada como hipertensão pulmonar do Grupo Dois.A Organização Mundial da Saúde classifica a hipertensão pulmonar em cinco grupos. O rato de estenose atrial esquerda é um grupo modelo animal dois hipertensão pulmonar que é a forma mais prevalente de hipertens...