Name: the left pneumonectomy combined with monocrotaline or sugen as a model of pulmonary hypertension in rats
Uploaded: 2019-03-08T14:30:00
Description: Watch this Scientific Journal Video about The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats at JoVE.com

This manuscript was supported by NIH grant 7R01 HL083078-10 grants from the American Heart Association AHA-17SDG33370112 and from the National Institutes of Health NIH K01 HL135474 to Y.S. and from the National Institutes of Health R01 HL133554 to L.H.

The procedures described below have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Icahn School of Medicine at Mount Sinai. All rats received humane care in compliance with the Mount Sinai &#8220;Guide for the Care and Use of Laboratory Animals&#8221;.
1. Preparation for Surgery
<ol>
	<li>Autoclave Cooley-Mayo curved scissors (large scissors), straight Iris scissors (small scissors), McPherson-Vannas Iris scissors (back scissors), Wangensteen tissue forceps (...

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	<li>Leopold, J., Maron, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Pulmonary+Vascular+Remodeling+in+Pulmonary+Arterial+Hypertension.">Molecular Mechanisms of Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.</a> International Journal of Molecular Sciences. 17 (5), 761 (2016).</li><li>Gomez-Arroyo, J. 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=The+monocrotaline+model+of+pulmonary+hypertension+in+perspective.">The monocrotaline model of pulmonary hypertension in perspective.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 302 (4), L363-L369 (2012).</li><li>van Albada, M. 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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=Plexiform-like+lesions+and+increased+tissue+factor+expression+in+a+rat+model+of+severe+pulmonary+arterial+hypertension.">Plexiform-like lesions and increased tissue factor expression in a rat model of severe pulmonary arterial hypertension.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 293 (3), L583-L590 (2007).</li><li>Samson, N., Paulin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics,+inflammation+and+metabolism+in+right+heart+failure+associated+with+pulmonary+hypertension.">Epigenetics, inflammation and metabolism in right heart failure associated with pulmonary hypertension.</a> Pulmonary Circulation. 7 (3), 572-587 (2017).</li></ol>

In PAH-affected lungs, vascular proliferation with neointimal formation and obliteration of the pulmonary arteries result in severe hemodynamic changes, right ventricular failure and early mortality7,8. The changes to the vessel walls increase resistance to blood flow, increasing arterial and right ventricular pressure. In the early stages of PAH, usually 3 weeks after administering MCT or Sugen, rats developed nonspecific histological changes like medial hypertr...

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by an increase in pulmonary blood flow, increased vascular resistance, inflammation, and remodeling of small pulmonary blood vessels1. This remodeling usually results in vascular lesions that obstruct and obliterate small pulmonary arteries, causing vasoconstriction and increasing right ventricle afterload2. Few successful pharmacological treatments of PAH exist; as a consequence, PAH-related mortality remains high. Recently, the focus of research on the pathobiology of pulmonary hypertension has moved towards a mechanism of angio-proliferation in which the role of increased pulmonary blood flow is considered as an important trigger in the development of pulmonary vascular remodeling3,4.
Animal models of pulmonary hypertension have provided critical insights that help to explain the pathophysiology of the disease and have served as a platform for drug, cell, gene, and protein delivery. Traditionally, the chronic hypoxia-induced pulmonary hypertension model and the MCT lung injury model have been the main models used to study PAH pathophysiology5. However, they are not sufficient to produce increased pulmonary blood flow and neointimal pattern of remodeling compared to alterations described in human patients. The chronic-hypoxia model in rodents results in thickening of vessel walls with hypoxic vasoconstriction without angio-obliteration of small pulmonary vessels6. Additionally, the hypoxia condition is reversible. Thus, the hypoxia model is also not sufficient to produce severe PAH. The MCT lung injury model does elicit some endothelial dysfunction but the complex vascular obliterative lesions found in humans with severe primary PAH do not develop in the rats2. Additionally, MCT-treated rats tend to die from MCT-induced lung toxicity, veno-occlusive liver disease and myocarditis rather than from PAH2. Finally, the pneumonectomy alone is not sufficient to produce neointimal lesions in the small pulmonary vessels in a short period of time. After the pneumonectomy, there is minimal elevation in pulmonary arterial pressure7. In humans, the pneumonectomy is well tolerated when the contralateral lung is healthy7.
However, the left pneumonectomy procedure combined with MCT or Sugen is advantageous since it mimics increased pulmonary blood flow and results in pulmonary vascular remodeling comparable to severe clinical PAH. The pneumonectomy is performed on the left lung, which has only 1 lobe, rather than on the right, which has four lobes. If the right lung was removed, the animal would be unable to compensate for the respiratory insufficiency. In the pneumonectomy-MCT model, neointimal pattern of remodeling develops in over 90% of operated-animals treated7. Similarly, the combination of Sugen and pneumonectomy results in severe PAH, characterized by angio-obliterative vascular lesions, proliferation, apoptosis, and RV dysfunction8. The left pneumonectomy procedure is also advantageous compared to other surgical procedures to induce PAH. Previously described models in rats to increase pulmonary blood flow to the lungs include the aorto-caval shunt or subclavian-pulmonary artery anastomosis. These models are extremely complicated7,9,10,11. To perform an aorto-caval shunt, the animal&#39;s abdomen has to be opened. The shunt is placed in the abdominal aorta, which increases blood flow to all abdominal organs instead of just the lungs, thus, PAH takes much longer to develop. Additionally, it is difficult to determine the blood flow through the shunt, whereas with the pneumonectomy the blood flow to the remaining lung doubles. The subclavian-pulmonary artery anastomosis also has many complications. The flow of arterial blood into the vein can lead to thrombosis of anastomosis and bleeding. Like the aorto-caval shunt, it is difficult to determine the blood flow through the anastomosis. Furthermore, it is an expensive and difficult technique that requires vascular surgical skills. The unilateral left pneumonectomy doubles blood flow and shear stress in the contralateral lung and, in combination with MCT or Sugen, causes the typical hemodynamic and histopathological findings of PAH which is endothelial cell damage8,12.
The novelty of this manuscript is presented in the very detailed and comprehensive surgical protocol of the left pneumonectomy in rats and in the discussion of the technical and physiological challenges of these models. Because this protocol is not currently available, many investigators believe the model is too difficult use. Investigators who have performed the left pneumonectomy have faced high mortality and morbidity rates associated with the unnecessary loss of animals, compromising scientific assessment. Instead, many will use classical models such as MCT injection, chronic-hypoxia, or just the pneumonectomy to create PAH. However, these models are much less effective than the combination of MCT or Sugen with the left pneumonectomy. The primary purpose of this article is to provide the first detailed and reproducible surgical protocol for the left unilateral pneumonectomy in rats and provide the best surgical model of PAH. Combining this protocol for left unilateral pneumonectomy with MCT or SU5416 will allow investigators to create a far more effective and clinically relevant model of severe PAH to study the pathogenesis of this fatal disease.

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			<td height="22" style="height:22px;width:223px;">Surgical Blade</td>
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			<td height="21" style="height:21px;width:223px;">BD Angiocath 16 G</td>
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			<td style="width:153px;">381157</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:223px;">BD 1 mL Insulin Syringe</td>
			<td style="width:111px;">BD</td>
			<td style="width:153px;">329652</td>
			<td>administer buprinex post-operatively</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Biogel Surgeons Surgical Gloves</td>
			<td style="width:111px;">Biogel</td>
			<td style="width:153px;">30460-01</td>
			<td>sterile surgical gloves</td>
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			<td height="42" style="height:42px;width:223px;">Wahl BravMini+ Trimmer</td>
			<td style="width:111px;">Braintree Scientific</td>
			<td style="width:153px;">CLP-41590 P</td>
			<td>shave surgical site</td>
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			<td height="42" style="height:42px;width:223px;">SU5416</td>
			<td style="width:111px;">Cayman Chemical</td>
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			<td>Sugen&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fiber Optic Illuminator</td>
			<td style="width:111px;">Cole-Parmer</td>
			<td style="width:153px;">EW-41723-02</td>
			<td>light for intubation</td>
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			<td height="21" style="height:21px;width:223px;">Surgipro II 4-0 Suture</td>
			<td style="width:111px;">Covidien</td>
			<td style="width:153px;">VP831X</td>
			<td>Closing intercostal muscles</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Polysorb 5-0 Suture</td>
			<td style="width:111px;">Covidien</td>
			<td style="width:153px;">GL-885</td>
			<td>Closing skin</td>
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			<td height="42" style="height:42px;width:223px;">Medium Slide Top Induction Chamber</td>
			<td style="width:111px;">DRE Veterinary</td>
			<td style="width:153px;">12570</td>
			<td>oxygen &#38; isoflurane delivery</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:223px;">DRE Compact 150 Rodent Anesthesia Machine</td>
			<td style="width:111px;">DRE Veterinary</td>
			<td style="width:153px;">373</td>
			<td>oxygen &#38; isoflurane delivery</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Small Vessel Cauterizer Kit</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:153px;">18000-00</td>
			<td>cauterizer to minimize bleeding</td>
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			<td height="42" style="height:42px;width:223px;">VentElite Small Animal Ventilator</td>
			<td style="width:111px;">Harvard Apparatus</td>
			<td style="width:153px;">55-7040</td>
			<td>ventilator</td>
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			<td height="42" style="height:42px;width:223px;">MouseSTAT Jr</td>
			<td style="width:111px;">Kent Scientific</td>
			<td style="width:153px;">MSTAT-JR</td>
			<td style="width:252px;">pulse oximeter &#38; heart rate monitor</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Mouse Paw Pulse Oximeter Sensor</td>
			<td style="width:111px;">Kent Scientific</td>
			<td style="width:153px;">SPO2-MSE</td>
			<td style="width:252px;">pulse oximeter &#38; heart rate paw sensor</td>
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			<td height="21" style="height:21px;width:223px;">PhysioSuite RightTemp</td>
			<td style="width:111px;">Kent Scientific</td>
			<td style="width:153px;">PS-02</td>
			<td>temperature pad</td>
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			<td height="21" style="height:21px;width:223px;">PVP Prep Solution</td>
			<td style="width:111px;">Medline</td>
			<td style="width:153px;">MDS093944</td>
			<td>Cleaning surgical site</td>
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			<td height="21" style="height:21px;width:223px;">Poly-lined Drape</td>
			<td style="width:111px;">Medline</td>
			<td style="width:153px;">NON21002Z</td>
			<td>cover animal</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:223px;">3 mL syringe</td>
			<td style="width:111px;">Medline</td>
			<td style="width:153px;">SYR103010</td>
			<td>administer fluids post-operatively</td>
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		<tr height="83">
			<td height="83" style="height:83px;width:223px;">Microsurgical Kits, Integra&#160;</td>
			<td style="width:111px;">Miltex</td>
			<td style="width:153px;">95042-540</td>
			<td style="width:252px;">surgical tools: plain wire speculum, double-ended probe, McPherson-Vannas Iris scissors straight, straight iris scissors</td>
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			<td height="42" style="height:42px;width:223px;">Hemostatic forceps - Micro-Jacobson-Mosquito</td>
			<td style="width:111px;">Miltex</td>
			<td style="width:153px;">17-2602</td>
			<td>mosquito</td>
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			<td height="42" style="height:42px;width:223px;">Buprenorphrine HCl 0.3 mg/mL</td>
			<td style="width:111px;">Par Pharmaceutical</td>
			<td style="width:153px;">NDC 42023-179-01</td>
			<td>Pain relief</td>
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			<td height="21" style="height:21px;width:223px;">Gerald Tissue forceps</td>
			<td style="width:111px;">Pilling</td>
			<td style="width:153px;">351900</td>
			<td>forceps</td>
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			<td height="21" style="height:21px;width:223px;">Wangesnsteen Tissue Forceps</td>
			<td style="width:111px;">Pilling</td>
			<td style="width:153px;">342929</td>
			<td>atraumatic forceps</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Pilling Thin Vascular Needle Holder</td>
			<td style="width:111px;">Pilling</td>
			<td style="width:153px;">354962DG</td>
			<td>needle holder</td>
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			<td height="21" style="height:21px;width:223px;">Crotaline</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:153px;">C2401-1G</td>
			<td>MCT</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Surflash 20 G IV Catheter</td>
			<td style="width:111px;">Terumo</td>
			<td style="width:153px;">SR*FF2051</td>
			<td style="width:252px;">For pressure reading during organ harvest</td>
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			<td height="42" style="height:42px;width:223px;">ADVantage PV System with 1.2 Fr Catheter</td>
			<td style="width:111px;">Transonic Inc</td>
			<td style="width:153px;">ADV500</td>
			<td style="width:252px;">Record pulmonary artery and right ventricle pressure</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Medium Hemoclip</td>
			<td style="width:111px;">Weck</td>
			<td style="width:153px;">523700</td>
			<td>ligate vessels</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Open Ligating Clip Applicator; Medium, curved</td>
			<td style="width:111px;">Weck Horizon</td>
			<td style="width:153px;">237081</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Surgical Microscope</td>
			<td style="width:111px;">Zeiss OPMI MD</td>
			<td style="width:153px;">1808</td>
			<td>magnification</td>
		</tr>
	</tbody>
</table>

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.

According to the accepted classification system, pulmonary hypertension is characterized by a mean pulmonary artery pressure (mPAP) exceeding the upper limits of normal pulmonary artery pressure (i.e., 25 mm Hg). In the pneumonectomy + MCT group, severe PAH developed by day 21 with an increased mPAP (Figure 1). The mPAP is calculated by the formula:
<img alt="Equation" src="/files/ftp_upload/59050/5...

Watch this Scientific Journal Video about The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats at JoVE.com

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats (Video) | JoVE

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.

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 ...

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Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery ALCAPA (Video) | JoVE

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ...

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

Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat (Video) | JoVE

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

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

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model (Video) | JoVE

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

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

A Model of Reverse Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease by Aortic Debanding in Rats (Video) | JoVE

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

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model (Video) | JoVE

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, ...