Name: the left pneumonectomy combined with monocrotaline or sugen as a model of pulmonary hypertension in rats
Uploaded: 2019-03-08T14:30:00.000+00:00
Duration: 7 min 29 s
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 (atraumatic forceps), Gerald tissue forceps, plain wire speculum (self-retaining retractor), small vessel cauterizer, gauze, double-ended probe, thin vascular needle holder, and Micro-Jacobson mosquito hemostatic forceps (mosquito).</li>
</ol>
2. Preparation and Intubation of Rats
<ol>
	<li>Administer a subcutaneous injection of buprenorphine analgesia (0.1 mg/kg) in 6&#8211;8 week old male, Sprague-Dawley rats, 30 min prior to surgery.</li>
	<li>Place the rat individually in a chamber exposed to isoflurane 4% and oxygen (1 L/min) for 3&#8211;4 min until the rat is properly anesthetized (i.e., unconscious, breathing slowly, and unresponsive to touch or sound).</li>
	<li>Intubation of Rats
	<ol>
		<li>Remove the rat from the chamber and tape the rat to a board so the rat lays flat on its back, with the mouth at the edge of the board. Use string to hold the rat by the two front teeth so that the neck remains flat/straight.</li>
		<li>Place a fiber optic goose light source above the neck over the larynx, lift and hold the tongue with fingers, and move the epiglottis up to visualize the vocal cords. The trachea should be brightly lit behind the vocal cords; adjust the position of the light until the trachea is bright.</li>
		<li>Intubate the rats by inserting a 16 G catheter into the larynx within 60 s&#160;of removing the rat from the chamber as they wake quickly. If the rat wakes before successful intubation, put the rat back and repeat.&#160;</li>
		<li>Immediately connect the rat to the ventilator to resume isoflurane and oxygen delivery. Confirm the intubation using a cold mirror to observe condensation of the humidity in the exhaled breath from the catheter. Make sure the animal is still breathing by the expansion of the chest.</li>
	</ol>
	</li>
	<li>Connect the rat to the ventilator. Set the oxygen to flow at 1 L/min. Set the ventilation rate to 70 breaths/min with a tidal volume of 0.35&#8211;0.45 mL. Set positive end expiratory pressure (PEEP) to 2&#8211;4 cm H2O. Reduce isoflurane to 2&#8211;3%.</li>
	<li>Place the rat under the microscope in the right decubitus position on a heating pad at 37 &#176;C. Tape the front legs down. Shave the left thorax behind the front leg until the end of the rib cage with clippers. Clean the surgical area with gauze and 10% povidone-iodine solution, followed by 70% ethanol (three times).</li>
	<li>Place the pulse oximeter on the rat&#8217;s foot and monitor the heart rate and oxygen saturation throughout the surgery. Heart rate should be between 200&#8211;500 bpm and oxygen saturation should be &#62;95%.</li>
</ol>
3. Preparation of Sterile Environment
<ol>
	<li>Visualize the surgical area under the microscope. This must be done first because the microscope cannot be adjusted once the environment is sterile.</li>
	<li>Put on sterile, surgical gloves. Do not touch anything besides the sterilized instruments, the surgical drapes, and the surgical area of the rat&#8217;s body.</li>
	<li>Place a sterile drape over the rat&#8217;s body and on the instrument tray to create a sterile environment.</li>
	<li>With sterile gloves, place sterile instruments on the sterile instrument tray.</li>
</ol>
4. Left Pneumonectomy Surgical Procedure
<ol>
	<li>Use Cooley-Mayo scissors and Gerald tissue forceps to cut a small hole in the surgical drape. The hole should be about 2&#8211;3 cm in diameter to fully visualize the incision but not too large so that only sterilized skin is exposed.</li>
	<li>Make a 2 cm long lateral incision in the left thorax using a surgical blade. Use gauze and the electrocautery pen to manage bleeding/maintain hemostasis. Use the Cooley-Mayo scissors to cut each layer of tissue until the ribs and intercostal muscles are exposed.</li>
	<li>Entering Intercostal Space
	<ol>
		<li>Use the mosquito to make a hole through the muscle of the third intercostal space.</li>
		<li>Use the double-ended probe to move the lung out of the way to visualize the pulmonary artery. The investigator may enter another intercostal space if the pulmonary artery is not accessible.</li>
		<li>Use iris scissors and McPherson-Vannas iris scissors (back scissors) to open the intercostal muscles to about 1&#8211;2 cm. Use the electrocautery pen to stop any bleeding.</li>
		<li>Place a small, self-retaining retractor to hold the ribs and muscles open.</li>
	</ol>
	</li>
	<li>Open only the left pleura and move the left lung down lower in the abdomen to allow access to the pulmonary artery and bronchus. Load a medium hemoclip into the ligating clip applicator.</li>
	<li>Ligation of the Left Main Bronchus and Left Main Pulmonary Artery
	<ol>
		<li>Lift the upper portion of the left lung carefully with the Wangensteen atraumatic forceps to expose the pulmonary artery.</li>
		<li>Clip the pulmonary artery by closing the clip and applicator around the artery. Be careful not to close or rupture the left azygos vein. Do not clamp too forcefully otherwise, the vessel can rupture.</li>
		<li>Open the incision further using the tonsil to separate the muscle fibers.</li>
		<li>Load another medium hemoclip into the ligating clip applicator so that it is ready.</li>
		<li>Use the atraumatic forceps to lift the lower portion of the lung out of the incision until the left main bronchus and left pulmonary vein are visualized. Atraumatic forceps must be used pull to move the lung up and out of the incision. Since the lung is too large to be pulled out in one motion without tearing the tissue, the issue forceps are necessary to hold the lung in place while the atraumatic forceps are used carefully to pull more lung out.</li>
		<li>Ligate the left main bronchus and left main pulmonary vein together by closing the applicator around them. Do not clamp the vein and bronchus too forcefully otherwise, the vessel can rupture.</li>
	</ol>
	</li>
	<li>Remove the lung with scissors. Be careful not to cut or tear out the clip. Use a small piece of sterile gauze to absorb any blood and make sure there is no bleeding.</li>
	<li>Closure of the Ribs and Intercostal Muscles
	<ol>
		<li>Before closing the intercostal muscles, insert a 16 G catheter into the thoracic cavity away from the surgical incision and into the seventh intercostal space. Make sure the investigator can visualize the needle so as not to puncture any vital organs or vessels.</li>
		<li>Immediately remove the needle, leaving the catheter in place. This will be used as a chest tube.</li>
		<li>Close the ribs and intercostal muscles with a 4-0 prolene suture.</li>
		<li>Close the skin and subcutaneous space with a running 5-0 suture (Table of Materials).</li>
		<li>Place a 5-0 suture (Table of Materials) into the skin and around the chest tube so when the chest tube is removed, the hole will be tied shut.</li>
	</ol>
	</li>
	<li>Evacuate the air from the pleural cavity with a 3 mL syringe through the catheter to regain the negative normal pressure in the thorax. Immediately clamp the catheter with a needle holder to prevent air from going back into the thoracic cavity. Quickly remove the catheter and tie the suture to close the hole.</li>
</ol>
5. Post-operative Recovery
<ol>
	<li>Place the animal in the sternal position. Turn off the isoflurane but continue giving the rat oxygen via endotracheal tube. Do not leave the animal unattended at any point until it has regained sufficient consciousness to ambulate and the animal is safely in its cage.</li>
	<li>Administer 2&#8211;3 mL of sterile lactated Ringer&#8217;s solution subcutaneously.</li>
	<li>Monitor the heart rate (&#8776;200&#8211;500 bpm), oxygen saturation (&#8805;95%), and animal color to confirm the animal is breathing well.</li>
	<li>When the animal starts making spontaneous breathing motions and neck movements and responds to physical stimuli (such as eyes widening, nose moving, and ears responding to sound), extubate the animal by pulling out the catheter and disconnect the ventilator.</li>
	<li>Return the animal to an empty cage, away from the company of other animals. The animal should be alone for at least 3 days.</li>
	<li>For pain control, administer 0.1 mg/kg of Buprenorphine subcutaneously every 12 h for 3 days.</li>
</ol>
6. Administration of &#8220;Second Hit&#8221; MCT or Sugen
<ol>
	<li>Administer 1 mL of MCT (60 mg/kg) or 1 mL of Sugen (25 mg/kg) via subcutaneous injection, 1 week after the surgery.</li>
</ol>
7. Terminal Harvest
<ol>
	<li>Seven weeks after MCT or Sugen administration, place the animal in a&#160;chamber with flowing oxygen (1 L/min) and 4% isoflurane.</li>
	<li>Connect the animal to the ventilator via nose cone. Keep oxygen flowing and lower the isoflurane to 2.5&#8211;3%. Lay the animal on his back and tape down all limbs.</li>
	<li>Shave the animal along the entire abdomen.</li>
	<li>Opening the Chest
	<ol>
		<li>Make an incision down the chest&#160;with a scalpel from the subclavian notch to the xyphoid.</li>
		<li>Use the tissue forceps to hold the xyphoid. Cut through the sternum with the Cooley-Mayo scissors until the heart and pulmonary artery are fully accessible. Use the electrocautery to stop the bleeding. Hold the ribs open with a retractor.</li>
	</ol>
	</li>
	<li>Identify the pulmonary artery. Adjust the orientation of the heart, if necessary, by adding gauze underneath. Use a 20 G IV catheter to puncture the pulmonary artery between the pulmonary valve and the bifurcation of the main pulmonary artery. Advance only the 20 G catheter into the artery and remove the needle. Advance the 1.2 Fr transonic pressure catheter into IV catheter that is in the pulmonary artery. Once the PA curve is steady, record the pressure for at least 10 s. Repeat these steps to record the pressure in the right ventricle.</li>
	<li>Remove the heart and lungs. Place pieces of the lung in formalin to be fixed in paraffin and stained with hematoxylin and eosin.</li>
</ol>

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The authors have no potential conflicts of interest.

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.

<table><tbody><tr><td>Surgical Blade</td><td>Bard-Parker</td><td>371215</td><td>Incision</td></tr><tr><td>Forane (Isoflurane, USP)</td><td>Baxter</td><td>NDC 10019-360-40</td><td>anesthesia</td></tr><tr><td>BD Angiocath 16 G</td><td>BD</td><td>381157</td><td>intubation tube, chest tube</td></tr><tr><td>BD 1 mL Insulin Syringe</td><td>BD</td><td>329652</td><td>administer buprinex post-operatively</td></tr><tr><td>Biogel Surgeons Surgical Gloves</td><td>Biogel</td><td>30460-01</td><td>sterile surgical gloves</td></tr><tr><td>Wahl BravMini+ Trimmer</td><td>Braintree Scientific</td><td>CLP-41590 P</td><td>shave surgical site</td></tr><tr><td>SU5416</td><td>Cayman Chemical</td><td>13342</td><td>Sugen&amp;nbsp;</td></tr><tr><td>Fiber Optic Illuminator</td><td>Cole-Parmer</td><td>EW-41723-02</td><td>light for intubation</td></tr><tr><td>Surgipro II 4-0 Suture</td><td>Covidien</td><td>VP831X</td><td>Closing intercostal muscles</td></tr><tr><td>Polysorb 5-0 Suture</td><td>Covidien</td><td>GL-885</td><td>Closing skin</td></tr><tr><td>Medium Slide Top Induction Chamber</td><td>DRE Veterinary</td><td>12570</td><td>oxygen &amp;amp; isoflurane delivery</td></tr><tr><td>DRE Compact 150 Rodent Anesthesia Machine</td><td>DRE Veterinary</td><td>373</td><td>oxygen &amp;amp; isoflurane delivery</td></tr><tr><td>Small Vessel Cauterizer Kit</td><td>Fine Science Tools</td><td>18000-00</td><td>cauterizer to minimize bleeding</td></tr><tr><td>VentElite Small Animal Ventilator</td><td>Harvard Apparatus</td><td>55-7040</td><td>ventilator</td></tr><tr><td>MouseSTAT Jr</td><td>Kent Scientific</td><td>MSTAT-JR</td><td>pulse oximeter &amp;amp; heart rate monitor</td></tr><tr><td>Mouse Paw Pulse Oximeter Sensor</td><td>Kent Scientific</td><td>SPO2-MSE</td><td>pulse oximeter &amp;amp; heart rate paw sensor</td></tr><tr><td>PhysioSuite RightTemp</td><td>Kent Scientific</td><td>PS-02</td><td>temperature pad</td></tr><tr><td>PVP Prep Solution</td><td>Medline</td><td>MDS093944</td><td>Cleaning surgical site</td></tr><tr><td>Poly-lined Drape</td><td>Medline</td><td>NON21002Z</td><td>cover animal</td></tr><tr><td>3 mL syringe</td><td>Medline</td><td>SYR103010</td><td>administer fluids post-operatively</td></tr><tr><td>Microsurgical Kits, Integra&amp;nbsp;</td><td>Miltex</td><td>95042-540</td><td>surgical tools: plain wire speculum, double-ended probe, McPherson-Vannas Iris scissors straight, straight iris scissors</td></tr><tr><td>Hemostatic forceps - Micro-Jacobson-Mosquito</td><td>Miltex</td><td>17-2602</td><td>mosquito</td></tr><tr><td>Buprenorphrine HCl 0.3 mg/mL</td><td>Par Pharmaceutical</td><td>NDC 42023-179-01</td><td>Pain relief</td></tr><tr><td>Cooley-Mayo curved scissors</td><td>Pilling</td><td>352090</td><td>Large scissors</td></tr><tr><td>Gerald Tissue forceps</td><td>Pilling</td><td>351900</td><td>forceps</td></tr><tr><td>Wangesnsteen Tissue Forceps</td><td>Pilling</td><td>342929</td><td>atraumatic forceps</td></tr><tr><td>Pilling Thin Vascular Needle Holder</td><td>Pilling</td><td>354962DG</td><td>needle holder</td></tr><tr><td>Crotaline</td><td>Sigma-Aldrich</td><td>C2401-1G</td><td>MCT</td></tr><tr><td>Surflash 20 G IV Catheter</td><td>Terumo</td><td>SR*FF2051</td><td>For pressure reading during organ harvest</td></tr><tr><td>ADVantage PV System with 1.2 Fr Catheter</td><td>Transonic Inc</td><td>ADV500</td><td>Record pulmonary artery and right ventricle pressure</td></tr><tr><td>Medium Hemoclip</td><td>Weck</td><td>523700</td><td>ligate vessels</td></tr><tr><td>Open Ligating Clip Applicator; Medium, curved</td><td>Weck Horizon</td><td>237081</td><td>hemoclip applicator</td></tr><tr><td>Surgical Microscope</td><td>Zeiss OPMI MD</td><td>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

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 ...

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Research

JoVE Journal

Medicine

10.7K Views.  Icahn School of Medicine at Mount Sinai. 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.

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

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Cancer Research

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 ...

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 ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

Bioengineering

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

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

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

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

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

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

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

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

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 ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...