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>

<ol>
	<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. E., 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+role+of+increased+pulmonary+blood+flow+in+pulmonary+arterial+hypertension.">The role of increased pulmonary blood flow in pulmonary arterial hypertension.</a> European Respiratory Journal. 26 (3), 487-493 (2005).</li><li>Dickinson, M. G., Bartelds, B., Borgdorff, M. A. J., Berger, R. M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+disturbed+blood+flow+in+the+development+of+pulmonary+arterial+hypertension:+lessons+from+preclinical+animal+models.">The role of disturbed blood flow in the development of pulmonary arterial hypertension: lessons from preclinical animal models.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 305 (1), L1-L14 (2013).</li><li>Stenmark, K. R., Meyrick, B., Galie, N., Mooi, W. J., McMurtry, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pulmonary+arterial+hypertension:+the+hope+for+etiological+discovery+and+pharmacological+cure.">Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 297 (6), L1013-L1032 (2009).</li><li>Cahill, E., 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+pathophysiological+basis+of+chronic+hypoxic+pulmonary+hypertension+in+the+mouse:+vasoconstrictor+and+structural+mechanisms+contribute+equally.">The pathophysiological basis of chronic hypoxic pulmonary hypertension in the mouse: vasoconstrictor and structural mechanisms contribute equally.</a> Experimental Physiology. 97 (6), 796-806 (2012).</li><li>Okada, K., 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+hemodynamics+modify+the+rat+pulmonary+artery+response+to+injury.+A+neointimal+model+of+pulmonary+hypertension.">Pulmonary hemodynamics modify the rat pulmonary artery response to injury. A neointimal model of pulmonary hypertension.</a> American Journal of Pathology. 151 (4), 1019-1025 (1997).</li><li>Happ&#233;, C. 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=Pneumonectomy+combined+with+SU5416+induces+severe+pulmonary+hypertension+in+rats.">Pneumonectomy combined with SU5416 induces severe pulmonary hypertension in rats.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 310 (11), L1088-L1097 (2016).</li><li>Tanaka, Y., Schuster, D. P., Davis, E. C., Patterson, G. A., Botney, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+vascular+injury+and+hemodynamics+in+rat+pulmonary+artery+remodeling.">The role of vascular injury and hemodynamics in rat pulmonary artery remodeling.</a> Journal of Clinical Investigation. 98 (2), 434-442 (1996).</li><li>Nishimura, T., Faul, J. L., Berry, G. J., Kao, P. N., Pearl, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+surgical+aortocaval+fistula+on+monocrotaline-induced+pulmonary+hypertension.">Effect of a surgical aortocaval fistula on monocrotaline-induced pulmonary hypertension.</a> Critical Care Medicine. 31 (4), 1213-1218 (2003).</li><li>Linardi, D., 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=Ventricular+and+pulmonary+vascular+remodeling+induced+by+pulmonary+overflow+in+a+chronic+model+of+pretricuspid+shunt.">Ventricular and pulmonary vascular remodeling induced by pulmonary overflow in a chronic model of pretricuspid shunt.</a> Journal of Thoracic and Cardiovascular Surgery. 148 (6), 2609-2617 (2014).</li><li>White, R. 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>

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 hypertrophy, adventitial thickening, and muscularization of the small arteries and arterioles. These changes are potentially reversible. In the later stages, about 6&#8211;8 weeks after administering MCT or Sugen, the rat&#39;s lungs have neointimal and plexiform vasculopathy that obstruct and obliterate medium and small pulmonary arteries and arterioles, cellular intimal proliferation, and concentric intimal fibrosis. Plexiform lesions usually consist of a plexus of channels lined by endothelial cells and myofibroblasts. In many cases, these changes are associated with interstitial edema and fibrosis, thromboembolic obstruction of distal small pulmonary arteries, partial recanalization of a thrombosed muscular vessels, and fibrinoid necrosis12.
As stated previously, rats in other PAH models do not develop vascular obliteration of the small vessels, neointimal and plexiform lesions, and high PA and RV pressures. Treatment with the chronic-hypoxia PAH model was found to produce some thickening of vessels but the vessels remain open and there are no vascular obliterative lesions and little inflammation5. RVSP and mPAP values are slightly elevated compared to control5. Similarly, treatment with either MCT or pneumonectomy resulted in pulmonary arterial pressures that were not significantly higher than control rats and slightly remodeled vessels7,9.
Conversely, we found that the left pneumonectomy combined with MCT or Sugen is an effective model to create severe PAH. Compared to the control group, the mean pulmonary arterial pressure (mPAP) and the right ventricular systolic pressure (RVSP) in rats with severe PAH nearly doubled (Figure 1). Additionally, these rats developed plexiform lesions, concentric medial and intimal thickening, thromboembolic obstruction of distal small pulmonary arteries, inflammation, and very elevated pulmonary artery and right ventricular pressures (Figure 2). Compared to the chronic-hypoxia, MCT injection, and pneumonectomy only models of PAH performed in other studies, the pneumonectomy combined with MCT or Sugen model creates a clinically relevant condition. Furthermore, this model, if performed correctly,&#160;has a nearly 0% intra-operative mortality rate and&#160;only a 10% mortality rate post-recovery from failure of the right lung to compensate respiratory insufficiency. Higher rates of either intra-operative or post-recovery mortality is usually indicative that human or equipment error has taken place.
In order to successfully perform the left pneumonectomy and create PAH, there are several critical steps in this protocol that must be completed. First, it is very important to monitor the animal&#39;s oxygen saturation, capnography, and counting heart rate throughout the entire procedure to ensure the animal is breathing and his heart is beating. Monitoring oxygen saturation further confirms proper endotracheal intubation. Intubating the animal is absolutely necessary for this procedure. When the animal&#39;s chest cavity is opened, the negative pressure that normally exists in the thoracic cavity is shifted to atmospheric positive pressure. Thus, rats need to be provided with positive pressure via artificial ventilation. Without proper intubation, the animal&#39;s lungs will collapse from the positive atmospheric pressure.
The surgical incision has to be made laterally or posterolaterally and in the third intercostal space. Depending on the rat&#39;s anatomy, the surgeon may have to open a different intercostal space in order to access the lung and visualize the vessels. Using other approaches will make it very difficult to reach the left pulmonary artery. When opening the chest cavity, it is very important to use an electrocautery pen to stop bleeding from the skin, muscles, and surrounding vessels. If the investigator skips this step, the animal will lose blood and less oxygen will circulate. The incision must be a minimum of 2 cm long so that there is enough space to take out the entire lung when ligating. Otherwise, the investigator will tear the tissue when trying to remove the lung from a small opening. It is also essential to use atraumatic forceps when moving the lung in order to avoid rupturing the tissue and hemorrhaging. The pulmonary artery is closed first to prevent bleeding from manipulation of the lungs. The investigator should be very careful when ligating the vessels to avoid closing the left azygos vein or tearing the vessels otherwise, the animal will die. Additionally, it is much easier for the surgeon to use titanium hemoclips rather than sutures to ligate the left pulmonary artery, left main bronchus, and left pulmonary veins with a suture. Because the investigator has to make a relatively long incision in the intercostal space, it is necessary to close the intercostal muscles and ribs with sutures. Once the chest is closed, air has to be evacuated from the chest to restore negative pressure and it is important to prevent contralateral lung and heart distortion with a closed pneumothorax.
Finally, recovery is one of the most critical steps after the rat pneumonectomy. A thoracotomy is considered a very painful surgery and analgesics are essential to promote adequate ventilation, improve lung excursions in the post-operative period, and reduce pain. Rats should be recovered in sternal position to maximize lung inflation. After 10 min, the investigator can decrease the ventilation rate to help stimulate the animal to breathe on its own and wake up. If the animal becomes cyanotic and oxygen saturation levels decrease, it is necessary to increase the oxygen, ventilation rate, and increase tidal volume to 5 mL or more depending on the capacity of the ventilator. Extubation of the rats should be done as late as possible, once the animal is fully awake. The investigator may put the extubated rat into a chamber with just flowing oxygen to further aid recovery.
If the technique is executed correctly and the aforementioned considerations are addressed, performing a left unilateral pneumonectomy combined with MCT or Sugen creates a reliable model of severe PAH than MCT alone, hypoxia or other methods. When the pneumonectomy is performed correctly, the animal survives, the procedure has a short duration (15&#8211;30 min), and the investigator does not need specific vascular surgical skills. Furthermore, the investigator is able to successfully create PAH. The limitations of this method are that a thoracotomy is an invasive procedure, endotracheal intubation is required, and the investigator does need some general surgical skills. The neointimal pulmonary vascular occlusive lesions and the pronounced increases in pulmonary artery pressure similar to human pulmonary hypertension patients are evident in rats after the combination of pneumonectomy with MCT or SU5416 injection7,8,9. The present model is a reliable method to study the role of pulmonary overflow in the contralateral lung and flow-induced pulmonary hypertension.
This protocol may be useful for studying other diseases. It is possible to use this model to study the compensatory growth of pulmonary tissue in the contralateral lung. This technique can also be used to study and develop treatments for right ventricular failure (RVF). Severe right ventricle hypertrophy develops in animals that develop neointimal lesions from increased arterial pressure7. RVF causes death in about 70% of patients with PAH13. Increasing pulmonary blood flow can be useful for studying and developing treatments for patients who suffer from congenital cardiac diseases as well.

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 border="0" cellpadding="0" cellspacing="0" style="width:739px;" width="739">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Surgical Blade</td>
			<td style="width:111px;">Bard-Parker</td>
			<td style="width:153px;">371215</td>
			<td style="width:252px;">Incision</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Forane (Isoflurane, USP)</td>
			<td style="width:111px;">Baxter</td>
			<td style="width:153px;">NDC 10019-360-40</td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">BD Angiocath 16 G</td>
			<td style="width:111px;">BD</td>
			<td style="width:153px;">381157</td>
			<td>intubation tube, chest tube</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">SU5416</td>
			<td style="width:111px;">Cayman Chemical</td>
			<td style="width:153px;">13342</td>
			<td>Sugen&#160;</td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cooley-Mayo curved scissors</td>
			<td style="width:111px;">Pilling</td>
			<td style="width:153px;">352090</td>
			<td>Large scissors</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
			<td>hemoclip applicator</td>
		</tr>
		<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/59050/59050eq1.jpg" />
Systolic and diastolic RV and PA pressures and were measured with the pressure catheter in the main pulmonary artery connected to the ADVantage PV system. The mean pulmonary arterial pressures (mPAP) was calculated using the formula above. In the control group (n=20), the mean PA pressure was 18.6&#177;1.76 mm Hg (Figure 1). In the pneumonectomy + MCT group (n=30), the mPAP increased 2.25 times more compared to the control group (41.9&#177;2.89 mm Hg) (Figure 1). In the pneumonectomy + Sugen group (n=30), the mPAP was three times higher than in the control group (53&#177;6.60 mm Hg). In both the MCT and Sugen groups, the RVSP was much higher compared to the control group (Figure 1).
Histopathology of rat lung tissue was performed using hematoxylin and eosin staining followed by imaging with an optical light microscope. In the normal lung, there is space between the alveoli and the alveolar structures are apparent. The vessels are clear and of normal thickness (Figure 2A). In the PAH lung, there is evidence of remodeling, thickening of vessel walls, severe constriction of vessels, inflammation and focal pulmonary arteritis (Figure 2B, C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59050/59050fig1.jpg" /> 
Figure 1: Severe pulmonary arterial hypertension in the left pneumonectomy combined with MCT model. Significant differences in mPAP and RVSP between the control group and pneumonectomy + MCT group and the control group and pneumonectomy + Sugen group. Data are presented as mean SEM. The p-values were calculated using a one-way ANOVA and Tukey post hoc test. <a href="https://www.jove.com/files/ftp_upload/59050/59050fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59050/59050fig2.jpg" /> 
Figure 2: Representative photomicrographs of H&#38;E staining of lung tissue. (A) Normal lung tissue. In the normal rat lung, the arteries (red arrow) are open and the vessel walls are normal sized. (B) Pathological vascular remodeling in pulmonary arterial hypertension rats treated with pneumonectomy and MCT. The lung shows focal pulmonary arteritis and inflammation, concentric medial thickening of the vessel walls (white arrow), and concentric intimal thickening of vessel walls (black arrow), resulting in severely constricted vessels. (C) Pathological vascular remodeling in PAH rats treated with pneumonectomy and Sugen. These lungs also show focal pulmonary arteritis and inflammation, concentric medial thickening of vessel walls (white arrow), and&#160;concentric intimal thickening of vessel walls (black arrow). The lumens of these vessels are severely constricted and/or entirely closed. <a href="https://www.jove.com/files/ftp_upload/59050/59050fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

the-left-pneumonectomy-combined-with-monocrotaline-or-sugen-as-model

Nanyang Technological University

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

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

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 ...

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 ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

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 ...

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

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 ischemia and infarction in children. If left untreated, it results in a 90% mortality rate in the first year of life. In patients who survive to the adulthood, the coronary steal phenomenon and retrograde left-sided coronary flow provide a substrate for chronic subendocardial ischemia, which may lead to left ventricular dysfunction, ischemic mitral regurgitation, malignant ventricular arrhythmias, and sudden cardiac death. The average age of life-threatening presentation is 33 years and of sudden cardiac death 31 years. Therefore, surgical correction is highly recommended as soon as the diagnosis is made, regardless of age. In adult-type ALCAPA originating from the right-facing sinus of the pulmonary artery, direct re-implantation of the ALCAPA into the aorta is the more physiologically sound repair technique to re-establish the dual-coronary perfusion system and is recommended. This protocol describes the technique of direct re-implantation of adult-type ALCAPA into the aorta.

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

Surgical correction of ALCAPA is highly recommended, regardless of age or the degree of intercoronary collateralization. This protocol presents a technique for the direct re-implantation of adult-type ALCAPA into the aorta to re-establish the dual-coronary perfusion. Whenever feasible, direct re-implantation is preferred to other surgical correction techniques.

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

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 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 ...

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

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

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