Name: hemodynamic characterization of rodent models of pulmonary arterial hypertension
Uploaded: 2016-04-11T14:00:00
Description: Watch this Scientific Journal Video about Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension at JoVE.com

SR is supported by NIH K08HL114643, Gilead Research Scholars in Pulmonary Arterial Hypertension and a Burroughs Wellcome Fund Career Award for Medical Scientists.

All procedures described follow the animal care guidelines of Duke University School of Medicine.
1. Prior to Starting the Procedure
Note: Prior to any animal procedures, ensure that appropriate institutional permission has been obtained. As with all procedures, use appropriate pain medication to ensure that there is no animal suffering.
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	<li>Flush catheters with heparinized sterile saline (100 U/ml) to ensure the patency. Mark a point from the tip of the cat...

<ol>
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The protocols outlined here describe a comprehensive characterization of hemodynamics and right ventricular function in rodent models of pulmonary hypertension. While right heart catheterization as described here is a terminal procedure, the mortality associated with echocardiography is minimal, which allows for screening and follow-up of disease progression. However, similar to patients with PH having markedly increased mortality with anesthesia17, in our experience, rats with severe PH do not tolerate anesth...

Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vasculature associated with inflammatory cell infiltration, smooth muscle proliferation and endothelial cell apoptosis. These changes result in obliteration of pulmonary arterioles, subsequently leading to right ventricular (RV) dysfunction and heart failure. In order to understand the pathophysiology underlying PAH and RV failure in PAH, a number of different models, including genetic and pharmacologic models, for studying this disease have been developed (reviewed elsewhere1,2).
Of these models, the most popular are hypoxia-induced (Hx) PAH in the mouse and the monocrotaline (MCT) and SU5416-hypoxia (SuHx) models in the rat. In the mouse Hx model, mice are exposed to 4 weeks of hypoxia (either normobaric or hypobaric, corresponding to an altitude of 18,000 feet with a FiO2 of 0.10), with the resultant development of medial proliferation, increased RV systolic pressures and the development of RV hypertrophy3. MCT at a single dose of 60 mg/kg results in injury to pulmonary endothelial cells through an unclear mechanism that then results in the development of PAH4. SU5416 is a an inhibitor of the vascular endothelial growth factor receptors (VEGFR) 1 and 2 blocker, and treatment with a single subcutaneous injection of 60 mg/kg followed by exposure to chronic hypoxia for 3 weeks results in permanent pulmonary hypertension with pathologic changes similar to that seen in the human disease, with the formation of obliterative vascular lesions5. In the past years, several transgenic mouse models for pulmonary hypertension have been developed. These include knockout and mutations of the bone morphogenetic protein receptor 2 (BMPR2), as BMPR2 gene mutations are found in both familial and idiopathic forms of PAH, heme oxygenase-1 knockout and IL-6 overexpression (reviewed elsewhere1,2).
These different rodent models of PH have different levels of pulmonary hypertension, RV hypertrophy and RV failure. While the hypoxia and various transgenic mouse models result in much milder PAH than the either rat model1, it does allow testing of different genetic mutations and their associated molecular signaling pathways. The MCT model does result in severe PAH, although MCT appears to be toxic to endothelial cells in multiple tissues4. The SuHx model is characterized by vascular changes more similar to that seen in idiopathic PAH in humans, although requires both pharmacologic manipulation and hypoxia exposure. Moreover, in all of these models, there may be a disconnection between the histopathologic changes, pulmonary pressures and RV function associated with the development of PAH. This is in contrast to the human disease, where there is usually a proportionate relationship between histopathologic changes, the severity of pulmonary hypertension and the degree of RV failure. Thus, a comprehensive characterization of these rodent models of PH is required, and involves assessments of RV function (typically by echocardiography), hemodynamics (by cardiac catheterization) and histopathology of the heart and lungs (from tissue harvesting).
In this protocol, we describe the basic techniques used for hemodynamic characterization of PAH models in the rat and the mouse. These general techniques can be applied to any study of the right ventricle and pulmonary vasculature and is not limited to models of PAH. Visualizing the RV by echocardiography is relatively straightforward in rats, but is more challenging in mice due to their size and the complex geometry of the RV. Moreover, some surrogates used for quantifying RV function, such as TAPSE, pulmonary artery (PA) acceleration time and PA Doppler waveform notching, are not well validated in humans and correlate only weakly with assessment of pulmonary hypertension and RV function by invasive hemodynamics. Determination of the RV hemodynamics is best done with a closed-chest, to maintain the effects of a negative intrathoracic pressure with inspiration, although open chest catheterization with an impedance catheter allows determination of pressure-volume (PV) loops and a more detailed hemodynamic characterization. As with any procedure, developing experience with the procedures is critical to experimental success.

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			<td>Alternative catheter available from Scisense FT111B (mouse) and FT211B (rat)</td>
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			<td>Alternative catheter available from Scisense FT112 (mouse)</td>
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			<td>Alternative catheter available from Scisense FT112 (mouse)</td>
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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.

As right heart catheterization in rodents is typically a terminal procedure that is not applicable to longitudinal follow-up, echocardiography is an excellent noninvasive alternative for screening and follow-up12. While pulmonary artery systolic pressure in human PAH on echocardiography is usually derived from tricuspid regurgitation that is usually straightforward to be obtained in the apical view, such a view is not reliably obtained in rodents, preventing the estimation of pulmonary artery systolic pressure...

Watch this Scientific Journal Video about Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension at JoVE.com

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

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 ...

The overall goal of echocardiography and right heart catheretization in rodents is to determine the function of the right heart in various cardiac and pulmonary disease models. This method can help answer key questions in cardiovascular physiology research, such as, what are the effects of genetic manipulations or drug effects on the development of pulmonary hypertension? The main advantage of this technique is that it provides a comprehensive analysis of right heart function in these models of cardiac and pulmonary disease.To view the parasternal long axis of a rodent heart, begin by placing an anesthetized rodent in the supine position. Confirm the appropriate level of sedation by toe pinch and apply veterinary ointment to the animal's eyes. Next, shave the chest and apply ultrasound gel.Select B-Mode to project a 2D live image. Then, align an ultrasound transducer with the appropriate frequency to the left parasternal line. Rotate the transducer counterclockwise 30 degrees, with the probe indicator pointing in the caudal direction.Then, angle the transducer slightly, rocking the instrument along the short axis of the transducer in the same tomographic plane to obtain a full left ventricle chamber view in the center of the screen. Locate and view the lumen of the left ventricle, the interventricular septum, the lumen of the right ventricle, the ascending aorta, and the left atrium. To view the parasternal short axis at the aortic level, in B-Mode, rotate the transducer 90 degrees and angle the transducer toward the cranium to identify the aortic valve cross section view.Next, holding the transducer steady at the same position, switch to the Pulse Wave Doppler mode. Then, place the sample volume proximal to the level of the pulmonary valve in the center of the right ventricular outflow tract and position the cursor parallel to the direction of blood flow through the vessel. Adjust the scale along the direction of the blood flow as necessary to obtain a good doppler envelope, that is, an image with white borders and a dark hollow inside, indicative of laminar blood flow, and record the doppler tracing.To perform a closed chest right heart catheterization, place the anesthetized rodent under a dissection microscope and incise the skin from the mandible to the sternum. Place a pair of retractors on each side of the incision to fully expose the cervical area. Then, separate the salivary glands by blunt dissection, followed by use of fine, blunt tip forceps to expose the right external jugular vein.Carefully isolate the right external jugular vein from the surrounding connective tissue, and place two pieces of silk suture beneath the right external jugular vein. Distally ligate the vein as close to the mandible as possible and tie a loose knot proximally. Using iris scissors, make a small incision in the external jugular vein proximal to the distal tied knot.Next, mark the catheter at a length roughly corresponding to that from the external jugular vein to the heart. Then, using forceps, insert the catheter into the incision and tighten the proximal knot. Gently push the catheter into the right heart, monitoring the depth of advancement according to the mark on the catheter and verifying the location of the catheter in situ by the pressure tracing on the computer monitor.Once a right ventricular pressure tracing has been identified, stabilize the catheter for data collection. To obtain right ventricular pressure volume loops, next intubate the rodent. Then, make an incision below the xiphoid process and bilaterally dissect the skin with scissors towards the flank.To open the abdominal cavity through the abdominal wall, make a bilateral dissection through the abdominal wall under the diaphragm. Then, open the diaphragm to expose the apex of the heart and bilaterally cut the rib cage. Carefully isolate the inferior vena cava from the surrounding connective tissue and place sterile gauze soaked in normal saline over the abdominal cavity and intubation sites.Then, using a small piece of PE 60 tubing, guide the puncture of the conductance catheter into the right ventricle apex and insert the catheter tip through the stab wound until all of the electrodes are inside the ventricle. Now monitor the pressure volume loop in the software, adjusting the position of the catheter to obtain consistently shaped loops that do not demonstrate significant respiratory variation as necessary. To obtain a family of pressure volume loops, gently apply pressure to the inferior vena cava to decrease the right ventricular filling.A number of hemodynamic parameters can then be calculated from these data. To perform inflation perfusion of the lung, first, connect inflation tubing to a ring stand. Next, bluntly dissect the trachea from the surrounding muscle and connective tissue, and place a piece of silk suture around the trachea.Then, tie a loose knot in the suture, press the head to gently stretch the trachea, and cut 70%of the circumference of the trachea close to the mandible. Keeping the trachea stretched, insert a tracheal cannula and secure the cannula with a suture. Connect the cannula to the inflation tubing, then stab the right ventricle free wall with a 10 milliliter syringe filled with PBS, and flush the lungs, injecting toward the pulmonary artery.When the lungs begin to blanch, nick the left atrium once. Then, cut the root of the aorta to harvest the heart. Finally, inflate the lung with 10%buffered neutral formalin for five minutes and remove the tracheal cannula.Then, ligate the trachea, excise the lung from the thorax, and fix the lung tissue with fresh 10%buffered neutral formalin. A parasternal short axis view at the aortic level can be easily visualized in rodents, enabling the recording and measurement of pulmonary arterial doppler tracings, the shapes of which have been associated with the degree of pulmonary hypertension. In a closed chest right heart catheterization of a mouse exposed to chronic hypoxia, the right ventricle systolic pressure is elevated to 45 millimeters of mercury, consistent with significant pulmonary hypertension.In an open chest right heart catheterization of a normal rat, the right ventricle systolic pressure is observed at a significantly lower level of 27 millimeters of mercury. The procedure can be just completely in few minutes if the technique can be mastered and performed properly. While attempting this procedure, it is important to remember to make sure that the rodent is adequately anesthetized and that there are no significant sources of bleeding or tissue drying that could result in hypervolemia.After watching this video, you should have a good understanding of how to perform a comprehensive hemodynamic characterization of the right heart. Practice is critical to mastering these techniques.

Research

JoVE Journal

Medicine

Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, ...

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 ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

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 ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...

Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

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 ...

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 ...

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 ...