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.

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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, fraction (FIO2) = 0.21) hypoxia (FIO2 = 0.125), and hyperoxia 
(FIO2 = 1.00). In addition, the physiological responses of the subject are monitored in the MR scan environment. MR images 
were obtained on a 1.5 T GE MRI scanner during a breath hold from a sagittal slice in the right lung at functional residual capacity. An arterial 
spin labeling sequence (ASL-FAIRER) was used to measure the spatial distribution of pulmonary blood flow 1,2 and a multi-echo fast 
gradient echo (mGRE) sequence 3 was used to quantify the regional proton (i.e. H2O) density, allowing the quantification 
of density-normalized perfusion for each voxel (milliliters blood per minute per gram lung tissue). 
With a pneumatic switching valve and facemask equipped with a 2-way non-rebreathing valve, different oxygen concentrations 
were introduced to the subject in the MR scanner through the inspired gas tubing. A metabolic cart collected expiratory gas via expiratory tubing. Mixed expiratory O2 and CO2 concentrations, oxygen consumption, carbon dioxide production, respiratory exchange ratio, 
respiratory frequency and tidal volume were measured. Heart rate and oxygen saturation were monitored using pulse-oximetry. 
Data obtained from a normal subject showed that, as expected, heart rate was higher in hypoxia (60 bpm) than during normoxia (51) or hyperoxia (50) and the arterial oxygen saturation (SpO2) was reduced during hypoxia to 86%. Mean ventilation was 8.31 L/min BTPS during hypoxia, 7.04 L/min during normoxia, and 6.64 L/min during hyperoxia. Tidal volume was 0.76 L during hypoxia, 0.69 L during normoxia, and 0.67 L during hyperoxia. 
Representative quantified ASL data showed that the mean density normalized perfusion was 8.86 ml/min/g during hypoxia, 8.26 ml/min/g during normoxia and 8.46 ml/min/g during hyperoxia, respectively. In this subject, the relative dispersion4, an index of global heterogeneity, was increased in hypoxia (1.07 during hypoxia, 0.85 during normoxia, and 0.87 during hyperoxia) while the fractal dimension (Ds), another index of heterogeneity reflecting vascular branching structure, was unchanged (1.24 during hypoxia, 1.26 during normoxia, and 1.26 during hyperoxia). 
Overview. This protocol will demonstrate the acquisition of data to measure the distribution of pulmonary perfusion noninvasively under conditions of normoxia, hypoxia, and hyperoxia using a magnetic resonance imaging technique known as arterial spin labeling (ASL). 
Rationale: Measurement of pulmonary blood flow and lung proton density using MR technique offers high spatial resolution images which can be quantified and the ability to perform repeated measurements under several different physiological conditions. In human studies, PET, SPECT, and CT are commonly used as the alternative techniques. However, these techniques involve exposure to ionizing radiation, and thus are not suitable for repeated measurements in human subjects.

A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.

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 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 ...

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. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

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 (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 ...

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. Portal hypertension (PHT) due to cirrhosis is responsible for the most severe complications in patients with liver disease. The full picture of the portal hypertensive syndrome is characterized by increased portal pressure (PP) due to the increased intrahepatic vascular resistance (IHVR), hyperdynamic circulation, and increased splanchnic blood flow. Progressive splanchnic arterial vasodilation and increased cardiac output with elevated heart rate (HR) but low arterial pressure characterizes the portal hypertensive syndrome.
Novel therapies are currently being developed that aim to decrease PP by either targeting IHVR or increased splanchnic blood flow &#8212; but side effects on systemic hemodynamics may occur. Thus, a detailed characterization of portal venous, splanchnic, and systemic hemodynamic parameters, including measurement of PP, portal venous blood flow (PVBF), mesenteric arterial blood flow, mean arterial pressure (MAP), and HR is needed for preclinical evaluation of the efficacy of novel treatments for PHT. Our video article provides the reader with a structured protocol for performing invasive hemodynamic measurements in cirrhotic rats. In particular, we describe the catheterization of the femoral artery and the portal vein via an ileocolic vein and the measurement of portal venous and splanchnic blood flow via perivascular Doppler-ultrasound flow probes. Representative results of different rat models of PHT are shown.

Here we describe a detailed protocol for invasive measurements of hemodynamic parameters including portal pressure, splanchnic blood flow, and systemic hemodynamics in order to characterize the portal hypertensive syndrome in 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 pulmonary hypertension. For a better understanding and treatment of this disease, precise hemodynamic monitoring of left and right ventricular parameters is important. For this reason, it is essential to establish experimental pig models of cardiac hemodynamics and measurements for research purpose. 
This article shows the induction of ARDS by using oleic acid (OA) and consequent right ventricular dysfunction, as well as the instrumentation of the pigs and the data acquisition process that is needed to assess hemodynamic parameters. To achieve right ventricular dysfunction, we used oleic acid (OA) to cause ARDS and accompanied this with pulmonary artery hypertension (PAH). With this model of PAH and consecutive right ventricular dysfunction, many hemodynamic parameters can be measured, and right ventricular volume load can be detected. 
All vital parameters, including respiratory rate (RR), heart rate (HR) and body temperature were recorded throughout the whole experiment. Hemodynamic parameters including femoral artery pressure (FAP), aortic pressure (AP), right ventricular pressure (peak systolic, end systolic and end diastolic right ventricular pressure), central venous pressure (CVP), pulmonary artery pressure (PAP) and left arterial pressure (LAP) were measured as well as perfusion parameters including ascending aortic flow (AAF) and pulmonary artery flow (PAF). Hemodynamic measurements were performed using transcardiopulmonary thermodilution to provide cardiac output (CO). Furthermore, the PiCCO2 system (Pulse Contour Cardiac Output System 2) was used to receive parameters such as stroke volume variance (SVV), pulse pressure variance (PPV), as well as extravascular lung water (EVLW) and global end-diastolic volume (GEDV). Our monitoring procedure is suitable for detecting right ventricular dysfunction and monitoring hemodynamic findings before and after volume administration.

We present a protocol of creating right ventricular dysfunction in a pig model by inducing ARDS. We demonstrate invasive monitoring of left and right ventricular cardiac output using flow probes around the aorta and the pulmonary artery, as well as blood pressure measurements in the aorta and pulmonary artery.

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 morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

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 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 ...

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.

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 ...

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 ...