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 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 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.
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.
2. Echocardiography
Note: A full description of rodent echocardiography is described elsewhere7. For the mouse, prior to anesthesia, images can be obtained on the awake, manually restrained animal. For the rat, anesthesia prior to echocardiography is preferred as rats are too large to be manually restrained while awake).
3. Right Heart Catheterization
4. Collection of Heart and Lung Samples
Note: As the procedures here are described as terminal, the animal must be euthanized after either closed- or open-chest right heart catheterization.
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 by Doppler. However, a PSAX view at aortic level can be easily visualized in rodents, which enables to record and measure the pulmonary arterial Doppler tracing, the shape of which has been associated with the degree of pulmonary hypertension12. Representative results of the echocardiography studies are demonstrated in Figure 3. In this protocol, sonographers were blinded to the treatments or procedures that animals received. The results were analyzed off line.
Right heart catheterization and measurement of RVSP, which serves as an accuracy estimation of pulmonary artery systolic pressure in the absence of pulmonary stenosis, is the gold standard for quantification of PAH in rodent models13,14. In this protocol, both the closed-chest approach for RV pressure measurement (Figure 5) and open-chest approach for RV P-V loop analysis (Figure 6, 7) are presented15,16. Advantages of the closed-chest approach is less invasive than the open-chest approach and animals are more stable for a longer period6. Also, positive pressure ventilation is not required with this approach nor is the thorax opened, preserving the normal right-sided filling pressures associated with breathing and negative intrathoracic pressure. The open-chest approach allows the use of conductance catheters and the determination of PV loops, from which important parameters of RV function can be calculated. Thus, these approaches are complementary as they have different strengths and weaknesses.
In the closed-chest data shown from a mouse Hx model, the RVSP is elevated at 45 mmHg, consistent with significant pulmonary hypertension (Figure 5). In the open-chest data shown from a normal rat, the RVSP is significantly lower, at 27 mmHg (Figure 7). The relative volume units (RVU) of the X axis can be converted to volume units after cuvette calibration, followed by saline calibration to remove the component of the conductance due to the heart wall6,8. This then allows a calculation of important parameters of cardiac function, such as contractility (usually as assessed by the end-systolic elastance, Ees), diastolic function (from the end-diastolic pressure volume relationship), arterial elastance (Ea) and preload-recruitable stroke work, calculations of which are discussed elsewhere6,8.
Figure 1: Preparation of rodent for procedure. Rats were anesthetized and the chest and neck were shaved. The red dash line denotes the incision that will be used for exposing the external jugular vein. Black lines represent the clavicles and sternum. The blue circle indicates the probe location for echocardiography.
Figure 2: Echo views of different anatomic structures. These representative images are from a normal mouse. (A) Parasternal long axis (PLAX) view. LA: Left atrium; LV: The lumen of the left ventricle; IVS: Interventricular septum; RV: The lumen of the right ventricle; AO: Ascending aorta (AO). (NOTE: Different imaging orientation on PLAX may result from differing imaging conventions.) (B) M-mode of the LV with LV systolic (LVs) and diastolic (LVd) diameters, and anterior (AWT) and posterior wall thickness (PWT) noted. Fractional shortening is calculated as (LVd-LVs)/LVd. (C) PW Doppler of the aorta demonstrating an aortic outflow signal. Please click here to view a larger version of this figure.
Figure 3: Parasternal short-axis (PSAX) and RVOT views. These representative images are from a rat with MCT PAH. (A) PSAX view at the mid-pap level of right ventricle. (B) PSAX view at the aortic level. RVOT: right ventricular outflow tract. PA: pulmonary artery. Ao: aorta. (C) PW Doppler Mode. The sample volume (yellow line) is placed in the center of the right ventricular outflow tract proximal to the level of pulmonary valve. Please click here to view a larger version of this figure.
Figure 4: Exposure of external jugular vein for catheterization of a rat. (A) An incision from the mandible to the sternum was made and a pair of retractors was placed to each side of the incision to expose the cervical area. The salivary is gland (SG) is overlying the external jugular vein (EJ). (B) Bluntly dissect to separate the salivary glands and surrounding connective tissue to fully mobilize the right external jugular vein. (C) Place distal and proximal 4-0 silk suture around the right external jugular vein. (D) A PE-50 tube used as the pressure catheter was inserted into the right EJ. SG: salivary gland; EJ: external jugular vein; DS: Distal suture; PS: proximal suture; Cath: Catheter.
Figure 5: Waveforms in different chambers during right heart catheterization. Representative sample traces of pressure changes during right heart catheterization of a mouse with hypoxia-induced PAH. Panel left, middle, and right show pressure changes (mmHg) over time (sec) in superior vena cava (venous), right atrium (RA), right ventricle (RV).
Figure 6: Open-chest approach for RV catheter placement. (A) View after intubation of the trachea, cutting through the abdominal wall, opening the diaphragm to expose the apex of the heart and bilaterally cut the rib cage. (B) Isolation and placement of a piece of suture around the IVC.; and (C) After insertion of the conductance catheter through the RV apical free wall.
Figure 7: Right ventricular pressure-volume loop analysis. (A) Channels in the software demonstrating conductance (RVU – relative volume units), RV pressure (mmHg) and heart rate (BPM). Smoothing of 7-11 beats is required for obtaining good signal. (B) Placement of the conductance catheter in an area that is prone to changes in respiration results in PV loops that are variable. (C) Stable PV loops with proper placement of the conductance catheter. (D) Representative family of PV loops after relieving pressure on the inferior vena cava. This family of curves allows a calculation of end-systolic elastance (Ees – a measure of cardiac contractility) and vascular elastance (Ea – a measure of pulmonary vascular elastance). Please click here to view a larger version of this figure.
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 anesthesia as well, due to decompensated right heart failure, decreased RV preload, and subsequently hypoxia-induced pulmonary vascular constriction. Therefore, slow induction and closely monitoring respiration are essential.
The video and illustrations demonstrate what our laboratory has found to be successful with regards to these procedures, although alternative approaches can also be utilized (reviewed in Pacher et al.6). In PH research, these techniques are required in order to properly characterize different models of disease and the effects of genetic or pharmacologic manipulations on them. These methods each have their inherent limitations: for echocardiography, views of the right ventricle are limited and are difficult to quantify; for closed-chest catheterization, only an estimate of PA pressure is provided by an RVSP and an assessment of RV function is limited to dP/dt; for open-chest catheterization, the thorax must be opened, which can have effects on venous return and right heart function.
Experience with these procedures is required to obtain reproducible and consistent results and there are a few critical steps where care is required. Determination of parameters from echocardiography requires standardized views, as small changes in probe angle can result in very different distance and Doppler measurements. During catheterization, introduction of a curve into the catheter can facilitate entry into the right ventricle; with polyethylene tubing, this can be done by heating the tubing gently; for micromanometer catheters, there are specialized catheters designed with a curve for this purpose. Dissection of the RV from the LV+S requires carefully en bloc harvest of the heart and the separation of the ventricles from the atria. As in any experiment, careful attention to detail with all of these procedures is critical for obtaining useful data. It is also important to be aware that animal respiration can affect the signal recording for pressure and PV loops. Identification and quantification of good and uniform pressure traces with consistently shaped loops are important in obtaining reliable data.
This protocol will be of utility in research with animal models of PH and the right ventricle. Such studies include models of RV pressure overload (pulmonary artery banding) and right heart failure. These different experimental approaches allow quantification of pulmonary hypertension and RV function that are complementary. Cardiac catheterization has been used for the assessment of RV pressures, and recently investigators have reported success in traversing the pulmonic valve in the rat with micromanometer catheters designed for the mouse, allowing a direct measurement of PA pressures18. Open-chest PV loops allow a quantification of RV function; for example, in the rat MCT model, losartan significantly reduced RV afterload, restored ventricular-arterial coupling and improved RV diastolic function16. Noninvasive parameters derived from echocardiography have been used to estimate stroke volume, cardiac output and an estimate of PA pressures19,20. In total, these different techniques allow an assessment of the hemodynamic severity of pulmonary vascular disease and the RV response.
The authors have nothing to disclose.
SR is supported by NIH K08HL114643, Gilead Research Scholars in Pulmonary Arterial Hypertension and a Burroughs Wellcome Fund Career Award for Medical Scientists.
Vevo 2100 Imaging System (120V) | VisualSonics, inc. | VS-11945 | |
Vevo 2100 Imaging Station | VisualSonics, inc. | ||
High-frequency Mechanical Transducers | VisualSonics, inc. | MS250, MS550D, MS400 | |
Ultrasound Gel Parker | Laboratories Inc. | 01-08 | |
PowerLab 4/35 | ADInstruments | ML765 | |
Labchart 8 | ADInstruments | ||
BP transducer with stopcock and cable | ADInstruments | MLT1199 | |
BP transducer calibration kit | ADInstruments | MLA1052 | |
Mikro-Tip Pressure Catheter for mouse | Millar | SPR-1000 | Alternative catheter available from Scisense FT111B (mouse) and FT211B (rat) |
Mikro-Tip Pressure Catheter for rat | Millar | SPR-513 | Alternative catheter available from Scisense FT111B (mouse) and FT211B (rat) |
Millar Mikro-Tip ultra-miniature PV loop catheter for mice | Millar | PVR-1035 | Alternative catheter available from Scisense FT112 (mouse) |
Millar Mikro-Tip ultra miniature PV loop catheter for rats | Millar | SPR-869 | Alternative catheter available from Scisense FT112 (mouse) |
Millar PV system MPVS-300 | Millar | MPVS-300 | |
4-0 Silk Black Braid 100 Yard Spool | Roboz Surgical | SUT-15-2 | |
6-0 Silk Black Braid 100 Yard Spool | Roboz Surgical | SUT-14-1 | |
Iris Scissors, Delicate, Integra Miltex | VWR | 21909-248 | |
VWR Dissecting Scissors, Sharp/Blunt Tip | VWR | 82027-588 | |
VWR Delicate Scissors, 4 1/2" | VWR | 82027-582 | |
Two star Hemostats, Excelta | VWR | 63042-090 | |
Neutral-buffered formalin | VWR | 89370-094 | |
Crotaline | Sigma | C2401 | |
SU5416 | Tocris Biosciences | 3037 | |
3.5X-45X Boom Stand Trinocular Zoom Stereo Microscope | AmScope | SM-3BX | |
PE (Polyethylene Tubing)-10 | Braintree Scientific Inc | PE10 36 FT | |
PE (Polyethylene Tubing)-50 | Braintree Scientific Inc | PE50 36 FT | |
PE (Polyethylene Tubing)-60 | Braintree Scientific Inc | PE60 36 FT | |
Tabletop Isoflurane Anesthesia Unit | Kent Scientific | ACV-1205S | |
Surgisuite multi-functional surgical platform | Kent Scientific | Surgisuite | |
Retractor set | Kent Scientific | SURGI-5002 | |
Anesthesia induction chamber | VetEquip | 941443 | |
Anesthesia Gas filter canister | Kent Scientific | ACV-2001 | |
Rodent nose cone | VetEquip | 921431 |