This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.
Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.
Elevated pulmonary pressure and right ventricular (RV) dysfunction are the hallmarks of pulmonary vascular disease in animal models and human patients with pulmonary arterial hypertension (PAH). Transgenic and toxic (e.g. monocrotaline or hypoxia) models of PAH are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease.
Echocardiography is the mainstay of the noninvasive assessment of ventricular function in rodent models1,2. Echocardiography has the advantage of clear translation to humans in whom the same tool is used. In addition, some genetic models exhibit incomplete penetrance3; the ability to noninvasively identify affected animals saves valuable time and resources. Noninvasive assessment of disease severity without sacrificing an animal also allows researchers to serially study the effects of investigative therapies. This is especially important given the rapidity with which translational therapies can progress to human trials4,5.
In humans, echocardiographic assessment of RV size and pulmonary hypertension is particularly challenging due to the retrosternal position and irregular shape of the RV6. Rodent models have the added challenges of small size and extremely rapid heart rates (300-700 beat/min). Recent advances including higher frame rates and smaller transducers have improved image quality and even allowed conscious imaging in some experimental protocols, though most rodent imaging is done under anesthesia7,8. Excellent experimental protocols of echocardiography in rat models of PAH have been described and validated against both MRI and invasive hemodynamics1,9. However, published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation and a model of isolated RV afterload after pulmonary artery banding; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We will describe animal preparation and detailed assessment of RV size and function as well as main pulmonary artery (PA) size. We also demonstrate the techniques and calculations needed to estimate stroke volume and cardiac output. Technical limitations preclude accurate Doppler estimates of pulmonary pressure, but we have applied a well-validated human surrogate, pulmonary artery acceleration time, to estimate PA pressure.
1. Equipment Preparation
2. Mouse Preparation Including Anesthesia, Hair Removal, and Positioning
3. Acquisition of Images: Imaging in Parasternal Long Axis View
4. Acquisition of Images: Imaging in Parasternal Short Axis View
The principal goals of this protocol are to quantify RV size and function, and to understand the degree to which the pulmonary vasculature is diseased. Appropriate preparation of both the mouse and echocardiography equipment is essential to obtaining accurate and reproducible results. Mice should have their chest depilated and limbs secured to the imaging platform with tape. Anesthesia, in this case isoflurane, is administered via nose cone. The transducer should be checked for defects, particularly air bubbles, which can degrade image quality. Obtaining good quality 4-chamber views of the heart is quite difficult in mice so this protocol focuses on RV assessment using the parasternal short and long axes. Relevant anatomy in these views is shown in Figures 1A and 1B.
RV size is best assessed in the parasternal long axis view and is measured as the distance from the free wall to the interventricular septum using M-mode (Figure 2). This measurement is only possible when the RV is dilated as the normal RV is very small. In mice, it is not possible to accurately measure the usual metrics of RV function in humans such as fractional area change and the tricuspid annular plane systolic excursion. These measurements require high quality views of the RV free wall which are very difficult to obtain in mice. However, using PW Doppler to measure the velocity time integral (VTI) at the level of the right ventricular outflow tract (RVOT) and the diameter of the pulmonary artery, it is possible to estimate RV stroke volume (Figure 3). Stroke volume and cardiac output are calculated from the formulae in Table 1. Heart rate is obtained from m-mode imaging.
Main PA diameter reflects PAH severity in humans10 and can be measured in mice in the parasternal short-axis view (Figure 3). It is important to have clear view of both sides of the main PA because this value is squared in the equation used to calculate cardiac output. If PA size cannot be accurately measured, left ventricular outflow tract diameter and LVOT VTI can be inserted into the equations above as RV and LV output are equal in the absence of shunting.
The RV VTI can be further interrogated to estimate PA pressure by measuring the time to peak velocity (pulmonary artery acceleration time [PAT], Figure 4). In humans PAT is used to dichotomize PA pressure as high or low11, and may be used to estimate PA pressure when a tricuspid regurgitant jet is not present.12
Figure 1. Echocardiographic Views of Murine Anatomy. Panel A shows normal anatomy in the parasternal long-axis view. Panel B shows anatomy in the parasternal short axis view. The right ventricle is enlarged in panel B. Click here to view larger image.
Figure 2. Measurement of Dilated Right Ventricle. This figure demonstrates (A) normal RV size in a control mouse (B) severe RV enlargement in a mouse that underwent pulmonary artery banding model. Click here to view larger image.
Figure 3. Measurement and Calculation of Right Ventricular Stroke Volume. This figure shows the measurements for both right ventricular VTI and pulmonary artery diameter. The method for calculating stroke volume with these data is also demonstrated. Click here to view larger image.
Figure 4. Measurement of PAT. Pulmonary acceleration time is measured as the time to peak velocity in the RVOT VTI. Click here to view larger image.
Table 1: Useful Calculations in Echocardiography.
Measurement | Formula |
Pulmonary artery/aortic area | π(diameter/2)2 |
Right Ventricular Stroke volume | PA area x VTI |
Cardiac Output | heart rate x stroke volume |
Cardiac Index | cardiac output / body surface area |
Fractional Shortening | (LV end-diastolic dimension – LV end-systolic dimension) / LV end-diastolic dimension |
Body surface area = 10.5 (grams)2/3 13
Mouse models of disease, either transgenic or toxin-related, require phenotypic validation that the model actually recapitulates the human disease it is intended to emulate. This validation can often be accomplished by the presence or absence of a particular feature, for example development of a tumor. However, models that result in hemodynamic abnormalities such as aortic constriction models of left ventricular hypertrophy or our transgenic model of PAH are more difficult to validate. These models require either terminal measurement of hemodynamics or tools to noninvasively measure hemodynamics and abnormalities in cardiac function. Echocardiography is critical to such models because it allows real-time quantification of hemodynamics and cardiac function without requiring sacrifice of diseased animals14. In addition, individual animals can be imaged serially to follow the natural history of a disease or response to therapy. We estimate that proficiency in echocardiography of the right heart according to this protocol can be gained after performing approximately 20 examinations.
The ability to estimate cardiac output on echocardiography is also critical to the calculation of pulmonary vascular resistance (PVR) at the time of sacrifice. Measurement of cardiac output using conductance catheters is often unreliable in our model because of the small size of the RV. At the time of sacrifice, we measure invasive PA systolic pressure using a conductance catheter and combine this with cardiac output from echocardiography to determine PVR (pulmonary wedge pressure is assumed to be low and is ignored). This allows us to further quantify the degree of pulmonary vascular disease in our model.
Theoretical and Practical Limitations of Echocardiography
It is important to recognize that the application of ultrasound physics to humans and live animals has limitations. Accurate measurement of blood velocity using Doppler is dependent on the angle of flow relative to the angle of insonation (angle at which transducer is aimed). For every degree those two angles are unaligned, the measurement of blood velocity will be decreased by the cos (θ)15. Clinically, if the two angles are off by more than 20° the measurement is felt to be unreliable. This has potentially important implications for this protocol in the measurement of the LVOT and RVOT VTI. If the PW angle cannot be well-aligned with the direction of blood flow in the LVOT and RVOT, the measured SV and cardiac output will be falsely low.
Another potential measurement error is in the calculation of PA and aortic area which are then used to calculate SV and cardiac output. Because the area of a circle is πr2, any inaccuracy in the measurement of the diameter of the aorta or pulmonary artery is squared and the error compounded. In humans, the RVOT and LVOT diameters are used to calculate SV instead of the diameter of the aorta and pulmonary arteries; however, in mice it is very difficult to accurately identify the LVOT and RVOT so we substitute the aorta and pulmonary artery areas. Provided the same technique is used in one animal to the next, this minor difference should not impact study results.
Right ventricular dysfunction is the major manifestation of pulmonary hypertension in humans. A number of practical limitations pertain to the noninvasive assessment of the right heart. In humans and mice, the right ventricle is situated adjacent to the chest wall. This close proximity to the transducer makes imaging the anterior RV free wall very difficult. The RV is an irregular crescent shape which precludes volumetric assumptions like those used to determine LV size and function. RV size in mice can usually only be determined as normal or enlarged due to the difficultly in seeing the RV free wall. However, this categorization is still helpful to validate the presence or absence of pulmonary vascular disease.
Echocardiography may be performed on mice with or without anesthesia. We prefer to use anesthesia to maximize the quality and accuracy of our measurements but recognize that anesthesia will lower the heart rate. When performed without anesthesia, image quality may suffer and the process is a source of stress for the animals which will elevate the heart rate and blood pressure. We perform all echocardiograms with an identical degree of anesthesia to allow comparison of results between and within mice.
Quantification of RV and pulmonary vascular function in mice relies on Doppler estimation of beat to beat flow across the pulmonary valve, the RVOT VTI. This can be used as a dichotomous variable (high/low), but when measured carefully can be used as a serial measurement in a mouse or compared between groups with different interventions. Advanced equipment is commercially available to use color Doppler assessment for the presence and velocity of a tricuspid regurgitation (TR) jet, which is used in humans to quantify the severity of pulmonary hypertension. In humans without a measureable TR jet, the PAT is used as a surrogate to determine whether pulmonary hypertension is present or absent11. PAT will shorten as PH worsens because RV ejection will stop sooner against increased pressure. This method has also been validated in rat models of PAH as an accurate estimate of the severity of pulmonary hypertension1. Finally, the shape of the RVOT VTI envelope can shed light on coupling between the RV and the pulmonary vasculature in humans16. Notching of the envelope is consistent with elevated pulmonary vascular resistance with later notching indicating higher resistance. However, we have not observed these patterns in mice in our model of PAH, even in mice subsequently confirmed to have severe PH by invasive measurement.
Aside from echocardiography, cardiac magnetic resonance imaging (CMR) is the only noninvasive alternative for the assessment of RV function. In rats, CMR provides accurate measurement of RV thickness, mass and volumes (and thus ejection fraction and cardiac output)17. In addition, CMR-measured PAT and flow-time curves (analogous to VTI) correlate strongly with echocardiography and invasively measured hemodynamics. Despite some obvious advantages, CMR is more expensive and time-intensive than echocardiography and for those reasons is rarely used in our experiments. To our knowledge no study has validated the echocardiographic measurements described here with invasive measurements or CMR. However, we routinely use the measurements presented in this protocol to assess disease penetrance and severity18-20.
The authors have nothing to disclose.
Vevo 770 High Resolution Micro-Ultrasound System | Visualsonics Inc. | get more info at www.visualsonics.com/products | |
RMV (Real-Time MicroVisualization) 704B 40 mH Scanhead w/ Encapsulated Transducer | Visualsonics Inc. | get more info at www.visualsonics.com/products | |
Vevo Integrated Rail System including the Physioogical Monitoring System | Visualsonics Inc. | get more info at www.visualsonics.com/products | |
Computer Monitor set up for use with the Vevo770 | DELL or other General Supplier | ||
Computer Mouse set up for use with the Vevo770 | General Supplier | ||
Vevo770 Cardiac Package Software | Visualsonics Inc. | get more info at www.visualsonics.com/products | |
VetEquip Portable Tabletop Anesthesia Machine with an Isoflurane Vaporizer | VetEquip | get more info at vetequip.com | |
Activated Charcoal Waste Gas Containers | VetEquip/Vaporguard | 931401 | get more info at vetequip.com |
Puralube Eye Ointment | Henry Schein | get more info at henryschein.com | |
Ecogel 100 Ultrasound Gel | EcoMed Pharmaceuticals | 30GB | get more info at ecomed.com |
3M Transpore Tape | Fisher Scientific | 1527-0 | get more info at fishersci.com |
Small Flathead Screwdriver | General Supplier | ||
Sterile H2O | DDI H2O from faucet and then autoclave | ||
6 in Cotton Tipped Applicators | Fisher Scientific | get more info at fishersci.com | |
Nair (depilatory cream) | General Supplier | ||
2 in x 2 in Gauze Sponges | Fisher Scientific | get more info at fishersci.com |