Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.
Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.
Historically, prognostic assessment of heart failure has focused on the LV, which is easy to image via echocardiography. Numerous studies on LV structure and function using echocardiography have led to the establishment of normal values for LV structure and function1,5,6. Measurements of LV size and systolic function obtained from two-dimensional and Color Doppler images are of great importance as they allow visual delineation of compartments and geometry in great detail for the LV7. M-Mode is often used for measuring LV dimensions and fractional shortening (FS) in mice. Inter-observer and intra-observer variability are low for diameter measurements using this mode, but wall thickness measurements tend to be quite variable7. Pulsed Doppler with color (PW or Color Doppler) has been used to evaluate valvular regurgitation8,9.
Similar to LV, the RV plays an important role and is a significant predictor of morbidity and mortality in patients afflicted with cardiopulmonary disease1,7,10. However, echocardiographic evaluation of RV is inherently challenging due to its complex shape5,11 and its retrosternal position that blocks the ultrasound waves8,9. RV is a crescent shaped structure wrapping around the LV and has a complex anatomy with thin walls that are accustomed to low pressure and resistance to pulmonary vasculature6. To overcome elevated vascular resistance (PVR), the RV first increases in size and undergoes hypertrophies. In chronic diseases like pulmonary hypertension or pulmonary vascular disease, RV undergoes progressive dilatation, eventually resulting in the deterioration of systolic and diastolic function4,5,10.
Echocardiography plays an important role in the screening and diagnosis of PAH despite some limitations present in its clinical diagnostic capability. The main advantage of TTE lies in that it is noninvasive and that it can be performed on lightly sedated, or even conscious animals9. TTE also provides a reasonable estimate of PA pressures, as well as an ongoing assessment of changes in RV structure and function12,13. Due to technical advances in TTE, which include the development of high-frequency mechanical probes, allowing axial resolution of approximately 50 μm at a depth of 5-12 mm, high frame rates (greater than 300 frame/sec), and high sampling rates, echocardiography is a choice tool for imaging the rapidly contracting small sized mouse heart8,11.
Longitudinal monitoring of RV function using multiple views, including 2-dimensional (2D) short and long axis, M-mode and Doppler acoustic windows provide complementary information of RV anatomy and function. Collectively, this methodology permits complete longitudinal assessment of RV hemodynamics in physiology and pathological setting4,7.
Herein, we provide a detailed step-by-step methodology of using noninvasive TTE to characterize RV anatomical and functional changes secondary to PAC in mice.
Surgical Procedure
1. Parasternal Long Axis (PLAX) M Mode View to Obtain RV Chamber Dimension, Fractional Shortening (FS), and RV Wall Thickness
2. Parasternal Short-axis View at Mid Papillary Level to Obtain Fractional Area Changes (FAC)
3. Parasternal Short-axis View at Aortic Valve Level (RV PSAX Aortic Level) to Obtain RV Wall Thickness and PA Peak Velocity
4. Modified Parasternal Long-axis View of RV and PA to Obtain PA Peak Velocity
5. Data Calculation and Analysis
6. Notes
In this study, baseline echocardiography was performed 48 hr prior to surgery. Mice were randomized into two groups. Mice received pulmonary artery occlusions (PAC) and sham operations (Sham). Echocardiography was performed at day 0, 3, and 7 following surgical procedure. The animals were euthanized immediately following the last echocardiography and hearts were harvested for histological assessment. Catheterization was conducted in subgroup (n=3 and 2 for day 0 and 7, respectively) of PAC mice to measure RVSP via pressure catheter.
All imaging data obtained was analyzed off line. Importantly, sonographers were blinded to the procedures that the animals underwent. The images presented in this study were taken by two independent imagers. The inter- and intra- observer variability was tested, and found to be less than 6 % and 11%, respectively. The measurements were obtained using all available acoustic windows- B Mode, M Mode and Color Doppler images taken together were used in assessment of RV structure and function. All measurements were averaged over 5 cardiac cycles. For each measurement, the mean value and standard deviation (SD) was obtained. Often similar measurements were performed from different imaging windows to obtain complementary information and multiple data-points for comparison of accuracy and reliability.
As shown in Figures 7A and 7B, systolic function of RV can be measured in PLAX view as %FS or in mid papillary muscle view as %FAC, respectively. While the decrease in FAC was already significant at day 0, the decrease in FS was only significant at day 7 (n=6, P<0.01). One major caveat of this view is that because of the retrosternal position of RV and occasionally due to the obstruction posed by the ribs, much care should be taken to obtain the RV image to accurately demonstrate the maximum diameter of the right ventricle without foreshortening the image. Small variations in RV diameter can mask small but significant changes in function. In contrast, %FAC is markedly decreased following PAC, even at day 0 right after the PA occlusion (n=6, P< 0.05) and decreased progressively overtime (n=6, P< 0.001). Thus, %FAC should be used as a primary measure of RV function and %FS as a secondary measure. It is noteworthy that %FAC has been shown to be a reliable predictor of heart failure, sudden death, stroke and/or mortality3,4,10,16.
The RV dilatation can be measured in the long- and short-axis as RV chamber dimension (RVIDd) and RV area in diastole (Figures 7C and 7D). The reliability of echo derived RVIDd in small rodents is indeed not as dependable as those measures in humans. This represents an important caveat in measuring RVID in mice. In small animals, the RVID is more clearly visualized in the long axis view, rather than the apical four-chamber view, as is commonly done in humans. Importantly, though, the endocardial definition of the anterior wall is often suboptimal under the long axis view and oblique imaging may underestimate size measures. We find that RV area measure in the mid papillary muscle view is a more reproducible and reliable surrogate for RV chamber dimension and RV dilatation in mice.
RV free wall thickness, as a marker of RV hypertrophy, can be determined accurately either using M Mode or the area-trace method (Figures 7E and 7F). Similarly, the PA peak velocity can also be obtained with either at PLAX or SAX mode (Figures 7G and 7H, respectively). Reliable measurements of PA peak velocity and thus, peak-pressure gradient within the PA can be obtained using Color Doppler in both short- and long- axis acoustic windows (Figures 7G and 7H). It should be noted that these velocity measurements are angle dependent and hence, it is recommended to obtain velocities using multiple views and with similar sweep speed for all tracings (greater than 100 mm/sec).
Lastly, Figure 8 shows that in noninvasive echocardiography is a viable alternative to the terminal right heart catheterization method used as the gold standard for RVSP measurement9. For the 5 animals, catheterization for comparison of RVSP measurement methods was performed, and calculations of pressure were highly comparable (Pearson correlation coefficient r=0.943, P>0.05). In echocardiography, the PA peak velocity is measured reliably, and it follows that the calculation from the PA peak velocity is also reproducible. Additionally, this method allows for serial measurement of the pulmonary pressure gradient over time.
In summary, the noninvasive echo-based imaging can be a useful tool to follow RV structural and functional remodeling longitudinally similar to what has been commonly used in LV.
Figure 1. Graphical illustrations of the imaging probe position. Red line indicating the position of the probe for obtaining A, parasternal long axis B, parasternal short axis, C, modified parasternal long-axis view and D, the x-y direction of the probe. Click here to view larger image.
Figure 2. Parasternal long axis (PLAX) view. Graphical illustration and representative PLAX images from A, sham and B, PAC mouse heart. Key landmarks seen in the view areas follows. 1: Right ventricle (RV), 2: Left ventricle (LV), 3: Aorta (Ao), 4: Mitral valve (MV), 5: Left atrium (LA), 6: Diastolic dimension of right ventricle (D), 7: Systolic dimension of right ventricle (S), 8: Right ventricular wall (RVW), 9: Interventricular septum (IVS). Click here to view larger image.
Figure 3. Parasternal short-axis view (PSAX) at mid-pap level of right ventricle (RV). Graphic illustration, representative image in PSAX at mid-papillary muscle level and H&E staining from A, sham and B, PAC mouse heart. Key landmarks seen in the view are as follows. 1: right ventricle (RV), 2: interventricular septum (IVS), 3: left ventricle (LV), and 4 & 5: papillary muscles. Click here to view larger image.
Figure 4. Parasternal short-axis view (PSAX) at aortic-level. Graphic illustration and representative B Mode images from A, sham and B, PAC mouse heart. Graphic illustration and Color Doppler images from C, sham and D, PAC mouse heart. Key landmarks seen in the view are as follows. 1: Right ventricular outflow tract (RVOT), 2: Tricuspid valve (TV), 3: Right atrium (RA), 4: Left atrium (LA), 5: Aortic valve (AV), 6: Pulmonary valve (PV), and 7: Pulmonary artery (PA). Click here to view larger image.
Figure 5. Modified parasternal long-axis (PLAX) view of right ventricle (RV) and pulmonary artery (PA).Graphical illustration, representative modified PLAX images, and H&E histology from A, sham and B, PAC mouse heart. Graphic illustration and Color Doppler images from C, sham and D, PAC mouse heart. Key landmarks seen in the view areas follows. 1: Right ventricle (RV), 2: Left ventricle (LV), 3: Aorta (Ao), 4: Left atrium (LA), and 5: Pulmonary artery (PA). Click here to view larger image.
Figure 6. RV wall-thickness from Parasternal short-axis view (PSAX) at aortic level view. Graphical illustration of PSAX-image of heart section at aortic-level. Measurement of RV wall thickness can be derived from the area/length. Pink shade indicates area of RV free wall and blue line indicates inner and outer circumferences of RV.
Figure 7. Structural and functional assessments of right ventricle (RV). A, Fractional shortening (FS) obtained using M mode at PLAX. B, Fractional area changes (FAC) obtained using PSAX at mid pap level. C, Right ventricular chamber dimension in diastole (RVIDd) obtained using M mode at PLAX. D, End diastolic right ventricular area obtained using PSAX at mid pap level. E, Right ventricular wall thickness at diastole obtained using M mode at PLAX and F, PSAX at aortic level. Pulmonary artery peak velocity obtained at G, modified PLAX at RV and PA view and H, PSAX at aortic level. Sham, n=6 and PAC, n=6, *, p<0.05. Click here to view larger image.
Figure 8. Correlation of pulmonary artery (PA) pressure measured using echocardiography (ECHO) and Millar microtip pressure catheter (Catheter). For echocardiography, peak-pressure gradient were calculated from PA peak velocities using modified Bernoulli’s equation. The peak-pressure gradients (measured at site of constriction) were consistent with RVSP via catheterization with a correlation coefficient 0.943 (n=5).
Full name | Abbreviation |
Left atrium | LA |
Left ventricle | LV |
Right atrium | RA |
Right ventricle | RV |
Aorta | Ao |
Pulmonary artery | PA |
Aortic valve | AV |
Mitral valve | MV |
Tricuspid valve | TV |
Pulmonary valve | PV |
Interventricular septum | IVS |
Papillary muscle | PM |
Fractional shortening | FS |
Fractional area change | FAC |
Parasternal long axis view | PLAX |
Parasternal short axis view | PSAX |
Transthoracic echocardiography | TTE |
Pulmonary artery constriction | PAC |
Right ventricular systolic pressure | RVSP |
Pulmonary arterial hypertension | PAH |
Right Ventricular outflow tract | RVOT |
Right ventricular internal dimension in diastole | RVIDd |
Table 1.
We demonstrate that TTE provides a sensitive and reproducible methodology for routine assessment of RV structure and function in mice. Before the advent of TTE, studies of the RV largely focused on RVSP measurement via right heart catheterization, a terminal and invasive procedure6,9,11,17.
Prior reports have described a variety of techniques for performing right heart measurements3,4,11,17-19. However, the majority of previous studies reported RV size and structural data in a predominantly qualitative rather than quantitative fashion5. A standardization of RV assessment is thus still in the beginning stages despite recent interest in RV function in the context of PAH and other models of diseases9,19.
Taken together, these data provide evidence that noninvasive method of imaging can be a reliable and valuable tool for early evaluation of RV dysfunction. We established an imaging methodology to noninvasively visualize RV structural and functional changes in real time using a number of complementary imaging windows, and benchmarked our echo-based method of pressure-gradients against the conventional gold standard RVSP measurement by catheterization.
When imaged longitudinally, following an acute injury such as PAC, the RV undergoes rapid remodeling and the dynamic changes can be captured reproducibly through imaging. The image data coupled with the steps outlined in this methodology, along with further progress in technology such as 2D strain imaging, 3D echocardiography, and use of speckle-training20 will improve a systematic echocardiographic evaluation of RV12,15. This could lead to increased therapeutic intervention in pathology of cardiopulmonary diseases by permitting earlier disease detection.
In summary, TTE can provide an essential first-step towards a comprehensive assessment of cardiac status and can serve as an effective discovery and assessment tool of physiological changes in structure and function. Because TTE is a noninvasive and widely accessible imaging modality, it offers the potential to aid investigations of cardiac diseases that require high-throughput and rapid data collection.
The authors have nothing to disclose.
We thank Fred Roberts and Chris White for exemplary technical support. We thank Brigham Women’s Hospital Cardiovascular Physiology Core for providing with the instrumentation and the funds for this work. This work was supported in part by NHLBI grants HL093148, HL086967, and HL 088533(RL), K99HL107642 and the Ellison Foundation (SC).
High Frequency Ultrasound | FUJIFILM VisualSonics, Inc. | Vevo 2100 | |
High-frequency Mechanical Transducer | FUJIFILM VisualSonics, Inc. | MS250, MS550D, MS400 | |
Millar Mikro Pressure Catheter | Millar | SPR-1000 |