The present study demonstrates that CO can be accurately estimated using echocardiography. Four echocardiographic methods were tested including M Mode-, 2D-, and pulmonary flow-calculated CO, and pulmonary VTI-derived CO. All echocardiographic methods correlated closely with flow probe-measured CO. Echocardiographic measurements overestimated CO measured by flow probe. The overestimation was greatest using the M Mode and lowest using 2D images. Intraobserver variability was similar using all algorithms however, interobserver variability was lowest using pulmonary VTI. In a model of mouse endotoxin-induced shock, the values of CO reported using M Mode were the highest. Values of CO reported using 2D images were the lowest. Pulmonary flow and pulmonary VTI detected a greater decrease in CO after endotoxic injection than 2D or M Mode.
Cardiac output is routinely measured using echocardiography in humans and large animals. Recently, CO was validated in rats by comparing echocardiographic measurements to thermodilution 19
. Measurement of the CO by diameter and VTI measurements at the level of the aortic annulus were found to have the greatest accuracy and the smallest bias. In mice, the absence of reproducible apical views precludes the accurate measurement of flow velocities at the level of LV outflow tract. The LV outflow tract velocities may be measured on the parasternal long axis view, however the orientation of the aortic root on that view is not parallel or close to parallel to the Doppler beam. In contrast, the pulmonary artery is easily visualized using a parasternal short axis view, the orientation of the pulmonary artery is parallel to the Doppler beam, and the pulmonary valve is seen, allowing to place the Doppler cursor reproducibly at the same location in all mice (tip of the leaflets). Similarly, the M Mode and parasternal long axis views of the LV may be easily obtained in all mice.
Cardiac output was measured using a flow probe positioned around the ascending aorta. This method has been used in mice by our team 1
and others 3, 4, 6
. Janssen et al.
found that CO measured using a flow probe in awake mice was of 16 ml/min 3
, a value greater than that found in our study. However, in a subsequent study, the same investigators reported a significant decrease in flow probe-measured CO using anesthesia, with values similar to our results 4
. Likewise, using a conductance catheter, Pacher et al. 7
reported values of CO between 6 and 10ml/min in anesthetized mice.
All ehocardiographic methods overestimated the flow probe-derived CO. Although the flow probe did not take into account the coronary artery flow, the underestimation of the CO linked to coronary flow represents approximately 10% of the CO 20
and cannot be the only explanation to the differences observed in our study.
As reported previously in rats 19
, the greatest overestimation of the flow probe-derived CO was noted with M Mode measurements. Similarly, the highest values of CO in mice before and after endotoxin injection were reported using M Mode. Calculation of LV volume using the M Mode and equating LV volume to the cube of its short axis assumes that the long axis of the LV has twice the length of the anteroposterior diameter. As shown by our results, this ratio appears to be valid in mice in diastole however it is much greater in systole, as the long axis of the LV does not decrease as much as the short axis. The LV end-systolic volume is therefore underestimated using the M Mode calculation and the CO overestimated. Another factor that may play a role in the overestimation observed using the M Mode is the fact that the LV may not be perfectly perpendicular to the ultrasonic beam, leading to an overestimation of both end-systolic and end-diastolic diameters. As the CO calculation is based on the cube of each diameter, the impact of this error will affect the larger volume (end-diastole) to a greater extent than the smaller volume (end-systole), inducing an overestimation of the stroke volume and of the CO.
The overestimation of the CO by the pulmonary flow measurement has also been reported in rats 19
. The major source of error in the measurement of pulmonary flow is the pulmonary artery diameter measurement, due to poor visualization of the walls of the main pulmonary artery 21
. The difficulty in the estimation of the pulmonary artery diameter is underscored in the present study by the greater inter-observer variability found in the calculation of pulmonary flow than in pulmonary VTI. However various loading changes did not significantly impact the measured diameter of the pulmonary artery since the greatest observed standard deviation for a unique observer was 10 μm, far below our spatial resolution. Pulmonary VTI was also able to detect a decrease in the mouse CO after endotoxin injection that was more pronounced and less variable than the other methods of CO measurements.
The degree of overestimation of the CO was less using the 2 dimensional images than that of the M Mode or pulmonary flow. Similarly, in mice after endotoxin injection, CO measurements using 2 dimensional images were lower than measurements using M Mode or pulmonary flow. Investigators had previously noted that algorithms based on 2D images were more accurate than those based on M Mode for measurement of LV mass in mice 22
. Two-dimensional echocardiography still overestimated the flow probe-measured CO, suggesting either an overestimation of the LV end-diastolic volume or an underestimation of LV end- systolic volume. Pacher et al. 7
reported end systolic volumes in mice between 9 and 20 μl using a conductance catheter; using echocardiography our volumes were smaller (from 2 to 11.5 μl). It is conceivable that analysis of the echocardiogram underestimates LV systolic volume as a very small LV cavity may not be totally accurately traced. Furthermore, on the parasternal long axis view of the mouse, it is difficult to avoid and exclude the papillary muscle from the area measured, in particular in systole. Not avoiding the papillary muscle would also underestimate LV end-systolic volume and participate in an overestimation of CO.
Limitations. Due to the presence of the flow probe in the suprasternal area, we were not able to reproducibly obtain aortic flow parallel to the Doppler beam. This limitation precluded us from comparing the methods tested to the measurement of CO using aortic flow 19
. For the 2D LV volume assessment, although the definition of the papillary muscles insertion may be seen better in a short axis plane, we used the parasternal long axis view. The parasternal long axis view allowed us to use area and LV length measured within the same cardiac cycle. Finally, due to the technical complexity of our model, we had to use a well-controlled open-chest model and variability of CO measurement could be expected to be higher in conscious mice.
In conclusion, echocardiography can noninvasively measure CO in the mouse, with close correlations to flow probe measures. Although overestimation of the flow probe-measured CO is noted whatever the echocardiographic method used, the close correlations noted between each echocardiographic method and flow probe-measured CO allow an acceptable estimation of CO and, more importantly, an accurate assessment of its changes within and between mice. Investigators may either choose to report CO calculated from the pulmonary VTI, a method with low variability or CO derived from 2D images, the approach that gives the least overestimation of the flow probe-measured CO.