We have demonstrated that pulmonary artery flow is readily detectable using echocardiography and can be used as a screening tool for the evaluation of PAH in a rat model. Furthermore, we have shown that parameters such as echo-TPVR, PAAT, and PAD are associated with the severity of PAH. Since rat models are increasingly used in studies evaluating various therapeutic interventions for PAH, these non-invasive parameters allow identification and quantification of disease progression at multiple time-points, replacing or at least complementing invasive data. Furthermore, we have developed algorithms for the evaluation of PAH that may also have value in the evaluation of PAH in patients.
Invasive PAPs in our study were similar to the values reported in earlier studies of rats with MCT-induced PAH [20
]. Distinction to the high yield of non-invasive echocardiography methods, we measured invasive PAP successfully in only 34 out of 47 cases (72%) with advanced PAH. This experience is in-line with the success rates reported by other investigators: 80% at day 21 and 50% at day 35 (personal communication, Dr Hirofumi Sawada, Stanford University, CA). The difficulty of performing invasive measurements in animals with advanced PAH is an important limitation of all invasive parameters and underscores the importance of innovative non-invasive measurements.
To our knowledge, TPVR has not been studied in rodents using echocardiography. In our study, echo-TPVR values predicted invasive TPVR values but were limited to animals with more advanced PAH with TR jet. Echo-TPVRs were studied only to check whether their use would offer relevant and easily detectable information in this animal model. Both Echo-TPVR formulas 1 and 4, which are simpler measurements, were found to be useful when compared to the diagnostic yield of echo-TPVR formula-2, which is a more complex calculation.
We found tissue Doppler parameter difficult to measure. This failure may be explained by the fact that rats have a much thinner right ventricular wall and a higher heart rate than humans. Additionally, we noticed that Doppler gain settings have a significant influence on IVRT’ values. Only 2–4 ms difference in IVRT’ corresponds to 10–20% change in this parameter. IVRT’ values corrected to RR-interval were similar to the levels observed by Boissiere et al. [26
] (data not shown). Recently, it has been reported that IVRT’ <40 ms has 100% negative predictive value for PAH in humans [14
]. Furthermore, IVRT’ is generally a measure of right ventricular diastolic function describing ventricular burden of PAH. Therefore, it has substantial limitations for use in the measurement of PAH. Other ultrasound systems with different filtering algorithms for tissue Doppler signals may have led to better diagnostic yield of tissue Doppler technique but this remains to be studied.
Pulmonary artery acceleration time values in our study were similar to the values observed in earlier studies of rats with MCT-induced PAH [30
]. Jones et al. [30
] reported the correlation of 0.84 between PAAT and invasive PAP only in animals with PAAT between 20 and 32 ms. In our study a comparable correlation was found even without excluding values outside this range. Short PAAT values are likely due to reduced capacitance and the increased impedance of the pulmonary vascular bed causing the deceleration to begin at early systole [32
]. Furthermore, increased stiffness of pulmonary artery may cause short PAAT and rapid PAD by increasing the pulse wave velocity and therefore causing earlier reflection from the periphery resulting in early deceleration [33
Previously, Jones et al. [30
] showed that quantification of TR velocity is usually unsuccessful in rats with systolic PAP below 65 mmHg. Our data supports this observation, as we were able to quantify TR velocity from only one rat with invasive systolic PAP below 65 mmHg. Jones et al. [30
] also demonstrated a good correlation (r
= 0.92) between rat PAP using this method compared to invasive measurement, but the technique succeeded only in six of 61 animals. In our study, the lack of significant correlation between TR velocity derived PAP compared to invasive PAP may be explained by the relatively small range of successful PAP measurements by this particular echo technique. No other studies have evaluated intra- or interobserver variabilities of the invasive PAP or TPVR measurement in the rat model of PAH. Jones et al. [30
] reported the intraobserver and interobserver variability between 3.0 and 3.5% for PAAT by a non-standard method of using twice SD. In our report, all the echocardiographic parameters were found to have low intraobserver and interobserver variability. The exception was IVRT’, which had a high interobserver variability and also the quite poor correlation with invasive PAP.
The algorithm of PAAT <28 ms and PAD >11.8 m/s2 has the best specificity of 97.6% and the algorithm of PAAT <28 ms or PAD >11.8 m/s2 has the best sensitivity of 82.6% of the studied parameters to detect significant PAH (of note, PAAT alone also has the same sensitivity). Parameter with a high sensitivity is advantageous for detecting established disease before treatment and parameter with a high specificity for excluding animals before further experiments due to failure in the induction of the disease. This is a critical issue in translational studies, as they are usually performed using relatively small treatment groups and are therefore, especially vulnerable for misleading results. The presented algorithms can be employed to ensure disease induction and improve the verity of the data collected.
There are a number of limitations to our study. First, echo-TPVR-formula-2 and invasive-TPVR were both calculated by cardiac outputs derived from non-invasive method. In this regard, it should be highlighted that the purpose of this study was to evaluate whether simple echocardiographic measurements such as those used for the calculation of echo-TPVR-1, 3 and 4 would have value when evaluating rat PAH. Based on our previous published work [25
] non-invasive cardiac output was comparable to the invasive measurement. Additionally, it is well recognized that the presence of moderate to severe TR, as observed in advanced PAH, may lead to inaccurate invasive thermodilution derived cardiac outputs whereas TR has no effect on the echocardiographically derived measurements and this may be preferable as an easier, more reproducible and non-invasive measure of cardiac output. Bland–Altman plots from two recent studies indicate that there may be more differences between thermodilution and Fick methods derived cardiac outputs compared to echocardiography and Fick [34
]. Despite this, we do recognize this point as a limitation of our manuscript but do not believe that the invasive measurements would have altered the results of our findings and in fact, since it is very challenging to make these measurements in rats with significant PAH, we propose that future studies use the non-invasive method proposed here to avoid high animal mortality and allow completion of the research studies in a timely and cost-effective manner. Second, agitated saline or contrast agent was not used to enhance the Doppler signal determining TR velocity, which might have improved yield of successful measurements. However, contrast agents in rats require tail vein cannulation and a separate infusion pump with agitation capability, and importantly it would have potentially influenced the non-invasive nature of our measurements. Third, a correction for heart rate in PAAT measurement was not used. Finally, echocardiographically determined TPVR (formula 1) was not adjusted for right ventricular outflow tract diameter. However, in another study, this correction did not lead to a significant change in the correlation between the methods (r
= 0.73 vs. r
= 0.71) [13
In addition, it should be noted that the Bland–Altman technique is considered ideal when evaluating agreement between two methods especially when one is comparing measurements, which are expected to give the same values in the ideal situation when the methods do not differ. In our study, only two variables were suitable for assessment with the Bland–Altman method, and this has been reported.
Finally, it should be emphasized that observed values of echocardiographic parameters in this study can be assumed valid only in rats. It has been shown that PAAT values are much higher in humans compared to rats due to lower heart rate [36
]. PAP was measured only in animals with chronic pulmonary hypertension, and our results may not apply to acute PAH.