A New Echo-Doppler Method of Assessing Pulmonary Arterial Wave Re�ection: In Vivo Validation

Background: Pulmonary arterial (PA) wave re�ection provides additional information for assessing right ventricular afterload, but its applications is hampered by the need for invasive pressure and �ow measurements. We tested the hypothesis that PA pressure and �ow waveforms estimated by Doppler echocardiography could be used to quantify PA wave re�ection. Methods: Doppler echocardiographic images of tricuspid regurgitation and right ventricular out�ow tract �ow used to estimate PA pressure and �ow waveforms were acquired simultaneously with direct measurements with a dual sensor-tipped catheter under various hemodynamic conditions in a canine model of pulmonary hypertension (n=8). Wave separation analysis was performed on echo-Doppler derived as well as catheter derived waveforms to separate PA pressure into forward (Pf) and backward (Pb) pressures and derive wave re�ection coe�cient (RC) de�ned as Pb divided by Pf. Results: RC by echo-Doppler agreed well with RC indices by catheter (RC: bias = 0.13, 95% limits of agreement = -0.25 to 0.26). RC correlated negatively with pulmonary arterial compliance and right ventricular systolic function. Conclusions: This echo-Doppler method yields accurate measurement of re�ected wave in the pulmonary circulation, paving the way to a more integrative assessment of pulmonary hemodynamics in the clinical setting.


Introduction
Pulmonary hypertension (PH) is a serious disease characterized by increased pulmonary artery pressure (PAP) associated with pulmonary vascular remodeling.It causes right-sided heart failure due to increased afterload on the right ventricle and has been shown to carry poor prognosis 1 .Mean PAP and pulmonary vascular resistance (PVR) are the most common hemodynamic measures used to diagnose PH and monitor treatment effects.However, mean PAP used in isolation cannot characterize the severity of disease or de ne the pathological process, since it changes depending on various hemodynamic factors such as cardiac output 2 , and as the disease progresses, an increase in PAP usually lag behind pathological changes in the pulmonary arterial tree, leading to a diagnostic delay 3 .Mean PAP and PVR have been shown to correlate, but not closely, with the degree of right ventricular dysfunction and adverse clinical outcomes 4,5 .These inherent limitations may be because they do not represent all components of load faced by the right ventricle.
Pulmonary blood ows are pulsatile in nature.A complete description of right ventricular afterload, therefore, should also include the load to pulsatile component.In the pulmonary circulation, it accounts for approximately 25% of the total workload of right ventricle 6 ; the proportion is much higher than that of the systemic circulation, because of lower vascular resistance in the pulmonary circulation.Once pathological changes develop in the pulmonary vasculature, they cause premature wave re ection from distal sites of pulmonary arterial trees, leading to an additional increase in pulsatile load 7 .Wave re ection has been studied in the systemic circulation, where altered wave re ection has been shown to be associated with the development of left ventricular hypertrophy and adverse clinical outcomes in leftsided heart failure 7,8 .Analysis of pulmonary arterial wave re ection could also provide additional information above and beyond that obtained from mean PAP and PVR, thereby helping better identify or treat patients with PH.
One promising method of estimating the magnitude of wave re ection is to separate pressure into its forward and backward components by wave intensity analysis 9 .This analysis requires measurements of both pressure (P) and ow velocity (U) waveforms.Previous animal and clinical studies measured these two waveforms directly with catheters 10,11 .In clinical practice, pulmonary hemodynamics is assessed using Doppler echocardiography, for example, by estimating PAP from tricuspid regurgitation (TR) velocity and evaluate the pattern of ow pro le at right ventricular out ow tract (RVOT) 12 .Accordingly, we tested the hypothesis that pulmonary arterial wave re ection can be assessed noninvasively using Doppler echocardiography.

Pulmonary arterial hemodynamics and wave re ection
The changes in pulmonary hemodynamics and right ventricular function induced by pulmonary arterial microembolization and hemodynamic manipulations are summarized in Table 1.The injection of dextran microsphere signi cantly increased mean PAP from 14 ± 3 to 34 ± 7 mmHg (p = 0.002) and PVR from 2.9 ± 1.0 to 13.7 ± 4.9 Woods unit (p < 0.001).Cardiac output decreased (p = 0.006).Right ventricular systolic function was also reduced, as demonstrated by a signi cant decrease in TAPSE (p = 0.01).There were alterations in RVOT ow pro le such as relatively shortened acceleration time (p = 0.12) and the presence of mid-systolic notching (p = 0.04); TR ow pro le exhibited high and late peaking pattern as shown by higher TR velocity (p < 0.001) and longer time to peak velocity (p = 0.001).
Fluid loading did not change PVR (at baseline, p = 0.75; at PH state, p = 0.53); it increased cardiac output only marginally at baseline (p = 0.048) or not at all at PH state (p = 0.26).It also did not affect right ventricular systolic function (at baseline, p = 0.19; at PH state, p = 0.42).Inotropic stimulation did not change PVR (at baseline, p = 0.39; at PH state, p = 0.76), although it signi cantly increased cardiac output (at baseline, p < 0.001; at PH state, p = 0.03).It enhanced right ventricular systolic function as demonstrated by a signi cant increase in TAPSE at baseline (p = 0.01), but not at PH state (p = 0.26).

Correlations of wave re ection indices by Doppler with those by catheter
Figure 1 compares sample separated waveforms obtained from direct P and U measurements and those obtained from echo-Doppler derived P and U measurements.At baseline, no wave re ection was observed.After the induction of PH, pulse pressure by Doppler dramatically increased from 7.7 to 29.2 mmHg with late-peaking pattern; Pb by Doppler started to rise 52 msec later than the onset of ejection and peaked at 12.1 mmHg.This comparison demonstrated that the new echo-Doppler method produced comparable Pb and Pf waveforms with those derived from direct measurements.
When the data for all load-state conditions were pooled, Pb, Pf and RC by Doppler correlated linearly with the corresponding indices by catheter with acceptable variabilities (Figure 2).WS by Doppler also signi cantly correlated with that by catheter; the regression line in the scatterplot was deviated slightly upward from the identical line, suggesting that the echo-Doppler method overestimated WS.When looking at individual change in wave re ection indices for each dog, the induction of PH increased the indices by Doppler similarly as did those by catheter (Figure 3).This indicated that the echo-Doppler method was able to track the alterations in pulmonary arterial wave re ection associated with the development of PH, with reasonable accuracy.

Wave re ection indices and effects of hemodynamic alterations
Overall, Pb by Doppler was minimal (1.4 ± 1.3 mmHg) at baseline and increased 7.5 times, up to 10.4 ± 3.9 mmHg, associated with the development of PH, even though mean PAP increased only 2.4 times, up to 34 ± 7 mmHg; its increase was substantially larger, compared to 1.9 times increase in Pf by Doppler (from 8.6 ± 2.5 to 16.1 ± 5.1 mmHg), resulting in a signi cant rise in RC by Doppler (from 0.16 ± 0.10 to 0.65 ± 0.13).WS by Doppler also signi cantly increased from 1.2 ± 0.3 to 3.3 ± 1.0 m/s.Table 2 summarizes the changes in wave re ection indices by Doppler induced by preload loading and inotropic stimulation.An increase in preload by uid loading, which caused a signi cant, but small increase in cardiac output (Table 1), did not alter the wave re ection indices (Pb, p = 0.30; Pf, p = 0.15; RC, p = 0.86; WS, p = 0.06).Inotropic stimulation produced a larger increase in cardiac output than uid loading (Table 1); it increased Pb (p = 0.01) as well as Pf (p = 0.04), but did not alter RC (p = 0.65), suggesting that both Pb and Pf were ow-dependent and the effect of ow status on Pb can be cancelled out by taking a ratio of it to Pf. Inotropic stimulation also did not alter WS.These trends were also con rmed for catheter-derived wave re ection indices (Supplemental table 1); uid loading did not change the indices; inotropic stimulation increased Pb (p = 0.01) and Pf (p = 0.03), but did not change RC (p = 0.64).

Hemodynamic determinants of wave re ection indices
The results of linear regression analyses identifying the determinants of echo-Doppler derived wave re ection indices are shown in Table 3. RC was correlated strongly with PAC and systolic PAP, moderately with PVR and mean PAP, and weakly with cardiac output.WS was also correlated strongly with PAC and PVR, moderately with cardiac output, systolic and mean PAP.The magnitude of the associations with both RC and WS was the highest for PAC (RC by Doppler, r = -0.85;WS by Doppler, r = -0.71).PAC was also identi ed as the strongest hemodynamic determinant for catheter-derived RC and WS (RC, r = -0.70;WS, r = -0.79,Supplemental table 2).
Right ventricular -pulmonary arterial coupling by wave re ection indices Figure 4 relates right ventricular systolic function measured by TAPSE with echo-Doppler derived wave re ection indices.Overall, TAPSE was negatively correlated with both RC (r = -0.40,p = 0.005) and WS (r = -0.49,p = 0.001).When analyzed separately for loading conditions, the relationships of TAPSE with RC and WS did not change by uid loading (RC, p = 0.48; WS, p = 0.28).However, the relationships were shifted upward by inotropic stimulation (RC, p = 0.06; WS, p = 0.008).When catheter-derived RC and WS were used instead for the above analysis, similar upward shifts by inotropic stimulation were observed (Supplemental gure 1).

Discussion
The present study demonstrated that pulmonary arterial wave re ection can be assessed noninvasively using Doppler echocardiography.This was feasible by performing wave intensity analysis to separate pulmonary arterial pressure waveform estimated from TR velocity pro le into its forward and backward components.The present study validated this method in vivo against invasive wave separation analysis, and showed that re ection coe cient and wave speed stayed unchanged by alterations in cardiac output and correlated with PAC and right ventricular systolic function, suggesting that the both wave re ection indices can serve as a ow-independent measure of pulsatile load on right ventricle.

Noninvasive assessment of pulmonary arterial wave re ection
To infer a diagnosis of PH, the effects of PH on the right heart are imaged with echocardiography in clinical practice.Some of such echocardiographic signs are related to the presence of pulmonary arterial wave re ection.For example, a late-peaking pattern on TR velocity pro le is caused by PAP augmentation by early arrival of re ected wave 13 .A mid-systolic notching on RVOT ow pro le represents an abrupt reduction in right ventricular ejection ow secondary to wave re ection 14 .Although these signs are reasonably speci c, the diagnostic sensitivity is generally low and these signs are absent in some patients 15 .The signs are also not sensitive enough to be used for monitoring the treatment course of patients.
To provide a more accurate assessment of wave re ection, wave separation analysis is performed on pulmonary arterial pressure and ow waveforms into their forward and backward components.This analysis is classically undertaken in the frequency domain, but it is fairly complicated and needs sophisticated software 16 .An alternative method, called wave intensity analysis, has been particularly attracting attention, because it is a time-domain based approach and therefore can be more easily applied to study the timing and magnitude of wave re ection. 10The results are easier to interpret in physiological terms.
Most previous studies of pulmonary arterial wave intensity analysis measured pressure and ow waveforms directly with catheters, which hinders its use in the clinical setting 9 .A recent study has attempted to obtain these waveforms noninvasively, using magnetic resonance imaging, where It measured the temporal changes in cross-sectional area of pulmonary artery for a surrogate for PAP 17 .The noninvasive method we introduce herein uses Doppler echocardiography to obtain pulmonary arterial pressure and ow waveforms from TR and RVOT ow pro les.These measurements are routinely performed for diagnosing PH or monitoring the treatment course, so this echo-Doppler method can be easily applied in the clinical setting.This echo-Doppler method involves a couple of assumptions.Firstly, right atrial pressure is assumed to be constant during systole.This method therefore may not be applicable to patients with severe TR which causes a signi cant rise in right atrial pressure in late systole.Secondly, RR interval on electrocardiogram is assumed to be constant throughout image acquisitions.In this method, pressure and ow waveforms are obtained from echo-Doppler images acquired at different cardiac cycle and the both waveforms are aligned in time with reference to the R wave on electrocardiogram.Therefore, this method cannot be applied to patients with large heart rate variability such as atrial brillation.Despite the above assumptions, the present study demonstrated that the echo-Doppler method can quantify pulmonary arterial wave re ection with reasonable accuracy and it is sensitive for detecting the changes in pulmonary arterial wave re ection associated with the development of PH.

Potential for clinical applications
Pulmonary arterial hypertension remains a fatal disease with a 1-year mortality of approximately 20%, despite advances in disease-speci c therapies 1 .This may be because the disease is often far established at diagnosis as demonstrated by contemporary disease registries reporting that mean PVR at diagnosis ranged between 8 and 10 Wood units 18 .Early initiation of the therapies may be effective, as suggested by a previous randomized control study including patients with mildly symptomatic pulmonary arterial hypertension 19 .At the early stage of disease, PAC falls considerably while PVR increases only slightly.Thus, the assessment of pulsatile load may allow an early diagnosis even when PVR stays within normal limits.In the present study, wave re ection, an important component of the pulsatile load, rose substantially despite a slight increase in mean PAP induced by pulmonary arterial microembolization.This nding is consistent with another study of wave intensity analysis, evidencing that a signi cant wave re ection was present even in PH patients with mildly elevated PAP 11 .Therefore, this echo-Doppler assessment of wave re ection may be able to offer an early diagnosis of PH.PH-speci c treatments do not always produce desirable clinical outcomes.For example, some patients with chronic thromboembolic pulmonary hypertension exhibit exercise intolerance even after pulmonary endarterectomy, often despite normalization of pulmonary arterial hemodynamics.This may partly be attributable to persistent pulmonary arterial wave re ection after the surgery.A previous study demonstrated using wave intensity analysis that there remained a large re ected pressure wave 3 months after the surgery, even in patients without residual PH 20 .Pulmonary arterial wave re ection could be an attractive target for the treatment of PH.However, it remained to be tested whether a reduction in pulmonary arterial wave re ection will lead to a signi cant improvement in clinical outcomes.

Limitations
To validate the echo-Doppler method, the present study created an animal model of PH which mimicked pulmonary arterial hypertension by embolizing peripheral pulmonary arterial with microsphere.The magnitude and timing of wave re ection vary depending on the site of narrowing in pulmonary vasculature; this was evidenced by previous studies of wave intensity analysis observing a larger re ected wave that arrived earlier during systole in chronic thromboembolic pulmonary hypertension than that in pulmonary arterial hypertension 21 .It needs to be investigated whether or not the echo-Doppler method can detect the alterations in the magnitude and timing of pulmonary arterial wave re ection by changes in the site of narrowing.Some studies questioned the accuracy of echocardiographic estimates of pulmonary arterial systolic pressure in the clinical setting, because TR velocity is sometimes di cult to measure or to obtain clear spectral Doppler envelopes 22 .Therefore, this echo-Doppler method should also be tested for feasibility and accuracy in patients before clinical application.

Conclusions
The new echo-Doppler method yields an accurate measurement of pulmonary arterial wave re ection and can sensitively detect the alterations associated with the development of PH.The results of the present study provide the basis for integrating this assessment of pulmonary arterial wave re ection for more detailed description of right ventricular afterload, thereby, diagnosis of PH and treatment monitoring in the clinical setting.

Methods
Principles of echo-Doppler method of assessing pulmonary arterial wave re ection Pulmonary arterial wave re ection can be assessed based on the concept of wave intensity analysis.This analysis determines the origin, type and timing of traveling waves in a circulation from combined P and U measurements and allows wave separation into forward-traveling and backward-traveling components.
Theoretical background and practical applications have been described elsewhere 9 .Brie y, wave speed (WS), which represents the elastic properties of the local artery, can be determined by P-U loop method 23 ; it takes advantage of the water hammer equation relating P and U on the condition that there is no wave re ection.c = (dP / dU) / ρ , where dP and dU are the changes in P and U, ρ is the density of blood (1050 kg/m 3 ) and c is WS.Pulse pressure can be separated into those attributed to forward-traveling (Pf) and backward-traveling (Pb) waves using equations 2 and 3.
dog was then placed in right lateral recumbency.Heparin sodium (100 IU/kg) was administered for preventing thrombosis.
A 4-Fr catheter (Atom nutrition catheter, Atom Medical, Tokyo, Japan) was inserted into the right femoral artery to monitor systemic arterial pressure.A 4.2-Fr multipurpose angiographic catheter (Goodtec angiographic catheter, GOODMAN, Aichi, Japan) was advanced retrograde into the left atrium through the left carotid artery to monitor left atrial pressure.Another 4.2-Fr multipurpose angiographic catheter was placed in the right atrium through the left jugular vein to monitor right atrial pressure.These uid-lled catheters were connected to pressure transducers (Life kit DX-360, Nihon Kohden, Tokyo, Japan) and pressure waveforms were displayed using a multi-channel monitor (Life Scope BSM-5192; Nihon Kohden, Tokyo, Japan).These two catheters were also used to sample blood from the left and right atriums to calculate cardiac output.
To obtain P and U waveforms as a reference for validating the new echo-Doppler method, a dual sensortipped pressure and ow wire (Combowire, Royal Philips, Amsterdam, Netherlands) was advanced to approximately 1 cm beyond the pulmonary valve through another 4.

Figures Figure 1
Figures

Figure 3 The
Figure 3

Figure 4 Relationship
Figure 4 2-Fr multipurpose angiographic catheter inserted from the left jugular vein.Careful catheter and wire manipulation ensured that signals were steadily obtained.SAP, systemic arterial pressure; LAP, left atrial pressure; PAP, pulmonary arterial pressure; RAP, right atrial pressure; HR, heart rate; CO, cardiac output; SV, stroke volume; PVR, pulmonary vascular resistance; PAC, pulmonary arterial compliance; LVEF, left ventricular ejection fraction; TAPSE, tricuspid annular plane systolic excursion; RVOT, right ventricular out ow tract; AcT, acceleration time; TR, tricuspid regurgitation; * denotes p value < 0.05 vs before challenge and † denotes p value < 0.05 vs baseline.Abbreviations: Pb, backward pressure; Pf, forward pressure; RC, re ection coe cient; WS, wave speed; * denotes p value < 0.05 vs before challenge Table 3. determinants of wave re ection indices