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Are Changes in Myocardial Integrated Backscatter Restricted to the Ischemic Zone in Acute Induced Ischemia? An In Vivo Animal Study
Are Changes in Myocardial Integrated Backscatter Restricted to the Ischemic Zone in Acute Induced Ischemia? An In Vivo Animal Study Bart Bijnens, PhD, Jan D’hooge, PhD, Maarten Schrooten, Sorin Pislaru, PhD, Cristina Pislaru, MD, Bruno De Man, MSc, Johan Nuyts, PhD, Paul Suetens, PhD, Frans Van de Werf, MD, PhD, George R. Sutherland, MD, PhD, and Marie-Christine Herregods, MD, PhD, Leuven, Belgium
Integrated backscatter (IB) from a myocardial region, calculated from radiofrequency echocardiographic data, has been proposed as a useful parameter for investigating changes in myocardial tissue induced by ischemia. In 10 closed-chest dogs, 5 minutes of myocardial ischemia was induced by either a proximal occlusion of the circumflex coronary artery (CX) (5 dogs), resulting in extensive ischemia in the posterior wall, or by occluding the distal CX vessel (5 dogs) to produce a small localized ischemic zone in the posterior wall. High-resolution digital radiofrequency data from the whole left ventricular myocardium, in the imaging plane during one complete heart cycle, were acquired with a whole-image realtime acquisition approach. Regions in the septum
and posterior wall (both ischemic tissue and, in the case of distal occlusions, tissue surrounding the ischemic zone) were chosen for analysis, and IB and cyclic variation (CV) of IB were calculated. Post occlusion, an increase in mean IB values was found in the ischemic segment. However, an increase in CV was also observed in the peri-ischemic zone for the distal CX occlusion and in the septum after proximal CX occlusion. These findings show that changes in CV are not restricted to the ischemic zone but may also occur in distal myocardium. This may be explained by changes in the regional contractile state and loading conditions of the “normal” myocardium, which are altered in response to the distal ischemia. (J Am Soc Echocardiogr 2000;13:306-15.)
INTRODUCTION
ultrasonic tissue characterization. Investigators have increasingly turned their attention to the investigation of the unprocessed RF data set to determine whether there is clinically relevant information in this potentially higher resolution signal.4 The parameter extracted from the RF data set that has been most studied is integrated backscatter (IB).1 Integrated backscatter is calculated by integrating the power spectrum of the received signal (after compensation for the spectrum of the transmitted pulse) over the meaningful bandwidth of the transducer. This implies that IB is a measure of the mean reflected ultrasonic energy from a particular region of tissue.4 Prior extensive clinical evaluation of the properties of IB has demonstrated that changes in its magnitude, its cycle-dependent variation, and the timing of peak and trough levels can all be influenced by a variety of disease processes.5-15 In these studies, IB values were measured either from off-line analysis of acquired RF signals or by using dedicated hardware incorporated in a standard clinical ultrasound
The characterization of the functional state and structural composition of the myocardium remains an important goal in clinical cardiology. In this respect, there is increasing interest in the use of parameters extracted from raw, unprocessed radiofrequency (RF) data from echocardiographic equipment to characterize changes in myocardial reflectivity induced by a variety of pathologic processes.1-3 A general consensus now exists that postprocessing analysis of video data acquired during cardiac ultrasonographic (ultrasound) studies is not ideal for From the Department of Cardiology and the Department of Nuclear Medicine, Medical Image Computing, Gasthuisberg University Hospital, Herestraat 49, B-3000 Leuven, Belgium. Reprint requests: Bart Bijnens, UZ Gasthuisberg, Department of Cardiology, Herestraat 49, B-3000 Leuven, Belgium (E-mail: [email protected]). Copyright © 2000 by the American Society of Echocardiography. 0894-7317/2000 $12.00 + 0 27/1/103595 doi:10.1067/mje.2000.103595
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machine. However, many of these studies have been limited in their ability to capture real-time data from the whole myocardium in the B-mode imaging plane and thus have been unable to compare changes in IB data for all myocardial segments. To improve on prior studies, we have developed an approach that enables the acquisition of RF data from a complete sector scan during a complete heart cycle.16 As indicated above, this approach differs from that of most prior researchers who either used Mmode data or implemented small time-gates to limit the amount of 2-dimensional (2D) data acquired.6,17-19 Our approach enables the investigation of the signal from the complete 2D image throughout a complete cardiac cycle. Thus it will allow the comparison of reflectivity parameters derived simultaneously from different regions of the myocardium. With this approach we have reinvestigated the value of IB measurements in acute myocardial ischemia induced in a canine model.Transthoracic echocardiographic data were acquired with a standard clinical ultrasound machine. This is in contrast to most work from prior investigators, who used laboratory echographic equipment, such as single-element piezo-electric crystals connected to dedicated pulse-receive electronics, instead of scanners designed for clinical work. To investigate the changes in IB and its cyclic variation (CV), we induced 5 minutes of ischemia in a series of dogs. In the first set of animals, transient localized ischemia was induced in the posterior wall with the use of a balloon occlusion of a distal branch of the circumflex coronary artery (CX). In the second set, severe widespread ischemia was induced by 5 minutes of occlusion of the proximal CX.
MATERIALS AND METHODS Animal Preparation Ten mongrel dogs (17 to 25 kg) were studied. All procedures performed were in accordance with the institutional guidelines set by the University of Leuven. All animals were sedated with xylazine (50 mg intramuscularly). After anesthesia with sodium-pentobarbital (15 mg/kg intravenously for induction and 0.1 mg/kg/min for maintenance), each dog was intubated (cuffed endotracheal tube) and mechanically ventilated with a mixture of oxygen (20%) and air (Bird respirator, Bird Products Corp, Palm Springs, Calif). Both carotid arteries, jugular veins, one femoral artery, and one femoral vein were dissected free and cannulated with arterial sheaths or infusion lines.After completion of the dissection, all dogs received heparin (100 IU/kg bolus intravenously plus continuous infusion of
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Figure 1 A typical coronary angiogram of the circumflex coronary artery is shown with indication of the position of the balloon during occlusion. Arrows indicate position for experiments with proximal occlusions (prox) and distal occlusions (dist).
20 IU/kg/h) and aspirin (5 mg/kg intravenous bolus). Ventilation was adjusted to maintain pH and arterial blood gasses within the physiologic range. Body temperature was maintained with a heating pad. Reperfusion arrhythmias were treated with lidocaine.
Measurements Both at baseline conditions and after 5 minutes of ischemia, a 2D echocardiographic study was performed with a Toshiba 160 (Tokyo, Japan) clinical ultrasound scanner. High-quality left ventricular 2D images were acquired with a transducer frequency of 5 MHz and a frame rate of 30 Hz with use of the standard parasternal left ventricular longaxis view.Video data and RF data were acquired and stored. In all dogs, an area of myocardial ischemia was induced in the posterior myocardial wall. In half (5/10) of the dogs, localized myocardial ischemia was induced by balloon occlusion (percutaneous transluminal coronary angioplasty balloon, Quick 2.5, Baxter, Volen, The Netherlands) of a marginal branch of the CX. In the other half, the perfusion of the complete posterior wall was interrupted by a balloon occlusion of the proximal CX. Coronary angiography was performed twice during each animal study. The first study was to assess coronary anatomy before balloon inflation. The second was to check the efficacy of the occlusion during the inflation. In Figure 1 a typical coronary angiogram of the CX is shown. The positions used for the proximal and distal occlusions are indicated. At the end of the experiments, the heart was arrested with saturated potassium chloride intravenously. For the 5 dogs with moderate ischemia, the vessel was occluded for a further 60 minutes after the RF data acquisition to stain
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Figure 2 The setup used to digitize the radiofrequency (RF) data from the echocardiographic examination. 3D, Three-dimensional.
the excised hearts with triphenyltetrazolium chloride (TTC) to achieve a delineation of the area that was ischemic at the time of data acquisition. In all 10 dogs, the left ventricle was subsequently sectioned parallel to its long axis in such a way that this section corresponded as much as possible to the echocardiographic parasternal long-axis plane. Enlarged pictures of the slices after TTC staining were obtained and visually assessed for the site, extent, and severity of infarction.
RF Data Acquisition The RF signal was digitized using the acquisition setup described by Bijnens et al.16 Figure 2 demonstrates the equipment used. Data acquisition was triggered using the R peak of the electrocardiogram (ECG).This resulted in 50 RF images at a frame rate of 30 images/s and a sample frequency of 40 MHz (corresponding to approximately 60 Mb of data for each heart cycle).
RF Processing To process the RF data, we used a software package (“Speqle”) developed in-house and previously described in our work.20 After reconstruction of the sector scans of the relevant part of the RF data, both endocardium and epicardium contours were manually drawn to allow segmentation of the complete left ventricular wall. Next, both contours (endocardium and epicardium) were “shrunk.” Their centerline was determined and the contours were moved 10% toward each other and perpendicular to the centerline. This was performed to avoid inclusion of the specular reflections from the tissue-blood border and pericardium during subsequent analysis.
For each 2D image sequence analysis, the position of the apex was indicated. The resulting delineation of the wall was divided into 24 segments: 12 “septal” from the apex and 12 “posterior.” Each of the segments was divided into 4 parts from endocardium to epicardium for future study of transmural behavior. Figure 3 shows how this process was effected.The ECG and the phonocardiogram were displayed in real time together with the images. End diastole and end systole were indicated on the data sets by the investigator. These were determined from the timing of events derived both from the synchronously sampled ECG and phonocardiogram and from the 2D images that used the timing of opening and closure of the mitral valve. From each defined region of interest, IB was calculated for all 50 consecutive images. Integrated backscatter was calculated in the time domain as the sum of the squares of all data points in the region of interest, normalized by the number of points.21,22 On the basis of end-diastolic and end-systolic frames, IB traces were resampled to get the same number of data points for each heart cycle (10 for diastolic phase and 10 for systolic phase) for each acquisition. To use as many RF data points as possible to reduce the effect of noise, comparable regions were merged for the IB analysis. In this work, the transmural IB values from the total septal wall were compared with the posterior wall (transmural). In the case of proximal occlusions, all regions from the posterior wall were merged, whereas in the case of occlusion of the distal CX, infarcted areas (as defined by data from the TTC–stained excised heart) were separated from normal regions and IB values were calculated for both infarcted and noninfarcted myocardium.
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Figure 3 A left ventricular parasternal long-axis view showing the segmentation into regions used to analyze the radiofrequency data. ECG, Electrocardiogram; Phono, phonocardiogram.
Table 1 Summary of the IB and CV measurements Distal occlusion Baseline
Mean IB septum (dB) Mean IB posterior (dB) CV septum (dB) CV posterior (dB)
–0.2 –0.1 5.2 3.7
± ± ± ±
1.4 0.1 2.0 0.9
Proximal occlusion
0.4 1.5 6.5 3.8
± ± ± ±
2.2 2.9* 2.8 0.9
Ischemic region
Peripheral region
–0.6 ± 2.8 3.9 ± 2.0*
1.0 ± 2.0 5.5 ± 0.6
4.4 ± 1.1
6.7 ± 2.7*
IB, Integrated backscatter; CV, cyclic variation. *Significantly different from baseline: P < .01.
All IB values were normalized to the mean value of the basal measurement (separately for septum and posterior wall).This provided an implicit attenuation correction, setting the basal mean values of the posterior wall measurements and the septum to unity.This implies that the only difference between posterior and septal IB values in basal conditions is caused by attenuation. To make our results comparable to those obtained by others in the literature, derived IB values were expressed in decibels. The CV of the IB was calculated as the difference between the maximum and the minimum value of the IB.
Statistical Analysis Integrated backscatter and CV values for the different groups and regions were compared and statistically ana-
lyzed with a combination of an analysis-of-variance approach with a Tukey “honestly significant difference”test (with the standardized range statistic). Differences with a statistical probability <.05 were considered significant.
RESULTS During basal conditions, IB values for each region of interest were normalized.The values obtained for all myocardial segments interrogated were within the range of normal values previously reported (IB septum: –0.2 ± 1.4 dB; IB posterior wall: –0.1 ± 0.1 dB). Cyclic variation values were also normal (CV septum: 5.2 ± 2.0 dB; CV posterior wall: 3.7 ± 0.9 dB)
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Figure 4 An overview of the mean integrated backscatter (IB) values and the cyclic variation (CV) (with their SDs) for these experiments. Left, Septum; right, posterior wall. Shown are the values at baseline (bas), after occlusion of the proximal (prox) circumflex coronary artery (CX), and after occlusion of the distal CX (dist). For the distal occlusions (after 60-min occlusion), the values for the ischemic area (isch) and the peri-ischemic area (peri) are separated.
(Figure 4 and Table 1).These values are similar to the normal CV values reported by other authors.1-3 For the severe generalized ischemic zone (proximal CX occlusions), IB values were significantly (P < .01) increased (1.5 ± 2.9 dB), but no significant blunting of CV occurred (3.8 ± 0.9 dB). In the peri-ischemic zone of the localized ischemia (distal CX occlusion), IB levels were only slightly increased (1.0 ± 2.0 dB). However, CV increased significantly (6.7 ± 2.7 dB) in this segment, which was hyperkinetic, whereas in the localized ischemic zone itself, IB levels increased much more (3.9 ± 2.0 dB), but CV remained comparable to baseline levels (4.4 ± 1.1 dB). The most interesting findings in this study were the changes recorded in the septum in animals with severe generalized ischemia of the posterior wall. In these animals, the septum was hyperkinetic on the 2D image, presumably in response to the hypokinesia or akinesia in the posterior wall. Although mean septal IB levels did not change at all compared with baseline, a clear increase was seen in CV (6.5 ± 2.8 dB) (Figure 4). In Figure 5, these findings are illustrated by the curves obtained from 2 studies. The normalized heart cycles, averaged over all experiments (10 basal, 5 proximal, and 5 distal occlu-
sion) are displayed in Figure 6. A total overview of the calculated mean IB values and CV is displayed in Figure 4.In the case of the occlusions of the distal CX,the posterior wall could be divided into an area at risk and a “peripheral”area, on the basis of the results of the TTC staining after 60 minutes of occlusion.The results for these two different “tissue-types” are separated and illustrated in the figure. Table 1 lists the measurements. The mean IB values for the septum (Figure 4, top left) were not statistically different. Analysis of the mean IB values from the posterior wall (Figure 4, top right) showed a significant (P < .01) difference between baseline values and all others and, for the distal occlusions, between the ischemic area and the peripheral area as well as between the ischemic area for the distal occlusion and the ischemic area for the proximal occlusions. For the CV values from the septum (Figure 4, bottom left) no statistically significant difference could be found, whereas for the CV values from the posterior wall, a significant difference was observed between the basal values and the peripheral area during distal occlusion as well as between the ischemic area for the proximal occlusions and these peripheral regions for the distal occlusions.
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Figure 5 Examples of integrated backscatter (IB) traces obtained from the experiments (dashed lines, basal; solid lines, after 5 minutes of occlusion). Top, Plots correspond to values from the septum; bottom, values from the posterior wall. Left, Plots from experiments that used an occlusion of the proximal circumflex coronary artery; right, plots that used distal occlusions. For each, the IB values for 50 consecutive frames are plotted. For each trace, this corresponds to 2 or more heart cycles. The IB values are expressed in dB.
DISCUSSION One of the major problems in the use of IB and its CV for tissue characterization is the lack of uniformity and standardization of data acquisition and processing. Important problems include the large amount of data to be acquired and the accuracy of data acquisition. In our approach, we have chosen to sample the complete ultrasound scan at a high sample frequency so that no data were lost.The only possible limitation is related to the dynamic range of the analog-to-digital converter (8 bit), but this was, in theory, of sufficient resolution to interrogate the myocardium. A better solution for any potential problems posed by this limited dynamic range will be offered by the new generation of fully digital echocardiographic equipment, which should enable the acquisition of digital RF data with a dynamic range up to 20 bit. Recent findings suggest that one of the major problems in investigating myocardial function is the limited frame rate of the previous generation of echocardiographic equipment. It has been shown
that a frame rate of at least 80 Hz should be used to resolve the fastest functional changes from the myocardium.23-25 Because the study described in this paper was performed with the use of a frame rate of 30 Hz, and if IB is related to functional changes (as suggested in this work), it should be noted that some of the data could have been undersampled. A further problem is the precise definition and methods used to calculate CV.26 We used the maximal excursion of the IB. In baseline conditions, this corresponds best to the difference between the enddiastolic and the end-systolic values. However, during ischemia, a phase shift of the IB can occur (Figure 6). In theory, this could be detected by fitting an appropriate function to the measured IB data.27 However, a question still remains regarding the type of function that should be fitted because there are difficulties in defining the origin of the CV and thus choosing a well-founded physical model. Another important factor to take into account is the problem of the inherent potential variability in measuring IB levels as demonstrated and discussed in Bijnens et al.28 In the worst case, it would be
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Figure 6 An overview of the average normalized heart cycles (from end diastole [ED] over end systole [ES] up to the next ED) for all cases. Left, Septal values; right, posterior wall values. Top, Baseline integrated backscatter (IB); middle, values for 5-min proximal (prox) occlusion (occl) of circumflex coronary artery (CX); bottom, values after 5 minutes of distal (dist) CX occlusion.
expected that variations of up to 0.9 dB from the mean (or 1.8 dB CV) could be observed, which were not related to the state of the tissue. This clearly explains the large standard deviations within our measurements. Because the inherent variations on IB are statistical deviations from the expected mean value caused by randomness in scatterer distributions, combining several experiments should lead to results that are related to the actual tissue. Also, measurements exceeding the expected variations should be regarded as relevant and related to tissue changes induced during the experiments. To explain the observed changes in IB and CV, the underlying mechanisms of interaction between ultrasound and tissue have to be understood. However, the structures in myocardium that are primarily responsible for the backscatter have not been definitively identified. Thus the causes of changes in IB during the cardiac cycle in normal conditions and changes induced by ischemia are very difficult to explain. Several mechanisms could influence changes in the backscattered signals: the change in size and distribution of the scatterers, the modification in
acoustic properties (eg, density, compressibility, and elasticity), as well as alterations in the anisotropy of the reflective structures.3,10,29,30 During ischemia, regional tissue parameters may change.Several studies suggest that the change in geometrical properties of the scattering structures is the predominant factor that accounts for the CV of the IB and that changes in elastic properties (as they occur during increased stress) are less important.29,31 Myocardial fiber orientation and angle of ultrasonic insonification are important determining factors for backscattered energy.32 Because in our experiments the same echocardiographic view and the same region on the myocardium are used and the global shape and orientation of the heart are not changed after the short period of ischemia (5 min), changes in fiber orientation cannot be proposed to explain the observed changes in IB. Prior reports have demonstrated that severe ischemia is associated with an increase in mean IB values and a blunting of the CV of the IB. From our studies (investigating short periods [5 min] of ischemia), we can make several new observations (Figure 4).
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First of all, when studying the measurements from the ischemic region in the posterior wall after occlusion of the distal CX, we noticed a clear increase in mean IB. The same observation (however less pronounced, but still significant) was made for posterior wall mean IB levels after a proximal occlusion. A (significant) increase was also found in mean IB levels in the myocardium surrounding the ischemic region during the distal occlusions. In such occlusions where only a limited area of ischemia was induced in the posterior wall, the mean IB values from the septal wall showed no change. Our findings for the ischemic zone correspond to the prior findings of other investigators in that mean IB levels increase. However, the clear increase of IB levels in the myocardium surrounding the ischemic region created by the distal occlusion was somewhat unexpected. This could be partially explained by the limitations induced by the use of the TTC coloring for the definition of the peripheral region. The TTC staining reflects the necrotic tissue after 60 min of occlusion and could be somewhat smaller than the actual ischemic region at 5 min of occlusion (caused by opening of collaterals, etc).This way, ischemic tissue could have been wrongly included in the regions indicated as peri-infarct. Another confounding factor might have been that the balloon catheter used to create the occlusion could have decreased the blood flow in the proximal perfusion territory of the CX. It was the analysis of the CV of the IB that showed much more interesting and controversial results.We did not find, as others have reported, a clear blunting of the CV during ischemia. In addition, we observed a clear increase of the CV, first in the region surrounding the ischemic zone after distal occlusion of the CX (statistically significant), and second in the septum after the proximal occlusion of the CX (which is not the feeding artery for the septum) (Figure 4). These findings indicate that the interpretation of the IB and especially of its CV may not be as simple and straightforward as previously suggested. Prior findings have suggested that an increase in IB is suggestive of ischemia within a myocardial segment. However, in this study the increase in IB was greater in distal occlusions where the ischemia might have been expected to be less severe (particularly because of the very well developed collateral circulation in dogs).Thus it seems that an increase in IB may not be directly related to the extent or severity of ischemia. For the CV, we clearly measured changes in myocardium that were not ischemic, but were nor-
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mocontractile or hypercontractile and working under altered loading conditions. Because the automatic regulatory function of the heart tries to preserve global pumping performance, compensation for the ischemic, noncontracting muscle must be made by the remaining nonischemic tissue. In the case of a distal occlusion, only a localized part of the posterior wall is ischemic and the cardiac performance is maintained by an increase in the working load of the remaining part of the posterior wall, leaving the septal contraction unchanged.This is shown in Figure 6. On the other hand, the proximal CX occlusion induced akinesia in the entire posterior wall, which was compensated for by an increase in contractility in the remaining myocardial walls, including the septum. This could explain the changes in the septal measurements during these proximal occlusions. Our findings suggest that changes in CV of IB can be altered by factors other than ischemia. This may be caused by the fact that CV more likely reflects the contractile state of the muscle (either active contraction or passive stretching by surrounding tissue). This finding has also been observed by others.7,15,18 It was noted that changes in CV of IB were related to changes in myocardial wall thickness, which reflects the systolic contractile performance of the muscle. Our findings suggest that CV is related not only to wall thickening and thinning changes but that IB levels and CV are related to the either active or passive contraction or stretching of the myocardium. These conclusions fit clearly with recent work that has used high frame rate data acquisition.23-25 This also could explain why we still observed CV in ischemic zones. It has been shown that in ischemia induced by short acute occlusions, even if the wall is akinetic, there is still important local deformation of the myocardium.33,34 Thus if CV reflects this deformation of the tissue after 5 min of total occlusion, the presence of a CV could still be expected. It is only in chronic ischemia and after long periods of severe ischemia, when the tissue is becoming necrotic and fibrotic, that there is much less local deformation and thus a blunting of the CV. Conclusion Our studies have confirmed that IB and its CV clearly change during ischemic conditions in canine myocardium. However, changes in IB and its CV were not restricted to the ischemic zone in this animal model. This new finding has important implications for the use of such measurements in clinical research. In addition, because the exact mechanism and origin of the IB and especially its CV remain poorly understood, we suggest that its precise role in defin-
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ing the degree of ischemic damage to the myocardium remains unclear. We confirmed that the magnitude of IB clearly increases under ischemic conditions, but it is unclear whether this increase is linearly related to the extent or the severity of ischemia. Furthermore, the magnitude of the CV seems to be influenced by the conditions under which the muscle has to contract and is not only altered by ischemia. This suggests a close relationship between CV and the interpositioning of fibers, the intercellular matrix and collagen, as well as the contractile state of the myofibers. In addition, when studying IB and CV, it is important to keep in mind the acquisition and analysis methodology. To reduce statistical variations, the myocardial regions to be interrogated should be as large as possible, but the merging of contiguous regions that correspond to tissues with different ischemic and loading conditions should be avoided. Consequently, this approach is difficult to use in clinical practice because the investigator will have little or no a priori knowledge of the extent and localization of the ischemia and of regional differences in workload and wall stress. Future research with the use of the new generation of echocardiographic equipment with high frame rates and high-resolution digital RF data will provide the opportunity to study the relation between IB and myocardial function in more depth.
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24. Bijnens B, D’hooge J, Pislaru C, Pislaru S, Kirkhorn J, Herregods M, et al. High frame rate, high resolution integrated backscatter levels in normal canine myocardium [abstract]. Eur Heart J 1998;19(Suppl):209. 25. Bijnens B, D’hooge J, Pislaru C, Pislaru S, Kirkhorn J, Herregods M, et al. High frame rate integrated backscatter in normal canine myocardium [abstract]. Circulation 1998;98 (Suppl):I642. 26. van der Steen A, Rijsterborgh H, Lancee C, Mastik F, Krams R, Verdouw P, et al. Influence of data processing on cyclic variation of integrated backscatter and wall thickness in stunned porcine myocardium. Ultrasound Med Biol 1997;23: 405-14. 27. Mohr G, Vered Z, Barzilai B, Perez J, Sobel B, Miller J. Automated determination of the magnitude and time delay (‘phase’) of the cardiac cycle dependent variation of myocardial ultrasonic integrated backscatter. Ultrason Imaging 1989;11:245-59. 28. Bijnens B, D’hooge J, Sutherland G, Herregods M, Nuyts J, Suetens P, et al. Robustness of integrated backscatter for myocardial tissue characterization. Ultrasound Med Biol 1999;25:95-103.
29. Wear KA, Shoup TA, Popp RL. Ultrasonic characterization of canine myocardium contraction. IEEE Transactions on Ultrasonics, Ferroelectric and Frequency Control 1986;33:347-53. 30. Wickline S, Thomas LJI, Miller JG, Sobel B, Perez J. A relationship between ultrasonic backscatter and myocardial contractile function. J Clin Invest 1985;76:2151-60. 31. Rijsterborgh H, van der Steen A, Krams R, Mastik F, Lancee C, Verdouw P, et al. The relationship between myocardial integrated backscatter, perfusion pressure and wall thickness during isovolumic contraction: an isolated pig heart study. Ultrasound Med Biol 1996;22:43-52. 32. Recchia D, Hall CS, Shepard RK, Miller JG. Mechanisms of the view-dependence of ultrasonic backscatter from normal myocardium. IEEE Transactions on Ultrasonics, Ferroelectric and Frequency Control 1995;42:91-8. 33. Sutherland G, Kukulski T, Hatle L. Tissue Doppler echocardiography: future developments. Echocardiography 1999;16: 509-20. 34. Leone B, Norris R, Safwat A, Foëx P, Ryder W. Effect of progressive myocardial ischemia on systolic function, diastolic dysfunction and load dependent relaxation. Cardiovasc Res 1992;26:422-9.
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Integrated backscatter (IB) from a myocardial region, calculated from radiofrequency echocardiographic data, has been proposed as a useful parameter for investigating changes in myocardial tissue induced by ischemia. In 10 closed-chest dogs, 5 minutes of myocardial ischemia was induced by either a proximal occlusion of the circumflex coronary artery (CX) (5 dogs), resulting in extensive ischemia in the posterior wall, or by occluding the distal CX vessel (5 dogs) to produce a small localized ischemic zone in the posterior wall. High-resolution digital radiofrequency data from the whole left ventricular myocardium, in the imaging plane during one complete heart cycle, were acquired with a whole-image realtime acquisition approach. Regions in the septum
and posterior wall (both ischemic tissue and, in the case of distal occlusions, tissue surrounding the ischemic zone) were chosen for analysis, and IB and cyclic variation (CV) of IB were calculated. Post occlusion, an increase in mean IB values was found in the ischemic segment. However, an increase in CV was also observed in the peri-ischemic zone for the distal CX occlusion and in the septum after proximal CX occlusion. These findings show that changes in CV are not restricted to the ischemic zone but may also occur in distal myocardium. This may be explained by changes in the regional contractile state and loading conditions of the “normal” myocardium, which are altered in response to the distal ischemia. (J Am Soc Echocardiogr 2000;13:306-15.)
INTRODUCTION
ultrasonic tissue characterization. Investigators have increasingly turned their attention to the investigation of the unprocessed RF data set to determine whether there is clinically relevant information in this potentially higher resolution signal.4 The parameter extracted from the RF data set that has been most studied is integrated backscatter (IB).1 Integrated backscatter is calculated by integrating the power spectrum of the received signal (after compensation for the spectrum of the transmitted pulse) over the meaningful bandwidth of the transducer. This implies that IB is a measure of the mean reflected ultrasonic energy from a particular region of tissue.4 Prior extensive clinical evaluation of the properties of IB has demonstrated that changes in its magnitude, its cycle-dependent variation, and the timing of peak and trough levels can all be influenced by a variety of disease processes.5-15 In these studies, IB values were measured either from off-line analysis of acquired RF signals or by using dedicated hardware incorporated in a standard clinical ultrasound
The characterization of the functional state and structural composition of the myocardium remains an important goal in clinical cardiology. In this respect, there is increasing interest in the use of parameters extracted from raw, unprocessed radiofrequency (RF) data from echocardiographic equipment to characterize changes in myocardial reflectivity induced by a variety of pathologic processes.1-3 A general consensus now exists that postprocessing analysis of video data acquired during cardiac ultrasonographic (ultrasound) studies is not ideal for From the Department of Cardiology and the Department of Nuclear Medicine, Medical Image Computing, Gasthuisberg University Hospital, Herestraat 49, B-3000 Leuven, Belgium. Reprint requests: Bart Bijnens, UZ Gasthuisberg, Department of Cardiology, Herestraat 49, B-3000 Leuven, Belgium (E-mail: [email protected]). Copyright © 2000 by the American Society of Echocardiography. 0894-7317/2000 $12.00 + 0 27/1/103595 doi:10.1067/mje.2000.103595
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machine. However, many of these studies have been limited in their ability to capture real-time data from the whole myocardium in the B-mode imaging plane and thus have been unable to compare changes in IB data for all myocardial segments. To improve on prior studies, we have developed an approach that enables the acquisition of RF data from a complete sector scan during a complete heart cycle.16 As indicated above, this approach differs from that of most prior researchers who either used Mmode data or implemented small time-gates to limit the amount of 2-dimensional (2D) data acquired.6,17-19 Our approach enables the investigation of the signal from the complete 2D image throughout a complete cardiac cycle. Thus it will allow the comparison of reflectivity parameters derived simultaneously from different regions of the myocardium. With this approach we have reinvestigated the value of IB measurements in acute myocardial ischemia induced in a canine model.Transthoracic echocardiographic data were acquired with a standard clinical ultrasound machine. This is in contrast to most work from prior investigators, who used laboratory echographic equipment, such as single-element piezo-electric crystals connected to dedicated pulse-receive electronics, instead of scanners designed for clinical work. To investigate the changes in IB and its cyclic variation (CV), we induced 5 minutes of ischemia in a series of dogs. In the first set of animals, transient localized ischemia was induced in the posterior wall with the use of a balloon occlusion of a distal branch of the circumflex coronary artery (CX). In the second set, severe widespread ischemia was induced by 5 minutes of occlusion of the proximal CX.
MATERIALS AND METHODS Animal Preparation Ten mongrel dogs (17 to 25 kg) were studied. All procedures performed were in accordance with the institutional guidelines set by the University of Leuven. All animals were sedated with xylazine (50 mg intramuscularly). After anesthesia with sodium-pentobarbital (15 mg/kg intravenously for induction and 0.1 mg/kg/min for maintenance), each dog was intubated (cuffed endotracheal tube) and mechanically ventilated with a mixture of oxygen (20%) and air (Bird respirator, Bird Products Corp, Palm Springs, Calif). Both carotid arteries, jugular veins, one femoral artery, and one femoral vein were dissected free and cannulated with arterial sheaths or infusion lines.After completion of the dissection, all dogs received heparin (100 IU/kg bolus intravenously plus continuous infusion of
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Figure 1 A typical coronary angiogram of the circumflex coronary artery is shown with indication of the position of the balloon during occlusion. Arrows indicate position for experiments with proximal occlusions (prox) and distal occlusions (dist).
20 IU/kg/h) and aspirin (5 mg/kg intravenous bolus). Ventilation was adjusted to maintain pH and arterial blood gasses within the physiologic range. Body temperature was maintained with a heating pad. Reperfusion arrhythmias were treated with lidocaine.
Measurements Both at baseline conditions and after 5 minutes of ischemia, a 2D echocardiographic study was performed with a Toshiba 160 (Tokyo, Japan) clinical ultrasound scanner. High-quality left ventricular 2D images were acquired with a transducer frequency of 5 MHz and a frame rate of 30 Hz with use of the standard parasternal left ventricular longaxis view.Video data and RF data were acquired and stored. In all dogs, an area of myocardial ischemia was induced in the posterior myocardial wall. In half (5/10) of the dogs, localized myocardial ischemia was induced by balloon occlusion (percutaneous transluminal coronary angioplasty balloon, Quick 2.5, Baxter, Volen, The Netherlands) of a marginal branch of the CX. In the other half, the perfusion of the complete posterior wall was interrupted by a balloon occlusion of the proximal CX. Coronary angiography was performed twice during each animal study. The first study was to assess coronary anatomy before balloon inflation. The second was to check the efficacy of the occlusion during the inflation. In Figure 1 a typical coronary angiogram of the CX is shown. The positions used for the proximal and distal occlusions are indicated. At the end of the experiments, the heart was arrested with saturated potassium chloride intravenously. For the 5 dogs with moderate ischemia, the vessel was occluded for a further 60 minutes after the RF data acquisition to stain
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Figure 2 The setup used to digitize the radiofrequency (RF) data from the echocardiographic examination. 3D, Three-dimensional.
the excised hearts with triphenyltetrazolium chloride (TTC) to achieve a delineation of the area that was ischemic at the time of data acquisition. In all 10 dogs, the left ventricle was subsequently sectioned parallel to its long axis in such a way that this section corresponded as much as possible to the echocardiographic parasternal long-axis plane. Enlarged pictures of the slices after TTC staining were obtained and visually assessed for the site, extent, and severity of infarction.
RF Data Acquisition The RF signal was digitized using the acquisition setup described by Bijnens et al.16 Figure 2 demonstrates the equipment used. Data acquisition was triggered using the R peak of the electrocardiogram (ECG).This resulted in 50 RF images at a frame rate of 30 images/s and a sample frequency of 40 MHz (corresponding to approximately 60 Mb of data for each heart cycle).
RF Processing To process the RF data, we used a software package (“Speqle”) developed in-house and previously described in our work.20 After reconstruction of the sector scans of the relevant part of the RF data, both endocardium and epicardium contours were manually drawn to allow segmentation of the complete left ventricular wall. Next, both contours (endocardium and epicardium) were “shrunk.” Their centerline was determined and the contours were moved 10% toward each other and perpendicular to the centerline. This was performed to avoid inclusion of the specular reflections from the tissue-blood border and pericardium during subsequent analysis.
For each 2D image sequence analysis, the position of the apex was indicated. The resulting delineation of the wall was divided into 24 segments: 12 “septal” from the apex and 12 “posterior.” Each of the segments was divided into 4 parts from endocardium to epicardium for future study of transmural behavior. Figure 3 shows how this process was effected.The ECG and the phonocardiogram were displayed in real time together with the images. End diastole and end systole were indicated on the data sets by the investigator. These were determined from the timing of events derived both from the synchronously sampled ECG and phonocardiogram and from the 2D images that used the timing of opening and closure of the mitral valve. From each defined region of interest, IB was calculated for all 50 consecutive images. Integrated backscatter was calculated in the time domain as the sum of the squares of all data points in the region of interest, normalized by the number of points.21,22 On the basis of end-diastolic and end-systolic frames, IB traces were resampled to get the same number of data points for each heart cycle (10 for diastolic phase and 10 for systolic phase) for each acquisition. To use as many RF data points as possible to reduce the effect of noise, comparable regions were merged for the IB analysis. In this work, the transmural IB values from the total septal wall were compared with the posterior wall (transmural). In the case of proximal occlusions, all regions from the posterior wall were merged, whereas in the case of occlusion of the distal CX, infarcted areas (as defined by data from the TTC–stained excised heart) were separated from normal regions and IB values were calculated for both infarcted and noninfarcted myocardium.
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Figure 3 A left ventricular parasternal long-axis view showing the segmentation into regions used to analyze the radiofrequency data. ECG, Electrocardiogram; Phono, phonocardiogram.
Table 1 Summary of the IB and CV measurements Distal occlusion Baseline
Mean IB septum (dB) Mean IB posterior (dB) CV septum (dB) CV posterior (dB)
–0.2 –0.1 5.2 3.7
± ± ± ±
1.4 0.1 2.0 0.9
Proximal occlusion
0.4 1.5 6.5 3.8
± ± ± ±
2.2 2.9* 2.8 0.9
Ischemic region
Peripheral region
–0.6 ± 2.8 3.9 ± 2.0*
1.0 ± 2.0 5.5 ± 0.6
4.4 ± 1.1
6.7 ± 2.7*
IB, Integrated backscatter; CV, cyclic variation. *Significantly different from baseline: P < .01.
All IB values were normalized to the mean value of the basal measurement (separately for septum and posterior wall).This provided an implicit attenuation correction, setting the basal mean values of the posterior wall measurements and the septum to unity.This implies that the only difference between posterior and septal IB values in basal conditions is caused by attenuation. To make our results comparable to those obtained by others in the literature, derived IB values were expressed in decibels. The CV of the IB was calculated as the difference between the maximum and the minimum value of the IB.
Statistical Analysis Integrated backscatter and CV values for the different groups and regions were compared and statistically ana-
lyzed with a combination of an analysis-of-variance approach with a Tukey “honestly significant difference”test (with the standardized range statistic). Differences with a statistical probability <.05 were considered significant.
RESULTS During basal conditions, IB values for each region of interest were normalized.The values obtained for all myocardial segments interrogated were within the range of normal values previously reported (IB septum: –0.2 ± 1.4 dB; IB posterior wall: –0.1 ± 0.1 dB). Cyclic variation values were also normal (CV septum: 5.2 ± 2.0 dB; CV posterior wall: 3.7 ± 0.9 dB)
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Figure 4 An overview of the mean integrated backscatter (IB) values and the cyclic variation (CV) (with their SDs) for these experiments. Left, Septum; right, posterior wall. Shown are the values at baseline (bas), after occlusion of the proximal (prox) circumflex coronary artery (CX), and after occlusion of the distal CX (dist). For the distal occlusions (after 60-min occlusion), the values for the ischemic area (isch) and the peri-ischemic area (peri) are separated.
(Figure 4 and Table 1).These values are similar to the normal CV values reported by other authors.1-3 For the severe generalized ischemic zone (proximal CX occlusions), IB values were significantly (P < .01) increased (1.5 ± 2.9 dB), but no significant blunting of CV occurred (3.8 ± 0.9 dB). In the peri-ischemic zone of the localized ischemia (distal CX occlusion), IB levels were only slightly increased (1.0 ± 2.0 dB). However, CV increased significantly (6.7 ± 2.7 dB) in this segment, which was hyperkinetic, whereas in the localized ischemic zone itself, IB levels increased much more (3.9 ± 2.0 dB), but CV remained comparable to baseline levels (4.4 ± 1.1 dB). The most interesting findings in this study were the changes recorded in the septum in animals with severe generalized ischemia of the posterior wall. In these animals, the septum was hyperkinetic on the 2D image, presumably in response to the hypokinesia or akinesia in the posterior wall. Although mean septal IB levels did not change at all compared with baseline, a clear increase was seen in CV (6.5 ± 2.8 dB) (Figure 4). In Figure 5, these findings are illustrated by the curves obtained from 2 studies. The normalized heart cycles, averaged over all experiments (10 basal, 5 proximal, and 5 distal occlu-
sion) are displayed in Figure 6. A total overview of the calculated mean IB values and CV is displayed in Figure 4.In the case of the occlusions of the distal CX,the posterior wall could be divided into an area at risk and a “peripheral”area, on the basis of the results of the TTC staining after 60 minutes of occlusion.The results for these two different “tissue-types” are separated and illustrated in the figure. Table 1 lists the measurements. The mean IB values for the septum (Figure 4, top left) were not statistically different. Analysis of the mean IB values from the posterior wall (Figure 4, top right) showed a significant (P < .01) difference between baseline values and all others and, for the distal occlusions, between the ischemic area and the peripheral area as well as between the ischemic area for the distal occlusion and the ischemic area for the proximal occlusions. For the CV values from the septum (Figure 4, bottom left) no statistically significant difference could be found, whereas for the CV values from the posterior wall, a significant difference was observed between the basal values and the peripheral area during distal occlusion as well as between the ischemic area for the proximal occlusions and these peripheral regions for the distal occlusions.
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Figure 5 Examples of integrated backscatter (IB) traces obtained from the experiments (dashed lines, basal; solid lines, after 5 minutes of occlusion). Top, Plots correspond to values from the septum; bottom, values from the posterior wall. Left, Plots from experiments that used an occlusion of the proximal circumflex coronary artery; right, plots that used distal occlusions. For each, the IB values for 50 consecutive frames are plotted. For each trace, this corresponds to 2 or more heart cycles. The IB values are expressed in dB.
DISCUSSION One of the major problems in the use of IB and its CV for tissue characterization is the lack of uniformity and standardization of data acquisition and processing. Important problems include the large amount of data to be acquired and the accuracy of data acquisition. In our approach, we have chosen to sample the complete ultrasound scan at a high sample frequency so that no data were lost.The only possible limitation is related to the dynamic range of the analog-to-digital converter (8 bit), but this was, in theory, of sufficient resolution to interrogate the myocardium. A better solution for any potential problems posed by this limited dynamic range will be offered by the new generation of fully digital echocardiographic equipment, which should enable the acquisition of digital RF data with a dynamic range up to 20 bit. Recent findings suggest that one of the major problems in investigating myocardial function is the limited frame rate of the previous generation of echocardiographic equipment. It has been shown
that a frame rate of at least 80 Hz should be used to resolve the fastest functional changes from the myocardium.23-25 Because the study described in this paper was performed with the use of a frame rate of 30 Hz, and if IB is related to functional changes (as suggested in this work), it should be noted that some of the data could have been undersampled. A further problem is the precise definition and methods used to calculate CV.26 We used the maximal excursion of the IB. In baseline conditions, this corresponds best to the difference between the enddiastolic and the end-systolic values. However, during ischemia, a phase shift of the IB can occur (Figure 6). In theory, this could be detected by fitting an appropriate function to the measured IB data.27 However, a question still remains regarding the type of function that should be fitted because there are difficulties in defining the origin of the CV and thus choosing a well-founded physical model. Another important factor to take into account is the problem of the inherent potential variability in measuring IB levels as demonstrated and discussed in Bijnens et al.28 In the worst case, it would be
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Figure 6 An overview of the average normalized heart cycles (from end diastole [ED] over end systole [ES] up to the next ED) for all cases. Left, Septal values; right, posterior wall values. Top, Baseline integrated backscatter (IB); middle, values for 5-min proximal (prox) occlusion (occl) of circumflex coronary artery (CX); bottom, values after 5 minutes of distal (dist) CX occlusion.
expected that variations of up to 0.9 dB from the mean (or 1.8 dB CV) could be observed, which were not related to the state of the tissue. This clearly explains the large standard deviations within our measurements. Because the inherent variations on IB are statistical deviations from the expected mean value caused by randomness in scatterer distributions, combining several experiments should lead to results that are related to the actual tissue. Also, measurements exceeding the expected variations should be regarded as relevant and related to tissue changes induced during the experiments. To explain the observed changes in IB and CV, the underlying mechanisms of interaction between ultrasound and tissue have to be understood. However, the structures in myocardium that are primarily responsible for the backscatter have not been definitively identified. Thus the causes of changes in IB during the cardiac cycle in normal conditions and changes induced by ischemia are very difficult to explain. Several mechanisms could influence changes in the backscattered signals: the change in size and distribution of the scatterers, the modification in
acoustic properties (eg, density, compressibility, and elasticity), as well as alterations in the anisotropy of the reflective structures.3,10,29,30 During ischemia, regional tissue parameters may change.Several studies suggest that the change in geometrical properties of the scattering structures is the predominant factor that accounts for the CV of the IB and that changes in elastic properties (as they occur during increased stress) are less important.29,31 Myocardial fiber orientation and angle of ultrasonic insonification are important determining factors for backscattered energy.32 Because in our experiments the same echocardiographic view and the same region on the myocardium are used and the global shape and orientation of the heart are not changed after the short period of ischemia (5 min), changes in fiber orientation cannot be proposed to explain the observed changes in IB. Prior reports have demonstrated that severe ischemia is associated with an increase in mean IB values and a blunting of the CV of the IB. From our studies (investigating short periods [5 min] of ischemia), we can make several new observations (Figure 4).
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First of all, when studying the measurements from the ischemic region in the posterior wall after occlusion of the distal CX, we noticed a clear increase in mean IB. The same observation (however less pronounced, but still significant) was made for posterior wall mean IB levels after a proximal occlusion. A (significant) increase was also found in mean IB levels in the myocardium surrounding the ischemic region during the distal occlusions. In such occlusions where only a limited area of ischemia was induced in the posterior wall, the mean IB values from the septal wall showed no change. Our findings for the ischemic zone correspond to the prior findings of other investigators in that mean IB levels increase. However, the clear increase of IB levels in the myocardium surrounding the ischemic region created by the distal occlusion was somewhat unexpected. This could be partially explained by the limitations induced by the use of the TTC coloring for the definition of the peripheral region. The TTC staining reflects the necrotic tissue after 60 min of occlusion and could be somewhat smaller than the actual ischemic region at 5 min of occlusion (caused by opening of collaterals, etc).This way, ischemic tissue could have been wrongly included in the regions indicated as peri-infarct. Another confounding factor might have been that the balloon catheter used to create the occlusion could have decreased the blood flow in the proximal perfusion territory of the CX. It was the analysis of the CV of the IB that showed much more interesting and controversial results.We did not find, as others have reported, a clear blunting of the CV during ischemia. In addition, we observed a clear increase of the CV, first in the region surrounding the ischemic zone after distal occlusion of the CX (statistically significant), and second in the septum after the proximal occlusion of the CX (which is not the feeding artery for the septum) (Figure 4). These findings indicate that the interpretation of the IB and especially of its CV may not be as simple and straightforward as previously suggested. Prior findings have suggested that an increase in IB is suggestive of ischemia within a myocardial segment. However, in this study the increase in IB was greater in distal occlusions where the ischemia might have been expected to be less severe (particularly because of the very well developed collateral circulation in dogs).Thus it seems that an increase in IB may not be directly related to the extent or severity of ischemia. For the CV, we clearly measured changes in myocardium that were not ischemic, but were nor-
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mocontractile or hypercontractile and working under altered loading conditions. Because the automatic regulatory function of the heart tries to preserve global pumping performance, compensation for the ischemic, noncontracting muscle must be made by the remaining nonischemic tissue. In the case of a distal occlusion, only a localized part of the posterior wall is ischemic and the cardiac performance is maintained by an increase in the working load of the remaining part of the posterior wall, leaving the septal contraction unchanged.This is shown in Figure 6. On the other hand, the proximal CX occlusion induced akinesia in the entire posterior wall, which was compensated for by an increase in contractility in the remaining myocardial walls, including the septum. This could explain the changes in the septal measurements during these proximal occlusions. Our findings suggest that changes in CV of IB can be altered by factors other than ischemia. This may be caused by the fact that CV more likely reflects the contractile state of the muscle (either active contraction or passive stretching by surrounding tissue). This finding has also been observed by others.7,15,18 It was noted that changes in CV of IB were related to changes in myocardial wall thickness, which reflects the systolic contractile performance of the muscle. Our findings suggest that CV is related not only to wall thickening and thinning changes but that IB levels and CV are related to the either active or passive contraction or stretching of the myocardium. These conclusions fit clearly with recent work that has used high frame rate data acquisition.23-25 This also could explain why we still observed CV in ischemic zones. It has been shown that in ischemia induced by short acute occlusions, even if the wall is akinetic, there is still important local deformation of the myocardium.33,34 Thus if CV reflects this deformation of the tissue after 5 min of total occlusion, the presence of a CV could still be expected. It is only in chronic ischemia and after long periods of severe ischemia, when the tissue is becoming necrotic and fibrotic, that there is much less local deformation and thus a blunting of the CV. Conclusion Our studies have confirmed that IB and its CV clearly change during ischemic conditions in canine myocardium. However, changes in IB and its CV were not restricted to the ischemic zone in this animal model. This new finding has important implications for the use of such measurements in clinical research. In addition, because the exact mechanism and origin of the IB and especially its CV remain poorly understood, we suggest that its precise role in defin-
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ing the degree of ischemic damage to the myocardium remains unclear. We confirmed that the magnitude of IB clearly increases under ischemic conditions, but it is unclear whether this increase is linearly related to the extent or the severity of ischemia. Furthermore, the magnitude of the CV seems to be influenced by the conditions under which the muscle has to contract and is not only altered by ischemia. This suggests a close relationship between CV and the interpositioning of fibers, the intercellular matrix and collagen, as well as the contractile state of the myofibers. In addition, when studying IB and CV, it is important to keep in mind the acquisition and analysis methodology. To reduce statistical variations, the myocardial regions to be interrogated should be as large as possible, but the merging of contiguous regions that correspond to tissues with different ischemic and loading conditions should be avoided. Consequently, this approach is difficult to use in clinical practice because the investigator will have little or no a priori knowledge of the extent and localization of the ischemia and of regional differences in workload and wall stress. Future research with the use of the new generation of echocardiographic equipment with high frame rates and high-resolution digital RF data will provide the opportunity to study the relation between IB and myocardial function in more depth.
REFERENCES 1. Perez J, Dávila-Román V, Miller J. Assessment of myocardial viability by ultrasonic tissue characterization. Coron Artery Dis 1995;6:613-8. 2. Miller J, Perez J, Sobel B. Ultrasonic characterization of myocardium. Prog Cardiovas Dis 1985;28:85-110. 3. Skorton D, Miller J, Wickline S, Barzilai B, Collins S, Perez J. Ultrasonic characterization of cardiovascular tissue. In: Marcus ML, Schelbert HR, Skorton DJ, Wolf GL, editors. Cardiac imaging (A companion to Braunwald’s heart disease). Philadelphia: WB Saunders; 1991. p. 538-56. 4. Wickline S, Perez J, Miller J. Cardiovascular tissue characterization in vivo. In: Shung KK, Thieme GA, editors. Ultrasonic scattering in biological tissues. Boca Raton, (FL): CRC Press; 1993. p. 313-45. 5. Mimbs J, Bauwens D, Cohen R, O’Donnel M, Miller J, Sobel B. Effects of myocardial ischemia on quantitative ultrasonic backscatter and identification of responsible determinants. Circ Res 1981;49:89-96. 6. Schnittger I, Veili A, Heiserman J, Director B, Billingham M, Ellis S, et al. Ultrasonic tissue characterization: detection of acute myocardial ischemia in dogs. Circulation 1985;72:193-9. 7. Rijsterborgh H, Mastik F, Lancee C, Sassen L, Verdouw P, Roelandt J, et al. The relative contributions of myocardial wall thickness and ischemia to ultrasonic myocardial integrated
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