| Backgrounds and ObjectivesIschemic heart disease is one kind of the most common heart diseases and coronary artery stenosis is the most common cause, which is called coronary artery disease (CAD). Chronic ischemia is more often occurred than the acute in clinical. Left ventricular (LV) systolic function is important in the diagnosis of CAD, and is related to the severity and range of myocardial ischemia and to effective blood perfusion. However, in the early phase of myocardial ischemia, when the regional wall motion has been changed, the global function may be still in the normal range owing to the compensation of the segment near to the ischemic territory and would be mistaken to normal. So the quantitative assessment of regional myocardial wall motion and blood perfusion plays important role in the diagnosis of CAD. Recent updates in the field of echocardiography have resulted in improvements in both image quality and techniques permitting simultaneous assessment of global and regional myocardial structure, function, and perfusion, enabling non-invasive assessment of coronary artery disease.When echocardiography quantifying LV systolic function, LV ejection fraction (EF) is the most commonly used index. Two-dimensional echocardiography (2DE) is the most common technique, but it has pitfalls when LV morphology changed obviously. Recently, real-time three-dimensional echocardiography (RT-3DE) could acquire full volume data within only several seconds, enabling truly real time three dimensional data acquisition. And full volume data could be obtained with probe located at apical region, avoiding measurement error coming from the change of probe location and the different cardiac cycle. Further more, RT-3DE displays directly LV three dimensional morphology and then calculate LV volume directly, need no geometry hypothesis, as is in the 2-DE, so it conquer the limitation of 2-DE. RT-3DE analyzes all segmental volumes during the same cardiac cycle, and calculates segmental EF (SEF) from segmental volume of end diastole and end systole.Rhythmic LV contraction and relaxation is necessary to maintain normal cardiac function. Cardiac resynchronization therapy has been proved effective in improving cardiac function of the patients of LV contractile dyssynchrony. But the problem is how to determine LV contractile dyssynchrony. RT-3DE outline LV endocardium and display directly the 3-dimensional morphology, reflect accurate LV volume change during cardiac cycle, so has potential superiority in evaluating LV synchronization.More recently, speckle tracking echocardiography, a new echocardiographic 2-dimensional (2D) strain technique based on automatic tracking the two-dimensional motion of characteristic speckle patterns in B-mode images, enables simultaneous valuation of regional myocardial strain in both the radial and circumferential directions (i.e., circumferential strain (CS), and radial strain (RS) ) from parasternal short-axis view. Two-dimensional strain is in principle angle independent, and has been shown to be repeatable and reproducible, and accurate and correlate well with sonomicrometry and magnetic resonance imaging tagging during ischemia.Normal LV myocardial contractility depends on sufficient blood perfusion. Ischemia leads to abnormal myocardial metabolism and then to abnormal myocardial contractility in CAD. Real-time myocardial contrast echocardiography (RT-MCE) is a newly developed technique to quantify myocardial blood perfusion, displaying the replenishment process of microbubbles into myocardium after microbubbles being destroyed by a high energy pulse. The replenishment of contrast can be characterized by a time-intensity curve (i.e. destruction–refilling curve), which can be fitted to a monoexponential function: y=A×(1-eβt)+C. The plateau value (A) of the replenishment curve reflects the myocardial blood volume, and the slope (β) reflects the reappearance rate of microbubbles (i.e. myocardial microbubble velocity). The product of A×βtherefore represents myocardial blood flow. Thus RT-MCE could analyze quantitively the microvascular blood perfusion in regions of interest in myocardium with specialist software.Conronary artery has powerful compensation capacity, so mild to moderate stenosed coronary vessel will supply adequate blood to maintain normal function at rest, so echocardigraphy may display no abnormality in LV wall motion and perfusion. Stress will induce an increase of myocardial consumption of oxygen, but in stenosed artery the blood supply could not increase accordingly, leading to ischemia in the victim territory, therefore abnormal wall motion and perfuison will be found in echocardiography. Dobutaming stress echocardiography (DSE) is one of the favorite stress methods, and is often applied to 2DE, RT-3DE or RT-MCE and combined with baseline, to evaluate the myocardial motion or blood perfusion in both stress and rest condition and further to evaluate the reserves, thus increases the technique's sensitivity and accuracy, and has potential to anticipate prognosis.In the present study, chronic myocardial ischemia was induced by placing an Ameroid constrictor in the left circumflex in swines, then RT-3DE and RT-MCE examination combining rest and DSE was performed to ascertain chronic ischaemic LV dysfunction and myocardial perfusion and reserves, and Vivid 7 was used to assess 2 dimensional strain and torsional anomalies, so as to validate the role of Echocardiography to recognize regional contractile variations and perfusional anomalies for the detection of ischemia.MethodsA chronic ischemic model was induced and echocardiography and coronary angiography was performed as described below before LCX constriction, every week after LCX constriction, until the LCX stenosis was of≥90% validated coronary angiographically. Then animals were killed, hearts were harvested and processed for hematoxylin and eosin (HE) stain, Poley stain and scanning electron microscopy.Chronic ischemic model and Animal preparationTen normal juvenile domestic swines in either gender, body weight 20 to 26Kg, were subjected to induce chronic ischemia by constricting the left circumflex artery (LCX) with Ameroid constrictor. Pigs were sedated with an intramuscular injection of 300mg ketamine (about 15mg/Kg) with 5mg midazolam and 1mg atropine, intubated, and maintained with combined anesthesia with inhalant enflurane and intravenous vecuronium bromide. A thoracotomy was performed through the fourth left intercostal space. The pericardium was opened and an Ameroid constrictor was placed around the proximal LCX just distal to the main stem of the left coronary artery. The chest was then closed, and the animals were allowed to recover and returned to their cages. This animal experiment was approved by the Animal Care Committee of Third Military Medical University. RT-3DE and RT-MCE Echocardiographic data acquisitionGeneral anesthesia was initiated with ketamine (15mg/Kg) with 1mg atropine intramuscular, and maintained with intravenous ketamine or intraperitoneal pentobarbital. Animals were investigated in the closed-chest state in the left lateral decubitus position. All RT-3DE Full Volume echocardiograms were obtained with iE33 ultrasound machine (Philips Medical Systems) with Probe X3-1, with Probe located at apical area. If the border was unclear, ZhiFuXian—a homemade contrast agent in our laboratory was infused to enhance the resolution. RT-MCE contrast images of parasternal short axis at PM level were acquired with iE33 machine, S5 probe. The LVO pattern was select and set as second harmonic (transmit/receive: 1.7/3.4 MHz), mechanic index 0.1, beats 20.Images were copied to QLAB 5.2 postprocess workstation, and 3DQ and 3DQA software was used to analyze Full Volume data sets, and LV global ejection fraction (EF) and EDV and ESV of 16 segments except for apex were yielded, and then segmental EF (SEF) were calculated with EXCEL. Software 3DQA provided the maxium difference of Tmsv (Tmsv-dif) and standard deviation (Tmsv-SD) among various segments and standard index (Tmsv-dif% and Tmsv-SD%), to evaluate LV dyssynchrony. ROI software were used to analyze contrast images, and yielded the plateau value A of the replenishment curve (A reflects the myocardial blood volume), the slopeβ(βreflects the myocardial velocity). Calculate the product of A×β( A×βrepresents myocardial blood flow).DSE images acquisition and data analysisAfter the RT-3DE and RT-MCE images were acquired, dobutamine was administered intravenous by micro-infusion pump according to the low-dose protocol (5μg/kg/min followed by 10μg/kg/min, each step lasting 5 min). When target heart rate increased to 20bpm higher than that of baseline, repeated the baseline image acquisition. The reserve was determined as the ratio of the data of stress to that of the baseline, including SEF reserve, A reserve,βreserve, and A×βreserve, reflecting the contractile and perfusion reserve function. Each data was an average of two measurements.Two-dimensional Strain imaging and data analysisAnimals anesthesia and prepare as before. A Vivid 7 (GE Medical Systems) was used to acquire 2D strain images data with a M3S Probe. B-mode second harmonic images (frame rate≥50 frames/s) were recorded in parasternal apical, middle and basal short axis views of three consecutive cardiac cycles. The digital data were stored and transferred to a computer for subsequent offline analyses.Digital data were transferred to a dedicated software EchoPac (GE Medical Systems) for subsequent offline analysis. Each level of LV was divided into six segments, and each segment was individually analyzed. Two-dimensional peak radial and circumferential strains during systole were measured. By tracing the endocardial contour on an end-diastolic frame, the software will automatically track the contour on subsequent frames. Adequate tracking can be verified in real-time and corrected by adjusting the region of interest semi-manually to ensure optimal tracking. Circumferential strain (CS) and radial strain (RS) were recorded. At the same time, left ventricular rotation at apical and basal level of each wall and LV torsion were analyzed. The rotation difference between apical level and mitral level reflected the LV torsional angle (Tor).Selective coronary angiographySelective coronary angiography was performed the same day as or the day after the echocardiography to confirm the stenosis degree of LCX. Quantitative coronary angiography was performed through cine review by two blinded experienced angiographers. The stenosis at site of Ameroid placement was calculated by the following equation: (1 ? lumen diameter stenotic site/lumen diameter reference)×100. The severity of LCX stenosis were grouped into mild stenosis (stenosis <50%), moderate stenosis (stenosis 50~75%) and severe stenosis (stenosis≥75%).Gated SPECT myocardium perfusion imaging and data analysis99mTc-MIBI Gated SPECT myocardium perfusion imaging was performed with Infinia Hawkeye SPECT instrument (GE Medical Systems) 1 to 3 day after Echocardiogram. Two day method was used to perform rest/stress scan, with scanning at rest performed 90 min after intravenous infusion 15mCi 99mTc-MIBI on first day, then scanning the same way at stress on the second day.Emory Cardiac Toolbox for cardiac was used to analyze the Dicom data. LV functional parameters and LV mass was yielded. Region of interest was set at the center of 6 segments at LV short axis corresponding to echogram, radioactive counting was then measured. Statistical analysisData were expressed as mean±SD. Statistical analysis was performed with SPSS 13.0 software. Measurements values were grouped by the severity of LCX stenosis verified by selective coronary angiography and their means were compared. Measurements of at rest and stress were compared by a paired Student t test. Multiple comparisons between different groups were performed using ONE-WAY ANOVA analysis followed by post-hoc least-significant difference test. A p<0.05 was considered statistically significant for all analysis.ResultsAnimal models and animal groupOf the 10 subjects, 8 were induced chronic ischemia successfully and each was showed severe stenosis of LCX 4 to 6 weeks after placement of an Ameroid constrictor verified by quantitative coronary angiography analysis. Total times of echocardiography examination of all the 8 pigs from 1 to 6 weeks post operation were 38, of which 3 were excluded owing to unclear images, and 35 times were included in analysis, of which mild, moderate and severe stenosis were 12, 12 and 11 times respectively, according to the coronary angiography. Ten normal pigs before operation served as controls.RT-3DE variablesCompared with baseline, the global ejection fraction (EF) and segmental ejection fraction (SEF) of most segments in severe stenosis decreased in stress condition, while that in control, mild and moderate stenosis increased (p<0.05, 0.01).LV SEF showed a tendency of gradually decrease according to the progressive LCX stenosis under both baseline and stress condition.SEF of LCX territory region compare among groups: under both baseline and stress condition, SEF of lateral and posterior wall at mitral and papillary muscle (PM) level decreased significantly in moderate and severe stenosis, compared with control and mild stenosis. At stress, SEF of lateral and inferior wall at apical level in severe stenosis and SEF of lateral and posterior wall at mitral level in mild stenosis decreased, compared with control. (p<0.05, 0.01).SEF of LAD and RCA territory region compare among groups: SEF of anterior and inferior wall at mitral and PM level decreased significantly in severe stenosis at stress, compared with control. (p<0.05). SEF reserve compare among groups: SEF reserve had a tendency of gradually decrease along with the progress of LCX stenosis. SEF reserve of lateral and posterior wall at all levels decreased significantly in moderate and severe stenosis, compared with control. Among the LAD and RCA segments, anterior and inferior wall at mitral and PM level and inferior wall at apical level showed marked decrease of SEF reserve in severe stenosis, compared with control (p<0.05, 0.01).Synchrony evaluation: RT-3DE analyzed quantitively the maximum difference and standard deviation of time to minimal segmental volume (Tmsv) among segments. The results indicated that RT-3DE in healthy controls showed highly synchronous contraction in contrast to widespread dyssynchrony found in subjects of moderate and severe stenosis, and dyssynchrony found only in basal segments in mild stenosis.RT-MCE variablesCompared with baseline, A and A×βof lateral wall and posterior wall decreased obviously, andβand A×βof other segments increased under stress in severe stenosis (p<0.05,0.01). By contrast, all perfusion variables (A,βand A×β) of all segments in other groups increased (p<0.05, 0.01).Perfusion parameters of LCX territory region compared among groups: all perfusion variables of lateral and posterior wall decreased significantly in moderate and severe stenosis at rest, compared with control. By contrast, those variables decreased in all stenosis groups at stress, and statistical difference was found among all stenosis groups. (p<0.05, 0.01).Perfusion parameters of other segments compare among groups: at rest A×βof anterior and inferior wall in severe stenosis increased significantly, compared with other groups (p<0.05, 0.01). At stress A×βof anterior wall in severe stenosis and A×βof anterior and inferior wall in moderate stenosis decreased significantly, compared with control (p<0.05).Perfusion reserve variables compare: A×βreserve of lateral and posterior wall in mild stenosis,βand A×βreserve in moderate stenosis and reserves of A,β, and A×βin severe stenosis decreased significantly, compared with control (p<0.05, 0.01). Of nonischemic segments, anterior wall demonstrated decreasedβand A×βreserve and inferior wall decreased A×βreserve in severe stenosis, compared with control (p<0.05).Under both rest and stress condition, myocardial blood flow of all segments in each group measured by RT-MCE correlated well with radioactive counting by SPECT (all p<0.05, 0.01).Two-dimensional Strain and torsion variables1. The circumferential strain curve was positive wave form, and radial strain curve was negative. The peak value of the circumferential strain curve (CS) and radial strain curve (RS) had a tendency of gradually decrease according to the progressive stenosis. LCX segments compare among groups: RS and CS of lateral and posterior wall decreased significantly at all levels in severe stenosis, compared with control and mild stenosis; and decreased significantly at all levels in moderate stenosis, and at mitral level in mild stenosis, compared with control (p<0.05, 0.01).LAD and RCA segments compare among groups: compared with control, RS and CS of anterior wall at all levels and RS of inferior wall at mitral and PM levels in severe stenosis decreased significantly, while RS of anterior and inferior wall at PM level in moderate stenosis decreased significantly (p<0.05, 0.01).2. At apical and mitral level, rotation curve of each segment of LV had homogeneous wave form, i.e., LV rotated counterclockwise at apical level (positive angle) and rotated clockwise at mitral level (negative angle). LCX stenosis groups had almost the same rotation direction at both apical and mitral level like that of control.LCX segments compare among groups: Rotation angle of lateral and posterior wall at all levels in severe stenosis, at mitral and PM level in moderate stenosis, and at mitral level in mild stenosis decreased significantly, compared with control (p<0.05, 0.01). Rotation angle of lateral and posterior wall at all levels in moderate and severe stenosis also decreased significantly, compared with mild stenosis (p<0.05,0.01).Nonischemic segments compare among groups: Rotation angle of anterior wall at mitral level in moderate and severe stenosis and of anterior wall at PM level in severe stenosis decreased significantly, compared with control (p<0.05).3. Torsional angle (Tor) compare: Torsional angle of lateral and posterior wall in moderate and severe stenosis decreased significantly, compared with control and mild stenosis (p<0.05). Compared with control, Tor of anterior wall in severe stenosis decreased significantly (p<0.05). Conclusions1. Chronic ischemia model induced by placing an Ameroid constrictor at the left circumflex artery (LCX) is angiographically documented.2. Quantitative analysis indicates that along with progressive stenosis, segmental ejection fraction and perfusion parameters of ischemic segments decrease gradually. There is statistical difference of those parameters in moderate and severe stenosis compared with control.3. A linear relationship exists between myocardial blood flow measured by RT-MCE and radioactive counting by SPECT. Normal coronary artery has sufficient reserve capacity. Although territories supplied by mild and moderate stenotic LCX still have some reserve ability, they are impaired according to reserve analysis. Segments supplied by severe stenotic LCX demonstrate decreased myocardial contractility and perfusion and exhausted reserve capacity, company by impaired reserve capacity of nonischemic regions.4. RT-3DE could evaluate LV dyssynchrony by analyzing the maximum difference and standard deviation and standard variables of time to minimal segmental volume, and standard variable is found more sensitive than the absolute time value.5. Along with progressive stenosis, circumferential strain, radial strain and rotation angle show progressive decrease tendency. Most segments of the victim regions in moderate and severe stenosis decrease markedly than that of control.6. Real-time three-dimensional echocardiography, real-time myocardial contrast echocardiography and speckle tracking echocardiography reflect various aspects of LV segmental wall motion and microvascular perfusion. Combining assessment parameters at baseline and dobutaming stress, they could evaluate the myocardial motion or blood perfusion during both rest and stress condition and further to evaluate the reserves, finding indications concealed at sole rest, thus increases the techniques' diagnostic sensitivity.
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