【作者】 付倩；

【导师】 谢明星；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2010， 博士

【摘要】 大鼠是心血管研究中的广泛使用实验动物模型之一,其心脏疾病的发展过程与人类相似。随着心肌保护治疗研究的深入,越来越多的基础研究采用大鼠制备心肌梗死模型,靶向给药后观察心室整体和局部功能以及代谢变化,使得超微结构特征改变与局部心肌功能的相关研究成为可能。超声心动图以无创性、价格经济、使用简便等优势,成为评价小型实验动物心脏形态和结构的首选方法。左室运动形式复杂,可分解为纵向、径向和环向三个方向上的运动,每种运动方式均显著的影响心脏功能。从不同层次和方向全面评价心肌功能,可帮助理解缺血心肌生物力学及生理学机制。近年推出的超声二维斑点追踪成像技术(two-dimensional speckle tracking imaging, STI)是一种可全面、准确定量评价左室整体和局部心肌功能的新方法,具有无声束角度和帧频依赖性的优势,可实时跟踪心肌内声学斑点的空间运动轨迹,通过逐帧运算重建心肌组织实时运动,以获取运动心肌在纵向、径向与环向上的形变特征。急性心肌缺血时局部心肌灌注迅速减低,导致心肌细胞电生理特性发生改变,诱导局部心肌应力、应变重新分布,最终引起心肌局部和整体功能障碍。心梗后及时准确地检测出受损节段,了解心肌梗死范围及损伤程度对于指导治疗和判定预后至关重要。急性心梗后肾素-血管紧张素系统和交感-肾上腺素能系统活性增高,诱发心肌细胞凋亡、细胞外基质断裂,刺激心肌纤维化,促进左室重构的发生。由于坏死心肌失去收缩功能,左室收缩运动严重不协调,导致心室腔内血液不能有效排空,心室内压力负荷增加,心动周期中心室壁受到的室壁应力增加,使得室壁反应性增厚、肌细胞拉长,最终引起左室明显扩张伴心室构型变化,表现为进行性的心脏增大和功能障碍。左室前壁心梗多为透壁心梗,梗死面积大,常累及心尖部,左室形态明显失常。研究表明,梗死面积在30%以上的前壁心梗可出现明显的左室重构和心功能不全。既往研究多通过评价心室整体形态和功能、室壁厚度、心室质量等判断心室重构,较少关注重构进程中局部心肌功能的变化。研究表明首次前壁心梗患者行再灌注治疗后早期即可出现梗死节段远端心肌功能障碍。冠心病患者心肌梗死后有严重功能障碍的心肌中50%以上都属于血流灌注正常的心肌,提出将这种局部收缩功能减弱但具有正常血流灌注的心肌称为“重构心肌”。心梗后梗死周边区域心肌收缩功能持续性降低,并延伸累及梗死远处区域,随着重构心肌的范围增大导致心室扩大及心力衰竭,位于梗死周边区域心肌功能障碍可能是左室重构进程的重要影响因素。因此心肌梗死后准确定量分析心肌梗死区、梗死周边区和梗死远端区的局部心肌功能,以及观察其动态变化过程对于评估心室重构进程具有重要意义。本研究采用STI技术测量大鼠急性心梗前后左室心肌节段收缩期峰值径向应变(RS)、径向应变率(RSr)、环向应变(CS)、环向应变率(CSr)以及左室整体环向应变(GCS)和环向应变率(GCSr)等参数,通过与组织学结果对照,旨在1.评价正常大鼠左室节段心肌收缩运动的特征,以及基于STI技术参数的可重复性；2.探讨STI技术评价大鼠急性心肌梗死后局部心肌功能改变的应用价值,筛选甄别可逆性与不可逆性功能障碍心肌节段的敏感参数；3.评价急性心梗后不同时点左室梗死区、梗死周边区和梗死远端区心肌收缩运动的变化特征。本研究分为以下三个部分：20只健康成年雄性Wistar大鼠行超声心动图检查。记录左室短轴乳头肌水平高帧频二维灰阶动态图像,EchoPAC工作站测量节段心肌的收缩期峰值径向应变(RS)、径向应变率(RSr)、环向应变(CS)、环向应变率(CSr)及左室整体环向应变(GCS)和左室整体环向应变率(GCSr)。解剖M型超声心动图测量左室舒张末期内径(LVIDd)、左室收缩末期内径(LVIDs)、短轴缩短率(FS)、射血分数(EF)。RS、RSr、CS、CSr参数分别行组内比较。GCS和GCSr值分别与EF值行相关性分析。重复测量基于STI技术的各项参数,行观察者间和观察者内一致性检验。结果显示：①左室短轴乳头肌水平各节段RS、RSr值分布均一,差异无统计学意义(P>0.05)；②左室短轴乳头肌水平各节段CS、CSr值分布存在不均一性,前间壁和前壁的CS测值最大,下壁的CS测值最小,差异有统计学意义(P<0.05)；③STI技术和解剖M型超声心动图测量左室整体收缩功能显著相关性,GCS和GCSr值与EF值的相关系数分别为0.84、0.92(P<O.05)；④基于STI技术的各项参数于观察者间和观察者内均具有高度一致性。25只Wistar大鼠随机两组：急性心肌梗死组15只,结扎冠状动脉左前降支45-60min以制备急性心肌梗死模型；假手术组10只,在冠状动脉左前降支钳夹部位绕线但不结扎。两组分别于基础状态和恢复灌注24h后行超声心动图检查。记录左室短轴乳头肌水平高帧频二维动态图像,EchoPAC工作站测量节段心肌的收缩期峰值径向应变(RS)、径向应变率(RSr)、环向应变(CS)、环向应变率(CSr)及左室整体收缩期峰值环向应变(GCS)和环向应变率(GCSr)。解剖M型超声心动图测量左室舒张末期内径(LVIDd)、左室收缩末期内径(LVIDs)、短轴缩短率(FS)、射血分数(EF)和室壁增厚率(TR)。实验结束后处死大鼠,取出心脏行左室短轴乳头肌水平TTC染色,并计算节段心肌的坏死面积百分比(NAR)以及左室短轴乳头肌水平整体坏死面积百分比(GNAR)。以TTC染色作为金标准,ROC曲线分析RS、RSr、CS、CSr及TR参数识别节段不可逆性功能障碍的准确性。结果显示：①与心肌梗死组基础状态及假手术组术后相比,心肌梗死组术后的LVIDd和LVIDs值均明显增加,FS、EF和前间壁TR值均明显降低(P<0.05)；②与心肌梗死组基础状态及假手术组术后相比,心肌梗死组术后的前间壁、前壁、前侧壁、后侧壁和后间壁的RS、RSr、CS、CSr值均明显减低,心肌梗死组术后前间壁、前壁和前侧壁的RS、RSr、CS、CSr值明显低于其他节段(P<0.05)；③STI技术所测前间壁、前壁、前侧壁的RS、RSr、CS、CSr参数均分别与对应NAR显著相关(P<0.001),GCS、GCSr分别与GNAR显著相关(P<0.001)；解剖M型超声心动图所测EF和前间壁TR值均与对应的GNAR、NAR相关性良好。④以TTC染色为金标准,ROC曲线分析显示RS、RSr、CS、CSr等STI参数的曲线下面积均高于解剖M型超声心动图所测TR参数,其中以CS的曲线下面积最大,故CS为判断心肌不可逆性功能障碍的最佳指标,CS值为临界值(-6.14%)时诊断心肌不可逆性功能障碍的灵敏度为93.75%,特异度为90.91%。25只Wistar大鼠随机分为两组：心肌梗死组15只,结扎冠状动脉左前降支45-60min以制备急性冠状动脉心肌梗死模型；假手术组10只,在冠状动脉左前降支钳夹部位绕线但不结扎。两组分别于术前及术后1周、4周、8周行超声心动图检查。记录左室短轴乳头肌水平高帧频二维动态灰阶图像,EchoPAC工作站测量节段心肌的收缩期峰值径向应变(RS)、径向应变率(RSr)、环向应变(CS)、环向应变率(CSr)及左室整体环向应变(GCS)和左室整体环向应变率(GCSr)。解剖M型超声心动图测量左室舒张末期内径(LVIDd)、左室收缩末期内径(LVIDs)、短轴缩短率(FS)和射血分数(EF)。检查结束后处死大鼠,取出心脏行左室短轴乳头肌水平TTC染色,并计算节段心肌的坏死面积百分比(NAR); VG染色观察心肌组织中的胶原纤维。各项超声心动图参数行组间和组内比较。结果显示：①基于TTC染色结果将左室短轴乳头肌水平分为3个区域：梗死心肌区(前间壁、前壁、前侧壁),梗死周边心肌区(后间壁、后侧壁),梗死远端心肌区(下壁)。②与心肌梗死组基础状态及同时期假手术组相比,心肌梗死组术后1周时梗死区、梗死周边区、梗死远端区节段心肌的RS、RSr、CS、CSr值均显著降低(P<0.05)。与心肌梗死组术后1周相比,术后4周时梗死区部分节段(前壁及前侧壁)以及梗死周边区部分节段(后侧壁)的RS、RSr、CS、CSr值显著降低(P<0.05),其它节段的RS、RSr、CS、CSr值无显著差异(P>0.05),但仍显著低于基础状态测值(P<0.05)。与心肌梗死组术后4周时相比,术后8周时各节段RS、RSr、CS、CSr值无显著差异(P>0.05),但仍显著低于基础状态测值(P<0.05)。③与心肌梗死组基础状态及同时期假手术组相比,心肌梗死组术后1周、4周和8周时心肌梗死组GCS和GCSr值显著降低(P<0.05),术后4周时GCS和GCSr值显著低于术后1周时(P<0.05),术后4周和8周的GCS和GCSr值差异无统计学意义(P>0.05)；④与心肌梗死组基础状态及同时期假手术组相比,心肌梗死组术后1周的LVIDd、LVIDs值显著增加(P<0.05),FS、EF测值显著降低(P<0.05)。与心肌梗死组术后1周相比,术后4周时LVIDd、LVIDs测值显著增加(P<0.05),但FS、EF测值无统计学差异(P>0.05),但仍显著低于基础状态测值(P<0.05)。与心肌梗死组术后4周相比,术后8周时LVIDd、LVIDs、FS、EF测值无显著差异(P>0.05)。⑤VG染色后,镜下观胶原纤维呈红色,肌肉、神经胶质、胞浆和红血球呈黄色,细胞核呈黑蓝色或棕色；梗死心肌、梗死周边和远端心肌组织内均出现了不同程度的胶原纤维沉着,以梗死心肌最多,其次为梗死周边心肌,梗死远端心肌组织内可见局灶状胶原纤维沉着。结论1.正常大鼠左室短轴乳头肌水平各节段心肌环向应变和应变率存在异质性,径向应变和应变率分布一致。2.大鼠急性心梗模型中左室局部心肌呈不同程度损伤,STI参数CS是识别心肌节段不可逆性功能障碍的最佳指标。3.大鼠急性心梗模型血流灌注正常的梗死周边区和远端区于术后1周出现心肌收缩功能障碍,并持续至术后8周。4.STI是一种评价大鼠心梗模型左室整体和局部收缩功能改变的敏感方法。更多还原

【Abstract】 BackgroundRats are widely used in cardiovascular research as experimental model and the development of cardiac diseases in rats is similar to that in humans. With the development of research in myocardial protective treatment, more and more basic research prepared myocardial infarction model in the rats. After the targeted drug delivery was given, the ventricular global and regional function and metabolism change were observed, making the evaluation of the correlation of ultra-structural characteristic and regional myocardial function to be possible. Echocardiography is the preferred method in assessing the cardiac structure and function in small experimental animal, for its advantages such as non-invasive, economic and convenient for use.The left ventricle moved in a complex and three dimensional patterns. The motion can be divided into longitudinal, radial and circumferential components. An adequate presentation of these components could enhance the understanding of the biomechanics and pathophysiology of ischemia myocardium. Two-dimensional speckle tracking imaging (STI), without angle and frame rate dependent, is a new method to quantify left ventricular global and regional function accurately and comprehensively. STI can track the location of the echo spots in the two-dimensional grey scale image and reestablish the real time movement of the myocardium through frame by frame algorithm to obtain the myocardial deformational information in radial, circumferential and longitudinal direction.Myocardial perfusion reduced immediately after myocardial ischemia occurred, which would lead to change electrophysiological properties of cardiac myocytes and induce redistribution of regional myocardial stress and eventually caused global and regional myocardial dysfunction. Timely and accurately detection of damaged segment after myocardial infarction is essential for guiding clinical treatment and prognosis as well as defining range of infarction and extension of injury.The activity of rennin-angiotensin system and sympathic-adrenergic system increased after myocardial infarction. This would induce a series of responses, including apoptosis of cardiac myocytes, rupture of extracellular matrix and fibrosis of myocardium and finally promoting ventricular reverse remodeling. As necrotic myocardium loss contractile function, left ventricular motion was lack of coordination and ventricular cavity blood could not be drained effectively. Wall stress increased caused by ventricular pressure overload could result hypertrophy of regional myocardium and stretch of myocytes. The left ventricle significantly expanded and ultimately changed the global geometry, clinically manifested as progressive enlargement and dysfunction of the heart. Left ventricular anterior wall infarction, often involving the apex, almost was transmural injury with large necrotic area and abnormal left ventricular shape. Studies demonstrated that anterior wall myocardial infarction with necrotic area over 30% often leaded to significantly ventricular remodeling and heart failure.Previous studies identified ventricular remodeling through the wall thickness and global morphology, function and mass of the left ventricle, while few research focused on the regional myocardial function change during the process of ventricular remodeling. Bogaert et al. demonstrated that patients with first anterior wall myocardial infarction occurred remote myocardial dysfunction early after myocardial reperfusion treatment. Narula et al. demonstrated that over 50% of the severely dysfunctional myocardium in patients with coronary disease had normal blood flow, and proposed this hypocontractile but normally perfused myocardium to be called "remodeling myocardium". Jackson et al. demonstrated that systolic function of segment in peri-infarcted region persisted decreased and remote region was involved. The extentsion of remodeling myocardium could lead to ventricular enlargement and heart failure. The dysfunction of peri-infarcted myocardial region may be the important factor in the left ventricular remodeling processing. Consequently, quantifing function of segments in the myocardial infarcted region, peri-infarcted region and remote region accurately and observing the dynamic course is important for evaluating the ventricular remodeling processing.This study measured the left ventricular segmental peak systolic radial strain(RS), radial strain rate(RSr), circumferential strain(CS), circumferential strain rate(CSr) and left ventricular global circumferential strain(GCS) and global circumferential strain rate(GCSr) of rats model at baseline and after myocardial infarction by STI and aimed to 1.assess the characteristics of left ventricular segmental systolic movement in normal rats and determine the reproducibility of parameters based on STI; 2.explore the value of STI in evaluating left ventricular regional myocardial function in rats after acute myocardial infarction, and select the sensitive parameter to distinguish segment with irreversible dysfunction from segment with reversible dysfunction; 3.assess the change of systolic function of segments in left ventricular infarcted region, peri-infarcted region and remote region. This study included three parts as following:Echocardiography was performed in 20 normal adult male Wistar rats. High frame rate of two-dimensional images were recorded from the left ventricular short-axis views at the papillary muscle level. Peak systolic radial strain(RS), radial strain rate(RSr), circumferential strain(CS) and circumferential strain (CSr) of each segment and left ventricular peak systolic global circumferential strain (GCS) and global circumferential strain rate(GCSr) were measured at EchoPAC work station. Left ventricular internal diameter at diastole (LVIDd), systole (LVIDs), fractional shortening (FS) and ejection fraction (EF) were measured with anatomical M-model echocardiography. The correlations between EF and GCS, EF and GCSr were analyzed respectively. For analysis of intraobserver and interobserver variability, all measurements based on STI were repeated by two observers. Results①Regional RS, as well as RSr, was similar in all segments of mid-ventricular in short-axis view, the difference had no statistics significance (P>0.05).②CS of each segment, as well as CSr, showed heterogeneity, while the anteroseptal and anterior wall showing the largest value and the inferior wall showing the lowest value, the difference had statistics significance (P<0.05).③The left ventricular global systolic function measured by STI and anatomical M-mode echocardiography correlated significantly, while the correlation coefficient between EF and GCS was 0.84 and the one between EF and GCSr was 0.92 (P<0.05).④The parameters based on STI showed good intraobserver and interobserver reproducibility.Twenty-five Wistar rats were randomly divided into acute myocardial infarction group (n=15) and sham-operation group (n=10). The left anterior descending coronary of the acute myocardial infarction group was occlusion by suture for 45-60 minutes, while the sham-operation group underwent an identical operation but the suture was not tired. Echocardiography was performed at baseline and 24 h after reperfusion. High frame rate two-dimensional images were recorded from the left ventricular short-axis views at the papillary muscle level. Peak systolic radial strain (RS), radial strain rate (RSr), circumferential strain (CS) and circumferential strain (CSr) of each segment and left ventricular peak systolic global circumferential strain (GCS) and global circumferential strain rate(GCSr) were measured at EchoPAC work station. Left ventricular internal diameter at diastole (LVIDd) and systole (LVIDs), fractional shortening (FS), ejection fraction (EF), wall thickening rate (TR) were measured with anatomical M-model echocardiography. After echocardiography was performed, the rats were sacrificed. The heart was excised and the left ventricular short-axis slice at the papillary muscle level was stained by triphenyl tetrazolium chloride (TTC). Necrosis area ratio (NAR) of each segment in the slice and global necrosis area ratio (GNAR) of the slice were measured after staining. Based on TTC staining, ROC curve was used to analyze the accuracy of RS, RSr, CS, CSr and TR in identifying segment with irreversible dysfunction. Results①Compared with acute myocardial infarction group at baseline and sham-operation group after operation, LVIDd and LVIDs of acute myocardial infarction group after operation increased significantly respectively (P<0.05), while FS, EF and TR of anteroseptal wall reduced significantly (P<0.05).②Compared with acute myocardial infarction group at baseline and sham-operation group after operation, RS, RSr, CS, CSr decreased significantly in anteroseptal, anterior, lateral, posterior and septal wall of the acute myocardial infarction group after operation (P<0.05), In the acute myocardial infarction group after operation, RS, RSr, CS, CSr of anterosepetal, anterior and lateral wall were significantly lower than the other segments (P<0.05).③RS, RSr, CS, CSr of anterosepetal, anterior and lateral wall measured by STI were significantly correlated with corresponding NAR respectively (P<0.001), while GCS and GCSr were significantly correlated with GNAR respectively (P<0.001). EF and AWTR measured by anatomic M-mode echocardiography were significantly correlated with GNAR and corresponding segmental NAR respectively (P<0.001).④Based on TTC staining, ROC analysis showed that the parameters based on STI, such as RS, RSr, CS, CSr, had larger area under curve(AUC) than that in TR which was measured by anatomic M-mode echocardiography. CS had the best ability to identify segment with irreversible dysfunction as defined by NAR>50%, as CS had the largest AUC. Using a cut-off of-6.14%, CS had a sensitivity of 93.75% and specificity of 90.91% for distinguishing segment with irreversible dysfunction from segment with reversible dysfunction.Twenty-five Wistar rats were randomly divided into acute myocardial infarction group (n=15) and sham-operation group (n=10). The left anterior descending coronary of the acute myocardial infarction group was occlusion by suture for 45-60 minutes, while the sham-operation group underwent an identical operation but the suture was not tired. Echocardiography was performed at baseline and 1 week,4 weeks and 8 weeks after operation. High frame rate two-dimensional images were recorded in the left ventricular short-axis views at the papillary muscle level. Peak systolic radial strain(RS), radial strain rate(RSr), circumferential strain(CS) and circumferential strain (CSr) of each segment and left ventricular peak systolic global circumferential strain (GCS) and global circumferential strain rate(GCSr) were measured at EchoPAC work station. Left ventricular internal diameter at diastole (LVIDd) and systole (LVIDs), fractional shortening (FS) and ejection fraction (EF) were measured by anatomical M-model echocardiography. After echocadrdiography was performed, the rats were sacrificed. The heart was excised and the left ventricular short-axis slice at the papillary muscle level was stained by triphenyl tetrazolium chloride (TTC). Necrotic area ratio (NAR) of each segment in the slice was measured after staining. Fibrosis of left ventricle myocardium was displayed using Van Gieson stain. The echocardiographic parameters were compared in the interior-group and inter-group. Results①Based on the TTC findings, the left ventricle of the acute myocardial infarction group was divided into three regions:infarcted myocardial region (anteroseptal, anterior, lateral wall), peri-infarcted myocardial region (posterior and septal wall) and remote myocardial region (inferior wall).②Compared with baseline and sham-operated group, segmental RS, RSr, CS, CSr in the infarcted, peri-infarcted and remote region of the acute myocardial infarction group significantly decreased at 1 week after operation (P<0.05). Compared with the acute myocardial infarction group at 1 week after operation, RS, RSr, CS, CSr of some segments in infarcted region (anterior and lateral wall) and peri-infarcted region (posterior wall) significantly decreased at 4 weeks after operation (P<0.05), while RS, RSr, CS, CSr of the other segments at 4 weeks after operation showed no significant reduction (P>0.05) but were still lower than baseline significantly (P<0.05). Compared with the acute myocardial infarction group at 4 week after operation, RS, RSr, CS, CSr of each segment in infarcted, peri-infarcted and remote region at 8 weeks after operation showed no significant reduction (P>0.05).③Compared with baseline and sham-operated group, GCS and GCSr of the myocardial infarction group decreased at 1,4,8 weeks after operation significantly (P<0.05). Compared with the acute myocardial infarction group at 1 week after operation, GCS and GCSr decreased significantly at 4 week after operation (P<0.05). GCS and GCSr of the acute myocardial infarction group at 8 week after operation showed no significant reduction compared with those at 8 week after operation (P>0.05).④Compared with baseline and sham-operated group, LVIDd and LVIDs of acute myocardial infarction group increased significantly at 1 week after operation, and FS and EF decreased significantly (P<0.05). Compared with the acute myocardial infarction group at 1 week after operation, LVIDd and LVIDs increased significantly at 4 week after operation (P<0.05), while FS and EF showed no significant reduction but were still lower than baseline significantly (P<0.05). Compared with the acute myocardial infarction group at 4 week after operation, LVIDd, LVIDs, FS and EF at 8 week after operation showed no significant change but were still lower than baseline significantly (P<0.05).⑤Microscopically, after VG staining the collagenous fibre stained red, whereas the muscle, neurospongium, cytoplasm and erythrocyte stained yellow and nucleolus stained black blue or brown. Analysis of the histologic sections from the infarcted, peri-infarcted and remote myocardial region demonstrated fibrosis in various degrees. The infarcted region showed large platy fibrosis. The peri-infarcted and remote region showed patchy and focal fibrosis respectively.Conclusions1. The circumferential strain and strain rate of segment in left ventricular short-axis views at the papillary muscle level showed significant heterogeneity, while the radial strain and strain rate were similar in all the segments.2. Varying damages were presented in left ventricular regional myocardium in rat model of acute myocardial infarction. CS based on two-dimensional speckle tracking imaging is the best parameter for distinguishing segment with irreversible dysfunction from segment with reversible dysfunction. 3. The myocardial peri-infarcted and remote region with normal perfusion in rat model of acute myocardial infarction revealed dysfunction at lweek after operation, and persisted to 8 weeks.4. Two-dimensional speckle tracking imaging is a sensitive method in evaluating left ventricular global and regional myocardial function in rat model of acute myocardial infarction.更多还原