【作者】 卫张蕊；

【导师】 张军； 苏海砾； 朱永胜； 刘丽文；

【作者基本信息】 第四军医大学 ， 影像医学与核医学， 2008， 博士

【摘要】 第一部分:速度向量成像对糖尿病大鼠左室局部功能的评价目的:研究速度向量成像(Velocity Vector Imaging, VVI)技术结合潘生丁负荷实验是否能够检测出糖尿病(Diabetes milletus, DM)大鼠左心室潜在的弥漫性室壁运动异常,为临床糖尿病心肌病(Diabetic Cardiomyopathy, DCM)心肌功能障碍的早期诊断和疗效监测提供一种可定量的、简便易行的检查方法。研究方法:23只雄性SD大鼠,禁食12小时后,腹腔内注射1%的链脲菌素(streptozotocin, STZ)溶液65mg/kg。于注射后第3天、第7天、第28天、第56天及第84天禁食4小时后,取尾静脉血测定血糖浓度,血糖浓度>16.7mmol/l,且具有明显的多饮、多食、多尿以及体质量减轻症状者选定为糖尿病模型(DM组, n=18,其中5只大鼠因STZ抵抗而剔除)。另选12只体质量匹配的雄性SD大鼠做为正常对照组,腹腔内注射等量的柠檬酸缓冲液。常规饲养12周后,充分麻醉状态下行气管、颈静脉插管,分别连接呼吸机和三通管,然后行左侧胸腔切开术。采用Sequoia 512C彩色多普勒超声诊断系统,14MHz线阵探头,经心包进行超声心动图检查,分别存储静息状态及潘生丁负荷(3.5mg/kg)后乳头肌水平左室短轴M型及动态二维图像。从M型图像中测量室壁的厚度并计算室壁增厚率。动态二维图像脱机后用syngo VVI软件进行分析。根据美国超声心动图学会制定的标准16节段法,将乳头肌水平左室短轴分为6个节段,在一帧静态图像上分别描记心内膜和心外膜下心肌的节段点,在动态过程中,软件自动追踪并描记心内膜和心外膜。从分析软件自动给出的速度、应变、应变率曲线图中测量各个节段心肌最大收缩期运动速度(Vs)、最大舒张期运动速度(Vd)、最大径向应变(εr)、最大切向应变(εc)、最大收缩(舒张)期切向应变率(SRc)以及径向应变率(SRr)。超声心动图检查完毕后处死动物,摘除心脏,一部分左室心肌组织做透射电镜切片,观察心肌细胞超微结构的改变;余心肌组织固定后行HE染色及CD31免疫组化染色。结果:无论在静息状态还是潘生丁负荷后,DM组大鼠左室壁各个节段之间的心肌运动速度、应变、应变率均无显著性差异(P > 0.05),这一特点与正常对照组大鼠的研究结果相一致。因此我们引入了各个指标6个节段的平均值进行进一步的分析。静息状态下,DM组收缩期和舒张期SRc较正常对照组显著减低(P < 0.05),其余指标与正常对照组没有统计学差异。潘生丁负荷后,尽管DM组和正常对照组室壁运动速度、应变、应变率均较静息状态下显著增高(P < 0.05);但DM组各指标却显著低于正常对照组(P < 0.05)。无论在静息状态还是在潘生丁负荷后,DM组和正常对照组之间室壁增厚率的差异没有统计学意义(P > 0.05)。HE染色结果显示DM组及正常对照组心外膜下的冠状动脉均没有明显的粥样硬化斑块形成。免疫组化结果表明DM组毛细血管密度较正常对照组显著减低(P < 0.05)。透射电镜结果显示DM组心肌细胞超微结构发生了一系列的改变:如毛细血管基底膜局灶性增厚,局部毛细血管管腔变窄呈“裂隙”状以及毛细血管内微血栓的形成;心肌细胞间连接受到破坏(局部闰盘的断裂),肌丝排列紊乱,部分断裂、融解,线粒体肿胀,嵴变短或消失呈“空泡”状,肌浆网肿胀等。结论:基于VVI的室壁运动速度、应变以及应变率的各项指标与负荷超声心动图相结合,可以比较全面的评价DM大鼠左室短轴的室壁运动,较早地检测出DM大鼠由于微循环功能障碍及心肌细胞超微结构改变造成的弥漫性室壁运动异常。收缩(舒张)期的最大切向应变率可能是更为敏感的指标,其在静息状态下便可以检测出这种室壁运动的异常。第二部分:心肌造影超声心动图对糖尿病大鼠心肌微循环的评价目的:研究心肌造影超声心动图(Myocardial Contrast Echocardiography, MCE)技术结合潘生丁负荷试验是否能够早期检测出DM大鼠左心室心肌微循环障碍,为临床DCM心肌微循环障碍的早期诊断和疗效监测提供一种可定量的、简便易行的检查方法。方法:35只雄性SD大鼠,分为糖尿病组(n=18,其中5只大鼠因STZ抵抗而剔除)和正常对照组(n=12),模型的制备同第一部分。常规饲养12周后,充分麻醉状态下行气管、颈静脉插管及左侧胸腔切开术。采用Sequoia 512C彩色多普勒超声诊断系统经心包行MCE检查。造影剂选用声诺维TM (SonoVueTM),输注方式为经颈静脉连续注射,输注速度为168ml.kg -1 .h -1。分别在静息状态和潘生丁负荷后行乳头肌水平左室短轴心肌造影并存储实时动态图像,脱机后用syngo ACQ软件进行分析。在心肌灌注第一帧短轴切面的前壁、侧壁、后壁、室间隔心肌组织及心腔内分别勾画出感兴趣区域,软件动态追踪并测量感兴趣区域内的平均声学强度,拟合成声学强度-时间曲线,并给出各个感兴趣区域峰值声学强度(Plateau Intensity, PI, A),造影剂灌注速率(β),声学强度达峰时间(Time to Plateau Intensity, TTP)及曲线的拟合度(Goodness of Fit, GOF)等指标。根据公式计算出心肌血流量(Myocardial Blood Flow, MBF)和心肌血流储备(Myocardial Flow Reserve, MFR):MBF=A×β;MFR =负荷后MBF/静息状态MBF。MCE检查完毕后,每组6只大鼠经颈静脉注射99m锝-甲氧基异丁基异腈( 99m Tc-MIBI) 0.6 mCi,代谢3小时后处死大鼠,迅速摘除心脏,取乳头肌水平的左室心肌组织,依照其空间位置将其分成前壁、侧壁、后壁及室间隔四个组织块,称重后进行γ计数。其余的心肌组织固定后行HE染色、CD31免疫组化染色以及电镜下观察心肌细胞超微结构的改变,同第一部分。结果:对两组大鼠左室各壁感兴趣区域的造影结果进行研究发现,同组大鼠前壁、侧壁和室间隔的心肌血流量之间没有显著差异(P > 0.05);后壁的心肌血流量较其他壁减低,且具有统计学意义(P < 0.05)。我们取前壁心肌为代表进行进一步研究,在静息状态和潘生丁负荷后,DM组的A、MBF均较正常对照组显著减低,(P < 0.05);MFR也较正常对照组显著减低(P < 0.05)。DM组的β和TTP在静息状态下与正常对照组没有显著差异(P > 0.05),但在潘生丁负荷后,β较正常对照组显著减低,TTP显著延长,且均具有统计学意义(P < 0.05)。99m Tcγ计数结果证明DM组各个室壁心肌组织的核素摄取量之间没有明显差异(P > 0.05),但均较正常对照组显著减低(P < 0.05)。病理及免疫组化结果同第一部分。结论:MCE检测的峰值声学强度,声学强度达峰时间,心肌血流量及心肌血流储备等指标可以敏感地检测出DCM早期的心肌微循环功能障碍。第三部分:糖尿病大鼠心肌功能变化与微循环状态改变的相关性研究目的:研究VVI检测的心肌运动速度、应变、应变率及其储备与MCE检测的MBF和MFR之间是否具有相关关系。方法:动物模型的建立、潘生丁负荷前后MCE和VVI数据采集和处理同第一、第二部分相关内容。将基础状态VVI检测的心肌运动速度、应变、应变率与MCE检测的MBF之间以及潘生丁负荷后心肌功能(运动速度、应变、应变率)储备与MFR之间进行相关分析。心肌功能(运动速度、应变、应变率)储备定义为潘生丁负荷前后各项指标之间的差值。结果:静息状态下DM组大鼠的心肌运动速度、应变、应变率与MBF之间均没有显著的相关性;潘生丁负荷后,DM组大鼠的心肌运动速度储备、应变储备、应变率储备均与MFR之间呈现显著的正相关性。结论:静息状态下,心肌血流量的变化并非心肌运动速度、应变、应变率等功能指标降低的主要决定因素;而潘生丁负荷后,心肌血流储备的降低可能是心肌功能储备降低的主要决定因素。更多还原

【Abstract】 Part one: Assessment of Left Ventricular Wall Motion in Diabetic Rats Using Velocity Vector Imaging Combined with Stress EchocardiographyObjective: The aim of this study was to investigate whether velocity vector imaging （VVI） combined with stress echocardiography could detect potential diffused myocardial impairment of the left ventricle （LV） in diabetic rats. Methods: Twenty-three male Sprague–Dawley rats weighing 230 to 270g were administered STZ at 65 mg/kg （1% STZ solution, diluted with 0.1M citrate buffer, pH 4.4 before injection） through an intraperitoneal injection after a 12-hour fast. Using an autoanalyzer （Surestep, Lifescan）, blood glucose was measured in the tail blood after four hours’fasting on days 3, 7, 28, 56 and 84 after injection. Rats with fasting blood glucose > 16.7mM and positive for characteristics of diabetes, such as weight loss and polydipsia were selected for the DM group （n = 18, five rats were excluded for STZ tolerance）. Another 12 weight-matched male rats were selected for the control group and given the same dosage of sodium citrate buffer. All rats were given a standardized portion of rat food and ad libitum access to tap water for 12 weeks. Twelve weeks after STZ injection, the rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital （1ml/kg）. After adequate anesthesia, all animals were intubated in a suAne position and ventilated with a rodent ventilator （Natime, Japan）. A thoracotomy was performed to obtain unrestricted visualization of all myocardial regions. Echocardiograms were performed over the pericardial sac with a linear-array transducer （14 MHz, Acuson Sequoia 512C system, Siemens, U.S.A）. Two-dimensional echocardiographic cine loops and M-mode images of three consecutive beats were obtained at rest and after dipyridamole stress （3.5 mg/kg） from the short-axis views at the mid-LV level. All data were stored on MO and analyzed off-line （Sygno VVI, Siemens）. In the present study, the LV wall at mid-level from the short-axis view was divided into six segments according to the standard 16-segment model of the American Society of Echocardiography. The segments of the LV wall were plotted, endocardial and epicardial borders were manually identified in a single frame of a cine-loop, and the borders in other frames were automatically generated, allowing operators to alter any of those contours. Next, segmental peak systolic velocity （Vs）, diastolic velocity （Vd）, radial strain （εr）, circumferential strain （εc）, systolic and diastolic radial strain rate （SRr） and circumferential strain rate （SRc） were obtained from velocity, strain and strain rate curves provided by Sygno VVI. LV wall thickness was measured online using M-mode image, and the percent wall thickening （WT %） was calculated. After echocardiograms were performed, the hearts were excised, washed quickly in PBS and cut into six short-axis slices from the apex to the base. Each slice was embedded in paraffin and cut into serial 4-μm sections for hematoxylin and eosin （HE） staining （eight to ten sections of each slice） to observe the coronary arteries and cardiocytes under light microscopy. An additional section or two were selected for CD31 immunohistochemistry staining to determine the capillary density. Myocardial Aeces from five rats in each group were selected for ultrastructural observations under electron microscopy. Results: No significant differences were found between the six walls in the Vs, Vd,εr,εc, systolic and diastolic SRr and SRc in each group （all P > 0.05）. Now that there were homogeneities of these parameters between six walls, the mean value of each of these parameters from the six walls was calculated as the index for comparison between the two groups. At rest, systolic and diastolic SRc in the DM group were significantly lower than those in the control group （both P < 0.05）. However, the other parameters were statistically comparable between the two groups. After dipyridamole stress, all VVI parameters in the DM group were significantly lower than those in the control group （all P < 0.05）, although these parameters increased significantly in both groups compared to those at rest （all P < 0.05）. However, there were no significant differences in WT% between the two groups either at rest or after dipyridamole stress （both P > 0.05）. No evident atherosclerotic plaques of coronary arteries under the epicardium were found, and cardiocytes appeared to arrange orderly in all sections in both groups. The capillary density decreased significantly in the DM group compared with the control group. Ultrastructural impairments of the capillaries and cardiocytes were observed in the DM group, such as destroyed basal laminars, slit-shaped cavities and microthrombosis of the capillaries, opened intercalated disks, swollen mitochondria and destroyed sarcomere structures of the cardiocytes. Conclusion: The VVI-derived Vs, Vd,εr,εc, systolic and diastolic SRr and SRc, combined with dipyridamole stress are all effective parameters in evaluating potential diffused myocardial impairment of the LV walls due to ultrastructural cardiocyte impairment and microcirculation disturbances in diabetic rats. Systolic and diastolic SRc might be more sensitive indices that can be used to detect myocardial impairment at rest.Part two: Assessment of Myocardial Microcirculation in Diabetic Rats Using Myocardial Contrast EchocardiographyObjective: The aim of this study was to investigate whether myocardial contrast echocardiography （MCE） combined with stress echocardiography could detect myocardial microcirculation disturbance of LV in diabetic rats. Methods: Twenty-three male Sprague–Dawley rats weighing 230 to 270g were selected for DM group as obviously mentioned in part one （n = 18, five rats were excluded for STZ tolerance）. Another 12 weight-matched male rats were selected for the control group and given the same dosage of sodium citrate buffer. All rats were given a standardized portion of rat food and ad libitum access to tap water for 12 weeks. Twelve weeks after STZ injection, the rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital （1ml/kg）. After adequate anesthesia, all animals were intubated in a supine position and ventilated with a rodent ventilator （Natime, Japan） and three-way joint were connected to the right jugular veins for administration of contrast agent and dipyridamole, etc. A thoracotomy was performed to obtain unrestricted visualization of all myocardial regions. MCE were performed over the pericardial sac with a 8MHz （14MHz linear-array transducer, Acuson Sequoia 512C system, Siemens, U.S.A） at a mechanical index of 0.25 with contrast pulse sequencing. SonoVueTM （Bracco, Italy） were selected in our study and infused intraveneously at 2.8ml.kg min with micro pump. Perfusion images were acquired in real time （frame rate of 25 Hz） after a sequence of a serial of high-energy frames （mechanical index of 1.9） -1 -1 from parasternal short-axis views at the papillary muscle level in all rats. After baseline images were acquired, dipyridamole (3.5 mg/kg, 0.2mg/ml, 2.8 ml.kg^-1 h^-1 ) was infused intravenously. After 4 minutes of continuous infusion, MCE images were acquired again. All data were stored on MO and analyzed off-line （Sygno ACQ, Siemens）. Regions of interest were positioned with the anterior, lateral, posteral, septal walls and within the LV cavity. Average signal intensity with the region of interest was measured automatically on each frame. A curve of signal intensity over time was obtained in each region of interest and fitted to an exponential function: y = A (1-e^-βt), where y is signal intensity at any given time,βis the initial slope of the curve, and A is the plateau intensity （A）. A,β, time to PI （TTP） were obtained from the curve and myocardial blood flow （MBF） and myocardial flow reserve （MFR）were estimated as the following formula: MBF = A *β, MFR = MBFstress / MBFbaseline. All these parameters were compared between the two groups after the PIs in the regions of interest in four walls were standardized to the PI in the LV cavity. After the performance of MCE, 6 rats in each group were administrated with 99m Tc-MIBI 0.6 mCi. The hearts were excised 3 hours later, the myocardium at the papillary level were selected and cut into 4 parts and weighed.γwell counting were performed at 4, 8, 12 and 24 hours after administration. The remained myocardium were prepared for HE staining, CD31 immunohistochemisry staining and ultrastructural observations under electron microscopy as mentioned in part one. Results: There was no significant difference in MBF between the regions of interest of anterior, lateral, septal wall beyond posteral wall. MCE values from anterior wall were selected as the index for comparison between the two groups. The PI and MBF in the DM group were significantly lower than those in the control group at baseline and after dipyridamole stress （all P < 0.05）; MFR in the DM group was also lower than that in the control group （P < 0.05）. The was no significant difference inβand TTP between the two groups at baseline, however, theβin the DM group was significantly lower and TTP was significantly longer after dipyridamole stress （P < 0.05）. The result of 99m TcγWell counting indicated that the nuclide intake of myocardial tissue in diffefent walls were similiar in the DM group, but they were all lower than those in the control group （P < 0.05）. The capillary density decreased significantly in the DM group compared with the control group. No evident atherosclerotic plaques were found of coronary arteries under the epicardium, and cardiocytes appeared to arrange orderly in all sections in both groups.Ultrastructural impairments of the capillaries and cardiocytes were observed in the DM group, such as destroyed basal laminars, slit-shaped cavities and microthrombosis of the capillaries, opened intercalated disks, swollen mitochondria and destroyed sarcomere structures of the cardiocytes. Conclusion: The A, TTP, MBF and MFR derived from MCE were all sensitive parameters in detecting the microcirculation disturbances in the ealier period of DCM.Part three: Study of the Correlation of Myocardial Microcirculation Disturbance and Mechanical Dysfunction in Diabetic Rats Using Myocardial Contrast Echocardiography and Velocity Vector ImagingObjective: The aim of this study was to investigate whether MBF, MFR derived from MCE correlates with the parameters of myocardial systolic function reserve （velocity, strain and SR reserve） derived from VVI. Methods: The selection of DM rats, data acquisition and analysis of VVI and MCE at baseline and after dypiridamole stress were performed as mentioned in part one and two. In the present study, the correlation between myocardial velocity, strain, strain rate and MBF at rest, and the correlation between myocardial velocity, strain, strain rate reserve and MFR after dipyridamole stress were analylized. The myocardial systolic function （velocity, strain, strain rate） reserve was calculated as the peak velocity （strain, strain rate） stress - peak velocity （strain, strain rate） at rest. Results: No significant correlations were found between myocardial velocity, strain, strain rate and MBF at rest in the DM group （r = -0.252, P = 0.314; r = -0.080, P = 0.754 and r = -0.191, P = 0.448）; however, there were significant correlations between myocardial velocity, strain, strain rate reserve and MFR after dipyridamole in the DM group （r = 0.653, P = 0.03; r = 0.769, P < 0.001 and r = 0.787, P < 0.001）. Conclusion: The decrease of MBF was not the predominant cause of the decrease of myocardial systolic function parameters such as velocity, strain and strain rate at rest; however, the decrease of MFR may greatly contribute to the decrease of myocardial systolic function reserve after dipyridamole stress.更多还原