| 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. |
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