| Objective:To establish a rabbit model of acute myocardial infarction (AMI) and perform myocardial contrast echocardiography (MCE) with targeted ultrasound contrast agent carrying interleukin-8 (EL-8) monoclonal antibody to quantitatively evaluate the myocardial microcirculation perfusion.Methods:Thirty Japanese white rabbits were randomized into two groups:acute myocardial infarction group (AMI group, n=15) and sham operation control group (Sham group, n=15). In the AMI group, left ventricular anterior wall and anteriorseptal myocardial infarctions were induced by ligation of the left anterior descending artery, whereas the Sham group was only treated with threading below the anterior descending branch without ligation after thoracotomy. Continuous electrocardiographic monitoring was obtained intraoperatively, and electrocardiograms (ECGs) were recorded before thoracotomy, at 10 min postoperatively and 1 h postoperatively, respectively. Blood samples (3 ml) were collected from the central auricular arteries of both groups of rabbits before thoracotomy, at 30 min,2 h and 6 hpostoperatively, respectively, and levels of serum creatine kinase (CK), CK-MB, lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were determined. Routine echocardiography were performed in both groups of rabbits before thoracotomy, at 30 min,2 h and 6 h postoperatively, respectively, followed by MCE with SonoVue and targeted ultrasound contrast agent carrying IL-8 monoclonal antibody. QLAB software was used for off-line analysis after dynamic images were obtained. Animals were sacrificed 6 h after postoperative examination. Rabbit hearts were sampled:three 2 mm thick left-heart sections were transected 5 to 10 mm below the coronary artery ligation or threading point for pathological examination.Results:1. Changes reflecting myocardial ischemia were observed in the AMI group on postoperative ECG, including ST segment elevation or depression, inverted, positive, negative, or biphasicT waves.2.In the AMI group, serum levels of CK, CK-MB, LDH and AST were slightly higher at 30 min and 2h postoperatively and markedly higher at 6 h postoperatively than those preoperatively, respectively. In the Sham group, no distinct change was observed at these three time pointspostoperatively. 3. Quantitative MCE analysis of left ventricular short axis at papillary muscle level1)MCE was performed in the same experimental rabbit with SonoVue. The postoperative peaksand means of ultrasoundcontrast agents were lower in the anterior wall and anteroseptum of two modeled segments than in the inferior wall and postseptum of the non-infarcted segments in the AMI group (all P<0.05), lower than in the corresponding segments in the Sham group (all P<0.05). On the contrary, opposite results were obtained when using targeted ultrasound contrast agent carrying IL-8 monoclonal antibody at 30 min postoperatively(all P<0.05).2)Starting with the first frame after the Flash:MCE was performed in the same experimental rabbit with SonoVue. The postoperativepeak time of contrast agents was later in the anterior wall and anteroseptum of two modeled segments than in the inferior wall and postseptum of the non-infarcted segmentsin the AMI group(all P<0.05), as well as in the corresponding segments in the Sham group (all.P<0.05). On the contrary, opposite results were obtained when using targeted ultrasound contrast agent carrying IL-8 monoclonal antibody at 30 min postoperatively(all P<0.05).3)Starting with the beginning of injection of contrast agent:MCE was performed in the same experimental rabbit with SonoVue. The postoperative myocardialcontrast arrival timewas later in the anterior wall and anteroseptum of two modeled segments than in the inferior wall and postseptum of the non-infarcted segmentsin the AMI group(all P<0.05), as well as in the corresponding segments in the Sham group (all P<0.05). On the contrary, opposite results were obtained when using targeted ultrasound contrast agent carrying IL-8 monoclonal antibody at 30 min postoperatively (all P<0.05).4)The time-intensity curve (TIC) of the contrast agent was plotted with the beginning of the post-Flash first frame and fitted with a linear negative exponential function [y (t) = A*(1-exp (-B*t)+C]. MCE was performed in the same experimental rabbit with SonoVue.The postoperative myocardial blood volume (A value), blood flow velocity (B value) and local myocardial blood perfusion (A*B value) were markedly lower in the anterior wall and anteroseptum of two modeled segments than in the inferior wall and postseptum of the non-infarcted segments in the AMI group (all P<0.05), lower than in the corresponding segments in the Sham group (all P<0.05). On the contrary, when using targeted ultrasound contrast agent carrying IL-8 monoclonal antibody, the postoperative plateau acoustic intensity (A value), the rise rate of the curve (B value) and the filling volume of the contrast agent per unit time (A*B) were slightly higher in the anterior wall and anteroseptum of two modeled segments than in the inferior wall and postseptum of the non-infarcted segments in the AMI group at 30 min postoperatively.4. Pathological sectionsHE stain:All cardiac segments of 13 rabbits in the sham group exhibited normal myocardia. The anterior walls and anterosepta ofmodeled cardiac segments of 10 rabbits in the AMI group were manifested as myocardial ischemia or infarction: myocardial tissues were disorganized and myocardial fibers were swollen; myocardial cells partially lysed and ruptured with karyorrhexis, karyolysis and hypochromatosis, resulting in patches ofnuclear-free zones; partial myocardial tissues exhibited coagulative necrosis, with partial lytic necrosis and vacuolar degeneration; granulation tissues and new capillaries were observed in the infarcted border zone, with massive neutrophil infiltration.Masson’s trichrome stain:All cardiac segments of 13 rabbits in the sham group exhibited normal myocardia:uniformmyocardial tissues, regularly-arranged and red muscle fibers, and occasionally fistuliform and blue capillaries. The anterior walls and anterosepta ofmodeled cardiac segments of 10 rabbits in the AMI group were manifested as myocardial ischemia or infarction:myocardial fibers were swollen and disorganized; perivascularlyscattered and strip-shaped blue collagen fibers replaced normal myocardial cells; a small part of myocardial tissues were manifested as disappearance of red muscle fibers and replaced by a mass ofblue collagen fibers.Conclusion:1.The method for quantitative analysis of myocardial microcirculation perfusionby real-time MCE (SonoVue) has confirmed that the myocardial blood volume, blood flow velocity and blood perfusion are markedly lower in the modeled segments than in the non-infarcted zonesin the AMI group, lower than in the corresponding segments in the sham group; the mean and peak video intensities of the ultrasound contrast agent are markedly lower in the modeled segments than in the non-infarcted segments, lower than in the corresponding segments in the sham group; the myocardial contrast arrival time and the peak time are later in the modeled segments than in the non-infarcted segments, as well as in the corresponding segments in the sham group. This suggests the sensitivity and reliability of quantitative evaluation of myocardial microcirculation perfusion by MCE.2. The MCE with targeted ultrasound contrast agent carrying IL-8 monoclonal antibody has found at 30 min postoperatively that:the postoperative peaks and means of the ultrasound contrast agent are higher in the modeled segments than in the non-infarcted segments in the AMI group, higher than in the corresponding segments in the Sham group at 30 min postoperatively; the arrival time and peak time of the ultrasound contrast agent are earlier in the modeled segments than in the non-infarcted segments, earlier than in the corresponding segments in the Sham group at 30 min postoperatively. This indicates the presence of the targeting effect of the targeted ultrasound contrast agent carrying IL-8 monoclonal antibody.3.Covalent couplingwith SPDP crosslinkeris a feasible way to conjugate the IL-8 monoclonal antibody to the surface of the SonoVue microbubble. Each experimental rabbit has received six myocardial contrast echocardiographies within 6 h in the state of tolerating thoracotomy without any abnormal response caused by the contrast agent, suggestive of the safety and feasibility of MCE.4.The inflammatory factor, IL-8, markedly increases in early AMI (at 30 min postoperatively). When using targeted ultrasound contrast agent carrying IL-8 monoclonal antibody to perform MCE, the IL-8 monoclonal antibody can neutralize inflammatory factors in regions of myocardial ischemia. |
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