| Part I Establishment and characterization of an Experimental Model of Coronary Thrombotic Microembolism in RatsObjective:To establish a model of coronary thrombotic microembolism in rats.Methods:5mg dried auto-microthrombotic particulates dissolved in0.2ml saline (CM E group) or0.2ml saline (SHAM group) was injected into temporarily clamped aorta of male Sprague-Dawley rats. After auto-microthrombotic particulates injection, serum c-troponin I was measured by electrochemistry and immunofluorescence method and von Willebrand factor (3hours,24hours,1days,28days) was determined by antibody in antigen-based sandwich enzyme-linked immunosorbent assay, myocardial leukocyte infiltration (24hours and7days,28days), percent of arterioles obstructed by thrombosis, early myocardial ischemia or infarct area (3hours),myocardial fibrosis (28days) were observed by HE, CARSTAIRS,HBFP and Masson staining in Light Microscopic Analysis. Cardiac function was evaluated by transthoracic echocardiography and hemodynamic measurements.Results:After automicrothrombotic particulate injection, serum c-troponin I and von Willebrand factor levels, myocardial leukocyte infiltration levels, the percentage of arterioles obstructed by thrombosis, and myocardial fibrosis were all significantly increased whereas cardiac function as evaluated by echocardiography and hemodynamic measurements were significantly reduced compared with the SHAM group.Conclusions:Injection of5mg homologous microthrombotic particle suspension into aorta when clamping the ascending aorta is an effective method to produce coronary microembolism in small animals. Part II The study on microcirculatory disturbance and no-reflow phenomenon mechanism after coronary artery autologous micro thromboembolism in ratsObjective:To research microcirculatory disturbance and no-reflow phenomenon mechanism in the model of coronary thrombotic microembolism in rats.Methods:5mg dried auto-microthrombotic particulates dissolved in0.2ml saline (CME group) was injected into temporarily clamped aorta of male Sprague-Dawley rats. After auto-microthrombotic particulates injection, serum c-troponin I a, von Willebrand factor and ET-level (3hours,24hours,1days,28days) was determined, no-flow area was evaluated by Thioflavin-S (3hours), myocardial leukocyte infiltration (24hours and7days,28days), myocardial expressions of TNFa and IL-6(24hours,1and28days) were measured by Immunohistochemical Analysis and Western Blot Analysis, Arteriole density (AD) was calculated by immunohistochemical analysis. Cardiac function was evaluated by transthoracic echocardiography and hemodynamic measurementsResults:After automicrothrombotic parti cul ate injection, serum c-troponin I and von Willebrand factor levels, the no-flow area as evaluated by Thioflavin S, myocardial leukocyte infiltration levels, myocardial expressions of tumor necrosis factor and interleukin-6, were all significantly increased whereas cardiac function as evaluated by echocardiography and hemodynamic measurements were significantly reduced compared with the SHAM group. Number of arterioles with diameter between10-50μm, especially for arterioles with diameter between20-50μm was significantly lower in CME group at3hours post injection.Conclusion:aortic automicrothrombotic particulate injection could induce coronary microembolism in rats, and this model could be of value in improving the understanding of pathophysiology of no-reflow phenomenon mechanism. Part III Effects of intravenous diltiazem in a rat model of experimental coronary thrombotic microembolismObjective:The objective of this study was to evaluate the feasibility of evaluating the therapeutic effects of intravenous diltiazem in a newly established rat model of coronary thrombotic microembolism (CME).Methods:CME was induced by injecting0.199ml saline containing5mg automicrothrombotic particulates (around10μm) into the aorta of Sprague-Dawley rats over10seconds using a tuberculin syringe with a28-gauge needle. CME rats were randomized to untreated (CME, n=38) group and diltiazem-treated (CME+DIL, n=38) group. Diltiazem (1mg/ml,50μg/min/Kg) was intravenously injected with an infusion pump through tail vein for175minutes at5minutes after automicrothrombotic particulates injection. Hemodynamic measurements, echocardiographic and pathohistogical examinations were performed at various time points (3hours,24hours,7days and28days) post operation.Results:Arteriole thrombosis, multi-focal myocardial necrosis, inflammatory cell infiltration with remarkably increased myocardial TNF-a, IL-6expression and reduced LV systolic function as well as increased plasma vWF, ET-1and c-TNI levels (indicating vascular endothelial injury and myocardial necrosis) were evidenced in CME rats. These pathologic responses in CME rats could be partly attenuated by intravenous diltiazem treatment.Conclusion:This CME model is suitable to evaluate the therapeutic effects of intravenous diltiazem and intravenous diltiazem treatment significantly improves cardiac function through alleviating inflammatory responses and microvascular thrombotic injury in this rat CME model. |
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