| Coronary microembolizaion (CME) is a frequent and important event in acute coronary syndromes and during coronary interventions. Effective therapies are limited except GPIIb/IIIa antagnosis and distal protective devices in acute phase. Inflammation plays an important role in CME-induced myocardial damage and ventricular impairment, and NF-κB is a key pro-infalmmation transcription factor. In the present study, our aims were to develop a reproducible and relatively stable model of coronary microembolization by use of homologous microthrombi in rats; to investigate the roles of inflammation and NF-κB activation in CME-induced myocardial and ventricular impairment; to evaluate the impacts of specific NF-κB Inhibitor pyrrolidine dithiocarbamate (PDTC) on inflammation reaction and cardiac function following CME.Part I A Novel Rat Model of CME with Homologous MicrothrombiObjective To develop a reproducible and relatively stable CME model by use of homologous microthrombi in rats. Methods After thoracotomy, 24 Sprague Dawley rats were subjected to sham operation and 96 rats underwent injection of homologous microthrombotic particle suspension into left ventricle when clamping the ascending aorta, which were divided into three groups according to injection of three dosages of 3, 5, 7mg of microthrombi. The microthrombotic particles were generated from rat clots sized by filtration through 38μm screen. The rats were sacrificed on day 1, 3, 7 and 14 post-operationally and each subgroup consisted of 8 rats. cTnI in serum, as an indicator of cardiac cytolysis, was determined with ELISA. The morphological characteristics were evaluated in sections with HE, HBFP staining 1 day after operation and Masson's trichrome staining 14 days post-operationally. CARSTAIRS staining was used to identify thrombosis in microvasculature. Cardiac function was evaluated echocardiographically and hemodynamically. Results All rats survived through the following procedure in sham group, 3 (9%), 7 (22%), 19 rats (59%) were died in CME3, CME5 and CME7 groups respectively. The rats in CME7 group showed depression, tachypnea, inactivity and intaking decrease. Histological examination showed multi-focal myocardial ischemia, necrosis with inflammatory cells infiltration and fibrosis with more involvement in subendocardial region. Compared with that of sham group, the total area of myocardial micro-infarction in CME3, CME5 and CME7 were (7.27±1.00)%, (16.70±2.07)% and (21.51±1.98)% vs (1.36±0.93)%, respectively(P<0.01). Scattered microthrombosis were observed in microvasculature (<100μm) after CME assessed by CARSTAIRS staining. Compared with the measurements in the sham group, LV systolic and diastolic function was significantly reduced with serum cTnI significantly increased in all CME groups, the degree of those was associated with the dosage of microthrombi. Conclusions Injection of 5mg homologous microthrombotic particle suspension into left ventricle when clamping the ascending aorta is an effective method to produce coronary microembolism in small animals.Part II Roles of Inflammation on ventricular impairment following CMEObjective To investigate the roles of inflammation and NF-κB activation on myocardial damage following CME. Methods 24 SD rats served as controls, CME model in 32 rats was created as described previously in part I. The rats were sacrificed on day 1, 3, 7 and 14 post-operationally. Locations and distributive expressions of TNFα, IL-6, ICAM-1 mRNA were determined qualitatively or semi-quantitatively by in situ hybridization histochemistry, and their dynamic alterations were quantitatively assessed by Real-time PCR. Protein expressions of TNFα, IL-6, ICAM-1 and IκBαwere analyzed by immunohistochemistry and western blotting. Immunohistochemistry was used to identify the source of NF-κB P65 and NF-κB DNA-binding activity was evaluated by electrophoretic mobility shift assay. Myocardial apoptosis rate was calculated in the sections with TUNEL-staining. Left ventricular function was evaluated echocardiographically and hemodynamically. Results (1) TNFα, IL-6, ICAM-1 mRNA were expressed few in normal myocardium. CME produced strong TNFα, IL-6 mRNA and protein expressions not only confined strictly at the myocytes in microinfarct or peri-microinfarct zone but also at vascular endothelium and partial"normal"myocytes. The expressions of ICAM-1 mRNA and protein were enhanced at myocytes in microinfarct or peri-microinfarct zone and vascular endothelium. The expression of ICAM-1 in myocardium by IOD analysis had a significant positive correlation with the number of infiltrating leukocytes (r=0.722, P<0.01). Compared with those of the sham group, the expressions of TNFα, IL-6 and ICAM-1 detected via western blotting were significantly increased in model group at each stage. The inflammation peaked during day 3 to 7 following CME. (2) Immunohistochemistry demonstrated that leukocytes adhered at, aggregated in or even blocked small vasculature, and migrated into vascular wall obviously. (3) NF-κB P65 expression and NF-κB DNA-binding activity in the nuclei of myocytes in CME group were significantly increased than those of sham group on day 1, 3, with no significant difference on day 14. IκBαexpression was decreased significantly in the myocardium on day 1, 3 following CME, but restored to the level of sham group on day 7, 14. (4) Expressions of TNFα, IL-6, ICAM-1 mRNA had significant positive correlations with NF-κB DNA-binding activity, respectively (r=0.72, P<0.05; r=0.94, P<0.01; r=0.62, P<0.05). (5) Compared to sham group, myocyte apoptosis rates in CME group were markedly increased at all stages. (6) In acute phase (within 7 days) the increased myocardial TNFαconcentration had a negative correlation with LV dp/dt max (r=-0.77,P<0.01) and EF((r=-0.65,P<0.01). Conclusions (1) CME produced amplified myocardial inflammation, which is not only confined to the microinfarct or peri-microinfarct zones but also expressed in"innocent"myocytes, namely"bystander effect". (2) IκBαdegradation induced NF-κB activation following CME; NF-κB activation up-regulating the transcription of TNFα, IL-6, ICAM-1 genes is a key acute event that may contribute to inflammatory responses and myocardial damage in CME. Part III Impacts of NF-κB Inhibitor PDTC on Myocardial Inflammation and Ventricular Impairment following CMEObjective To investigate the impacts of specific NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) on CME-induced myocardial inflammation and ventricular impairment, and further to identify the roles of NF-κB activation. Methods 24 SD rats served as sham group, 128 coronary microembolized rats were randomized to 4 groups, including untreated CME group, PDTC low dose group, PDTC moderate dose group, PDTC high dose group, which were administered intraperitoneally 50 mg.kg-1.day-1, 100 mg.kg-1.day-1 or 200 mg.kg-1.day-1 respectively. The rats were sacrificed on day1, 3, 7 and 14 post-operationally. The severity of the myocardial damage was evaluated by microinfarct size, inflammatory cells infiltration and the level of serum cTnI. The fraction of collagen fiber was measure in sections with Masson staining. NF-κB DNA-binding activity were assessed by electrophoretic mobility shift assay. Immunohistochemistry and western blot were used to assessed the levels of TNFα, IL-6, ICAM-1, IκBαprotein, and Real-time PCR was performed to analyze the expression of TNFα, IL-6, ICAM-1 mRNA between different groups. TUNEL staining was employed to identify myocyte apoptosis and cardiovascular echocardiographical and hemodynamic index was performed to evaluate cardiac function. Results (1) PDTC therapy attenuated microinfarct size, inflammatory cells infiltration,the level of serum cTnI, and decreased collagen fiber in myocardial interstitium; there was significant difference between P200, P100 and P50 group (P<0.05), no difference between P200 and P100 group (P>0.05). (2) PDTC markedly suppressed NF-κB DNA-binding activity in a dose-dependent manner and reduced degradation of IκBαon day 1, 3. The level of IkBa protein in the cytoplasm in PDTC groups became markedly increased than that in sham group on day 7 (P<0.05), and reduced to normal level on day 14. (3) PDTC significantly suppressed TNFα, IL-6 protein and mRNA expression in a dose-related manner. Reductions in ICAM-1 protein and mRNA expression were also dose-related, except at a PDTC concentration of 200 mg.kg-1.day-1. PDTC of 200 mg.kg-1.day-1 caused less inhibition of ICAM-1 protein and mRNA expression than that of 100 mg.kg-1.day-1at different points. (4) PDTC markedly reduced myocyte apoptosis rate (P<0.05), but the rate of P200 group was higher than that of P100 group on day 7 and no difference existed after withdrawing therapy. (5) PDTC therapy significantly improved cardiac function; there was significant difference between P200, P100 and P50 group (P<0.05), no different between P200 and P100 group (P>0.05). Conclusions PDTC inhibited NF-κB and transcription system activation in a dose-dependent manner, effectively attenuated myocardial amplified inflammation and ameliorated cardiac function, although the higher concentration may produced greater apoptosis of myocyte.
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