| It has been shown that it is extremely important to maintain epicardial infarct-related artery patency in patients with acute myocardial infarction (AMI).But low or no distal tissue-level perfusion,namely no-reflow phenomenon,may lead to poor reverse clinical prognosis.Therefore,more and more people pay close attention to functional status of myocardial microcirculation. By now,the mechanisms of no-reflow phenomenon have not been completely distinct,however,microcirculation disturbance which is induced by neutrophil adhesion,release of inflammatory mediators, fibrin thrombus formation,platelet aggregation,endothelial cell injury in structure and function,is the important factor for no-reflow.From the experimental and clinical researches for more than thirty years,it has indicated that inflammation is an independent risk factor for the development of atherosclerosis and mediates all stages of atherosclerosis.Inflammatory injury run through the whole course of myocardial cell injury and impact on other injury factors during the process of myocardial ischemia reperfusion (I/R).Interleukin-6 (IL-6),a circulating cytokine,has been identified as a marker of inflammation in coronary atherosclerotic plaque.Serum levels of IL-6 increase in response to AMI,unstable angina,percutaneous coronary intervention(PCI),and late restenosis.IL-6 stimulates platelet aggregation and expression of tissue factor,high sensitive C reaction protein (hs-CRP),and fibrinogen.Renin-angiotensin system (RAS),related to atherosclerosis and Inflammation,can damage the vessel endothelium.In addition to the significant proinflammatory reactions of angiotensin (Ang) II within the vessel wall, Ang II activity within circulating cells has been linked to some cardiovascular risk factors,such as plasma glucose concentration,plasma insulin levels.By contrast,antagonists of Ang II actions in vitro and in vivo lessen proinflammatory and proatherogenic factors,including reactive oxygen species generation, nuclear factor [kappa]B and plasma hs-CRP.By interrupting the expression of adhesion molecules and cytokines,angiotensin-converting enzyme (ACE) inhibitors exert antiinflammatory effects during the development of atherosclerosis. However,the relations among inflammation,RAS and endothelial function have not been completely known.Thus,in order to evaluate changes of inflammation,RAS and endothelial function during coronary occlusion and reperfusion and to investigate the mechanisms and managements of no-reflow phenomenon, we planned this study in a rabbit model of coronary occlusion and reperfusion so as to find a new way for clinical prevention and treatment of no-reflow.Objectives1. To observe the changes of inflammation reaction in circulation and myocardium,and the relations between inflammation and no-reflow phenomenon in a rabbit model of AMI and reperfusion.To evaluate the effects of atorvastatin on inflammation and on prevention and treatment of no-reflow phenomenon.2. To observe the changes of RAS activity in circulation and myocardium,and the relations between RAS and on no-reflow phenomenon in a rabbit model of AMI and reperfusion;To evaluate the effects of benazepril on RAS and no-reflow.3. To investigate the changes and the effects of endothelium function on no-reflow phenomenon in a rabbit model of AMI and reperfusion;To evaluate the effects of atorvastatin and sodium ferulate on endothelium function and no-reflow.Methods1. Animal protocolFifty-six New-Zealand white rabbits were randomized into 8 study groups:8 in sham-operated group,8 in AMI1h group,8 in AMI4h group (control group),8 in atorvastatin group 1 (10mg·kg-1, once 12h before operation),8 in atorvastatin group 2 (5mg·kg-1·d-1 for 3 days before operation),8 in benazepril group (3mg·kg-1·d-1 for 3 days before operation), 8 in sodium ferulate group (20mg·kg-1, infusion 5 min before operation for 6 hours).The AMI1h group were subjected to 1h of left circumflex coronary artery (LCX) occlusion followed by 2h of reperfusion. The AMI4h group and the three treated groups were subjected to 4h of coronary occlusion followed by 2h of reperfusion. In the sham-operated animals, the LCX was only encircled by a suture after operation,but not occluded. 2. Histopathological evaluationAt the end of the protocol,size of no-reflow (SNR) was delineated by intra-atrial injection of 1ml·kg?1 of 60 g/L thioflavin S (dissolved in 0.9% saline and then 1,500 rpm centrifuged for 5 min).After about 1 min, the LCX was reoccluded and 1ml·kg?1 Evans blue dye was injected into the left atrium to determine ligation size (LS).The animal was then killed and the heart was removed.The left ventricle was cut into 5–7 transverse sections parallel to the atrioventricular groove.Under a ultraviolet ray (UV)light in a dark room,the areas not perfused by thioflavin S were identified.LS was defined as the region unstained by Evans blue,whereas area of no-reflow (SNR) was defined as the non-fluorescent area within the LS.The outlines of the LV wall area(LVWA), LS and SNR were calculated.The LA was expressed as a percentage of LS over the LVWA, and ANR was expressed as a percentage of SNR over LS.Finally,the slices were put into 1% solution of triphenyltetrazolium chloride(TTC) and hatched for 15 minutes in 37℃.The red area was identified as non-infarcted myocardium and the non-colored area was infarcted myocardium.The necrosis area(NA) was expressed as a percentage of infarcted area over LS.3. Data of haemodynamics were collected by putting a 4F catheter into the right carotid artery with a multi-lead electrophysiogical reader.4. The serum levels of IL-6 were measured by enzyme linked immunosorbent assay (ELISA).5. The expressions of interferon gamma (IFN-γ),P-selectin (Ps) and ACE2 were detectd by immunohistochemical (IHC) method.6. The levels of AngⅡand ET-1 in blood sample, normal flow,infarcted reflow and no-reflow myocardium were determined by radioimmunoassay(RIA).7. ACE mRNA in the myocardium was evaluated by reverse transcription-polymerase chain reaction (RT-PCR).Results1. Data of haemodynamicsThe baselines of heart rate(HR),systolic blood pressure(SBP) and diastolic blood pressure(DBP) in each group were similar.Compared with the baselines,all of HR,SBP,DBP in AMI1h and AMI4h group were significantly declined after 60 or 4hutes of coronary occlusion and 2hutes of reperfusion(all P<0.01).At the time point of AMI and reperfusion, the declines of SBP and DBP in AMI4h group were more obvious than that in AMI 1h group(P< 0.05 or P< 0.01).Compared with the control group (AMI4h group),the SBP and DBP were significantly lower in atorvastatin group 1 and 2 (P<0.01).2.Activities of inflammation mediators and the protective effects of atorvastatin against inflammation and no-reflow phenomenon(1)Compared with the sham-operated group,the levels of serum IL-6 in AMI1h group were significantly increased at the end of 1h of LCX occlusion and 2h of reperfusion ( all P<0.01).Compared with the baseline,the levels of IL-6 in blood sample were significantly increased at the end of 1h of LCX occlusion and 2h of reperfusion, showing time-dependant increase tendency( all P<0.01).The changes of IL-6 in AMI4h group were similar with those in the AMI1h group,but the levels of IL-6 were higher in AMI4h group than those in AMI 1h group (P< 0.05 or P< 0.01).In the treated group, the levels of IL-6 (except baseline) were significantly lower than those in control group (P< 0.01 or P< 0.05) after AMI.(2)It was shown by HE staining that in AMI 1h group,the cardiomyocytes in reflow myocardium present denaturalization and light inflammatory reaction,but no obvious necrosis,while the inflammatory reaction and necrosis in no-reflow myocardium were more severe.Degeneration,necrosis and inflammatory reaction in reflow or no-reflow myocardium were more severe in AMI4h group than in AMI 1h group,but less severe in treated groups.(3) It was shown by IHC that the expression of Ps was detected in vascular endothelial cells of myocardium. The expressions of Ps in reflow and no-reflow area were stronger than that in the normal myocardium in AMI 1h an 4h groups ,and also stronger than that in sham-operated groups(all P<0.01).The expression of Ps in no-reflow myocardium was stronger than that in reflow myocardium(all P<0.01). In AMI 4h group, the expressions of Ps in reflow and no-reflow myocardium were more obvious than those in the AMI 1h group(P <0.05 and P=0.01).In the two atorvastatin treated groups,the expression in reflow and no-reflow myocardium attenuated significantly than those in the control group respectively(P<0.05 or P <0.01).(4)It was shown by IHC that strong expressions of IFN-γin vascular endothelial cells of reflow and no-reflow myocardium were detected in the two AMI groups, and the expressions were weaker in normal myocardium(P<0.05). The Ps expression in no-reflow myocardium was stronger than that in reflow myocardium(all P<0.01).In AMI 4h group, the expressions in reflow and no-reflow myocardium were stronger than those in the AMI 1h group respectively(P <0.01 or P<0.05).In the two atorvastatin treated groups, the expressions in reflow and no-reflow myocardium attenuated significantly than those in the control group respectively (all P <0.01).(5)Between AMI 4h group and AMI 1h group, the LA was similar(36.87±2.16% vs 36.20±1.67%,P > 0.05),but the ANR was significantly increased in the former than that in the later(85.67±4.94% vs 75.26±3.27%,P < 0.01 ) , and the NA was also significantly increased(96.56±2.26% vs 87.24±4.95%,P < 0.01 ). There were no significant differences in LA between the control group(36.87±2.16%)and the two atorvastatin treated groups(37.45±3.25% and 37.92±3.92%,) respectively(both P >0.05).ANR of atorvastatin group 1 and 2(47.01±6.89% and 44.52±4.24%) decreased significantly than that of the control group(85.67±4.94%)(both P< 0.01 ). The NA of atorvastatin group 1 and 2 (85.94±7.01%,87.21±4.46%) also decreased significantly than that of the control group(96.56±2.26%)( both P< 0.01 ).3. Activities of RAS in circulation and tissue,and the effects of benazepril on RAS and no-reflow phenomenon(1)Compared with the sham-operated group and the baseline, the levels of serum AngⅡin AMI 1h and 4h groups significantly increased at the end of 1h or 4h of LCX occlusion and 2h of reperfusion( all P<0.01),showing time-dependant increase tendency ( all P<0.01). In AMI 4h group, the levels of AngⅡwere significantly higher than those in AMI 1h group(all P< 0.01). In benazepril group, levels of AngⅡwere significantly lower than those in control group (all P< 0.01).(2)In both AMI4h and AMI1h groups, compared with that in normal myocardium, AngⅡin both reflow and no-reflow myocardium significantly increased (both P<0.01), while levels of AngⅡin no-reflow myocardium were significantly higher than those in reflow myocardium (P<0.01). In AMI 4h group, levels of AngⅡin ischemic myocardium were significantly higher than those in the AMI 1h group (P < 0.01).In benazepril group ,the levels of AngⅡin both reflow and no-reflow myocardium were significantly lower than those in control group.(3)It was shown by HE staining that in AMI 1h group,cadiomyocytes in reflow myocardium underwent degeneration and light inflammatory reaction, but no necrosis, and which in no-reflow myocardium was more severe. In AMI 4h group, degeneration , necrosis and inflammatory reaction in reflow and no-reflow myocardium were more severe than those in the AMI 1h group. In benazepril group, degeneration and necrosis in reflow and no-reflow myocardium attenuated significantly than those in the control group.(4)It was shown by IHC that ACE2 powerfully expressed in vascular endothelial cells of reflow and no-reflow myocardium in AMI 1h and 4h groups, and more powerfully than normal myocardium and sham-operated groups(all P<0.01), and its expression in no-reflow myocardium was more powerful than that in reflow myocardium(all P<0.01).In AMI 4h group, its expression in reflow and no-reflow myocardium was respectively more powerful than that in AMI 1h group(all P <0.05). In benazepril group, its expression in reflow and no-reflow myocardium respectively attenuated significantly than that in the control group(all P<0.05).(5)It was shown by IHC that IFN-γpowerfully expressed in vascular endothelial cell of reflow and no-reflow myocardium in AMI groups, and more powerfully than normal myocardium and sham-operated groups(all P<0.01), and its expression in no-reflow myocardium was more powerful than that in reflow myocardium(all P<0.01). In AMI 4h group, its expression in reflow and no-reflow myocardium was respectively significantly powerful than that in AMI 1h group(all P <0.01).(6)In AMI groups,expression of ACE mRNA in the reflow myocardium was significantly higher than that in the normal myocardium,while expression of ACE mRNA in the no-reflow myocardium was significantly lower than that in the normal and reflow myocardium(all P< 0.01).Compared AMI4h group with AMI1h group,expression of ACE mRNA only in the reflow myocardium significantly increased (P<0.05).Compared benazepril group with the control,expression of ACE mRNA only in the reflow myocardium shown a decreased tendency, but not significantly (P=0.052).(7)Compared the benazepril with control group,LA were similar(37.62±2.90% vs 36.87±2.16%,P > 0.05),ANR was significantly decreased(45.12±4.62% vs 85.67±4.94%,P < 0.01 ) ,and NA was also significantly decreased (86.05±5.22% vs 96.56± 2.26%,P < 0.01 ).4.Endothelium dysfunction during ischemia,reperfusion and no-reflow,and the antagonistic effects of atorvastatin and sodium ferulate(1)Compared with the sham-operated group,serum ET-1 in AMI1h group significantly increased at the end of 1h of LCX occlusion and 2h of reperfusion( all P<0.01);compared with the baseline,serum ET-1 in AMI1h group significantly increased at the end of 1h of LCX occlusion and 2h of reperfusion,showing an increasing tendency ( all P<0.01). In AMI4h group,the changes of ET-1 were the same as those in AMI1h group.In AMI 4h group,levels of ET-1 were significantly higher than those in the AMI 1h group. In the treated groups,levels of ET-1 were significantly lower than those in control group (all P< 0.01).(2)In both AMI 1h and AMI 4h groups, compared with that in normal myocardium, ET-1 in both ischemic and no-reflow myocardium significantly increased (both P<0.01), while levels of ET-1 in no-reflow myocardium was significantly higher than that in reflow myocardium (P<0.01).In AMI 4h group,levels of ET-1 were significantly higher than those in AMI 1h group.In the treated groups,the level of ET-1 only in reflow myocardium was significantly lower than that in the control group.(3)It was shown by HE staining that in AMI 1h group,cardiocytes in reflow myocardium generated degeneration and light inflammatory reaction, but no necrosis, and which in no-reflow myocardium was more severe. In AMI 4h group, degeneration,necrosis and inflammatory reaction in reflow and no-reflow myocardium were significantly more severe than those in AMI 1h group. In two treated groups,degeneration,necrosis and inflammatory reaction in reflow and no-reflow myocardium attenuated significantly than those in the control group.(4)Values of LA in atorvastatin group 1, group 2,sodium ferulate group and the control group were 37.45±3.25%, 37.92±3.92%, 38.03±4.05% and 36.87±2.16% respectively,among which there were no significant diferences(all P >0.05).The values of ANR in atorvastatin group 1, 2 and sodium ferulate group(47.01±6.89%,44.52±4.24% and 78.12±6.13% respectively),decreased significantly than the control group(85.67±4.94%)respectively (all P< 0.01 ).The values of NA in atorvastatin group 1 or group 2(85.94±7.01%, 87.21±4.46%) were decreased significantly than the control group(96.56 ±2.26%)(both P< 0.01 ), but sodium ferulate group(92.00±2.99% )didn't (P > 0.05).Conclusions1. Inflammation reaction occurs in ischemic myocardium after AMI and reperfusion in rabbits, and the severity of inflammation is associated with infarct time.The inflammation could be one of the important mechanisms of no-reflow phenomenon.Atorvastatin,either in short-term or in one-dose,is effective in anti-inflammation, preventing no-reflow and decreasing myocardial necrosis area during AMI and reperfusion in rabbits.2. In a rabbit model of AMI and reperfusion, myocardial ischemia and oxygen deficiency could induce activation of RAS which could lead to no-reflow phenomenon.The activation of RAS may be one of the important mechanisms of no-reflow phenomenon. Benazepril is effective in preventing no-reflow and decreasing myocardial necrosis area by inhibiting activation of RAS.3. In a rabbit model of acute myocardial infarction and reperfusion, myocardial ischemia and oxygen deficiency could induce releasing of endothelin-1.Dysfunction of endothelial cells cold be one of the important mechanisms of no-reflow phenomenon. Atorvastatin is effective in protecting endothelial cell from injury,preventing myocardial no-reflow and decreasing myocardial necrosis area. Sodium ferulate is effective in protecting endothelum function, and in preventing myocardial no-reflow, but not effective in decreasing myocardial necrosis area . |
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