Myocardial Protective Effects Of Extracorporeal Cardiac Shock Wave Combined With Sulfur Hexafluoride Microbubble Post-treatment On Myocardial Ischemia/Reperfusion Injury In Rats

Acute myocardial infarction(AMI)is one of the leading causes of death and disability worldwide.The most effective treatment intervention is to restore blood flow to the ischemic area as soon as possible in the early stage of myocardial infarction.However,some patients with myocardial infarction may further aggravate the death of ischemic cardiomyocytes during myocardial reperfusion.This phenomenon is called myocardial ischemiareperfusion injury(MI/RI).MI/RI can cause coronary microvascular dysfunction and obstruction.Studies have shown that even if epicardial vessels are successfully opened,80% of patients still have varying degrees of coronary microcirculation dysfunction.The degree of coronary microcirculation dysfunction is closely related to the prognosis of patients.Persistent coronary microcirculation dysfunction after PCI in patients with acute ST segment elevation segment elevated myocardial infarction,STEMI can lead to myocardial infarction area enlargement,poor left ventricular remodeling,heart failure and high mortality.At present,studies have shown that some drugs can reduce myocardial ischemia-reperfusion injury and improve myocardial microcirculation,but the effect is not satisfactory.Extracorporeal cardiac shock wave is a new technique for the treatment of myocardial ischemia in coronary heart disease,which can increase the concentration of nitric oxide(NO)and the expression of vascular endothelial growth factor(VEGF)in myocardial tissue.Ultrasonic microbubble sulfur hexafluoride blasting can also increase the concentration of vascular endothelial nitric oxide synthase,increase the rate of blood perfusion,increase local blood circulation and improve myocardial blood supply.Both of them have their common mechanism and can improve myocardial blood flow.In vitro experiments,we found that extracorporeal cardiac shock waves can destroy ultrasonic microbubbles and cause inertial cavitation,which is more significant after ultrasonic blasting with higher mechanical index.In this study,the combination of extracorporeal cardiac shock wave and sulfur hexafluoride microbubbles was used for the first time to observe whether the combination of the two will cause additional damage,such as whether the damage is related to the concentration of sulfur hexafluoride microbubbles,and to explore the appropriate microbubble concentration.The appropriate concentration of sulfur hexafluoride microbubbles and extracorporeal cardiac shock wave were used to post-treat the rat model of myocardial ischemiareperfusion injury,and its myocardial protective effect was observed in order to provide a new therapeutic idea for the treatment of myocardial microcirculation disturbance after myocardial ischemia.Part one Echocardiographic evaluation of myocardial microcirculation perfusion and left ventricular function after thrombolysis and /or interventional therapy in patients with acute STEMIObjective: Myocardial contrast echocardiography(MCE)was used to evaluate myocardial microcirculation perfusion and left ventricular function in patients with acute STEMI after thrombolysis and / or intervention.Methods:1.This study included 69 STEMI patients who underwent MCE examination after thrombolysis and / or PCI treatment in the second Hospital of Hebei Medical University from September 2021 to November 2022.2.Echocardiography and MCE were performed within 24 hours after thrombolysis and / or PCI.3.Myocardial perfusion was observed by MCE.According to the results of MCE,the patients were divided into normal myocardial perfusion group and abnormal myocardial perfusion group.4.The diameter of each chamber was measured by conventional twodimensional echocardiography,and the left ventricular end-systolic volume(LVESV),left ventricular end-diastolic volume(LVEDV)and left ventricular ejection fraction(LVEF)were measured by Simpson double plane method.Left ventricular long-axis strain(LVGLS)was evaluated by two-dimensional speckle tracking technique.5.The clinical data,coronary angiography and laboratory results of the patients were collected,including the peak of serum troponin I(Tn I)and NTpro BNP.Results:1.The incidence of 24-hour myocardial perfusion abnormality in 69 STEMI patients treated with thrombolysis and / or PCI was 53.17%.In the group with normal myocardial perfusion,there were fewer smokers(42.2% vs.55.5%,P=0.036).The plasma Tn I level of the patients with normal myocardial perfusion was lower(68.8 ±35.1vs.78.3 ±31.7,P=0.046).The heart rate at admission in the normal myocardial perfusion group was lower than that in the abnormal myocardial perfusion group(66.78 ±10.6vs.75.4±18.9,P=0.025).There was no significant difference in other variables,P >0.05.2.Compared with the normal myocardial perfusion group,the proportion of STEMI in the anterior wall was higher in the abnormal myocardial perfusion group.The proportion of anterior descending artery in abnormal myocardial perfusion group was higher than that in normal myocardial perfusion group,while the proportion of right coronary artery in abnormal myocardial perfusion group was lower than that in normal myocardial perfusion group(P < 0.05).Compared with the normal myocardial perfusion group,the proportion of single vessel disease was higher in the abnormal myocardial perfusion group,while the proportion of two-vessel disease and three-vessel disease was lower in the abnormal myocardial perfusion group(P< 0.05).3.There was no significant difference in left ventricular diameter between abnormal myocardial perfusion group and normal myocardial perfusion group(P > 0.05).The left ventricular end-systolic volume and left ventricular end-diastolic volume in the abnormal myocardial perfusion group were higher than those in the normal myocardial perfusion group(P < 0.05).The left ventricular end-systolic volume and left ventricular end-diastolic volume in the abnormal myocardial perfusion group were higher than those in the normal myocardial perfusion group(P < 0.05).Conclusions:1.Even with thrombolysis and / or PCI therapy,the incidence of coronary microcirculation disturbance in patients with STEMI was still high(53.17%).2.Most of the culprit vessels in patients with myocardial microcirculation disturbance were left anterior descending vessel and single vessel lesion.3.The patients with myocardial microcirculation disturbance had worse left ventricular function,lower left ventricular ejection fraction and lower left ventricular global longitudinal strain.4.MCE examination can detect the disturbance of myocardial microcirculation early,and early clinical intervention has become the focus of research.Part two Effects of cavitation from extracorporeal shock wave combined with sulfur hexafluoride microbubble on myocardial ultrastructure in ratsObjective: In the present study,the effects of extracorporeal cardiac shock waves combined with different concentrations of sulfur hexafluoride ultrasound microbubbles on myocardial ultrastructure in rats were observed.Methods:1.Thirty-six rats were randomly divided into 6 groups: normal control group(N),extracorporeal cardiac shock wave group(SW)and extracorporeal cardiac shock wave combined with different concentrations(0.225 m L/Kg.min,0.45 m L/Kg.min,0.9m L/Kg.min,1.8m L/Kg.min)sulfur hexafluoride microbubble group(SW+MB0.225 group,SW+MB0.45 group,SW+MB0.9group,SW+MB1.8 group).2.The rats in the extracorporeal heart shock wave combined with sulfur hexafluoride microbubble group received shock wave while receiving intravenous infusion of sulfur hexafluoride microbubble,and the other groups were given the same volume of normal saline.3.The heart rhythm,heart rate,blood pressure,cardiac troponin I(cTnI),nitric oxide(NO),vascular endothelial growth factor(VEGF)and cardiac ultrasound indexes were observed.4.The effects of myocardial inflammation and fibrosis were observed by immunohistochemical method.5.The ultrastructural changes of myocardial tissue in each group were observed by transmission electron microscope.Results:1.The combination of extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubbles of different concentrations had no significant effect on hemodynamic indexes and left ventricular function in rats.2.There were significant differences in cTnI and NO among different groups.Histopathology showed that inflammatory cells infiltrated in the SW+MB0.9 and SW+MB1.8 group.3.Myocardial ultrastructurl injury score of SW+MB1.8 group was significantly higher than that of N group,SW group,SW+MB0.225 group and SW+MB0.45 group.The score of SW+MB0.9 group was higher than that of the control group(P=0.009).4.Western blot results showed that the expression of VEGF and e NOs protein in the rats treated with extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubbles of different concentrations was higher than that in the N group and SW group,with SW+MB0.45 group had the strongest expression.Conclusions:1.Although myocardial ultrastructure damage occurs when high concentrations sulfur hexafluoride microbubbles are present,a proper concentration of sulfur hexafluoride microbubbles could promote the cavitation effect of extracorporeal cardiac shock waves,but the myocardial injury occurs when the concentration of sulfur hexafluoride microbubbles reaches 1.8 m L/kg.min.2.When the microbubble concentration is in 0.45 mL/kg.min,the levels of NO and VEGF in myocardial tissue are the best.Part three Effect of extracorporeal cardiac shock wave combined with ultrasound microbubble postconditioning on cardiomyocyte apoptosis in rats with myocardial ischemia / reperfusion injuryObjective: At present,there is no effective drug and treatment for myocardial injury caused by reperfusion treatment of acute myocardial infarction.in this study,extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubble treatment was used to observe the myocardial protective effect on myocardial ischemia-reperfusion injury in rats,so as to provide theoretical support for clinical treatment of myocardial ischemiareperfusion injury.Methods:1.Preparation of rat MI/RI model.According to the experimental design,rats were divided into four groups with 8 rats in each group: sham operation group(Sham),myocardial ischemia-reperfusion injury group(IR),extracorporeal cardiac shock wave treatment group(IR+SW),extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubble treatment group(IR+SW+MB).2.IR+SW group and IR+SW+MB group were treated with extracorporeal cardiac shock wave and extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubbles on the 1st,3rd and 5th day after modeling,while Sham group and IR group received no treatment.3.On the 7th day after intervention,myocardial fibrosis was observed by Masson staining and cardiomyocyte apoptosis was observed by TUNEL staining.4.The parameters of left ventricular systolic function.(LVEDD,LVESD,EF,FS)were measured by echocardiography before and after treatment.5.The myocardial apoptotic proteins Bcl2,BAX,Cleaved-Caspase3 and Cleaved-Caspase9 in the infarct boundary area were detected by Western blot.Results:1.The combination of extracorporeal shock wave and sulfur hexafluoride microbubbles had no significant effect on heart rhythm and heart rate in rats,and no arrhythmia occurred in all rats during the treatment.2.Extracorporeal shock wave combined with sulfur hexafluoride microbubble therapy can reduce myocardial fibrosis in MI/RI rats.Compared with IR group,the degree of fibrosis in IR+SW group and IR+SW+MB group was alleviated.3.Extracorporeal shock wave combined with sulfur hexafluoride microbubble treatment could reduce cardiomyocyte apoptosis in MI/RI rat model.Compared with IR group,the proportion of cardiomyocyte apoptosis in IR+SW group and IR+SW+MB group decreased P<0.001(59.25±3.77 vs.43.77±4.71 vs.35.25±4.57).Comparison between IR+SW group and IR+SW+MB group,The proportion of cardiomyocyte apoptosis decreased in IR+SW+MB group,P<0.001(43.77 ±4.71 vs.35.25 ±4.57).4.The results of Western blot detection showed that after treatment with extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubbles,the level of apoptosis-inhibiting protein Bcl2 increased,the level of apoptosis-promoting protein Bax decreased,and the activation levels of Caspase-3 and Caspase-9 proteins decreased.5.Extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubbles can improve left ventricular remodeling and left ventricular systolic function in MI/RI rats.The results showed that there was no significant difference in LVEDD among Sham group,IR group,IR+SW group and IR+SW+MB group before treatment,but there was significant difference in LVESD,EF and FS between IR group,IR+SW group and IR+SW+MB group compared with Sham group,and there was no significant difference among IR group,IR+SW group and IR+SW+MB group.There were significant differences in LVEDD,LVESD,EF and FS after treatment.After treatment,LVEDD and LVESD in IR+SW+MB group were lower than those in IR group,while EF and FS increased.Conclusions:1.MCE examination showed that some patients with acute STEMI h ad myocardial microcirculation disturbance and left ventricular dysfunctio n after thrombolysis and / or intervention,which needed early clinical i ntervention.2.Extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubble therapy is safe and feasible.3.Extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubble therapy can inhibit cardiomyocyte apoptosis.4.Extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubble therapy can improve left ventricular remodeling and left ventricular systolic function.