| Objective:To explore the biological effects of different intensity ultrasound microbubble destruction technology on rabbit cardiomyocytes,and to optimize the optimal irradiation conditions affecting myocardial cells.Based on this,a rabbit model of myocardial ischemia/reperfusion(MI/RI)was established.Targeting of MI/RI was studied by ultrasonic microbubble destruction with interleukin 8(IL-8)monoclonal antibody(MAB).Methods:1.Biological effects of ultrasound irradiation on rabbit myocardial cells:Twenty Japanese big-eared white rabbits were randomly divided into 4 groups,5 in each group.0.2ml microbubble contrast agent(UCA)was injected through the ear vein and irradiated with different ultrasound intensity((1)0.5W/cm2 group;(2)0.75W/cm2 group;(3)1.0W/cm2 group;(4)1.5W/cm2 group)rabbit myocardial tissue,irradiation time 30s,intermediate interval 10s,continuous irradiation lmin.After the end of ultrasound irradiation,animals were sacrificed and local myocardial tissue was taken for histopathological Haematoxylin and eosin(HE)staining to observe the changes of myocardial cells in rabbits.2.Preparation and determine the rabbit model of myocardial ischemia reperfusion:90 Japanese rabbits were randomly divided into 3 groups:close-chest control group(A group,15 only,randomly divided into U0,U1,and U2 groups)and open-chest group(B group,15 only,randomly divided into U0,U1,and U2 groups)and I/R groups(C group,60 only,divided into T2-5 periods,each period was divided into U0,U1,and U2 groups).C group of homemade blocking devices used to open the chest to block left anterior descending(LAD)coronary artery blood flow,and in 30 Minute(min)of ischemia lifting block,reperfusion began.In group B,the same site was not blocked after thoracotomy;group A received no treatment.Three groups of rabbits were checked by the electrocardiogram and conventional echocardiography before operation,ischemia for 30 min,reperfusion for 30 min,60 min,120 min and 180 min,respectively.3.Targeted acoustic contrast agent preparation and identification:The IL-8 MAB was conjugated to the surface of Targestar SA microbubbles by biotin method.microscope micro bubble size,shape and dispersion and through the glass agglutination experiment and fluorescence dyeing experiments show coupling was successful.4.Ultrasound microbubble destruction process:Routine echocardiography was performed on the three groups of rabbits,and then each group of rabbits in the Ui and U2 groups was subjected to myocardial contrast echocardiography using a ready-made contrast agent containing IL-8 monoclonal antibody.3 min later,The low-power ultrasonic bubble burster was used for microbubble blasting.The irradiation time was 30 seconds,the intermediate interval was 10 seconds,and the continuous irradiation time was 1 minute.The pre-and post-irradiation dynamic images were obtained and analyzed by Quantification software(QLAB).5.Enzyme linked immunosorbent assay(ELISA)analysis:Each group of rabbits was sacrificed to take myocardial tissue for ELISA test to detect the IL-8 content in myocardial tissue.Results:1.Biological effects of ultrasound irradiation on myocardial tissue of rabbits:Myocardium without edema and hemorrhage after ultrasound irradiation with intensity of 0.5 W/cm2;Myocardial tissue slight edema after 0.75 W/cm2 intensity ultrasound irradiation,no bleeding;After 1.0 W/cm2 intensity ultrasound irradiation,there was slightly hemorrhage in the myocardial tissue,but a small amount of inflammatory cells infiltrated;1.5 W/cm2 intensity ultrasound destroyed microbubbles and the cardiac tissue was hemorrhaged,and the inflammatory cells were infiltrated to a certain extent.2.The rabbit myocardial ischemia-reperfusion model:preoperative observing three groups of rabbit two-dimensional ultrasound,ecg are no exception.Electrocardiogram:C group rabbits ischemia 30 min ST segment elevation,reperfusion 45 min began to ST is fall of and the heart rate was slightly increased;B group of rabbit heart rate is a bit faster,ST segment saw no change.Ultrasound observation:C group rabbit ultrasound wall motion amplitude compared with preoperative and A,B group were reduced;B group sham operation group wall motion compared with preoperative no obvious abnormalities.3.Targeted acoustic contrast agent preparation and identification:glass agglutination experiment and fluorescence dyeing experiments confirmed that IL-8 MAB has been successfully coupled to the surface of Targestar SA microbubbles.The coupled microbubbles were similar to unconjugated microbubbles in terms of their size,shapeand properties,indicating that there was no significant change in the physical and biological properties of the ultrasound contrast agent prepared by the covalent coupling method.4.Ultrasound microbubble destruction treatment:Before ultrasound irradiation,There was no significant difference between the ultrasound video intensity of the anterior wall of the myocardial template segment of the close-chest group and the open-chest group and the side wall of the undamaged segment(both P>0.05).which was smaller than that of the I/R group(both P<0.05).In the ischemic reperfusion group,the ultrasound intensity of the anterior wall of the model segment increased with the extension of reperfusion time,and the intensity of ultrasound video increased continuouslyand was significantly higher than that of the undamaged segmen(both P<0.05).The intensity of the ultrasound video intensity peaked at Reperfusion 120 min(Rep 120 min).There was no significant change in the video intensity of the uninjured segmental sidewall ultrasound video intensity(UVI)with the extension of reperfusion time(both P>0.05).After ultrasonic irradiation,there was no significant difference in UVI between the 0.5W/cm2 group and the 0.75W/cm2 group in the close-chest and the open-chest group(P>0.05);the closed-chest group,the open-chest group,and the I/R group in the non-lesioned myocardial segment was no significant difference in UVI between0.5 W/cm2 group in the 0.75 W/cm2 group(both P>0.05).In I/R group,At the same reperfusionsame time,different irradiation intensity was used to form the anterior wall of the segment,and the UVI in the 0.75W/cm2 group was lower than 0.5W/cm2 group(P<0.05);and all were lower than the pre-irradiation(P<0.05).At the same irradiation intensity,the UVI increases with the extension of reperfusion time,and the highest isat Rep 120 min.Ultrasonic Video Intensity Difference(VID):There was no significant difference in the VID value of different ultrasound intensity of the anterior wall of the myocardium in the close-chest group and the open-chest group(P>0.05).However,At the same irradiation intensity,the close-chest group and the open-chest group of VID were smaller than those in I/R group(both P<0.05).I/R group of the same time period under different intensity ultrasound irradiation,the 0.75W/cm2 VID was higher than that of the 0.5W/cm2 group underunder different intensity ultrasound irradiation(P<0.05);However,under the same irradiation conditions,there was no significant difference in VID with prolonged perfusion time(P>0.05).Ultrasound video intensity change rate:At the same reperfusion time during the same period of the myocardial model segment,the 0.75W/cm2 VID was higher than that of the 0.5W/cm2 group underunder different intensity ultrasound irradiation(P<0.05);However,under the same irradiation conditions,the rate of change of ultrasound video intensity gradually decreases with the extension of reperfusion time(P>0.05).5.ELIS A of myocardial tissue analysis:Without ultrasound irradiation(U0 group):The levels of IL-8 in the myocardial tissue of the close-chest group were lower than open-chest group,and the difference was not statistically significant(P>0.05).The levels of IL-8 in myocardial tissue of the close-chest group and the open-chest group were lower than I/R group(both P<0.05).It was confirmed that IL-8 was secreted from myocardial cells after reperfusion injury by ligation of LAD.The levels of IL-8 in the untreated myocardial tissue of I/R group increased with the prolongation of myocardial reperfusion time,and peaked at Reperfusion 120 min.Cardiomyocytes treated with reperfusion injury release large amounts of IL-8.After ultrasonic irradiation(U1,U2 group):The levels of IL-8 in myocardial tissue at 0.75w/cm2 were lower than 0.5w/cm2 in the close-chest group and the open-chest group,but the difference was not statistically significant(both P>0.05).At the same reperfusion time in the I/R group,the levels of IL-8 in the 0.75w/cm2 group was less than 0.5w/cm2(P<0.05).After the same intensity,the levels of IL-8 in the myocardial tissue was increased with the prolongation of reperfusion time.Difference of myocardial IL-8 by ultrasound irradiation:There was no significant difference in the level of IL-8 in myocardial tissue between close-chest group and open-chest group(P>0.05).The same intensity of ultrasound irradiation,the differences of IL-8 levels in the close-chest group and theopen-chest group were lower than those in the I/R group(both P<0.05).In the I/R group,The difference of IL-8 content was higher in the 0.75 w/cm 2 group than in the 0.5 w/cm 2 group,under different intensity of ultrasound irradiation in the same period(P<0.05).Neutralization rate of myocardial IL-8 by ultrasound irradiation:There was no significant difference in the IL-8 neutralization rate between0.75w/cm2 group and the 0.5w/cm2 group at the close-chest group and the open-chest control group(P>0.05).In the I/R group,the intensity of IL-8 neutralization was higher in the 0.75 w/cm 2 group than in the 0.5 w/cm 2 group(P<0.05),and the same irradiation intensity,the IL-8 neutralization rate gradually decreases with the extension of reperfusion time.6.The correlation analysis between the difference in the levels of IL-8 in the myocardial tissue(D)and the mean value of the ultrasound video intensity(VID)found that the correlation coefficient(rU1 = 0.645,rU2=0.734),indicates that there is a strong correlation between the two.Conclusion:1.Biological effects of ultrasound irradiation on rabbit myocardial tissues:Different intensity ultrasound destruction of microbubble contrast agent can produce different degrees of biological effects in rabbit myocardial tissue;ultrasound intensity of 1.0-1.5 w/cm2 can cause a certain degree of inflammatory response when mild myocardial injury.2.The rabbit myocardial ischemia-reperfusion model:By blocking LAD coronary artery of the rabbit after 30 minutes,the animal model of I/R can be successfully established,which provides an animal model for the next ultrasound targeted microbubble blasting experiment.3.Targeted acoustic contrast agent preparation and identification:Through glass agglutination experiment and fluorescence dyeing experiments,IL-8 MAB has been successfully coupled to the surface of the Targestar S A microbubble shell.4.The ultrasound intensity of the anterior wall of the model group in the I/RI group was higher than that of the uninjured section side wall,the closed-chest group,and the open-chest group,and the video intensity peaked at Rep 120 min,which indicating IL-8 MAB specifically binds to IL-8,and the IL-8 antibody achieves high aggregation in the inflammatory region.5.Ultrasound irradiation treatment found:The anterior wall of the I/R group,the greater of ultrasound intensity,the greater difference in VID,the higher the rate of change of UVI,and the greater the amount of neutralized IL-8;The same irradiation time,with the increase of the degree of myocardial injury,the difference of VID was not significant,and there was no significant difference in the amount of IL-8,but the change rate of ultrasound video intensity gradually decreased.It was demonstrated that the ultrasound microbubble destruction treatment successfully targeted the IL-8 monoclonal antibody into the inflammatory region of MI/RI,and at the same time,it could inhibit the inflammatory reaction(neutralization of IL-8 antibody),reduce reperfusion injury,and The greater irradiatethe intensity,the sooner the treatment is intervened,the effectthe more obvious.6.ELISA of Myocardial tissueanalysis found:With the increase of myocardial cell injury,the leves of IL-8 in cardiomyocytes gradually increased and peaked at Rep 120 min.Ultrasound irradiation treatment,I/R group in the same period,the greater ultrasound irradiatio intensity,the moreamount of IL-8 in the myocardial tissue;At the same irradiation intensity,with the increase of the degree of injury,there was no significant difference in the neutralized amount of IL-8 in myocardial tissue,but the IL-8 neutralization rate gradually decreased.It shows that the greater intensity of ultrasound irradiation,the earlier the intervention time,the more obvious effect of reducing inflammation.7.The correlation analysis between neutralization of IL-8 level in myocardial tissue and VID value in myocardium tissue found that the correlation coefficient rU1 =0.803,rU2 = 0.857,indicating that there is a strong correlation between them. |
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