Study On The Effect Of Inflammation Mediated Targeted Ultrasound Microbubbles On The Stability Of Abdominal Aortic Plaque In Rabbits

The First PartAn improved method of establishing atherosclerotic plaque model of abdominal aorta in rabbitsObjective:Use an improved early feedng-balloon injury-immune injury combined method to establish the atherosclerotic plaque model of abdominal aortic in rabbits and evaluate the effect of modeling.Looking for an efficient way to build plaque model and lay the foundation for subsequent contrast-enhanced ultrasound and targeted treatment of the atherosclerotic plaque.Methods:120 male New Zealand rabbits were recruited and divided into early feeding group and late feeding group,60 rabbits in each group.Early feeding group:60 1-month experimental rabbits were included.30 rabbits as the EF(early feeding)group and only fed with high-fat and underwent balloon injury.30 rabbits as the EFI(early feeding and immune)group and underwent immune injury also.Late feeding group:60 4-month experimental rabbits were included.30 rabbits as the LF(late feeding)group and only fed with high-fat and underwent balloon injury 30 rabbits as the LFI(late feeding and immune)group and underwent immune injury also.High-fat feeding was performed on all of the experimental rabbits for the first 12 weeks.Balloon injury of the intima of the abdominal aorta was performed at the end of 12th week,followed by immune injury of experimental rabbits in EFI and LFI groups by injecting bovine serum albumin.High-fat feeding was continued on all of the experimental rabbits for the next 8 weeks.Kaplan-meier survival analysis was performed on the survival condition of experimental rabbits in four groups during the modeling time of 20 weeks.All the experimental rabbits in four groups underwent blood lipid examination of triglyceride(TG),total cholesterol(TC)and low density lipoprotein-C(LDL-C)before modeling,after high-fat feeding(at the end of 12th week)and after molding(at the end of 20th week).At the end of 20th week,two-dimensional ultrasound imaging,color doppler flow imaging(CDFI)and pathological examination were performed on the abdominal aorta plaques of experimental rabbits in four groups.Results:1.Model preparation93 of 120 experimental rabbits survived and 80 of them had atherosclerotic plaques.The success rates of EF group,EFI group,LF group and LFI group were 70.0%,66.7%,66.7%and 63.3%,respectively.2.Kaplan-Meier survival analysisThere were no significant difference in the survival rate between EF group and LF group,EFI group and LFI group,EF group and EFI group,LF group and LFI group(P>0.05).3.Histopathological examinationThe order of plaque area ratio(plaque area/total area of intima)showed by Gross Oil red O staining was:EFI group>EF group>LFI group>LF group(P<0.05).Oil red O staining,Sirius red staining and Masson staining showed that there were lipid cells,fiber and smooth muscle components in the plaques.The order of plaque area ratio(the surface area of plaque/the surface area of intima)under the microscope was:EFI group>EF group>LFI group>LF group(P<0.05).4.Blood lipidsThe blood lipids(TC,TG,LDL-C)of the rabbits in the same group between different feeding times(before modeling,after high-fat feeding and after molding)were compared,the differences were statistically significant(P<0.05).The blood lipids(TC,TG,LDL-C)increased with the prolongation of the feeding(P<0.05).At the same feeding time,there were no significant differences in blood lipids(TC,TG and LDL-C)between different groups(EF group,EFI group,LF group and LFI group)(P>0.05).5.Two-dimensional ultrasound evaluation of plaqueAt the end of 20th week,there was no significant difference in the intimal-media thickness(IMT)of the abdominal aorta between four groups(P>0.05).There were significant difference in the maximum plaque thickness(Thickness)between four groups(P<0.05).The order of Thickness was:EFI group>EF group>LFI group>LF group(P<0.05).Conclusions:Early high-fat feeding and immune injury had no effect on the survival rate of the experimental rabbits.The effect of modeling of early high-fat feeding was better than that of late feeding and adding of immune injury was better than not.That is to say,the combined method of early high-fat feeding-balloon injury-immune injury is an efficient method for establishing plaque model.The second partThe effect of inflammation mediated targeted microbubbles on plaque stabilityObjective:To prepare the targeted microbubbles carrying interleukin-8(IL-8)monoclonal antibody and detect the specific adhesion efficiency of the microbubbles.To explore the value of targeted microbubbles in evaluating the inflammation of abdominal aortic plaque in rabbits.To explore the effect of ultrasound-targeted microbubble destruction(UTMD)carrying IL-8 monoclonal antibody on plaque stability.Methods:1.The biotinylated IL-8 monoclonal antibody was coupled to the USphere Labeler microbubbles by biotin avidin bridging method to prepare the targeted microbubbles carrying IL-8 monoclonal antibody.The specific adhesion efficiency of the targeted microbubbles was detected by the parallel plate flow chamber experiment for future targeted contrast-enhanced ultrasound.2.The rabbits which were successfully modeling(at the end of 20th week)in the same modeling group of the first part were randomly divided into targeted groups(EF1 group,EFI1 group,LF1 group and LFI1 group)and bared microbubble groups(EF0 group,EFI0 group,LFo group and LFI0 group).Targeted groups:Used the targeted microbubbles carrying IL-8 monoclonal antibody as contrast agent.Bared microbubble groups:Used the bared USphere Labeler microbubbles as contrast agent.The time-signal intensity curve in the plaques were analyzed by TOMTEC software after contrast-enhanced ultrasound(CEUS).The maximumintensity(IMAX)and time to peak(TTP)of the plaques were obtained.The immunohistochemical stainings were used to observe and count the microvessel density(MVD)(anti-CD34 immunohistochemical staining)and the macrophages(anti-CD68 immunohistochemical staining)in the plaques.3.After contrast-enhanced ultrasound and specimen collection,all the surviving experimental rabbits were randomly divided into three groups:the control group,the simple irradiation group and the targeted treatment group.Simple irradiation group:Underwent UTMD operation once as targeted treatment every other week from the end of 20th to the end of 28th week.The bared USphere Labeler microbubbles was injected.Targeted treatment group:Underwent UTMD operation once as targeted treatment every other week from the end of 20th to the end of 28th week.The targeted microbubbles carrying IL-8 monoclonal antibody was injected.Control group:Received no operation.During the treatment,all the rabbits were fed with common feed.4.The blood lipids,content of IL-8 in serum,two-dimensional and CEUS parameters and pathological parameters of plaques in each group were evaluated before treatment(at the end of 20th week),during treatment(at the end of 24th week)and after treatment(at the end of 28th week).The CEUS parameters were obtained after 30 minutes of the targeted treatment,targeted microbubbles carrying IL-8 monoclonal antibody were used in all the rabbits.Results:1.Evaluation of inflammation in plaque by targeted CEUS1.1 Evaluation of the specific adhesion efficiency of the targeted microbubblesThe targeted microbubbles carrying IL-8 monoclonal antibody can specificly adhere to the IL-8 antigens in the parallel plate flow chamber.The specific adhesion efficiency of the targeted microbubbles was the highest when the shear stress was 0.2 dyn/cm2.The adhesion efficiency decreased obviously when the shear stress was 0.4 dyn/cm2 and decreased to the minimum when the shear stress was 0.5 dyn/cm2.1.2 Contrast-enhanced ultrasound of plaqueIn the same modeling group,IMAX of the plaques in targeted groups were higher than those in bared microbubble groups(P<0.05)while there were no significant difference in TTP between two groups(P>0.05).Between different modeling groups,the order of IMAX between targeted groups was:EFI1 group>EF1 group>LFI1 group>LF1 group(P<0.05)and the order of TTP was:LF1 group>LFI1 group>EF1 group>EFI1 group(P<0.05);The order of IMAX between bared microbubble groups was:EFI0 group>EF0 group>LFI0 group>LF0 group(P<0.05)and the order of TTP was:LFo group>LFI0 group>EF0 group>EFI0 group(P<0.05).1.3 Histopathological examinationThe order of MVD and macrophage count in the plaques between different modeling groups was:EFI group>EF group>LFI group>LF group(P<0.05).1.4 Correlation analysisThere were strong correlation between IMAX and MVD,IMAX and macrophage count,IMAX and macrophage count in each plaque(P<0.05).2.Effect of UTMD on plaque stability2.1 Blood lipidsIn the same experimental group,there were significant differences in blood lipids(TC,TG,LDL-C)between different treatment stages(the 20th,24th and 28th week)(P<0.05).All the blood lipids decreased with the prolongation of the treatment:The 20th week>24th week>28th week(P<0.05).At the same treatment stage,there were no significant difference in blood lipids(TC,TG,LDL-C)between different experimental groups(control,simple irradiation and targeted treatment group)(P>0.05).2.2 Evaluation of plaque by two-dimensional ultrasoundIn the same experimental group(the control and the simple irradiation group),there were no significant difference in IMT and Thickness between different treatment stages(the 20th,24th and 28th week)(P>0.05).The two parameters(IMT and Thickness)of the 28th week were lower than those of the 20th week in the targeted treatment group(P<0.05).There were no significant differences in two parameters(IMT and Thickness)between the 20th and 24th week,the 24th and 28th week in the targeted treatment group(P>0.05).At the same treatment stage(the 20th and 24th week),there were no significant difference in IMT and Thickness between different experimental groups(control,simple irradiation and targeted treatment group)(P>0.05).At the 28th week,the two parameters(IMT and Thickness)of the targeted treatment group were lower than those in the control and simple irradiation group(P<0.05).There were no significant difference in two parameters(IMT and Thickness)between the control and the simple irradiation group at the 28th week(P>0.05).2.3 Evaluation of plaque by targeted CEUSIn the same experimental group(the control and the simple irradiation group),there were no significant difference in IMAX and TTP of the plaques between different treatment stages(the 20th,24th and 28th week)(P>0.05).IMAX decreased(The 20th week>24th week>28th week)and TTP increased(The 20th week<24th week<28th week)with the prolongation of treatment in the targeted treatment group(P<0.05).There were no significant difference in IMAX and TTP of plaques between different experimental groups(control,simple irradiation and targeted treatment group)at the 20th week(P>0.05).At the same treatment stage(the 24th and 28th week),IMAX of plaques in the targeted treatment group were lower than those in the control and the simple irradiation group(P<0.05).TTP of plaques in the targeted treatment group were higher than those in the control and simple irradiation group(P<0.05).There were no significant differences in the two CEUS parameters(IMAX and TTP)of plaques between the control and simple irradiation group in these two treatment stages(P>0.05)2.4 IL-8 content in serumIn the same experimental group(the control and the simple irradiation group),there were no significant difference in the IL-8 content in serum of the rabbits between different treatment stages(the 20th,24th and 28th week)(P>0.05).The IL-8 content in serum of the rabbits decreased with the prolongation of treatment in the targeted treatment group:The 20th week>24th week>28th week(P<0.05)There were no significant difference in the IL-8 content in serum of the rabbits between different experimental groups(control,simple irradiation and targeted treatment group)at the 20th week(P>0.05).At the same treatment stage(the 24th and 28th week),the IL-8 content in serum of the rabbits in the targeted treatment group were lower than those in the control and the simple irradiation group(P<0.05).There were no significant difference in the IL-8 content in serum of the rabbits between the control and simple irradiation group in these two treatment stages(P>0.05).2.5 Histopathological examinationIn the same experimental group(the control and the simple irradiation group),there were no significant difference in MVD and macrophage count between different treatment stages(the 20th,24th and 28th week)(P>0.05).MVD and macrophage count decreased with the prolongation of the treatment in the targeted treatment group:The 20th week>24th week>28th week(P<0.05).There were no significant difference in MVD and macrophage count between different experimental groups(control,simple irradiation and targeted treatment group)at the 20th week(P>0.05).At the same treatment stage(the 24th and 28th week),the MVD and macrophage count in the targeted treatment group were lower than those in the control and simple irradiation group(P<0.05).There were no significant difference in two parameters(MVD and macrophage count)between the control and simple irradiation group in these two treatment stages(P>0.05).Conclusions:The targeted microbubbles carrying IL-8 monoclonal antibody can be successfully prepared by biotin avidin bridge method.The specific adhesion efficiency of the targeted microbubbles is satisfactory.Targeted contrast-enhanced ultrasound can indirectly reflect the MVD and macrophage count in plaque by quantitatively analyzing the IMAX of the plaque,so as to realize the noninvasive and quantitative evaluation of the degree of inflammation in plaque.Inflammation mediated UTMD can effectively reduce the inflammatory response,inhibit the formation of neovascularization and reduce the number of macrophages in plaque by neutralizing the inflammatory factors.It provides a new idea for finding safe and efficient method to stabilize plaque.