| Part 1 Experimental Study: Effects of the Microbubble-enhanced Ultrasound Combined with Prothrombin on Microwave Ablation in Rabbit LiverObjective: To investigate the effect of the Microbubble-enhanced Ultrasound(MEUS)combined with prothrombin on microwave ablation(MWA)in rabbit liver.Methods: Forty healthy New Zealand rabbits were randomly divided into 4 groups(10 per group): Group A(sham therapeutic ultrasound exposure with normal saline group,sham+NS),Group B(sham therapeutic ultrasound exposure with normal saline and prothrombin group,sham+NS+P),Group C(therapeutic ultrasound with microbubble group,MEUS)and Group D(therapeutic ultrasound plus microbubble with prothrombin group,PMEUS).After treatment,the targeted liver tissues were ablated with microwave(MWA)and the volumes of coagulated areas were measured.Tissues in ablated areas,transition areas and surrounding areas were examined under light microscopy with HE stain and transmission electron microscopy to compare the effects among 4 groups,respectively.Results: After MWA,coagulated volumes in Group D(3.13±0.78cm~3)were larger than those in remaining 3 groups(P<0.05).The coagulated volumes in Group C(1.65±0.51cm~3)were larger than those in Group A(0.53±0.10 cm~3)and Group B(0.46±0.09 cm~3)(P<0.05).There was no significant difference in the coagulated volumes between Group A and Group B.The border between unablated areas and ablated areas was clearly,and ablated areas in each group were detected without extensive necrotic tissues under light microscopy with HE stain.Transmission electron microscopy showed that the cellular ultrastructure disorder was most severe in Group D.Conclusion: MEUS combined with prothrombin can significantly expand ablation volume and improve the therapeutic effects of low power MWA on rabbit liver compared with the control group.Part 2 Experimental Study: Effects and Mechanisms of Microbubble-enhanced Ultrasound Combined with Prothrombin on Blocking the Microvessels in Rabbit VX2 Liver TumorsObjective : To investigate the effects and mechanisms of microbubble-enhanced ultrasound(MEUS)combined with prothrombin on blocking and damaging regional circulation in rabbit VX2 liver tumors.Methods:Fourteen days after the surgical implantation of VX2 tumors in livers of rabbits,32 survival animals were randomly divided into four groups.They were 8 with ultrasound plus normal saline(Group A),8 with ultrasound plus prothrombin(Group B),8 treated with MEUS alone(Group C)and 8 with MEUS and prothrombin(PMEUS)(Group D).Corresponding treatment were used in 4 groups: Contrast-enhanced ultrasound(CEUS)was performed on the exposed liver tumors before and after treatment,and acoustic quantification was done and Peak value was obtained to assess the perfusion of liver tumors.Then,the liver tumors were harvested and examined under light microscopy with HE stain and transmission electron microscopy to compare the effects of treatment among all groups.Results:CEUS images and Peak values of Group A and Group B showed no statistical significance(P>0.05).Both of Group C and Group D showed defects in the contrast filtration,but it was more obvious in Group D.Peak value of Group D decreased significantly(72.94±10.27 to 15.74±8.50,P<0.05).Peak of Group C declined from68.79±6.48 to 29.57±4.31(P<0.05).Piecemeal hemorrhage and intercellular edema were occasionally found in Group C under light microscopy.Obvious cellular edema and hematoma were observed in Group D.Transmission electron microscopy showed that microvascular walls were disrupted and the basement membranes were incomplete in Group C and Group D.Conclusions:PMEUS promotes the endothelial damage and produces larger thrombotic area in liver tumor.It is also verified that prothrombin can selectively cause the injured microvascular of liver tumor to form thrombus and block blood flow quickly with targeting the tumor tissues and it enhanced thermal ablation effects.Part 3 The Effects of Microbubble-enhanced Ultrasound Combined with Prothrombin on Microwave Ablation in Rabbit VX2 Liver Tumors :Assessment with Ultrasound ImagingObjective: To investigate the effect of the microbubble cavitation combined with prothrombin on microwave ablation(MWA)in rabbit VX2 liver tumor,using two-dimensional(2D)gray-scale ultrasonography(US),color Doppler flow imaging(CDFI)and contrast enhanced ultrasonography(CEUS).Methods:Thirty-two rabbits with VX2 tumors were randomly divided into four groups:8 with ultrasound plus normal saline(Group A),8 with ultrasound plus prothrombin(Group B),8 treated with MEUS alone(Group C)and 8 with MEUS and prothrombin(PMEUS)(Group D).After treatment,the targeted liver tumor in all groups were ablated with microwave(MWA).2D gray-scale ultrasonography(US),CDFI and CEUS were performed before and after treatment.Results:Before treatment,tumor volumes shown by 2D gray-scale US and by CEUS had no significant differences(P>0.05).After MWA,2D gray-scale US demonstrated diffuse hyper-echo in ablated areas.Coagulated areas had no vessels;while diffused residual vessels existed in periphery coagulated areas on CDFI in Group A,Group B and Group C.Coagulated areas presented perfusion defect on CEUS.2D Gray-scale US showed that coagulated volumes in Group D were larger than other groups(P <0.05).On CEUS,the coagulated volumes in Group D were also larger than those in remaining 3 groups(P<0.05).The coagulated volumes were larger shown by CEUS than those by 2D gray-scale US(P <0.05).Conclusions:2D gray-scale US and CEUS both can show and measure tumor volume and ablated areas.CEUS can more accurate to show the volume of tumor and ablated areas,to find residual tissue in ablated areas,and it proved to assess the effect accurately of ablation in rabbit VX2 liver tumor.Part 4 The Outcomes and Bioactivities of Microbubble-enhanced Ultrasound Combined with Prothrombin on Microwave Ablation in Rabbit VX2 Liver TumorsObjective:To investigate sequential biological effects and outcomes of microbubbleenhanced ultrasound(MEUS)combined with prothrombin on microwave ablation(MWA)in rabbit VX2 liver tumors,using examination of transmission electron microscopy,histopathology and immunohistochemistry.Methods:80 rabbits with VX2 liver tumors were randomly divided into four groups(20per group):Group A(sham therapeutic ultrasound exposure plus normal saline group,sham+NS);Group B(sham therapeutic ultrasound exposure plus normal saline plus prothrombin group,sham+NS+P);Group C(therapeutic ultrasound plus microbubble group,MEUS)and Group D(therapeutic ultrasound plus microbubble plus prothrombin group,PMEUS).After treatment,the targeted liver tumors in all groups were ablated with MWA.On the 0,3,7 and 14 d,the volumes of coagulated areas of 5 rabbits of each group randomly were measured using contrast enhanced ultrasonography(CEUS).Tissues in ablated areas,transition areas and surrounding areas were examined under light microscopy with HE stain,immunohistochemical stain for PCNA expression,terminal deoxynucleotidyl transferase-mediated biotin-d UTP nick end labeling(TUNEL)stain for apoptosis and transmission electron microscopy to observe the differences among 4groups.Tumor metastasis was graded using visual method.Results: On the 0,3,7 and 14 d,the coagulated volumes(3.059±0.476 cm~3,2.734±0.407cm~3,2.796±0.482 cm~3,10.159±1.444 cm~3)in Group D were larger than those in remaining3 groups(P<0.05);and the coagulated volumes(1.086±0.337 cm~3,0.919±0.174cm~3,1.087±0.354 cm~3,3.707±0.850 cm~3)in Group C were larger than those in Group A(0.447±0.180 cm~3,0.454±0.161 cm~3,0.472±0.154 cm~3,0.765±0.204 cm~3)and Group B(0.467±0.168 cm~3,0.290±0.075 cm~3,0.304±0.080 cm~3,0.652±0.151 cm~3)(P <0.05).On the 7 d and 14 d,the tumor volumes(1.166±0.468 cm~3,1.445±0.593 cm~3)in Group D were smaller than those in remaining 3 groups(P <0.05),and the volumes of tumor(2.447±0.793 cm~3,5.477±1.703 cm~3)in Group C were smaller than those in Group A(6.859±1.126 cm~3,10.698±4.910 cm~3)and Group B(6.475±1.068 cm~3,11.821±1.885 cm~3)(P<0.05).Under light microscopy with HE stain,the ablated areas in 4 groups were detected without extensive necrotic tissues on the 0 d and tissue necrosis after 3 days.In transition area,a fibra band was detected after 3 days and the fibra band in Group D was wider than that in remaining 3 groups(P<0.05).The observations under transmission electron microscopy showed that the cellular ultrastructure disorder in ablated area on the0 d and mitochondrial injury in transition area on the 7 d were more severe in Group D than those in remaining 3 groups.In transition areas,PCNA-positive cells and apoptosis cells were detected.On the 0,3,7 and14 d,PCNA-positive index in transition areas in Group C and Group D were lower than those in Group A and Group B(P <0.05),and the indice in 4 groups reached the peak values on the 7 d.On the 0,3 and 7 d,the apoptosis index in transition areas in Group D was higher than that in remaining 3 groups(P <0.05),and the indice in 4 groups reached the peak values on the 3 d.Compared with the remaining 3 groups,the degree of tumor metastasis in Group D was less,and the time of ocurrence is later.Conclusions : MEUS combined with prothrombin can significantly expand ablation volume,enhance the necrosis of ablated tissues,inhibit tumor growth and metastasis and improve the therapeutic effect of MWA on rabbit VX2 liver tumors.Moreover,it can improve the apoptosis and inhibit cell proliferation in transition areas.The fibra bands in the transition areas can promote the organization and repair the surrounding tissue in the ablation areas. |
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