| Part 1 Establish of A VX2 Rabbit Liver Tumor Model and The Role of Ultrasonography for Implantation and Sequential EvaluationObjective: To compare percutaneous US-guided implantation with the surgical implantation for establishing VX2 rabbit liver tumor model and to investigate the role of ultrasonography in the implantation process and the sequential evaluation.Methods: VX2 tumor tissue was obtained from the thigh of a tumor-bearing rabbit. Fifty healthy New Zealand rabbits were randomly divided into two groups (25 per group). Animals in group I underwent surgical implantation, while those in group II received percutaneous US-guided implantation. On 7 d, 14 d, 21 d, and 28 d after implantation, respectively, five rabbits in each group were examined using conventional 2-dimensional gray-scale ultrasonography, color Doppler flow imaging (CDFI), contrast enhanced pulsed-inversion harmonic ultrasonography (CE PIH US) and contrast enhanced color Doppler ultrasonography (CE CD US). Pathological examination was performed using haematoxylin-eosin (HE), nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and succinic dehydrogenase (SDH) stain. Results: During 28-day sequential period, 22 rabbits survived in group I and 23 in group II. The time duration of implantation in group II was 16.9±3.4 min (mean±SD), while that in group I was 21.5±4.1 min (P < 0.05). The tumor volume measured by conventional 2-dimensional gray-scale ultrasonography increased from 0.28±0.14 cm3 on 7 d to 16.49±5.50 cm3 on 28 d in group I, and from 0.31±0.19 cm3 to 19.79±4.70 cm3 in group II, while no significant difference existed between two groups (P > 0.05). On CDFI, CE PIH US and CE CD US, most tumors were hypervascular before 14 d, and after 14 d they were detected with peripheral vessels and the central hypovascular areas which were demonstrated as necrosis areas by HE stain, NADPH-d stain and SDH stain.Conclusions: Compared with the surgical implantation, the percutaneous US-guided implantation achieved a similar success rate with convenient inoculation performance and demanded shorter implantation duration . Conventional 2-dimensional gray-scale ultrasonography, CDFI, CE PIH US and CE CD US were useful in sequential evaluation of tumor growth and characteristic vascularity. Part 2 Experimental Studies of HIFU Ablation Enhanced by Microbubbles on Normal Rabbit LiverStudy 1 Histological and Ultrastructure Changes on Normal Rabbit Liver after HIFU Ablation Enhanced by MicrobubblesObjective: To explore the histological findings and ultrasturcture changes of normal rabbit liver after high-intensity focused ultrasound (HIFU) ablation enhanced by microbubbles of ultrasound contrast agent SonoVue. Methods: Forty-five rabbits were randomly divided into three groups (15 per group). Animals in group III were given injections of 0.2 mL SonoVue intravenously and 2 mL 0.9 % saline; those in group II were given 2 mL 0.9 % saline; and those in group I were not given injections as control. HIFU abltaion was set on a single dot with exposure time at 2 s and acoustic power at 600 W. After ablations, volumes of coagulated areas were measured. Tissues in ablated areas, transition areas (surrounding ablated areas within 3 mm)and surrounding areas (surrounding ablated areas beyond 3 mm) were examined under light microscopy with HE stain and the bubbles per 1000μm2 in ablated areas were calculated. Transmission electron microscopy was used and the percentage of interrupted nuclear membranes (the number of interrupted nuclear membranes/ the total number of nuclei) in ablated areas were calculated.Results: Coagulated volumes in group III (2.41±0.44 cm3, mean±SD) were larger than those in group I (0.83±0.16 cm3) and group II (0.80±0.13 cm3) (P < 0.05). Separated from unablated areas with a clear demarcation line, ablated areas in each group were detected without extensive necrotic tissues under light microscopy with HE stain. A few interrupted cell membranes and nuclei were detected in ablated areas in group III. More cavitation bubbles were observed in ablated areas in group III than those in the other two groups (P < 0.05). Transmission electron microscopy showed obvious coagulated necrosis in ablated areas in each group. More severe cell ultrastructure disorder, including the higher percentage of interrupted cell nuclear membranes, in group III than in the other two groups (P < 0.05). In each group, transition areas apeared lightly hydropic degeneration and surrounding areas were not affected. Conclusions: Ultrasound contrast agent can substantially increase the coagulated volume of HIFU and enhance the necrosis of ablated tissues, suggesting that microbubble be useful for improving HIFU efficiency.Study 2 Sequential Histopathological Findings, Apoptosis and Cell Proliferation after HIFU Ablation Enhanced by Microbubbles on Normal Rabbit LiversObjective: To sequentially observe histopathological findings, apoptosis and the expression of proliferative cellular nuclear antigen (PCNA) in the rabbit liver tissue after HIFU ablation enhanced by ultrasound contrast agent SonoVue. Methods: Fifty rabbits were divided into two groups randomly (25 per group). Rabbits in group II received injections with 0.2 mL ultrasound contrast agent SonoVue and 2 mL 0.9 % saline before HIFU ablation, and those in group I received injections with only 2 mL 0.9 % saline. HIFU ablation was set on a 1- cm line with exposure time 4.8 s and acoustic power 600 W. Five rabbits in each group were killed, respectively, on 0 d, 1 d, 3 d, 7 d and 14 d after HIFU ablation. Tissues in ablated areas, transition areas (surrounding ablated areas within 3 mm) and surrounding areas (surrounding ablated areas beyond 3 mm) were excised for HE stain, immunohistochemical stain for PCNA expression, terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling (TUNEL) stain for apoptosis,and observation under transmission electron microscopy.Results: On 0 d, 1 d, 3 d, 7 d and 14 d , the coagulated volume in group II (0.39±0.11 cm3,0.42±0.14 cm3,0.36±0.10 cm3,0.45±0.14 cm3,0.43±0.11 cm3, mean±SD) was larger than that in group I (0.15±0.06 cm3,0.16±0.07 cm3,0.16±0.04 cm3,0.17±0.07 cm3,0.16±0.07 cm3) (P < 0.05). Under light microscopy, a fibra band composed of fibrocytes and capillary vessels was detected in transition area after 3 days, and the fibra band in group II was wider than that in group I (P < 0.05). Apoptosis cells and PCNA-positive cells were detected in transition areas, but not in other areas. The apoptosis index in transition areas in group II was higher than that in group I (P < 0.05), and the indexes in both groups reached the peak values on 3 d. PCNA-positive index in transition areas in group II was higher that that in group I (P < 0.05), and the indexes in both groups reached the peak values on 7 d. Transmission Electron microscopy showed ablated areas in both groups were necrotic, and fibra bands were detected in transition areas. The cell structure in ablated areas in group II were destroyed more severely than that in group I.Conclusions: Compared with single HIFU, HIFU ablation enhanced by ultrasound contrast agent can improve the organization of coagulated areas, the apoptosis and cell proliferation in transition areas sequentially after ablation, which may be explored to benefit enhanced-HIFU treatment. Part 3 Experimental Studies of HIFU ablation Enhanced by Microbubbles on Rabbit VX2 Liver TumorStudy 1 HIFU Ablation Enhanced by Microbubbles on Rabbit VX2 Liver Tumors: Assessment with Ultrasound ImagingObjective: We investigated effects of HIFU enhanced by contrast agent SonoVue on rabbit VX2 liver tumors by using conventional 2-dimensional gray-scale ultrasonography, CDFI, power Doppler imaging (PDI), CE CD US, and CE PIH US. Methods: Fourteen days after US-guided implantation of VX2 tumors in livers of 50 rabbits, animals were randomly separated into two groups. Based on principles of HIFU, the volume of the tumor was divided into several parallel''planes''to be ablated. Before ablation on each''plane,''0.2 mL SonoVue was injected in bolus followed with 2 mL 0.9 % saline via ear veins of rabbits ingroup II and 2 mL 0.9 % saline was administrated in group I. Acoustic power of HIFU exposure was set at 600 W. Conventional gray-scale US, CD US, PDI, CE CD US, and CE PIH US were performed before and 1 h after ablation.Results: Twenty-three surviving rabbits in each group underwent HIFU ablation. Conventional gray-scale US showed ablated areas diffusely hyperechoic. On CE PIH US, coagulated areas presented perfusion defect. Conventional gray-scale US showed the ablated volume in group II (2.23±0.83 cm3)was larger than that in group I(1.22±0.55 cm3) (P < 0.05). CE PIH US also showed the ablated volume in group II (2.96±0.99 cm3)was larger than that in group I(1.41±0.47 cm3) (P < 0.05). CDFI and PDI demonstrated residual vessels in periphery ablated areas in group I, but no residual vessels in group II. CE CD US and CE PIH US depicted less residual vessels in periphery ablated areas in group II than those in group I (P < 0.05).Conclusion: By enlarging ablated volume and reducing residual vessels, effects of HIFU ablation on rabbit VX2 liver tumors were enhanced by ultrasound contrast agent.Study 2 HIFU Ablation Enhanced by Microbubbles on Rabbit VX2 Liver Tumors: Sequential Pathological Findings Objective: We investigated sequential effects of HIFU ablation enhanced by ultrasound contrast agent SonoVue using examination of histopathology, immunohistochemistry and enzyme histochemistry.Methods: Fourteen days after US-guided implantation of VX2 tumors in livers of 50 rabbits, animals were randomly separated into two groups. With HIFU algorithm, the tumor volume was divided into'planes'for ablating. Before ablation on each'plane', 0.2 mL SonoVue was administrated in bolus in group II followed with 2 mL 0.9 % saline, and only 2 ml 0.9 % saline were injected in group I . Acoustic power of HIFU was set at 600 W. On 0 d, 3 d, 7 d and 14 d after ablation, the effects on ablated areas, transition areas (surrounding ablated areas within 3 mm) and surrounding areas (surrounding ablated areas beyond 3 mm) were assessed in terms of coagulated volume measurement, histopathological examination, immunohistochemistry of Ki 67, Bcl-2, CD 54 and MMP-2, and enzyme histochemistry of NADPH-d and SDH. Results: On 0 d, 3 d, 7 d and 14 d after ablation, the volume of coagulated areas in group II was larger than that in group I (P < 0.05). In both groups, HE stain demonstrated tissue necrosis in ablated areas and surrounding fibra bands after 3 d. Increased expression of Ki 67, Bcl-2, CD 54 and MMP-2 in transient areas was detected by immunohistochemistry, while no significant difference was detected between two groups (P > 0.05). The stain of NADPH-d and SDH showed enzyme activities in ablated areas decreased dramatically compared to surrounding tissues, while more animals with residual viable tissues in ablated areas in group I were detected than those in group II (P < 0.05).Conclusion: Contrast agent SonoVue can benefit effects of HIFU ablation on rabbit VX2 liver tumors by enlarging the coagulated volume, decreasing the residual tumors and reducing the risk of recurrence and metastasis.
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