微泡增强HIFU消融生物学效应的实验研究
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摘要
第一部分兔VX2肝肿瘤模型的建立及超声成像技术在建模过程中的作用
目的:比较传统开腹瘤块种植法与超声引导下经皮穿刺种植法在兔VX2肝肿瘤模型建立中的应用,并探讨超声成像技术在肿瘤种植及后续观察评价中的作用。
方法: VX2肿瘤组织取自荷瘤种兔腿部VX2肿瘤。50只健康新西兰大白兔随机分为I组和II组,每组25只。I组动物接受传统开腹种植法,将VX2瘤块直接植入肝脏。II组动物接受超声引导下经皮穿刺法,将瘤块植入肝脏。种植后7 d、14 d、21 d、28 d,分别对每组5只动物进行二维灰阶超声成像,彩色多普勒血流成像(Color Doppler flow imaging,CDFI),造影增强脉冲反相谐波超声(Contrast enhanced pluse inversion harmonic ultrasonography, CE PIH US)及造影增强彩色多普勒超声(Contrast enhanced color Doppler ultrasonography, CE CD US)观察。而后处死动物,取出肝脏,进行大体病理学观察,苏木精-伊红(Hematoxylin-eosin,HE)染色、尼克酰胺腺嘌呤二核苷酸-黄递酶(Nicotinamide adenine dinucleotide phosphate-diaphorase,NADPH-d)染色和琥珀酸脱氢酶(Succinic Dehydrogenase,SDH)染色。
结果:种植后28 d的观察期内,I组存活荷瘤动物21只,II组22只。I组种植过程所用时间为21.5±4.1 min (mean±SD),II组种植过程所用时间为16.9±3.4 min (P < 0.05)。采用二维灰阶超声成像测量肿瘤大小,从7 d至28 d,I组肿瘤体积从0.28±0.14 cm3增长至16.49±5.50 cm3, II组肿瘤体积从0.31±0.19 cm3增长至19.79±4.70 cm3,两组间未见显著统计学差异(P > 0.05)。CDFI,CE PIH US及CE CD US示,14 d前大部分肿瘤内部具有丰富血流分布,14 d后血流主要分布在肿瘤边缘部分,而肿瘤内部血供较少,出现坏死组织,并通过HE染色,NADPH-d和SDH染色得以证实。
结论:超声引导下经皮穿刺法建立兔VX2肝肿瘤模型,与传统开腹瘤块种植法相比,种植时间缩短,操作相对简便,且具有相似的成瘤率和肿瘤体积生长变化。二维灰阶超声成像,CDFI,CE PIH US及CE CD US能够在建立肿瘤模型过程中和后续评价肿瘤生长及血供特征方面发挥积极的作用。
第二部分微泡增强HIFU消融正常兔肝脏的实验研究
实验一微泡增强HIFU消融正常兔肝脏的组织学及超微结构改变
目的:初步探讨微泡型超声造影剂SonoVue增强高强度聚焦超声(High intensity focused ultrasound,HIFU)消融正常兔肝的组织学及超微结构变化。
方法:45只健康新西兰大白兔分为I、II、III组,每组15只。I组接受单纯HIFU辐照。II组接受HIFU辐照前,经耳缘静脉注射2 mL 0.9 %生理盐水。III组接受HIFU辐照前,经耳缘静脉以团注法注射0.2 mL超声造影剂SonoVue,并随后迅速推注2 mL 0.9 %生理盐水。采用单点辐照模式,辐照时间2 s,辐照强度600 W。辐照后立即处死动物,将肝脏取出体外,大体病理学测量消融区体积。取消融区、过渡区(距消融区边缘3 mm以内)及周围区(距消融区边缘3 mm以外)组织,HE染色,光镜下计算消融区每1000μm2面积中的空化泡个数。透射电镜观察细胞超微结构变化,计算消融区细胞核膜破坏率(破坏的细胞核膜个数/总的细胞核数目)。
结果:III组兔肝凝固坏死体积(2.41±0.44 cm3,mean±SD)大于I组(0.83±0.16 cm3)及II组(0.80±0.13 cm3)凝固坏死体积(P < 0.05)。HE染色示消融区与未消融区域分界明显,三组消融区均未见大片凝固坏死。III组可见部分细胞膜及胞核破坏并较多空化泡分布(P < 0.05)。电镜下三组消融区细胞内均未见完整细胞器,呈现不可逆凝固坏死,I组及II组消融区细胞膜性结构较III组完整, III组消融区细胞核膜破坏率高于另两组(P < 0.05)。三组中,过渡区均见轻度细胞水样变性,周围区未见明显损伤。结论:微泡型超声造影剂能够稳定增加HIFU消融区体积并更进一步破坏组织细胞结构,从而增强HIFU消融作用。
实验二微泡增强HIFU消融正常兔肝脏后的连续性组织病理学、细胞凋亡和增殖变化
目的:探讨微泡型超声造影剂SonoVue增强HIFU消融正常兔肝脏后一段时间内细胞组织结构、细胞凋亡和增殖细胞核抗原(Proliferative cellular nuclear antigen,PCNA)表达的连续性变化。
方法:50只健康新西兰大白兔分为I、II组,每组25只。I组接受HIFU辐照前,经耳缘静脉注射2 mL 0.9 %生理盐水。II组接受HIFU辐照前,经耳缘静脉以团注法注射0.2 mL超声造影剂SonoVue,并随后迅速推注2 mL 0.9 %生理盐水。HIFU消融靶区设定为长度1 cm线状区域,辐照时间4.8 s,辐照强度600 W。两组在辐照后0 d、1 d、3 d、7 d、14 d分别处死5只动物。取消融区、过渡区(距消融区边缘3 mm以内)及周围区(距消融区边缘3 mm以外)组织,进行大体病理学观察,HE染色光镜观察和透射电镜观察。脱氧核苷酸末端转移酶介导的生物素化脱氧三磷酸尿苷末端缺口标记法(Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling,TUNEL)检测细胞凋亡,免疫组织化学方法检测细胞PCNA表达。
结果:辐照后0 d、1 d、3 d、7 d、14 d,II组消融区体积(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)明显大于I组(0.15±0.06 cm3,0.16±0.07 cm3,0.16±0.04 cm3,0.17±0.07 cm3,0.16±0.07cm3)(P < 0.05)。HE染色示,辐照后3 d、7 d、14 d ,II组过渡区纤维包裹带宽度大于I组(P < 0.05)。辐照后1 d、3 d、7 d、14 d,两组过渡区出现凋亡细胞和PCNA阳性细胞。II组过渡区凋亡指数较I组增高(P < 0.05),两组均在3 d达到峰值。II组过渡区PCNA阳性细胞指数较I组增高(P < 0.05),两组均在7 d达到峰值。透射电镜示辐照后两组消融区细胞坏死,过渡区可见纤维包裹带,II组消融区细胞结构较I组破坏严重。
结论:与单纯HIFU相比,微泡型超声造影剂增强了HIFU消融体积及消融后凝固坏死区机化包裹,对不同时间段过渡区细胞凋亡和增殖能力有不同程度促进趋势,为进一步提高HIFU疗效提供了实验依据。
第三部分微泡增强HIFU消融兔VX2肝肿瘤的实验研究
实验一微泡增强HIFU消融兔VX2肝肿瘤的超声影像学研究
目的:采用常规二维灰阶超声成像、CDFI、能量多普勒成像(Power Doppler imaging,PDI)、CE CD US及CE PIH US,探讨微泡型超声造影剂增强HIFU消融兔VX2肝肿瘤的作用。
方法:50只健康新西兰大白兔接受超声引导下经皮穿刺种植法建立兔VX2肝肿瘤模型。VX2肝肿瘤种植14 d后,所存活荷瘤动物分为两组。采用HIFU消融系统计算机化控制程序将每个肿瘤“消融体”分割为多个“消融面”。II组接受每个“消融面”的辐照前,经耳缘静脉注射0.2 mL SonoVue,并随后迅速推注2 mL 0.9 %生理盐水。I组接受每个“消融面”的辐照前注射2 mL 0.9 %生理盐水作为对照。辐照前及辐照后1 h,进行常规二维灰阶超声成像,CDFI、PDI、CE CD US及CE PIH US观察。
结果:种植后14 d,共存活46只动物,每组23只,均接受HIFU辐照。HIFU辐照后,常规二维灰阶超声成像示消融区弥漫性高强回声光点分布,CE PIH US示消融区内超声造影剂灌注缺失。二维灰阶超声成像示II组消融区体积(2.23±0.83 cm3,mean±SD)较I组消融区体积(1.22±0.55 cm3)明显扩大(P < 0.05)。CE PIH US也显示II组消融区体积(2.96±0.99 cm3)较I组消融区体积(1.41±0.47 cm3)明显扩大(P < 0.05)。CDFI和PDI示I组消融区边缘部分存在残留血流分布,但是II组消融区内未发现残留血流。CE CD US和CE PIH US均显示II组消融区边缘部分残留血流分布较I组明显减少(P < 0.05)。
结论:通过扩大消融区体积和减少消融区内残留血流,微泡型超声造影剂能够加强HIFU消融兔VX2肿瘤的作用。
实验二微泡增强HIFU消融兔VX2肝肿瘤的连续性病理学变化
目的:通过组织病理学、免疫组织化学及酶组织化学染色方法,探讨微泡型超声造影剂增强HIFU消融兔VX2肝肿瘤后一段时间内的连续性生物学效应变化。
方法:50只新西兰大白兔接受超声引导下经皮穿刺种植法建立兔VX2肝肿瘤模型。种植14 d后,所存活荷瘤动物分为两组。HIFU消融系统计算机化控制程序将每个肿瘤“消融体”分割为多个“消融面”。II组接受每个“消融面”的辐照前,经耳缘静脉注射0.2 mL SonoVue溶液,并随后迅速推注2 mL 0.9 %生理盐水。I组在接受HIFU辐照前注射2 mL 0.9 %生理盐水作为对照。辐照后0 d、3 d、7 d及14 d每组各处死5只动物。通过大体病理学方法测量肿瘤及消融区大小。对消融区,过渡区(距消融区边缘3 mm以内的区域)及周围区(距消融区边缘3 mm以外的区域)组织进行HE染色,Ki 67、Bcl-2、CD 54和MMP-2免疫组化染色、NADPH-d及SDH染色。
结果:辐照后0 d、3 d、7 d及14 d,所测量II组消融体积明显大于I组消融体积(P < 0.05)。辐照后0 d,HE染色示两组消融区内未见大片凝固样坏死。3 d后,HE染色示两组消融区内细胞坏死,过渡区有纤维包裹带生成。免疫组化染色示两组的过渡区Ki 67、Bcl-2、CD 54和MMP-2表达增加,两组间未见明显差异(P > 0.05)。辐照后0 d,NADPH-d及SDH染色即示消融区内酶活性丧失,染色呈阴性。在辐照后连续观察中,NADPH-d及SDH染色显示II组消融区残留活性肿瘤组织明显少于I组(P < 0.05)。结论:通过扩大消融范围,减少肿瘤残留及降低肿瘤复发转移风险,微泡型超声造影剂能够有效增强HIFU对兔VX2肿瘤的消融作用。
Part 1 Establish of A VX2 Rabbit Liver Tumor Model and The Role of Ultrasonography for Implantation and Sequential Evaluation
Objective: 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 Liver
Study 1 Histological and Ultrastructure Changes on Normal Rabbit Liver after HIFU Ablation Enhanced by Microbubbles
Objective: 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 Livers
Objective: 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 Tumor
Study 1 HIFU Ablation Enhanced by Microbubbles on Rabbit VX2 Liver Tumors: Assessment with Ultrasound Imaging
Objective: 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 in
group 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.
目的:比较传统开腹瘤块种植法与超声引导下经皮穿刺种植法在兔VX2肝肿瘤模型建立中的应用,并探讨超声成像技术在肿瘤种植及后续观察评价中的作用。
方法: VX2肿瘤组织取自荷瘤种兔腿部VX2肿瘤。50只健康新西兰大白兔随机分为I组和II组,每组25只。I组动物接受传统开腹种植法,将VX2瘤块直接植入肝脏。II组动物接受超声引导下经皮穿刺法,将瘤块植入肝脏。种植后7 d、14 d、21 d、28 d,分别对每组5只动物进行二维灰阶超声成像,彩色多普勒血流成像(Color Doppler flow imaging,CDFI),造影增强脉冲反相谐波超声(Contrast enhanced pluse inversion harmonic ultrasonography, CE PIH US)及造影增强彩色多普勒超声(Contrast enhanced color Doppler ultrasonography, CE CD US)观察。而后处死动物,取出肝脏,进行大体病理学观察,苏木精-伊红(Hematoxylin-eosin,HE)染色、尼克酰胺腺嘌呤二核苷酸-黄递酶(Nicotinamide adenine dinucleotide phosphate-diaphorase,NADPH-d)染色和琥珀酸脱氢酶(Succinic Dehydrogenase,SDH)染色。
结果:种植后28 d的观察期内,I组存活荷瘤动物21只,II组22只。I组种植过程所用时间为21.5±4.1 min (mean±SD),II组种植过程所用时间为16.9±3.4 min (P < 0.05)。采用二维灰阶超声成像测量肿瘤大小,从7 d至28 d,I组肿瘤体积从0.28±0.14 cm3增长至16.49±5.50 cm3, II组肿瘤体积从0.31±0.19 cm3增长至19.79±4.70 cm3,两组间未见显著统计学差异(P > 0.05)。CDFI,CE PIH US及CE CD US示,14 d前大部分肿瘤内部具有丰富血流分布,14 d后血流主要分布在肿瘤边缘部分,而肿瘤内部血供较少,出现坏死组织,并通过HE染色,NADPH-d和SDH染色得以证实。
结论:超声引导下经皮穿刺法建立兔VX2肝肿瘤模型,与传统开腹瘤块种植法相比,种植时间缩短,操作相对简便,且具有相似的成瘤率和肿瘤体积生长变化。二维灰阶超声成像,CDFI,CE PIH US及CE CD US能够在建立肿瘤模型过程中和后续评价肿瘤生长及血供特征方面发挥积极的作用。
第二部分微泡增强HIFU消融正常兔肝脏的实验研究
实验一微泡增强HIFU消融正常兔肝脏的组织学及超微结构改变
目的:初步探讨微泡型超声造影剂SonoVue增强高强度聚焦超声(High intensity focused ultrasound,HIFU)消融正常兔肝的组织学及超微结构变化。
方法:45只健康新西兰大白兔分为I、II、III组,每组15只。I组接受单纯HIFU辐照。II组接受HIFU辐照前,经耳缘静脉注射2 mL 0.9 %生理盐水。III组接受HIFU辐照前,经耳缘静脉以团注法注射0.2 mL超声造影剂SonoVue,并随后迅速推注2 mL 0.9 %生理盐水。采用单点辐照模式,辐照时间2 s,辐照强度600 W。辐照后立即处死动物,将肝脏取出体外,大体病理学测量消融区体积。取消融区、过渡区(距消融区边缘3 mm以内)及周围区(距消融区边缘3 mm以外)组织,HE染色,光镜下计算消融区每1000μm2面积中的空化泡个数。透射电镜观察细胞超微结构变化,计算消融区细胞核膜破坏率(破坏的细胞核膜个数/总的细胞核数目)。
结果:III组兔肝凝固坏死体积(2.41±0.44 cm3,mean±SD)大于I组(0.83±0.16 cm3)及II组(0.80±0.13 cm3)凝固坏死体积(P < 0.05)。HE染色示消融区与未消融区域分界明显,三组消融区均未见大片凝固坏死。III组可见部分细胞膜及胞核破坏并较多空化泡分布(P < 0.05)。电镜下三组消融区细胞内均未见完整细胞器,呈现不可逆凝固坏死,I组及II组消融区细胞膜性结构较III组完整, III组消融区细胞核膜破坏率高于另两组(P < 0.05)。三组中,过渡区均见轻度细胞水样变性,周围区未见明显损伤。结论:微泡型超声造影剂能够稳定增加HIFU消融区体积并更进一步破坏组织细胞结构,从而增强HIFU消融作用。
实验二微泡增强HIFU消融正常兔肝脏后的连续性组织病理学、细胞凋亡和增殖变化
目的:探讨微泡型超声造影剂SonoVue增强HIFU消融正常兔肝脏后一段时间内细胞组织结构、细胞凋亡和增殖细胞核抗原(Proliferative cellular nuclear antigen,PCNA)表达的连续性变化。
方法:50只健康新西兰大白兔分为I、II组,每组25只。I组接受HIFU辐照前,经耳缘静脉注射2 mL 0.9 %生理盐水。II组接受HIFU辐照前,经耳缘静脉以团注法注射0.2 mL超声造影剂SonoVue,并随后迅速推注2 mL 0.9 %生理盐水。HIFU消融靶区设定为长度1 cm线状区域,辐照时间4.8 s,辐照强度600 W。两组在辐照后0 d、1 d、3 d、7 d、14 d分别处死5只动物。取消融区、过渡区(距消融区边缘3 mm以内)及周围区(距消融区边缘3 mm以外)组织,进行大体病理学观察,HE染色光镜观察和透射电镜观察。脱氧核苷酸末端转移酶介导的生物素化脱氧三磷酸尿苷末端缺口标记法(Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling,TUNEL)检测细胞凋亡,免疫组织化学方法检测细胞PCNA表达。
结果:辐照后0 d、1 d、3 d、7 d、14 d,II组消融区体积(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)明显大于I组(0.15±0.06 cm3,0.16±0.07 cm3,0.16±0.04 cm3,0.17±0.07 cm3,0.16±0.07cm3)(P < 0.05)。HE染色示,辐照后3 d、7 d、14 d ,II组过渡区纤维包裹带宽度大于I组(P < 0.05)。辐照后1 d、3 d、7 d、14 d,两组过渡区出现凋亡细胞和PCNA阳性细胞。II组过渡区凋亡指数较I组增高(P < 0.05),两组均在3 d达到峰值。II组过渡区PCNA阳性细胞指数较I组增高(P < 0.05),两组均在7 d达到峰值。透射电镜示辐照后两组消融区细胞坏死,过渡区可见纤维包裹带,II组消融区细胞结构较I组破坏严重。
结论:与单纯HIFU相比,微泡型超声造影剂增强了HIFU消融体积及消融后凝固坏死区机化包裹,对不同时间段过渡区细胞凋亡和增殖能力有不同程度促进趋势,为进一步提高HIFU疗效提供了实验依据。
第三部分微泡增强HIFU消融兔VX2肝肿瘤的实验研究
实验一微泡增强HIFU消融兔VX2肝肿瘤的超声影像学研究
目的:采用常规二维灰阶超声成像、CDFI、能量多普勒成像(Power Doppler imaging,PDI)、CE CD US及CE PIH US,探讨微泡型超声造影剂增强HIFU消融兔VX2肝肿瘤的作用。
方法:50只健康新西兰大白兔接受超声引导下经皮穿刺种植法建立兔VX2肝肿瘤模型。VX2肝肿瘤种植14 d后,所存活荷瘤动物分为两组。采用HIFU消融系统计算机化控制程序将每个肿瘤“消融体”分割为多个“消融面”。II组接受每个“消融面”的辐照前,经耳缘静脉注射0.2 mL SonoVue,并随后迅速推注2 mL 0.9 %生理盐水。I组接受每个“消融面”的辐照前注射2 mL 0.9 %生理盐水作为对照。辐照前及辐照后1 h,进行常规二维灰阶超声成像,CDFI、PDI、CE CD US及CE PIH US观察。
结果:种植后14 d,共存活46只动物,每组23只,均接受HIFU辐照。HIFU辐照后,常规二维灰阶超声成像示消融区弥漫性高强回声光点分布,CE PIH US示消融区内超声造影剂灌注缺失。二维灰阶超声成像示II组消融区体积(2.23±0.83 cm3,mean±SD)较I组消融区体积(1.22±0.55 cm3)明显扩大(P < 0.05)。CE PIH US也显示II组消融区体积(2.96±0.99 cm3)较I组消融区体积(1.41±0.47 cm3)明显扩大(P < 0.05)。CDFI和PDI示I组消融区边缘部分存在残留血流分布,但是II组消融区内未发现残留血流。CE CD US和CE PIH US均显示II组消融区边缘部分残留血流分布较I组明显减少(P < 0.05)。
结论:通过扩大消融区体积和减少消融区内残留血流,微泡型超声造影剂能够加强HIFU消融兔VX2肿瘤的作用。
实验二微泡增强HIFU消融兔VX2肝肿瘤的连续性病理学变化
目的:通过组织病理学、免疫组织化学及酶组织化学染色方法,探讨微泡型超声造影剂增强HIFU消融兔VX2肝肿瘤后一段时间内的连续性生物学效应变化。
方法:50只新西兰大白兔接受超声引导下经皮穿刺种植法建立兔VX2肝肿瘤模型。种植14 d后,所存活荷瘤动物分为两组。HIFU消融系统计算机化控制程序将每个肿瘤“消融体”分割为多个“消融面”。II组接受每个“消融面”的辐照前,经耳缘静脉注射0.2 mL SonoVue溶液,并随后迅速推注2 mL 0.9 %生理盐水。I组在接受HIFU辐照前注射2 mL 0.9 %生理盐水作为对照。辐照后0 d、3 d、7 d及14 d每组各处死5只动物。通过大体病理学方法测量肿瘤及消融区大小。对消融区,过渡区(距消融区边缘3 mm以内的区域)及周围区(距消融区边缘3 mm以外的区域)组织进行HE染色,Ki 67、Bcl-2、CD 54和MMP-2免疫组化染色、NADPH-d及SDH染色。
结果:辐照后0 d、3 d、7 d及14 d,所测量II组消融体积明显大于I组消融体积(P < 0.05)。辐照后0 d,HE染色示两组消融区内未见大片凝固样坏死。3 d后,HE染色示两组消融区内细胞坏死,过渡区有纤维包裹带生成。免疫组化染色示两组的过渡区Ki 67、Bcl-2、CD 54和MMP-2表达增加,两组间未见明显差异(P > 0.05)。辐照后0 d,NADPH-d及SDH染色即示消融区内酶活性丧失,染色呈阴性。在辐照后连续观察中,NADPH-d及SDH染色显示II组消融区残留活性肿瘤组织明显少于I组(P < 0.05)。结论:通过扩大消融范围,减少肿瘤残留及降低肿瘤复发转移风险,微泡型超声造影剂能够有效增强HIFU对兔VX2肿瘤的消融作用。
Part 1 Establish of A VX2 Rabbit Liver Tumor Model and The Role of Ultrasonography for Implantation and Sequential Evaluation
Objective: 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 Liver
Study 1 Histological and Ultrastructure Changes on Normal Rabbit Liver after HIFU Ablation Enhanced by Microbubbles
Objective: 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 Livers
Objective: 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 Tumor
Study 1 HIFU Ablation Enhanced by Microbubbles on Rabbit VX2 Liver Tumors: Assessment with Ultrasound Imaging
Objective: 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 in
group 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.
引文
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