高频双极脉冲不可逆电穿孔消融猪肝组织的安全性和有效性研究

Analysis of safety and efficacy of irreversible electroporation hepatic ablation with high-frequency bipolar pulse in swine

目的:探讨高频双极脉冲不可逆电穿孔(IRE)消融猪肝组织的安全性和有效性。方法:采用实验研究方法。采用陆军军医大学实验中心巴马小型猪18只,雌雄不限,月龄为(6.8±0.8)个月,月龄范围为5.5~8.0个月。18只巴马小型猪按随机数字表法分为实验组(15只)和对照组(3只),实验组巴马小型猪行高频双极脉冲IRE消融,于消融结束后即刻、消融后第3、7、14、28天各取3只行增强CT检查后处死,取肝组织行组织病理学检查;对照组巴马小型猪行单极脉冲IRE消融,于消融后第3天行增强CT检查后处死,取肝组织行组织病理学检查。检测指标:(1)两组巴马小型猪肌肉收缩强度比较。(2)实验组巴马小型猪IRE消融后CT增强检查影像学表现。(3)实验组巴马小型猪IRE消融后组织病理学表现。(4)两组巴马小型猪IRE消融后消融区肝组织细胞凋亡指数比较。正态分布的计量资料以Mean±SD表示,组间比较采用独立样本t检验。结果:(1)两组巴马小型猪肌肉收缩强度比较:两组巴马小型猪均成功完成IRE消融。实验组巴马小型猪术中肌肉收缩强度为(9.8±0.4)m/s^2,对照组为(48.6±0.5)m/s2,两组比较,差异有统计学意义(t=-163.50,P<0.05)。(2)实验组巴马小型猪IRE消融前后CT增强检查影像学表现:IRE消融结束后即刻、消融后第7天,实验组巴马小型猪CT增强检查见消融区呈低密度影,边界清晰,消融区内及其毗邻大血管未见明显异常,消融后均未出现严重并发症。随消融后时间延长,实验组消融区与正常肝组织边界逐渐模糊,消融区逐渐被正常肝组织替代,消融后第28天增强CT检查可见消融区明显缩小甚至消失。实验组巴马小型猪IRE消融后消融区最长径:消融后即刻为(1.81±0.17)cm、消融后第3天为(1.75±0.19)cm、消融后第7天为(1.32±0.22)cm、消融后第14天为(0.65±0.14)cm、消融后第28天为(0.28±0.10)cm。(3)实验组巴马小型猪IRE消融后组织病理学表现:消融后即刻,实验组巴马小型猪苏木素伊红染色组织病理学检查示消融区细胞肿胀,排列紊乱,部分针道周围可见出血;消融后第3天HE染色组织病理学检查示消融区内胆管与血管形态完整,可见大量深染细胞核、部分溶解或裂开的细胞核和凋亡小体,消融区周围可见大量炎性细胞浸润;消融后第3天血管性血友病因子染色组织病理学检查示完整的血管内皮细胞;消融后第3天原位末端标记法染色检查示消融区内大量核深染的凋亡细胞显著多于消融区外;消融后第3天Von Kossa染色检查示部分黑褐色钙盐沉积;消融后第7、14、28天均可见大量新生的肝细胞从消融区周边向中心生长,且随时间延长呈逐渐增多趋势;消融后第14、28天均可见平滑肌细胞增生;消融后第28天消融区基本被新生细胞替代。(4)两组巴马小型猪IRE消融后消融区肝组织细胞凋亡指数比较:实验组和对照组巴马小型猪IRE消融后第3天消融区肝组织细胞凋亡指数分别为76.67%±0.04%和64.03%±0.05%,两组比较,差异有统计学意义(t=4.79,P<0.05)。结论:高频双极脉冲IRE消融猪肝组织安全、有效,且比单极脉冲IRE消融更加彻底。

Objective:To investigate the safety and efficacy of irreversible electroporation (IRE) hepatic ablation with high-frequency bipolar pulse in swine. Methods:The experimental study was conducted. A total of 18 swines of either gender, aged (6.8+0.8)months with a range of 5.5-8.0 months, were collected from Animal Laboratory Center of Army Medical University. were randomly divided into 15 in experimental group and 3 in control group. The swines in experimental group underwent IRE hepatic ablation with high-frequency bipolar pulse, and 3 swines were chose randomly and underwent enhanced CT examination immediately after ablation, and at 3, 7, 14, and 28 days after ablation. The liver tissues were taken for histopathological examination. The swines in the control group underwent IRE hepatic ablation with high-frequency monopolar burst, and was performed enhanced CT examination at 3 days after ablation. Liver tissues were taken for histopathological examination. Observation indicators:(1) comparison of muscle contraction of siwnes between two groups;(2) imaging performance on enhanced CT after IRE ablation in the experimental group;(3) hepatic histopathological findings after IRE ablation in the experimental group;(4) comparison of apoptotic index in the ablation zone between two groups. The measurement data with normal distribution were expressed as Mean±SD, and comparison between groups was performed by the independent sample t test. Results:(1) Comparison of muscle contraction between two groups: swines in both groups underwent ablation successfully. The degree of muscle contraction was (9.8±0.4)m/s2 and (48.6±0.5)m/s^2 in the experimental group and in the control group, respectively, showing statistically significant difference between the two groups (t=-163.50, P<0.05).(2) Imaging performance on enhanced CT after IRE ablation in the experimental group: the enhanced CT examination of swines immediately after IRE ablation showed a low-density shadow and clear boundary in the ablation zone. There was no obvious abnormality in the ablation zone and its adjacent large vessels. No serious complications occurred after the ablation. The boundary between the ablation zone and the normal liver tissue of the experimental group gradually became blurred over time, and the ablation zone was gradually replaced by normal liver tissue. The ablation zone at the 28 days after ablation was significantly reduced or even disappeared on imaging of enhanced CT examination.The maximum diameter of the ablation zone was (1.81±0.17)cm immediately after ablation,(1.75±0.19)cm at the 3 days after ablation,(1.32±0.22)cm at the 7 days after ablation,(0.65±0.14)cm at the 14 days after ablation,(0.28±0.10)cm at the 28 days after ablation, respectively.(3) Hepatic histopathological findings after IRE ablation in the experimental group: the HE staining of ablated tissue immediately after ablation showed that the cells in the ablation zone were swollen, arranged disorderly, and bleeding was observed around some of the needles.The bile ducts and blood vessels were intact in the ablation zone, and a large number of deeply stained nuclei were seen at 3 days after ablation, some of the nucleus and apoptotic bodies were partially dissolved or cleaved. A large number of inflammatory cell were infiltrated around the ablation zone. Intact vascular and biliary endothelial cells were observed by von Willebrand factor staining, a larger number of apoptotic cells with deeply stained nuclei in the ablation zone were observed by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining, and partial deposited dark brown calcium salt was seen by Von Kossa staining. More newborn hepatocytes were observed growing from the periphery of the ablation zone to the center at the 7, 14, 28 days after ablation. Smooth muscle cell proliferation was observed at 14 and 28 days after ablation. The ablation zone was replaced by new cells on 28 days after ablation.(4) Comparison of apoptotic index in the ablation zone between two groups: the apoptotic index of the ablation zone was significantly higher in the experimental group than in the control group on the 3 days after operation (76.67%±0.04% vs. 64.03%±0.05%, t=4.79, P<0.05). Conclusion:IRE hepatic ablation of swine using high-frequency bipolar pulse is safe and reliable, and it has more apoptotic cells than IRE ablation with high-frequency monopolar burst.