超声引导不可逆性电穿孔消融山羊肝脏组织的实验研究
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摘要
电穿孔(electroporation)是指一定场强(kV/cm级)、脉宽(μs-ms级)的脉冲电场作用下,细胞膜的脂质双分子层重新排列形成了一些被称为微孔的亲水性通道,使许多平时不能穿越的亲水性大分子能顺利通过。根据外加脉冲电场撤消后细胞膜能否恢复到正常生理状态,电穿孔可分为可逆性电穿孔(reversible electroporation,RE)和不可逆性电穿孔(irreversible electroporation,IRE)。RE是电化学疗法(electrochemotherapy,ECT)和基因电转染技术的理论基础;IRE是在ECT基础上发展起来的,通过提高电场能量使处理区细胞膜发生不可逆性穿孔,达到杀灭肿瘤细胞的目的,为摆脱化疗药物的耐药性、毒副反应,单独使用脉冲电场治疗肿瘤带来了希望。微秒级不可逆性电穿孔技术细胞效应机制独特,前期研究已经证实该方法的有效性及其广阔的应用前景,但电场的施加必须以电极针为传导介质,既往研究多数是针对体表病变或开腹进行消融治疗,一定程度上阻碍了IRE技术的临床应用。基于无创的医学影像技术为IRE的发展带来了新的思路,超声、CT、MRI在深部肿瘤的局部治疗中能起到引导、定位、监测等作用,其中超声因其使用方便、价格经济而使用广泛。为推进IRE技术的临床化进程,本实验在已有细胞及小动物实验的基础上,选择大型动物山羊为对象,试图采用超声影像引导,实时监测电极针穿刺,靶向消融肝脏组织,分别从组织定位的准确性、靶向组织的损伤、超声影像学变化特点、超声测量靶区径线的准确性进行评估,并探讨消融术后靶区的组织学变化特点等,对微创IRE治疗技术的可行性、有效性及安全性进行较全面的实验研究,解决IRE临床应用难题。本研究在国内尚属空白,有一定的创新性。
第一部分:超声引导不可逆性电穿孔消融山羊肝脏组织的可行性及安全性探讨
第一节:IRE消融正常山羊肝脏组织的参数筛选
目的:筛选固定场强、脉宽及频率的IRE消融正常山羊肝脏组织的最适宜电场参数。
方法:采用针距2cm的两针电极,固定脉宽100μs、频率1Hz、电压2000V,梯度改变脉冲个数30-150个脉冲,每次递增30个脉冲的IRE作用于开腹后的山羊肝脏,肉眼观察处理区轮廓与仿真电场相的吻合度,多路温度巡检仪测量处理前后处理区温度变化。24h后处死动物,处理区TTC染色,游标卡尺测量不着色区最大坏死径线。
结果:消融区径线随着脉冲个数的增加逐渐扩大,围绕电极针的两个圆形消融区逐步融合,当脉冲增加至120个时,消融范围趋于稳定,坏死区域轮廓与针状电极的有限元仿真电场FEMLAB分布基本吻合。120个脉冲组与30个、60个、90个脉冲组最大坏死径线均有统计学差异(P<0.05),而120个脉冲处理组与150个脉冲组之间的最大坏死轮廓径线大小无统计学意义(P>0.05),120个脉冲可能是该参数下IRE剂量作用的一个饱和点。消融区温度在IRE作用前后无明显差别(P>0.05),均在组织热损伤温度以下。
结论:针距2cm的两针电极,固定脉宽100μs、频率1Hz和电压2000V的条件下,IRE消融在体山羊肝脏的最佳脉冲个数是120个。IRE技术具有非热效应,呈现出与传统的温度依赖的肿瘤消融技术不同的独特优势,不受血液流动热沉效应的影响。
第二节:超声引导IRE消融山羊肝脏组织的可行性及B超影像学变化特点
目的:探讨超声引导经皮穿刺微创IRE消融山羊肝脏组织的可行性以及B超影像学变化特点。
方法:固定脉宽100μs,频率1Hz,电压2000V,脉冲数120个,彩色超声探头引导电极针经皮穿刺山定位靶区羊肝脏组织,施加脉冲电场。观察IRE处理后即刻、24小时、3天、7天、14天靶区肝脏组织大体标本以及超声灰度变化。
结果:超声引导经皮IRE消融靶区组织定位准确,电极针显影清楚。IRE消融后即刻与正常肝脏组织回声强度相比较,治疗区域呈低强度回声,靠近电极针处回声稍高,处理区域界限清晰。治疗后24h,回声强度变化范围较处理后即刻有所缩小,回声强度增高,呈稍高强度回声。IRE消融后3d,处理区回声强度较24h略有升高,边界更加清晰。IRE消融术后7天复查B超,处理区较前缩小,呈高回声,此时回声强度最高,消融边界在新增生的纤维结缔组织的牵扯下变得不规则。术后14天复查B超时,消融区较前明显缩小,回声强度也有所下降,呈稍高回声表现,超声回声杂乱,不均质,其内见点状高回声,消融区坏死组织被机化吸收。各时间点的肝脏靶区超声回声变化区域轮廓与大体标本基本一致。
结论:采用无创性的超声影像监测引导技术,实验中可以清楚观察到电极针的进针深度及位置,定位准确。在IRE消融后即刻以及术后,靶区超声影像存在规律性的变化,超声有希望作为IRE消融肝脏组织术中监测以及术后进行随访的有效手段。将超声引导与不可逆性电穿孔治疗结合使IRE微创消融体内深部肿瘤成为可能,确保了治疗的安全性。
第三节:超声引导IRE消融山羊肝脏组织的安全性探讨
目的:研究IRE消融山羊肝脏对山羊全身情况的影响,探讨IRE消融技术的安全性。
方法:固定脉宽100μs,频率1Hz,电压2000V,脉冲数120个。彩色超声探头引导电极针经皮定位穿刺处理山羊肝脏组织,施加脉冲电场,比较术前、术后即刻的心率、呼吸、心电生理(ECG)变化。同时经耳缘静脉抽血,评价IRE处理后即刻、24小时、3天、7天、14天不同时间点术后并发症、肝功能、肾功能、全血细胞计数、心肌酶谱(CK-MB)变化。
结果:脉冲电场实施过程中,山羊的呼吸频率和心率较处理前平静时有所增高,治疗结束后,呼吸节律和心率逐渐恢复正常。消融前和消融后即刻,山羊自身前后比较差异无显著性(P>0.05)。IRE消融后24小时至3天山羊肝功ALT、AST呈一过性增高,至术后7天逐渐降低至正常水平,所有肝功能、肾功能、全血细胞计数、心肌酶谱(CK-MB)指标治疗前后差异无显著性(P>0.05)。IRE消融术后山羊复查ECG结果正常,所有山羊无术后心率失常发生、未发现腹腔内出血、感染、肝脏周围邻近器官(胆囊、大网膜、肠管等)损伤。
结论:超声引导IRE微创消融靶区组织安全可行,消融定位准确、范围可控、边界清楚且不会对周围邻近器官造成损伤。
第二部分:超声对IRE消融山羊肝脏靶区的监测意义
目的:研究通过超声影像学变化特点,利用超声实时监测经皮穿刺微创IRE消融山羊肝脏组织靶区范围的准确性和可控性。
方法:固定脉宽100μs、频率1Hz、电压2000V,电极针距2cm,脉冲个数120个。超声探头引导电极针经皮定位穿刺处理山羊肝脏组织,测量电场处理后即刻靶区回声变化范围的最大径线D1。消融后24小时复查B超,测量IRE后24小时靶区回声变化范围的最大径线D2。过量麻醉剂处死山羊,电子游标卡尺测量处理区实际最大消融径线D3。分别比较以上三种测量径线之间的关系并且与第一部分第一节山羊开腹直视下相同参数消融24h后测得最大消融径线D4进行比较分析。
结果:①山羊开腹直接消融后24h测得的120脉冲组最大消融直径D4[(35.96±2.52)mm]较超声引导IRE经皮微创消融后24h山羊标本的实际最大消融直径D3无显著差异(P>0.05)。②D1、D2、D3三组数据中,IRE处理后即刻超声测量回声变化范围最大径线D1[(39.58±2.13)mm]数值最大,其次为24h后超声回声变化最大径线D2[(37.07±3.51)mm],两者之间差异具有统计学意义(P<0.05);处理后24h肝脏取材测量的实际最大消融径线D3[(36.44±2.04)mm]最小,但是与D2没有显著差异(P>0.05)。IRE处理后即刻超声回声变化最大径线D1与处理后24h肝脏实际测量到的最大消融径线D3呈线性相关(r=0.949)。
结论:超声引导电极针经皮穿刺定位消融山羊肝脏靶区与直视下消融效果一致。在对靶区消融范围实时监测的过程中,超声用于测量靶区实际消融径线准确可行。IRE后即刻,超声测量消融范围径线较IRE后24h超声测量及实际标本测量径线显著偏大;但是和实际测量的消融范围径线之间存在良好的线性关系。通过实时B超监测可以对消融目标的有效范围进行较为准确的控制。
第三部分:IRE消融山羊肝脏的组织学观察
第一节:IRE术后肝脏组织学及肝细胞超微结构变化特点
目的:评价IRE消融山羊肝脏组织的有效性,探讨IRE消融术后不同时间点肝细胞组织学及电镜超微结构变化特点。
方法:固定脉宽100μs,频率1Hz,电压2000V,脉冲数120个,彩色超声探头引导电极针经皮定位穿刺处理山羊肝脏组织。于治疗后即刻、24小时、3天、7天、14天处死动物,取材,行HE、葡萄糖-6-磷酸酶( Glucose-6-phosphatse,G-6-P )、琥珀酸脱氢酶(Succinodehydrogenase, SDH)、毛细胆管吡啶银及网状纤维组织化学染色,观察组织细胞形态、两种酶活性产物的变化及IRE处理区毛细胆管及网状纤维的病理改变。电子显微镜观察IRE术后即刻、24小时及3天肝脏超微结构变化。
结果:①HE染色显示,IRE处理后即刻靶区内肝细胞胞浆内空泡形成、细胞明显水肿;24小时后观察到处理区肝细胞完全坏死,与周围正常细胞分界清楚,并出现大量炎症细胞浸润;IRE处理后14天,处理区坏死组织主要被肉芽组织填充、机化。②肝脏超微结构证实IRE处理后肝脏组织遭到严重破坏,表现为肝细胞胞膜破裂,线粒体肿胀坏死、粗面内质网扩张、肝血窦毛细血管内皮细胞连续性破坏等改变。③靶区肝细胞酶组织化学染色显示,IRE作用后即刻,G-6-P和SDH活性无明显变化;而24小时后,活性显著降低;3天后活性最低。消融区内毛细胆管结构破坏消失,网状纤维断裂破碎。
结论:G-6-P、SDH两种细胞酶学染色,从细胞内功能的改变与HE染色及电镜超微结构观察到肝细胞发生的形态学变化相互印证,充分证实了IRE对靶区细胞的完全非选择性消融效果。IRE可以实现病变区域具有细胞膜性结构成分组织的广泛性作用,同时对其内细胞骨架结构产生较强破坏效果。
第二节:IRE对消融区域内血管结构的影响
目的:探讨IRE对消融区域内血管结构的影响。
方法:固定脉宽100μs,频率1Hz,电压2000V,脉冲数120个,彩色超声探头引导电极针经皮消融山羊肝脏组织。处理后即刻、24小时、3天、7天、14天复查超声了解消融区内血管二维超声及多普勒血流影像学变化。逐层水平切开处理区肝脏组织,肉眼观察到消融区内包含有血管结构即取材,4%多聚甲醛固定,石蜡包埋,行HE、VG及Masson染色组织染色。
结果:标本取材后肉眼观察,IRE作用范围区域内血管结构完整(包括管径在10mm以上的肝内大血管和管径在2mm左右的肝内小血管)。血管二维影像提示消融区内的血管连续性完整,走行清晰,CDFI可以观察到血管内彩色多普勒血流频谱。血管组织学染色示内皮细胞连续性完好,血管周围弹性纤维、血管平滑肌结构完整。
结论:IRE非热消融治疗后病灶内滋养血管和毛细血管网大量破坏,而靶区内较大血管无损伤或栓塞。脉冲电场不可逆性电穿孔保留了消融区重要的管道结构(如动脉、静脉、胆管),血管周围的弹性纤维、胶原纤维结构完整。IRE不受血液流动热沉效应的影响,保证电场能量在靶区组织内的有效传递和积累,为生长在大血管周围、手术进行有困难的肿瘤消融提供可能。
第三节:IRE致消融区组织细胞凋亡的实验观察
目的:观察IRE消融山羊肝脏组织靶区肝细胞坏死、凋亡与电场分布的关系。
方法:固定电压2000V、脉宽100μs、频率1Hz,经彩色超声探头引导电极针经皮定位肝脏,施加120个脉冲电场。24h后处死动物,取电极针周围肝脏组织标本(包括脉冲电场处理区域中心部分和处理区域与正常组织交界区)和正常组织(作为对照)。将标本进行处理后,分别行HE染色观察组织的病理学变化;采用DNA ladder和TUNEL法检测组织细胞凋亡,采用流式细胞仪分析细胞周期。
结果:病理学观察可见,消融处理区的肝细胞完全坏死,围绕电极针的中心区域呈现凝固性坏死。TUNEL法显示凋亡细胞(细胞核为深黑色,胞质呈淡棕黄色)主要集中在处理区与正常组织的交界区。DNA ladder法在处理区与正常组织交界区组织检测到400-1000bp的梯状条带;流式细胞术分析显示,该区细胞DNA二倍体峰前面出现亚二倍体凋亡峰。
结论:采用IRE消融山羊肝脏组织,细胞在电场强度分布最高处(即围绕电极针的中心区域)呈现凝固性坏死;在电极针周边处理区域,随电场强度的衰减,交界区肝细胞还呈现出凋亡途径的细胞程序性死亡(programmed cell death,PCD)。肿瘤组织在脉冲电场消融时被“超范围切除”,可以有益于降低治疗后的复发率及对周边正常组织的侵犯、转移。
Electroporation occurs when the application of an electric field with electric field intensity of kV/cm and pulse duration ofμs to ms grade across a cell alters the transmembrane potential. Thus, the lipid bilayer structure is disrupted and small nanopores are created in the cell membrane, and the nanopores then allow micromolecules and macromolecules to be transported into and out of the cells. Electroporation could be used as reversible electroporation (RE) or irreversible electroporation (IRE) due to the nanopores recover or not. This process when used in RE has been used in medicine and research for drug or macromolecule delivery into cells, such as electrochemotherapy (ECT). With sufficiently high voltage, these pores become permanent and contribute to cell death by interfering with cell homeostasis.This is the mechanism of IRE ablation method. IRE tumor treatment process doesn’t depend on chemotherapeutics which might take some disadvantages of toxicity and drug resistance. In the past findings, researchers had fully certified the extensive application prospect of IRE though tumor cells and solid tumor ablation treatment experiments. But in the past, IRE only could be used on body surface or superficial tumor ablation, because lack of some equipment to help for the location of the electrodes placement accurately. With non-thermal cell death and a markedly decreased treatment time, IRE provides a potential new ablation method that can be operated in a well-controlled and focused manner under image monitoring (such as ultrasound). In this study, we used an in vivo goat liver model to evaluate the clinical feasibility of IRE imagine-guided by US, the mechanism of cell death by using histochemical analysis, the effectiveness of tissue destruction near bile ducts and vessels, and the correlation of ablation areas as measured by using US versus gross section examination.
Part 1 Investigation of the feasibility and safety with ultrasound imaging guided irreversible electroporation on goat livers
Chapter 1 Parameter selection of goats’liver ablation with IRE
Objective:To select the most suitable IRE ablation parameters under fixed electric field intensity, permanent duration and frequency.
Methods:IRE induced by 30 to 150 pieces of pulses(Pulse electric fields with a 2-cm probe distance, permanent duration 100μs, frequency 1Hz and voltage 2000V) were performed on goats’livers directly after abdomen discission. 30 pieces of pulses were increased for each group. Temperature of the target area was measured immediately after IRE ablation. All the animals were scarified 24 hours post IRE. Specimens were collected respectively for TTC stain. The largest ablation diameters of specimens were measured by electronic sliding caliper.
Results:It was shown that there was dose-dependence result between the dosage of the electric field and diameters of the ablation zone. Necrosis area enlarged gradually with the increasing of the pulse pieces of electric field. When the hepatic tissue was exposed to pulsed electric fields with 120 pieces of pulses, permanent duration 100μs, frequency 1Hz and voltage 2000V, the diameters of ablation area appeared to be obvious. Necrospy-based measurement demonstrated highly consistency with FEM-anticipated ablation zones. The dose in group 120 pieces of pulses may be considered as the optimal dosage for the hepatic tissue ablation in goat’s livers. There was no significant difference between 120 pulses and 150 pulses group in diameter comparison of the ablation zones (P>0.05). And 120 pulses group had significant difference when compared with 30 pulses, 60 pulses and 90 pulses group separately (P<0.05). The temperature of the injuring tissues was measured at the same time, and there was no significant difference before and after IRE ablation procedure (P>0.05).The temperature of the ablation area was under the regular injure level instantly post IRE.
Conclusion:120 pieces of pulses was the most optimal IRE ablation parameters under fixed pulse electric fields with 2-cm probe distance, permanent duration 100μs, frequency 1Hz and voltage 2000V. Electric field created by IRE was devoid of any joule heating and therefore, non-thermal ablation was achieved. Since it does not depend on heat, IRE can create a focal tumor ablation area which was independent of any heat sink effect.
Chapter 2 Feasibility investigations and characteristic of US imaging changes with ultrasound imaging guided IRE on goat livers
Objective: To investigate the proper method of percutaneous irreversible electroporation (IRE) on goat livers under ultrasonic guidance, and observe the characteristic of ultrasound imaging changes.
Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) applied on the electrodes and placement of the electrodes had been localized into goats’liver under ultrasound guidance though the animal skin to the target area. The treated area was observed by real-time ultrasound scanning and gross appearance observation at the time of 0, 24hours, 3d, 7d and 14d after IRE ablation.
Results: Ultrasound imaging guidance was accurate to focus on the target area. Imagines captured by the ultrasound after IRE procedure were quite different compared with the normal liver imaging. Real-time US images obtained during IRE show hypoechoic areas of ablation with hyperechoic region in close proximity to the probes. Sharp demarcation of IRE-ablated zone is well visualized immediately after the procedure. This ablated area was well correlated with pathologic measurements. US images obtained after 24 hours showed that area of hypoechogenicity becomes hyperechogenic, and seems to have shrunk when compared with US images obtained istantly. US images obtained after 3 days post IRE shows that gray scale of ablation area became more hyperechogenic, and boundary of the ablation area was quite clear. US images obtained after 7 days shows the most hyperechogenic gray scale of ablation area. Meanwhile, the circumsciption injured zone became irregular for hyperplasia of the fibrous connective tissue. 14 days post IRE, US images obtained showed insignificant hyperechogenic images with high point-like echo in the center. The ablation area shrunk obviously. US images correlated well with gross appearance observation at the time of 0, 24hours, 3d, 7d and 14d after IRE ablation.
Conclusion: With real-time monitoring by ultrasonography and well-controlled ablation of the target tissue, percutaneous IRE can provide a novel and unique ablative method for IRE tumor ablation. There was regular changement of US images after IRE ablation. Image-guided provides the fundamental experimental work for future studies on IRE clinical application and follow-up investigation.
Chapter3 Security investigation with ultrasound imaging guided irreversible electroporation on goat livers
Objective: To investigate the influence of IRE ablating to the liver of goat on general condition, and the security of IRE ablation method.
Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) applied on the electrodes and placement of the electrodes had been localized into goats’liver under ultrasound guidance though the animal skin to the target area. Heart rate, respiration frequency, and electrophysiology (ECG) changes between pre and post IRE ablation were compared. Postoperative complications and venous blood analyzing including liver function, kidney function, complete blood count and myocardial enzyme (CK - MB) were compared 0 hour, 24 hours, 3 days, 7 days and 14 days after IRE ablation.
Results: The respiratory frequency and heart rate of goat increased in the process of IRE ablation compared with in rest preoperation, and resumed to the condition of preoperation after IRE ablation. No significant statistic difference was showed between preoperation and postoperation (P>0.05). The ALT and AST progressively increased in postoperation 24h to 3rd day, and returned to normal level in postoperation 7th day. No significant statistic difference was showed between pre and post ablation in liver function, kidney function, complete blood count and myocardial enzyme (CK-MB)level (P>0.05). Instantly after IRE ablation, the ECG showed normal and no arrhythmia of electrophysiology changes. No complications including intraperitoneal hemorrhage, infection, and neighbouring organs damages (gallbladder, greater omentum, intestinal tract, etc) have been found.
Conclusion: IRE ablation was security and validity, and no obvious complications was observed.
Part 2 Intraprocedural US accuracy for IRE ablation area monitoring
Objective: To evaluate the difference between intraprocedural US and gross section measurements and to analysis the accuracy and controllability of monitoring the ablation zones by US.
Methods: Pulsed electric fields (PEFs) with a 2-cm probe distance, permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) applied on the electrodes and placement of the electrodes had been localized into goats’liver under ultrasound guidance though the animal skin to the target area.The ablation zone was monitored and measured in real time with US, which was repeated both immediately after the IRE procedure (D1) and again 24 hours after the IRE ablation procedure (D2), and the gross section (D3) was measured by electric vernier caliper after the animal was sacrificed by taken an overdose of anesthetic agent. Correlations between D1 and D2, D2 and D3, D1 and D3, D3 and D4(24 hours post IRE ablation when 120 pieces of pulses were performed on goat livers directly after abdomen discission ) were statistically analyzed separately.
Results: There was no significant statistic difference showed between D3 and D4[(35.96±2.52)mm](24 hours post IRE ablation when 120 pieces of pulses were performed on goat livers directly after abdomen discission)(P>0.05). Among the three sets of data (D1, D2, and D3), the maximum diameter measured by intraprocedural US immediately after IRE ablation procedure [D1,(39.58±2.13)mm] was the largest diameter, and followed was the data measured by US 24 hours after the procedure [D2(,37.07±3.51)mm]. The two sets were statically different(P<0.05). The gross section measurement [D3,(36.44±2.04)mm] which was measured 24 hours after IRE ablation was the smallest, but it’s not significant statistic difference between D2 and D3(P>0.05). D1 showed a well linear correlation with D3(r=0.949).
Conclusion:Ultrasound-guided percutaneous IRE on goats’liver ablation had the same effect with IRE ablation areas when pulses were performed on goat livers directly after abdomen discission. In the percutaneous IRE ablation procedure, data measured by intraprocedural US correlated well with gross section measurement. Data measured by intraprocedural US immediately after IRE ablation procedure was larger than the same set of data measured by US and though gross section measurement 24 hours post IRE ablation, but correlated well with the latter one. US provided an accurate and available method for IRE target area monitoring.
Part 3 Research on histological features of goat livers post IRE ablation
Chapter 1 Histopathology and ultra-structure changes of goat livers post IRE ablation
Objective: To investigate the histopathology and ultra-structure changes of goat livers with percutaneous irreversible electroporation (IRE) treatment.
Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) were performed on the electrodes which were placed under ultrasound guidance through the animal skin to the target area. The treated area was observed by hematoxylin and eosin (HE) stain, Glucose-6-phosphatse (G-6-P)stain, Succinodehydrogenase (SDH) stain, Cholangioles Pyridine silver stain and reticular fibers silver stain at the time of 0hour, 24hours, 3days, 7days, 14days after IRE ablation. The function of G-6-P and SDH enzymes and histological feature of liver cells, cholangioles and reticular fibers were all observed by light microscope. Ultra-structure changes of liver cells were observed by transmission electron microscope at the time of 0hour, 24hours, and 3days after IRE ablation.
Results: Complete hepatic cell death was observed after the IRE treatment and there was a sharp demarcation between the ablated zone and the non-ablated zone. By 14 days the ablated lesions was almost completely replaced by fibrous scartissue. Ultra-structure changes of liver cells observed by TEM showed that there were rupture of cell membranes, swelling and necrosis of mitochondrion, extension of rough endoplasmic reticulum and destruction of capillaries continuity in the ablated zone. Stain of G-6-P, SDH and cholangioles were all negative 24 hours post IRE. Endothelial cells of the cholangioles and capillaries and reticular cells were ablated at the same time.
Conclusion: Percutaneous IRE can provide a novel and unique method in ablating living cells with lipid bilayer membranes, such as liver cells, endothelial cells of the capillaries, cholangioles or reticular cells. The activity function of G-6-P and SDH in hepatic cells was lost at the same time.
Chapter 2 Effect of IRE ablation on the structure of blood vessels
Objective: To investigate the effect of IRE ablation on the structure of blood vessels.
Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) were performed on the electrodes which were placed under ultrasound guidance through the animal skin to the target area. The treated area was observed by real-time ultrasound scanning and color doppler flow imaging at the time of 0hour, 24hours, 3d, 7d and 14d after IRE ablation. At gross section examination, blood vessel sections in the ablation area were fixed in 4% paraformaldehyde and embedded with paraffin. Histological changes of the treated area were observed by HE stain, VG stain and Masson stain.
Results: Gross section examination showed that structural integrity of vascular within IRE-ablated zone (including the intrahepatic large vessels with diameters of more than 10mm and small intrahepatic vessels with diameters of less than 2mm). The imaging of two-dimensional ultrasound showed that blood vessels were intact and there was color flow angiography in blood vessels. Endothelial cells of were blood vessels not ablated by IRE. The blood vessel wall structures which mainly formed by vascular elastic and collagenous structures, peri-cellular matrix proteins and vascular smooth muscle structures were not damaged by IRE ablation.
Conclusion: IRE-ablation destroyed capillaries and cholangioles, but retained important vital structure such as hepatic arteries, hepatic veins and bile duct and perivascular elastic fiber structure and collagen fiber structure in the ablation zone. It suggested that IRE would not be affected by the heat sink effect of blood flow, which ensured effective transmission and accumulation of electric field energy. Thus, IRE could produce a more complete ablation effect within the lesion area, even with a large vessel traversing the ablation zone.
Chapter 3 Apoptosis of goat livers induced by irreversible electroporation
Objective:To investigate the impact of IRE ablation on apoptosis and necrosis of goat liver cells, and the relationship between these biological changes and distribution of electric fields.
Methods:IRE induced by 120 pieces of pulses(Pulsed electric fields (PEFs) with permanent duration 100μs, frequency 1Hz and voltage 2000V) were performed on goats livers. The electrodes were placed under ultrasound guidance via the animal skin to the target area. All the animals were scarified 24 hours after IRE ablation. Specimens were collected respectively from the center of ablated zone and the margin of ablated zone. Normal hepatic tissue was also collected as control. The specimens were observed by hematoxylin and eosins (HE) stain. Apoptosis was detected by DNA ladder and TUNEL. Hepatic cells were analyzed with flow cytometry (FCM) for cell cycle changes.
Results:Complete hepatic cell death were observed after IRE treatment. In the center of ablated zone (electrode point), coagulative necrosis was obvious. Apoptotic cells stained by TUNEL were found mainly at the margin of the ablated zone. Nucleus and cytoplasm of apoptotic cells were positively stained as pitch black and light buffy color respectively. DNA ladder analysis detected ladder strap with a length of 400 to 1000bp at the margin of ablated zone. Hypodiploid peak was found with FCM analysis in the hepatic cells from this area.
Conclusion:IRE induced liver cell death was by the means of coagulative necrosis in the center of ablation area (electrode point), while programmed cell death by the means of apoptosis was gradually involved as the decay of electric field strength at the margin of ablated zone. IRE could ablate tumor beyond the area of macroscopic observation. It is benefit to reducing recurrence and metastasis in surrounding area after IRE tumor ablation.
第一部分:超声引导不可逆性电穿孔消融山羊肝脏组织的可行性及安全性探讨
第一节:IRE消融正常山羊肝脏组织的参数筛选
目的:筛选固定场强、脉宽及频率的IRE消融正常山羊肝脏组织的最适宜电场参数。
方法:采用针距2cm的两针电极,固定脉宽100μs、频率1Hz、电压2000V,梯度改变脉冲个数30-150个脉冲,每次递增30个脉冲的IRE作用于开腹后的山羊肝脏,肉眼观察处理区轮廓与仿真电场相的吻合度,多路温度巡检仪测量处理前后处理区温度变化。24h后处死动物,处理区TTC染色,游标卡尺测量不着色区最大坏死径线。
结果:消融区径线随着脉冲个数的增加逐渐扩大,围绕电极针的两个圆形消融区逐步融合,当脉冲增加至120个时,消融范围趋于稳定,坏死区域轮廓与针状电极的有限元仿真电场FEMLAB分布基本吻合。120个脉冲组与30个、60个、90个脉冲组最大坏死径线均有统计学差异(P<0.05),而120个脉冲处理组与150个脉冲组之间的最大坏死轮廓径线大小无统计学意义(P>0.05),120个脉冲可能是该参数下IRE剂量作用的一个饱和点。消融区温度在IRE作用前后无明显差别(P>0.05),均在组织热损伤温度以下。
结论:针距2cm的两针电极,固定脉宽100μs、频率1Hz和电压2000V的条件下,IRE消融在体山羊肝脏的最佳脉冲个数是120个。IRE技术具有非热效应,呈现出与传统的温度依赖的肿瘤消融技术不同的独特优势,不受血液流动热沉效应的影响。
第二节:超声引导IRE消融山羊肝脏组织的可行性及B超影像学变化特点
目的:探讨超声引导经皮穿刺微创IRE消融山羊肝脏组织的可行性以及B超影像学变化特点。
方法:固定脉宽100μs,频率1Hz,电压2000V,脉冲数120个,彩色超声探头引导电极针经皮穿刺山定位靶区羊肝脏组织,施加脉冲电场。观察IRE处理后即刻、24小时、3天、7天、14天靶区肝脏组织大体标本以及超声灰度变化。
结果:超声引导经皮IRE消融靶区组织定位准确,电极针显影清楚。IRE消融后即刻与正常肝脏组织回声强度相比较,治疗区域呈低强度回声,靠近电极针处回声稍高,处理区域界限清晰。治疗后24h,回声强度变化范围较处理后即刻有所缩小,回声强度增高,呈稍高强度回声。IRE消融后3d,处理区回声强度较24h略有升高,边界更加清晰。IRE消融术后7天复查B超,处理区较前缩小,呈高回声,此时回声强度最高,消融边界在新增生的纤维结缔组织的牵扯下变得不规则。术后14天复查B超时,消融区较前明显缩小,回声强度也有所下降,呈稍高回声表现,超声回声杂乱,不均质,其内见点状高回声,消融区坏死组织被机化吸收。各时间点的肝脏靶区超声回声变化区域轮廓与大体标本基本一致。
结论:采用无创性的超声影像监测引导技术,实验中可以清楚观察到电极针的进针深度及位置,定位准确。在IRE消融后即刻以及术后,靶区超声影像存在规律性的变化,超声有希望作为IRE消融肝脏组织术中监测以及术后进行随访的有效手段。将超声引导与不可逆性电穿孔治疗结合使IRE微创消融体内深部肿瘤成为可能,确保了治疗的安全性。
第三节:超声引导IRE消融山羊肝脏组织的安全性探讨
目的:研究IRE消融山羊肝脏对山羊全身情况的影响,探讨IRE消融技术的安全性。
方法:固定脉宽100μs,频率1Hz,电压2000V,脉冲数120个。彩色超声探头引导电极针经皮定位穿刺处理山羊肝脏组织,施加脉冲电场,比较术前、术后即刻的心率、呼吸、心电生理(ECG)变化。同时经耳缘静脉抽血,评价IRE处理后即刻、24小时、3天、7天、14天不同时间点术后并发症、肝功能、肾功能、全血细胞计数、心肌酶谱(CK-MB)变化。
结果:脉冲电场实施过程中,山羊的呼吸频率和心率较处理前平静时有所增高,治疗结束后,呼吸节律和心率逐渐恢复正常。消融前和消融后即刻,山羊自身前后比较差异无显著性(P>0.05)。IRE消融后24小时至3天山羊肝功ALT、AST呈一过性增高,至术后7天逐渐降低至正常水平,所有肝功能、肾功能、全血细胞计数、心肌酶谱(CK-MB)指标治疗前后差异无显著性(P>0.05)。IRE消融术后山羊复查ECG结果正常,所有山羊无术后心率失常发生、未发现腹腔内出血、感染、肝脏周围邻近器官(胆囊、大网膜、肠管等)损伤。
结论:超声引导IRE微创消融靶区组织安全可行,消融定位准确、范围可控、边界清楚且不会对周围邻近器官造成损伤。
第二部分:超声对IRE消融山羊肝脏靶区的监测意义
目的:研究通过超声影像学变化特点,利用超声实时监测经皮穿刺微创IRE消融山羊肝脏组织靶区范围的准确性和可控性。
方法:固定脉宽100μs、频率1Hz、电压2000V,电极针距2cm,脉冲个数120个。超声探头引导电极针经皮定位穿刺处理山羊肝脏组织,测量电场处理后即刻靶区回声变化范围的最大径线D1。消融后24小时复查B超,测量IRE后24小时靶区回声变化范围的最大径线D2。过量麻醉剂处死山羊,电子游标卡尺测量处理区实际最大消融径线D3。分别比较以上三种测量径线之间的关系并且与第一部分第一节山羊开腹直视下相同参数消融24h后测得最大消融径线D4进行比较分析。
结果:①山羊开腹直接消融后24h测得的120脉冲组最大消融直径D4[(35.96±2.52)mm]较超声引导IRE经皮微创消融后24h山羊标本的实际最大消融直径D3无显著差异(P>0.05)。②D1、D2、D3三组数据中,IRE处理后即刻超声测量回声变化范围最大径线D1[(39.58±2.13)mm]数值最大,其次为24h后超声回声变化最大径线D2[(37.07±3.51)mm],两者之间差异具有统计学意义(P<0.05);处理后24h肝脏取材测量的实际最大消融径线D3[(36.44±2.04)mm]最小,但是与D2没有显著差异(P>0.05)。IRE处理后即刻超声回声变化最大径线D1与处理后24h肝脏实际测量到的最大消融径线D3呈线性相关(r=0.949)。
结论:超声引导电极针经皮穿刺定位消融山羊肝脏靶区与直视下消融效果一致。在对靶区消融范围实时监测的过程中,超声用于测量靶区实际消融径线准确可行。IRE后即刻,超声测量消融范围径线较IRE后24h超声测量及实际标本测量径线显著偏大;但是和实际测量的消融范围径线之间存在良好的线性关系。通过实时B超监测可以对消融目标的有效范围进行较为准确的控制。
第三部分:IRE消融山羊肝脏的组织学观察
第一节:IRE术后肝脏组织学及肝细胞超微结构变化特点
目的:评价IRE消融山羊肝脏组织的有效性,探讨IRE消融术后不同时间点肝细胞组织学及电镜超微结构变化特点。
方法:固定脉宽100μs,频率1Hz,电压2000V,脉冲数120个,彩色超声探头引导电极针经皮定位穿刺处理山羊肝脏组织。于治疗后即刻、24小时、3天、7天、14天处死动物,取材,行HE、葡萄糖-6-磷酸酶( Glucose-6-phosphatse,G-6-P )、琥珀酸脱氢酶(Succinodehydrogenase, SDH)、毛细胆管吡啶银及网状纤维组织化学染色,观察组织细胞形态、两种酶活性产物的变化及IRE处理区毛细胆管及网状纤维的病理改变。电子显微镜观察IRE术后即刻、24小时及3天肝脏超微结构变化。
结果:①HE染色显示,IRE处理后即刻靶区内肝细胞胞浆内空泡形成、细胞明显水肿;24小时后观察到处理区肝细胞完全坏死,与周围正常细胞分界清楚,并出现大量炎症细胞浸润;IRE处理后14天,处理区坏死组织主要被肉芽组织填充、机化。②肝脏超微结构证实IRE处理后肝脏组织遭到严重破坏,表现为肝细胞胞膜破裂,线粒体肿胀坏死、粗面内质网扩张、肝血窦毛细血管内皮细胞连续性破坏等改变。③靶区肝细胞酶组织化学染色显示,IRE作用后即刻,G-6-P和SDH活性无明显变化;而24小时后,活性显著降低;3天后活性最低。消融区内毛细胆管结构破坏消失,网状纤维断裂破碎。
结论:G-6-P、SDH两种细胞酶学染色,从细胞内功能的改变与HE染色及电镜超微结构观察到肝细胞发生的形态学变化相互印证,充分证实了IRE对靶区细胞的完全非选择性消融效果。IRE可以实现病变区域具有细胞膜性结构成分组织的广泛性作用,同时对其内细胞骨架结构产生较强破坏效果。
第二节:IRE对消融区域内血管结构的影响
目的:探讨IRE对消融区域内血管结构的影响。
方法:固定脉宽100μs,频率1Hz,电压2000V,脉冲数120个,彩色超声探头引导电极针经皮消融山羊肝脏组织。处理后即刻、24小时、3天、7天、14天复查超声了解消融区内血管二维超声及多普勒血流影像学变化。逐层水平切开处理区肝脏组织,肉眼观察到消融区内包含有血管结构即取材,4%多聚甲醛固定,石蜡包埋,行HE、VG及Masson染色组织染色。
结果:标本取材后肉眼观察,IRE作用范围区域内血管结构完整(包括管径在10mm以上的肝内大血管和管径在2mm左右的肝内小血管)。血管二维影像提示消融区内的血管连续性完整,走行清晰,CDFI可以观察到血管内彩色多普勒血流频谱。血管组织学染色示内皮细胞连续性完好,血管周围弹性纤维、血管平滑肌结构完整。
结论:IRE非热消融治疗后病灶内滋养血管和毛细血管网大量破坏,而靶区内较大血管无损伤或栓塞。脉冲电场不可逆性电穿孔保留了消融区重要的管道结构(如动脉、静脉、胆管),血管周围的弹性纤维、胶原纤维结构完整。IRE不受血液流动热沉效应的影响,保证电场能量在靶区组织内的有效传递和积累,为生长在大血管周围、手术进行有困难的肿瘤消融提供可能。
第三节:IRE致消融区组织细胞凋亡的实验观察
目的:观察IRE消融山羊肝脏组织靶区肝细胞坏死、凋亡与电场分布的关系。
方法:固定电压2000V、脉宽100μs、频率1Hz,经彩色超声探头引导电极针经皮定位肝脏,施加120个脉冲电场。24h后处死动物,取电极针周围肝脏组织标本(包括脉冲电场处理区域中心部分和处理区域与正常组织交界区)和正常组织(作为对照)。将标本进行处理后,分别行HE染色观察组织的病理学变化;采用DNA ladder和TUNEL法检测组织细胞凋亡,采用流式细胞仪分析细胞周期。
结果:病理学观察可见,消融处理区的肝细胞完全坏死,围绕电极针的中心区域呈现凝固性坏死。TUNEL法显示凋亡细胞(细胞核为深黑色,胞质呈淡棕黄色)主要集中在处理区与正常组织的交界区。DNA ladder法在处理区与正常组织交界区组织检测到400-1000bp的梯状条带;流式细胞术分析显示,该区细胞DNA二倍体峰前面出现亚二倍体凋亡峰。
结论:采用IRE消融山羊肝脏组织,细胞在电场强度分布最高处(即围绕电极针的中心区域)呈现凝固性坏死;在电极针周边处理区域,随电场强度的衰减,交界区肝细胞还呈现出凋亡途径的细胞程序性死亡(programmed cell death,PCD)。肿瘤组织在脉冲电场消融时被“超范围切除”,可以有益于降低治疗后的复发率及对周边正常组织的侵犯、转移。
Electroporation occurs when the application of an electric field with electric field intensity of kV/cm and pulse duration ofμs to ms grade across a cell alters the transmembrane potential. Thus, the lipid bilayer structure is disrupted and small nanopores are created in the cell membrane, and the nanopores then allow micromolecules and macromolecules to be transported into and out of the cells. Electroporation could be used as reversible electroporation (RE) or irreversible electroporation (IRE) due to the nanopores recover or not. This process when used in RE has been used in medicine and research for drug or macromolecule delivery into cells, such as electrochemotherapy (ECT). With sufficiently high voltage, these pores become permanent and contribute to cell death by interfering with cell homeostasis.This is the mechanism of IRE ablation method. IRE tumor treatment process doesn’t depend on chemotherapeutics which might take some disadvantages of toxicity and drug resistance. In the past findings, researchers had fully certified the extensive application prospect of IRE though tumor cells and solid tumor ablation treatment experiments. But in the past, IRE only could be used on body surface or superficial tumor ablation, because lack of some equipment to help for the location of the electrodes placement accurately. With non-thermal cell death and a markedly decreased treatment time, IRE provides a potential new ablation method that can be operated in a well-controlled and focused manner under image monitoring (such as ultrasound). In this study, we used an in vivo goat liver model to evaluate the clinical feasibility of IRE imagine-guided by US, the mechanism of cell death by using histochemical analysis, the effectiveness of tissue destruction near bile ducts and vessels, and the correlation of ablation areas as measured by using US versus gross section examination.
Part 1 Investigation of the feasibility and safety with ultrasound imaging guided irreversible electroporation on goat livers
Chapter 1 Parameter selection of goats’liver ablation with IRE
Objective:To select the most suitable IRE ablation parameters under fixed electric field intensity, permanent duration and frequency.
Methods:IRE induced by 30 to 150 pieces of pulses(Pulse electric fields with a 2-cm probe distance, permanent duration 100μs, frequency 1Hz and voltage 2000V) were performed on goats’livers directly after abdomen discission. 30 pieces of pulses were increased for each group. Temperature of the target area was measured immediately after IRE ablation. All the animals were scarified 24 hours post IRE. Specimens were collected respectively for TTC stain. The largest ablation diameters of specimens were measured by electronic sliding caliper.
Results:It was shown that there was dose-dependence result between the dosage of the electric field and diameters of the ablation zone. Necrosis area enlarged gradually with the increasing of the pulse pieces of electric field. When the hepatic tissue was exposed to pulsed electric fields with 120 pieces of pulses, permanent duration 100μs, frequency 1Hz and voltage 2000V, the diameters of ablation area appeared to be obvious. Necrospy-based measurement demonstrated highly consistency with FEM-anticipated ablation zones. The dose in group 120 pieces of pulses may be considered as the optimal dosage for the hepatic tissue ablation in goat’s livers. There was no significant difference between 120 pulses and 150 pulses group in diameter comparison of the ablation zones (P>0.05). And 120 pulses group had significant difference when compared with 30 pulses, 60 pulses and 90 pulses group separately (P<0.05). The temperature of the injuring tissues was measured at the same time, and there was no significant difference before and after IRE ablation procedure (P>0.05).The temperature of the ablation area was under the regular injure level instantly post IRE.
Conclusion:120 pieces of pulses was the most optimal IRE ablation parameters under fixed pulse electric fields with 2-cm probe distance, permanent duration 100μs, frequency 1Hz and voltage 2000V. Electric field created by IRE was devoid of any joule heating and therefore, non-thermal ablation was achieved. Since it does not depend on heat, IRE can create a focal tumor ablation area which was independent of any heat sink effect.
Chapter 2 Feasibility investigations and characteristic of US imaging changes with ultrasound imaging guided IRE on goat livers
Objective: To investigate the proper method of percutaneous irreversible electroporation (IRE) on goat livers under ultrasonic guidance, and observe the characteristic of ultrasound imaging changes.
Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) applied on the electrodes and placement of the electrodes had been localized into goats’liver under ultrasound guidance though the animal skin to the target area. The treated area was observed by real-time ultrasound scanning and gross appearance observation at the time of 0, 24hours, 3d, 7d and 14d after IRE ablation.
Results: Ultrasound imaging guidance was accurate to focus on the target area. Imagines captured by the ultrasound after IRE procedure were quite different compared with the normal liver imaging. Real-time US images obtained during IRE show hypoechoic areas of ablation with hyperechoic region in close proximity to the probes. Sharp demarcation of IRE-ablated zone is well visualized immediately after the procedure. This ablated area was well correlated with pathologic measurements. US images obtained after 24 hours showed that area of hypoechogenicity becomes hyperechogenic, and seems to have shrunk when compared with US images obtained istantly. US images obtained after 3 days post IRE shows that gray scale of ablation area became more hyperechogenic, and boundary of the ablation area was quite clear. US images obtained after 7 days shows the most hyperechogenic gray scale of ablation area. Meanwhile, the circumsciption injured zone became irregular for hyperplasia of the fibrous connective tissue. 14 days post IRE, US images obtained showed insignificant hyperechogenic images with high point-like echo in the center. The ablation area shrunk obviously. US images correlated well with gross appearance observation at the time of 0, 24hours, 3d, 7d and 14d after IRE ablation.
Conclusion: With real-time monitoring by ultrasonography and well-controlled ablation of the target tissue, percutaneous IRE can provide a novel and unique ablative method for IRE tumor ablation. There was regular changement of US images after IRE ablation. Image-guided provides the fundamental experimental work for future studies on IRE clinical application and follow-up investigation.
Chapter3 Security investigation with ultrasound imaging guided irreversible electroporation on goat livers
Objective: To investigate the influence of IRE ablating to the liver of goat on general condition, and the security of IRE ablation method.
Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) applied on the electrodes and placement of the electrodes had been localized into goats’liver under ultrasound guidance though the animal skin to the target area. Heart rate, respiration frequency, and electrophysiology (ECG) changes between pre and post IRE ablation were compared. Postoperative complications and venous blood analyzing including liver function, kidney function, complete blood count and myocardial enzyme (CK - MB) were compared 0 hour, 24 hours, 3 days, 7 days and 14 days after IRE ablation.
Results: The respiratory frequency and heart rate of goat increased in the process of IRE ablation compared with in rest preoperation, and resumed to the condition of preoperation after IRE ablation. No significant statistic difference was showed between preoperation and postoperation (P>0.05). The ALT and AST progressively increased in postoperation 24h to 3rd day, and returned to normal level in postoperation 7th day. No significant statistic difference was showed between pre and post ablation in liver function, kidney function, complete blood count and myocardial enzyme (CK-MB)level (P>0.05). Instantly after IRE ablation, the ECG showed normal and no arrhythmia of electrophysiology changes. No complications including intraperitoneal hemorrhage, infection, and neighbouring organs damages (gallbladder, greater omentum, intestinal tract, etc) have been found.
Conclusion: IRE ablation was security and validity, and no obvious complications was observed.
Part 2 Intraprocedural US accuracy for IRE ablation area monitoring
Objective: To evaluate the difference between intraprocedural US and gross section measurements and to analysis the accuracy and controllability of monitoring the ablation zones by US.
Methods: Pulsed electric fields (PEFs) with a 2-cm probe distance, permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) applied on the electrodes and placement of the electrodes had been localized into goats’liver under ultrasound guidance though the animal skin to the target area.The ablation zone was monitored and measured in real time with US, which was repeated both immediately after the IRE procedure (D1) and again 24 hours after the IRE ablation procedure (D2), and the gross section (D3) was measured by electric vernier caliper after the animal was sacrificed by taken an overdose of anesthetic agent. Correlations between D1 and D2, D2 and D3, D1 and D3, D3 and D4(24 hours post IRE ablation when 120 pieces of pulses were performed on goat livers directly after abdomen discission ) were statistically analyzed separately.
Results: There was no significant statistic difference showed between D3 and D4[(35.96±2.52)mm](24 hours post IRE ablation when 120 pieces of pulses were performed on goat livers directly after abdomen discission)(P>0.05). Among the three sets of data (D1, D2, and D3), the maximum diameter measured by intraprocedural US immediately after IRE ablation procedure [D1,(39.58±2.13)mm] was the largest diameter, and followed was the data measured by US 24 hours after the procedure [D2(,37.07±3.51)mm]. The two sets were statically different(P<0.05). The gross section measurement [D3,(36.44±2.04)mm] which was measured 24 hours after IRE ablation was the smallest, but it’s not significant statistic difference between D2 and D3(P>0.05). D1 showed a well linear correlation with D3(r=0.949).
Conclusion:Ultrasound-guided percutaneous IRE on goats’liver ablation had the same effect with IRE ablation areas when pulses were performed on goat livers directly after abdomen discission. In the percutaneous IRE ablation procedure, data measured by intraprocedural US correlated well with gross section measurement. Data measured by intraprocedural US immediately after IRE ablation procedure was larger than the same set of data measured by US and though gross section measurement 24 hours post IRE ablation, but correlated well with the latter one. US provided an accurate and available method for IRE target area monitoring.
Part 3 Research on histological features of goat livers post IRE ablation
Chapter 1 Histopathology and ultra-structure changes of goat livers post IRE ablation
Objective: To investigate the histopathology and ultra-structure changes of goat livers with percutaneous irreversible electroporation (IRE) treatment.
Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) were performed on the electrodes which were placed under ultrasound guidance through the animal skin to the target area. The treated area was observed by hematoxylin and eosin (HE) stain, Glucose-6-phosphatse (G-6-P)stain, Succinodehydrogenase (SDH) stain, Cholangioles Pyridine silver stain and reticular fibers silver stain at the time of 0hour, 24hours, 3days, 7days, 14days after IRE ablation. The function of G-6-P and SDH enzymes and histological feature of liver cells, cholangioles and reticular fibers were all observed by light microscope. Ultra-structure changes of liver cells were observed by transmission electron microscope at the time of 0hour, 24hours, and 3days after IRE ablation.
Results: Complete hepatic cell death was observed after the IRE treatment and there was a sharp demarcation between the ablated zone and the non-ablated zone. By 14 days the ablated lesions was almost completely replaced by fibrous scartissue. Ultra-structure changes of liver cells observed by TEM showed that there were rupture of cell membranes, swelling and necrosis of mitochondrion, extension of rough endoplasmic reticulum and destruction of capillaries continuity in the ablated zone. Stain of G-6-P, SDH and cholangioles were all negative 24 hours post IRE. Endothelial cells of the cholangioles and capillaries and reticular cells were ablated at the same time.
Conclusion: Percutaneous IRE can provide a novel and unique method in ablating living cells with lipid bilayer membranes, such as liver cells, endothelial cells of the capillaries, cholangioles or reticular cells. The activity function of G-6-P and SDH in hepatic cells was lost at the same time.
Chapter 2 Effect of IRE ablation on the structure of blood vessels
Objective: To investigate the effect of IRE ablation on the structure of blood vessels.
Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) were performed on the electrodes which were placed under ultrasound guidance through the animal skin to the target area. The treated area was observed by real-time ultrasound scanning and color doppler flow imaging at the time of 0hour, 24hours, 3d, 7d and 14d after IRE ablation. At gross section examination, blood vessel sections in the ablation area were fixed in 4% paraformaldehyde and embedded with paraffin. Histological changes of the treated area were observed by HE stain, VG stain and Masson stain.
Results: Gross section examination showed that structural integrity of vascular within IRE-ablated zone (including the intrahepatic large vessels with diameters of more than 10mm and small intrahepatic vessels with diameters of less than 2mm). The imaging of two-dimensional ultrasound showed that blood vessels were intact and there was color flow angiography in blood vessels. Endothelial cells of were blood vessels not ablated by IRE. The blood vessel wall structures which mainly formed by vascular elastic and collagenous structures, peri-cellular matrix proteins and vascular smooth muscle structures were not damaged by IRE ablation.
Conclusion: IRE-ablation destroyed capillaries and cholangioles, but retained important vital structure such as hepatic arteries, hepatic veins and bile duct and perivascular elastic fiber structure and collagen fiber structure in the ablation zone. It suggested that IRE would not be affected by the heat sink effect of blood flow, which ensured effective transmission and accumulation of electric field energy. Thus, IRE could produce a more complete ablation effect within the lesion area, even with a large vessel traversing the ablation zone.
Chapter 3 Apoptosis of goat livers induced by irreversible electroporation
Objective:To investigate the impact of IRE ablation on apoptosis and necrosis of goat liver cells, and the relationship between these biological changes and distribution of electric fields.
Methods:IRE induced by 120 pieces of pulses(Pulsed electric fields (PEFs) with permanent duration 100μs, frequency 1Hz and voltage 2000V) were performed on goats livers. The electrodes were placed under ultrasound guidance via the animal skin to the target area. All the animals were scarified 24 hours after IRE ablation. Specimens were collected respectively from the center of ablated zone and the margin of ablated zone. Normal hepatic tissue was also collected as control. The specimens were observed by hematoxylin and eosins (HE) stain. Apoptosis was detected by DNA ladder and TUNEL. Hepatic cells were analyzed with flow cytometry (FCM) for cell cycle changes.
Results:Complete hepatic cell death were observed after IRE treatment. In the center of ablated zone (electrode point), coagulative necrosis was obvious. Apoptotic cells stained by TUNEL were found mainly at the margin of the ablated zone. Nucleus and cytoplasm of apoptotic cells were positively stained as pitch black and light buffy color respectively. DNA ladder analysis detected ladder strap with a length of 400 to 1000bp at the margin of ablated zone. Hypodiploid peak was found with FCM analysis in the hepatic cells from this area.
Conclusion:IRE induced liver cell death was by the means of coagulative necrosis in the center of ablation area (electrode point), while programmed cell death by the means of apoptosis was gradually involved as the decay of electric field strength at the margin of ablated zone. IRE could ablate tumor beyond the area of macroscopic observation. It is benefit to reducing recurrence and metastasis in surrounding area after IRE tumor ablation.
引文
[1] Lee EW, Chen C, Prieto VE, et al. Advanced hepatic ablation technique for creating complete cell death: irreversible electroporation [J]. Radiology, 2010, 255(2): 426-433.
[2] Rockwell AD. The medical and surgical uses of electricity, including the X-ray, Finsen light, vibratory therapeutics, and high-frequency currents [J]. New ed. New York: E. B. Treat & Company, 1903.
[3] Okino M, Mohri H. Optimal electric conductions in electrical impulse chemotherapy [J]. Jpn J Cancer Res. 1992. 83(10): 1095-1101.
[4] G. A. Hofmann, S. B. Dev, S. Dimmer, and G. S. Nanda, Electroperation Therapy: A New Approach for the Treatment of Head and Neck Cancer [J]. IEEE Trans. on Biomedical Engineering, 1999, (46)6: 752-759.
[5] Lluis M. Mir, Julie Gehl, Gregor Sersa, and etal.Standard operating procedures of the electrochemotherapy: Instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the CliniporatorTM by means of invasive or non-invasive electrodes [J].EJC Supplement, 2006, 4:14-25.
[6] Weaver JC. Electroporation of cells and tissues [J]. IEEE Trans Plasma Sci, 2000, 28(1): 24-33.
[7] Boris Rubinsky, Gary Onik, Paul Mikus. Irreversible Electroporation: A New Ablation Modality– Clinical Implications [J]. Technol Cancer Res Treat. 2007, 6(1): 37-48
[8] Boris Rubinsky. Irreversible Electroporation in Medicine [J]. Technology in Cancer Research and Treatment. 2007, 6(4): 255-259.
[9] Gary Onik, Paul Mikus, and Boris Rubinsky, Irreversible Electroporation: Implications for Prostate Ablation [J]. Technology in Cancer Research and Treatment,2007,6(4):295-300
[10] Boris Rubinsky. Irreversible Electroporation [M]. Springer-Verlag Berlin Heidelberg, 2010: 235-247.
[11]周玮,熊正爱,刘颖,等.不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究[J].第三军医大学学报, 2010, 32(18): 1941-1944.
[12]凌晓娟,熊正爱,李成祥,等.电脉冲在活体兔肝组织中的电场分布研究[J].第三军医大学学报,2007,29(19):1916-1917.
[13]顾华妍,熊正爱,李成祥,等.陡脉冲电场的生物学效应[J].中国组织工程研究与临床康复,2008,12(22):4201-4204.
[14]吴静,熊正爱,李成祥,等.B超监测下陡脉冲作用兔肝肿瘤的实验研究[J].西南大学学报(自然科学版),2009,31 (1):111-115.
[15]杨晓燕,熊正爱,李成祥,等.陡脉冲不可逆性电击穿治疗兔肝脏肿瘤研究[J].西南大学学报(自然科学版),2009, 31(4): 120-125.
[1] Boris Rubinsky. Irreversible Electroporation [M]. Springer-Verlag Berlin Heidelberg, 2010: 235-247
[2] Rubinsky J, Onik G, Mikus P, et al. Optimal Parameters for the Destruction of Prostate Cancer Using Irreversible Electroporation [J]. J Urol, 2008, 180(6): 2668-2674.
[3] Al-Sakere B, Andre F, Bernat C, et al. Tumor Ablation with Irreversible Electroporation [J]. PLoS ONE, 2007, 2 (11): e1135.
[4] Lee EW, Chen C, Prieto VE, et al. Advanced hepatic ablation technique for creating complete cell death: irreversible electroporation [J]. Radiology, 2010, 255(2): 426-433.
[5] Rubinsky B, Onik G, Mikus P. Irreversible electroporation: a new ablation modality--clinical implications [J]. Technol Cancer Res Treat, 2007, 6(1):37-48.
[6]周玮,熊正爱,刘颖,等.不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究[J].第三军医大学学报, 2010, 32(18): 1941-1944.
[7]喻金梅,熊正爱,凌晓娟等.陡脉冲对离体牛肝量效关系的实验研究[J].第三军医大学报,2006,7(13):2006,28(24):2419-242.
[8]凌晓娟,熊正爱,李成祥,等.电脉冲在活体兔肝组织中的电场分布研究[J].第三军医大学学报,2007,29(19):1916-1917.
[9]顾华妍,熊正爱,李成祥,等.陡脉冲电场的生物学效应[J].中国组织工程研究与临床康复,2008,12(22):4201-4204.
[10]吴静,熊正爱,李成祥,等.B超监测下陡脉冲作用兔肝肿瘤的实验研究[J].西南大学学报(自然科学版),2009,31 (1):111-115.
[11]杨晓燕,熊正爱,李成祥,等.陡脉冲不可逆性电击穿治疗兔肝脏肿瘤研究[J].西南大学学报(自然科学版),2009, 31(4): 120-125.
[12] Matsuki N, Ishikawa T, Imai Y, et al. Low voltage pulses can induce apoptosis[J]. Cancer Lett, 2008, 269(1):93-100.
[13]喻金梅.陡脉冲电场治疗剂量学实验研究[D].重庆:重庆医科大学,2006年.
[14] Damijan M, Dejan S, Halima M, et al. A Validated Model of in Vivo Electric Field Distribution in Tissues for Electrochemotherapy and for DNA Electrotranfer for Gene Therapy [J]. Biochimica Biophysica Acta. 2000, 15(19): 73-83.
[15] Rubinsky J, Onik G, Mikus P, et al. Optimal Parameters for the Destruction of Prostate Cancer Using Irreversible Electroporation [J]. J Urol, 2008, 180(6): 2668-2674.
[16] Hatfield RH, Mendelow AD, Perry RH, et al. Triphenylte- trazoliumchloride (TTC) as a marker for ischaemic changes in rat brain following permanent middle cerebral artery occlusion [J]. Neuropathol Appl Neurobiol,1991,17:61-67.
[17]常淑芳,顾美礼,伍烽等.三苯基四氮唑染色在高强度超声生物学焦域观测中的应用[J].中国超声医学杂志,2000,16(9):641-645.
[18] Zupanic A, Ribaric S, Miklavcic D. Increasing the repetition frequency of electric pulse delivery reduces unpleasant sensations that occur in electrochemotherapy [J]. Neoplasma, 2007, 54(3):246-250.
[19]刘颖,周玮,熊正爱,等.超声引导不可逆电穿孔消融山羊肝脏的实验研究[J].解放军医学杂志, 2011, 36(3): 254-256.
[20] Goldberg SN, Solbiati L, Hahn PF, et al. Large-volume tissue ablation with radio frequency by using a clustered, internally cooled electrode technique: laboratory and clinical experience in liver metastases [J]. Radiology, 1998,209 (2):371- 379.
[21] Solbiati L, Ierace T, Tonolini M, Osti V, Cova L. Radiofrequency thermal ablation of hepatic metastases[J]. Eur J Ultrasound 2001, 13(2):149-158.
[22] Reilly JP. Applied bioelectricity: from electrical stimulation to electropathology [M]. Springer, NewYork, 1998.
[23] LaveeJ, OnikG, MikusP, et al. A novel nonthermal energy source for surgical epicardial atrial ablation: irreversible electroporation [J].Heart Surg Forum, 2007, 10(2):162-167.
[24]熊兰,孙才新,王士彬等.陡脉冲电场分布仿真与在体兔肝组织的实验研究[J].中国生物医学工程学报,2007,26(2):208-213.
[25]王鸿利,仲人前,陈丽梅,等.实验诊断学[M],人民卫生出版社,2001:171-175.
[1]张泽宝主编.医学影像物理学[M].人民卫生出版社. 2000
[2]谢敬霞,杜湘珂主编.医学影像学[M].北京医科大学出版社. 2002
[3]喻金梅.陡脉冲电场治疗剂量学实验研究[D].重庆:重庆医科大学,2006年.
[4]吴静.超声监测陡脉冲治疗兔肝肿瘤的可行性研究D].重庆:重庆医科大学,2009年.
[5] Carey RI, Leveillee RJ. First prize: direct real-time temperature monitoring for laparoscopic and CT-guided radiofrequency ablation of renal tumors between 3 and 5cm. J Endourol, 2007, 21(8):807-813.
[6] Mast TD, Pucke DP, Subramanian SE, et al. Ultrasound monitoring of in vitro radio frequency ablation by echo decorrelation imaging. J Ultrasound Med, 2008, 27(12) : 1685–1697.
[7] Lee EW, Chen C, Prieto VE, et al. Advanced hepatic ablation technique for creating complete cell death: irreversible electroporation [J]. Radiology, 2010, 255(2): 426-433.
[1]高英茂,徐昌芬,王亚平等.组织学与胚胎学[M],人民卫生出版社,2001:40.
[2] SCHOENBACH K H, NUCCITELLI R, BEEBE S J. Zap [J].IEEE Spectrum, 2006, 43(8):22-26.
[3] Boris Rubinsky, Gary Onik, Paul Mikus. Irreversible Electroporation: A New Ablation Modality– Clinical Implications [J]. Technology in Cancer Research and Treatment.2007, 6(1):37-48.
[4] Deodhar A, Monette S, Single GW Jr, et al. Renal tissue ablation with irreversible electroporation: preliminary results in a porcine model [J].Urology.2011, 77(3):754-760.
[5] Maeshima A. Label-retaining cells in the kidney: origin of regeneration cells after renal ischemia. Clin Exp Nephrol. 2007, 11(4):269-274.
[6] Romagnani P, Kalluri R. Possible mechanisms of kidney repair. Fibrogen Tissue Repair. 2009, 2(1):3.
[7]陈平,韩本立,陈娟,等.肝部分切除术后肝细胞DNA合成及细胞周期变化的实验研究[J].中华肝胆外科杂志,1998,4(3):147-149.
[8]王泊云,李玉松,黄高升,等.病理学技术[M],人民卫生出版社,2000:147-219.
[9]邹晓毅,叶彬,王俊安,等.高强度聚焦超声波对细粒棘球绦虫原头节酶活性的影响[J].中国人兽共患病学报,2007,23(11):1097-1100.
[10]邹文兵,牛陵川,向理科,等.高强度聚焦超声消融兔乳腺移植瘤效果的酶化学检测[J].中国超声医学杂志,2010,26(5):392-395.
[11]牛陵川,邹文兵,张炼,等.高强度聚焦超声消融兔VX2种植性乳腺肿瘤的病理学变化[J].肿瘤,2010,30(2):105-108.
[12]高英茂,徐昌芬,王亚平等.组织学与胚胎学[M],人民卫生出版社,2001:223-230.
[13]刘田福,李春华,王树党,等.不同鼠龄Wistar大鼠肝组织内网状纤维分布的计量形态学研究[J].中国比较医学杂志,2003,13(3):166-168.
[14] Lee EW, Chen C, Prieto VE, et al. Advanced hepatic ablation technique for creating complete cell death: irreversible electroporation [J]. Radiology, 2010,255(2): 426-433.
[15] Ajita D, Sébastien M, GordonW, et al. Renal Tissue Ablation With Irreversible Electroporation: Preliminary Results in a Porcine Model [J].Urology, 2011,77(3):754-760.
[16] Boris Rubinsky, Gary Onik, Paul Mikus. Irreversible Electroporation: A New Ablation Modality– Clinical Implications [J]. Technol Cancer Res Treat. 2007, 6(1): 37-48
[17] Gary Onik, Paul Mikus, and Boris Rubinsky, Irreversible Electroporation: Implications for Prostate Ablation [J]. Technology in Cancer Research and Treatment, 2007, 6(4):295-300
[18] Boris Rubinsky. Irreversible Electroporation [M]. Springer-Verlag Berlin Heidelberg, 2010: 235-247.
[19]姚陈果,孙才新,米彦,等.陡脉冲对恶性肿瘤细胞不可逆性电击穿的实验研究[J].中国生物医学工程学报, 2004, 23(1): 92-97.
[20]周玮,熊正爱,刘颖,等.不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究[J].第三军医大学学报, 2010, 32(18): 1941-1944.
[21] Lee EW, Chen C, Prieto VE, et al. Advanced Hepatic Ablation Technique for Creating Complete Cell Death: Irreversible Electroporation [J]. Radiology, 2010, 255(2): 426-433.
[22] Miklavcic D, Beravs K, Semrov D, et al. The importance of electric field distribution for effective in vivo electroporation of tissues [J]. Biophys J, 1998, 74 (5): 2152-2158.
[23] Miklavcic D, Semrov D, Mekid H, et al. A validated model of in vivo electric field distribution in tissues for electrochemotherapy and for DNA electrotransfer for genetherapy [J]. Biochim Biophys Acta, 2000, 1523(1): 73-83.
[24] Kranjc S, Cemazar M, Grosel A, et al. Radiosensitising effect of electrochemotherapy with bleomycin in LPB sarcoma cells and tumors in mice [J]. BMC Cancer, 2005, 5: 115.
[25]彭黎明,王曾礼.细胞凋亡的基础与临床[M].北京:人民卫生出版社, 2000.
[26]周玮,熊正爱,刘颖,等.不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究[J].第三军医大学学报, 2010, 32(18): 1941-1944.
[27] Joza N, Susin SA, Daugas E, et al. Essential role of the mitochondrialapoptosis-inducing factor in programmed cell death [J].Nature, 2001, 410(6828): 549-554.
[28] Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy [J]. Oncogene, 2006, 25(34): 4798-4811.
[29] Bovanovic F, Bootman MD, Berridge MJ, et al. Elementary [Ca++]i signals generated by electroporati on functionally mimic those evoked by hormonal stimulation[J]. FASEB J, 1999, 13(2):365–376.
[1] Lee EW, Chen C, Prieto VE, et al. Advanced hepatic ablation technique for creating complete cell death: irreversible electroporation [J]. Radiology, 2010, 255(2): 426-433.
[2] Kranjc S, Cemazar M, Grosel A, et al. Radiosensitising effect of electrochemotherapy with bleomycin in LPB sarcoma cells and tumors in mice[J]. BMC Cancer, 2005, 5: 115.
[3] RubinskyB. Irreversible electroporation in medicine [J]. Technol Cancer Res Treatm, 2007, 6(4): 255-260.
[4] Fuller GW, Louisville Water Company (Louisville Ky.). Report on the investigations into the purification of the Ohio River water: at Louisville, Kentucky, made to the president and directors of the Louisville Water Company [J]. New York: D. Van Nostrand Company, 1898.
[5] Rockwell AD. The medical and surgical uses of electricity, including the X-ray, Finsen light, vibratory therapeutics, and high-frequency currents [J]. New ed. New York: E. B. Treat & Company, 1903.
[6] Okino M, Mohri H. Optimal electric conductions in electrical impulse chemotherapy [J]. Jpn J Cancer Res. 1992. 83(10): 1095-1101.
[7] G. A. Hofmann, S. B. Dev, S. Dimmer, and G. S. Nanda, Electroperation Therapy: A New Approach for the Treatment of Head and Neck Cancer [J]. IEEE Trans. on Biomedical Engineering, 1999, (46)6: 752-759.
[8] Lluis M. Mir, Julie Gehl, Gregor Sersa, and etal.Standard operating procedures of the electrochemotherapy: Instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the CliniporatorTM by means of invasive or non-invasive electrodes [J].EJC Supplement, 2006, 4:14-25.
[9] Weaver JC. Electroporation of cells and tissues [J]. IEEE Trans Plasma Sci, 2000, 28(1): 24-33.
[10] Boris Rubinsky, Gary Onik, Paul Mikus. Irreversible Electroporation: A New Ablation Modality– Clinical Implications [J]. Technol Cancer Res Treat. 2007, 6(1): 37-48
[11] Boris Rubinsky. Irreversible Electroporation in Medicine [J]. Technology inCancer Research and Treatment. 2007, 6(4): 255-259.
[12] Gary Onik, Paul Mikus, and Boris Rubinsky, Irreversible Electroporation: Implications for Prostate Ablation [J]. Technology in Cancer Research and Treatment,2007,6(4):295-300
[13] Boris Rubinsky. Irreversible Electroporation [M]. Springer-Verlag Berlin Heidelberg, 2010: 235-247.
[14]刘颖,周玮,熊正爱,等.超声引导不可逆电穿孔消融山羊肝脏的实验研究[J].解放军医学杂志, 2011, 36(3): 254-256.
[15] Goldberg SN, Solbiati L, Hahn PF, et al. Large-volume tissue ablation with radio frequency by using a clustered, internally cooled electrode technique: laboratory and clinical experience in liver metastases [J]. Radiology, 1998,209 (2):371- 379.
[16] Solbiati L, Ierace T, Tonolini M, Osti V, Cova L. Radiofrequency thermal ablation of hepatic metastases[J]. Eur J Ultrasound, 2001, 13(2):149-158.
[17] Ajita D, Sébastien M, GordonW, et al. Renal Tissue Ablation With Irreversible Electroporation: Preliminary Results in a Porcine Model [J].Urology, 2011,77(3):754-760.
[18]姚陈果,孙才新,米彦,等.陡脉冲对恶性肿瘤细胞不可逆性电击穿的实验研究[J].中国生物医学工程学报, 2004, 23(1): 92-97.
[19]周玮,熊正爱,刘颖,等.不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究[J].第三军医大学学报, 2010, 32(18): 1941-1944.
[20] Karl HS, Sunao K, Robert HS.et a1. Bioelectrics—new applications for pulsed power technology J]. IEEE Transactions on Plasma Science,2002,30(1):293
[21]唐均英,吴晓娟,姚陈果,等.纳秒级陡脉冲对人卵巢癌细胞SKOV3凋亡及细胞内ca2+浓度的影响[J].现代妇产科进展,2007,16(11):827-831
[22]刘颖,周玮,熊正爱,等.不可逆性电穿孔致山羊肝脏组织凋亡与坏死的实验研究[J].解放军医学杂志, 2011, 36(4): 352-356.
[23] Yue Zhang, Yang Guo, Ann B, et al. MR Imaging to Assess Immediate Response to Irreversible Electroporation for Targeted Ablation of Liver Tissues: Preclinical Feasibility Studies in a Rodent Model [J]. Radiology, 2010, 256(2): 424-432.
[2] Rockwell AD. The medical and surgical uses of electricity, including the X-ray, Finsen light, vibratory therapeutics, and high-frequency currents [J]. New ed. New York: E. B. Treat & Company, 1903.
[3] Okino M, Mohri H. Optimal electric conductions in electrical impulse chemotherapy [J]. Jpn J Cancer Res. 1992. 83(10): 1095-1101.
[4] G. A. Hofmann, S. B. Dev, S. Dimmer, and G. S. Nanda, Electroperation Therapy: A New Approach for the Treatment of Head and Neck Cancer [J]. IEEE Trans. on Biomedical Engineering, 1999, (46)6: 752-759.
[5] Lluis M. Mir, Julie Gehl, Gregor Sersa, and etal.Standard operating procedures of the electrochemotherapy: Instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the CliniporatorTM by means of invasive or non-invasive electrodes [J].EJC Supplement, 2006, 4:14-25.
[6] Weaver JC. Electroporation of cells and tissues [J]. IEEE Trans Plasma Sci, 2000, 28(1): 24-33.
[7] Boris Rubinsky, Gary Onik, Paul Mikus. Irreversible Electroporation: A New Ablation Modality– Clinical Implications [J]. Technol Cancer Res Treat. 2007, 6(1): 37-48
[8] Boris Rubinsky. Irreversible Electroporation in Medicine [J]. Technology in Cancer Research and Treatment. 2007, 6(4): 255-259.
[9] Gary Onik, Paul Mikus, and Boris Rubinsky, Irreversible Electroporation: Implications for Prostate Ablation [J]. Technology in Cancer Research and Treatment,2007,6(4):295-300
[10] Boris Rubinsky. Irreversible Electroporation [M]. Springer-Verlag Berlin Heidelberg, 2010: 235-247.
[11]周玮,熊正爱,刘颖,等.不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究[J].第三军医大学学报, 2010, 32(18): 1941-1944.
[12]凌晓娟,熊正爱,李成祥,等.电脉冲在活体兔肝组织中的电场分布研究[J].第三军医大学学报,2007,29(19):1916-1917.
[13]顾华妍,熊正爱,李成祥,等.陡脉冲电场的生物学效应[J].中国组织工程研究与临床康复,2008,12(22):4201-4204.
[14]吴静,熊正爱,李成祥,等.B超监测下陡脉冲作用兔肝肿瘤的实验研究[J].西南大学学报(自然科学版),2009,31 (1):111-115.
[15]杨晓燕,熊正爱,李成祥,等.陡脉冲不可逆性电击穿治疗兔肝脏肿瘤研究[J].西南大学学报(自然科学版),2009, 31(4): 120-125.
[1] Boris Rubinsky. Irreversible Electroporation [M]. Springer-Verlag Berlin Heidelberg, 2010: 235-247
[2] Rubinsky J, Onik G, Mikus P, et al. Optimal Parameters for the Destruction of Prostate Cancer Using Irreversible Electroporation [J]. J Urol, 2008, 180(6): 2668-2674.
[3] Al-Sakere B, Andre F, Bernat C, et al. Tumor Ablation with Irreversible Electroporation [J]. PLoS ONE, 2007, 2 (11): e1135.
[4] Lee EW, Chen C, Prieto VE, et al. Advanced hepatic ablation technique for creating complete cell death: irreversible electroporation [J]. Radiology, 2010, 255(2): 426-433.
[5] Rubinsky B, Onik G, Mikus P. Irreversible electroporation: a new ablation modality--clinical implications [J]. Technol Cancer Res Treat, 2007, 6(1):37-48.
[6]周玮,熊正爱,刘颖,等.不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究[J].第三军医大学学报, 2010, 32(18): 1941-1944.
[7]喻金梅,熊正爱,凌晓娟等.陡脉冲对离体牛肝量效关系的实验研究[J].第三军医大学报,2006,7(13):2006,28(24):2419-242.
[8]凌晓娟,熊正爱,李成祥,等.电脉冲在活体兔肝组织中的电场分布研究[J].第三军医大学学报,2007,29(19):1916-1917.
[9]顾华妍,熊正爱,李成祥,等.陡脉冲电场的生物学效应[J].中国组织工程研究与临床康复,2008,12(22):4201-4204.
[10]吴静,熊正爱,李成祥,等.B超监测下陡脉冲作用兔肝肿瘤的实验研究[J].西南大学学报(自然科学版),2009,31 (1):111-115.
[11]杨晓燕,熊正爱,李成祥,等.陡脉冲不可逆性电击穿治疗兔肝脏肿瘤研究[J].西南大学学报(自然科学版),2009, 31(4): 120-125.
[12] Matsuki N, Ishikawa T, Imai Y, et al. Low voltage pulses can induce apoptosis[J]. Cancer Lett, 2008, 269(1):93-100.
[13]喻金梅.陡脉冲电场治疗剂量学实验研究[D].重庆:重庆医科大学,2006年.
[14] Damijan M, Dejan S, Halima M, et al. A Validated Model of in Vivo Electric Field Distribution in Tissues for Electrochemotherapy and for DNA Electrotranfer for Gene Therapy [J]. Biochimica Biophysica Acta. 2000, 15(19): 73-83.
[15] Rubinsky J, Onik G, Mikus P, et al. Optimal Parameters for the Destruction of Prostate Cancer Using Irreversible Electroporation [J]. J Urol, 2008, 180(6): 2668-2674.
[16] Hatfield RH, Mendelow AD, Perry RH, et al. Triphenylte- trazoliumchloride (TTC) as a marker for ischaemic changes in rat brain following permanent middle cerebral artery occlusion [J]. Neuropathol Appl Neurobiol,1991,17:61-67.
[17]常淑芳,顾美礼,伍烽等.三苯基四氮唑染色在高强度超声生物学焦域观测中的应用[J].中国超声医学杂志,2000,16(9):641-645.
[18] Zupanic A, Ribaric S, Miklavcic D. Increasing the repetition frequency of electric pulse delivery reduces unpleasant sensations that occur in electrochemotherapy [J]. Neoplasma, 2007, 54(3):246-250.
[19]刘颖,周玮,熊正爱,等.超声引导不可逆电穿孔消融山羊肝脏的实验研究[J].解放军医学杂志, 2011, 36(3): 254-256.
[20] Goldberg SN, Solbiati L, Hahn PF, et al. Large-volume tissue ablation with radio frequency by using a clustered, internally cooled electrode technique: laboratory and clinical experience in liver metastases [J]. Radiology, 1998,209 (2):371- 379.
[21] Solbiati L, Ierace T, Tonolini M, Osti V, Cova L. Radiofrequency thermal ablation of hepatic metastases[J]. Eur J Ultrasound 2001, 13(2):149-158.
[22] Reilly JP. Applied bioelectricity: from electrical stimulation to electropathology [M]. Springer, NewYork, 1998.
[23] LaveeJ, OnikG, MikusP, et al. A novel nonthermal energy source for surgical epicardial atrial ablation: irreversible electroporation [J].Heart Surg Forum, 2007, 10(2):162-167.
[24]熊兰,孙才新,王士彬等.陡脉冲电场分布仿真与在体兔肝组织的实验研究[J].中国生物医学工程学报,2007,26(2):208-213.
[25]王鸿利,仲人前,陈丽梅,等.实验诊断学[M],人民卫生出版社,2001:171-175.
[1]张泽宝主编.医学影像物理学[M].人民卫生出版社. 2000
[2]谢敬霞,杜湘珂主编.医学影像学[M].北京医科大学出版社. 2002
[3]喻金梅.陡脉冲电场治疗剂量学实验研究[D].重庆:重庆医科大学,2006年.
[4]吴静.超声监测陡脉冲治疗兔肝肿瘤的可行性研究D].重庆:重庆医科大学,2009年.
[5] Carey RI, Leveillee RJ. First prize: direct real-time temperature monitoring for laparoscopic and CT-guided radiofrequency ablation of renal tumors between 3 and 5cm. J Endourol, 2007, 21(8):807-813.
[6] Mast TD, Pucke DP, Subramanian SE, et al. Ultrasound monitoring of in vitro radio frequency ablation by echo decorrelation imaging. J Ultrasound Med, 2008, 27(12) : 1685–1697.
[7] Lee EW, Chen C, Prieto VE, et al. Advanced hepatic ablation technique for creating complete cell death: irreversible electroporation [J]. Radiology, 2010, 255(2): 426-433.
[1]高英茂,徐昌芬,王亚平等.组织学与胚胎学[M],人民卫生出版社,2001:40.
[2] SCHOENBACH K H, NUCCITELLI R, BEEBE S J. Zap [J].IEEE Spectrum, 2006, 43(8):22-26.
[3] Boris Rubinsky, Gary Onik, Paul Mikus. Irreversible Electroporation: A New Ablation Modality– Clinical Implications [J]. Technology in Cancer Research and Treatment.2007, 6(1):37-48.
[4] Deodhar A, Monette S, Single GW Jr, et al. Renal tissue ablation with irreversible electroporation: preliminary results in a porcine model [J].Urology.2011, 77(3):754-760.
[5] Maeshima A. Label-retaining cells in the kidney: origin of regeneration cells after renal ischemia. Clin Exp Nephrol. 2007, 11(4):269-274.
[6] Romagnani P, Kalluri R. Possible mechanisms of kidney repair. Fibrogen Tissue Repair. 2009, 2(1):3.
[7]陈平,韩本立,陈娟,等.肝部分切除术后肝细胞DNA合成及细胞周期变化的实验研究[J].中华肝胆外科杂志,1998,4(3):147-149.
[8]王泊云,李玉松,黄高升,等.病理学技术[M],人民卫生出版社,2000:147-219.
[9]邹晓毅,叶彬,王俊安,等.高强度聚焦超声波对细粒棘球绦虫原头节酶活性的影响[J].中国人兽共患病学报,2007,23(11):1097-1100.
[10]邹文兵,牛陵川,向理科,等.高强度聚焦超声消融兔乳腺移植瘤效果的酶化学检测[J].中国超声医学杂志,2010,26(5):392-395.
[11]牛陵川,邹文兵,张炼,等.高强度聚焦超声消融兔VX2种植性乳腺肿瘤的病理学变化[J].肿瘤,2010,30(2):105-108.
[12]高英茂,徐昌芬,王亚平等.组织学与胚胎学[M],人民卫生出版社,2001:223-230.
[13]刘田福,李春华,王树党,等.不同鼠龄Wistar大鼠肝组织内网状纤维分布的计量形态学研究[J].中国比较医学杂志,2003,13(3):166-168.
[14] Lee EW, Chen C, Prieto VE, et al. Advanced hepatic ablation technique for creating complete cell death: irreversible electroporation [J]. Radiology, 2010,255(2): 426-433.
[15] Ajita D, Sébastien M, GordonW, et al. Renal Tissue Ablation With Irreversible Electroporation: Preliminary Results in a Porcine Model [J].Urology, 2011,77(3):754-760.
[16] Boris Rubinsky, Gary Onik, Paul Mikus. Irreversible Electroporation: A New Ablation Modality– Clinical Implications [J]. Technol Cancer Res Treat. 2007, 6(1): 37-48
[17] Gary Onik, Paul Mikus, and Boris Rubinsky, Irreversible Electroporation: Implications for Prostate Ablation [J]. Technology in Cancer Research and Treatment, 2007, 6(4):295-300
[18] Boris Rubinsky. Irreversible Electroporation [M]. Springer-Verlag Berlin Heidelberg, 2010: 235-247.
[19]姚陈果,孙才新,米彦,等.陡脉冲对恶性肿瘤细胞不可逆性电击穿的实验研究[J].中国生物医学工程学报, 2004, 23(1): 92-97.
[20]周玮,熊正爱,刘颖,等.不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究[J].第三军医大学学报, 2010, 32(18): 1941-1944.
[21] Lee EW, Chen C, Prieto VE, et al. Advanced Hepatic Ablation Technique for Creating Complete Cell Death: Irreversible Electroporation [J]. Radiology, 2010, 255(2): 426-433.
[22] Miklavcic D, Beravs K, Semrov D, et al. The importance of electric field distribution for effective in vivo electroporation of tissues [J]. Biophys J, 1998, 74 (5): 2152-2158.
[23] Miklavcic D, Semrov D, Mekid H, et al. A validated model of in vivo electric field distribution in tissues for electrochemotherapy and for DNA electrotransfer for genetherapy [J]. Biochim Biophys Acta, 2000, 1523(1): 73-83.
[24] Kranjc S, Cemazar M, Grosel A, et al. Radiosensitising effect of electrochemotherapy with bleomycin in LPB sarcoma cells and tumors in mice [J]. BMC Cancer, 2005, 5: 115.
[25]彭黎明,王曾礼.细胞凋亡的基础与临床[M].北京:人民卫生出版社, 2000.
[26]周玮,熊正爱,刘颖,等.不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究[J].第三军医大学学报, 2010, 32(18): 1941-1944.
[27] Joza N, Susin SA, Daugas E, et al. Essential role of the mitochondrialapoptosis-inducing factor in programmed cell death [J].Nature, 2001, 410(6828): 549-554.
[28] Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy [J]. Oncogene, 2006, 25(34): 4798-4811.
[29] Bovanovic F, Bootman MD, Berridge MJ, et al. Elementary [Ca++]i signals generated by electroporati on functionally mimic those evoked by hormonal stimulation[J]. FASEB J, 1999, 13(2):365–376.
[1] Lee EW, Chen C, Prieto VE, et al. Advanced hepatic ablation technique for creating complete cell death: irreversible electroporation [J]. Radiology, 2010, 255(2): 426-433.
[2] Kranjc S, Cemazar M, Grosel A, et al. Radiosensitising effect of electrochemotherapy with bleomycin in LPB sarcoma cells and tumors in mice[J]. BMC Cancer, 2005, 5: 115.
[3] RubinskyB. Irreversible electroporation in medicine [J]. Technol Cancer Res Treatm, 2007, 6(4): 255-260.
[4] Fuller GW, Louisville Water Company (Louisville Ky.). Report on the investigations into the purification of the Ohio River water: at Louisville, Kentucky, made to the president and directors of the Louisville Water Company [J]. New York: D. Van Nostrand Company, 1898.
[5] Rockwell AD. The medical and surgical uses of electricity, including the X-ray, Finsen light, vibratory therapeutics, and high-frequency currents [J]. New ed. New York: E. B. Treat & Company, 1903.
[6] Okino M, Mohri H. Optimal electric conductions in electrical impulse chemotherapy [J]. Jpn J Cancer Res. 1992. 83(10): 1095-1101.
[7] G. A. Hofmann, S. B. Dev, S. Dimmer, and G. S. Nanda, Electroperation Therapy: A New Approach for the Treatment of Head and Neck Cancer [J]. IEEE Trans. on Biomedical Engineering, 1999, (46)6: 752-759.
[8] Lluis M. Mir, Julie Gehl, Gregor Sersa, and etal.Standard operating procedures of the electrochemotherapy: Instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the CliniporatorTM by means of invasive or non-invasive electrodes [J].EJC Supplement, 2006, 4:14-25.
[9] Weaver JC. Electroporation of cells and tissues [J]. IEEE Trans Plasma Sci, 2000, 28(1): 24-33.
[10] Boris Rubinsky, Gary Onik, Paul Mikus. Irreversible Electroporation: A New Ablation Modality– Clinical Implications [J]. Technol Cancer Res Treat. 2007, 6(1): 37-48
[11] Boris Rubinsky. Irreversible Electroporation in Medicine [J]. Technology inCancer Research and Treatment. 2007, 6(4): 255-259.
[12] Gary Onik, Paul Mikus, and Boris Rubinsky, Irreversible Electroporation: Implications for Prostate Ablation [J]. Technology in Cancer Research and Treatment,2007,6(4):295-300
[13] Boris Rubinsky. Irreversible Electroporation [M]. Springer-Verlag Berlin Heidelberg, 2010: 235-247.
[14]刘颖,周玮,熊正爱,等.超声引导不可逆电穿孔消融山羊肝脏的实验研究[J].解放军医学杂志, 2011, 36(3): 254-256.
[15] Goldberg SN, Solbiati L, Hahn PF, et al. Large-volume tissue ablation with radio frequency by using a clustered, internally cooled electrode technique: laboratory and clinical experience in liver metastases [J]. Radiology, 1998,209 (2):371- 379.
[16] Solbiati L, Ierace T, Tonolini M, Osti V, Cova L. Radiofrequency thermal ablation of hepatic metastases[J]. Eur J Ultrasound, 2001, 13(2):149-158.
[17] Ajita D, Sébastien M, GordonW, et al. Renal Tissue Ablation With Irreversible Electroporation: Preliminary Results in a Porcine Model [J].Urology, 2011,77(3):754-760.
[18]姚陈果,孙才新,米彦,等.陡脉冲对恶性肿瘤细胞不可逆性电击穿的实验研究[J].中国生物医学工程学报, 2004, 23(1): 92-97.
[19]周玮,熊正爱,刘颖,等.不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究[J].第三军医大学学报, 2010, 32(18): 1941-1944.
[20] Karl HS, Sunao K, Robert HS.et a1. Bioelectrics—new applications for pulsed power technology J]. IEEE Transactions on Plasma Science,2002,30(1):293
[21]唐均英,吴晓娟,姚陈果,等.纳秒级陡脉冲对人卵巢癌细胞SKOV3凋亡及细胞内ca2+浓度的影响[J].现代妇产科进展,2007,16(11):827-831
[22]刘颖,周玮,熊正爱,等.不可逆性电穿孔致山羊肝脏组织凋亡与坏死的实验研究[J].解放军医学杂志, 2011, 36(4): 352-356.
[23] Yue Zhang, Yang Guo, Ann B, et al. MR Imaging to Assess Immediate Response to Irreversible Electroporation for Targeted Ablation of Liver Tissues: Preclinical Feasibility Studies in a Rodent Model [J]. Radiology, 2010, 256(2): 424-432.