The nanosecond pulsed electric field is an emerging locoregional electric ablative therapy that can be used to treat a variety of solid tumors, including melanoma , breast cancer , colon cancer , osteosarcoma , pancreatic cancer  and hepatocellular carcinoma . The nonthermal characteristic and avoidance of heatsink effects are two of the most significant features of nsPEF ablation, which makes it possible to apply nsPEF ablation on liver tumors located near vital structures such as the porta hepatis. Operation in this area is recognized as complicated for surgeons and is considered as a relative contraindications for RFA [22, 23].
In this study, we demonstrated for the first time that nsPEF achieved complete ablation of liver tissues and preserved the large vessels within ablation areas well. Conventional thermal ablations such as RFA relies on excessive heat energy to cause necrosis of liver tissues. They cannot discriminate parenchymal hepatic tissues from matrix tissue, such as vascular fibers, which may lead to vascular damages . However, nsPEF uses electric pulses to generate irreversible nanopores on the cell membrane and does not damage other types of molecules, which explains why it has the potential to preserve the cellular matrix of the large vessels. As the short-term and mid-term pathological follow-up shows, the large vessels, including veins and bile ducts, in the ablated area maintained a complete vascular structure and the perivascular tissues were ablated accurately with no sparing. It was confirmed that the large vessels were protected from electric damage. Although some of the large vessels exhibited mild muscularis layer damage and endothelial loss, the presence of the extracellular matrix greatly facilitated the reendothelialization process within 14 days. This may also contributes to the significant immune responses and rapid tissue healing observed in a series of nsPEF treatment studies [16, 18, 21, 25]. Another interesting phenomenon is that compared with veins, bile ducts maintained a more complete structure, and the biliary epithelial cells were better preserved. According to previous studies about RFA, it is assumed that bile ducts are more vulnerable to thermal damage and less vulnerable to electric stimulations [9, 26, 27].
The protective effects on vasculature systems, especially for bile ducts, were also validated by the blood test results. Serum bilirubin, including TB and DB, showed no obvious changes during the whole procedure, indicating that nsPEF treatment did not cause biliary complications. However, biliary complications, such as cholestasis secondary to bile duct strictures that resulted from the thermal damage, was of high incidence after RFA . In addition, TBA underwent mild changes immediately after the treatment but then remained steady, suggesting that nsPEF may slightly disturb lipid metabolism. Notably, the number of blood platelets (PLT) increased posttreatment , which raises the concern of thrombosis formation. Although the thrombosis was not found in the histopathologic results, and the PLT went down at 14 days posttreatment, the increasing trend of PLT indicated a procoagulant effect of nsPEF. Previous studies reported that thrombogenicity is one of the risk factors after thermal ablation, and can lead to the administration of heparin . For the above reasons, coagulation function of the patients should be monitored after nsPEF treatment.
Aminotransferases (ALT and AST) underwent a temporary increase, which was assumed to be resulted from hepatic parenchymal cells death. Their subsequent recovery confirmed the safety of nsPEF. Additionally, TP and Alb proved that nsPEF does not impair the capacity of albumin synthesis in the liver. According to the routine blood tests, RBC and Hb remained at the same level, proving that no massive hemorrhage occurred during the treatment. Furthermore, the results for inflammation, such as the percentages of neutrophils, did not change significantly, which indicated that strict asepsis during the operation could effectively avoid the risks of infection during the ultrasound-guided puncture process.
The effect of the electric ablation on the cardiovascular and skeletal muscle systems was investigated in this study. Previous studies proved that the release of high voltage energy increases cell membrane permeability and opens a path for ion transport, which can induce cardiac arrythmias and defibrillation, and may lead to unpredictable cardiac complications [30-32]. In addition, the electric stimulation of excitable tissues, such as motor nerves can cause involuntary contraction of the muscles of subjects, which may hinder the treatment [33-35]. However, in this study, we adopted the synchronization pulse generating system, which would automatically stop if the ECG detected abnormal heart activities, effectively protecting cardiovascular muscles from electric damage. The slight increase in the myocardial enzymes CK and CK-MB was caused by muscle puncture rather than myocardial injury, and the results for Cardiac troponin I confirmed this assumption. CK-MB-related muscle damage and mild increases in LDH and HBDH are tolerable in liver cancer patients. In addition, the use of the general anesthesia and insulated electrode needles prevented the contraction of skeletal muscles. These results proved that the appropriate operation of the synchronization pulse generating system and general anesthesia in nsPEF treatment is necessary.
However, certain problems remain to be solved in the future experiments. (1) It is difficult to find the accompanying arteries of the large veins in the hepatic hilar area under ultrasound guidance and even harder to include the three kinds of vessels (hepatic veins, arteries and bile ducts) in the ablation zone at one time. Therefore, our research mainly focused on the effect of nsPEF ablation on large hepatic veins and bile ducts and some of the minor arteries. However, the effect of nsPEF on large arteries requires further investigation. (2) The risk of thrombosis formation, as reflected by PLT, should be evaluated in the long-term follow-up studies. Such evaluation requires not only the reexamination of coagulation functions, but also the regular ultrasonic monitoring of the large vascular.