Comparison between cryoablation and irreversible electroporation of rabbit livers at a location close to the gallbladder

In total, 24 healthy female New Zealand white rabbits, weighing (2.5 ± 0.2) kg each, were provided by the Animal Experimental Center of Jinan University. They were maintained in a clean, and mechanically-ventilated environment at a constant temperature. The rabbits were randomly assigned to the cryoablation group (n = 12) and IRE group (n = 12). All animals were fasted for 24 h before surgery, and intravenous access was established for intraoperative drug administration. The study was approved by the Research Animal Care and Use Committee of Guangzhou Fuda Cancer Hospital (approval number, LL201402).

A cryoablation system (Cryocare^TM, Endocare, Irvine, CA), composed of the main body, argon/ helium gas container and cryoprobes (CRYO-42; Endocare), was used for cryoablation. Rabbits were anesthetized by the intramuscular injection of 44 mg/kg·bw ketamine, intubated and connected to a respirator for mechanically controlled respiration. Intraoperative anesthesia was maintained with 1.5% – 2% isoflurane.

General anesthesia was induced and maintained by the intramuscular injection of 44 mg/kg·bw ketamine. The surgeon had 17 years of experience in cryoablation. The rabbits were fixed in a supine position on the operating table and shaved. The skin was disinfected with iodine, and an abdominal incision was made to expose the liver and gallbladder under sterile conditions. The gallbladder was pressed on the liver, and a cryoprobe with a diameter of 1.4 mm was inserted into the liver until its tip was approximately 0.5 cm away from the gallbladder. The location of the tip was monitored using an ultrasound system (DP-50Vet, Mindray, Shenzhen, China; Figure 1A). When it was confirmed that the cryoprobe was connected to the main equipment, the argon gas was released to freeze the tissue. The cryoablation protocol was a double freeze-thaw cycle consisting of a 2-min freeze and 1-min thaw; this protocol ensured that part of the gallbladder was included within the ablation zone of the ice ball (Figure 1B). No animal died during the cryoablation process. After the cryoablation, the cryoprobe was retrieved. The liver was put back into the abdominal cavity, and conventional abdominal wall sutures were placed to complete the cryoablation. The cryoablation tract was filled in with a thrombin-soaked gelatin sponge and closed with a short suture.

General anesthesia was induced with an intramuscular injection of 44 mg/kg·bw ketamine and maintained with 1.5% – 2% isoflurane. Respiration was controlled using a respirator during the operation (tidal volume: 30 ml/time, respiratory rate: 30/min, oxygen concentration: 100%). Before the IRE operation, 0.12 mg/kg·bw pancuronium was intramuscularly injected to block muscle contraction. IRE was performed using the Angiodynamics Nanoknife system (AngioDynamics, Latham, NY). The Nanoknife system consists of a main body and electrode probes (catalog no. 20400104, AngioDynamics), which were used for electroporation ablation. Monopolar probes were used, which could be changed to achieve 1 – 4 cm of electrode exposure. The size of the ablation zone created depended on the exposed length of the applicator as well as the distance between the probes. When the gallbladder was exposed, two monopolar probes were carefully inserted into the liver (Figure 2A). The probes were inserted to a depth of 0.5 cm into the liver parenchyma under ultrasound guidance (DP-50Vet, Mindray, Shenzhen, China), and were kept 1.5 cm apart and 0.5 cm away from the gallbladder (Figure 3). The monopolar probes were fastened with a spacer device. The ablation parameters consisted of nine groups of ten electrical pulses (total: 90 pulses), and each electrode pulse was 70-μs long; the output voltage was set at 1500 V/cm. This protocol ensured that the electric field covered part of the gallbladder (Figure 2B). Pulse delivery was synchronized with the R waves of the cardiac cycle in case of arrhythmia. One rabbit died during the IRE ablation because of tracheal intubation failure; we replaced this rabbit with another rabbit. After the IRE ablation, muscle relaxation was reversed with intramuscular injections of 5 μg/ kg·bw neostigmine and 5 μg/kg·bw atropine.

The 24 experimental animals returned to consciousness under the monitoring of a senior veterinarian and were sent back to the specific pathogen-free experimental room, which was filled with fresh air and maintained at a constant temperature. All the rabbits were under meticulous care, and pain was managed with intramuscular buprenorphine (0.01 mg/kg) and oral meloxicam (0.4 mg/kg). In addition, the rabbits received daily intramuscular injections of 40 mg/kg·bw cephazolin for 3 days after the surgery in order to prevent infection. For 7 days after the surgery, the animals were observed by veterinarians, who assessed whether the rabbits had suffered any postoperative complications. Feeding was gradually restored over 3 days after the surgery in both groups of animals; however, both food and water intake were lower in the cryoablation group than in the IRE group.

We collected blood (2 ml, from an ear marginal vein) 1 day before the operation and on days 1, 3, 5 and 7 after the ablation. The blood samples were centrifuged to obtain serum. Hepatic function was measured using an automatic biochemical analyzer (Hitachi-7100; Hitachi, Tokyo, Japan). Alanine aminotransferase/aspartate aminotransferase (ALT/ AST) and serum bilirubin levels were determined with kits for in vitro diagnostic use (Biosino Biotechnology and Science Ltd, Beijing, China) by the velocity method.

No animal died after the surgery. At 7 days after the ablation, all the animals were euthanized with an overdose of intravenous pentobarbital sodium (120 mg/kg, Sleepaway; Fort Dodge Animal Health, Fort Dodge, IA). The liver tissues from the ablation tract to the gallbladder were harvested, and the ablation lesions were cut and sectioned from the center. The maximum diameter was measured, and the lesions were photographed. Then, the t issue samples, including the abnormal and adjacent normal tissues, were fixed in 10% neutral buffered formalin, processed routinely for histology, embedded in paraffin, cut into 5-μm-thick slices and stained with hematoxylin and eosin. Pathological analyses were performed by an attending surgical pathologist.

All analyses were performed using GraphPad Prism 5 software (GraphPad Inc., San Diego, CA). The size of the ablation lesions and the serum ALT, AST and bilirubin levels were analyzed using the Student t-test. A P value of < 0.05 was considered to indicate a statistical difference, while P values of < 0.01 and < 0.001 indicated significant differences.

In the cryoablation group, there were severe inflammatory adhesions between the ablation sites in the liver and gallbladder, the abdominal wall and the mesentery (12/12); the liver ablation zone was khaki-colored and oval. The lesion area had a narrow and sharp edge of light pink edematous tissue, and the ablation range obviously covered the gallbladder. There was a large gray area of necrosis adjacent to the gallbladder, and gallbladder perforation was found in four animals (Figure 4). In the IRE g roup, there were f ewer inflammatory adhesions than in the cryoablation group (5/12 vs.12/12). The lesion area was yellow and oval with sharp edges. The ablated gallbladder tissue appeared grayish-white, but none of the 12 experimental animals showed gallbladder perforation (Figure 5). Details of the sizes of the ablation lesions are presented in Table 1; there were no differences in lesion size between the two groups. As gallbladder perforation was found in four animals in the cryoablation group, we recorded the major diameter of the gallbladder lesions in only eight animals in this group.

There were no intact hepatic cells in the liver slices in both the cryoablation and IRE groups. From the cryoablation tract to the liver edge, the cryolesion appeared as a pink-stained area of disintegrating tissue that included hemorrhagic congestion and tissue necrosis, and it showed nuclear disappearance, no intact cellular structures, a band of local bleeding, and abundant inflammatory cell infiltration (Figure 6A). In the IRE group, the main pathological changes consisted of small bile duct, acute granulation tissue hyperplasia, rare congestion, hemorrhage band and a small amount of inflammatory cell infiltration (Figure 6B). The most conspicuous difference between the two groups was the appearance of the gallbladder: in the cryoablation group, full-thickness gallbladder wall necrosis, muscular collapse and mucosal loss were found, and the necrotic area was large and uniformly stained red. In the IRE group, there was mucosal epithelial necrosis but no full-thickness gallbladder wall necrosis, and serous layer edema were largely concentrated in the area of the gallbladder wall near the liver.

In both groups, the serum aminotransferase and bilirubin levels obviously increased on day 3, gradually decreased thereafter and recovered to normal in a week. At each time point, the serum aminotransferase and serum bilirubin levels were higher in the cryoablation group than in the IRE group (Table 2).

This study was funded by the Medical and Health Fund of Guangdong Province (2013kw051), China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.