Fig 1.
Ablation apparatus and the inoculation of rabbit VX2 liver tumors.
A: The nsPEF ablation instrument is on the left, and the radiofrequency ablation instrument is on the right. B: Device for treating cells with nsPEFs. C: Ablation electrodes in animal experiments. D: The pulse waveform generated by nsPEFs. E: Preparation of the needles and tweezers. F: Percutaneous puncture under ultrasound guidance. nsPEFs: nanosecond pulsed electric fields.
Fig 2.
Hep3B cell behavior experiments.
A: Hep3B cells were treated in cuvettes as shown in Fig 1B with 2.5 × 106 cells with pulses with durations of 0 and 300 ns and pulse numbers of 800, respectively. At days 1, 2, and 3 after treatment cell viability was determined by the CCK-8 assay. B: Hep3B cells were treated with pulsed electric field under the above parameters and inoculated into the confocal plate at a density of 10 × 105 cells per well, and then a cell proliferation experiment was conducted. C-F: Hep3B cells treated with the pulsed electric field for 2 h were observed by transmission electron microscopy (C and D, original magnification × 2500; E and F, original magnification × 10000). Data are expressed as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001. nsPEFs: nanosecond pulsed electric fields.
Fig 3.
HE-stained sections of nsPEF-ablated and unablated regions of normal rat livers(n = 6).
The two electrodes of nsPEFs were inserted into the two sides of the liver lobe of anesthetized healthy rats at a distance of 1 cm. Pulse parameters were set as field intensity of 10 kV/cm, frequency of 2 Hz, pulse number of 800 times, pulse duration of 300 ns. At 24 h after ablation, HE staining was performed on the melted tissue. The light-colored areas in the image on the left are ablated areas, and the unablated areas are in the image on the right. As the red arrowheads show, at 24 h after nsPEF ablation, most hepatocytes were large and swollen, with pale eosinophilic cytoplasm, nucleolysis and necrosis. nsPEFs: nanosecond pulsed electric fields.
Fig 4.
Ultrasound images of nsPEFs and RFA of VX2 liver tumor models.
After the rabbit was anesthetized, the sonographer inserted two electrodes around the tumor under ultrasound guidance. Pulse parameters were set as field intensity of 10 kV/cm, frequency of 2 Hz, pulse number of 800 times, pulse duration of 300 ns. The ultrasonic probe was placed at the level of the xiphoid process. After 30 min ablation, contrast-enhanced ultrasound was performed to observe whether there was abnormal blood perfusion in the ablation area. A and B represent ultrasound images taken prior to ablation. The red arrow points to the region before nsPEFs ablation. C shows the two electrodes of the nanosecond pulse ablation system. The red arrow points to the electrode needle of the nsPEFs ablation device. D represents the ultrasound image 30 min after nsPEF ablation. Red arrows point to the region after nsPEFs ablation. E and F represent the ultrasound images before and 30 min after RFA, respectively. Red arrows point to the area before and after RFA. nsPEFs: nanosecond pulsed electric fields; RFA: radiofrequency ablation.
Fig 5.
HE staining and Masson staining images and analysis of tumor tissue after ablation.
A and C: HE and Masson staining were performed on the tissues at days 1, 2, 5 and 7 after nsPEF ablation and 5 days after RFA (n = 6). B: The percentage of nuclear area was calculated in the total high magnification field at different time points and groups (Days 1, 2, 5, and 7 were compared with the hypothesis at 0). D: The IOD percentage of the Masson staining area was calculated at different time points and groups (Days 1, 2, 5, and 7 and RFA groups were each compared with the tumor group). Data are expressed as the mean ± SD. ** P < 0.01; *** P < 0.001. nsPEFs: nanosecond pulsed electric fields; RFA: radiofrequency ablation; IOD: integrated optical density.
Fig 6.
Immunohistochemistry of Ki67, PCNA, and α-SMA was performed on the VX2 tumor rabbit model at days 1, 2, 5 and 7 after nsPEF ablation.
After 5 days RFA ablation, immunohistochemistry of Ki67, PCNA and α-SMA was performed on the rabbit VX2 liver tumor model. A: Representative immunohistochemical staining of Ki67, PCNA and α-SMA in liver sections from the different groups. B-D: Quantitative analysis of Ki67, PCNA and α-SMA staining in the different groups. Data are expressed as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001. PCNA: proliferating cell nuclear antigen; α-SMA: alpha-smooth muscle actin; nsPEFs: nanosecond pulsed electric fields; RFA: radiofrequency ablation.