Histopathological and Ultrastructural Changes after Electroporation in Pig Liver Using Parallel-Plate Electrodes and High-Performance Generator

Abstract

Irreversible electroporation (IRE) has gained attention as a new non-thermal therapy for ablation with important benefits in terms of homogeneous treatment and fast recovery. In this study, a new concept of high voltage generator is used, enabling irreversible electroporation treatment in large tissue volume using parallel plates. Unlike currently available generators, the proposed versatile structure enables delivering high-voltage high-current pulses. To obtain homogeneous results, 3-cm parallel-plates electrodes have also been designed and implemented. IRE ablation was performed on six female pigs at 2000 V/cm electric field, and the results were analysed after sacrifice three hours, three days and seven days after ablation. Histopathological and ultrastructural studies, including transmission and scanning electron microscopy, were carried out. The developed high-voltage generator has proved to be effective for homogeneous IRE treatment using parallel plates. The destruction of the membrane of the hepatocytes and the alterations of the membranes of the cellular organelles seem incompatible with cell death by apoptosis. Although endothelial cells also die with electroporation, the maintenance of vascular scaffold allows repairing processes to begin from the third day after IRE as long as the blood flow has not been interrupted. This study has opened new direction for IRE using high performance generators and highlighted the importance of taking into account ultrastructural changes after IRE by using electron microscopy analysis.

Figure 1

Electroporation. Reversible (RE, left) and irreversible (IRE, right) processes.

Figure 2

Example of application of IRE electrodes on human liver tissue using parallel-plate electrodes. “BodyParts3D liver” by Lambchops is licensed under CC-BY SAExample of application of IRE electrodes on human liver tissue using parallel-plate electrodes 2.1JP (This image has been modified).

Figure 3

Electric field generated using 3-cm parallel plates. Electrodes are situated in the upper and lower part, and liver tissue is in the middle. Homogeneous field distribution is found in the central part, providing known and repeatable results for this study. Simulated with COMSOL Multiphysics v. 5.2, Stockholm, Sweden.

Figure 4

Versatile high-voltage pulse generator. The combination of isolated output full-bridge inverter cells provides high-voltage capabilities.

Figure 5

Main current and voltage waveforms during IRE treatment. Current and conductivity increases due to cell permeabilization. Full treatment train of pulses comprising 100 pulses of 100 µs (a) and detail of one pulse (b). Voltage polarity is changed between pulses and absolute values are plotted for effective representation. Besides, gap between pulses has been omitted for illustrative purposes.

Figure 6

Experimental test. 3-cm stainless-steel parallel-plate electrodes placed embracing a lobe of the pig liver. Histological samples obtention. Three samples are obtained from the electroporated area (1), interphase with normal not treated tissue (2) and healthy tissue (3).

Figure 7

Digital image analysis of the electroporated area. The measured area is in the yellow box (165 mm2) and encompasses the necrotic area. The green zone corresponds to the regenerative zones.

Figure 8

Histopathology of the liver after IRE ablation with H&E stain. Three hours (3 h) after IRE the lobular architecture is preserved although with visible hemorrhagic, congestion, and edema(a–d). After three (3d) and seven days (7d), two zones can be distinguished: one necrotic (nz) and the other with signs of cellular regeneration (rz) (e,i). The hepatocytes of the electroporated area appear irregular and with disfigurement of the membranes, apparently intact nuclei although some of them with karyolysis (arrows, (c,d and h)). The centrilobular vein (cv) and the hepatocyte trabeculae are distorted with loss of hepatocytes (arrows) and endothelial cells (b,f and j). The reparative area show: new bile ducts and vessels (asterisks, (g,k)); mesenchymal spindle cells infiltrating the necrotic area (arrowheads, (h)) and regenerative nodules of hepatocytes with acinar arrangement and mitotic figures (arrows, (l)).

Figure 9

Electron micrograph of pig liver after IRE. Portion of a liver lobule showing plates of hepatocytes damaged but preserving its structure (b–d) 2,000X. Under TEM, the hepatocytes show lysis of the plasma membrane with the release of organelles, dilatation of the granular endoplasmic reticulum and mitochondrial alterations but the nuclear envelope is preserved (arrowhead, f,g). After 7 days (7d), phagocytic cells are frequent, phagocytizing cell debris (h). SEM of large blood vessel, after 3 hours (3 h) and 3 days (3d), illustrating the lack of endothelial cells and their substitution by platelets (arrows) (j,k) 2,000X. After 7 days, endothelial cells are distinguished in some vessels (l). Controls were taken in healthy tissue (a,e and i). Scale bar (a–d,i–l) 10 μm.

Figure 10

TEM 3 days after IRE. The plasma membrane of the hepatocytes shows small pores (arrows) together with large areas in which the membrane has disappeared (arrowheads). Amounts of chromatin are expelled from the nucleus to extracellular space.