Research Article: A Vivens Ex Vivo Study on the Synergistic Effect of Electrolysis and Freezing on the Cell Nucleus

Date Published: December 22, 2015

Publisher: Public Library of Science

Author(s): Franco Lugnani, Fabrizio Zanconati, Thomas Marcuzzo, Cristina Bottin, Paul Mikus, Enric Guenther, Nina Klein, Liel Rubinsky, Michael K. Stehling, Boris Rubinsky, Christof Markus Aegerter.


Freezing—cryosurgery, and electrolysis—electrochemical therapy (EChT), are two important minimally invasive surgery tissue ablation technologies. Despite major advantages they also have some disadvantages. Cryosurgery cannot induce cell death at high subzero freezing temperatures and requires multiple freeze thaw cycles, while EChT requires high concentrations of electrolytic products—which makes it a lengthy procedure. Based on the observation that freezing increases the concentration of solutes (including products of electrolysis) in the frozen region and permeabilizes the cell membrane to these products, this study examines the hypothesis that there could be a synergistic effect between freezing and electrolysis in their use together for tissue ablation. Using an animal model we refer to as vivens ex vivo, which may be of value in reducing the use of animals for experiments, combined with a Hematoxylin stain of the nucleus, we show that there are clinically relevant protocols in which the cell nucleus appears intact when electrolysis and freezing are used separately but is affected by certain combinations of electrolysis and freezing.

Partial Text

Surgery is the field of medicine that employs manual and instrumental techniques to treat medical conditions. Minimally invasive and non-invasive surgery have emerged as an important branch of surgery. They employ a variety of biophysical based techniques, each with their specific advantages, disadvantages and applications. Enlarging the armamentarium of minimally invasive surgery technologies can provide new tools for treating diseases. This study introduces and explores a possible new tissue ablation technique that combines two biophysical processes, freezing and electrolysis. It is interesting to note that both processes originate from the early 19th century work of Faraday on technologies for lowering temperature and on technologies for generating electrochemical reactions, respectively.

Liver tissue from 200 kg pigs were treated within 10 minutes from animal death. The livers were obtained from a certified and regulated commercial abattoir and the experiments were carried out at an adjacent location outside the abattoir. Cryosurgery was delivered with a cryosurgery system, Cryo Electric S model CO2 driven Metrum Cryoflex Warsaw, Poland using commercial 2 mm, G20 type reusable cryoprobes, Trocar type, Cryoflex Warsaw, Poland. Electrolysis was delivered using a laboratory power supply (HCS 3304 USB, Manson, Hong Kong), whose electric output was attached to steel rods, with the same diameter as the cryosurgery probes, namely 2 mm.

The two panels on the right hand side on Fig 1 were used to establish the base line for the histological evaluation. The top panel shows the cellular morphology of pig liver tissue from an in vivo study. The bottom panel shows the cellular morphology of a vivens ex vivo pig liver from an area which we used as control. With regards to the nucleus, it has a similar morphology in both panels. The nuclei are transparent and round, with normal chromatin distribution, and the nucleolus remain clearly visible.

Obviously, our analysis is limited by the nature of the model we have used. Nevertheless, we believe that there is value in using the vivens ex vivo model in a first feasibility study. In fact it is a requirement of the 3R principle. We believe that this study shows that despite its limitations, the model can generate important information.