Molecular and histological study on the effects of non-thermal irreversible electroporation on the liver

Abstract

Non-thermal irreversible electroporation (NTIRE) is a biophysical phenomenon in which certain electric fields delivered across the cell membrane in tissue, cause cell death, without affecting the extracellular matrix. "Minimally invasive regenerative surgery" is a new medical modality for treatment of end-stage organ or tissue failure in which exogenous cells are implanted in a decellularized niche in tissue, formed by the delivery of NTIRE electric fields across a targeted volume of tissue. We anticipate that the success of the procedure will depend on the time of implantation relative to the application of NTIRE. This study was performed to elucidate the histological and molecular events that occur within 24 h after NTIRE, in the context of optimal criteria for the time of implantation. To this end, we examined the histology of NTIRE treated rat liver with H&E, Masson trichrome and TUNEL staining. Western blot was used to examine pro and cleaved caspase-3 (marker for apoptosis), pro and cleaved caspase-1 and gasdermin D (markers for pyroptosis), and RIP3 and MLKL (markers for necroptosis). The key findings are that, complete hepatocytes disintegration within an intact extracellular matrix is seen at 6 h and, new hepatocytes are seen in the treated region at 24 h, after NTIRE. There is no evidence of apoptotic cell death from NTIRE, contrary to commonly made claims in the NTIRE literature. However, molecular pathways of pyroptosis and necroptosis, programed necrosis associated with inflammation, are activated at 6 h after NTIRE and are not evident at 24 h after NTIRE. These are fundamental new findings of basic value to the field of NTIRE in all its applications. Taken together the results suggest the hypothesis that an optimal time for implantation is about 24 h after NTIRE. Future studies in which exogenous cells are implanted at different times after NTIRE are required to examine this hypothesis.

Dynamics of Cell Death After Conventional IRE and H-FIRE Treatments

“…The absence of Caspase 3/7 expression in cells treated with conventional IRE is consistent with recent studies that report RCD mechanisms independent of Caspase 3/7 expression such as pyroptosis or necroptosis. 25,55 In the case of H-FIRE, histological examination of tissues found activation of Caspase 3, 29,43 consistent with our results. However, another study showed evidence of pyroptotic and necroptotic cell death 35 which would contradict this study.…”

“…7,20,23,53,54 However, our understanding of the death mechanisms has quickly evolved in recent years and more recent studies point towards other forms of RCD such as pyroptosis or necroptosis rather than apoptosis. 25,55 Currently, very little is known regarding the cell death dynamics and mechanisms of H-FIRE and how they differ from those of conventional IRE treatments. The pulse lengths that make up H-FIRE bursts have been relatively unexplored 49 and the cell death dynamics and mechanisms after the delivery of bipolar pulses have been scarcely studied.…”

Analysis of damage-associated molecular pattern molecules due to electroporation of cells in vitro

“… 11 , 12 Both pathways were confirmed also in microsecond pulse treatment. 13 , 14 , 15 , 16 , 17 , 18 , 19 Nevertheless, in recent studies new cell death types were also detected like pyroptosis 20 , necroptosis 20 , 21 and immunogenic cell death. 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 …”

Irreversible Electroporation in Liver Cancers and Whole Organ Engineering

“…Nevertheless, this analysis demonstrated the utility of IRE in whole segments of liver tissue. More recently, Zhang et al performed a molecular and histological analysis on IRE-treated rat liver, seeking to elucidate the cell-level events that occur up to 24 h after ablation [16]. The investigators noted complete hepatocyte disintegration at 6 h post-treatment and by 24 h, new hepatocytes were seen in the treated region.…”

“…The non-thermal nature of this ablation technique distinguishes it from other commonly used thermal methods like microwave and radiofrequency ablation techniques. The exact mechanism of cell death in IRE is under debate and apoptosis, necroptosis, and pyroptosis have been observed in the laboratory [15,16,17].…”