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.

Keywords: Cell death mechanism; Liver; Minimally invasive regenerative surgery; Non thermal irreversible electroporation.

Figure 1. The histology of the H&E stained liver at different locations relative to the treatment zone and at different times after treatment

The columns from left are: macroscopic cross section; untreated region; the interface between the untreated (left side) and treated (right side) regions; core of the treated region at (×20); core of the treated region (×40). The rows are, from top to bottom: 1h, 3h, 6h, and 24h after treatment. Scale bar given in the figures. Thin arrow, triangle arrow, arrowhead, arrow with flat tail, point to hepatocytes, endothelial cells, Kupffer cell and vacuolated hepatocyte without nuclei structure, respectively. Arrows marked with S point to wide sinusoids. Arrow marked with C&H point to areas of congestion and hemorrhagic change. Arrow marked with B point to bi-nucleate hepatocytes. CV means central vein. BD means bile duct.

Figure 2. Histology with Mason’s trichrome stain for evaluation of collagen after NTIRE

The columns from left are: macroscopic cross section; untreated region; the interface between the untreated (left side) and treated (right side) regions; core of the treated region at (×20); core of the treated region (×40). The rows are, from top to bottom: 1h, 3h, 6h, and 24h after treatment. Scale bar in the micrographs. Thin arrow points to collagen. PV means portal vein, HA – hepatic artery. BD means bile duct. CV means central vein.

Figure 3. Histology with TUNEL stain

The first column shows a macroscopic cross section through the treated lobe, the second column shows micrographs from the center of the treated lesion at ×10 and the third column shows the same site at × 40. The rows are for 1h, 3h, 6h, and 24h after the treatment. Scale bar in the micrographs.

Figure 4. Western blot

Response to antibodies against pro Caspase 3, cleaved Caspase 3, pro Caspase 1, cleaved Caspase 1, GSDMD, cleaved GSDMD, RIP3 and MLKL are shown GAPDH was used as a loading control. The figure shows, from right to left, activities of not treated controls (NC) and NTIRE treated tissues, 1h, 3h, 6h and 24h after the treatment. The columns and rows are labeled.