Electron microscopic demonstration and evaluation of irreversible electroporation-induced nanopores on hepatocyte membranes

Abstract

Purpose: To demonstrate, evaluate, and verify the existence of irreversible electroporation (IRE)-ablation induced nanopores on the plasma membrane of hepatocytes.

Materials and methods: On animal research committee approval, four New Zealand rabbits and two Yorkshire swine underwent IRE ablation of the liver (90 pulses, 100 μs per pulse at 2,500 V), and selected ablated liver tissues were harvested, fixed, and air-dried according to the electron microscopy (EM) protocol. A scanning electron microscope (SEM; Nova 230 NanoSEM [FEI, Hillsboro, Oregon] with 80 picoamperes and 10-kV acceleration) was used to visualize and verify IRE-created nanopores. Using NIH image (Bethesda, Maryland) and ImageScope (Aperio Inc., Vista, California), 21 ablated tissues (16 rabbit, 5 swine) were evaluated. Corresponding hematoxylin and eosin (H&E) evaluation was performed to verify IRE-induced cell death.

Results: In all 21 IRE-ablated tissues, the SEM was able to show numerous, well-circumscribed, round, and concave-shaped pore defects disturbing the hepatocyte plasma membranes. These pores were not seen in normal liver. The size of the nanopores ranged from 80-490 nm with the greatest frequency of pores in bimodal distribution. The highest frequency of pore size was noted at the size range 340-360 nm.

Conclusions: IRE induces nanopores on hepatocyte membranes, as shown by SEM. The pore diameters are larger than nanopores created by reversible electroporation (RE), which may have implications for irreversibility or permanency.