title: Electron microscopic demonstration and evaluation of irreversible electroporation-induced nanopores on hepatocyte membranes.
abstract: OBJECTIVE
  To demonstrate, evaluate, and verify the existence of irreversible electroporation (IRE)-ablation induced nanopores on the plasma membrane of hepatocytes.
  
  
  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.
authors.affiliation: Division of Interventional Radiology, Department of Radiology, Ronald Reagan Medical Center at UCLA Medical Center, 757 Westwood Plaza, Suite 2125, Los Angeles, CA 90095-743730, USA. EdwardLee@mednet.ucla.edu
authors.forename: Edward W
authors.initials: EW
authors.lastname: Lee
authors.forename: Daphne
authors.initials: D
authors.lastname: Wong
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Prikhodko
authors.forename: Alejandro
authors.initials: A
authors.lastname: Perez
authors.forename: Cassidy
authors.initials: C
authors.lastname: Tran
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Loh
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Kee
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure; D018274:Electroporation / Q000379:methods*; D022781:Hepatocytes / Q000648:ultrastructure*; D008099:Liver / Q000166:cytology / Q000601:surgery*; D008855:Microscopy, Electron, Scanning; D058608:Nanopores / Q000648:ultrastructure; D011817:Rabbits; D013552:Swine