The second phase of bipolar, nanosecond-range electric pulses determines the electroporation efficiency

Abstract

Bipolar cancellation refers to a phenomenon when applying a second electric pulse reduces ("cancels") cell membrane damage by a preceding electric pulse of the opposite polarity. Bipolar cancellation is a reason why bipolar nanosecond electric pulses (nsEP) cause weaker electroporation than just a single unipolar phase of the same pulse. This study was undertaken to explore the dependence of bipolar cancellation on nsEP parameters, with emphasis on the amplitude ratio of two opposite polarity phases of a bipolar pulse. Individual cells (CHO, U937, or adult mouse ventricular cardiomyocytes (VCM)) were exposed to either uni- or bipolar trapezoidal nsEP, or to nanosecond electric field oscillations (NEFO). The membrane injury was evaluated by time-lapse confocal imaging of the uptake of propidium (Pr) or YO-PRO-1 (YP) dyes and by phosphatidylserine (PS) externalization. Within studied limits, bipolar cancellation showed little or no dependence on the electric field intensity, pulse repetition rate, chosen endpoint, or cell type. However, cancellation could increase for larger pulse numbers and/or for longer pulses. The sole most critical parameter which determines bipolar cancellation was the phase ratio: maximum cancellation was observed with the 2nd phase of about 50% of the first one, whereas a larger 2nd phase could add a damaging effect of its own. "Swapping" the two phases, i.e., delivering the smaller phase before the larger one, reduced or eliminated cancellation. These findings are discussed in the context of hypothetical mechanisms of bipolar cancellation and electroporation by nsEP.

Using Nanosecond Shocks for Cardiac Defibrillation

“…The pulse shapes typically used in whole heart 29 and single cell experiments 58 are approximately trapezoidal, with rise and fall time in the range of 5-20% of the pulse duration; we do not expect that differences in pulse shape are responsible for substantial changes in stimulation results as those already discussed.…”

Paradoxical effects on voltage-gated Na+ conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses

“…This is analogous to the phenomenon of bipolar cancellation whereby the application of a second nanosecond pulse of opposite polarity can attenuate or abolish a cellular response (Ca 2+ transient, electronanoporation, fluorescent dye uptake, etc.) triggered by the corresponding unipolar pulse [25][26][27][28][29][30][31][32][33].…”

“…One plausible hypothesis is that twin NEPs produced a transient suppression of the number of active channels through perturbations of the lipid bilayer closely interacting with the channel protein. This suggestion is based on a number of indirect observations from our group and others: 1) theoretical molecular dynamics studies [34][35][36] demonstrated that high intensity NEPs are capable of triggering the formation of hydrophilic nanopores in the phospholipid bilayer that lower the energy barrier for ion conduction across the membrane; 2) single or multiple NEPs of various durations are known to cause important perturbations of the lipid bilayer, a process called electronanoporation, that allows the membrane to become permeable not only to ions [37,38] but in some cell types also to fluorescent organic molecules such as propidium iodide and YO-PRO-1 [25,26,29,33,[38][39][40][41][42]. Even though the application of up to ten 5 ns pulses does not lead to YO-PRO-1 uptake in chromaffin cells [23], a non-selective membrane conductance (I leak ) permeable to Na + could be elicited by a single 5 ns pulse in chromaffin cells clamped near the resting membrane potential (-70 mV) in these cells [5], which has also been detected for longer NEPs in GH3 and NG108 cells [7,8] and also in bovine chromaffin cells [8].…”

High-voltage 10 ns delayed paired or bipolar pulses for in vitro bioelectric experiments

“…Similarly, traditional effects of nsPEF were cancelled by the exposure to asymmetric bipolar nsPEF when the amplitude and the duration of the second pulse were reduced to 35% [2] and 33% [10] of the front pulse, respectively. Recently, Pakhomov et al [11] evaluated the efficiency of electroporation as a function of the second phase of the bipolar nsPEF with durations between 200 ns and 830 ns. It was evident that maximum cancellation, or in other words the least electroporation, occurred when the amplitude of the pulse's second polarity was 50% of the first polarity.…”

“…Results of these studies strongly suggest that duration, amplitude, and delay of the reversed second pulse play an important role in determining the efficacy of cancellation. However, despite several formulated hypothesis [11], the mechanism responsible for cancellation is still under investigation. In addition, bipolar cancellation is characteristic of the nanosecond range, as it was not observed in conventional electroporation.…”