Plasma membrane permeabilization by trains of ultrashort electric pulses

Radio Frequency Radiation Branch, 711th Human Performance Wing, Air Force Research Laboratory, Brooks City Base, San Antonio, TX, United States.
Bioelectrochemistry (Amsterdam, Netherlands) (Impact Factor: 4.17). 08/2010; 79(1):114-21. DOI: 10.1016/j.bioelechem.2010.01.001
Source: PubMed


Ultrashort electric pulses (USEP) cause long-lasting increase of cell membrane electrical conductance, and that a single USEP increased cell membrane electrical conductance proportionally to the absorbed dose (AD) with a threshold of about 10 mJ/g. The present study extends quantification of the membrane permeabilization effect to multiple USEP and employed a more accurate protocol that identified USEP effect as the difference between post- and pre-exposure conductance values (Deltag) in individual cells. We showed that Deltag can be increased by either increasing the number of pulses at a constant E-field, or by increasing the E-field at a constant number of pulses. For 60-ns pulses, an E-field threshold of 6 kV/cm for a single pulse was lowered to less than 1.7 kV/cm by applying 100-pulse or longer trains. However, the reduction of the E-field threshold was only achieved at the expense of a higher AD compared to a single pulse exposure. Furthermore, the effect of multiple pulses was not fully determined by AD, suggesting that cells permeabilized by the first pulse(s) in the train become less vulnerable to subsequent pulses. This explanation was corroborated by a model that treated multiple-pulse exposures as a series of single-pulse exposures and assumed an exponential decline of cell susceptibility to USEP as Deltag increased after each pulse during the course of the train.


Available from: Bennett L Ibey, Dec 25, 2013
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    • "Nanoporation, the generation of very small (<2 nm) pores in the plasma membrane, is hypothesized to result from exposures of very short (<1 µs) electric pulses in the megavolt/meter range [1] [2] [3]. The exposure to nsEP is not a " clean " insult, making determination of the mechanism of nanoporation quite difficult. "
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    ABSTRACT: Despite 30 years of research, the mechanism behind the induced breakdown of plasma membranes by electrical pulses, termed electroporation, remains unknown. Current theories treat the interaction between the electrical field and the membrane as an entirely electrical event pointing to multiple plausible mechanisms. By investigating the biophysical interaction between plasma membranes and nanosecond electrical pulses (nsEP), we may have identified a non-electric field driven mechanism, previously unstudied in nsEP, which could be responsible for nanoporation of plasma membranes. In this investigation, we use a non-contact optical technique, termed probe beam deflection technique (PBDT), to characterize acoustic shockwaves generated by nsEP traveling through tungsten wire electrodes. We conclude these acoustic shockwaves are the result of the nsEP exposure imparting electrohydraulic forces on the buffer solution. When these acoustic shockwaves occur in close proximity to lipid bilayer membranes, it is possible that they impart a sufficient amount of mechanical stress to cause poration of that membrane. This research establishes for the first time that nsEP discharged in an aqueous medium generate measureable pressure waves of a magnitude capable of mechanical deformation and possibly damage to plasma membranes. These findings provide a new insight into the longunanswered question of how electric fields cause the breakdown of plasma membranes.
    SPIE Photonics West 2014; 02/2014
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    • "For shorter pulses (b 60 ns), the Ca 2+ release is believed to result from intracellular Ca 2+ stores. However, for longer or greater numbers of pulses, nanopore formation in the plasma membrane has been demonstrated [2] [3] [4] [5] [6]. The nsPEF-induced Ca 2+ rise appears similar to the calcium rise observed after purinergic P 2 Y 6 G q/11 coupled receptor stimulation [7], and thus may signify phosphoinositide signaling induction. "
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    ABSTRACT: Exposure to nanosecond pulsed electrical fields (nsPEFs) results in a myriad of observable effects in mammalian cells. While these effects are often attributed to the direct permeabilization of both the plasma and organelle membranes, the underlying mechanism(s) are not well understood. We hypothesize that nsPEF-induced membrane disturbance will initiate complex intracellular lipid signaling pathways, which ultimately lead to the observed multifarious effects. In this article, we show activation of one of these pathways - phosphoinositide signaling cascade. Here we demonstrate that nsPEF initiates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis or depletion from the plasma membrane, accumulation of inositol-1,4,5-trisphosphate (IP3) in the cytoplasm and increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. All of these events are initiated by a single 16.2kV/cm, 600ns pulse exposure. To further this claim, we show that the nsPEF-induced activation mirrors the response of M1-acetylcholine Gq/11-coupled metabotropic receptor (hM1). This demonstration of PIP2 hydrolysis by nsPEF exposure is an important step toward understanding the mechanisms underlying this unique stimulus for activation of lipid signaling pathways and is critical for determining the potential for nsPEFs to modulate mammalian cell functions.
    Bioelectrochemistry (Amsterdam, Netherlands) 05/2013; 94C:23-29. DOI:10.1016/j.bioelechem.2013.05.002 · 4.17 Impact Factor
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    • "The amplitude of the electric field was estimated using a three dimensional Finite Difference Time Domain (FDTD) model (Fig. 1C) [30], [31], as previously reported by Ibey et al. [26]. The model included a pair of two 125 µm diameter tungsten electrodes separated by 100 µm, angled at 30°, positioned 50-µm above the 180 µm thick glass coverslip and immersed in a 0.9% homogenous saline solution. "
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    ABSTRACT: High-amplitude, MV/m, nanosecond pulsed electric fields (nsPEF) have been hypothesized to cause nanoporation of the plasma membrane. Phosphatidylserine (PS) externalization has been observed on the outer leaflet of the membrane shortly after nsPEF exposure, suggesting local structural changes in the membrane. In this study, we utilized fluorescently-tagged Annexin V to observe the externalization of PS on the plasma membrane of isolated Chinese Hamster Ovary (CHO) cells following exposure to nsPEF. A series of experiments were performed to determine the dosimetric trends of PS expression caused by nsPEF as a function of pulse duration, τ, delivered field strength, ED, and pulse number, n. To accurately estimate dose thresholds for cellular response, data were reduced to a set of binary responses and ED50s were estimated using Probit analysis. Probit analysis results revealed that PS externalization followed the non-linear trend of (τ*ED (2))(-1) for high amplitudes, but failed to predict low amplitude responses. A second set of experiments was performed to determine the nsPEF parameters necessary to cause observable calcium uptake, using cells preloaded with calcium green (CaGr), and membrane permeability, using FM1-43 dye. Calcium influx and FM1-43 uptake were found to always be observed at lower nsPEF exposure parameters compared to PS externalization. These findings suggest that multiple, higher amplitude and longer pulse exposures may generate pores of larger diameter enabling lateral diffusion of PS; whereas, smaller pores induced by fewer, lower amplitude and short pulse width exposures may only allow extracellular calcium and FM1-43 uptake.
    PLoS ONE 04/2013; 8(4):e63122. DOI:10.1371/journal.pone.0063122 · 3.23 Impact Factor
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