Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.     

Transmembrane molecular transport during versus after extremely large, nanosecond electric pulses

Recently there has been intense and growing interest in the non-thermal biological effects of nanosecond electric pulses, particularly apoptosis induction. These effects have been hypothesized to result from the widespread creation of small, lipidic pores in the plasma and organelle membranes of cells (supra-electroporation) and, more specifically, ionic and molecular transport through these pores. Here we show that transport occurs overwhelmingly after pulsing. First, we show that the electrical drift distance for typical charged solutes during nanosecond pulses (up to 100 ns), even those with very large magnitudes (up to 10 MV/m), ranges from only a fraction of the membrane thickness (5 nm) to several membrane thicknesses. This is much smaller than the diameter of a typical cell (∼16 μm), which implies that molecular drift transport during nanosecond pulses is necessarily minimal. This implication is not dependent on assumptions about pore density or the molecular flux through pores. Second, we show that molecular transport resulting from post-pulse diffusion through minimum-size pores is orders of magnitude larger than electrical drift-driven transport during nanosecond pulses. While field-assisted charge entry and the magnitude of flux favor transport during nanosecond pulses, these effects are too small to overcome the orders of magnitude more time available for post-pulse transport. Therefore, the basic conclusion that essentially all transmembrane molecular transport occurs post-pulse holds across the plausible range of relevant parameters. Our analysis shows that a primary direct consequence of nanosecond electric pulses is the creation (or maintenance) of large populations of small pores in cell membranes that govern post-pulse transmembrane transport of small ions and molecules.     

Nanosecond electric pulses cause mitochondrial membrane permeabilization in Jurkat cells

Nanosecond, high‐voltage electric pulses (nsEP) induce permeabilization of the plasma membrane and the membranes of cell organelles, leading to various responses in cells including cytochrome c release from mitochondria and caspase activation associated with apoptosis. We report here evidence for nsEP‐induced permeabilization of mitochondrial membranes in living cells. Using three different methods with fluorescence indicators—rhodamine 123 (R123), tetramethyl rhodamine ethyl ester (TMRE), and cobalt‐quenched calcein—we have shown that multiple nsEP (five pulses or more, 4 ns duration, 10 MV/m, 1 kHz repetition rate) cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. These effects could be a consequence of nsEP permeabilization of the inner mitochondrial membrane or the activation of mitochondrial membrane permeability transition pores. Plasma membrane permeabilization (YO‐PRO‐1 influx) was detected in addition to mitochondrial membrane permeabilization. Bioelectromagnetics 33:257–264, 2012. © 2011 Wiley Periodicals, Inc.     

Experimental study and mechanism analysis on bioeffects by nanosecond electromagnetic pulses

The athermal bioeffects caused by nanosecond electromagnetic pulses with body cells was studied by using a broad band transverse EM-wave cell (BTEM CELL). The experimental system and preliminary mechanism analysis were presented.     

Activation of the JNK pathway by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.     

Recruitment of the intracellular Ca by ultrashort electric stimuli: The impact of pulse duration

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca²⁺ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca²⁺ imaging with Fura-2. In a Ca²⁺-free medium, 10-, 60-, and 300-ns stimuli evoked Ca²⁺ transients by mobilization of Ca²⁺ from the endoplasmic reticulum. With 2 mM external Ca²⁺, the transients included both extra- and intracellular components. The recruitment of intracellular Ca²⁺ increased as the stimulus duration decreased. At the threshold of 200–300 nM, the transients were amplified by calcium-induced calcium release. We conclude that nanosecond stimuli mimic Ca²⁺ signaling while bypassing the usual receptor- and channels-mediated cascades. The recruitment of the intracellular Ca²⁺ can be controlled by the duration of the stimulus.     

Ultrashort electric pulse induced changes in cellular dielectric properties

The interaction of nanosecond duration pulsed electric fields (nsPEFs) with biological cells, and the models describing this behavior, depend critically on the electrical properties of the cells being pulsed. Here, we used time domain dielectric spectroscopy to measure the dielectric properties of Jurkat cells, a malignant human T-cell line, before and after exposure to five 10ns, 150kV/cm electrical pulses. The cytoplasm and nucleoplasm conductivities decreased dramatically following pulsing, corresponding to previously observed rises in cell suspension conductivity. This suggests that electropermeabilization occurred, resulting in ion transport from the cell's interior to the exterior. A delayed decrease in cell membrane conductivity after the nsPEFs possibly suggests long-term ion channel damage or use dependence due to repeated membrane charging and discharging. This data could be used in models describing the phenomena at work.     

Differences in the electric birefringence of spectrin dimers and tetramers as shown by the fast reversing electric pulse method

The electric birefringence of purified Spectrin has been examined in medium of low ionic strength ai 20°C and for electric fields smaller than 4 × 10⁴ V m^?1. using the reversing electric pulse method. This technique allows study of the permanent and induced dipole electric moment of macromolecules more easily than in measurements using only rectangular pulses. We show that spectrin heterodimers and heterotetramers have different electro-optical properties. The relaxation time of the tetramer (7 μs) is significantly longer than that of the dimer (4.5 μs). Tetramers and dimers have also different polarizability parameters.     

Aspects of plant plasmalemma charging induced by external electric field pulses

The charging of the plasmalemma is a necessary condition for permeabilization of the plasma membrane (electroporation) in response to external electric field exposure. Common theories explain this permeabilization by formation of pores in the lipid bilayer. Using pulsed laser fluorescence microscopy, we measured the charging process of the membrane during the application of an external electric field with a temporal resolution of 5 ns. Visualization of the charging process of protoplasts plasma membrane (Nicotiana tabacum Bright Yellow 2) was achieved by staining of the plasma membrane with the voltage-sensitive fluorescent dye ANNINE-6. Measurements on membranes exhibiting negligible membrane permeabilization confirm the sine-shaped azimuthal distribution of the membrane voltage predicted by the relation of Cole. At higher membrane voltages, enhanced pore formation allows for the exchange of charge carriers, leading to deviations from the sine-shaped curve progression, i.e., a saturation of the membrane voltage at membrane segments facing the electrodes. Additionally, measurements on protoplasts exposed to multiple successive pulses indicate that the recovery of the membrane seems to be a fast process, occurring within seconds after termination of the external electric field pulse.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

电脉冲和布吡卡因增强A型肉毒毒素DNA核酸疫苗的免疫效果

目的：考查DNA疫苗注射免疫后电脉冲和布吡卡因佐剂化DNA疫苗递送方式对A型肉毒毒素DNA核酸疫苗免疫效果的影响。方法：A型肉毒毒素DNA复制子疫苗和传统DNA疫苗肌肉注射免疫小鼠后电脉冲和布吡卡因佐剂化DNA后再肌肉注射免疫小鼠;检测免疫小鼠的抗体和细胞水平,并分析抗体亚类。结果：电脉冲和布吡卡因这二种递送方式均增强DNA复制子疫苗和传统DNA疫苗的体液免疫和细胞免疫效果;电脉冲提高DNA疫苗免疫效果更为明显,并且电脉冲和布吡卡因组合这种递送方式增强DNA疫苗体液免疫和细胞免疫水平最高;与传统DNA疫苗相比,A型肉毒毒素DNA复制子疫苗在这些递送方式下均诱导产生了更好的特异性体液免疫和细胞免疫应答,并且这些递送方式没有改变DNA疫苗的Th1/Th2免疫应答特性,即DNA复制子疫苗诱导产生Th1/Th2混合免疫应答但偏向于Th2途经,而传统DNA疫苗则完全偏向于Th2途经。结论：电脉冲和布吡卡因增强DNA复制子疫苗和传统DNA疫苗的免疫效果,是提高DNA疫苗免疫原性的良好策略。     

Effect of actin cytoskeleton disruption on electric pulse‐induced apoptosis and electroporation in tumour cells

Electric pulses are known to affect the outer membrane and intracellular structures of tumour cells. By applying electrical pulses of 450 ns duration with electric field intensity of 8 kV/cm to HepG2 cells for 30 s, electric pulse‐induced changes in the integrity of the plasma membrane, apoptosis, viability and mitochondrial transmembrane potential were investigated. Results demonstrated that electric pulses induced cell apoptosis and necrosis accompanied with the decrease of mitochondrial transmembrane potential and the formation of pores in the membrane. The role of cytoskeleton in cellular response to electric pulses was investigated. We found that the apoptotic and necrosis percentages of cells in response to electric pulses decreased after cytoskeletal disruption. The electroporation of cell was not affected by cytoskeletal disruption. The results suggest that the disruption of actin skeleton is positive in protecting cells from killing by electric pulses, and the skeleton is not involved in the electroporation directly.     

Calcium modulates conformational changes in F-actin induced by smooth muscle heavy meromyosin

The effect of Ca²⁺ on conformational changes in rhodamine-phalloidin-labeled F-actin induced by binding of smooth muscle heavy meromyosin (HMM) with either phosphorylated or dephosphorylated regulatory light chains (LC₂₀) was studied by polarized fluorimetry. LC₂₀ phosphorylation caused alterations in the F-actin structure typical of the force-producing (strong-binding) state, while dephosphorylation of the chains led to alterations typical of the formation of non-force-producing (weak-binding) state of the actomyosin complex. The presence of Ca²⁺ enhanced the effect of LC₂₀ phosphorylation and weakened the effect of LC₂₀ dephosphorylation. These data suggest that Ca²⁺ modulates actin-myosin interaction in smooth muscle by promoting formation of the strong-binding state.     

Current densities induced in swine and rat models by power-frequency electric fields

Measurements have been made of vector current densities induced by vertical, uniform, 60-Hz electric fields in the torsos of homogeneous models of swine and rats. The observed data were a strong function of the five grounding configurations invested: all four feet grounded, only front feet grounded, only rear feet grounded, left front and right rear feet grounded, and right front and left rear feet grounded. In the first configuration and with an exposure field strength of 10 kV/m, average total current densities induced in the torsos of pigs and rats were 34 nA/cm2 and 20 nA/cm2, respectively. The corresponding value for human exposure is about 250 nA/cm2, 7.3 and 12.5 times larger than for swine and rats, respectively. Current densities measured at 60 Hz can be linearly extrapolated to frequencies in a range extending from at least 1 Hz to 1 MHz. Human and animal current-density data can provide an improved rationale for extrapolating biological data across species. In addition, these data can be used to validate the predictions of numerical models.     

Receptor- and store-operated mechanisms of calcium entry during the nanosecond electric pulse-induced cellular response

Nanosecond electric pulses have been shown to open nanopores in the cell plasma membrane by fluorescent imaging of calcium uptake and fluorescent dyes, including propidium (Pr) iodide and YO-PRO-1 (YP₁). Recently, we demonstrated that nsEPs also induce the phosphoinositide intracellular signaling cascade by phosphatidylinositol-4,5-bisphosphate (PIP₂) depletion resulting in physiological responses similar to those observed following stimulation of G_q11-coupled receptors. In this paper, we explore the role of receptor- and store-operated calcium entry (ROCE/SOCE) mechanisms in the observed response of cells to nsEP. We show that addition of the ROCE/SOCE and transient receptor potential channel (TRPC) blocker gadolinium (Gd³⁺, 300 μM) slows PIP2 depletion following 1 and 20 nsEP exposures. Lipid rafts, regions of the plasma membrane rich in PIP₂ and TRPC, are also disrupted by nsEP exposure suggesting that ROCE/SOCE mechanisms are likely impacted. Reducing the expression of stromal interaction molecule 1 (STIM1) protein, a key protein in ROCE and SOCE, in cells exposure to nsEP resulted in a reduction in induced intracellular calcium rise. Additionally, after exposure to 1 and 20 nsEPs (16.2 kV/cm, 5 Hz), intracellular calcium rises were significantly reduced by the addition of GD³⁺ and SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl-1H-imidazole hydrochloride, 100 μM), a blocker of STIM1 interaction. However, using similar nsEP exposure parameters, SKF-96365 was less effective at reducing YP₁ uptake compared to Gd³⁺. Thus, it is possible that SKF-96365 could block STIM1 interactions within the cell, while Gd³⁺ could acts on TRPC/nanopores from outside of the cell. Our results present evidence of nsEP induces ROCE and SOCE mechanisms and demonstrate that YP₁ and Ca²⁺ cannot be used solely as markers of nsEP-induced nanoporation.     

Inactivation of bacteria and spores by pulse electric field and high pressure CO2 at low temperature

The common methods for inactivation of bacteria involve heating or exposure to toxic chemicals. These methods are not suitable for heat-sensitive materials, food, and pharmaceutical products. Recently, a complete inactivation of many microorganisms was achieved with high-pressure carbon dioxide at ambient temperature and in the absence of organic solvent and irradiation. The inactivation of spores with CO(2) required long residence time and high temperatures, such as 60 degrees C. In this study the synergistic effect of pulsed electric field (PEF) in combination with high-pressure CO(2) for inactivation was investigated. The bacteria Escherichia coli, Staphylococcus aureus, and Bacillus cereus were suspended in glycerol solution and treated in the first step with PEF (up to 25 KV/cm) and then with high-pressure CO(2) not higher than 40 degrees C and 200 bar. The inactivation efficiency was determined by counting the colony formation units of control and sample. Samples of the cells subjected to PEF treatment alone and in combination with CO(2) treatment were examined by scanning electron microscopy to determine the effect of the processes on the cell wall. Experimental results indicate that the viability decreased with increasing electrical field strength and number of pulses. A further batch treatment with supercritical CO(2) lead to complete inactivation of bacterial species and decreased the count of the spores by at least three orders of magnitude, the inactivation being enhanced by an increase of contact time between CO(2) and the sample. A synergistic effect between the pulsed electric field and the high-pressure CO(2) was evident in all the species treated. The new low temperature process is an alternative for pasteurization of thermally labile compounds such as protein and plasma and minimizes denaturation of important nutrient compounds in the liquid media.     

Calcium release of heat-shocked porcine oocytes induced by thimerosal or inositol 1,4,5-trisphosphate (IP3)

The objective of this study was to determine the effect of heat shock (HS) on the Ca(2+) release and the subsequent development in matured porcine oocytes. Oocytes were matured in vitro and randomly allocated to different heat treatments at 41.5 degrees C for 1 (HS1h), 2 (HS2h) or 4h (HS4h). Control groups of oocytes were cultured for 0 or 4h without HS (39 degrees C, C0h, C4h). In Experiment 1 (eight replicates), matured oocytes were activated by thimerosal (200 microM, 10 min) following HS. Among all heated groups, maximal intracellular calcium concentration ([Ca(2+)](i)) was the highest in the HS2h. The lowest [Ca(2+)](i) peak among HS groups was observed in the HS4h, but it was higher than that in the non-heated C4h group (P<0.05). In Experiment 2 (12 replicates), each matured oocyte was injected with IP(3) (0.5mM) and the Ca(2+) transient was recorded. The peak [Ca(2+)](i) in the C4h group was still the lowest among all groups (P<0.05). Total Ca(2+) release in HS2h appeared the highest among all treatments, and it was significantly higher than that in HS1h and C4h groups (P<0.05). In order to clarify the effect of incubation time in vitro (Experiment 3), matured oocytes were cultured at 39 degrees C for 0, 2 and 4h prior to treatment with thimerosal or injected with IP(3) (three replicates). The Ca(2+) release of matured oocytes declined with the prolonged culture (P<0.05). Finally, the development of HS-oocytes was evaluated after parthenogenetic activation (Experiment 4, three replicates), and the proportion of embryos developing to the blastocysts were lower (P<0.05) in the HS groups (31+/-7% to 33+/-1%) than in the control groups (52+/-11% to 56+/-9%). We conclude that HS alters the Ca(2+)-releasing ability of matured pig oocytes, and that heat-shocked oocytes with greater Ca(2+) release incur a low developmental competence after parthenogenetic activation.