Nanosecond pulsed electric field thresholds for nanopore formation in neural cells.

The persistent influx of ions through nanopores created upon cellular exposure to nanosecond pulse electric fields (nsPEF) could be used to modulate neuronal function. One ion, calcium (Ca(2+)), is important to action potential firing and regulates many ion channels. However, uncontrolled hyper-excitability of neurons leads to Ca(2+) overload and neurodegeneration. Thus, to prevent unintended consequences of nsPEF-induced neural stimulation, knowledge of optimum exposure parameters is required. We determined the relationship between nsPEF exposure parameters (pulse width and amplitude) and nanopore formation in two cell types: rodent neuroblastoma (NG108) and mouse primary hippocampal neurons (PHN). We identified thresholds for nanoporation using Annexin V and FM1-43, to detect changes in membrane asymmetry, and through Ca(2+) influx using Calcium Green. The ED50 for a single 600 ns pulse, necessary to cause uptake of extracellular Ca(2+), was 1.76  kV/cm for NG108 and 0.84  kV/cm for PHN. At 16.2  kV/cm, the ED50 for pulse width was 95 ns for both cell lines. Cadmium, a nonspecific Ca(2+) channel blocker, failed to prevent Ca(2+) uptake suggesting that observed influx is likely due to nanoporation. These data demonstrate that moderate amplitude single nsPEF exposures result in rapid Ca(2+) influx that may be capable of controllably modulating neurological function

nsPEF-induced PIP2 depletion, PLC activity and actin cytoskeletal cortex remodeling are responsible for post-exposure cellular swelling and blebbing

Cell swelling and blebbing has been commonly observed following nanosecond pulsed electric field (nsPEF) exposure. The hypothesized origin of these effects is nanoporation of the plasma membrane (PM) followed by transmembrane diffusion of extracellular fluid and disassembly of cortical actin structures. This investigation will provide evidence that shows passive movement of fluid into the cell through nanopores and increase of intracellular osmotic pressure are not solely responsible for this observed phenomena. We demonstrate that phosphatidylinositol-4,5-bisphosphate (PIP2) depletion and hydrolysis are critical steps in the chain reaction leading to cellular blebbing and swelling. PIP2 is heavily involved in osmoregulation by modulation of ion channels and also serves as an intracellular membrane anchor to cortical actin and phospholipase C (PLC). Given the rather critical role that PIP2 depletion appears to play in the response of cells to nsPEF exposure, it remains unclear how its downstream effects and, specifically, ion channel regulation may contribute to cellular swelling, blebbing, and unknown mechanisms of the lasting “permeabilization” of the PM

neural

Nanosecond pulsed electric field thresholds for nanopore formation i

Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP)

<div><p>Nanosecond electrical pulse (nsEP) exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s) between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.</p></div

Oxidative stress related protein levels in U937 and Jurkat cells exposed to nsEP.

<p>A) Levels of oxidative stress proteins for U937 at 4 h post exposure. B) Levels of oxidative stress proteins for Jurkat at 4 h post exposure. C) Levels of oxidative stress proteins for U937 at 8 h post exposure. D) Levels of oxidative stress proteins for Jurkat at 8 h post exposure. E) Levels of oxidative stress proteins for U937 at 12 h post exposure. F) Levels of oxidative stress proteins for Jurkat at 12 h post exposure. Error bars represent standard deviation (SD).</p

Scatter dot plot for each qRT-PCR validation sample.

<p>A) Comparison of the expression levels of JUN for U937 cells exposed to nsEP. B) Comparison of the expression levels of JUN for Jurkat cells exposed to nsEP. C) Comparison of the expression levels of DUSP10 for U937 cells exposed to nsEP. D) Comparison of the expression levels of DUSP10 for Jurkat cells exposed to nsEP. E) Comparison of the expression levels of HPRT1 for U937 cells exposed to nsEP. F) Comparison of the expression levels of HPRT1 for Jurkat cells exposed to nsEP. Mean and standard deviation are plotted as the green and black lines respectively.</p

Genes up- or down-regulated in common by nsEP exposure in Jurkat and U937 cells.

<p>Genes up- or down-regulated in common by nsEP exposure in Jurkat and U937 cells.</p

Blumlein line cuvette-based, 10 ns pulser apparatus.

<p>A) Drawing of the complete 10-ns set up, including the Tektronix ocilloscope, Glassman high voltage power supply and custom contol module for regulating the pressure of SF6 in the spark gap chamber. B) This is an enhanced view of the cuvette and its placement/orientation in regards to the pulser.</p