Nanosecond pulsed electric fields (nsPEF) have proven useful for transporting cargo across cell membranes and selectively activating cellular pathways. The chemistry and biophysics governing this cellular response, however, are complex and not well understood. Recent studies have shown that the conductivity of the solution cells are exposed in could play a significant role in plasma membrane permeabilization and, thus, the overall cellular response. Unfortunately, the means of detecting this membrane perturbation has traditionally been limited to analyzing one possible consequence of the exposure – diffusion of molecules across the membrane. This method has led to contradictory results with respect to the relationship between permeabilization and conductivity. Diffusion experiments also suffer from “saturation conditions” making multi-pulse experiments difficult. As a result, this method has been identified as a key stumbling block to understanding the effects of nsPEF exposure. To overcome these limitations, we recently developed a nonlinear optical imaging technique based on second harmonic generation (SHG) that allows us to identify nanoporation in live cells during the pulse in a wide array of conditions. As a result, we are able to explore and fully test whether lower conductivity extracellular solutions could induce more efficient nanoporation. This hypothesis is based on membrane charging and the relative difference between the extracellular solution and the cytoplasm. The experiments also allow us to test the noise floor of our methodology against the effects of ion leakage. The results emphasize that the electric field, not ionic phenomenon, are the driving force behind nsPEF-induced membrane nanoporation.
We recently developed a nonlinear optical imaging technique based on second harmonic generation (SHG) to identify membrane disruption events in live cells. This technique was used to detect nanoporation in the plasma membrane following nanosecond pulsed electric field (nsPEF) exposure. It has been hypothesized that similar poration events could be induced by the thermal gradients generated by infrared (IR) laser energy. Optical pulses are a highly desirable stimulus for the nervous system, as they are capable of inhibiting and producing action potentials in a highly localized but non-contact fashion. However, the underlying mechanisms involved with infrared neural stimulation (INS) are not well understood. The ability of our method to non-invasively measure membrane structure and transmembrane potential via Two Photon Fluorescence (TPF) make it uniquely suited to neurological research. In this work, we leverage our technique to understand what role membrane structure plays during INS and contrast it with nsPEF stimulation. We begin by examining the effect of IR pulses on CHO-K1 cells before progressing to primary hippocampal neurons. The use of these two cell lines allows us to directly compare poration as a result of IR pulses to nsPEF exposure in both a neuron-derived cell line, and one likely lacking native channels sensitive to thermal stimuli.
Second Harmonic Generation (SHG) imaging is a useful tool for examining the structure of interfaces between bulk materials. Recently, this technique was applied to detecting subtle perturbations in the structure of cellular membranes following nanosecond pulsed electric field (nsPEF) exposure. Monitoring the cell’s outer membrane as it is exposed to nsPEF via SHG has demonstrated that nanoporation is likely the root cause for size-specific, increased cytoplasmic membrane permeabilization. It is theorized that the area of the membrane covered by these pores is tied to pulse intensity or duration. The extent of this effect along the cell’s surface, however, has never been measured due to its temporal brevity and minute pore size. By enhancing the SHG technique developed and elucidated previously, we are able to obtain this information. Further, we vary the pulse width and amplitude of the applied stimulus to explore the mechanical changes of the membrane at various sites around the cell. By using this unique SHG imaging technique to directly visualize the change in order of phospholipids within the membrane, we are able to better understand the complex response of living cells to electric pulses.
Exposure of cells to very short (<1 μs) electric pulses in the megavolt/meter range have been shown to cause disruption of the plasma membrane. This disruption is often characterized by the formation of numerous small pores (<2 nm in diameter) in the plasma membrane that last for several minutes, allowing the flow of ions into the cell. These small pores are called nanopores and the resulting damage to the plasma membrane is referred to as nanoporation. Nanosecond electrical pulse (nsEP) exposure can impart many different stressors on a cell, including electrical, electro-chemical, and mechanical stress. Thus, nsEP exposure is not a “clean” insult, making determination of the mechanism of nanoporation quite difficult. We hypothesize that nsEP exposure creates acoustic shock waves capable of causing nanoporation. Microarray analysis of primary adult human dermal fibroblasts (HDFa) exposed to nsEP, indicated several genes associated with mechanical stress were selectively upregulated 4 h post exposure. The idea that nanoporation is caused by external mechanical force from acoustic shock waves has, to our knowledge, not been investigated. This work will critically challenge the existing paradigm that nanoporation is caused solely by an electric-field driven event and could provide the basis for a plausible explanation for electroporation.
Nonlinear optical probes, especially those involving second harmonic generation (SHG), have proven useful as sensors for near-instantaneous detection of alterations to orientation or energetics within a substance. This has been exploited to some success for observing conformational changes in proteins. SHG probes, therefore, hold promise for reporting rapid and minute changes in lipid membranes. In this report, one of these probes is employed in this regard, using nanosecond electric pulses (nsEPs) as a vehicle for instigating subtle membrane perturbations. The result provides a useful tool and methodology for the observation of minute membrane perturbation, while also providing meaningful information on the phenomenon of electropermeabilization due to nsEP. The SHG probe Di- 4-ANEPPDHQ is used in conjunction with a tuned optical setup to demonstrate nanoporation preferential to one hemisphere, or pole, of the cell given a single square shaped pulse. The results also confirm a correlation of pulse width to the amount of poration. Furthermore, the polarity of this event and the membrane physics of both hemispheres, the poles facing either electrode, were tested using bipolar pulses consisting of two pulses of opposite polarity. The experiment corroborates findings by other researchers that these types of pulses are less effective in causing repairable damage to the lipid membrane of cells.
Application of nanosecond pulsed electric fields (nsPEF) to various biological cell lines has been to shown to cause
many diverse effects, including poration of the plasma membrane, depolarization of the mitochondrial membrane,
blebbing, apoptosis, and intracellular calcium bursts. The underlying mechanism(s) responsible for these diverse
responses are poorly understood. Of specific interest in this paper are the long-term effects of nsPEF on cellular
processes, including the regulation of genes and production of proteins. Previous studies have reported transient
activation of select signaling pathways involving mitogen-activated protein kinases (MAPKs), protein
phosphorylation and downstream gene expression following nsPEF application. We hypothesize that nsPEF
represents a unique stimulus that could be used to externally modulate cellular processes. To validate our
hypothesis, we performed a series of cuvette-based exposures at 10 and 600ns pulse widths using a custom Blumlien
line pulser system. We measured acute changes in the plasma membrane structure using flow cytometry by tracking
phosphatidylserine externalization via FITC-Annexin V labeling and poration via propidium iodide uptake. We then
compared these results to viability of the cells at 24 hours post exposure using MTT assay and changes in the
MAPK family of proteins at 8 hours post-exposure using Luminex assay. By comparing exposures at 10 and 600ns
duration, we found that most MAPK family-protein expression increased in Jurkat and U937 cell lines following
exposure and compared well with drops in viability and changes in plasma membrane asymmetry. What proved
interesting is that some MAPK family proteins (e.g. p53, STAT1), were expressed in one cell line, but not the other.
This difference may point to an underlying mechanism for observed difference in cellular sensitivity to nsPEFinduced