Experimental studies on single cells have shown that application of pulsed voltages, with submicrosecond pulse duration and an electric field on the order of 10 kV/cm, causes sudden alterations in the intracellular free calcium concentration, followed by immobilization of the cell. In order to examine electrical stimulation and incapacitation with such ultrashort pulses, experiments on anesthetized rats have been performed. The effect of single, 450 nanosecond monopolar pulses have been compared with that of single pulses with multi-microsecond duration (TASER pulses). Two conditions were explored: 1. the ability to elicit a muscle twitch, and, 2. the ability to suppress voluntary movement by using nanosecond pulses. The second condition is relevant for neuromuscular incapacitation. The preliminary results indicate that for stimulation microsecond pulses are advantageous over nanosecond pulses, whereas for incapacitation, the opposite seems to apply. The stimulation effects seem to scale with electrical charge, whereas the disruption effects don't follow a simple scaling law. The increase in intensity (time of incapacitation) for a given pulse duration, is increasing with electrical energy, but is more efficient for nanosecond than for microsecond pulses. This indicates different cellular mechanisms for incapacitation, most likely subcellular processes, which have been shown to become increasingly important when the pulse duration is shortened into the nanosecond range. If further studies can confirm these initial results, consequences of reduced pulse duration are a reduction in weight and volume of the pulse delivery system, and likely, because of the lower required energy for neuromuscular incapacitation, reduced safety risks.
The charging of mammalian cell plasma membranes in response to ultrashort pulsed electric fields of 60 ns and field strengths up to 100 kV/cm was investigated. Membranes of Jurkat cells were stained with a potential-sensitive dye, Annine-6 and placed in a microreactor mounted on an inverted fluorescence microscope. Images of changes in the fluorescence intensity during the exposure were recorded with a high-sensitivity CCD-camera. A temporal resolution of 5 ns was achieved by illuminating the cells with a 5 ns laser pulse from a dye-laser. The laser pulse was synchronized with the high voltage pulse to record images at specific times before, during and after exposure to the electric field. When exposing Jurkat cells to a 60 ns, 100 kV/cm pulse, each hemisphere of the plasma membrane (as oriented with respect to the electrodes) responded uniquely to the applied field. From these observations it is possible to draw conclusions on the charging time of the membrane, maximum transmembrane voltages and the onset of poration.
Nanosecond, high intensity pulsed electric fields [nsPEFs] that are below the plasma membrane [PM] charging time constant have decreasing effects on the PM and increasing effects on intracellular structures and functions as the pulse duration decreases. When human cell suspensions were exposed to nsPEFs where the electric fields were sufficiently intense [10-300ns, ≤300 kV/cm.], apoptosis signaling pathways could be activated in several cell models. Multiple apoptosis markers were observed in Jurkat, HL-60, 3T3L1-preadipocytes, and isolated rat adipocytes including decreased cell size and number, caspase activation, DNA fragmentation, and/or cytochrome c release into the cytoplasm. Phosphatidylserine externalization was observed as a biological response to nsPEFs in 3T3-L1 preadipocytes and p53-wildtype and -null human colon carcinoma cells. B10.2 mouse fibrosarcoma tumors that were exposed to nsPEFs ex vivo and in vivo exhibited DNA fragmentation, elevated caspase activity, and reduced size and weight compared to contralateral sham-treated control tumors.
When nsPEF conditions were below thresholds for apoptosis and classical PM electroporation, non-apoptotic responses were observed similar to those initiated through PM purinergic receptors in HL-60 cells and thrombin in human platelets. These included Ca2+ mobilization from intracellular stores [endoplasmic reticulum] and subsequently through store-operated Ca2+ channels in the PM. In addition, platelet activation measured as aggregation responses were observed in human platelets. Finally, when nsPEF conditions followed classical electroporation-mediated transfection, the expression intensity and number of GFP-expressing cells were enhanced above cells exposed to electroporation conditions alone.
These studies demonstrate that application of nsPEFs to cells or tissues can modulate cell-signaling mechanisms with possible applications as a new basic science tool, cancer treatment, wound healing, and gene therapy.