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.
KEYWORDS: Calcium, Phase modulation, Cell death, Picosecond phenomena, Tissues, Luminescence, Colon, Tumors, In vivo imaging, Green fluorescent protein
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.
KEYWORDS: Bacteria, Microorganisms, Organisms, Capacitance, Cooling systems, Oceanography, Water, Information operations, Medical research, Energy efficiency
Previous studies on the effect of microsecond pulsed electric fields on bacteria have shown that the lethality increases linearly with pulse duration and exponentially with electric field strength. In order to determine the validity of this law for submicrosecond pulses, we applied pulses of fifty nanosecond duration to two strains of E. coli and to a marine crustacean. The results indicated that even at this short pulse duration, the empirical law not only holds for bacteria, but also for more complicated organisms. Theoretical considerations, however, and the observation of a pronounced difference in the field induced lethality of two strains of E. coli led us to believe that a change in the effect can be expected when the pulse duration is reduced further. The observed dependance of micro-organism lethality or temporary damage on field strength and pulse duration allows us to improve the energy efficiency of systems which make use of the effect. Examples are sterilizers (e.g., for food and water) and electrical filters for the prevention of biofouling in cooling systems.
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