Intracellular Effects of Nanosecond, High Field Electrical Pulses

Abstract

Mitochondria, which play a crucial role in apoptosis, release several apoptosis-inducing factors into the cytoplasm, presumably through the mitochondrial permeability transition pore (MTP). Under certain conditions, nanoelectropulses can manipulate mitochondrial structure and permeabilize the mitochondrial membrane without permanent damage to the plasma membrane. In this work we investigate the effects of nanoelectropulses on mitochondrial membrane permeabilization, assess changes in mitochondrial transmembrane potential, and monitor plasma membrane integrity under the same pulse conditions. 4 ns electrical pulses were applied to living human Jurkat T lymphoblasts in an electrode microchamber on a microscope slide. Changes in mitochondrial transmembrane potential were evaluated with rhodamine 123 (R123), a lipophilic cationic fluorescent dye that is accumulated within mitochondria. For assessing MTP opening, a calcein-cobalt quenching method was used. Calcein-AM is an anionic fluorochrome that enters cells freely and labels cytoplasmic as well as mitochondrial regions following esterase removal of the acetoxymethyl group. Because cobalt ions do not readily pass through mitochondrial membrane, mitochondria can be specifically identified by the cobalt quenching of cytoplasmic, not mitochondrial, calcein fluorescence, and MTP opening can be recognized by the decrease of calcein fluorescence within mitochondria. Finally, cell membrane integrity was evaluated with propidium iodide (PI), which is excluded from the cell interior by intact cell membranes. When the cell membrane is permeabilized, PI enters the cell, binds to double-stranded nucleic-acid molecules, and exhibits red fluorescence. The effects of different pulse amplitudes and pulse numbers on mitochondrial membrane permeability will be reported, providing a framework for an analysis of pulse doses and exposure conditions which lead to mitochondrial modifications while minimizing effects on the plasma membrane. We will also discuss the interpretation of data from fluorescence microscopic imaging analysis using R123 and cobalt-quenched intracellular calcein fluorescence intensity and the influx of PI.