Breitenbach, Thomas7; Sinks, Louise, E.4; Vionogradov, Sergej A.5; Lambert, John D. C.8; Ogilby, Peter Remsen7
1 Department of Chemistry, Science and Technology, Aarhus University2 Department of Biomedicine - Physiology and Biophysics, Department of Biomedicine, Health, Aarhus University3 Interdisciplinary Nanoscience Center, Science and Technology, Aarhus University4 University of Pennsylvania, Dept. of Biochemistry and Biophysics5 University of Pennsylvania, Dept. of Biochemistryand Biophysics6 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University7 Department of Chemistry, Science and Technology, Aarhus University8 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University
Several models to mimic oxidative stress of cells have been reported. However, these models are often limited to known ROS (e.g. H2O2) or exposure times, which may exceed the pathophysiological stimulation. We have previously investigated neuronal functioning following controlled production of 1O2, following light absorption of a photosensitizer (PS) at specific subcellular localizations. Here we use a hydrophilic PS based on a palladium porphyrin protected with a dendritic structure, rendering it impermeable to the cell membrane. Whole-cell electrophysiological recordings in HEPES-buffered medium (ABM) were made from cultured rat cortical neurons to provide insight into the events following extracellular generation of 1O2. Membrane resistance (Rm), capacitance (Cm), holding current (Ihold), and firing properties were monitored throughout. The V/I relationship was investigated with 1 s duration current steps of 0.1 nA (-0.4 - 1 nA). The PS, dissolved in ABM (10 µM), was administered by local application directly to the neuron monitored. The intensity of the applied light at 455 nm was adjusted by neutral density filters. Phosphorescence at 700 nm proved the presence of the PS, which was absent after washing, showing that PS had not associated with the neuron. Both controls: a) application of ABM alone with high irradiance (~7 J/cm2); b) application of PS without light, did not alter the investigated properties. The application of light in the presence of the PS affected the passive properties in an irradiance-dependent manner. After a delay of 5-20 s, Ihold had increased (indicative of a depolarization) and Rm decreased, while Cm was relatively unaffected. The firing frequency was little altered after lower irradiance (~0.2 J/cm2). However, after longer time delays (min.) or after relatively high irradiance (~4 J/cm2), the firing frequency and AP-shape were altered markedly relative to pre-irradiation patterns. All responses with extracellular release of 1O2 occur on a much longer timescale and irradiance (500x) relative to a lipophilic PS localized in the membrane. Due to the short lifetime of 1O2 (3µs), its sphere of action is limited to ~200 nm. Thus, in contrast to lipophillic PS, the probable target for ROS action is the external leaflet of the membrane.
Singlet Oxygen; Electrophysiology; PDT; Photodynamic Therapy; Neurons; Cancer