The observed increase in pillar cell mitochondrial membrane potential and redistribution within short- and long-NAD(P)H lifetime pools does, however, suggest some GM entry into pillar cells may have occurred [Figs.?1(d), 9(e), and 9(f)]. mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and best in high-frequency OHCs. is usually reduced to fluorescent NADH) and NADH utilization by the electron transport chain (NADH is usually oxidized to produce nonfluorescent asphyxiated postnatal day 6 (and 80% along the length of each cochlear explant, respectively.47 Unless otherwise noted, reagents and solutions were obtained from Sigma-Aldrich (St. Louis, Missouri). All animal care and use procedures were approved by the Creighton University Animal Care and Use Committee. 2.2. Gentamicin Uptake in Sensory and Supporting Cells To verify the uptake and accumulation of gentamicin (GM) in cochlear cells, explants were imaged by confocal microscopy while bathed in a solution made up of GM and GM conjugated to Texas Red (GTTR), as described in Dai et al.48 GTTR was single photon excited using a 543-nm HeNe laser focused through a bandpass filter and de-scanned through a one Airy unit pinhole, as described previously.45 Images were acquired at 10-min intervals to monitor the accumulation of GM in cochlear cells. 2.3. Metabolic Imaging Methods Fluorescence intensity and lifetime imaging of two-photon-excited NAD(P)H were performed using the 740-nm mode-locked pulse train of a Spectra Physics Mai Tai Ti:S laser (Newport Corporation, Irvine, California) and a Zeiss LSM 510 NLO META multiphoton microscope (Carl Zeiss, Oberkochen, Germany). Intrinsic cellular fluorescence was measured using a bandpass filter (Chroma Technology, Bellows Falls, Vermont), RGFP966 and detected with a Hamamatsu H7422p-40 photon-counting PMT (Hammamatsu, Hammamatsu City, Japan) and a time-correlated single-photon counting module (830 SPC, Becker and Hickl, Berlin, Germany).32,43,45 Cochlear explants were imaged in modified tyrodes imaging buffer containing 135?mM NaCl, 5?mM KCl, 1?mM during imaging using a warmed platform and heat controller (Warner Devices, Hamden, Connecticut). Previous studies have used room heat cochlear preparations, which have improved viability compared with preparations maintained at 37C.43GM, a representative AG antibiotic. This dose is within the range of AG doses that are frequently used to study AG ototoxicity.49carbonyl cyanide-sodium cyanide (NaCN). These concentrations have previously RGFP966 been shown to be sufficient to cause maximal NADH oxidation and reduction in cochlear hair cells, respectively.46 To determine if acute GM alters mitochondrial membrane potential in sensory and supporting cells, control and GM-exposed cochlear explants were incubated with tetramethylrhodamine-ethyl-ester-perchlorate (TMRE, a fluorescent mitochondrial membrane potential indicator) and MitoTracker Green (MTG, a membrane potential-independent fluorescent mitochondrial label) at 37C and 5% for 30 RGFP966 and 20?min, respectively. All fluorophores were obtained from Molecular Probes (Eugene, Oregon). Cochlear explants were maintained at and immediately imaged using a Leica TCS SPC830 multiphoton confocal microscope and an IRAPO depth intervals throughout each cochlear preparation, then averaged to determine mean cell-specific fluorescence intensities for TMRE and MTG. 2.5. Metabolic Imaging Analysis NAD(P)H fluorescence intensity and RPS6KA5 FLIM analyses were performed as described in Vergen et al.32 Briefly, individual sensory and supporting cells were analyzed as separate RGFP966 regions of interest (ROIs) using Becker and Hickl SPC Image software (SPC Image, Becker and Hickl, Berlin, Germany). Common ROIs consisted of 200 to 250?pixels for pillar cells and OHCs and approximately 350 pixels for inner hair cells (IHCs). The measured fluorescence decay at each pixel within an ROI, is the total concentration for the pixel. Separate concentration-weighted fluorescence lifetime histograms were compiled for each cell type and fitted to a sum of Gaussians (OriginLab, Northampton, Massachusetts) to determine the fluorescence lifetimes and fraction of the total concentration associated with each lifetime pool. The results from unique lifetime pools identified in each preparation were averaged by cell type. NAD(P)H intensity and fluorescence lifetime measurements were averaged for IHCs (8 to GTTR. (d)?GM significantly increases the mitochondrial membrane potential in sensory and supporting cells. Color-coded asterisks represent the significant differences (*of nine or more replicates (to 63; to 19, to 17). Color-coded asterisks represent significant differences (*of nine or more replicates (to 63; to 19, and to 17). Significance color coding is the same as in Fig.?1 (*of nine or more replicates (to 63, to 19, and to 17). 3.3. Ototoxic Antibiotic.