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In accordance with the manufacturer’s protocol. Complementary DNA was synthesized from total RNA (1 mg).Final results Base-to-apex gradient hair cell harm brought on by gentamicin Organ of Corti explants from 4 regions of P3 rat cochlea (apex, upper-middle, lower-Glycodeoxycholic Acid supplier middle and base) had been treated with 300 mM gentamicin for 24 h. The explants have been stained with phalloidin RITC (Figure 1Aa, b) and DAPI (Figure 1Ac, d) and observed under a fluorescent microscope. TRITCphalloidin-stained control explants exhibited a standard pattern of three OHC rows and also a single row of IHCs (Figure 1Aa). All OHCs exhibited V-shaped stereocilia bundles and typical Dabcyl acid Purity nuclei (Figure 1Aa, c). Nevertheless, gentamicin exposure induced apparent stereocilia bundle harm. Interestingly, basal turn IHCs and OHCs showed the greatest degree of harm,Experimental Molecular MedicineTRPV channels in gentamicin uptake J-H Lee et alFigure 1 Hair cell death caused by gentamicin within a time- and dose-dependent manner. (A) Cochlear explant cultures from postnatal day three rats were maintained in the absence (a, c) or presence (b, d) of 300 mM gentamicin for 24 h. Cultures have been stained with phalloidintetramethylrhodamine isothiocyanate (TRITC; a, b) and 40 ,6-diamidino-2-phenylindole (DAPI; c, d) and observed under a fluorescent microscope. Outer hair cells (OHCs): arrow, inner hair cells (IHCs): arrowhead, and Hensen’s cells: star. (B) Quantitative evaluation of OHC loss in explants treated for 24, 36 and 48 h with a variety of doses (50, 100, 200, 300, 400, 500, 600 and 700 mM) of gentamicin. The percentage of hair cells missing at numerous gentamicin doses was considerably different from that in the control. Data are imply .d. of three samples. Po0.05 and Po0.01 by one-way evaluation of variance (ANOVA), compared with each turn of handle group not treated with gentamicin.followed by hair cells in the middle and apical turns (Figure1Ab). The nuclei of handle IHCs and OHCs had been round shaped, but the nuclei of gentamicin-exposed IHCs and OHCs were fragmented and disappeared (Figure 1Ac, d). This base-to apex gradient damage caused by gentamicin was additional confirmed by treating the cochlear explants with 5000 mM gentamicin for 24, 36 and 48 h. Intact hair cells have been counted in a section corresponding to 10 IHCs at three diverse zones positioned around the apical, middle and basal turns of every organ of Corti. Hair cell survival decreased considerably after gentamicin exposure in a time- and dose-dependentExperimental Molecular Medicinemanner (Figure 1B). We also observed base-to-apex gradient hair cell harm (Figure 1B). In vitro gentamicin uptake into cochlear explants Complete cochlear explants on a collagen matrix had been treated with TR (1.8 mM) or GTTR (500 mM) for 30 min and fixed to directly observe in vitro gentamicin uptake. The explants were embedded in paraffin and cut into 4-mm-thick sections. For observing, specimens were deparaffinized and incubated with DAPI to observe nuclei. As shown in Figure 2Ab, sturdy red fluorescence was observed inside the IHCs and OHCs ofTRPV channels in gentamicin uptake J-H Lee et alFigure 2 Distribution of gentamicin-conjugated Texas Red (GTTR) in cochlear explants right after remedy in vitro. (A) Complete cochlear explants on a collagen matrix were treated with (a) Texas Red (TR; 1.8 mM) or (b) GTTR (500 mM total including unconjugated gentamicin) for 30 min and fixed. The explants have been embedded in paraffin and cut into 4-mm-thick sections. Specimens have been deparaffinized and incubate.