Animals and dissection
Newborn Wistar rats (p3-p5) were used to prepare cochlear organotypic cultures. All studies were performed in accordance with the German Prevention of Cruelty to Animals Act and were approved by the Berlin Senate Office for Health (T0234/00).
The dissection procedure is similar to that described by Sobkowicz et al. [20]. After decapitation, the heads were cleaned with 70% ethanol and positioned with ventral surface down. The scalp was removed and the skull was transected along mid-sagittal plane. The brain was scooped out to expose the posterior fossa. The temporal bones were freed from the posterior hemi-skulls and transferred into Petri dishes containing cold, sterile, buffered saline glucose solution (BSG, glucose 11.4 mM). Under a stereomicroscope (Stemi, SV6, Zeiss, Germany), the tympanic membrane and annulus were laterally peeled away and the surrounding cartilages were removed exposing the cochlear capsule. The cochlear capsules were bit off in small pieces from the oval window to the apex or shucked off from the base integrally. The stria vascularis and spiral ligament were stripped away as a single piece from the base to the apex, and the OC was separated away from the modiolus.
OC explant culture
The OC explants were incubated in 4-well culture dishes (4 × 1.9 cm2, Nunc, Wiesbaden, Germany) containing 500 μl medium. For different further use, OC explants were cultured in two different ways. For subsequent reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), whole OC tissue was incubated free-floating in the medium. For subsequent histological use, the OC was cut into three parts consisting of apical, middle and basal turns, and then the OC segments were positioned on the bottom of dishes as a flat surface preparation and were held in place by the surface tension of the culture medium. The culture medium consisted of DMEM/F12 (1:1) (Gibco, Karlsruhe, Germany) supplemented with 10% FBS (Biochrom AG, Berlin, Germany), 0.6% glucose, 2 μl/ml insulin-transferrin-Na-Selenit-Mix (Roche Diagnostics GmbH, Mannheim, Germany), 24 ng/ml recombinant human insulin-like growth factor-I (rhIGF-I, R&D Systems, Wiesbaden-Nordenstadt, Germany), and 100 U/ml penicillin (Grünenthal GmbH, Aachen, Germany). The explants were placed in an incubator (SANYO MCO-16AIC, 37°C, 5% CO2) for 24 h to condition them before further treatments.
GA and gentamicin treatment
GA (Cat. # Ant-g1, Invivogen, Toulouse, France) was dissolved in DMSO to make 1 mg/ml stock solutions. After initial 24 h of culture, the medium was exchanged with new medium containing a specific concentration of GA (0.5, 1 or 2 μM) for up to 24 h. Then, OC explants were fixed for staining or lysed for RNA isolation and protein extraction.
Gentamicin sulfate (G1264, CAS # 1405-41-0, Sigma-Aldrich, Munich, Germany) was dissolved in distilled water to prepare 50 mM stock solution and diluted into culture medium at final concentration of 500 μM. After initial 24 h, OC explants were incubated in culture medium containing 2 μM GA for 4 h and then exposed to 500 μM gentamicin for 24 h, or were treated simultaneously with GA and gentamicin for 24 h.
Total RNA samples were isolated from OC cultures using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and on-column DNase digested with RNase-free DNase Set (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. The isolated RNA was quantified spectrophotometrically with Ribogreen® RNA Quantitation Reagent (Molecular Probes, Göttingen, Germany) and then stored at -80°C until needed.
cDNA preparation
First-strand cDNA was synthesized from 100 ng of total RNA in a thermocycler (Perkin Elmer-applied biosystems thermal cycler 9600, Foster City, USA). The RNA was denatured by heating at 70°C for 5 min and quickly cooled at 4°C. The reaction mixture contained 0.5 mM dNTP Mix (Invitrogen GmbH, Karlsruhe, Germany), 3.8 μM Oligo(dT) (Biotez, Berlin, Germany), 26 U RNasin (Promega Co., Madison, WI, USA) and 25 U MMLV Reverse Transcriptase (Promega Co., Madison, WI, USA) in a final volume of 20 μl of MMLV Reaction Buffer (Promega Co., Madison, WI, USA). The RT reaction was performed at 42°C for 60 min followed by enzyme inactivation at 95°C for 5 min and cooling at 4°C. To exclude cross-reaction of PCR primers with containing DNA in the following PCR experiment, negative RT controls containing all reverse transcription components, including RNA samples, were prepared by carrying out the reaction in the absence of MMLV RT. In addition, another negative control containing nuclease-free water instead of RNA was set up.
Real-time quantitative PCR
The Master Mix (containing FastStart Taq DNA Polymerase, reaction buffer, SYBR Green I dye and MgCl2) for real-time quantitative PCR (qPCR) was prepared from solution 1a (Enzyme) and solution 1b (Reaction Mix) (LightCycler FastStart DNA Masterplus SYBR Green I, Roche Dianostics GmbH, Penzberg, Germany) according to the manufacturer's instructions. The PCR mixture contained 2 μl of cDNA, 10 pmol each reverse and forward specific primers (BioTez, Berlin, Germany), 4 μl Master Mix and PCR degree water in a final volume of 20 μl. A control PCR reaction, which involved PCR master mix and the primers, but no cDNA template, was used as a blank PCR reaction. Housekeeping gene, encoding ribosomal protein S16 (rS16) was used as internal control. The sequence of primers used in this study was as follows: rS16 (forward GGG TCC GCT GCA GTC CGT TC and reverse CGT GCG CGG CTC GAT CAT CT); HSP70 (Hsp70-2) (forward 5'-ACC AGG ACA CTG TTG AGT TC-3' and reverse 5'-ACT CAT CTC CGA GTT CAC AC-3'). PCR was initiated by preincubation at 95°C for 10 min followed by 35 cycles consisting of denaturation at 95°C for 10 sec, annealing at 65-70°C for 10 sec and extension at 72°C for 10-15 sec. Melting curves were obtained by final incubation from 60 to 95°C with a heating rate of 0.1°C/s and ended with cooling down to 4°C in LightCycler System 2.0 (Roche Diagnostics, Basel, Switzerland).
To visualize amplicons, 2 μl of each PCR product was separated by electrophoresis on a 2-3% agarose gel (Gibco BRL, Life Technologies, Paisley, Scotland) stained by GelStar® Nuleic Acid Gel Stain (Cambrex Bio Science, Rockland, USA). The amplicons were visualized under ultraviolet light using a Syngene-Gene Genius imaging system (Synoptics Inc., USA), and the product size were confirmed by comparison with DNA ladder Marker V (Roche Diagnostics, Mannheim, Germany).
Threshold cycle (Ct) values acquired in real-time qPCR were normalized to rS16 that served as an endogenous reference and calibrated to the control. Relative expression level (fold change) of the target genes in each experimental sample was calculated using 2-ΔΔC tmethod [21], where ΔC t = C t (target gene) - C t (reference gene) and ΔΔC t = ΔC t (treated) - ΔC t (control).
ELISA
The ELISA kit, Human/Mouse/Rat Total HSP70 DuoSet® IC (Cat. # DYC1663-5, R&D Systems, Wiesbaden-Nordenstadt, Germany), was used to quantify HSP70 protein expression in OC explants. Free-floating OC explants were lysed and the concentration of HSP70 protein in lysates was measured according to the manufacturer's instructions. HSP70 concentration in OC lysates obtained from ELISA was normalized to total protein concentration. Relative expression level of HSP70 in each experimental sample was calculated as picogram of HSP70 per 1 μg of total protein. Six OCs were used per each time point.
Immunofluorescence
HSP70 staining
OC explants were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered solution (PBS) at room temperature for 30 min. Next, the fragments were washed two times with PBS and permeabilized with 0.2% Triton X-100 in PBS for 30 min. After two washes in PBS, the fragments were incubated in blocking solution (0.8% goat serum, 0.4% Triton and 2% bovine serum albumin in PBS) at room temperature for 3 h and then incubated overnight at 4°C with mouse anti-HSP70 monoclonal antibody (Cat. # SPA-810, Stressgen Bioreagents, Ann Arbor, USA) (1:200 dilution in blocking solution, 5 μg/ml). In the negative control samples, mouse anti-HSP70 monoclonal antibody was substituted with an isotype control (mouse IgG1, Dianova, Hamburg, Germany) (1:40 dilution in blocking, 5 μg/ml). The fragments were washed three times in PBS, incubated for 3 h at room temperature with goat anti-mouse IgG conjugated with fluorescence isothiocyanate (FITC) (Dianova, Hamburg, Germany) (1:200 dilution in blocking solution, 7.5 μg/ml) and washed three times in PBS. The fragments were mounted on glass slides in Prolong Gold® antifade reagent (P36930, Molecular Probes (Invitrogen, Karlsruhe, Germany) and examined using a confocal microscope (Leica TCS SPE, Wetzlar, Germany).
Hair cell quantification
OC explants were fixed for 30 min in 4% paraformaldehyde in 0.1 M PBS at room temperature. Then, the fragments were washed two times with PBS and permeabilized with 0.2% Triton X-100 in PBS for 30 min. The fragments were washed two times with PBS and incubated with 5 μg/ml solution of phalloidin conjugated with tetramethyl rhodamine isothiocyanate (TRITC) (P1951, Sigma-Aldrich, Munich, Germany) at room temperature for 30 min. After washes with PBS, the fragments were mounted with mounting medium containing 1,4-diazabicyclo[2.2.2]octane (DABCO) (D2522, Sigma-Aldrich, Munich, Germany).
The OC fragments were examined under a fluorescence microscope (Leica DMIL, Wetzlar, Germany) with filters appropriate for TRITC (excitation: 544 nm, emission: 572 nm). There are three rows of OHCs and one row of IHCs along the whole OC. The hair cell numbers were counted over a longitudinal distance of 100 μm in five separated regions of each cochlear part (magnification 400×). Cells were considered missing when there was a gap in the normal arrays and no stereocilia or cuticular plates were to be seen. A mean value was calculated for each explant and at least four explants were used for each experimental condition. The fragments were photographed with a digital camera (Canon PowerShot S40). The contrast and brightness of images were adjusted by using Adobe Photoshop (version 9.0) software.
Statistical analyses
Means ± standard errors of the mean (SEM) were calculated for all parameters measured. The effect of GA on hair cell viability, HSP70 induction or gentamicin-induced hair cell loss were tested by two-way analysis of variance (ANOVA) followed by Scheffé's post-hoc test. To analyze the effect of GA on gentamicin-induced hair cell loss, the codes of the grouping variable (controls, gentamicin, GA before gentamicin, and GA and gentamicin simultaneously) were selected altogether and in pairs. A p-value of less than 0.05 was considered statistically significant. All statistical test and graphics were made using the software package Statistica 7.1 (StatSoft).