Integrity and regeneration of mechanotransduction machinery regulate aminoglycoside entry and sensory cell death.

Animals

Cdh23 v2J transgenic mice in C57Bl/6 background (Stock number 002552, Jackson Laboratory, Bar Harbor, ME) were used with genotypes determined by sequencing. Wildtype mouse (C57Bl/6) pups were obtained from Charles River laboratory (Cambridge, MA). All procedures involving these animals were approved by the Stanford University administrative panel on laboratory animal care (Assurance number A3213-01, Protocol ID 18606).

Organotypic Cochlear Cultures

Cochleae were isolated and cultured as previously described [6] , [30] . Briefly, cochleae were isolated under sterile conditions in Hank’s Balanced Salt Solution (HBSS, Cellgro, Manassas, VA) from postnatal (P) three-day-old mice. Stria vascularis and modiolus were removed before organs were attached onto 10 mm glass coverslips pre-coated with CellTak (BD Bioscience, San Jose, CA). Each organ was cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Invitrogen, Carlsbad, CA) culture media containing 10% fetal bovine serum (FBS)(Omega Scientific, Tarzana, CA) and ampicillin (50 µg/ml; Sigma, St. Louis, MO) in 4-well 35 mm tissue culture dishes (Greiner Bio-one, Monroe, NC) for 1–3 days at 37°C in a 5% CO 2 atmosphere [31] , [32] . Media was refreshed every 24–48 hr. After an overnight culture period, cochleae were exposed to gentamicin (Gentamicin solution, Hospira, Lake Forrest, IL,) for 1 hr, after that they were washed and incubated in fresh culture media for an additional 48 hr. In separate experiments, gentamicin-conjugated Texas Red (GTTR)(1 µM), which was synthesized from gentamicin sulfate (Sigma) and succinimidyl esters of Texas Red dye (Invitrogen) as previously described [33] , was also added for 1 hr after an overnight culture period. Separately, cochlear organs that had been cultured overnight were bathed in FM1-43 (5 µM in HBSS×15 sec)(Invitrogen) then imaged under an Olympus SZX10 microscope (Olympus, Center Valley, PA) with epifluorescent optics and FITC filters (488 nm excitation and 520 nm emission) [34] . For calcium chelation experiments, BAPTA (5 mM in DMEM/F12, containing 1.05 mM calcium chloride (Invitrogen) with 10% FBS and ampicillin) was added to cochleae as a steady stream aiming at the organ, followed by incubation for 15 min at 37°C with gentle stirring every 5 min. As previously shown this BAPTA solution breaks >95% of the tip-links resulting in the loss of MET current responses [16] , [23] , [35] . Subsequently, organs were incubated in BAPTA-free media for 10 min, 6 hr, or 24 hr before gentamicin or GTTR treatment. We found that adding BAPTA-containing media as a steady stream aiming at the organ generated more consistent results than when it was added as droplets onto the cochleae or steady stream directed away from the cochleae ( Figure S1 ). Therefore, the former method of application was used for all remaining experiments.

Immunohistochemistry

Procedures have been previously described [ 30 ] . Briefly, at the end of defined culture periods, tissues were fixed in 4% paraformaldehyde (in phosphate-buffered saline (PBS), pH 7.4) for 30 min, rinsed with PBS (3X), and immersed in blocking solution (5% normal goat serum, 0.1% Triton X-100, 1% bovine serum albumin, and 0.02% sodium azide in PBS, pH 7.4) for 60 min. Primary antibodies diluted in blocking solution were applied overnight at 4°C. The next day, tissues were rinsed with PBS (3X) and then exposed to fluorescent secondary antibodies diluted in PBS with 0.1% Triton X-100, 1% bovine serum albumin, and 0.02% sodium azide for 60–120 min. After washing in PBS (3X), organs were mounted in fluorescent mounting media (Dako, Carpinteria, CA) and analyzed. The following antibodies were used: anti-myosin7a antibody (1∶1000; Proteus Bioscience, Ramona, CA); anti-gentamicin antibody (1∶200; QED Bioscience, San Diego, CA); and corresponding secondary antibodies (Alexa Fluor 488 and 546; 1∶500; Invitrogen).

Live Imaging of Gentamicin Uptake Using Two-Photon Microscopy

We followed previously described procedures for live imaging [6] . Briefly, acutely isolated cochleae were secured onto sterile 35×10 mm tissue culture dishes (Greiner Bio-one). Organs were cultured overnight, then washed 3X with L-15 before two-photon imaging. GTTR (3 µM) was directly added to cochlear cultures. An Olympus BX-61 microscope (Olympus) with a 100X, 1.0 numerical aperture water immersion objective (Olympus LUMPlan) was used to image the middle turn of the cochlea. A 520 nm long-pass dichroic (Chroma Technology, Bellows Falls, VT) was used to separate the fluorescence emission into two channels and detected by photomultiplier tubes. Using Prairieview software at 1.4X digital magnification (Prairie Technologies, Middleton, WI), we ran 60 min time series at 1 min intervals. A z-position between the nuclei and apical surface of outer hair cells was selected. Femtosecond pulses of 900 nm illumination from a tunable Chameleon XR laser (Coherent Inc., Santa Clara, CA) were scanned across the sample with an average power of 3±0.5 mW to excite the Texas Red fluorophores. Red fluorescence originating from GTTR was isolated using a custom made bandpass filter (620/60, Chroma Technology). Prior to adding GTTR, we scanned the sample at 740 nm and detected blue NADH intrinsic fluorescence with a custom made bandpass filter (480/30) to confirm the health of the sample [36] . Samples with low NADH signals were discarded. To quantify the fluorescence intensity, ten individual cells were selected as regions of interest (ROIs) in each T-series.

Using Image J software

(NIH), the pixels within the ROIs were averaged to obtain a fluorescence intensity measure for each cell. The average background intensity was negligible and therefore was not subtracted from the total fluorescence. The fluorescence for the ten cells was averaged to create a single average fluorescence per T-series. The average of each T-series was normalized to the maximum fluorescence and this normalized average and the standard deviation was plotted as a function of time (OriginLab, Northampton, MA) to determine the average increase in cellular fluorescence over time after GTTR administration.

Image Analyses and Statistics

Tissues were imaged using a Zeiss Axiovert LSM 5 Pascal confocal microscope. The cochlea was divided into apical, middle and basal turns ( Figure 1 ), which were separately analyzed. For hair cell quantification, myosin7a-positive hair cells were counted per cochlear length using the cell counter in Image J software. For the gentamicin dose-response curve and experiments on the Cdh23 v2J mice, hair cells from the middle turn of the cochleae were counted per 400 µm. In experiments testing BAPTA treatment, hair cells from all three turns of the cochleae were counted over a 225 µm length of the organ of Corti. 10.1371/journal.pone.0054794.g001 Figure 1 Gentamicin causes cochlear hair cell loss in a dose-dependent manner. A) Low magnification image of a postnatal 3-day-old (P3) mouse cochlea immunolabeled for myosin7a, a specific marker for inner and outer hair cells. B) Cochleae were isolated from P3 mice and cultured overnight. The following day, cultures were bathed in gentamicin (0–0.5 mM) for 1 hr at 37°C followed by a 48 hr recovery period in AG-free culture media. C) Schematic of aminoglycoside (AG, red) entry into hair cells via mechanotransduction channels (MET, green) located at the apical end of stereocilia, and proposed mechanism of MET channel closure preventing AG entry following tip-link (yellow) breakage. D–G) Representative images of the middle turn of cochleae treated with varying doses of gentamicin and labeled with anti-myosin7a. (D) Control. (E) 0.1 mM gentamicin (F) 0.25 mM gentamicin (G) 0.5 mM gentamicin. H) Myosin7a-positive hair cells per 400 µm middle turn were counted and 0.23 mM gentamicin was determined to cause a 50% hair cell loss, mostly among outer hair cells. Error bars = S.D., scale bars = 100 µm in A, 25 µm in D–G. To quantify GTTR fluorescence in fixed tissues, images were captured from each turn using a Zeiss LSM5 Confocal microscope, with a 40X 1.3NA Plan Neofluor oil immersion objective and identical microscope settings (546 nm laser excitation, emission filter BP 560–615 nm, pinhole at 1 Airy unit, detector gain at 763). The identical settings were used for experimental and control groups from all trials. The cytoplasm of myosin7a-positive hair cells between the level of the cuticular plate and nucleus was selected as the ROI’s. As above, ROI’s were averaged using Image J and intensities of GTTR fluorescence were normalized to the brightest cell in each of the three culture durations. To assess the time course of GTTR uptake, best fit curves were produced using the formula Y = Ae (−x/t) , from which the time constants (t 1/2 ) were derived. Normalized intensity histograms (10% bin widths) were generated and fit with Gaussian functions to identify intensity peaks and full width half maximums. Either single or double Gaussian fits were used. Image preparation for figures was performed using Photoshop (Adobe Systems, San Jose, CA) software. Graph preparation and data analyses were done using Microsoft Excel (Redmond, WA) and Origin softwares. Two-tailed Student’s t-test (unpaired) was used for statistical comparison and p