Stereocilia

After completing the ABR measurements at 1, 3, 6, and 9 month of age at The Jackson Laboratory the inner ears were dissected out, immersed in 4% paraformaldehyde and shipped to the University at Buffalo for analyses of the cochlea and vestibular system. Our procedures for preparing cochleograms showing the percentage of missing inner hair cells (IHC) and outer hair cells (OHC) as a function of percent distance from the apex have been described in detail previously [14 (link), 15 (link), 122 ]. Mice evaluated by ABR at the Jackson Lab were euthanized by CO₂ asphyxiation and decapitated. The temporal bones were removed, immersed in 4% paraformaldehyde, and shipped to the University at Buffalo for analysis. Cochleae were stained with Ehrlich's hematoxylin solution, the organ of Corti dissected out as a flat surface preparation, mounted in glycerin on glass slides and coverslipped. A person, blind to the results, dissected the cochleae and prepared the surface preparation. A second person blind to the experimental conditions counted the hair cells using a light microscope (Zeiss Standard, 400X magnification). By raising and lowering the focal plane, the investigator can determine if the hair cell nucleus, cuticular plate and stereocilia bundle were present. A hair cell was counted as present if both the cuticular plated and nucleus were clearly visible and considered missing if either were absent. OHC and IHC were counted along successive 0.12-0.24 mm intervals from the apex to the base. Using lab norms and custom software, the percentage of missing IHC and OHC were determined for each animal and a cochleogram was constructed showing the percentage of missing OHC and IHC as a function of percent distance from the apex of the cochlea. Position in the cochlea was related to frequency using a mouse tonotopic map [33 (link)]. In some cases, the cochlear surface preparations were photographed with a digital camera (SPOT Insight, Diagnostic Instruments Inc.) attached to a Zeiss Axioskop microscope, processed with imaging software (SPOT Software, version 4.6) and Adobe Photoshop 5.5.
To evaluate the condition of the cochlea and vestibular sensory epithelium in more detail, some inner ears were embedded in plastic using procedures described in our earlier publications [32 (link), 122 , 123 (link)]. Following fixation, inner ears were decalcified (Decal, Baxter Scientific Products), rinsed in phosphate buffered saline, dehydrated through a graded series of EtOH and then embedded in Epon 812 (Electron Microscopy Sciences). Sections were cut parallel to the modiolus of the cochlea at a thickness of three μm on an ultramicrotome, stained with 0.5% toluidine blue, mounted on glass slides, examined with a Zeiss microscope (Axioskop) and photographed with a digital camera as above. Sections (3 μm) were also taken from the utricle, saccule and crista ampullaris following similar procedures.

The cochlea of the inner ear was isolated from mouse E18.5 embryos and attached to the transparent PET membrane of the cell culture insert (353096, Corning). They were then fixed with PBS containing 4% PFA for 1 h at room temperature, followed by three washes with PBS. Immunostained samples were visualized using a confocal microscope (LSM710; Zeiss, Oberkochen, Germany). The individual stereocilia angle was determined as previously described^{35 (link)}, and measured using the Fiji angle tool. Data for different genotypes was obtained from at least 100 cells in each hair cell row of mice from three different litters.

The cultured explants were fixed for 20 min with 4% paraformaldehyde in PBS and permeabilized with 5% Triton^TM X-100 (Sigma, St. Louis, MO) in PBS with 10% FBS for 10 min. The specimens were stained with a rhodamine-phalloidin probe (1:100; Invitrogen, Carlsbad, CA) at 15-25°C for 1 h. Phalloidin binds F-actin with nanomolar affinity and labels stereociliary arrays and cuticular plates of hair cells. The specimens were observed under a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan). If no stereocilia or cuticular plate were observed with phalloidin staining, the hair cells were considered missing. Quantitative results were obtained by evaluating 30 outer hair cells associated with 10 inner hair cells [20 (link)]. The average of three separate counts was representative of each culture. The residue of hair cells was measured as a percentage.

High-magnification fluorescent images were acquired using a 63 × objective on a Leica SP8 confocal microscope, while whole utricle images were captured using a 20 × objective on the same confocal microscope. Images were analyzed using Fiji software (v1.53, NIH, Bethesda, Maryland, USA). For utricles and saccules, the numbers of HCs were manually counted per 100 × 100 μm² area in both the striolar and extrastriolar region (Fig. 2C). For every crista, cell counts were performed per 100 × 100 μm² area in the central and peripheral regions. We chose HCs with bundles randomly and measured the heights of its tallest stereocilia in each cells in the utricular striola and extrastriola. Each cochlea was divided into the apical, middle, and basal turns, and two regions of 100 μm length in each turn were randomly chosen and the average number of HCs was calculated for each sample. The number of spiral ganglion neurons in Rosenthal’s canal was counted in each section, and data were obtained from five separate sections in each cochlea to obtain a mean value.