Animals
Forty-five CBA/J mice (male, 6 weeks old) weighing 17–20 g were purchased from the Japan Charles River (Yokohama, Japan). The animals were provided free access to water and were fed a regular diet. Moreover, they were individually housed and maintained at 23–25 °C. In this study, all procedures on mice were performed under general anesthesia induced by intramuscular injection of ketamine (75 mg/kg) and medetomidine (1 mg/kg). All experimental procedures reported herein were approved by the Institutional Animal Care and Use Committee of the National Defense Medical College and were performed in accordance with the guidelines of the National Institutes of Health and the Ministry of Education, Culture, Sports, Science and Technology of Japan (approval #18050). All efforts were made to minimize the number of animals used and their suffering.
LISW irradiation of the inner ear
LISWs were generated as described previously [5]: a laser target was irradiated with a 532-nm Q-switched neodymium-doped yttrium aluminum garnet (Nd: YAG) laser (Brilliant b, Quintal, Les Ulis Cerdex, France; pulse width, 6 nanoseconds). Specifically, the laser target was a 10-mm in diameter, 0.5-mm thick black natural rubber disk, and a 1.0-mm thick transparent polyethylene terephthalate sheet was bonded to the top of the target area to confine the laser-induced plasma, by which the LISW impulse was increased. The laser pulse was focused to a 4.0-mm diameter spot on the laser target using a plano-convex lens. The laser fluence on the target was set at 2.0 J/cm2, as described in a previous report [5], to generate the cochlear synaptopathy model without hair cell loss. Temporal pressure waveforms of LISWs were measured with a hydrophone (HNR-1000, Onda Co., Sunnyvale, CA, USA). The signals of the hydrophone were recorded using a digital oscilloscope (DPO4104B, Tektronix, Tokyo, Japan; bandwidth, 1 GHz), and they were calibrated using software provided by the manufacturer of the pressure sensor.
Administration of Y-27632, a ROCK inhibitor, to the inner ear
Y-27632 (257-00511, Wako Pure Chemical Industries Ltd, Osaka, Japan), was dissolved in water, and the concentration was adjusted to 1 mM and 10 mM. PBS was used as the sham control. The following three groups were compared to investigate the effects of the ROCK inhibitor, Y-27632, on the LISW-induced cochlear neuropathic model: 1. sham surgery group (treated with PBS); 2. ROCK inhibition group (1 mM Y-27632); and 3. ROCK inhibition group (10 mM Y-27632). To administer Y-27632 to the inner ear, the left postauricular region of the mice was positioned under a stereomicroscope. After a 20-mm postauricular skin incision was made, subcutaneous tissues and superficial fascia were dissected, and the otic bulla was exposed. Tympanotomy was performed using microforceps, and the hole was enlarged to allow clear observation of the round window niche. Then, 1 µL of PBS or ROCK inhibitor was injected into the round window niche using a Nanoject III Programmable Nanoliter injector (3-000-207, Drummond Scientific Company, Broomall, PA, USA).
Cochlear function tests
Cochlear function tests were performed in each animal at six log-spaced frequencies (half-octave steps from 5.6 to 32.0 kHz) before and 1 day, 7 days, and 28 days after blast exposure. Mice were anesthetized with ketamine (75 mg/kg i.p.) and medetomidine hydrochloride (1 mg/kg i.p.). For ABRs, needle electrodes were inserted at the vertex and pinna, with the ground near the tail. ABRs were evoked with 5-ms tone pips (0.5-ms rise-fall with a cos2 onset envelope delivered at 35/s). The response was amplified, filtered, and averaged using a LabVIEW-driven data-acquisition system. The sound level was raised in 5 dB steps from ≥10 dB to <80 dB SPL. At each sound level, 1024 responses were averaged (with alternating stimulus polarity). On visual inspection of stacked waveforms, the “ABR threshold” was defined as the lowest SPL level at which any wave could be detected, which usually corresponds to the level step just below that at which the peak-to-peak response amplitude rose significantly above the noise floor. When no response was observed at the highest sound level available, the threshold was designated as 5 dB greater than that level so that statistical tests could be performed. For amplitude versus level functions, the wave I peak was identified by visual inspection at each sound level, and the peak-to-peak amplitude was computed.
For the measurement of DPOAEs at 2f1–f2, the primary tones were set such that the frequency ratio (f2/f1) was 1.2 and the f2 level was 10 dB below the f1 level. For each f2/f1 primary pair, levels were swept in 5 dB steps from 20 to 80 dB SPL (for f2). At each level, both waveform and spectral averaging were used to increase the signal-to-noise ratio of the recorded ear-canal sound pressure, and the amplitude of the DPOAE at 2f1–f2 was extracted from the averaged spectra, along with the noise floor at nearby points in the spectrum. Iso-response curves were interpolated from the plots of the DPOAE amplitude versus sound level. The threshold was defined as the f1 level required to produce a DPOAE at 0 dB SPL.
Quantitative RT-PCR
Harvested cochlear tissues were collected and stored until further analysis (Ambion, Austin, TX, USA). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Quantitative RT-PCR was performed on a Thermal Cycler Dice Real Time System using the One Step SYBR PrimeScript PLUS RT-PCR Kit (RR096A; TaKaRa Bio, Shiga, Japan). Forward (F) and reverse (R) primer sequences were RhoA-F, 5′-AGCTTGTGGTAAGACATGCTTG-3′ and RhoA-R, 5′-GTGTCCCATAAAGCCAACTCTAC-3′; ROCK1-F, 5′-GACTGGGGACAGTTTTGAGAC-3′ and ROCK1-R, 5′-GGGCATCCAATCCATCCATCCAGC-3′; and ROCK2-F, 5′-TTGGTTCGTCATAAGGCATCAC-3′ and ROCK2-R, 5′-TGTTGGCAAAGGCCATAATATCT-3′. PCR cycling conditions included 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Relative mRNA expression levels were determined using the ΔΔCt method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. All reactions were performed in duplicate. For assessment, relative mRNA expression levels were standardized to cochlear samples extracted before LISW.
Cochlear processing and immunohistochemistry
Mice were perfused transcardially with 0.5 mL/g of lactated Ringer’s solution followed by 1 mL/g of 4% paraformaldehyde (PFA)/0.1 M phosphate buffer (PB) at room temperature. After decapitation, the cochlea was dissected out, and small holes were made at the round window, oval window, and apex of the cochlea; it was then bathed in 4% PFA/PB at 4 °C overnight. After decalcification with 0.5 M ethylenediaminetetraacetic acid (EDTA) (Decalcifying Soln. B (EDTA method); Wako Pure Chemical Industries Ltd, Osaka, Japan) for 4 days at 4 °C with shaking, each cochlea was microdissected into four pieces for whole mount preparation. For immunostaining, cochlear pieces were blocked and permeabilized with 5% normal horse serum in PBS and 0.3% Triton X-100 for 1 h at room temperature, followed by overnight incubation at 37 °C with the following primary antibodies diluted in 1% normal horse serum with 0.3% Triton X-100. Chicken anti-NF 200 (1:500, Millipore, Bedford, MA, USA: AB5539) was used as an auditory nerve marker, rabbit anti-Myo7a (1:500, Proteus Biosciences Inc. Ramona, CA, USA: 25-6790) as a hair cell marker, mouse (IgG1) anti-CtBP2 (1: 500 BD Biosciences, San Jose, CA, USA: 612044) as a presynaptic marker, mouse (IgG2a) anti-GluA2 (1: 2000 Millipore, Bedford, MA, USA: MAB397) as a postsynaptic marker, and rabbit anti-ROCK1 (1:100 abcam, Cambridge, UK: ab134181) and rabbit anti-ROCK2 (1:100 abcam: ab125025). After washing with PBS, the following secondary antibodies diluted 500-fold were incubated at room temperature for 2 h: Alexa Fluor 350-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA: A21068), Alexa 488-conjugated goat anti-mouse IgG2a ( A21131; Invitrogen), Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen, A11034), Alexa 568-conjugated goat anti-mouse IgG1 (Invitrogen: A21124), Alexa 647-conjugated goat anti-chicken IgY (Invitrogen: A21449), and rhodamine phalloidin (1:500 Invitrogen, Carlsbad, CA, USA: R415). After washing with PBS, samples were mounted with fluorescence mounting media (Dako, Santa Clara, CA, USA: S3023) and observed.
Quantitative analysis of synapses and hair cells
The fluorescent-labeled organ of Corti was observed using a BZ-X700 fluorescence microscope (Keyence Corporation, Osaka, Japan) with a water-immersion 60× objective and 3× digital zoom. Optical sections in the x–y plane (z- sections) were recorded at 0.2 μm intervals along the z-axis. The resulting confocal image series (z-stack) contained a three-dimensional image of the entire volume of the cochlear tissues. The z-stack was reconstructed (to view a plane perpendicular to the x–y plane) using ImageJ (https://imagej.nih.gov/ij/) or PhotoShop CC (Adobe, San Jose, CA, USA).
To count the number of outer hair cells (OHCs) and IHCs, confocal microscopy was performed at 5.6, 8.0, 11.3, 16.0, 22.6, and 32.0 kHz while focusing on the presynaptic ribbons in the basolateral portion of IHCs; an oil-immersion 100× or water-immersion 60× objective and a 0.2-μm z-step were used. For each frequency region, z-stacks were acquired in each cochlea at three adjacent areas, each containing ~10 IHCs in a row. The number of OHCs and IHCs per 200 µm was counted at each point, as described above. The densities of OHCs and IHCs per 200 µm were calculated and compared at each site. The numbers of IHC synaptic ribbons (CtBP2-positive puncta) and glutamate-receptor patches (GluA2 puncta) per 200 µm were counted at 5.6, 8, 11.3, 16, 22.6, and 32 kHz, as described above. The number of synaptic ribbons and glutamate-receptor patches per IHC was calculated and compared at each site. To minimize bias, the counts were performed by three different individuals who were blinded to the experimental groups.
Statistical analysis
Statistical analyses were conducted using Prism software (version 7.0; GraphPad software, Inc., La Jolla, CA, USA). For analyses of histological and cochlear function results, two-way analysis of variance (ANOVA), followed by Bonferroni correction for multiple comparisons, was used. The two-tailed Mann–Whitney U test was employed to compare differences in mRNA expression. The statistical power and the sample size were determined before and after data collection using PS: Power and Sample Size Calculation, Ver. 3.1.6 (Department of Biostatistics, Vanderbilt University, Nashville, TN, USA). Statistical significance was set at p < 0.05. Error bars represent standard deviation of the mean (SEM).