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.

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 ns). 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.

Y-27632 (257-00511, Wako Pure Chemical Industries Ltd, Osaka, Japan) was dissolved in water, and the concentration was adjusted to 1 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 at a rate of 20 nL/sec using a Nanoject III Programmable Nanoliter injector (3-000-207, Drummond Scientific Company, Broomall, PA, USA).

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