Age-related hearing los in Nox4 knockout mouse

[Description of methods used for collection/generation of data] Auditory function (ABR data): Auditory brainstem responses (ABR) recordings were performed on a TDT ABR and DPOAE acquisition system, with a RZ6 processor (Tucker‐Davis Technologies, Alachua, FL, USA). In brief, mice were anesthetized with ketamine (100 mg/kg; Imalgene 1000; Merial, Lyon, France) and xylazine (10 mg/kg; Rompun 2%; Bayer, Leverkusen, Germany) by intraperitoneal injection and the ABR tests were performed in a sound‐attenuating chamber. Two different sound stimuli, clicks and tone bursts, were generated with SigGenRZ software (TDT). Stimuli were calibrated using SigCalRZ software and a PCB 377C01 precision condenser microphone, with a 426B03 preamplifier and a 480C02 signal conditioner. Click (duration 0.1 ms) and tone burst (duration 5 ms, 2.5 ms each for rise and decay, without plateau) at 4, 8, 16, 24, 32 and 40 kHz stimuli were delivered by a MF1 open field magnetoelectrostatic speaker (TDT) at 30 (click) or 50 (tone bursts) pulses per second, and from 90 to 10 dB SPL, in 5–10 dB steps. The evoked response was collected with stainless steel needle electrodes placed at the vertex (active), ventrolateral to the right ear (reference) and tail base (ground), promediated, and analyzed with BioSigRZ software (TDT). Cochlear gene expresión: Inner ear dissection was performed and samples were frozen in RNAlater® solution (Ambion, Foster City, CA, USA). Cochlear RNA was extracted using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) automated on the Qiacube (Qiagen, Hilden, Germany). Quality determination and cDNA generation from pooled cochlear RNA extracts (3 cochlea from different animals per group) were performed. Quantitative amplification was performed in triplicate on a Quant Studio 7 Flex PCR System (Applied Biosystems, Foster City, CA, USA) using either commercial TaqMan probes (Nox4, Nox3, Nrf2, Nlrp3, Il1b, Tnfa). Data were collected after each amplification step and analyzed with QuantStudio™ Real-Time PCR software 1.3 (Applied Biosystems). Hprt1 gene was used as a housekeeping gene, and the n-fold differences were calculated using the 2−ΔΔCt method.