Rz6 workstation

The hearing status of mice was detected by the auditory brainstem response (ABR) threshold. In brief, animal were anesthetized with pentobarbital intraperitoneal injection (8.4 mg/100 g). Place the mouse on a constant temperature pad and keep the body temperature at 37°C. Insert electrodes on the top, subcutaneously at the outer auricle, and back of the mouse’s skull. RZ6 workstation and BioSig software (Tucker-Davis Technologies, Inc., Alachua, FL, United States) were used for ABR data collection and generation. At each sound level, 512 responses are sampled and averaged. The minimum detectable threshold of all ABR waves is determined by gradually reducing the stimulus intensity from 90 dB SPL to 10 dB SPL, so as to obtain the ABR threshold of each animal.

ABRs were recorded from mice anesthetized with chloral hydrate (480 mg/kg, IP) (Sigma Aldrich-Fluka, St. Louis, MO, USA). Body temperature was maintained at 37°C throughout recording with a Homeothermic Monitoring System (Harvard Apparatus, 55-7020). Three needle electrodes were positioned sub-dermally at the vertex (active), right mastoid region (reference), and the left shoulder (ground). A short toneburst of 3 ms duration with 1 ms rise and fall time was generated by the RZ6 workstation (Tucker–Davis Technologies, Alachua, FL, USA). Stimulus sounds were presented free-field via an MF1 speaker (TDT) placed 10cm away from the vertex. Stimulus frequencies of 32 kHz to 4 kHz were presented in half-octave step. The sound level was decreased from 90 to 0 dB SPL in 5-dB steps. Stimulus presentation rate is 20 per second. 400 responses were averaged at each frequency each level. Thresholds were determined by minimal stimulus level that evoked any one of the initial four peaks. Near threshold recordings were repeated to confirm the findings.
All latencies and amplitudes of ABR peak I were measured and analyzed by using BioSigRZ software (TDT). Latency referred to the time from the onset of the stimulus signal to the peak, while amplitude was determined by averaging the △V of both sides of the peak.

ABRs were performed at baseline and 1 day and 14 days after the repeated noise exposure procedure. Mice were anesthetized with xylazine (20 mg/kg) and ketamine (100 mg/kg) through intraperitoneal injection, and the body temperature was maintained near 37°C using a heating blanket (Harvard Apparatus, USA, 55-7020). Recordings were performed using three subcutaneous needle electrodes at the vertex (active), left mastoid area (reference), and right shoulder (ground), respectively. Short tone burst stimuli of 3 ms duration with 1 ms rise/fall times were generated by the RZ6 workstation (Tucker-Davis Technologies, USA). Stimulus sounds were delivered free-field via an MF-1 speaker placed 10 cm away from the vertex, in front of the mouse. Stimulus roved over frequencies of 32, 22.6, 16, 11.3, 8, and 4 kHz, and the sound level started from 90 to 0 dB sound pressure level (SPL) in 5 dB steps. For each ABR waveform, 400 responses were collected and averaged. ABR thresholds were identified as the minimal stimulus level that evoked any noticeable recording of waveforms at each frequency. Wave I amplitudes (μV) were measured by averaging the ΔV of both sides of the peak using the BioSigRZ software (Tucker-Davis Technologies, USA). Thresholds and amplitudes were measured by a researcher who was blind to the information of mice in groups.

Mice were placed on an isothermal pad to keep the body temperature at 37°C during the whole experiment. A RZ6 workstation and BioSig software (Tucker Davis Technologies) were used for stimulus generation, presentation, ABR acquisition, and data management. After the mice were anesthetized by intraperitoneally injecting 5% chloral hydrate for 0.5 ml/100 g body weight, electrodes were inserted subcutaneously at the vertex and pinna as well as near the tail. Acoustic stimuli (clicks or pure-tone bursts) of decreasing sound level from 90 dB SPL in 10 dB SPL steps were generated using high-frequency transducers. At each sound level, 512 responses were sampled and averaged. Hearing threshold of each mouse was determined as the lowest sound level at which all ABR waves were detectable.

Mice were anesthetized intraperitoneally with 8.4 mg/100g Pentobarbital. Body temperature was maintained at 37 °C by placing the mice on an isothermal pad during testing and recovery from anesthesia. Electrodes were inserted subcutaneously at the vertex and pinna, and a ground electrode was placed near the tail. The stimulus generation, presentation, ABR acquisition and data management were coordinated using RZ6 workstation and BioSig software (Tucker Davis Technologies, Inc.). Specific acoustic stimuli (clicks or tone bursts of 4, 8, 16 and 32 kHz) were generated using high frequency transducers. At each sound level, 512 responses were sampled and averaged. ABR thresholds were obtained for each animal by reducing the stimulus intensity in 10 dB SPL steps from 90 dB SPL to identify the lowest intensity at which all ABR waves were detectable.