Baseline and Endpoint auditory tests (ABR and DPOAE): Sustained depletion of macrophages via PLX3397 provided complete protection against cisplatin-induced hearing loss and OHC dysfunction (Experiment 2). (Manuscript: Figure 5_Supp Fig 5-1_Supp Fig 5-2_Supp Fig 5-3)

Prior to the start of PLX3397 chow pre-treatment and at the end of the cisplatin administration protocol, mice underwent two types of auditory tests: auditory brainstem responses (ABRs; measure hearing sensitivity) and distortion product otoacoustic emissions (DPOAEs; indirectly measure outer hair cell function). These data suggest that sustained depletion of macrophages via PLX3397 provided complete protection against cisplatin-induced hearing loss and OHC dysfunction (Experiment 2).For <b>ABR</b> measurements, subcutaneous needle electrodes (Rhythmlink, Columbia, SC, USA) were placed behind left pinna of the test ear (reference), vertex (active), and near the tail of the mouse (ground). Tone-burst stimuli (Cos2, 3 msec, 0.5 msec rise/fall in alternating polarity) were presented at a rate of 29.9/sec<b> </b>at 8, 11.2, 16, 22.4, 32, and 40 kHz starting at 90dB SPL. At each sound level, 1024 waveforms were averaged, amplified (20x), and filtered (HP: 300Hz, LP: 3kHz, NT: 60Hz). At near-threshold sound pressure levels (SPL), the ABR waveforms were recorded twice, and two waveforms were superimposed for comparison. ABR threshold was defined as the lowest stimulus intensity that resulted in a reproducible waveform displaying identifiable peaks.<b>DPOAEs</b> were measured in response to two primary pure tones, f1 and f2, generated by Multi Field 1 speakers. Two primary tones were presented at 6 frequency pairs, where f2 corresponded to ABR test frequencies (f2 = 8, 11.2, 16, 22.4, 32, 40kHz; f2/f1 = 1.25). The sound level was increased in 5dB steps from 30dB to 90dB. At each sound level, 512 responses were averaged. DPOAE at 2f1-f2 was recorded in the mouse inner ear canal using an ER-10B+ microphone (Etymotic, Elk Grove Village, IL, USA) connected to a modified pipette tip to fit the mouse external ear canal. Biological noise floors and amplitudes were calculated for each treatment group and plotted relative to each other.