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 ABR 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 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.DPOAEs 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.

在PLX3397饲料预处理开始前以及顺铂给药方案结束时，小鼠接受了两类听觉测试：听性脑干反应（auditory brainstem responses, ABR，用于评估听觉敏感度）以及畸变产物耳声发射（distortion product otoacoustic emissions, DPOAEs，用于间接检测外毛细胞（outer hair cell, OHC）功能）。上述数据表明，通过PLX3397持续耗竭巨噬细胞，可完全预防顺铂诱导的听力损失与外毛细胞功能障碍（实验2）。

针对听性脑干反应的测量，将皮下针状电极（Rhythmlink，美国南卡罗来纳州哥伦比亚市）分别放置于受试耳左耳廓后方（参考电极）、颅顶（活动电极）以及小鼠尾部附近（接地电极）。采用Cos2型调制短声刺激，时长3ms，升降沿时长0.5ms且极性交替，以29.9次/秒的速率在8、11.2、16、22.4、32及40kHz频率下呈现，初始声压级设为90dB SPL。在每个声压级下，对1024个采集得到的波形进行平均、放大20倍并滤波（高通滤波截止频率300Hz，低通滤波截止频率3kHz，陷波滤波频率60Hz）。在近阈值声压级下，ABR波形被重复记录两次，随后将两组波形叠加以进行比对分析。ABR阈值被定义为可引出带有可识别峰的稳定重复波形的最低刺激强度。

畸变产物耳声发射（DPOAEs）的检测以Multi Field 1扬声器生成的两个基频纯音f1与f2为刺激信号。设置6组频率对，其中f2的频率与ABR测试频率一致（f2=8、11.2、16、22.4、32、40kHz；f2/f1比值为1.25）。声压级以5dB为步进从30dB递增至90dB。在每个声压级下，对512个响应信号进行平均叠加。使用连接经改装移液管尖端以适配小鼠外耳道的ER-10B+麦克风（Etymotic，美国伊利诺伊州埃尔克格罗夫村），在小鼠内耳道中记录2f1-f2成分的DPOAE。为每个处理组计算生物本底噪声与信号振幅，并进行组间相对绘图。