Baseline and Endpoint auditory tests (ABR and DPOAE): Macrophage ablation followed by partial repopulation protected against cisplatin-induced hearing loss and OHC dysfunction (Experiment 1) (Manuscript: Fig. 2; Fig. S2-1, Fig. S2-2_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 partial macrophage ablation protected against cisplatin-induced hearing loss and OHC dysfunction (Experiment 1).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.

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，可间接检测外毛细胞功能）。本实验数据表明，部分巨噬细胞消融可预防顺铂诱导的听力损失与外毛细胞（outer hair cell, OHC）功能障碍（实验1）。针对听性脑干反应（ABR）的测量，将皮下针状电极（Rhythmlink，美国南卡罗来纳州哥伦比亚市）分别安置于受试耳耳廓后方（参考电极）、小鼠头顶（活动电极）以及尾部附近（接地电极）。采用Cos2型短声刺激，刺激时长3毫秒，交替极性下上升/下降沿为0.5毫秒，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个波形进行平均、放大（20倍）并滤波（高通：300Hz，低通：3kHz，陷波：60Hz）。在接近阈值的声压级下，ABR波形被记录两次，并将两个波形叠加以进行对比分析。ABR阈值被定义为可引出具有可识别峰的可重复波形的最低刺激强度。畸变产物耳声发射（DPOAEs）的测量采用Multi Field 1扬声器生成的两个初始纯音f1与f2。设置6组频率配对，其中f2与ABR测试频率保持一致（f2=8、11.2、16、22.4、32、40kHz；f2/f1=1.25）。声压级以5dB为步长从30dB递增至90dB。在每个声压级下，对512个响应信号进行平均。使用连接至适配小鼠外耳道的改良移液管吸头的ER-10B+麦克风（Etymotic，美国伊利诺伊州埃尔克格罗夫村市），在小鼠外耳道内记录2f1-f2频率的DPOAE。为每个处理组计算生物学本底噪声与振幅，并进行组间相对绘图。