改进小鼠噪声性听力损失的实验条件及听力功能和外毛细胞损伤的评估
Summary
在这里，我们提出了噪声诱导听力损失（NIHL）小鼠模型的方案。为了诱导NIHL，我们开发了一种使用波纹塑料，捕鼠笼和扬声器的新型简单设备。采用听觉脑干反应和免疫荧光成像分别评估听力功能和外毛细胞损伤。
Abstract
噪声性听力损失（NIHL）的动物模型有助于病理学家、治疗师、药理学家和听力研究人员彻底了解NIHL的机制，并随后优化相应的治疗策略。本研究旨在为开发NIHL小鼠模型创建改进的方案。本研究使用雄性C57BL / 6J小鼠。将未麻醉的小鼠暴露于响亮的噪音（1和6kHz，以115-125db spl-A同时呈现）连续6小时，连续5天。使用听觉脑干反应（ABR）在噪声暴露后1天和1周评估听觉功能。ABR测量后，处死小鼠，收集其Corti器官进行免疫荧光染色。从听觉脑干反应（ABR）测量中，在噪声暴露后1天观察到明显的听力损失。1周后，实验小鼠的听力阈值降至~80 dB SPL，仍明显高于对照小鼠（~40 dB SPL）。从免疫荧光成像的结果来看，外毛细胞（OHC）被证明是受损的。总之，我们使用雄性C57BL / 6J小鼠创建了NIHL模型。开发并采用了一种新的简单设备，用于产生和传递纯音噪声。听力阈值的定量测量和OHC损伤的形态学确认都表明，施加的噪声成功地诱发了预期的听力损失。
Introduction
全世界约有13亿人因接触噪音而遭受听力损失1。在这项研究中，我们旨在建立一个明确的分步过程来诱导和确认噪声引起的听力损失（NIHL）。NIHL 由毛细胞 （HC） 和螺旋神经节神经元 （SGN） 的变性/破坏、HC 立体纤毛损伤和/或耳蜗内部 HC 和 SGN 之间的突触缺失引起。除NIHL外，此类异常还可能导致耳鸣和言语感知受损（特别是在复杂的声学环境中）。社会、心理和认知功能可能依次受到这些生理缺陷的影响2,3,4,5,6。
在基于小鼠的NIHL相关临床前研究中，最受欢迎的小鼠品系是CBA / CaJ 2,3,6,7和C57BL / 6 4,5,8。此外，雄性3,4,7小鼠比雌性小鼠更常用，因为雌激素对听力具有保护作用。因此，我们在这项研究中只使用了雄性小鼠9。参考文献后，我们选择了1 kHz和6 kHz作为施加噪声的频率。施加噪声的强度为115 dB SPL-A（笼子周围）至125 dB SPL-A（笼子中心）。在将实验小鼠连续暴露于噪音中6小时，连续5天后，听力阈值的最佳增加表明在实验小鼠中产生了NIHL的最佳程度。处理动物、构建实验装置和诱导噪音的操作都在提供的方案中逐步清楚地描述。
Protocol
Representative Results
Discussion
NIHL可分为两种类型：临时NIHL，其显示听力阈值的时间偏移，以及永久性NIHL，其特征是永久性听力阈值偏移。我们在第 6天（噪音暴露后1天）观察到的听力损失被认为是这两种类型的组合。在这种情况下，由于听力损失的时间成分，听力阈值将随着时间的推移逐渐恢复。在我们的初步实验研究中，在相同的设置和动物下获得的结果，2天噪声暴露产生的听力损失在2周内完全恢复，表明永久…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我們感謝台灣政府科技部（MOST）的撥款（MOST 110-2314-B-715-005，MOST 111-2314-B-715-009-MY3），以及麥凱醫學院的校內研究基金（MMC-RD-110-1B-P030，MMC-RD-111-CF-G002-03）。
Materials
| 1/4" CCP Free-field Standard Microphone Set | GRAS | 428158 | For noise exposure |
| Amplifier Input Module, AMI100D | BIOPAC | For auditory brainstem response | |
| Bio-amplifier, BIO100C | BIOPAC | For auditory brainstem response | |
| Bovine Serum Albumin | SIGMA | A9647 | Immunofluorescence staining |
| Cellsens software | Olympus life science | Image acquisition | |
| Corrugated plastic | |||
| DAPI fluoromount | SouthernBiotech | 0100-20 | Immunofluorescence staining |
| Ethylenediaminetetraacetic acid | SIGMA | E5134 | Decalcification |
| Evoked Response Amplifier, ERS100C | BIOPAC | For auditory brainstem response | |
| Formaldehyde | APLHA | F030410 | Fixation of cochlear |
| High Performance Data Acquisition System, MP160 | BIOPAC | For auditory brainstem response | |
| Modular Extension Cable, MEC110C | BIOPAC | For auditory brainstem response | |
| Myo7A primary antibody | Proteus | 25-6790 | Immunofluorescence staining |
| Myo7A secondary antibody | Jackson immunoresearch | 711-545-152 | Immunofluorescence staining |
| Needle Electrode, Unipolar 12 mmTp, EL452 | BIOPAC | For auditory brainstem response | |
| phalloidin antibody | Alexa Fluor | A12381 | Immunofluorescence staining |
| phosphate-buffered saline | SIGMA | P4417 | |
| Rat trap cage | 14 cm x 17 cm x 24cm | ||
| ROMPUN- xylazine injection, solution | Bayer HealthCare, LLC | ||
| Sound amplifier, MT-1000 | unika | For noise exposure | |
| Sound generator/analyzer/miscellaneous, FW-02 | CLIO | 620300719 | For noise exposure |
| Soundproof chamber | IEA Electro-Acoustic Technology | For noise exposure and ABR | |
| Speaker | IEA Electro-Acoustic Technology | For noise exposure | |
| Stimulator Module, STM100C | BIOPAC | For auditory brainstem response | |
| Triton X-100 | SIGMA | T8787 | Immunofluorescence staining |
| Tubephone Set, OUT101 | BIOPAC | For auditory brainstem response | |
| Upright Microscope, BX53 | Olympus | Image acquisition | |
| Zoletil | Virbac |
References
- World Report on Hearing. World Health Organization Available from: https://www.who.int/publications/i/item/9789240020481 (2021)
- Fernandez, K. A., et al. Noise-induced cochlear synaptopathy with and without sensory cell loss. 神经科学. 427, 43-57 (2020).
- Kujawa, S. G., Liberman, M. C. Adding insult to injury: cochlear nerve degeneration after "temporary" noise-induced hearing loss. The Journal of Neuroscience. 29 (45), 14077-14085 (2009).
- Wang, J., et al. Overexpression of X-linked inhibitor of apoptosis protein protects against noise-induced hearing loss in mice. Gene Therapy. 18 (6), 560-568 (2011).
- Takeda, S., Mannström, P., Dash-Wagh, S., Yoshida, T., Ulfendahl, M. Effects of aging and noise exposure on auditory brainstem responses and number of presynaptic ribbons in inner hair cells of C57BL/6J mice. Neurophysiology. 49 (5), 316-326 (2017).
- Rouse, S. L., Matthews, I. R., Li, J., Sherr, E. H., Chan, D. K. Integrated stress response inhibition provides sex-dependent protection against noise-induced cochlear synaptopathy. Scientific Reports. 10 (1), 18063 (2020).
- Amanipour, R. M., et al. Noise-induced hearing loss in mice: Effects of high and low levels of noise trauma in CBA mice. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2018, 1210-1213 (2018).
- Edderkaoui, B., Sargsyan, L., Hetrick, A., Li, H. Deficiency of duffy antigen receptor for chemokines ameliorated cochlear damage from noise exposure. Frontiers in Molecular Neuroscience. 11, 173 (2018).
- Hultcrantz, M., Simonoska, R., Stenberg, A. E. Estrogen and hearing: a summary of recent investigations. Acta Oto-laryngologica. 126 (1), 10-14 (2006).
- Tsai, S. C. -. S., et al. The intravenous administration of skin-derived mesenchymal stem cells ameliorates hearing loss and preserves cochlear hair cells in cisplatin-injected mice: SMSCs ameliorate hearing loss and preserve outer hair cells in mice. Hearing Research. 413, 108254 (2022).
- Choi, M. Y., Yeo, S. W., Park, K. H. Hearing restoration in a deaf animal model with intravenous transplantation of mesenchymal stem cells derived from human umbilical cord blood. Biochemical and Biophysical Research Communications. 427 (3), 629-636 (2012).
- Yu, S. -. K., et al. Morphological and functional evaluation of ribbon synapses at specific frequency regions of the mouse cochlea. Journal of Visualized Experiments. (147), e59189 (2019).
