星形胶质细胞在噪声损伤后小鼠耳蜗核突触修复中的作用

doi:10.3969/j.issn.1674-8115.2024.04.005

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (4): 454-461.doi: 10.3969/j.issn.1674-8115.2024.04.005

• 论著 · 基础研究 • 上一篇

星形胶质细胞在噪声损伤后小鼠耳蜗核突触修复中的作用

周卫军¹^,²^,³(), 刘思迪¹^,²^,³, 蔡瑞捷¹^,²^,³, 刘宏超¹^,²^,³, 王美建¹^,²^,³, 吴皓¹^,²^,³, 刘辉辉¹^,²^,³(), 汪照炎¹^,²^,³()

^1.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011
^2.上海交通大学医学院耳科学研究所，上海 200125
^3.上海市耳鼻疾病转化医学重点实验室，上海 200125

收稿日期:2023-11-26 接受日期:2024-01-26 出版日期:2024-04-28 发布日期:2024-04-28
通讯作者: 刘辉辉,汪照炎 E-mail:drzhouwj@126.com;liuhent9h@163.com;wzyent@163.com
作者简介:周卫军（1998—），男，硕士生；电子信箱：drzhouwj@126.com。
基金资助:
国家自然科学基金青年科学基金项目(82101211);黄浦区产业扶持基金(XK2019011);上海市科学技术委员会“科技创新行动计划”基础研究领域项目(21JC1404000);上海市耳鼻疾病转化医学重点实验室(14DZ2260300)

Role of astrocytes in the repair of auditory synapses in the cochlear nucleus after noise damage

ZHOU Weijun¹^,²^,³(), LIU Sidi¹^,²^,³, CAI Ruijie¹^,²^,³, LIU Hongchao¹^,²^,³, WANG Meijian¹^,²^,³, WU Hao¹^,²^,³, LIU Huihui¹^,²^,³(), WANG Zhaoyan¹^,²^,³()

^1.Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
^3.Shanghai Key Laboratory of Translation Medicine on Ear and Nose Disease, Shanghai 200125, China

Received:2023-11-26 Accepted:2024-01-26 Online:2024-04-28 Published:2024-04-28
Contact: LIU Huihui,WANG Zhaoyan E-mail:drzhouwj@126.com;liuhent9h@163.com;wzyent@163.com
Supported by:
National Natural Science Foundation of China(82101211);Huangpu District Industrial Support Fund(XK2019011);Science and Technology Innovation Action Plan of Shanghai Municipal Science and Technology Commission(21JC1404000);Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases(14DZ2260300)

摘要/Abstract

摘要：

目的·通过形态学分析和分子生物学技术，探究噪声引起的耳蜗核损伤的病理生理变化以及星形胶质细胞对损伤的调节功能。方法·将48只C57BL/6J雄性小鼠随机分成2组暴露在110 dB声压级（sound pressure level，SPL）的宽频噪声下，持续2 h。然后在噪声暴露后的第1、7、14、30和90日对小鼠进行听性脑干反应（auditory brainstem response，ABR）测试；并对耳蜗核组织进行免疫荧光染色观察耳蜗核神经元、听觉突触损伤情况以及星形胶质细胞激活程度；此外，通过蛋白质印迹法（Western blotting）进一步验证噪声对耳蜗核神经元与神经突触的损伤情况。结果·噪声暴露后，腹侧耳蜗核中丛细胞数量显著减少。Western blotting结果显示耳蜗核神经元中神经纤维严重丢失，表明噪声对耳蜗核神经元造成了严重的损害。囊泡谷氨酸转运蛋白1（vesicular glutamate transporter 1，Vglut1）标记的听觉神经突触明显丧失，其中在噪声暴露后的第14日丧失最为严重，随后在第90日呈缓慢恢复趋势。此外，在噪声暴露后，耳蜗核中星形胶质细胞呈现出明显的聚集和激活。通过胶质酸性纤维蛋白（glial fibrillary acidic protein，GFAP）染色，提示在噪声暴露之前，大多数星形胶质细胞分布在耳蜗核的周边、颗粒细胞区和听觉神经根周围，并且形态较小。然而，在噪声暴露后的第14日，腹侧耳蜗核中大量激活的星形胶质细胞积聚，并且它们都表现出在突触周围生长的模式。结论·噪声暴露导致耳蜗核神经突触损伤，星形胶质细胞可能参与其损伤和修复过程。这些研究结果将为进一步理解耳蜗核中声音信号分析、整合和神经可塑性的机制提供关键基础。

关键词: 噪声性聋, 耳蜗核, 星形胶质细胞, 突触可塑性

Abstract:

Objective ·To investigate the pathological and physiological changes underlying noise-induced cochlear nucleus damage and the regulating function of astrocytes on the damage, using a combination of morphological analysis, and molecular biology techniques. Methods ·Forty-eight male C57BL/6J mice were randomly divided into two groups and exposed to 110 dB SPL (sound pressure level) broadband noise for 2 hours. Auditory brainstem response (ABR) tests were performed on the mice on days 1, 7, 14, 30, and 90 after the noise exposure. Immunofluorescence staining of cochlear nuclear tissue was conducted to observe cochlear nuclear neurons and auditory synapses, as well as astrocyte activation levels. In addition, the damage to the cochlear nuclear neurons and synapses caused by noise was verified through Western blotting. Results ·A significant decrease in cochlear nuclear Bushy cells after noise exposure was observed. The Western blotting results showed that there was severe loss of nerve fibers in cochlear nuclear neurons, indicating that noise caused significant damage to cochlear nucleus neurons. Moreover, a significant loss of auditory synapses labeled with vesicular glutamate transporter 1 (Vglut1) was observed, which was the severest on day 14 after noise exposure and slowly recovered on day 90. Interestingly, astrocytes in the cochlear nucleus displayed obvious clustering and activation after noise exposure. By staining with glial fibrillary acidic protein (GFAP), most astrocytes were distributed around the cochlear nucleus, granule cell area, and auditory nerve root before noise exposure, and they had a small size. However, on day 14 after noise exposure, a large number of activated astrocytes aggregated in the ventral cochlear nucleus, and they all showed a pattern of growth around the synapses. Conclusion ·Noise exposure leads to significant damage in the cochlear nucleus, and it is possible that astrocytes are involved in its damage and repair processes. These findings will provide a crucial foundation for further understanding the mechanisms of sound signal analysis, integration, and neural plasticity in the cochlear nucleus.

Key words: noise-induced hearing loss, cochlear nucleus, astrocytes, synaptic plasticity

中图分类号:

R764.43⁺3

周卫军, 刘思迪, 蔡瑞捷, 刘宏超, 王美建, 吴皓, 刘辉辉, 汪照炎. 星形胶质细胞在噪声损伤后小鼠耳蜗核突触修复中的作用[J]. 上海交通大学学报（医学版）, 2024, 44(4): 454-461.

ZHOU Weijun, LIU Sidi, CAI Ruijie, LIU Hongchao, WANG Meijian, WU Hao, LIU Huihui, WANG Zhaoyan. Role of astrocytes in the repair of auditory synapses in the cochlear nucleus after noise damage[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(4): 454-461.

图/表 4

图1 噪声性损伤小鼠模型建立Note： Comparison of mouse auditory brainstem response (ABR) thresholds after noise exposure. ①P=0.000, compared with the Pre (control) group.

Fig 1 Establishment of a noise-induced hearing loss mouse model

图2 噪声暴露导致耳蜗核神经元变性丢失Note： A. Western blotting and grayscale analysis of cochlear nucleus in mice after noise exposure, showing significant downregulation of neurofilament-associated proteins, indicating neuronal degeneration. B. Immunofluorescence staining (MafB) of bushy cells in mice after noise exposure and corresponding statistical analysis. ①P=0.000, ②P=0.004, ③P=0.001, ④P=0.016, ⑤P=0.013, ⑥P=0.005, compared with the D0 (control) group.

Fig 2 Noise exposure leads to degeneration and loss of cochlear nucleus neurons

图3 噪声暴露导致耳蜗核丛细胞听觉突触丢失Note：A. Immunofluorescence staining (Vglut1) of cochlear nucleus auditory nerve synapses after noise exposure. B. Immunofluorescence staining (×63) of cochlear nucleus auditory nerve synapses after noise exposure, showing auditory nerve synapses (Vglut1), Ⅰa-type auditory nerve (Calretinin), and cell nuclei (DAPI). C. Statistical analysis of the proportion of Ⅰa-type auditory nerve synapses after noise exposure. ①P=0.000, ②P=0.023, ③P=0.024, compared with the D0 (control) group.

Fig 3 Noise exposure leads to the loss of auditory synapses in the cochlear nucleus bushy cells

图4 噪声暴露导致耳蜗核星形胶质细胞激活Note： A. Immunofluorescence staining (GFAP) of cochlear nucleus astrocytes after noise exposure. B. Immunofluorescence staining (×63) of cochlear nucleus auditory nerve synapses and astrocytes after noise exposure, showing auditory nerve synapses (Vglut1), astrocytes (GFAP), and cell nuclei (DAPI). C. Western blotting results of cochlear nucleus after noise exposure. ①P=0.001, ②P=0.130, ③P=0.929, ④P=0.019, ⑤P=0.048, compared with the D0 (control) group.

Fig 4 Noise exposure leads to the activation of cochlear nucleus astrocytes

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星形胶质细胞在噪声损伤后小鼠耳蜗核突触修复中的作用

Role of astrocytes in the repair of auditory synapses in the cochlear nucleus after noise damage

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