中等强度带通噪声对C57小鼠内毛细胞形态与功能的影响
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摘要
目的探讨C57小鼠在8-16kHz,98dB SPL,2h的噪声暴露前后听力以及耳蜗内的病理改变。方法 1.以98dB SPL,8-16kHz的带通噪声对C57小鼠噪声暴露2小时。2.分别在噪声暴露前、噪声暴露后第一天及第十五天进行听觉脑干反应测试(Auditory Brainstem Response, ABR)以观测噪声造成的损伤及恢复。3.将非噪声小鼠设为对照组,噪声暴露后第15天的小鼠设为实验组,取对照组与实验组小鼠耳蜗基底膜进行免疫荧光染色,对内外毛细胞以及内毛细胞突触前结构Ribbon进行计数。4.取对照组与实验组小鼠耳蜗基底膜,对8-11kHz频率区域的内毛细胞进行全细胞膜片钳记录,比较两组内毛细胞钙电流大小以及突触囊泡的释放能力。结果噪声暴露后第十五天C57小鼠中高频阈值没有完全恢复。在22kHz,实验组小鼠ABR I波波幅显著下降,潜伏期显著延长。免疫荧光染色发现噪声后外毛细胞在高频区域有部分丢失,突触前Ribbon在中圈存在部分丢失,在底圈显著丢失。膜片钳记录结果显示实验组小鼠内毛细胞钙电流幅值无明显改变。长时程去极化刺激时,实验组小鼠内毛细胞ΔCm显著低于对照组。结论 8-16kHz,98dB SPL噪声暴露使C57小鼠产生轻度永久性阈移,噪声后基底膜受损区域主要集中在中高频区。相对于外毛细胞来说,噪声后内毛细胞突触结构与功能损伤的频率范围更加广泛。
Objective To investigate the effect of noise on hair cells in C57 mice, including pathological changes of hair cells before and after noise exposure. Methods 1. C57 mice were exposed to band-passed(8-16 kHz) noise for 2 h at98 dB SPL. 2.Auditory brainstem responses(ABRs) were tested before and on the 1 st and 15 th day after noise exposure.3. Mice on the 15 th day after noise exposure were used as the study group, while those without noise exposure were used as controls. The cochlea was dissected for whole mount immunostaining. The number of hair cells and presynaptic ribbons was counted. 4.The Basilar membrane was dissected, and whole cell patch clamp recording was performed on inner hair cells of the 8-11 kHz region to measure calcium current and synaptic transmitter release. Results High-frequency ABR thresholds did not fully recover on the 15 th day after noise exposure. Following noise exposure, wave I amplitudes were significantly reduced and latencies significantly delayed at 22 kHz. In the study group, there were significant inner hair cells loss in high frequencies region, and the loss of presynaptic ribbon mainly concentrated in the middle and high frequencies regions. There was no significant difference in calcium current amplitude between noise exposed and control mice. With long time depolarization stimulation, the ΔCm in noise exposed mice was significantly smaller than in control mice. Conclusion Band passed(8-16 kHz) noise at 98 dB SPL can cause moderate permanent threshold shift in C57 mice, with damage mainly concentrated in middle and high frequencies regions. Compared with outer hair cells,the range of inner hair cell damage is more extensive.
Objective To investigate the effect of noise on hair cells in C57 mice, including pathological changes of hair cells before and after noise exposure. Methods 1. C57 mice were exposed to band-passed(8-16 kHz) noise for 2 h at98 dB SPL. 2.Auditory brainstem responses(ABRs) were tested before and on the 1 st and 15 th day after noise exposure.3. Mice on the 15 th day after noise exposure were used as the study group, while those without noise exposure were used as controls. The cochlea was dissected for whole mount immunostaining. The number of hair cells and presynaptic ribbons was counted. 4.The Basilar membrane was dissected, and whole cell patch clamp recording was performed on inner hair cells of the 8-11 kHz region to measure calcium current and synaptic transmitter release. Results High-frequency ABR thresholds did not fully recover on the 15 th day after noise exposure. Following noise exposure, wave I amplitudes were significantly reduced and latencies significantly delayed at 22 kHz. In the study group, there were significant inner hair cells loss in high frequencies region, and the loss of presynaptic ribbon mainly concentrated in the middle and high frequencies regions. There was no significant difference in calcium current amplitude between noise exposed and control mice. With long time depolarization stimulation, the ΔCm in noise exposed mice was significantly smaller than in control mice. Conclusion Band passed(8-16 kHz) noise at 98 dB SPL can cause moderate permanent threshold shift in C57 mice, with damage mainly concentrated in middle and high frequencies regions. Compared with outer hair cells,the range of inner hair cell damage is more extensive.
引文
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2 Bielefeld EC.Protection from noise-induced hearing loss with Src inhibitors.Drug Discov Today.2015;20(6):760-5.
3 Kil J,Lobarinas E,Spankovich C,et al.Safety and efficacy of ebselen for the prevention of noise-induced hearing loss:a randomised,double-blind,placebo-controlled,phase 2 trial.Lancet.2017;390(10098):969-979.
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5 Liberman LD,Suzuki J,Liberman MC.Dynamics of cochlear synaptopathy after acoustic overexposure.J Assoc Res Otolaryngol.2015;16(2):205-19.
6 Richter CP,Young H,Richter SV,et al.Fluvastatin protects cochleae from damage by high-level noise.Sci Rep.2018;8(1):3033.
7 Chen H,Xing Y,Xia L,et al.AAV-mediated NT-3 overexpression protects cochleae against noise-induced synaptopathy.Gene Ther.2018;25(4):251-259.
8 Huang M,Kantardzhieva A,Scheffer D,et al.Hair cell overexpression of Islet1 reduces age-related and noise-induced hearing loss.J Neurosci.2013;33(38):15086-94.
9 Meehan DT,Delimont D,Dufek B,et al.Endothelin-1 mediated induction of extracellular matrix genes in strial marginal cells underlies strial pathology in Alport mice.Hear Res.2016.
10陈超,吴玮,王刚,等.低频强噪声急性暴露对巴马香猪耳蜗毛细胞损伤的观察[J].听力学及言语疾病杂志,2018,26(02):172-176.Chen C,Wu W,Wang G,et al.Low frenquency noise of acute exposure to bama fragrant pig hair cell damage[J].Journal of Audiology and Speech Pathology,2018,26(02):172-176.
11王顶,柳柯,张悦,等.120dB白噪声对C57BL/6J小鼠听力损失的观察研究[J].中华耳科学杂志,2017,15(02):217-221.Wang D,Liu K,Zhang Y et al.Effects of high level white noise exposure on hearing function in C57BL/6J Mice.[J].Chinese Journal of Otology,2017,15(02):217-221.
12 Graydon CW,Cho S,Li GL,et al.Sharp Ca2+nanodomains beneath the ribbon promote highly synchronous multivesicular release at hair cell synapses.J Neurosci.2011;31(46):16637-50.
13 Yamane H,Nakai Y,Takayama M,et al.Appearance of free radicals in the guinea pig inner ear after noise-induced acoustic trauma.Eur Arch Otorhinolaryngol.1995;252(8):504-8.
14 Yamashita D,Jiang HY,Schacht J,et al.Delayed production of free radicals following noise exposure.Brain Res.2004;1019(1-2):201-9.
15 Harman D.The free radical theory of aging.Antioxid Redox Signal.2003;5(5):557-61.
16 Brown KD,Maqsood S,Huang JY,et al.Activation of SIRT3 by the NAD+precursor nicotinamide riboside protects from noise-induced hearing loss.Cell Metab.2014;20(6):1059-68.
17 Liberman MC,Dodds LW.Single-neuron labeling and chronic cochlear pathology.III.Stereocilia damage and alterations of threshold tuning curves.Hear Res.1984;16(1):55-74.
18 Di Clemente L,Coppola G,Magis D,et al.Nociceptive blink reflex and visual evoked potential habituations are correlated in migraine.Headache.2005;45(10):1388-93.
19 Yamane H,Nakai Y,Takayama M,et al.The emergence of free radicals after acoustic trauma and strial blood flow.Acta Otolaryngol Suppl.1995.
20 Fridberger A,Flock A,Ulfendahl M,et al.Acoustic overstimulation increases outer hair cell Ca2+concentrations and causes dynamic contractions of the hearing organ.Proc Natl Acad Sci U SA.1998;95(12):7127-32.
21 Puel JL,Pujol R,Tribillac F,et al.Excitatory amino acid antagonists protect cochlear auditory neurons from excitotoxicity.JComp Neurol.1994;341(2):241-56.
22 Hu BH,Cai Q,Manohar S,et al.Differential expression of apoptosis-related genes in the cochlea of noise-exposed rats.Neuroscience.2009;161(3):915-25.
23 Vicente-Torres MA,Schacht J.A BAD link to mitochondrial cell death in the cochlea of mice with noise-induced hearing loss.JNeurosci Res.2006;83(8):1564-72.
24 Wichmann C,Moser T.Relating structure and function of inner hair cell ribbon synapses.Cell Tissue Res.2015;361(1):95-114.
25 Schmitz F,K?nigstorfer A,Südhof TC.RIBEYE,a component of synaptic ribbons:a protein's journey through evolution provides insight into synaptic ribbon function.Neuron.2000;28(3):857-72.
26 Platzer J,Engel J,Schrott-Fischer A,et al.Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+channels.Cell.2000;102:89-97.
27 Safieddine S,El-Amraoui A,Petit C.The auditory hair cell ribbon synapse:from assembly to function.Annu Rev Neurosci.2012.
28 Neef J,Jung S,Wong AB,et al.Modes and regulation of endocytic membrane retrieval in mouse auditory hair cells.J Neurosci.2014;34(3):705-16.
2 Bielefeld EC.Protection from noise-induced hearing loss with Src inhibitors.Drug Discov Today.2015;20(6):760-5.
3 Kil J,Lobarinas E,Spankovich C,et al.Safety and efficacy of ebselen for the prevention of noise-induced hearing loss:a randomised,double-blind,placebo-controlled,phase 2 trial.Lancet.2017;390(10098):969-979.
4 Kujawa SG,Liberman MC.Adding insult to injury:cochlear nerve degeneration after"temporary"noise-induced hearing loss.J Neurosci.2009;29(45):14077-85.
5 Liberman LD,Suzuki J,Liberman MC.Dynamics of cochlear synaptopathy after acoustic overexposure.J Assoc Res Otolaryngol.2015;16(2):205-19.
6 Richter CP,Young H,Richter SV,et al.Fluvastatin protects cochleae from damage by high-level noise.Sci Rep.2018;8(1):3033.
7 Chen H,Xing Y,Xia L,et al.AAV-mediated NT-3 overexpression protects cochleae against noise-induced synaptopathy.Gene Ther.2018;25(4):251-259.
8 Huang M,Kantardzhieva A,Scheffer D,et al.Hair cell overexpression of Islet1 reduces age-related and noise-induced hearing loss.J Neurosci.2013;33(38):15086-94.
9 Meehan DT,Delimont D,Dufek B,et al.Endothelin-1 mediated induction of extracellular matrix genes in strial marginal cells underlies strial pathology in Alport mice.Hear Res.2016.
10陈超,吴玮,王刚,等.低频强噪声急性暴露对巴马香猪耳蜗毛细胞损伤的观察[J].听力学及言语疾病杂志,2018,26(02):172-176.Chen C,Wu W,Wang G,et al.Low frenquency noise of acute exposure to bama fragrant pig hair cell damage[J].Journal of Audiology and Speech Pathology,2018,26(02):172-176.
11王顶,柳柯,张悦,等.120dB白噪声对C57BL/6J小鼠听力损失的观察研究[J].中华耳科学杂志,2017,15(02):217-221.Wang D,Liu K,Zhang Y et al.Effects of high level white noise exposure on hearing function in C57BL/6J Mice.[J].Chinese Journal of Otology,2017,15(02):217-221.
12 Graydon CW,Cho S,Li GL,et al.Sharp Ca2+nanodomains beneath the ribbon promote highly synchronous multivesicular release at hair cell synapses.J Neurosci.2011;31(46):16637-50.
13 Yamane H,Nakai Y,Takayama M,et al.Appearance of free radicals in the guinea pig inner ear after noise-induced acoustic trauma.Eur Arch Otorhinolaryngol.1995;252(8):504-8.
14 Yamashita D,Jiang HY,Schacht J,et al.Delayed production of free radicals following noise exposure.Brain Res.2004;1019(1-2):201-9.
15 Harman D.The free radical theory of aging.Antioxid Redox Signal.2003;5(5):557-61.
16 Brown KD,Maqsood S,Huang JY,et al.Activation of SIRT3 by the NAD+precursor nicotinamide riboside protects from noise-induced hearing loss.Cell Metab.2014;20(6):1059-68.
17 Liberman MC,Dodds LW.Single-neuron labeling and chronic cochlear pathology.III.Stereocilia damage and alterations of threshold tuning curves.Hear Res.1984;16(1):55-74.
18 Di Clemente L,Coppola G,Magis D,et al.Nociceptive blink reflex and visual evoked potential habituations are correlated in migraine.Headache.2005;45(10):1388-93.
19 Yamane H,Nakai Y,Takayama M,et al.The emergence of free radicals after acoustic trauma and strial blood flow.Acta Otolaryngol Suppl.1995.
20 Fridberger A,Flock A,Ulfendahl M,et al.Acoustic overstimulation increases outer hair cell Ca2+concentrations and causes dynamic contractions of the hearing organ.Proc Natl Acad Sci U SA.1998;95(12):7127-32.
21 Puel JL,Pujol R,Tribillac F,et al.Excitatory amino acid antagonists protect cochlear auditory neurons from excitotoxicity.JComp Neurol.1994;341(2):241-56.
22 Hu BH,Cai Q,Manohar S,et al.Differential expression of apoptosis-related genes in the cochlea of noise-exposed rats.Neuroscience.2009;161(3):915-25.
23 Vicente-Torres MA,Schacht J.A BAD link to mitochondrial cell death in the cochlea of mice with noise-induced hearing loss.JNeurosci Res.2006;83(8):1564-72.
24 Wichmann C,Moser T.Relating structure and function of inner hair cell ribbon synapses.Cell Tissue Res.2015;361(1):95-114.
25 Schmitz F,K?nigstorfer A,Südhof TC.RIBEYE,a component of synaptic ribbons:a protein's journey through evolution provides insight into synaptic ribbon function.Neuron.2000;28(3):857-72.
26 Platzer J,Engel J,Schrott-Fischer A,et al.Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+channels.Cell.2000;102:89-97.
27 Safieddine S,El-Amraoui A,Petit C.The auditory hair cell ribbon synapse:from assembly to function.Annu Rev Neurosci.2012.
28 Neef J,Jung S,Wong AB,et al.Modes and regulation of endocytic membrane retrieval in mouse auditory hair cells.J Neurosci.2014;34(3):705-16.