Name: modified experimental conditions for noise-induced hearing loss in mice and assessment of hearing function and outer hair cell damage
Uploaded: 2023-02-10T12:30:00
Description: Watch this Scientific Journal Video about Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage at JoVE.com

We thank the grants from the Ministry of Science and Technology (MOST) of the Taiwan Government (MOST 110-2314-B-715-005, MOST 111-2314-B-715-009-MY3), and intramural research grants from Mackay Medical College (MMC-RD-110-1B-P030, MMC-RD-111-CF-G002-03).

Animal experiments in this study were approved by the Animal Care Committee of Mackay Medical College. Eight-week-old Male C57BL/6J mice were purchased from the National Laboratory Animal Center (New Taipei City, Taiwan). All mice were bred and housed in accordance with the standard animal protocol.
1. Induction of NIHL in mice
<ol>
	<li>Prepare the cage for the experimental mice
	<ol>
		<li>To do so, use a rat trap cage with dimensions of 14 cm &#215; 17 cm &#215; 24 cm. Cu...

<ol>
	<li>World Report on Hearing. World Health Organization Available from: <a target="_blank" href="https://www.who.int/publications/i/item/9789240020481">https://www.who.int/publications/i/item/9789240020481</a> (2021)</li><li>Fernandez, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+cochlear+synaptopathy+with+and+without+sensory+cell+loss.">Noise-induced cochlear synaptopathy with and without sensory cell loss.</a> Neuroscience. 427, 43-57 (2020).</li><li>Kujawa, S. G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adding+insult+to+injury:+cochlear+nerve+degeneration+after+&amp;#34;temporary&amp;#34;+noise-induced+hearing+loss.">Adding insult to injury: cochlear nerve degeneration after &amp;#34;temporary&amp;#34; noise-induced hearing loss.</a> The Journal of Neuroscience. 29 (45), 14077-14085 (2009).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+X-linked+inhibitor+of+apoptosis+protein+protects+against+noise-induced+hearing+loss+in+mice.">Overexpression of X-linked inhibitor of apoptosis protein protects against noise-induced hearing loss in mice.</a> Gene Therapy. 18 (6), 560-568 (2011).</li><li>Takeda, S., Mannstr&#246;m, P., Dash-Wagh, S., Yoshida, T., Ulfendahl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+aging+and+noise+exposure+on+auditory+brainstem+responses+and+number+of+presynaptic+ribbons+in+inner+hair+cells+of+C57BL/6J+mice.">Effects of aging and noise exposure on auditory brainstem responses and number of presynaptic ribbons in inner hair cells of C57BL/6J mice.</a> Neurophysiology. 49 (5), 316-326 (2017).</li><li>Rouse, S. L., Matthews, I. R., Li, J., Sherr, E. H., Chan, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+stress+response+inhibition+provides+sex-dependent+protection+against+noise-induced+cochlear+synaptopathy.">Integrated stress response inhibition provides sex-dependent protection against noise-induced cochlear synaptopathy.</a> Scientific Reports. 10 (1), 18063 (2020).</li><li>Amanipour, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+hearing+loss+in+mice:+Effects+of+high+and+low+levels+of+noise+trauma+in+CBA+mice.">Noise-induced hearing loss in mice: Effects of high and low levels of noise trauma in CBA mice.</a> Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2018, 1210-1213 (2018).</li><li>Edderkaoui, B., Sargsyan, L., Hetrick, A., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+duffy+antigen+receptor+for+chemokines+ameliorated+cochlear+damage+from+noise+exposure.">Deficiency of duffy antigen receptor for chemokines ameliorated cochlear damage from noise exposure.</a> Frontiers in Molecular Neuroscience. 11, 173 (2018).</li><li>Hultcrantz, M., Simonoska, R., Stenberg, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+and+hearing:+a+summary+of+recent+investigations.">Estrogen and hearing: a summary of recent investigations.</a> Acta Oto-laryngologica. 126 (1), 10-14 (2006).</li><li>Tsai, S. C. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Hearing Research. 413, 108254 (2022).</li><li>Choi, M. Y., Yeo, S. W., Park, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+restoration+in+a+deaf+animal+model+with+intravenous+transplantation+of+mesenchymal+stem+cells+derived+from+human+umbilical+cord+blood.">Hearing restoration in a deaf animal model with intravenous transplantation of mesenchymal stem cells derived from human umbilical cord blood.</a> Biochemical and Biophysical Research Communications. 427 (3), 629-636 (2012).</li><li>Yu, S. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+functional+evaluation+of+ribbon+synapses+at+specific+frequency+regions+of+the+mouse+cochlea.">Morphological and functional evaluation of ribbon synapses at specific frequency regions of the mouse cochlea.</a> Journal of Visualized Experiments. (147), e59189 (2019).</li></ol>

NIHL can be divided into two types: temporary NIHL, which shows a temporal shift of the hearing threshold, and permanent NIHL, which is featured by a permanent hearing-threshold shift. The hearing loss that we observed on the 6th day (1 day after the noise exposure) is believed to be a combination of these two types. In this case, the hearing threshold would show a gradual recovery over time owing to the temporal component of hearing loss. In our preliminary experimental studies, the results acquired with the ...

About 1.3 billion people worldwide suffer from hearing loss due to noise exposure1. In this study, we aimed to establish a clear step-by-step process for inducing and confirming noise-induced hearing loss (NIHL). NIHL results from a degeneration/destruction of the hair cells (HCs) and spiral ganglion neurons (SGNs), damage in the HC stereocilia, and/or loss of synapses between the cochlear inner HCs and SGNs. Such abnormalities may also cause tinnitus and impaired speech perception (especially in a complex acoustic environment) besides NIHL. Social, psychological, and cognitive functions may be sequentially affected by these physiological deficiencies2,3,4,5,6.
In NIHL-related preclinical studies based on mice, the most popular mouse strains are CBA/CaJ2,3,6,7 and C57BL/64,5,8. The male3,4,7 mice, furthermore, are more commonly used than the female ones, as estrogen has a protective effect on hearing. Therefore, we only used male mice in this study9. After referring to the literature, we chose 1 kHz and 6 kHz as the frequencies of the applied noise. The intensity of the applied noise was 115 dB SPL-A (surrounding the cage) to 125 dB SPL-A (in the center of the cage). After exposing the experimental mice to the noise continuously for 6 h per day, for 5 consecutive days, an optimum increase in hearing threshold indicated an optimum extent of NIHL was generated in the experimental mice. The operations for handling the animals, building the experimental setup, and inducing noise are all clearly described step-by-step in the provided protocol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;1/4&#34; CCP Free-field Standard Microphone Set</td>
			<td>GRAS</td>
			<td>428158</td>
			<td>For noise exposure</td>
		</tr>
		<tr>
			<td>Amplifier Input Module, AMI100D</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Bio-amplifier, BIO100C</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A9647</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Cellsens software</td>
			<td>Olympus life science</td>
			<td></td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Corrugated plastic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI fluoromount</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>SIGMA</td>
			<td>E5134</td>
			<td>Decalcification</td>
		</tr>
		<tr>
			<td>Evoked Response Amplifier, ERS100C</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>APLHA</td>
			<td>F030410</td>
			<td>Fixation of cochlear</td>
		</tr>
		<tr>
			<td>High Performance Data Acquisition System, MP160</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Modular Extension Cable, MEC110C</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Myo7A primary antibody</td>
			<td>Proteus</td>
			<td>25-6790</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Myo7A secondary antibody</td>
			<td>Jackson immunoresearch</td>
			<td>711-545-152</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Needle Electrode, Unipolar 12 mmTp, EL452</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>phalloidin antibody</td>
			<td>Alexa Fluor</td>
			<td>A12381</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>phosphate-buffered saline</td>
			<td>SIGMA</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat trap cage</td>
			<td></td>
			<td></td>
			<td>14 cm x 17 cm x 24cm</td>
		</tr>
		<tr>
			<td>ROMPUN- xylazine injection, solution&#160;</td>
			<td>Bayer HealthCare, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sound amplifier, MT-1000</td>
			<td>unika</td>
			<td></td>
			<td>For noise exposure</td>
		</tr>
		<tr>
			<td>Sound generator/analyzer/miscellaneous, FW-02</td>
			<td>CLIO</td>
			<td>620300719</td>
			<td>For noise exposure</td>
		</tr>
		<tr>
			<td>Soundproof chamber</td>
			<td>IEA Electro-Acoustic Technology</td>
			<td></td>
			<td>For noise exposure and ABR</td>
		</tr>
		<tr>
			<td>Speaker&#160;</td>
			<td>IEA Electro-Acoustic Technology</td>
			<td></td>
			<td>For noise exposure</td>
		</tr>
		<tr>
			<td>Stimulator Module, STM100C</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>SIGMA</td>
			<td>T8787</td>
			<td>Immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Tubephone Set, OUT101</td>
			<td>BIOPAC</td>
			<td></td>
			<td>For auditory brainstem response</td>
		</tr>
		<tr>
			<td>Upright Microscope, BX53</td>
			<td>Olympus</td>
			<td></td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

modified experimental conditions for noise-induced hearing loss in mice and assessment of hearing function and outer hair cell damage

An animal model of noise-induced hearing loss (NIHL) is useful for pathologists, therapists, pharmacologists, and hearing researchers to thoroughly understand the mechanism of NIHL, and subsequently optimize the corresponding treatment strategies. This study aims to create an improved protocol for developing a mouse model of NIHL. Male C57BL/6J mice were used in this study. Unanesthetized mice were exposed to loud noises (1 and 6 kHz, presented simultaneously at 115-125 dB SPL-A) continuously for 6 h per day for 5 consecutive days. Auditory function was assessed 1 day and 1 week after noise exposure, using auditory brainstem response (ABR). After the ABR measurement, the mice were sacrificed, and their organs of Corti were collected for immunofluorescence staining. From the auditory brainstem response (ABR) measurements, significant hearing loss was observed 1 day after noise exposure. After 1 week, the hearing thresholds of the experimental mice decreased to ~80 dB SPL, which was still a significantly higher level than the control mice (~40 dB SPL). From the results of immunofluorescence imaging, outer hair cells (OHCs) were shown to be damaged. In summary, we created a model of NIHL using male C57BL/6J mice. A new and simple device for generating and delivering pure-tone noise was developed and then employed. Quantitative measurements of hearing thresholds and morphological confirmation of OHC damage both demonstrated that the applied noise successfully induced an expected hearing loss.

A shift in ABR hearing threshold 
The hearing threshold of the mice was measured using tone-burst ABR either 1 day or 1 week after the noise exposure. A significant increase in the hearing threshold at all three tested frequencies was observed (12 kHz: 84.29 &#177; 2.77 dB SPL; 24 kHz: 91.43 &#177; 0.92 dB SPL; 32 kHz: 98.57 &#177; 1.43 dB SPL) 1 day after the noise exposure (i.e., the 6th day). Partial hearing recovery occurred 1 week after the noise exposure (i.e., the 13th d...

Watch this Scientific Journal Video about Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage at JoVE.com

Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage

Here, we present a protocol for a mouse model of noise-induced hearing loss (NIHL). To induce NIHL, we developed a new and simple device using corrugated plastic, a rat trap cage, and a speaker. Auditory brainstem response and immunofluorescence imaging were employed to assess the hearing function and outer hair cell damage, respectively.

An animal model of noise-induced hearing loss (NIHL) is useful for pathologists, therapists, pharmacologists, and hearing researchers to thoroughly ...

Although there have been studies on noise-induced hearing loss, none have recorded the method in such detail, making this an important procedure. This method effectively caused noise-induced hearing loss in mice, and observes the actual changes in the hearing threshold by ABR. An animal model of noise-induced hearing loss is useful for scientists to thoroughly understand the mechanism of noise-induced hearing loss, and subsequently optimize the corresponding treatment strategies.Demonstrating the procedure will be Dian-Shiue Lee, a very talented student from my laboratory. To prepare the cage for the experimental mice, use a rat trap cage and cut four pieces of corrugated plastic boards into appropriate sizes, making them fit into the cage. To prevent the mice from getting their feet cut by the mesh grid, place two of the pieces at the bottom and on the back side, respectively.Place the other two pieces perpendicularly interlocked with each other, to divide the space within the cage into quarters Open the CLIO application software. Move the cursor to the speaker icon, and click on TwoSin. Enter and change the value of Frequency 1 to 1, 000 hertz, Frequency 2 to 6, 000 hertz, and click OK to start playing the sound.Under the Leq tab, change dBV to dBSPL. After setting the time on the software interface, click the green triangle button to play the sound. Place a microphone in front of the speaker at a distance of 8.5 centimeters to calibrate the noise level.Adjust the noise level to 125 decibels SPL-A, and continuously monitor for at least three minutes to ensure that the noise level is sufficiently stabilized. Use a generator, an analyzer, and an amplifier to create and control the noise. Place four male C57 black 6J mice into the cage, one for each quarter, for noise exposure.Randomly assign the mice to each quarter during the noise exposure. Place a microphone on the top of the cage to monitor the noise level during the noise exposure. Place the cage in front of the speaker inside a soundproof box, ensuring the speaker is positioned 8.5 centimeters away from the cage.Expose the mice to the noise at frequencies of one kilohertz and six kilohertz continuously for six hours per day, for five consecutive days. Measure the hearing thresholds of the mice on the 6th and 13th days by measuring the ABR. Use a commercial ABR testing system, specifically designed for small animals for ABR measurements, and maintain the animal under general anesthesia.Place 12-millimeter subdermal needle electrodes at the vertex behind the pinna of the left ear, and back near the tail, to measure the hearing threshold. After placing the speaker one centimeter away from the animal's left ear, present acoustic stimuli using the speaker. Choose Sine Wave for the stimuli, and 10 k for the window scale.Turn the frequency knob to obtain the desired frequency of the acoustic stimuli. Adjust the stimulus intensity by turning the AMLP knob on the function generator. Obtain the desired stimulus intensity by turning the AMLP knob to a suitable voltage calculated from calibration.Collect the ABR measurements under a series of stimulus intensities from 10 to 100 decibels SPL, with a 10-decibel step size. Mark the minimum stimulus intensity level that could give rise to a discernible Wave V as the ABR threshold. Sacrifice the mice after ABR measurements, and harvest the cochleae from the euthanized mice.Immediately after harvesting the tissues, immerse them in 10%formaldehyde for fixation, and let them rest for at least eight hours at four degrees Celsius. Use EDTA for decalcification, as described in the text. Place the cochleae in a Petri dish filled with PBS.For tissue staining, cut the spiral-shaped organ of Corti, or OC, into three sections:the basal turn, middle turn, and apical turn. Under a dissecting microscope at a magnification of 8x to 35x, remove the bony structures that include scala vestibuli, scala tympani, and modiolus of the decalcified cochleae, and obtain the soft tissues, including the OC.A significant increase in the hearing threshold was observed at 12 kilohertz, 24 kilohertz, and 32 kilohertz one day after the noise exposure. Partial hearing recovery occurred one week after the noise exposure, but the hearing thresholds were still elevated by more than 30 decibels at all frequencies compared to the control groups.In this study, the hearing was more damaged at high frequencies. A significant difference was observed in the hearing threshold between the control and experimental groups on both the 6th and 13th days, at 12 kilohertz and 32 kilohertz. A loss of outer hair cells was consistently observed in the microscopic images acquired from the NIHL mice, compared to those from the control mice.While the inner hair cells remained intact in all of the images, the outer hair cells in the basal and middle turns of the OC were severely damaged. In contrast, the outer hair cells in the apical turn were almost intact. The most important step in this protocol includes noise exposure, ABI measurement, harvesting the cochlea.This procedure can be applied to investigate the effect of noise exposure at different frequencies on hearing loss by using various types of sound.

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