Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage
February 10th, 2023
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.
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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Overview
This study presents a detailed protocol for inducing noise-induced hearing loss (NIHL) in a mouse model. Utilizing a novel noise exposure device and auditory assessments, it explores the mechanistic insights into hearing loss and the subsequent effects on outer hair cells.
Key Study Components
Area of Science
- Neuroscience
- Auditory studies
- Animal models
Background
- Existing protocols for NIHL have lacked detail.
- Understanding NIHL can inform treatment strategies.
- Auditory brainstem response (ABR) effectively measures hearing threshold changes.
Purpose of Study
- To develop a straightforward protocol for NIHL in mice.
- To assess the effects of noise exposure on hearing thresholds and outer hair cell integrity.
- To provide a detailed method that can be adapted for related research.
Methods Used
- The protocol involves using a rat trap cage and calibrated noise exposure.
- Male C57BL/6J mice serve as the model organism for assessing NIHL.
- Key steps include noise exposure setup, ABR measurement after exposure, and cochlea harvesting for histological analysis.
- Mice are exposed to noise at multiple frequencies for set durations, followed by auditory testing.
Main Results
- Significant increases in hearing thresholds were observed, particularly at high frequencies.
- Outer hair cells showed extensive damage, while inner hair cells remained intact.
- Partial recovery of hearing thresholds was noted a week post-exposure, highlighting the temporal dynamics of auditory recovery.
Conclusions
- This protocol enables detailed examination of NIHL mechanisms in mice.
- It provides a framework for future studies aimed at optimizing therapeutic strategies for hearing loss.
- Insights obtained could be significant for understanding auditory system plasticity and damage response.
Frequently Asked Questions
Executive Industry Relevance
Robust animal models of noise-induced hearing loss (NIHL) are essential for de-risking early-stage auditory drug discovery and validating mechanistic hypotheses around cochlear injury. This protocol enables quantitative assessment of hearing function and outer hair cell integrity, supporting translational continuity from preclinical studies to therapeutic strategy optimization. The detailed workflow enhances predictive confidence for portfolio decisions targeting auditory system disorders.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of NIHL mechanisms through controlled noise exposure and quantitative auditory assessment.
- Supports functional target validation by correlating auditory brainstem response (ABR) thresholds with cellular damage.
- Facilitates biological de-risking by distinguishing outer hair cell vulnerability across cochlear regions.
- Provides a reproducible model for evaluating candidate interventions in hearing loss.
Screening & Assay Development
- Establishes a validated in vivo system for downstream efficacy screening of otoprotective compounds.
- Delivers standardized ABR and immunofluorescence readouts for quantitative comparison across studies.
- Enables reproducible assessment of hearing thresholds and cellular outcomes post-intervention.
- Supports assay scalability and platform reuse for auditory research pipelines.
Translational & Preclinical Research
- Aligns with disease-relevant endpoints by modeling clinically observed patterns of high-frequency hearing loss.
- Provides continuity from mechanistic discovery to preclinical validation of therapeutic hypotheses.
- Enables risk-adjusted advancement of candidates based on quantitative functional and morphological data.
- Supports biomarker alignment through ABR and histological outputs.
Pipeline & Workflow Integration
This protocol integrates into the discovery-to-preclinical continuum by enabling hypothesis-driven NIHL modeling, quantitative functional assessment, and morphological validation in a single workflow.
- Discovery Biology: Supports mechanistic hypothesis testing and pathway clarification for auditory injury.
- Screening: Provides standardized, quantitative ABR and histological outputs for compound evaluation.
- Analytics: Enables statistical comparison of hearing thresholds and cellular damage across experimental groups.
- Translational Research: Bridges discovery and preclinical validation with disease-relevant functional and morphological endpoints.
- Enterprise Reuse: Offers a reusable, detailed protocol adaptable for diverse auditory research applications.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in auditory target validation.
- Operational Value: Delivers standardized, reproducible, and scalable in vivo auditory assessments.
- Strategic Value: Informs go/no-go decisions and enhances capital efficiency by providing robust preclinical data.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of hearing loss therapeutic candidates.
Implementation Considerations
- Requires expertise in auditory physiology, animal handling, and ABR measurement.
- Needs access to calibrated noise generation, ABR instrumentation, and histological analysis infrastructure.
- Demands rigorous cross-team standardization of exposure and measurement protocols.
- Adaptable to different mouse strains or noise parameters for broader model relevance.
- Limitations include the need for precise calibration and monitoring to ensure reproducibility.
Biopharma FAQs
Chapters in this video
0:04
Introduction
1:04
Induction of Noise‐Induced Hearing Loss (NIHL) in Mice
3:07
Auditory Brainstem Response (ABR)‐Based Assessment of Hearing Threshold
4:19
Microscope Examination
5:18
Results: Assessing Hearing Function and Outer Hair Cell Damage in NIHL Mice
6:32
Conclusion
