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 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.
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.
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
2. Auditory brainstem response (ABR)-based assessment of hearing threshold
3. Microscopic examination
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 ± 2.77 dB SPL; 24 kHz: 91.43 ± 0.92 dB SPL; 32 kHz: 98.57 ± 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 day), but the hearing thresholds were still elevated by more than 30 dB at all frequencies (12 kHz: 72.86 ± 2.86 dB SPL; 24 kHz: 84.29 ± 2.77 dB SPL; 32 kHz: 87.14 ± 4.21 dB SPL) compared to the control groups (12 kHz: 41 ± 0 dB SPL; 24 kHz: 51 ± 0 dB SPL; 32 kHz: 51 ± 0 dB SPL). In this study, the hearing was more damaged at high frequencies (Figure 5). A two-way ANOVA test was used for analysis, and Bonferroni correction was used for post-tests. A significant difference (p < 0.001) was observed between the control and experimental groups on both the 6th and 13th days. Comparison between the hearing thresholds measured on the 6th and 13th days showed a significant difference (p < 0.05) at the frequencies of 12 kHz and 32 kHz.
Outer hair cell loss
A loss of OHCs was consistently observed in the microscopic images acquired from the NIHL mice, compared to those from the control mice. By contrast, the inner hair cells were observed to be intact in all the images. In addition, the OHCs in the basal and middle turns of the organ of Corti were damaged more severely, while the OHCs in the apical turn were almost intact (Figure 6).
Figure 1: Noise exposure setup. A microphone was placed in front of the speaker at a distance of 8.5 cm to calibrate the noise level. The noise level was adjusted to 125 dB SPL-A, which is similar to the level of a nearby siren. Please click here to view a larger version of this figure.
Figure 2: The rat trap cage adapted to this study. Three male C57BL/6J mice were randomly assigned to each quarter during the noise exposure. The microphone was stuck to the top of the cage to monitor noise levels during noise exposure. The sound pressure level was measured several times at multiple positions. These positions are marked in the figure. Please click here to view a larger version of this figure.
Figure 3: Experimental timeline for the test and control groups. The mice were exposed to the noise at the frequencies of 1 and 6 kHz continuously for 6 h per day, for 5 days. After 5 consecutive days of noise exposure, the hearing thresholds of the experimental mice were measured with ABR on the 6th day. The ABR measurement was performed in the experimental mice again and in the control mice on the 13th day, followed by the sacrifice of all the involved mice to harvest their cochleae. Please click here to view a larger version of this figure.
Figure 4: ABR measurement of hearing. Representative ABR results at 12 kHz, collected on the 13th day (1 week after the noise exposure). The Wave V at each intensity is labeled if discernable. Please click here to view a larger version of this figure.
Figure 5: Hearing thresholds measured on the 6th and 13th days. The hearing threshold at frequencies of (A) 12 kHz, (B) 24 kHz, and (C) 32 kHz. A two-way ANOVA test was used for analysis, followed by Bonferroni correction. *p < 0.05, ***p < 0.001. Please click here to view a larger version of this figure.
Figure 6: Immunofluorescence imaging results obtained from OC. (A) Image obtained from the apical turn of the cochlea. (B) Image obtained from the middle turn of the cochlea. (C) Image obtained from the base turn of the cochlea. Blue: cell nuclei stained with DAPI; Green: hair cells stained with Myo7A; Red: cytoskeleton stained with phalloidin. Arrows indicate the loss of OHCs. Scale bar= 20 µm. Please click here to view a larger version of this figure.
Supplementary file 1: Voltage calculation from calibration. For each specific frequency, the value of the selected voltage (horizontal axis) will be input into the calibration curve for obtaining the corresponding sound level (vertical axis). Please click here to download this file.
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 same setup and animals, the hearing loss generated by 2 day noise exposure completely recovered in 2 weeks, indicating that permanent NIHL was not actually generated. On the contrary, in this study, the hearing loss generated by 5 day noise exposure is suggested to include a permanent component, as the hearing threshold was still significantly higher than the control level on the 13th day (1 week after the noise exposure).
One of the limitations of the current protocol is that we used the hearing threshold detected at 12 kHz to represent the low-frequency hearing threshold of the mice, which corresponds to the apical turn of the cochlea. Strictly speaking, the apical turn of the cochlea is more sensitive to sound at 4 kHz to 8 kHz12. However, this limitation hardly reduces the value of this study and protocol, as the presented protocol provides every detail of the operation steps and employs devices involved in the model creation. Most of the presented parameters, such as noise-exposure durations, noise frequencies, stimulus frequencies for ABR, and when to perform the ABR tests and animal sacrifice, can be altered and further optimized for different purposes in future studies.
The authors have nothing to disclose.
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).
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 |