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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 defic....

Protokół

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

1. Prepare the cage for the experimental mice
   1. To do so, use a rat trap cage with dimensions of 14 cm × 17 cm × 24 cm. Cut four pieces of corrugated plastic boards into appropriate sizes, making them fit into the cage (13 cm × 23 cm and 13 cm × 16 cm).
   <....

Wyniki

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 6^th day). Partial hearing recovery occurred 1 week after the noise exposure (i.e., the 13^th d.......

Dyskusje

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 6^th 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 .......

Materiały

Name	Company	Catalog Number	Comments
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