Research Application of Laser-Induced Shock Wave for Studying Blast-Induced Cochlear Injury

Published: March 01, 2024

doi:

Takaomi Kurioka, Kunio Mizutari, Katsuki Niwa, Eiko Kimura, Satoko Kawauchi, Yasushi Kobayashi, Shunichi Sato

¹Department of Otolaryngology, Head and Neck Surgery,National Defense Medical College, ²Division of Bioinformation and Therapeutic Systems,National Defense Medical College, ³Department of Biochemistry,National Defense Medical College

Summary

Here, we describe experimental protocols for creating an animal model of blast-induced cochlear injury using laser-induced shock wave (LISW). Exposure of the temporal bone to LISW allows the reproduction of blast-induced cochlear pathophysiology. This animal model could be a platform for elucidating cochlear pathology and exploring potential treatments for blast injuries.

Abstract

The ear is the organ most susceptible to explosion overpressure, and cochlear injuries frequently occur after blast exposure. Blast exposure can lead to sensorineural hearing loss (SNHL), which is an irreversible hearing loss that negatively affects the quality of life. Detailed blast-induced cochlear pathologies, such as the loss of hair cells, spiral ganglion neurons, cochlear synapses, and disruption of stereocilia, have been previously documented. However, determining cochlear sensorineural deterioration after a blast injury is challenging because animals exposed to blast overpressure usually experience tympanic membrane perforation (TMP), which causes concurrent conductive hearing loss. To evaluate pure sensorineural cochlear dysfunction, we developed an experimental animal model of blast-induced cochlear injury using a laser-induced shock wave. This method avoids TMP and concomitant systemic injuries and reproduces the functional decline in the SNHL component in an energy-dependent manner after LISW exposure. This animal model could be a platform for elucidating the pathological mechanisms and exploring potential treatments for blast-induced cochlear dysfunction.

Introduction

Hearing loss and tinnitus are among the most prevalent disabilities, reported in up to 62% of veterans¹. Several blast-induced auditory complications, including sensorineural hearing loss (SNHL) and tympanic membrane perforation (TMP), have been reported in individuals exposed to blast overpressure². Moreover, research on individuals exposed to blasts suggests that blast exposure frequently results in defects in auditory temporal resolution, even when the hearing thresholds are within normal range, which is known as "hidden hearing loss (HHL)"³. It is well established that there is a substantial loss of cochlear synapses between inner hair cells (IHCs) and auditory neurons (ANs) in blast-related cochlear pathology⁴. Synaptic degeneration results in impaired auditory processing and is a major contributing factor in the development of HHL⁵. Thus, auditory organs are fragile components containing complex and highly organized structures. However, the precise mechanism by which blast waves affect the inner ear at the cellular level remains unclear. This is because of the challenges in replicating the precise clinical and mechanical intricacies of blast injuries in laboratory settings and the complexity of blast-induced cochlear pathologies.

The primary component of a blast injury is the shock wave (SW), characterized by a rapid and high increase in peak pressure⁶. The complexity of blast injuries has been extensively investigated in numerous retrospective studies⁷^,⁸^,⁹. There are various devices for blast generation, such as compressed gas¹⁰, shock tubes¹¹, and small-magnitude explosives¹², at different levels of pressure. The pressure waveform of the SW generated by recently developed devices closely resembled that of an actual explosion. An important concept in establishing an animal model of blast-induced sensorineural hearing loss is to minimize concomitant injuries, other than auditory damage, to reduce animal death. Thus, blast injury studies have been developed in which shock tubes have been miniaturized and the output can be precisely controlled so that exposed animals rarely die. However, although these animal models usually develop complications, such as TMP, evaluation of cochlear function is difficult because of concurrent conductive hearing loss². We previously performed an ear-protected animal study on blast injury using earplugs and found no incidence of TMP¹³. The earplugs could partially attenuate severe cochlear damage but not central auditory neurodegeneration or tinnitus development. Thus, earplugs protect the cochleae as well as the tympanic membrane. However, an animal model of blast-induced pure cochlear damage without TMP is required to study the cochlear pathophysiology caused by blast injuries.

We previously developed a topical blast injury model of the inner ear in rats and mice using a laser-induced shock wave (LISW)¹⁴^,¹⁵. This method can be safely and easily performed at a standard laboratory level and has been used to generate models of lung and head blast injuries¹⁶^,¹⁷. The energy of the LISW can be adjusted by changing the laser type and power, allowing control over the degree of cochlear damage. The LISW-induced cochlear injury model is valuable for studying the mechanisms of SNHL caused by blast injuries and investigating potential treatments. In this study, we describe detailed experimental protocols for creating a mouse model of blast-induced cochlear damage using LISW and demonstrate cochlear degeneration, including the loss of hair cells (HCs), cochlear synapses, and spiral ganglion neurons (SGNs), in an energy-dependent manner in mice following LISW exposure.

Protocol

All experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Defense Medical College (approval #18050) and performed in accordance with the guidelines of the National Institutes of Health and the Ministry of Education, Culture, Sports, Science, and Technology of Japan. All efforts were made to minimize the number of animals and their suffering. 1. Animals Use 8-week-old male CBA/J mice to follow this protocol. Before …

Representative Results

LISW waveform The reproducibility of the LISW pressure waveform was measured 5x at 2.0 J/cm2 as follows. The waveforms were generally similar and stable and showed a sharp increase with time width, peak pressure, and impulse of 0.43±0.4 µs, 92.1 ± 6.8 MPa, and 14.1 ± 1.9 Pa∙s (median ± SD), which corresponds to SW characteristics (Figure 1B). LISWs are characterized by a fast rise time, high peak pressure, short duration, and p…

Discussion

This study aimed to validate a mouse model of blast-induced cochlear damage using LISW. Our findings demonstrated that following LISW application through the temporal bone, the exposed mice ear exhibited a consistent pathological and physiological decline in the cochlea, which was accompanied by an increase in LISW overpressure. These results indicate that this mouse model is appropriate for replicating various cochlear pathologies by adjusting the LISW output. Specifically, this LISW-induced cochlear dysfunction mouse m…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

This work was supported by two grants from JSPS KAKENHI (Grant Numbers 21K09573 (K.M.) and 23K15901 (T.K.)).

Materials

532 nm Q-switched Nd:YAG laser	Quantel	Brilliant b
ABR peak analysis software	Mass Eye and Ear	N/A	EPL Cochlear Function Test Suite
Acrylic resin welding adhesive	Acrysunday Co., Ltd	N/A
confocal fluorescence microscopy	Leica	TCS SP8
cryosectioning compound	Sakura	Tissue-Tek O.C.T
CtBP2 antibody	BD Transduction	#612044
Dielectric multilayer mirrors	SIGMAKOKI CO.,LTD	TFMHP-50C08-532	M1-M3
Digital oscilloscope	Tektronix	DPO4104B
Earphone	CUI	CDMG15008-03A
Hydrophone	RP acoustics e.K.	FOPH2000
Image J software plug-in	NIH	measurement line	https://myfiles.meei.harvard.edu/xythoswfs/webui/_xy-e693768_1-t_wC4oKeBD
Light microscope	Keyence Corporation	BZ-X700
Myosin 7A antibody	Proteus Biosciences	#25–6790
Neurofilament antibody	Sigma	#AB5539
Plano-convex lens	SIGMAKOKI CO.,LTD	SLSQ-30-200PM
Prism software	GraphPad	N/A	ver.8.2.1
Scanning electron microscope	JEOL Ltd	JSM-6340F
Small digital endoscope	AVS Co. Ltd	AE-C1
Ultrasonic jelly	Hitachi Aloka Medical	N/A
Variable attenuator	Showa Optronics Co.	N/A	Currenly avaiable successor: KYOCERA SOC Corporation, RWH-532HP II
Water-soluble encapsulant	Dako	#S1964

Referencias

Arun, P., et al. Blast exposure causes long-term degeneration of neuronal cytoskeletal elements in the cochlear nucleus: A potential mechanism for chronic auditory dysfunctions. Front Neurol. 12, 652190 (2021).
Kurioka, T., Mizutari, K., Satoh, Y., Shiotani, A. Correlation of blast-induced tympanic membrane perforation with peripheral cochlear synaptopathy. J Neurotrauma. 39 (13-14), 999-1009 (2022).
Liberman, M. C., Epstein, M. J., Cleveland, S. S., Wang, H., Maison, S. F. Toward a differential diagnosis of hidden hearing loss in humans. PLoS One. 11 (9), e0162726 (2016).
Koizumi, Y., et al. Y-27632, a rock inhibitor, improved laser-induced shock wave (lisw)-induced cochlear synaptopathy in mice. Mol Brain. 14 (1), 105 (2021).
Parthasarathy, A., Kujawa, S. G. Synaptopathy in the aging cochlea: Characterizing early-neural deficits in auditory temporal envelope processing. J Neurosci. 38 (32), 7108-7119 (2018).
Kurioka, T., et al. Characteristics of laser-induced shock wave injury to the inner ear of rats. J Biomed Opt. 19 (12), 125001 (2014).
Paik, C. B., Pei, M., Oghalai, J. S. Review of blast noise and the auditory system. Hear Res. 425, 108459 (2022).
Bryden, D. W., Tilghman, J. I., Hinds, S. R. Blast-related traumatic brain injury: Current concepts and research considerations. J Exp Neurosci. 13, 1179069519872213 (2019).
Ma, X., Aravind, A., Pfister, B. J., Chandra, N., Haorah, J. Animal models of traumatic brain injury and assessment of injury severity. Mol Neurobiol. 56 (8), 5332-5345 (2019).
Shao, N., et al. Central and peripheral auditory abnormalities in chinchilla animal model of blast-injury. Hear Res. 407, 108273 (2021).
Ou, Y., et al. Traumatic brain injury induced by exposure to blast overpressure via ear canal. Neural Regen Res. 17 (1), 115-121 (2022).
Cho, S. I., et al. Mechanisms of hearing loss after blast injury to the ear. PLoS One. 8 (7), e67618 (2013).
Kurioka, T., Mizutari, K., Satoh, Y., Kobayashi, Y., Shiotani, A. Blast-induced central auditory neurodegeneration affects tinnitus development regardless of peripheral cochlear damage. J Neurotrauma. , (2023).
Niwa, K., et al. Pathophysiology of the inner ear after blast injury caused by laser-induced shock wave. Sci Rep. 6, 31754 (2016).
Kimura, E., et al. Effect of shock wave power spectrum on the inner ear pathophysiology in blast-induced hearing loss. Sci Rep. 11 (1), 14704 (2021).
Satoh, Y., et al. Pulmonary blast injury in mice: A novel model for studying blast injury in the laboratory using laser-induced stress waves. Lasers Surg Med. 42 (4), 313-318 (2010).
Kawauchi, S., et al. Effects of isolated and combined exposure of the brain and lungs to a laser-induced shock wave(s) on physiological and neurological responses in rats. J Neurotrauma. 39 (21-22), 1533-1546 (2022).
Cassinotti, L. R., et al. Cochlear neurotrophin-3 overexpression at mid-life prevents age-related inner hair cell synaptopathy and slows age-related hearing loss. Aging Cell. 21 (10), e13708 (2022).
Kujawa, S. G., Liberman, M. C. Adding insult to injury: Cochlear nerve degeneration after "temporary" noise-induced hearing loss. J Neurosci. 29 (45), 14077-14085 (2009).
Hickman, T. T., Smalt, C., Bobrow, J., Quatieri, T., Liberman, M. C. Blast-induced cochlear synaptopathy in chinchillas. Sci Rep. 8 (1), 10740 (2018).
Jiang, S., Sanders, S., Gan, R. Z. Hearing protection and damage mitigation in chinchillas exposed to repeated low-intensity blasts. Hear Res. 429, 108703 (2023).
Nakagawa, A., et al. Mechanisms of primary blast-induced traumatic brain injury: Insights from shock-wave research. J Neurotrauma. 28 (6), 1101-1119 (2011).
Wu, P. Z., Liberman, M. C. Age-related stereocilia pathology in the human cochlea. Hear Res. 422, 108551 (2022).
Kurabi, A., Keithley, E. M., Housley, G. D., Ryan, A. F., Wong, A. C. Cellular mechanisms of noise-induced hearing loss. Hear Res. 349, 129-137 (2017).
Kim, J., Xia, A., Grillet, N., Applegate, B. E., Oghalai, J. S. Osmotic stabilization prevents cochlear synaptopathy after blast trauma. Proc Natl Acad Sci U S A. 115 (21), E4853-E4860 (2018).
Hu, N., Rutherford, M. A., Green, S. H. Protection of cochlear synapses from noise-induced excitotoxic trauma by blockade of ca(2+)-permeable ampa receptors. Proc Natl Acad Sci U S A. 117 (7), 3828-3838 (2020).
Wan, G., Gomez-Casati, M. E., Gigliello, A. R., Liberman, M. C., Corfas, G. Neurotrophin-3 regulates ribbon synapse density in the cochlea and induces synapse regeneration after acoustic trauma. Elife. 3, e03564 (2014).