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JARO: Journal of the Association for Research in Otolaryngology logoLink to JARO: Journal of the Association for Research in Otolaryngology
. 2022 Jul 29;23(5):603–616. doi: 10.1007/s10162-022-00862-2

Mitigation of Hearing Damage After Repeated Blast Exposures in Animal Model of Chinchilla

Shangyuan Jiang 1, Paige Welch 1, Sarah Sanders 1, Rong Z Gan 1,✉
PMCID: PMC9613841  PMID: 35906449

Abstract

High-intensity sound or blast-induced hearing impairment is a common injury for Service members. Epidemiology studies revealed that the blast-induced hearing loss is associated with the traumatic brain injury (TBI), but the mechanisms of the formation and prevention of auditory injuries require further investigation. Liraglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, has been reported as a potential treatment strategy for TBI-caused memory deficits; however, there is no study on therapeutics of GLP-1R for blast-induced hearing damage. This paper reports our current study on progressive hearing damage after repeated exposures to low-level blasts in the animal model of chinchilla and the mitigation of hearing damage using liraglutide. Chinchillas were divided into three groups (N = 7 each): blast control, pre-blast treatment, and post-blast treatment. All animals were exposed to six consecutive blasts at the level of 3–5 psi (21–35 kPa) on Day 1. The auditory brainstem response (ABR) was measured on Day 1 (pre- and post-blast) and Days 4, 7, and 14 after blast exposure. Upon the completion of the experiment on Day 14, the brain tissues of animals were harvested for immunofluorescence studies. Significant damage was revealed in blast-exposed chinchillas by increased ABR thresholds, decreased ABR wave I amplitudes, and cell apoptosis in the inferior colliculus in the blast control chinchillas. Treatment with liraglutide appeared to reduce the severity of blast-induced hearing injuries as observed from the drug-treated chinchillas comparing to the blast controls. This study bridges the gap between TBI and hearing impairment and suggests a possible intervention for blast-induced hearing loss for Service members.

Keywords: blast exposure, hearing damage, liraglutide, hearing function test, chinchilla

Introduction

Hearing loss and tinnitus are the top two most prevalent service-connected disabilities among veterans in the US with over 3 million victims (Veterans Benefits Administration 2019). Repeated exposures to blasts are considered to be intrinsic situations faced by military personnel involved in combat and occupational training, such as breaching exercises and grenade courses (Carr et al. 2016; Dougherty et al. 2013; Sajja et al. 2019). Epidemiologic studies revealed that the hearing complaints from Service members and Veterans were associated with their histories of blast exposures and traumatic brain injury (TBI) (Gallun 2017; Oleksiak et al. 2012; Theodoroff et al. 2015). However, the detailed mechanisms of the formation, prevention, and restoration of blast-induced auditory injuries require further investigations.

Blast overpressure (BOP) is a high-intensity disturbance in the ambient air pressure and both peripheral and central auditory systems are susceptible to blast exposures. When the BOP waves propagate through the peripheral auditory system (PAS), the rupture of tympanic membrane (TM) and disruption of ossicular chain can result in severe conductive hearing loss (Gan et al. 2016; Mayorga 1997; Patterson and Hamernik 1997), while hair cell loss and excitotoxicity to the ribbon synapses and spiral ganglion neurons in the cochlea could induce sensorineural hearing loss (Cho et al. 2013; Liberman and Kujawa 2017; Patterson and Hamernik 1997). BOP can also induce injuries in the central auditory system (CAS) through shearing and stretching of the brain in regions such as the brainstem and auditory cortex, sharing a similar mechanism with the TBI, even in cases of low-intensity blast exposure (Fausti et al. 2009; Gallun et al. 2012; Lew et al. 2007; Song et al. 2018). Currently, treatment to cure blast-induced auditory injury is mostly focused on the PAS, typically including TM repair and hair cell regeneration (Kozin et al. 2016; Zheng and Zuo 2017). Despite some pathophysiological mechanisms that have been identified, treatment strategies aimed at correcting the CAS dysfunction resulting from blasts have not been well investigated.

The glucagon-like peptide-1 (GLP-1) is an incretin produced by enteroendocrine L cells, and its primary function is to induce the release of insulin and regulate glucose levels (Drucker and Nauck, 2006). With the discovery of the neurotrophic and neuroprotective function of GLP-1 receptor (R) in the central nervous system, the potential of GLP-1R agonists as a treatment strategy for neurological disorders was reported (Athauda and Foltynie 2016; Salcedo et al. 2012). Recent studies have assessed the GLP-1R agonists such as liraglutide, which is an FDA-approved drug prescribed for the treatment of type 2 diabetes, as potential treatment strategies for TBI-induced memory and cognitive deficits in animal models of stroke and blast-induced TBI (Briyal et al. 2014; Li et al. 2015; Rachmany et al. 2013; Tweedie et al. 2013, 2016). Based on the similarities between the TBI and blast-induced hearing damage, a question is raised: whether liraglutide has therapeutic benefits against blast-induced auditory damage?

Smith et al. (2020) and Chen et al. (2019) have reported a chinchilla animal model of repeated-blast-induced auditory injuries at high (15–20 psi or 103–138 kPa) and low (3–5 psi or 21–35 kPa) BOP levels, respectively. Acutely and permanently impaired hearing functions and biomarkers of damage in neurons in CAS were observed in chinchillas 7 or 14 days after the blast exposures, and the severity of the damage was positively related to the BOP level. Such damage could be possibly ameliorated by the activation of GLP-1R, which was widely expressed in many regions of the CAS containing brainstem, thalamic, and cortical areas (Gu et al. 2013; Salcedo et al. 2012). Therefore, an immunohistochemistry study was conducted in our lab (reported by Gan et al. in 2019 ARO Midwinter meeting) to determine if the GLP-1R expression can be found in cochlea spiral ganglion neurons (SGNs), inferior colliculus (IC), and auditory cortex (AC) of the chinchilla. The tissues were harvested, decalcified (for cochlea), and embedded in paraffin following the standard procedure of histology study in chinchillas (Guan et al. 2015; Jiang et al. 2016). The tissue was then stained using the rabbit-source GLP-1R primary antibody (6 μg/ml, #188,605, Abcam, Cambridge, MA) and SPlink HRP detection kit for rabbit primary antibody (D03-6, GBI Labs, Bothell, WA). The positive staining displayed in Fig. 1A–C indicates that GLP-1R is wildly expressed in the chinchilla cochlea SGNs, IC, and AC. The low-magnification images shown in Fig. 1D, E, and F were a general approximation of locations of the SGNs, IC, and AC images in Fig. 1A, B, and C, respectively. Note that Fig. 1D was reported by Gan et al. in 2019 ARO Midwinter meeting, and Fig. 1E and F were taken from www.brainmuseum.org. This preliminary result suggested that GLP-1R agonist administration could potentially have neuroprotection and neurotrophic functions in the auditory system. Therefore, it is conceivable that the liraglutide may have a therapeutic function for hearing restoration after blast exposures.

Fig. 1.

Fig. 1

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Immunohistochemistry staining of the GLP-1R expression in chinchilla: A spiral ganglion neurons (SGNs), B inferior colliculus (IC), and C auditory cortex (AC). D The lower magnification image showing the structure of the cochlea and the location of SGNs is marked. E and F Chinchilla brain sections (#640 and #400) images provided by brainmuseum.org used to demonstrate the location of IC and AC, respectively. The location of IC and AC were highlighted by red circles. The distribution of IC and AC is bilaterally symmetric. (Gan et al. in 2019 ARO Midwinter meeting; http://www.brainmuseum.org)

This paper reports our recent study in chinchillas to characterize the low-level BOP-induced hearing damage and investigate the therapeutic function of liraglutide to mitigate the process of auditory injury after multiple blast exposures. This study bridges the gap between traumatic brain injury and hearing impairment and suggests a possible intervention for blast-induced hearing loss for Service members.

Methods

Animal Protocol and Liraglutide (Drug) Administration

Young adult chinchillas (Chinchilla laniger) with mixed gender provided by Ryerson Chinchilla Ranch (Plymouth, OH) were included in this study. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Oklahoma following the guidelines of the National Institutes of Health and the US Department of Agriculture. Twenty-one chinchillas were randomly separated into three groups: pre-blast drug treatment, post-blast drug treatment, and blast control (N = 7 each). To assess the effect of the liraglutide treatment itself and repeated sedation on the results, we also included drug control (N = 3) and sham control (N = 2) groups, which did not experience any blast exposures. Figure 2 shows the time course and experimental procedures for the pre-blast and post-blast drug treatment groups. Key procedures such as blast exposures, hearing function tests, and euthanasia are emphasized by arrows. Animals in the pre-blast treatment group were injected with liraglutide (Victoza, Novo Nordisk inc. Plainsboro, NJ) 2 days before the blast exposure and in the consecutive 7 days. The pre-blast treatment group was designed to examine the damage prevention function of the liraglutide. In the post-blast treatment group, the liraglutide treatment started 2 h after the blast on Day 1 and in consecutive 7 days. The blast control chinchillas underwent only blast exposures but no drug treatment. The drug control chinchillas experienced only 7-day-long liraglutide treatment without blasts while the sham control chinchillas only experienced hearing function tests without either blasts or drug treatment. Liraglutide was daily administered to chinchillas via subcutaneous injections with a dose of 246.7 μg/kg/day, which was equivalent to the human dose (20 μg/kg/day) normalized to body surface area across species and determined after considering the dose used for murine animal models (Hakon et al. 2015; Li et al. 2015). The hearing function tests were performed before and after the blast on Day 1 and on Days 4, 7, and 14. Upon the completion of the experiment on Day 14, the chinchilla was euthanized and the brain was harvested for histological studies.

Fig. 2.

Fig. 2

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Schematic diagram of the time course and experimental procedure for pre- and post-blast treatment groups

Experimental Setup for Blast Exposure

Each chinchilla was anesthetized with an intramuscular injection of 35 mg/kg Ketamine (Henry Schein Animal Health, Dublin, OH) and 3 mg/kg Xylazine (Akorn Inc., Lake Forest, IL) to ensure the animals were sedated during blast exposures. The experimental setup for blast exposure was based on our previous studies as reported by Smith et al. (2020) and Chen et al. (2019). Briefly, the chinchilla was placed in a specifically designed L-shape animal holder and fixed using straps to position the top of its head facing the center of the blast source (Fig. 3). The nose of the chinchilla pointed to the front and the pinna remained unfolded to ensure the ear canal was naturally open during the blast exposure. A pressure sensor (Model 102B16, Piezotronics, Depew, NY) was fixed to the animal holder and the measuring surface was next to the chinchilla ear to monitor the blast pressure at the entrance of the ear canal.

Fig. 3.

Fig. 3

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Schematic of the animal experimental setup with blast apparatus. The top of the chinchilla’s head was facing the blast source and the nose of the chinchilla pointed to the front

A well-controlled compressed nitrogen-driven blast apparatus located inside an anechoic chamber (Fig. 3) was used to generate BOPs (Chen et al. 2019; Gan et al. 2016; Smith et al. 2020). Polycarbonate films (McMaster-Carr, Atlanta, GA) of 0.25 mm were used to generate the BOP at a peak pressure level of 3–5 psi or 21–35 kPa monitored at the entrance of the chinchilla ear canal. Animals experienced 6 consecutive blasts at time intervals of approximately 5 min between blasts. The pressure signals from the sensor were processed by a cDAQ 7194 and A/D converter 9215 (National Instruments Inc., Austin, TX) at a sampling rate of 100 k/s (10 ms dwell time). The LabVIEW software package (National Instruments Inc., Austin, TX) was used for data acquisition and analysis.

Hearing Function Measurements

The hearing function tests for each chinchilla were conducted pre- and post-blast on Day 1 and on Days 4, 7, and 14, respectively as shown in Fig. 2. On Day 1, the hearing function tests were performed prior to blast exposure to measure the hearing baseline for each ear and immediately after the completion of blast exposures with the animal sedated under ketamine and xylazine. The time elapsed between the end of the pre-blast hearing assessment and the blast exposure was approximately 10 min. The blast exposures took 15 min, and the post-blast assessment started 10 min after the completion of the blast exposures. The hearing function measurement was completed within 2 h. For hearing tests performed on Days 4, 7, and 14, the chinchillas were sedated by isoflurane (covetrus, Dublin, OH) at a concentration of 1–3 % in the oxygen at a flow rate of 1 L/min. Ketamine and xylazine were used on Day 1 instead of isoflurane as the equipment used to provide isoflurane sedation could not work in the blast chamber to maintain sedation during blast exposure.

The conditions of the ear canal and TM of the animal ear were examined using a 3.9-mm digital otoscope (NTE 390, ScopeAround, Irvine, CA), and the middle ear status was checked by the wide-band tympanometry (Titan, Interacoustics, Denmark) following the otoscopic exam. These exams were performed each time before the hearing function tests. The TM of every animal was ruptured after the blasts. TM repair was not noted obviously in any of the animals in either the treatment or control group within 14 days. Auditory brainstem responses (ABRs) were recorded pre- and post-blast on Day 1 and on Days 4, 7, and 14. During the function tests, animals remained under anesthetic as described above.

Auditory Brainstem Response Measurements

ABR provides general information about hearing sensitivity by recording threshold values measured under pure tone stimuli at different frequencies (Henry et al. 2011). The ABR measurements were recorded bilaterally using a TDT system III (Tucker-Davis Technologies, Alachua, FL) following the protocol established in our previous studies on the chinchilla animal model of blast-induced hearing damage (Chen et al. 2019; Gan et al. 2016; Smith et al. 2020). Under anesthesia, stainless steel needle electrodes were placed subcutaneously at the vertex of the skull and ventrolateral surfaces of the ear, while a ground electrode was placed in the rear leg. Tone burst stimuli of 0.5-ms rise-fall time and 4-ms duration with alternating polarity at frequencies of 1, 2, 4, 6, and 8 kHz were used (Gan et al. 2016; Henry et al. 2011; Zhong et al. 2014). The stimuli were designed by the SigGenRP and BioSigRP software, created by RP2.1 signal processor with power amplifier TYPE 2718 (BRUEL & KJAER, Nærum, Denmark), generated by the MF1 multi-field magnetic speaker, and delivered into the chinchilla ear canal through a 10 cm standard tube at a presentation rate of 23/s. All measuring components were included in the TDT system III except for the power amplifier. The sound pressure level in the ear canal was monitored by a probe microphone (ER-7C, Etymotic Research, Elk Grove Village, IL). ABR recordings with a length of 10 ms were averaged 150 times. Note that this was a relatively small number used for ABR measurement compared to 300, 600, or 1000 which were used in similar studies (Henry et al. 2011; Land et al. 2016; Race et al. 2017). The 150 used in this study was due to the limited time length for a series of measurements, especially on Day 1. Prior to data collection, an initial analysis indicated that 150 was an adequate number of sweeps to produce replicable ABR waveforms and allowed for threshold and peak picking analysis in our experimental paradigm (Fig. 10 in Appendix). The band-pass filter for ABR signal was set to be 300 Hz to 3 kHz. ABR waveforms were recorded in descending 5 dB SPL intervals from the maximum amplitude of 100 dB SPL until 20 dB SPL.

Fig. 10.

Fig. 10

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Screenshots of ABR waveforms measured with different numbers of epochs. The ABR was measured at 8 kHz, 95 dB

ABR thresholds were determined by visually examining the prominent ABR peaks (usually waves I and V) to determine the lowest sound stimulation level at which reproducible waveforms were observed. Two independent observers who are blind to the treatment status of the animals were involved to read the thresholds from the ABR waveforms and their results were consistent with each other. When discrepancies between two scores happened, the average of two values was used as the threshold. If an ABR response was not detected at the maximum acoustic stimulation, the threshold was arbitrarily set to be 100 dB. If the ABR signal continued to appear at 20 dB, the threshold was arbitrarily set to be 20 dB. The ABR threshold shifts on Days 1, 4, 7, and 14 were calculated by subtracting the threshold measured pre-blast from the threshold measured at the following time points, respectively.

The ABR wave I was generated by the auditory nerve, and the suprathreshold amplitude of ABR wave I can be predictive of the damage in the auditory nerves, inner hair cell, or cochlear synaptopathy (Henry et al. 2011; Hickman et al. 2018; Liberman and Kujawa 2017). However, the amplitude of the ABR wave I could also be affected by the input to the auditory nerve induced by conductive hearing loss or loss of outer hair cells, which were lesions located at the upstream of the ascending auditory pathway. In this study, the ABR wave I amplitudes at the stimulus levels between 80 and 100 dB at 8 kHz were extracted from the waveform and recorded to characterize the injuries in the ascending auditory pathway at the locations up to the auditory nerve.

Immunofluorescence Study

Upon the completion of hearing function measurements on Day 14, the animals were euthanized and perfused transcardially with saline solution followed by 4 % paraformaldehyde in 9.6 g/L phosphate-buffered saline (PBS). The brains were then harvested and fixed in 4 % paraformaldehyde in 9.6 g/L PBS for 72 h at 4 °C. Following a standard histology protocol, the brains were dehydrated in ethanol with gradient concentration, decolored in xylene, and embedded in paraffin. Sections at a thickness of 10 μm were obtained from a rotatory microtome (Leitz 1512, Leitz, German) (Jiang et al. 2016; Smith et al. 2020). The sectioning plane of each specimen was perpendicular to the front-posterior central axis of the brain. Since the hearing function tests used in this study (ABR) reflected the function of the structures in the ascending auditory pathway up to the IC, we decided to observe the activity of caspase-3, a direct biomarker of caspase-dependent apoptosis, in IC using immuflouresence (IF) (Li et al. 2015). The location of IC was determined based on the anatomy of the chinchilla cerebral hemispheres and brainstem (http://www.brainmuseum.org; Irimescu et al. 2014; Kelley et al. 1992).

The antigen retrieval was performed by processing the slides in 95–100 °C 0.1 M sodium citrate buffer for 30 min. The tissues were then processed by 3 % H2O2 solution, blocked with 10 % goat serum (Sigma-Aldrich, Saint Louis, MO) containing 0.03 % TritonX-100 and incubated overnight at 4 °C with the rabbit-source cleaved caspase-3 primary antibody (1:200, #9661, Cell Signaling Technology Inc., Danvers, MA). The omission of the caspase-3 primary antibody was served as a negative control. The IF was performed using goat anti-mouse IgG secondary antibody (1:1000, Alexafluor 594, Thermo Fisher Scientific, Rockford, IL), and nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI) (D9542, Sigma-Aldrich, Saint Louis, MO). The immunofluorescence images were collected by an fluorescence microscope (EVOS M7000 Imaging System, Thermo Fisher Scientific, Waltham, MA). Fluorescence from two channels was collected for each section: blue: 405 nm (DAPI) and red: 594 nm (caspase-3). Sections obtained from the blast-control groups were used as references for adjusting the exposure times, and the exposure time was then fixed for the acquisition of other images. The images were post-processed using ImageJ software. The rolling ball radius for background subtraction was set to be 30 pixels, and the brightness and contrast were set to be identical for all images obtained from three groups. The integrated density of the caspase-3 signal (red channel) was calculated by the sum of the values of the pixels in the image using ImageJ.

Statistical Analysis

The ABR threshold and ABR wave I measurement data were expressed as the mean ± SEM and plotted in GraphPad Prism (GraphPad Software Inc., Version 9.0). Two-way repeated-measures ANOVA tests (α = 0.05) were utilized to check the significance (P < 0.05) of the time, drug treatment’s effects, and the interaction, which was the combined effect of two factors, on the hearing function of chinchillas. Treatment groups were compared in a pairwise fashion using Tukey’s post hoc multiple comparison tests subsequent to a significant main effect of a given ANOVA. The statistical analysis was also performed in GraphPad Prism. The measured ABR threshold shift at each frequency and ABR wave I amplitude shift at 100 dB stimulus level at 8 kHz were included in the statistical analysis. A one-way ANOVA test was performed on integrated density of the caspase-3 IF signal measured from chinchilla IC histology sections using GraphPad Prism (GraphPad Software Inc., Version 9.0) between three treatment groups followed by Tukey’s post hoc multiple comparison tests (α = 0.05) to check the significance of drug treatment’s effects (P < 0.05).

Results

BOP Waveform and Examinations of the Middle Ear

Figure 4 shows typical waveforms of BOP in units of psi (1 psi = 6.9 kPa) measured at the entrance of the ear canal over a duration of 10 ms. Waveforms obtained from six repeated measurements were consistent with each other. Six recorded waveforms displayed a single positive peak at 1 ms with a mean peak pressure level of 3.7 ± 0.3 psi (25.6 ± 1.9 kPa). The pressure then quickly decreased to a negative value of approximately 0.8 psi (5.5 kPa) before 2 ms and then returned to 0 with minor fluctuations at amplitudes lower than 0.2 psi (1.4 kPa) for the rest of the time. The BOP waveform was repeatable for each blast test.

Fig. 4.

Fig. 4

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Six examples of (n = 6) BOP waveforms recorded at the entrance of the ear canal from an animal experiment with a mean peak pressure level of 3.7 ± 0.3 psi or 25.6 ± 1.9 kPa (mean ± SD)

Otoscopic examination indicated all of the chinchilla TMs were ruptured after blasts and remained not completely cured in 14 days. The wide-band tympanometry measurements also showed the energy absorbance was not changing with the pressure variation and demonstrated the TM was ruptured after blasts and over 14 days. No signs of infection or fluid in the ear canal and middle ear were observed during the 14-day-long experiment.

ABR Waveforms and Threshold Shift

Figure 5 shows examples of ABR waveforms recorded from a blast control (20–3-15L in Fig. 5A) and a post-blast treatment (20–3-8L in Fig. 5B) ear. Since the pre-blast treatment ABR waveform was similar to the post-blast treatment, it was not shown due to limited space. ABR waveforms with a length of 10 ms measured at 8 kHz were plotted from top to bottom with the stimulus level decreased from 100 to 20 dB at a step size of 5 dB. Results measured pre-blast, post-blast, and on Day 14 were included to demonstrate the blast-induced acute damage and the post-blast recovery process. The pre-blast ABR waveforms from both blast control and post-blast treatment ears clearly showed five major peaks with decreasing amplitudes and increasing latency from 100 to 20 dB. At the stimulus level of 100 dB, the five peaks of ABR were highlighted in the pre-blast result in Fig. 5A and B. The wave I was highlighted in all six sets of results and the peak-to-peak amplitude of wave I was indicated only in three blast-control results. The pre-blast threshold was 25 dB in both blast control and post-blast treatment ears which indicated the hearing sensitivity was the same in both ears before the blast. Severe hearing damage induced by 6 consecutive blasts could be observed from a great reduction of the amplitudes and increase of thresholds in both ears. The post-blast threshold was 80 dB in the blast control ear and 75 dB in the post-blast treatment ear. On Day 14, the post-blast treatment ear showed much better recovery than the blast control in both amplitude and latency of the ABR. At the stimulus level between 70 and 100 dB on Day 14, the first three peaks of ABR showed amplitudes close to the pre-blast level in the post-blast treatment ear, while the waveform of the blast control ear was almost as damaged as the post-blast results. The threshold on Day 14 was 75 dB in the blast control ear and 60 dB in the post-blast treatment ear. ABR waveforms in Fig. 5 demonstrated examples of blast-induced waveform changes for hearing damage in blast control and post-blast treatment animal ears. The drug-treated animal ear showed better recovery than the control one 14 days after blast exposures.

Fig. 5.

Fig. 5

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ABR waveforms at 8 kHz measured pre- and post-blast and on Day 14 from (A) a blast control ear (20–3-15L) and (B) a post-blast treatment ear (20–3–8) at the stimulus levels that ranged from 100 to 20 dB with a step size of 5 dB. Five ABR peaks were numbered on the top of pre-blast waveforms at 100 dB. The red arrows indicate wave I peak to trough amplitudes

The mean and SEM of the ABR thresholds measured from pre-blast treatment, post-blast treatment, and blast control groups (N = 14 ears in each group) were plotted in Fig. 6A, B, and C, respectively. The threshold shifts on Days 1, 4, 7, and 14 were plotted in each figure and represented by different colors. On Day 1, the mean threshold shift ranged between 35 and 50 dB in all three groups with a lower shift at 1 kHz and higher shifts at 4–8 kHz. In all three groups, the threshold shifts gradually decreased with time from Day 1 to 14 and the amount of recovery between adjacent time points decreased with time. On Day 14, the mean threshold shifts in the pre-blast treatment group shown in Fig. 6A ranged from 5 dB at 1 kHz to approximately 20 dB at 6 kHz. The mean threshold shifts in the post-blast treatment group shown in Fig. 6B were approximately 5 to 15 dB. The blast-control group results shown in Fig. 6C, however, showed greater threshold shifts on Day 14 than the other two liraglutide-treated groups with the mean value increased from 15 dB at 1 kHz to 30 dB at 8 kHz. The ABR threshold shift indicated the blast-induced acute damage on Day 1 was approximately the same in all three groups and high-frequency hearing (4–8 kHz) was more severely damaged than the low-frequency hearing (1–2 kHz).

Fig. 6.

Fig. 6

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ABR threshold shifts (mean ± SEM, n = 14 ears in each group) measured on Days 1, 4, 7, and 14 from: A pre-blast treatment group; B post-blast treatment group; C blast control group. ABR threshold shifts from three chinchilla groups are plotted against time at 2 (D) and 8 (E) kHz. The statistically significant effect of drug treatment detected by 2-way ANOVA test was labeled on the title and significant difference detected by Tukey’s post hoc test was highlighted by brackets between the groups. (* P < 0.05; ** P < 0.01)

The two-way ANOVA analysis on ABR threshold data performed at each frequency point indicated that the main effect of time was significant at all frequencies (1 kHz: F (3, 117) = 80.47, P < 0.0001; 2 kHz: F (3, 117) = 108.30, P < 0.0001; 4 kHz: F (3, 117) = 50.06, P < 0.0001; 6 kHz: F (3, 117) = 35.08, P < 0.0001; 8 kHz: F (3, 117) = 34.82, P < 0.0001). The main effect of treatment group was significant only at 2 and 8 kHz (1 kHz: F (2, 39) = 3.05, P = 0.055; 2 kHz: F (2, 39) = 6.23, P = 0.0035; 4 kHz: F (2, 39) = 1.32, P = 0.28; 6 kHz: F (2, 39) = 2.24, P = 0.12; 8 kHz: F (2, 39) = 4.83, P = 0.012). Therefore, the threshold shifts at 2 and 8 kHz (statistical significance labeled at the figure title) at each time point of measurement were plotted in Fig. 6D and E with the pre-treatment, post-treatment, and control groups highlighted in red, blue, and black, respectively. Tukey’s post hoc tests on 2 and 8 kHz indicated that significance differences were observed between the control and pre-treatment groups at Day 7 (P = 0.0032) and Day 14 (P = 0.013) at 2 kHz, and at Day 7 (P = 0.015) and Day 14 (P = 0.046) at 8 kHz as highlighted by the brackets in Fig. 6D and E. In addition, significant time × treatment interaction effects were also present at 2 (F (6, 117) = 2.28, P = 0.039) and 8 kHz (F (6, 117) = 2.62, P = 0.019), indicating the effect of drug treatment changed over time.

ABR Wave I Amplitude

The ABR wave I amplitudes (peak-to-peak) measured from animals of pre-blast treatment, post-blast treatment, and blast control groups at 8 kHz are shown in Fig. 7. The mean and SEM values were plotted against the level of acoustic stimulus from 80 to 100 dB SPL. The results measured at different time points were represented by different colors as shown in the legend of Fig. 7A, B, and C. The results obtained from pre-blast treatment, post-blast treatment, and blast control groups were shown in Fig. 7A, B, and C, respectively with N = 14 ears in each group.

Fig. 7.

Fig. 7

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ABR wave I amplitude (mean ± SEM, n = 14 ears in each group) in response to stimulus level from 80 to 100 dB SPL at 8 kHz measured pre-and post- blast and on Days 4, 7, and 14 from A pre-blast treatment group, B post-blast treatment group, and C blast control group. D Statistical results in which significant difference detected by Tukey’s post hoc test was highlighted by brackets between the groups. (* P < 0.05)

In all three groups, the mean of the pre-blast wave I amplitudes ranged between 1 and 2 μV and the value increased with the stimulus level. A major reduction in the wave I amplitude could be observed from pre- to post-blast in all chinchillas. The major recovery of the wave I amplitude happened between Day 1 and Day 4 and the recovery after Day 4 was limited in all three groups. In the pre-blast and post-blast treatment groups, the recovered ABR on wave I amplitude on Day 14 was close (> 80 %) to the level before the blast, while the recovery in the blast control group was lower (< 60 %) than the pre-blast level.

The ABR wave I amplitude shifts on Days 1, 4, 7, and 14 were calculated by subtracting the wave I amplitudes measured at the different time points from the values measured pre-blast on Day 1, respectively. Results of two-way ANOVA on the amplitude shifts were plotted in Fig. 7D. The main effect of time was significant (F (3, 117) = 15.00, P < 0.001), and the main effect of treatment group was also significant (F (2, 39) = 5.50, P = 0.008). The interaction, which indicated the combined effect of drug and time, was not significant (F (6, 117) = 1.00, P = 0.412). Tukey’s post hoc test results indicated that significant differences were observed on Day 4 between control and two drug treatment groups (Pre: P = 0.048; Post: P = 0.026), on Day 7 between control and post-treatment group (P = 0.037), and on Day 14 between control and post-treatment group (P = 0.044).

Immunofluorescence Results

Representative images obtained from the IF study on chinchilla ICs are shown in Fig. 8. The example IC sections from the pre-blast treatment, post-blast treatment, and blast control chinchillas are shown in Fig. 8A, B, and C, respectively. The caspase-3 was highlighted in red, and the cell nuclei were highlighted in blue by DAPI. The activity of caspase-3 could be observed in the IC of the blast controls while the expression level in the two groups of drug-treated chinchillas was lower than the blast control. The negative control with the omission of the caspase-3 primary antibody was obtained from the blast control group and showed no signal of caspase-3 (Fig. 8D). The high-magnification image of the chinchilla brain tissue (Fig. 8E) demonstrated a compartmentalized and punctate labeling pattern of the caspase-3, suggesting of a membrane or mitochondrial location within the cell which was similar to the pattern of caspase-3 observed in rat brain tissues (Clark et al. 2000). A quantitative analysis showing the mean and SEM of the caspase-3 signal (red) integrated density (N = 3 in each group) measured from the pre-blast treatment, post-blast treatment, and blast control groups was plotted in 8F. One-way ANOVA indicated the liraglutide treatment significantly reduced the caspase-3 expression level in chinchilla ICs 14 days after the blast exposures (F (2, 6) = 7.80, P = 0.021). Tukey’s post hoc test results indicated that significant differences were observed between the blast control and post-blast treatment group (Post vs. Control: P = 0.020) as highlighted in Fig. 8F. The quantitative results are consistent with the example images shown in Fig. 8A to C. The current IF results provided evidence to support that the liraglutide administration reduced the level of caspase-3 activity in the IC 14 days after the blast exposure.

Fig. 8.

Fig. 8

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The immunofluorescence staining of caspase-3 in chinchilla inferior colliculus from A pre-blast treatment, B post-blast treatment, and C blast control chinchillas. D The negative control with the omission of the primary antibody. E A high-magnification image of chinchilla brain tissue with the caspase-3 at the membrane or mitochondrial location. F Mean and SEM of the caspase-3 signal (red) integrated density (N = 3 in each group). The statistically significant effect of drug treatment detected by one-way ANOVA test was labeled on the title of the y-axis and significant difference detected by Tukey’s post hoc test was highlighted by brackets between the groups. (* P < 0.05). The caspase-3 was stained in red, and the nuclear counterstain was DAPI (blue)

Discussions

Characterization of Hearing Damage Incurred by Repeated Low-Level BOPs

Currently, there are a limited number of published animal studies on blast-induced auditory injuries, most of which focus on the damage in the PAS (especially in the cochlea and using murine models with only a few using chinchillas) (Cho et al. 2013; Gan et al. 2016; Hickman et al. 2018; Mao et al. 2011; Niwa et al. 2016; Race et al. 2017). As more evidence showing the correlation between hearing loss and TBI was revealed, the blast-induced damage in the CAS and the respective hearing function measurements such as middle latency response and magnetic resonance imaging have gradually drawn more attention (Mao et al. 2011; Race et al. 2017). In this study, the ABR threshold, wave I amplitude, and IC histology were first-time reported in chinchillas after experiencing 6 repeated blast exposures of 3–5 psi (21–35 kPa). These multi-parameter measurements comprehensively characterize the damage and recovery process that occurred at different levels of the auditory system over 14 days after the exposure.

The ABR thresholds were substantially elevated after 6 repeated blast exposures and recovered partially over time in blast control chinchillas shown in Figs. 5 and 6. This suggested the hearing function was severely damaged by the blasts and the spontaneous recovery of the auditory system was insufficient. Comparing to the results reported by Chen et al. (2019), a similar 7-day long study in which chinchillas were exposed to 3 blasts at the same BOP level, the results of the present study (Fig. 6) showed that the ABR threshold shift in 6-blast ears was close to that of 3-blast ears on the first day, but greater than (approximately 10 dB in mean) that of 3-blast on Day 7 over the entire frequency range. This might suggest that the severity of the long-term hearing damage was likely to be more related to the number of blasts than the acute damage. Comparing to the results reported by Smith et al. (2020) which was a 14-day study on chinchillas exposed to 3 blasts at 15–20 psi (103–238 kPa), the ABR threshold shift in the present study was lower (approximately 10 dB in mean) than that of the high-level BOP results at 8 kHz at all time points of measurement. The current data, in the context of these previous reports, suggest that both the BOP and the number of blasts are important variables that can affect the severity of damage done to the auditory system. The ABR threshold shifts in the present and previous studies showed the same increasing trend with the frequency at all time points of measurement, which showed good consistency among the results obtained from the animal model of chinchilla for blast-induced hearing damage.

Amelioration of the Blast-Induced Auditory Injuries

The present study demonstrates that the GLP-1R agonist, liraglutide, improves the recovery of the hearing function after repeated exposures to blast in chinchillas. The ABR threshold reflected the general hearing sensitivity, which reflected the function of both the PAS and CAS. As shown in Figs. 5 and 6, although the hearing loss measured immediately after the blast in all the chinchillas was at the similar severity level, the hearing sensitivity of the liraglutide-treated chinchillas recovered better at 2 and 8 kHz than the blast controls over a time of 14 days. These ABR threshold results indicate the liraglutide treatment positively affects the post-injury recovery process in the auditory system instead of protecting against acute blast-induced auditory injuries. The significant effect of drug treatment was observed at 2 and 8 kHz, indicating both low and high-frequency hearing could be potentially improved by the liraglutide. Figure 6D and E show that only the pre-treatment group showed significantly better ABR thresholds while no difference between post-treatment and controls was reported or shown, which suggested that pre-blast treatment potentially had some advantages over the post-blast treatment.

The ABR wave I amplitude was generated by the auditory nerve and was used as a predictive indicator of cochlear synaptopathy in chinchilla ears damaged by blast or noise (Henry et al. 2011; Hickman et al. 2018; Liberman and Kujawa 2017). It could be also affected by lesions that occurred along the ascending auditory pathway below the auditory nerve such as conductive hearing loss, damage to cochlear structures, or myelination defects in the auditory nerve (Wan and Corfas 2017). Figures 5 and 7 show significant improvement of the ABR wave I amplitude in liraglutide-treated ears. Since the TMs were ruptured in the present study, the improvement in ABR wave I did not necessarily indicate the functional recovery of the ribbon synapses or spiral ganglion neurons in the cochlea. The liraglutide treatment could potentially facilitate the post-blast recovery in structures in the ascending auditory pathway below or at the auditory nerve in the PAS, and such a positive effect is evident at 8 kHz.

The IF results shown in Fig. 8A to C demonstrated caspase-3 expression in ICs from one example chinchilla in each of the three groups. The caspase-3 is the most important executioner protein for apoptosis which cleaves various substrates in the cell and ultimately causes the morphological and biochemical changes seen in apoptotic cells (Elmore 2007). Caspase-3 activation was proved to contribute to brain tissue loss and downstream biochemical events that executed programmed cell death after TBI (Clark et al. 2000). Therefore, caspase-3 was used as a biomarker of blast-induced damage in brain tissues, and the lack of caspase-3 was used to assess the neurotrophic and neuroprotective functions of liraglutide treatment against blast-induced TBI (Li et al. 2015). In Fig. 8, lower caspase-3 levels in drug-treated chinchilla IC indicated that the caspase-driven apoptosis in IC could be possibly inhibited by the liraglutide treatment, which protected the central auditory system against blast-induced damage. However, the impact of the liraglutide’s neuroprotective function in the CAS on the hearing function of the animal still requires further investigation. Regarding the acoustic evoked potentials (AEPs), the ABR arises from regions of the brainstem auditory pathway below the IC and the generators of later peaks of ABR vary across different species (Blatchley et al. 1987; Chen and Chen 1991; Laumen et al. 2016; Wada and Starr 1983). There is a correlation between the ABR threshold and the function of CAS, but its relation to the specific region of IC is unclear (Race et al. 2017). Therefore, the lower cell apoptosis level observed in Fig. 8 could potentially have contributed to the lower ABR threshold shift in the drug-treated chinchillas. However, the current IF results were preliminary and only provided a possible direction for future studies.

Limitation and Future Studies

The first limitation of this study was that the values of ABR threshold and waveform could be potentially altered by the different sedation method used at different time points, including the dose, duration, and species (Bielefeld 2014; Cederholm et al. 2012; Ruebhausen et al. 2012; Thiele and Köppl 2018). However, the aim of this study was to investigate the effect of liraglutide by comparing chinchillas between the drug-treated and blast-control and identical protocols were used for all groups at each time point of measurement. Therefore, the conclusion obtained from the intra-treatment-group comparisons should be valid and not significantly affected by the anesthesia methods. To address this concern, hearing function data measured from sham and drug control chinchillas were also included to examine the effect of sedation. The ABR threshold measured from drug control (mean ± SEM, N = 6 ears) and sham control (mean ± SEM, N = 4 ears) groups are plotted in Fig. 9A and B, respectively. The sedation protocol, drug treatment, and hearing function tests for drug control chinchillas were the same as the post-blast treatment group but no blast exposure. Therefore, the results were only reported pre-blast and on Days 4, 7, and 14. Similarly, the experiment for sham control chinchillas experienced neither blast nor liraglutide treatment, and the ABR was reported pre-blast and on Days 4 and 7. Figure 9A suggests that the ABR threshold change induced by liraglutide only is very limited and the small variation on Day 14 was still within the range of SEM. Figure 9B indicates that repeated sedation using ketamine and xylazine or isoflurane does not change the ABR threshold in the present study. Therefore, we believe our AEP results correctly reflected the therapeutic function of ABR in blast-damaged chinchilla ears.

Fig. 9.

Fig. 9

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The ABR threshold was measured from A drug control (mean ± SEM, n = 6 ears) and B sham control (mean ± SEM, n = 4 ears) groups. In the drug control group, the measurement was conducted on Days 1(Pre), 4, 7, and 14. In the sham control group, the measurement was only conducted on Days 1(Pre), 4, and 7

The second limitation was the measurement and analysis could not thoroughly explain the detailed mechanism of how the post-blast hearing restoration was facilitated by the liraglutide treatment. Given the great complexity of the blast-induced hearing damage which might include the rupture of TM, disarticulation of the ossicular chain, loss of hair cells, excitotoxicity to the spiral ganglion neurons, and damage to the auditory nerve and CAS, our characterization was rough, and the results were preliminary (Cho et al. 2013; Han et al. 2020; Hickman et al. 2018; Race et al. 2017). Although the ABR threshold, wave I amplitude, and IC histology showed that the hearing restoration was improved possibly in PAS and CAS, we could neither evaluate the contribution of each part of the auditory system nor explain the correlation between them. Our histological results, in addition, are qualitative and from only three animals per group and therefore require further improvement to provide solid evidence on drug treatment-induced changes along the auditory pathway. Histological studies from a larger sample size and on spiral ganglion neurons, hair cells, ribbon synapses, middle genicular body, and auditory cortex could provide a series of pathological evidence to support our findings observed from ABR. Moreover, the distortion product otoacoustic emission (DPOAE) was also a critical measurement to assess the blast-damaged hearing function, but it relied on the TM situation, which was ruptured in the present study. If the condition of the TM allows, we will include the DPOAE measurements in future studies. The statistical plan will be further improved to investigate the correlation between different results and the contribution of each factor to the hearing function change using the mixed-effect model.

Conclusions

The present study aimed to establish an animal model of chinchilla for repeated low-level blast exposures and use this model to investigate the potential therapeutic function of liraglutide. The ABR was measured in blast control and pre- and post-blast treated animals on Day 1 and over a time period of 14 days, and the brain tissues (IC) were used for histology study. Results indicated that 6 blasts of 3–5 psi (or 21–35 kPa) were able to induce solid damage in the chinchilla auditory system with significant but limited self-recovery over 14 days. The severity of hearing damage reflected by the ABR threshold elevation, ABR wave I amplitude reduction, and levels of apoptosis in the inferior colliculus measured within 14 days after blast exposures from liraglutide-treated animals were lower than those measured from the blast controls, which indicated that the liraglutide ameliorated the hearing function in blast-damaged chinchillas at a certain level. This statement was supported by statistically significant effects of the liraglutide treatment on ABR threshold shift at 2 and 8 kHz increased from Day 7 to Day 14 and ABR wave I amplitude reduction on Days 4, 7, and 14. AEP measurements and histology results indicated the liraglutide treatment potentially facilitated the functional recovery of multiple regions in the auditory system, but the detailed mechanism and contribution of different regions required further investigation. This study bridges the gap between TBI and hearing impairment and suggests a possible intervention for blast-induced hearing loss for Service members.

Acknowledgements

We gratefully acknowledge Dr. Emi Kiyotake, Dr. John Clegg, and Dr. Michael Detamore from Stephenson School of Biomedical Engineering at the University of Oklahoma for sharing their equipment, environment, and technology for the IF study presented in this paper.

Appendix

Figure 10 presents the screenshots of the ABR waveforms measured at different epoch numbers ranging from 16 to 908. The waveforms were recorded at 8 kHz, 95 dB. As can be seen from the figure, some random fluctuations were observed at 16, but it was hard to observe changes with the epoch number changing from 142 to 908. Therefore, 150 was an adequate number of sweeps to produce replicable ABR waveforms and allowed for threshold and peak picking analysis in our experimental paradigm.

Funding

This study was supported by the Department of Defense (DOD) grant W81XWH-19–1-0469.

Declarations

Conflict of Interest

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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