Due to the prevalence of war and increased numbers of terrorist attacks, blast injury is a critical issue in hearing research. Organs of the auditory system, including the tympanic membrane, cochlea, and central auditory pathways, are most commonly damaged by blast overpressure. For example, tympanic membrane perforation, temporary and permanent hearing loss, tinnitus, and hyperacusis have all been reported as consequences of blast exposure.  To study pathophysiology of the inner ear after blast injury, several animal models have been established. Mammals, such as rats and mice, are commonly used as blast-induced hearing loss models. [2–4] However, no model of hearing recovery or regeneration after blast wave-induced hearing loss has been reported thus far.
Zebrafish have emerged as a powerful vertebrate model to study regeneration, as most zebrafish organs and tissues can regenerate after injury. For example, a zebrafish can replace up to one-fifth of its heart ventricle after injury, [5–7] and they have seemingly unlimited potential to regenerate caudal fins following amputation. [8,9] In addition, the adult zebrafish brain exhibits a remarkable ability to restore the central nervous system (CNS). [10,11] Importantly, zebrafish possess hair cells in the inner ear and lateral line system. [12,13] These two types of hair cells can regenerate and replace themselves throughout their lives, making zebrafish a good model for hair cell regeneration research.
To study inner ear hair cell regeneration, ototoxic agents, such as aminoglycoside antibiotics and platinum-based cancer therapeutics, and acoustically-induced zebrafish or goldfish models have been established. [7,14,15] However, the former require repeated intraperitoneal administration and the latter involve long-term exposure, generally 36 to 60 hours. [16–18]
The aim of this study was to establish a new animal model for hearing loss and regeneration research.
Materials and methods
The wild type AB line zebrafish [19,20] (China Zebrafish Resource Center, Wuhan, China) were bred and reared in filtered aquaria at 28 ± 1°C in our fish colony. Fish were maintained under a 12:12-hour light-dark cycle and fed twice per day. In this study, 150 mixed sex adult fish (4–6 months of age) were used and randomly assigned to 5 groups (G15, G20, G25, G30 and control) with 30 fish in each group. All animal experiments were approved by the Shanghai 6th Hospital Animal Care and Use Committee, China (approval No. 2017-0196) on February 28, 2017 and performed in agreement with the National Institutes of Health guidelines (NIH Publication No. 85–23, revised 1996). All efforts were made to minimize both animal suffering and the number of animals used for experiments.
Blast wave exposure
We developed an underwater blast wave exposure apparatus, which includes a computer system, power control, blast wave generator, tank, and zebrafish cage (Fig. 1A). Blast intensities produced by the generator were measured using a hydrophone (8105; Brüel & Kjær, Nærum, Denmark). All blast wave exposures were carried out in a rectangular water tank (200 cm × 150 cm × 120 cm) in a sound-attenuating booth. Zebrafish were secured in a gauze cage (20 cm × 20 cm × 20 cm) positioned 25 cm away from the blast wave generator and 30 cm underwater (Fig. 1A). Based on the results of preliminary experiments testing different intensities, we determined appropriate exposure parameters. We used 4 experimental groups, which were subjected to 15 (G15), 20 (G20), 25 (G25), or 30 (G30) blasts, respectively, with 1-second intervals at 300J, and a control group (Con) without treatment. The exposure procedure generally lasted 1 to 2 minutes.
Auditory evoked potential recordings
Auditory evoked potential (AEP) recordings of zebrafish were performed according to a previously described method.  Zebrafish were temporarily anesthetized and briefly immobilized in a solution of 0.01% tricaine methanesulfonate (MS-222; Sigma-Aldrich, St. Louis, MO, USA) before being mounted on a fixation device. Three electrodes (tungsten electrode, 0.005-inch diameter; 5-MΩ resistance; A-M Systems Inc., Carlsborg, WA, USA) were used for recording. The recording electrode was inserted into the dorsal surface of the fish, just behind the brainstem, while the reference electrode was inserted into the muscles of the dorsal fin and the grounding electrode was inserted near the tail (Additional Figure 1, http://links.lww.com/JR9/A5). Stimuli consisted of tone bursts at 100, 200, 400, 600, 800, 1000, 2000, 4000, 6000, and 8000 Hz.
Fish were immersed and euthanized in a solution of MS-222. Their heads were removed and fixed with 4% paraformaldehyde solution overnight. Heads were rinsed four times for a duration of 10 minutes in 0.1 M phosphate-buffered saline (PBS), before the inner ears were dissected from them. The auditory system of teleost fishes consists of three sensory otolithic end organs, the saccule, lagena, and utricle. [22–24] Previous studies have suggested that the saccule is the major auditory organ in teleost fish, while the lagena plays roles in both hearing and orientation, and the utricle is primarily a vestibular organ. [25,26] Sensory epithelia of the inner ear were dissected according to a previous report.  Epithelia were permeabilized using 1% Triton X-100 solution at room temperature for 2 hours. Next, samples were blocked with 10% goat serum in PBS for 1hour. To assay for cell death, TUNEL staining was performed in accordance with the manufacturer's instructions (Roche, Basel, Switzerland). TUNEL-positive (TUNEL+) cells in sensory epithelia or brain sections were counted using ZEN 2011 software (Carl Zeiss Microimaging, Oberkochen, Germany) after imaging.
Cell proliferation assay
To assay for cell proliferation, fish were anesthetized in 0.02% MS-222 and injected intraperitoneally with 10 μL of 10 mg/mL 5-ethynyl-2′-deoxyuridine (EdU) using a WPI UltraMicroPump III (WPI, Worcester, MA, USA). Sensory epithelia of the inner ear were prepared as described above. EdU analysis of sensory epithelia in the inner ear or brain sections was performed according to the manufacturer's protocol for ClickIt EdU Imaging (Invitrogen, Carlsbad, CA, USA), 24 hours after injection. EdU-positive (EdU+) cells in sensory epithelia or brain sections were counted using using ZEN 2011 software (Carl Zeiss Microimaging, Oberkochen, Germany) after imaging.
Hair bundles were stained with Alexa Fluor 488-conjugated phalloidin (Invitrogen). Sensory epithelia were incubated in 1:100 fluorescein phalloidin solution (Invitrogen) in PBS in a dark box at room temperature for 20 minutes. Following incubation, sensory epithelia were mounted on glass slides using Prolong Gold Antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen).
Brains were dissected from the skull and incubated in 4% paraformaldehyde at 4°C for 3 hours. Next, they were washed in PBS, and cryoprotected twice: first in 10% sucrose overnight, and then in 20% sucrose overnight at 4°C. Brains were embedded in tissue freezing medium (TFM, Triangle Biomedical Sciences Inc., Durham, NC, USA) and stored at −80°C until sectioning. Frozen sections were cut using a cryostat (CM 3050S; Leica, Wetzlar, Germany) at 16-μm thickness for TUNEL and EdU assays.
Image acquisition and processing
Confocal images were acquired using a Zeiss LSM 710 microscope (Carl Zeiss Microimaging, Oberkochen, Germany) using a 20 × or 100 × oil immersion objective lens. DAPI (405 nm), Alexa Fluor 488 (488 nm), and DyLight 549 after TUNEL and/or EdU labeling were visualized via blue-violet diode, argon ion, and green helium-neon excitation, respectively. Adobe Photoshop CS4 (Adobe Inc, San Jose, CA, USA) was used to generate figures.
To analyze the significance of TUNEL+ and EdU+ cell numbers between exposed and control groups, P values were determined with GraphPad Prism (Version 5.01; GraphPad Software Inc., San Diego, CA, USA) using an unpaired Student's t-test.
Blast wave exposure leads to systemic injury
In this study, an underwater blast wave generator was prepared and zebrafish were subjected to blast wave exposure (Fig. 1A). When the energy was set at 300J, the first pulse peak pressure was recorded as 224 dB and 160 kPa, measured at 25 cm away from the front of the transmitting end of the generator (Fig. 1B). Following exposure, fish were able to maintain their balance, but swam slowly and seemed insensitive to knocking sounds compared with the control group (Additional video 1, http://links.lww.com/JR9/A6). They recovered normal swimming behavior by 3 to 4 days post blast wave exposure (dpb). Zebrafish in the four exposed groups showed varying injures. In the G30, blast wave exposure led to abdominal hemorrhage in 95% of zebrafish, with a 2% mortality rate (Fig. 2 and Additional video 1, http://links.lww.com/JR9/A6).
Blast wave exposure causes hearing loss
To investigate hearing loss post blast wave exposure, the hearing sensitivity of zebrafish was examined by analyzing AEPs. The AEP testing apparatus used herein is a system that we developed for zebrafish hearing testing, and has been described previously  (Additional Figure 1, http://links.lww.com/JR9/A5). We examined the hearing ability of zebrafish using frequency-specific AEPs from 100 to 8000 Hz in the four groups (G20, G25, G30, and control; please note G15 was not included for AEP analysis) at 1, 7, 14, and 35 dpb (Fig. 3A). There was a dose-effect relationship between blast intensity and AEP threshold. Indeed, thresholds of the three exposed groups were significantly higher than those of the control group, and their hearing frequency response ranges were also narrower than controls. G30 showed no response to stimuli of any frequency at 1 dpb, and responded only to a 2000-Hz stimulus at 7 dpb. AEP and hearing range thresholds of each group recovered gradually over 35 dpb. Of the three exposed groups, G30 did not respond to stimuli below 400 Hz at 35 dpb, while G25 returned to a normal hearing range with significantly higher thresholds than the control group (P < 0.05), and G20 recovered an almost normal hearing range and thresholds (P > 0.05; Fig. 3A).
Figure 3B shows AEP waveforms at 1 dpb for G20 and control groups at 600 Hz and 140 dB, as well as G20, G25, and control groups at 2000 Hz and 145 dB. At these two frequencies, the latency of exposed groups was prolonged (600 Hz, Con vs G20, P < 0.001; 2000 Hz, Con vs G20, P < 0.001; Con vs G25, P < 0.05) and the amplitude was significantly decreased compared with the control group (600 Hz, Con vs G20, P < 0.05; 2000 Hz, Con vs G20, P < 0.05; Con vs G25, P < 0.01; Fig. 3B & C).
Cell apoptosis after blast wave exposure
To characterize apoptosis, we performed a TUNEL assay on the three sensory epithelia of the inner ear, taken from G30 and control groups at 12 hours post blast wave exposure (hpb) and 1, 3, and 5 dpb (Figs. 4 and 5). At 12 hpb, numbers of saccular and lagenar apoptotic cells reached their peaks, and then decreased but remained significantly higher than observed in the control group until 3 dpb (P < 0.01; Figs. 4G and 5E). In Figure 4J–L, we can see that TUNEL+ cells include both supporting cells and hair cells. For utricular epithelia, the apoptotic peak also occurred at 12 hpb, but returned to normal level at 3 dpb (P > 0.05), which was earlier compared with the saccule and lagena (Fig. 5F). At G30, TUNEL+ cells gathered at the edge of saccular and lagenar epithelia, whereas few apoptotic cells were observed in the control group (Figs. 4A–C & 5A–D).
Cell proliferation after blast wave exposure
We also observed cell proliferation in the inner ear. Figure 6 shows saccular and lagenar sensory epithelia at 3 dpb. It is important to note that zebrafish hair cells regenerate throughout their lives. The control group showed a normal proliferation rate, while exposed groups showed significant differences compared with controls (Fig. 6A–F and 6G–J). Proliferation of saccular and lagenar sensory epithelia peaked at 3 dpb (P < 0.001), and then returned to a normal rate by 7–10 dpb (Fig. 6K–L).
As blast wave exposure can also result in brain injury, we investigated cell apoptosis and proliferation further in zebrafish brain cryosections at 3 dpb. Our results demonstrated that blast wave exposure led to increased number of TUNEL+ cells, with up to a 748% increase compared with controls (P < 0.001). Results also suggested blast wave exposure suppressed the proliferative capacity of cells by 347% in middle brain slices compared with controls (P < 0.001; Fig. 7).
In this study, we developed a zebrafish model induced by underwater blast wave exposure. Using the AEP technique, we examined hearing ability changes in zebrafish and assessed cell death and regeneration of sensory epithelia of inner ear and brain tissues following exposure. Our zebrafish model underwent severe hearing loss as a result of peripheral hearing damage and central deficits, followed by the recovery of hearing because of its great regeneration potential.
Animal models induced by blast wave exposure
The ear is the most common organ affected by blast injury because it is the most sensitive pressure transducer found in the body. Blast overpressure to the ear results in sensorineural hearing loss, which is untreatable and often associated with a decline in quality of life. [28,29] Therefore, animal models are necessary to investigate biological mechanisms involved in blast wave injury to the cochlea and brain.
Existing blast wave generators are predominantly designed for mammals. For example, Newman et al  reported a low-cost method of generating blast waves in a typical laboratory setting. In that study, blast wave exposure caused a significant reduction in distortion product otoacoustic emission amplitude, extensive hair cell loss, and long-term suppression of hippocampal neurogenesis in blast-exposed rats. Niwa et al  introduced a laser-induced blast wave rat model and observed pathophysiology of the inner ear following exposure. The results revealed that threshold elevation of the auditory brainstem response after blast exposure primarily resulted from hair cell dysfunction induced by stereociliary bundle disruption. Other studies have used mice and chinchillas to mimic blast trauma. [4,32] However, no model of hearing recovery or regeneration after blast wave-induced hearing loss has been reported thus far.
Hair cell regeneration, a major event in hearing recovery, is a core issue in hearing research. Although limited hair cell regeneration has been reported in mammalian utricular sensory epithelia, [33,34] hair cell regeneration does not appear to take place in postnatal cochlear sensory epithelia in mammals.  Therefore, model organisms capable of regenerating are of utmost importance for understanding the mechanisms of regeneration programs. Zebrafish have great potential to regrow injured organs and tissues, including hair cells. Two commonly used models for hair cell regeneration are induced by ototoxic drugs or acoustic overstimulation. [14,17] Drug-induced models are generally used to study lateral line hair cells after exposure to drugs in vitro.  However, this method is inconvenient for targeting adult inner ear hair cells because it requires injection in vivo. In this context, researchers must administrate ototoxic drugs via intraperitoneal injection, one by one, repeatedly. Alternatively, generating noise-induced hair cell regeneration models takes a considerable amount of time, usually more than 36 hours.  As such, there are currently only a few published studies exploring noise exposure paradigms in zebrafish. [16,18,35,37] We previously used a noise-induced zebrafish model to evaluate their hearing regeneration capability. The traumatizing tone, which was 150 Hz at 160 dB per Pa for 60 hours, was delivered via an underwater speaker. Threshold shifts were primarily observed on the same day as noise exposure and were partially recovered 2 days later.  However, this model entails serious consumption or damage of hardware, such as speakers and Tucker-Davies Technologies systems. More importantly, this model exhibits weak and transient hearing loss phenotypes. Therefore, we hoped that blast injury could generate a model with more distinct peripheral hearing damage and central deficits than noise-induced models.
There are four different mechanisms of damage after blast exposure. [38–40] Primary blast injury, which is caused by the direct effect of the high overpressure wave on tissue, is the main mechanism in our zebrafish model. Gas-filled structures, such as the inner ear, lungs, and gastrointestinal tract in mammals, are most susceptible to primary blast injury as a result of their interactions with the surrounding air. In this study, 95% of zebrafish exhibited abdominal hemorrhage, with a 2% mortality rate at G30 (Fig. 2 and Additional Movie 1, http://links.lww.com/JR9/A6), and almost every exposed fish showed varying degrees of hearing loss (Fig. 3). These data suggest that the blast injury in this model was uniform, both intra-group and inter-batch.
Hearing loss and recovery
Previous studies on zebrafish hearing following acoustic trauma did not employ auditory electrophysiological techniques, and instead only reported the morphology of sensory epithelia in the inner ear. [16,18] in a study of goldfish, a close relative of zebrafish, hearing following noise overstimulation at different frequencies (100, 800, 2000, and 4000 Hz) elicited increases in all AEP thresholds compared with a control, although the hearing sensitivity range was unchanged (100–4000 Hz).  An aminoglycoside-induced hair cell death model of adult zebrafish also exhibited significantly elevated AEP thresholds with an unaltered hearing sensitivity range (100–4000 Hz).  Similarly, a significant auditory threshold elevation between 300 and 600 Hz with an unchanged frequency range (100–3000 Hz) was observed in a Gentamicin-treated goldfish model.  In contrast, our results showed distinct functional changes or deficits in hearing ability post blast injury and during recovery. AEP analysis demonstrated that blast wave exposure causes hearing damage, especially at G30, in which zebrafish only responded to 2000-Hz stimuli at 7 dpb (Zebrafish have the best hearing ability from 600 to 1000 Hz, so it is unclear why 2000 Hz is exceptional in this regard). In the present study, the observed functional changes and longer recovery period, generally more than 35 days, indicate more severe systemic damage compared with drug or noise-induced models. Furthermore, AEP threshold recovery pattern ranged from middle-frequency sounds (approximately 2000 Hz) to both ends of the normal range. Notably, the ability to hear high frequencies (4000–8000 Hz) seemed to recover faster than the ability to hear low frequencies (100–600 Hz). These patterns of functional damage and recovery, which have not been previously reported, also suggest that damage existed not only in the inner ear but also in the brain or other auditory pathways, as hair cell proliferation was restored to normal levels within 7 to 10 dpb in this study.
In a noise-induced zebrafish model, saccular epithelia exhibited hair bundle damage and reduced stereocilia density immediately after sound exposure.  In addition, a variety of abnormal structures were observed in the region of hair bundle loss during recovery, including short or thin hair bundles, putative lesions, and bundle-less cuticular plates. Differential susceptibility of sensory hair cells to treatments was demonstrated in noise and drug-induced fish models,  and was also observed in rats subjected to blast injury. Niwa et al  showed that disturbed stereocilia present after blast exposure were mostly observed in the outermost row, whereas stereocilia in the inner and middle rows remained intact. However, we found no significant stereocilia damage in our blast wave-induced zebrafish model, indicating that noise and blast waves have different damage mechanisms. [42,43]
Although we observed no damage to hair cell bundles, we found substantial cell death following blast wave exposure. TUNEL+ staining revealed a unique pattern of apoptotic cells (Fig. 4) gathered at the edge of both saccular and lagenar sensory epithelia, which may indicate that these marginal cells are more sensitive to blast wave exposure. However, this pattern of apoptosis was not observed in the utricle, where fewer TUNEL+ cells were found compared with saccular and lagenar epithelia. These results indicate that blast waves affect tissues differently across species, depending on ear structure. Alternatively, it is well known that unlike mammals, zebrafish have an inner ear without external or middle ears, which may be one reason why damage phenotypes differ from mammal models.
According to a previous report, an aminoglycoside-induced hearing loss zebrafish model exhibited approximately 70 TUNEL+ cells in the saccule at 4 hours post treatment, and 60 TUNEL+ cells at 24 hours post treatment. In contrast, our blast-induced model demonstrated many more TUNEL+ cells, approximately 436 at 12 hpb, suggesting more serious hearing impairment. However, as no data quantifying apoptotic cells in the inner ear are available from noise-induced zebrafish models, we cannot make a direct comparison.
Zebrafish continue to add to and replace inner ear hair cells and neuromasts throughout their lives. As expected, low levels of proliferation were observed in the inner ear of untreated controls. In noise-exposed zebrafish, cell proliferation peaked at 2 dbp with approximately 60 EdU-labeled saccular cells,  whereas our model reached a regenerative peak at 3 dpb, with approximately 515 EdU+ cells, indicative of a more active proliferating state.
Hair cell regeneration in our model generally recovered to a normal level within 10 dpb, although AEP thresholds remained higher than observed in the control group. This indicates that other tissue damage was present, for example in the conducting system or CNS. As such, we also examined the brain and found reduced neurogenesis and/or regeneration in G30 brain sections at 3 dpb compared with controls.
Altogether, we have developed a reliable, convenient, and efficient blast wave-induced zebrafish model for studying hearing loss and regeneration.
SY and HS conceived and designed the research. JW and ZY performed the experiment and wrote the manuscript. DY, YX, and JW calibrated the speakers and analyzed the results. KL made the illustration.
This work was supported by the State Key Program of National Natural Science Foundation of China (No. 81530029 to SY), International Cooperation and Exchange of the National Natural Science Foundation of China (No. 8171001156 to SY), National Natural Science Foundation of China (No. 81771007 to JW), the Major Program of Shanghai Committee of Science and Technology of China (No. 14DJ1400202 to SY).
Institutional review board statement
All experiments were approved by the Shanghai 6th Hospital Animal Care and Use Committee, China (approval No. 2017-0196) on February 28, 2017.
Conflicts of interest
The authors declare that they have no conflicts of interest.
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blast wave; zebrafish; hearing loss; brain injury; auditory evoked potential; hair cell; apoptosis; regeneration
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