Near-infrared-light pre-treatment attenuates noise-induced hearing loss in mice

Auditory brainstem responses

Frequency-specific auditory brainstem responses (ABR) were recorded for 5 kHz spaced frequencies, between 5 and 40 kHz inclusive, in adult female mice of the NMRI strain (Charles River Laboratories, Erkrath, Germany). The mice were aged 10 to 11 weeks. All mice were caged in groups of 6 animals. In addition to a resting house, each cage contained enrichment features (plastic tunnels and nesting material). The animals had permanent access to food and water and were kept in a 12/12 h dark/light regime. All mice were divided among the experimental groups so that no animal survived. The study protocol stipulated that animals with a weight loss of more than 20% should be euthanatized by a single injection of 180 mg/kg ketamine and 18 mg/kg xylazine. No animal reached this criterion during the study.

ABR recordings were made under anaesthesia (60 mg/kg ketamine, 6 mg/kg xylazine) two days before and two weeks after a noise exposure. Tone stimuli were presented binaurally at different sound pressure levels (SPL) using a sine-wave generator (FG 250 D, H-Tronic, Germany) and were adjusted with an audio amplifier (AMP-50, Tangent, Denmark). ABR waveforms were collected from the output of a recording amplifier (USB-ME16-FAI-System, Multi-Channel Systems, Germany). Subdermal needle electrodes were placed (Fig. 1A) on the forehead (reference), mastoid (active) and at one foot (ground). For each individual frequency, the peak-to-peak amplitudes within the maximum deflection of wave III/IV were measured for different sound intensities (Fig. 1B). The ABR threshold per frequency was estimated by visual inspection of the series of recordings for each frequency and determining when the wave III/IV was no longer visible.

From these data, threshold differences for each frequency (mean threshold shifts) were calculated between the controls and the noise-exposed animals using the group average values. Results are represented as mean relative hearing loss (±S.E.) in dB for each experimental group compared to the control group.

NIR pre-treatment

Two control groups were formed, one without any noise exposure or NIR pre-treatment (control; n = 7) and one with noise-exposure but without NIR-treatment (noise only; n = 16). The remaining animals were unilaterally treated with NIR-light immediately before noise exposure of different durations, depending on the experimental group: 5 min NIR-treatment (5′ NIR; n = 16), 10 min NIR-treatment (10 ′NIR; n = 16), 20 min NIR-treatment (20 ′NIR; n = 7), 30 min NIR-treatment (30 ′NIR; n = 8), 40 min NIR-treatment (40 ′NIR; n = 8). An adjustable isolated point laser module (DB808-120-3(22 ×65), Picotronic, Germany) was used for the application of near-infrared light having a wavelength of 808nm and a power of 120mW. The laser module was placed at the outer ear canal at exactly the angle that allowed a total coverage of the cochlea with the laser beam of approximately seven mm diameter (Fig. 1C). The power density of the NIR-light was 312 mW/cm². Safety in terms of tissue damage in general, and to the tympanic membrane in particular, has been verified by several other studies using higher power densities. For example Rhee et al. (2013) used a power density of 900 mW/cm², delivered for 60 min/day over10 days). Moreover, Moon et al. (2016) demonstrated tympanic membrane damage resulting from NIR application for power densities above at least 1,137 mW/cm² applied for 30 minutes/day over 14 consecutive days. This previous work dispelled any safety concerns for the NIR power density applied in the present study (Moon et al., 2016).

Noise exposure

Just after receiving its NIR pre-treatment, each animal was exposed for 30 min to noise in a soundproof chamber measuring 0.8 m × 0.8 m × 0.8 m, having a minimal sound attenuation of 60 dB). Noise exposure was made using a broad-band white noise with corner frequencies of 5 and 20 kHz and having a level of 115 dB SPL. Noise was delivered binaurally from a loudspeaker (HTC 11.19, Visaton, Germany) placed above the animal’s head. The speaker was connected to an audio amplifier, which provided the broad-band noise from a DVD source (DK DVD-438, DK, Germany). The noise level was calibrated by using a sound level meter (Voltcraft 329, Voltcraft, Germany) placed close to the animal’s ear. Anaesthesia was controlled through observation via a video camera inside of the lighted chamber. Body temperature was maintained at a constant level of 37 °C using a heating pad.

Histological analysis

After its post-noise exposure ABR recording, each animal was perfused via the left heart chamber with 4% paraformaldehyde (PFA, pH 7.4, Sigma, Germany) in order to fixate the neural tissue. Inner ears were carefully removed from the skull and stored in PFA at 4 °C until further processing. Both cochleae of each animal were prepared for staining. After decalcification of the cochleae for 8 h with the solution renewed after 4 h, using 0.4 M ethylenediaminetetraacetic acid (EDTA, pH 8.0, Roth, Germany), cochleae were dissected twice horizontally to obtain large segments of half turns. Half turns were stained by using the Alexa-fluor-488-phalloidin staining to visualise F-actin compartments in the cytoskeleton. Cochleae were placed in a 24-well plate and washed with 2 ml phosphate buffered saline (PBS, Gibco, USA). Two ml of 0.2% Triton-X (Sigma, Germany) in PBS was added to the cochleae as permeabilization solution and incubated for 30 min on a shaker at room temperature. After washing with 2 ml PBS, 80 µl Alexa-Fluor-488-phalloidin dilution (1:40 in PBS, Molecular Probes, USA) was added and incubated for 60 min on a shaker at room temperature in the dark. All cochlear segments were embedded with RotiMount Fluorocare and diamidino phenylindole (DAPI, Roth, Germany) on microscope slides with coverslips over night at room temperature in the dark.

The stained cochleae were magnified microscopically with fluorescence channels for phalloidin (480 nm) and DAPI (340 nm). The images were digitized with the AxioCam ICc1 (Zeiss, Germany) and the ZEN 2.3 camera software (Zeiss, Germany). The number of missing hair cells was counted by hand on printouts of overlaid DAPI and phalloidin staining pictures. The gaps between cells were marked using a pen so that the number of cells that were missing could be counted.

The basilar membrane’s length was estimated using the ImageJ 1.47d software (National Institutes of Health, USA).

Statistical procedures

The hearing threshold data and cochlear hair cell counts were analysed and compared between the experimental and the control groups by comparing the results using the U-test (not normally distributed data) or the t-test (normally distributed data). Data distribution was tested using the Kolmogoroff-Smirnoff-test. The SPSS software (IBM SPSS Statistics Version 25, IBM Corp., USA) was used for all statistical analyses. The level of significance for all statistical tests was set at p < 0.05. A Bonferroni alpha correction was applied for the use of multiple comparisons.

The Landesamt für Gesundheit und Soziales, Berlin, Germany approved the study protocol (approval number LAGeSo-G 0146/14).

Effect on ABR-threshold shift

All NIR pre-treated groups showed a lower group mean hearing loss following the noise exposure compared to the “noise only”-control group (Fig. 2). A frequency-specific analysis showed significant changes between the controls and the 5′NIR-group for the 5; 35 and 40 kHz frequencies. All other pre-treated groups differed significantly from the noise only controls for every frequency tested (Fig. 2). No statistically significant differences between ABR-thresholds were found for the 10′; 20′; 30′and 40′ NIR-groups. The “control”-group showed only a small, negligible hearing loss.

Effect on amplitude increase of ABR wave IV

Due to the ABR recording electrode positions, ABR-wave IV was the only wave that was robust and prominent in all animals and experimental groups. This is why more detailed ABR-analysis was limited to this wave. Additional wave IV analysis aimed to identify any alteration in central suprathreshold processing resulting from noise exposure, with or without NIR pre-treatment. Wave IV represents bilateral activation of the auditory midbrain level, hence any changes in stimulus-related responses could point to modified central auditory processing. The slope of the wave IV amplitude growth function’s linear part was calculated and the post-exposure slope was subtracted from the pre-exposure slope (Fig. 3). The average slope difference over all frequencies tested for the “10′-NIR”-group (slope difference 0.004  ±  0.005 µV/dB) was more than 10 times lower than for the “noise only”-group (slope difference 0.053 ± 0.005 µV/dB). A higher slope difference indicates a smaller post-noise exposure slope for the amplitude growth function.

Apart from the values of the “5′-NIR”-group (0.047 ± 0.009 µV/dB) all experimental groups showed significantly lower slope differences compared to the “noise-only”-group (Fig. 3).

Effect on cochlear hair cells

No loss of inner hair cells could be detected bilaterally for any of the groups. Figure 4 shows a sample image of the whole cochlear immunofluorescence staining mount. For the noise only control group, a significant decrease in outer hair cell counts was found for both ears (Figs. 5 and 6), compared to the control group where no noise exposure was given. However, the percentage of missing outer hair cells was only 6.5% compared to a normal cochlea (Burda, Ballast & Bruns, 1988). No significant differences were found between the number of absent outer hair cells in any of the NIR pre-treated experimental groups and the “noise only”-group (Fig. 5). This also holds true for the non-pre-treated side (ear) of the animals (Fig. 6). In addition, the mean number of absent outer hair cells was plotted for the entire length of the organ of corti (Fig. 7). These graphs indicate that hair cell loss decreased from base to apex without any specific correlation to the functional analysis (ABR-thresholds). This was found for all experimental and control groups.

Efficiency of NIR pre-treatment

The noise-induced elevation of hearing thresholds, as determined by ABR-measurements, was reduced on average by 24 dB in the NIR-pre-treated groups when calculated across all effective treatment groups and the whole frequency range investigated. This shows that a single pre-treatment has a similar effect to a daily post-traumatic NIR-treatment delivered over 12 consecutive days (Rhee et al., 2012a). However, the energy density of the NIR-treatment in the present study was twice as that used by Rhee et al. (2012a). It should also worth considering that the exposure time in the earlier investigation was 12 ×  60 min (total of 720 min), instead of a single 10 min treatment in the present study. Thus, the total NIR-dosage applied in the post-treatment experiments was much higher than for this pre-treatment study. Another consideration is that the noise exposure differed greatly between the two studies: 115 dB SPL broad-band white noise (5–20 kHz) for 30 min in this study, compared to a 116 dB SPL narrow band noise (centred at 16 kHz (bandwidth 1 kHz) for 6 h in the post-treatment study. However, the effect on hearing thresholds was quite similar for both studies, showing a threshold shift of around 40 dB. This could be related to the different species used in both studies (rats and mice) and their possibly different susceptibility to noise.

Thus, a pre-treatment regime appears to be much more effective than several post-treatments. The reason for this is largely unknown. Earlier studies have shown that pre-conditioning using a very low NIR dose induced a significant decrease in superoxide radicals and NO production for cultured outer hair cells (Bartos et al., 2016). This could have resulted in an enhanced intracellular ATP level compared to the non-pre-treated cells, since superoxide radicals react with NO to form peroxynitrite, which blocks cellular respiration (Srinivasan & Avadhani, 2012). Furthermore, recent studies have revealed that the nuclear factor NF- κB is activated by NIR-treatment. This leads to protection against inducible NO synthase-triggered oxidative stress and, subsequently, to a reduced caspase-3-mediated apoptosis (Tamura et al., 2016). The time point of NIR-treatment plays an important role in the activation or inactivation of NF- κB. The difference being the presence or absence of inflammation in the cells at the time of the treatment (Chen et al., 2011). Pro inflammatory cytokines are activated during a noise exposure (Nakamoto et al., 2012). Thus, the effects of NIR pre-treatment are not affected by these inflammatory processes. This could explain why NIR pre-treatment is much more efficient than post-traumatic treatment.

Effect on cochlear hair cells

Apart from the quite similar outcome found for hearing preservation in our study, there are some differences in outer hair cell counts compared to earlier investigations. Former studies described a significant reduction of outer hair cells and no loss of inner hair cells due to noise exposure (Rhee et al., 2012a; Tamura et al., 2015). These reports are in line with our present results. However, NIR post-treatment led to a significant reduction of outer hair cell loss in the medial part of the cochlea. The basal and apical parts showed no statistically significant protective effect from NIR-light on hair cell loss (Rhee et al., 2012a). Another study which applied the NIR treatment after noise exposure found a protection of outer hair cells in all parts of the cochlea (Tamura et al., 2015). However, both studies applied a more intense noise trauma than in the present investigation. The relative loss of outer hair cells was very small in the present study compared to the earlier work: 6.5% vs approx. 30% (Tamura et al., 2015). Hence, a significant rescue of outer hair cells was harder to achieve in the present study. The present results show a trend for hair cell protection, even if no correlation between hair cell loss along the cochlea and frequency specific ABR-thresholds could be found. The bilateral effect is not contradictory for this hypothesis since both sides (left and right cochleae) are likely simultaneously treated due to the surprisingly good penetration of NIR-light of 808 nm through the small mouse skull (Lee et al., 2016a). Additionally, the discrepancy between hair cell counts and hearing thresholds, as found in the present study, is also well known for both animals and humans (Le Calvez et al., 1998; Landegger, Psaltis & Stankovic, 2016). The correlation coefficient between hearing threshold shift and cochlear hair cell loss was described as only around 0.5 with 1.0 being a perfect correlation (Landegger, Psaltis & Stankovic, 2016).

It is possible that functional damage to hair cells occurred in the present experiments (e.g., stereocilia Wang, Hirose & Liberman, 2002), or that other cochlear structures are responsible for the hearing loss and the NIR-related preservation observed. Suitable candidates which are known to be influenced by NIR-light are spiral ganglion cells, pre- and postsynaptic structures at the inner hair cells (ribbon synapse), cochlear neurofilaments and fibrocytes of the lateral wall (Tamura et al., 2016; Lee et al., 2016b; Lee et al., 2019; Chang et al., 2019). A first sign of such effects could be the relationship between the amplitude of a summating potential, which is generated within the auditory brainstem/midbrain (e.g., ABR wave IV), and the stimulus intensity. The significant differences that we describe between the slopes of this relationship in animals with and without a NIR-pre-treatment supports this hypothesis. The lower slope difference in NIR pre-treated animals indicates a near-normal sensitivity to an increased stimulus intensity and thus a near-normal loudness growth. This could be related to a preservation of cochlear structures by NIR-light.