Brain Research 1646 (2016) 467–474
Contents lists available at ScienceDirect
Brain Research
journal homepage: www.elsevier.com/locate/brainres
Research report
Photobiomodulation rescues the cochlea from noise-induced hearing
loss via upregulating nuclear factor κB expression in rats
Atsushi Tamura a,n, Takeshi Matsunobu a, Risa Tamura b, Satoko Kawauchi c, Shunichi Sato c,
Akihiro Shiotani a
a
Department of Otolaryngology – Head and Neck Surgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-0042, Japan
Department of Physiology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-0042, Japan
c
Division of Bioinformation and Therapeutic Systems, National Defense Medical College Research Institute, 3-2 Namiki, Tokorozawa, Saitama 359-0042,
Japan
b
art ic l e i nf o
a b s t r a c t
Article history:
Received 20 April 2016
Received in revised form
16 June 2016
Accepted 20 June 2016
Available online 21 June 2016
Photobiomodulation (PBM) is a noninvasive treatment that can be neuroprotective, although the underlying mechanisms remain unclear. In the present study, we assessed the mechanism of PBM as a novel
treatment for noise-induced hearing loss, focusing on the nuclear factor (NF)-κB signaling pathway.
Sprague–Dawley rats were exposed to 1-octave band noise centered at 4 kHz for 5 h (121 dB). After noise
exposure, their right ears were irradiated with an 808 nm diode laser beam at an output power density of
165 mW/cm2 for 30 min a day for 5 consecutive days. Measurement of the auditory brainstem response
revealed an accelerated recovery of auditory function in the groups treated with PBM compared with the
non-treatment group at 4, 7, and 14 days after noise exposure. Immunofluorescent image analysis for
inducible nitric oxide synthase and cleaved caspase-3 showed lesser immunoreactivities in outer hair
cells in the PBM group compared with the non-treatment group. However, immunofluorescent image
analysis for NF-κB, an upstream protein of inducible nitric oxide synthase, revealed greater activation in
the PBM group compared with the naïve and non-treatment groups. Western blot analysis for NF-κB also
showed stronger activation in the cochlear tissues in the PBM group compared with the naïve and nontreatment groups (p o0.01, each). These data suggest that PBM activates NF-κB to induce protection
against inducible nitric oxide synthase-triggered oxidative stress and caspase-3-mediated apoptosis that
occur following noise-induced hearing loss.
& 2016 Elsevier B.V. All rights reserved.
Keywords:
Photobiomodulation
Noise-induced hearing loss
Nuclear factor κB
Inducible nitric oxide synthase
Cleaved caspase-3
1. Introduction
Noise-induced hearing loss (NIHL) is a major source of hearing
disability in adults worldwide (Nelson et al., 2005). At the cellular
level, noise-induced cochlear damage consists of metabolic disruption, which includes ischemia (Nuttall, 1999), excitotoxic damage (Puel et al., 1998), and metabolic exhaustion (Chen et al.,
2000). Hearing loss caused by noise damage to auditory hair cells is
normally irreversible, as mammalian hair cells do not regenerate.
Once activated, this metabolic disruption results in permanent hair
cell death, which can occur via apoptosis (Nicotera et al., 2003).
Abbreviations: ABR, auditory brainstem response; iNOS, inducible nitric oxide
synthase; PBM, photobiomodulation; NF-κB, nuclear factor kappa B; NIHL, noiseinduced hearing loss; OHC, outer hair cell; p-Akt, phospho-Akt; ROS, reactive
oxygen species; RNS, reactive nitrogen species; SGC, spiral ganglion cell
n
Corresponding author.
E-mail address: [email protected] (A. Tamura).
http://dx.doi.org/10.1016/j.brainres.2016.06.031
0006-8993/& 2016 Elsevier B.V. All rights reserved.
Photobiomodulation (PBM), also referred to as low-level laser
therapy, has previously been applied as a noninvasive treatment
for promoting cell regeneration and repair (Huang et al., 2009;
Tata and Waynant, 2011). The US Food and Drug Administration
has approved PBM for the treatment of wound healing, chronic
pain, musculoskeletal complications, and other diseases (Conlan
et al., 1996; Streeter et al., 2004). The power of low-level lasers
varies from 10 to 1000 mW/cm2 in the continuous mode. The
wavelengths used for treatment extend from the visible
(λ ¼400 nm) to the near-infrared (λ ¼ 1000 nm) ends of the
spectrum.
The exact protective mechanisms of PBM remain unclear. Recently, we reported that following noise overstimulation, PBM can:
(1) accelerate recovery of auditory function; (2) attenuate loss of
outer hair cells (OHCs), which function as acoustic pre-amplifiers;
(3) inhibit inducible nitric oxide synthase (iNOS), which produces
large amounts of neurotoxic reactive oxygen species (ROS) and
reactive nitrogen species (RNS); and (4) suppress the activation of
468
A. Tamura et al. / Brain Research 1646 (2016) 467–474
caspase-3, the major apoptotic effector protease (Tamura et al.,
2015). Recent studies have suggested that PBM affects the activation of nuclear factor (NF)-κB transcription factor (Assis et al.,
2012; Rizzi et al., 2006), which plays an important role in iNOS
expression after noise exposure (Masuda et al., 2006; Yamamoto
et al., 2009). However, the exact role of NF-κB in the cochlea following noise exposure, and the changes in NF-κB after PBM for the
treatment of acoustic trauma, are unknown. Based on previous
reports (Assis et al., 2012; Rizzi et al., 2006), we hypothesized that
PBM modulates the activation of NF-κB after noise overstimulation. Thus, in the present study, we investigated the effects
of PBM on the NF-κB signaling pathway in a rat model of NIHL.
2. Results
2.1. Auditory brainstem response threshold shift
Immediately after noise exposure (day 0), the threshold shifts
were found in non-treatment and PBM groups (Fig. 1). At day 4,
the threshold shift was significantly lower in the PBM group
compared with the non-treatment group at 12, 16, and 20 kHz
(p o0.01 for each) (Fig. 1). At day 7, PBM groups showed a decreased threshold shift compared with the non-treatment group at
12 kHz (p o0.05) and at 16 and 20 kHz (p o0.01 for each) (Fig. 1).
At day 14, PBM groups showed a decreased threshold shift compared with the non-treatment group at 16 (p o0.01) and 20 kHz
(p o0.05) (Fig. 1). Statistically significant differences in threshold
shift were not found at day 28 (Fig. 1). These results indicate that
PBM accelerates recovery of auditory function.
2.2. Immunofluorescent image analysis for iNOS
To investigate the changes of expression of iNOS after noise
exposure, we performed immunofluorescent image analysis of
OHCs in all groups. At 1 h after noise exposure, we observed strong
immunoreactivity for iNOS in the basal, middle and apical turns of
OHCs in the surface preparations (non-treatment group), whereas
less immunoreactivity was observed in the PBM group (Fig. 2).
This indicates that PBM attenuates expression of iNOS at 1 h after
noise exposure.
2.3. Immunofluorescent image analysis for cleaved caspase-3
To investigate the changes of expression of cleaved caspase-3
after noise exposure, we performed immunofluorescent image
analysis of OHCs in all groups. At 8 h after noise exposure, we
observed strong immunoreactivity for cleaved caspase-3 in the
basal, middle and apical parts of OHCs in the surface preparations
(non-treatment group), whereas less immunoreactivity was observed in the PBM group (Fig. 3). These results indicate that PBM
attenuates expression of cleaved caspase-3 at 8 h after noise
exposure.
2.4. Immunofluorescent image analysis for NF-κB
In the organs of Corti, we observed weak NF-κB immunoreactivity in the basal turn of OHCs in the non-treatment and
PBM groups; no obvious differences were observed between the
groups (Fig. 4). In the basal turn of the fibrocytes of the lateral wall
and spiral ganglion cells (SGCs), stronger immunoreactivity for NFκB was observed at 1 h after noise exposure in the PBM group
compared with the non-treatment group (Fig. 4).
Subsequently, we quantified the numbers of NF-κB-positive
Fig. 1. Auditory brainstem response threshold shift is attenuated by photobiomodulation (PBM). Naïve (blue), Non-treatment (red), and PBM (green). PBM attenuates the
noise-induced threshold shift. In the PBM group, PBM significantly attenuated the noise-induced threshold shift at 12, 16, and 20 kHz at days 4 and 7. The values represent
the mean 7 SD. *po 0.05.
A. Tamura et al. / Brain Research 1646 (2016) 467–474
469
Fig. 2. Immunofluorescent image analysis for inducible nitric oxide synthase (iNOS) in surface preparation at 1 h after noise exposure. iNOS (green), F-actin (red), and DAPI
(blue). Strong immunoreactivity for iNOS was detected in the OHCs in the non-treatment group. In contrast, less immunoreactivity was observed in the PBM group. IHC,
inner hair cell; OHC, outer hair cell; PC, pillar cell. Scale bars ¼50 mm.
Fig. 3. Immunofluorescent image analysis for cleaved caspase-3 in surface preparation at 8 h after noise exposure. Cleaved caspase-3 (green), F-actin (red), and DAPI (blue).
Strong immunoreactivity for cleaved caspase-3 was detected in the OHCs in the non-treatment group. In contrast, less immunoreactivity was observed in the PBM group.
IHC, inner hair cell; OHC, outer hair cell; PC, pillar cell. Scale bars ¼ 50 mm.
470
A. Tamura et al. / Brain Research 1646 (2016) 467–474
Fig. 4. Nuclear factor (NF)-κB immunofluorescent image analysis in cryostat sections of the organs of Corti, lateral wall, and SGCs in the basal turn at 1 h after noise exposure.
NF-κB (green), F-actin (red), and DAPI (blue). In the organs of Corti, slight immunoreactivity for NF-κB was observed in the outer hair cell (arrows) in both the non-treatment
and PBM groups. In the fibrocytes of the lateral wall, stronger immunoreactivity for NF-κB was observed (arrows) in the PBM group compared with that in the naïve and nontreatment groups. In the SGCs, stronger immunoreactivity for NF-κB was observed (arrows) in the PBM group compared with that in the naïve and non-treatment groups.
Scale bar¼ 50 mm.
nuclei of OHCs, fibrocytes of the lateral wall, and SGCs in each
group (Fig. 5). In the OHCs, the number of NF-κB-positive nuclei
was increased in the PBM group compared with the naïve and
non-treatment groups, though there was no significant difference
between the non-treatment group and the PBM group (Fig. 5). In
the fibrocytes of the lateral wall and the SGCs, the number of NFκB-positive nuclei was significantly increased in the PBM group
compared with the naïve (p o0.01) and non-treatment groups
(p o0.01) (Fig. 5). These data suggest that PBM activates NF-κB in
the cochlea at 1 h after noise exposure.
2.5. Western blotting for NF-κB and phospho-Akt
We examined NF-κB and phosphorylated serine-threonine kinase (p-Akt) quantitatively because p-Akt is the active form that
regulates several nuclear transcription factors, including NF-κB,
and one of the pro-survival molecules (Kurioka et al., 2014a; Yamauchi et al., 2006). Therefore, in the present study, we evaluated
p-Akt, which is an upstream protein of NF-κB, at 1 h after noise
exposure to compare the effects of PBM between the non-treatment group and the PBM group.
Western blot analysis of NF-κB at 1 h after noise exposure
showed increased NF-κB levels in the non-treatment and PBM
groups (Fig. 6A). NF-κB levels in the PBM group were significantly
higher compared with those in the naïve (p o0.01) and nontreatment groups (p o0.01) (Fig. 6B). By contrast, significant
changes in the p-Akt levels were not observed among all groups
(Fig. 6A). These data suggest that PBM mediates cochlea protection
via NF-κB activation without the p-Akt pathway at 1 h after noise
exposure.
3. Discussion
Little is currently known about the effects of PBM on the cochlea. To our knowledge, this is the first study to demonstrate the
effects of PBM on NF-κB signaling in NIHL. We previously reported
that PBM improved auditory functions, and that these improvements were accompanied by a lower percentage of OHC loss (Tamura et al., 2015). We also reported that PBM (165 mW/cm2) inhibited expression of iNOS and activation of caspase-3 in the organs of Corti and the lateral wall after noise exposure (Tamura
et al., 2015). Based on our previous results, in the present study we
used a laser power density of 165 mW/cm2.
Recently, PBM was reported to modulate NF-κB activation,
which is an upstream protein of iNOS (Assis et al., 2012; Rizzi et al.,
2006). However, the exact role of NF-κB in the cochlea following
noise exposure, and the changes in NF-κB after PBM for the
treatment of acoustic trauma, have not been elucidated. Therefore,
in this study, we mainly investigated the mechanism of PBM, focusing on NF-κB expression, after intense noise stimulation.
The transcription factor NF-κB is induced in response to many
A. Tamura et al. / Brain Research 1646 (2016) 467–474
471
Fig. 5. NF-κB-positive nuclei were quantified in the OHCs, the fibrocytes of the lateral wall, and SGCs in the basal turn. The number of NF-κB-positive nuclei was significantly
increased in the PBM group compared with the naïve and non-treatment groups in the fibrocytes of the lateral wall and SGCs. In the OHCs, the number of NF-κB-positive
nuclei was significantly increased in the PBM group compared with the naïve group. The error bars represent the mean (SD) in all graphs. *po 0.05,**p o0.01.
signals that lead to cell growth, differentiation, inflammatory responses, and regulation of apoptosis (Assis et al., 2012; Masuda
et al., 2006; Rizzi et al., 2006; Yamamoto et al., 2009). NF-κB is
composed of homo- and heterodimers of Rel proteins including
p65 and p50. NF-κB is located in the cytoplasm in an inactivated
state through interaction with inhibitor of κB. Diverse stimulants
lead to the degradation of inhibitor of κB and the activation of NFκB. The activated NF-κB dimer is translocated into the nucleus
where it activates target genes by binding with high affinity to κB
in their promoters. Because activation of NF-κB induces both cytoprotective and cytotoxic proteins, the role of NF-κB in sensorineural hearing loss from noise exposure and administration of
ototoxic drugs is controversial (Jiang et al., 2005; Nagy et al., 2005;
Masuda et al., 2006; Yamamoto et al., 2009).
A previous study reported no differences in the immunofluorescent staining pattern of NF-κB p65 versus p50 after intense noise exposure (Masuda et al., 2006). Therefore, we performed immunofluorescent image analysis for NF-κB using the
p65 antibody. Our immunofluorescent image analysis of NF-κB
revealed strong immunoreactivity in the fibrocytes of the lateral
wall and SGCs in the non-treatment group at 1 h after noise exposure. Our previous and current studies showed strong immunoreactivity for iNOS in the fibrocytes of the lateral wall and
OHCs in the non-treatment group at 1 h after noise exposure
(Tamura et al., 2015). We also showed strong immunoreactivity
for cleaved caspase-3 in the fibrocytes and OHCs in the nontreatment group at 8 h after noise exposure (Tamura et al., 2015).
These results indicate that noise trauma activates NF-κB signaling to produce iNOS-triggered ROS/RNS in the cochlea. These
findings also support previous reports of a link between NF-κB
activation and iNOS expression (Assis et al., 2012; Masuda et al.,
2006; Rizzi et al., 2006; Yamamoto et al., 2009).
Based on previous reports (Assis et al., 2012; Rizzi et al., 2006),
we initially hypothesized that PBM suppresses NF-κB activation,
following the inhibition of iNOS expression and apoptosis. However, we observed that PBM markedly increased NF-κB immunoreactivity in the fibrocytes of the lateral wall and SGCs
especially compared with the non-treatment group. Fibrocytes
play a crucial role in maintaining electrochemical homeostasis in
the cochlea (Hsu et al., 2004). In the organs of Corti, we also detected more immunoreactivity for NF-κB in the PBM group compared with the non-treatment group, although there was no significant difference between these two groups. In the cochlea,
acoustic overstimulation causes morphological and physiological
alterations in the lateral wall, and causes NIHL (Hirose and Liberman. 2003). However, we previously demonstrated that PBM led
to significant decreases of iNOS and cleaved caspase-3 expression
in the lateral wall, and prevented cochlear damage after noise
exposure and NIHL (Tamura et al., 2015). We suggest that PBM
causes activation of NF-κB for protection of the fibrocytes of the
lateral wall and SGCs after noise overstimulation and maintains
electrochemical homeostasis in the cochlea.
Furthermore, western blotting revealed that PBM increased
NF-κB activation in the inner ear. NF-κB activation was significantly greater in the PBM group compared with the naïve and
non-treatment groups. Thus, our data and those from our previous
study (Tamura et al., 2015) suggest that PBM activates NF-κB for
cytoprotection, which is followed by inhibition of iNOS expression
and subsequent caspase-3-mediated apoptosis.
We also investigated Akt, one of the upstream proteins of NFκB (Madrid et al., 2000; Romashkova and Makarov, 1999), to
confirm whether Akt was activated before NF-κB activation for cell
protection following PBM. Akt is a crucial component of a signaling
pathway that regulates cell proliferation and survival via gene
expression and post-transcriptional mechanisms. Akt activation
leads to phosphorylation of numerous downstream proteins and
472
A. Tamura et al. / Brain Research 1646 (2016) 467–474
Fig. 6. Representative western blot for NF-κB and p-Akt. (A) Western blots for NFκB revealed a strong increase after PBM at 1 h after noise exposure. NF-κB expression level was higher in the PBM group compared with that in the naïve and
non-treatment groups. P-Akt western blots revealed a strong increase at 1 h after
noise exposure, with no difference in p-Akt expression levels among all groups. βActin served as the control for protein loading. (B) Expression levels of NF-κB. At 1 h
after noise exposure, NF-κB expression was significantly higher in the PBM group
compared with the naïve and non-treatment groups. The intensities of the NF-κB
bands were divided by their corresponding loading control levels (β-actin). Error
bars represent the mean (SD) in all graphs. **p o 0.01.
also contributes to cochlea survival (Sha et al., 2010). Furthermore,
Akt is considered an important anti-apoptotic factor in many different cell death paradigms (Paez and Sellers, 2003). To determine
possible cytoprotective mechanisms, we examined the phosphorylation of a pro-survival molecule, p-Akt, by western blotting
analysis (Kurioka et al., 2014a; Yamauchi et al., 2006). Our result
revealed that there were no significant differences among the
naïve group, non-treatment group and PBM group, suggesting that
Akt activation might not contribute to the NF-κB activation for
cytoprotection in this experimental condition. Therefore, in our
study, we speculate that the activation of NF-κB by PBM as demonstrated herein might be one explanation for the protective
effects against noise overstimulation.
Previous reports on the effects of PBM on NF-kB expression
have been contradictory (Chen et al., 2011a, 2011b), making it
unclear if PBM produces or reduces NF-κB activation. The difference between these reports is the presence or absence of inflammation in the cells at the time of laser irradiation. We performed PBM within 1 h after noise exposure for 5 h. A previous
study reported that proinflammatory cytokines, such as IL-6 and
IL-1β, are strongly activated at 3 h after noise exposure (Nakamoto
et al., 2012). Therefore, we think it is likely that inflammation had
not occurred or was weak in our experimental condition and that
NF-κB was activated significantly in PBM group compared with
non-treatment group (Chen et al., 2011a).
Additionally, inflammation is induced by NF-κB activation,
depending on the time after stimulation to cells (Chen et al.,
2011b). Thus, we speculate that there is also a condition where
inflammation is suppressed by reduction of NF-κB activation after
PBM. In our present study, the evaluation of inflammation was
very difficult because this was an in vivo study and evaluated very
topical cells. Thus, we could not evaluate the inflammation in the
cochlea after PBM. Previous reports were in vitro studies (Chen
et al., 2011a, 2011b) and used different cells and stimuli from the
present study. This makes it difficult to compare between these
two reports and our study.
Importantly, noise-induced oxidative stress begins early, becoming substantial over time. ROS/RNS production increases
within 1–5 h after noise exposure for up to 7 days, and an antioxidant drug administered for 3 days after noise exposure attenuates ROS/RNS formation and reduces hair cell death and NIHL
(Le Prell et al., 2007; Sha et al., 2001; Takumida et al., 2001; Takumida and Anniko, 2002). Therefore, PBM was provided within
1 h after noise exposure in our studies, and performed at 5 days
after noise exposure for 30 min a day to prevent auditory deficits
(Tamura et al., 2015). In addition, the NF-κB signaling pathway
plays a role in many processes, and we could not evaluate the
influence of PBM on almost all processes in our present study.
Further studies are needed to identify strategies for treatment
frequency and the duration of effect after noise overstimulation,
the precise mechanism underlying the effects of PBM and to validate the therapeutic potential of PBM resuscitation in the clinical
setting.
To summarize, our present data suggest that PBM activates NFκB signaling for cytoprotection, and that PBM can protect against
iNOS-triggered ROS/RNS production and caspase-3-mediated
apoptosis that occur following NIHL. Thus, PBM may be a new
candidate treatment strategy for NIHL.
4. Experimental procedures
4.1. Animals
A total of 69 male Sprague–Dawley rats (150–200 g) with normal Preyer's reflexes were obtained from Japan SLC Inc. (Shizuoka,
Japan) for use in this study. The experimental protocol was reviewed by the National Defense Medical College's Committee for
Ethics in Animal Experiments (Notification No. 13088). All experimental protocols were performed in accordance with their
guidelines and EC Directive 86/609/EEC for animal experiments.
Animals were maintained on a 12-h light/dark cycle (lights on
from 7:00 a.m. to 7:00 p.m.) with room temperature at 21 71 °C.
Animals had ad libitum access to water and food.
The animals were divided into three groups for the assessment
of auditory function: naïve (no noise exposure, n¼5), non-treatment (noise exposure only, n ¼5), and PBM (noise exposure plus
laser irradiation, n¼5). We also divided the animals into three
groups for immunohistochemistry of iNOS: naïve (n ¼5), nontreatment (n¼ 5), and PBM (n ¼5); cleaved caspase-3: naïve
(n¼ 5), non-treatment (n ¼5), and PBM (n ¼5); and NF-κB: naïve
(n¼ 5), non-treatment (n ¼5), and PBM (n ¼5); and for NF-κB and
p-Akt western blotting: naïve (n ¼3), non-treatment (n¼3), and
PBM (n ¼3).
4.2. Noise exposure
Rats were anesthetized by intraperitoneal injection of ketamine
(50 mg/kg) and medetomidine (1.0 mg/kg) for the duration of
noise exposure. They were exposed to 1-octave band noise centered at 4 kHz for 5 h (121 dB) in a ventilated sound exposure
A. Tamura et al. / Brain Research 1646 (2016) 467–474
chamber, following the procedure in our previous studies (Kurioka
et al., 2014b; Tamura et al., 2015). The sound chamber was fitted
with speakers (Model 2380A; JBL, Northridge, CA, USA) driven by a
noise generator (DANAC-31; Danajapan, Tokyo, Japan) and a power
amplifier (D-45; Crown International, Elkhart, IN, USA). Sound
levels were calibrated (Type 6224 Precision Sound Level Meter;
Aco Instruments, Tokyo, Japan) to ensure uniformity within 1 dB at
multiple locations in the sound chamber.
4.3. Auditory brainstem responses
Auditory brainstem responses (ABRs) were measured using a
signal recorder (Synax 1200; NEC, Tokyo, Japan) before and immediately after noise exposure to evaluate auditory function. All
subsequent steps were performed under general anesthesia.
Stainless steel needle electrodes were placed subcutaneously at
the vertex and ventrolateral to the left and right ears. Tone burst
stimuli, 0.2 ms rise/fall time, and 1 ms flat segments at frequencies
of 8, 12, 16, and 20 kHz were generated. The amplitudes were
specified by a sound generator and were attenuated using a realtime processor and programmable attenuator (RP2.1 and PA5;
Tucker–Davis Technologies, Alachua, FL, USA). The sound stimuli
were produced by a coupler-type speaker (ES1spc; Bio Research
Center, Nagoya, Japan). ABR waveforms were recorded for 12.8 ms
at a sampling rate of 40,000 Hz using 50–5000 Hz bandpass filter
settings; waveforms from 256 stimuli at a frequency of 9 Hz were
averaged. ABR waveforms were recorded in descending 5 dB increments from the maximum amplitude until no waveform could
be visualized. The ABR threshold was defined as the lowest stimulus intensity that produced a wave III or IV. Peak III is the
manifestation of neural activity in the cochlear nucleus, and the
anatomical location of the neural generator of peak IV is probably
the superior olivary complex (Kurioka et al., 2014b). Thresholds
obtained immediately before noise exposure were used as the
baseline thresholds for estimating noise-induced threshold shifts.
4.4. Photobiomodulation
PBM was performed under general anesthesia and was initiated
within 1 h after noise exposure, following the procedure in our
previous study (Tamura et al., 2015). An 808-nm continuous wave
diode laser beam (B&W Tek, Newark, DE, USA), transmitted
through an optical fiber, was applied to the right tympanic
membrane through the external auditory canal. The optical fiber
tip was positioned 6 mm from the right tympanic membrane. The
duration of laser irradiation was 30 min a day for 5 consecutive
days in each animal (Tamura et al., 2015). We set the power
density of PBM to 165 mW/cm2, following previous reports (Rhee
et al., 2012; Tamura et al., 2015). We also set spot size to 2 mm, the
total power of PBM to 2.9 mW, and the total energy to 26 J. The
laser power was checked using a photodiode-type laser power
meter (PD300; Ophir Optronics, Jerusalem, Israel) before and after
irradiation.
4.5. Immunofluorescent image analysis for iNOS and cleaved caspase-3 in surface preparations
The peak expression of iNOS can be observed in the organs of
Corti and the fibrocytes of the lateral wall at 1 h after noise exposure (Tamura et al., 2015). Additionally, the peak expression of
caspase-3 can be observed in the organs of Corti and the fibrocytes
of the lateral wall at 8 h after noise exposure (Tamura et al., 2015).
Therefore, in our present study, animals were examined for the
presence of iNOS at 1 h and for cleaved caspase-3 at 8 h after exposure in the presence or absence of PBM. The PBM group received
a single PBM session (165 mW/cm2, 30 min) (Tamura et al., 2015).
473
We also set the spot size to 2 mm, the total power of PBM to
2.9 mW, and the total energy to 5.2 J. The animals in the nontreatment group and the PBM group were exposed to the previously described experimental noise conditions. After decapitation with pentobarbital (100 mg/kg), the temporal bones were
quickly removed and the bullae were opened. The samples were
transferred to 4% paraformaldehyde in 0.1 M phosphate-buffered
saline (PBS; pH 7.4). The bones near the apex and the round and
oval windows were opened, followed by gentle local perfusion
with 2 1 mL 4% paraformaldehyde in PBS. The tissues were kept
in fixative overnight. The cochleae were dissected by removing the
lateral wall bone, lateral wall tissues, and the tectorial membranes.
After washing with PBS, the remaining parts of the cochlea were
incubated in 0.3% Triton X-100 in PBS for 15 min, washed three
times, and incubated in a blocking solution of 0.25% casein in PBS
(Dako, Glostrup, Denmark) to block any nonspecific reactions.
Immunolabeling was carried out overnight at 4 °C with a rabbit
polyclonal iNOS antibody (1:1000; BML-SA200, Enzo Life Sciences,
Farmingdale, NY, USA) or a rabbit polyclonal cleaved caspase-3
antibody (1:100; #9661, Cell Signaling Technology, Danvers, MA,
USA). The tissues were washed three times in PBS and were incubated with a secondary antibody (1:200; Alexa Fluor 488 goat
anti-rabbit; Invitrogen, Eugene, OR, USA). To double stain for
F-actin, we used 1% rhodamine–phalloidin (Invitrogen, Carlsbad,
CA, USA) for 1 h. After rinsing in PBS, the organs of Corti were
mounted on slides containing an anti-fade medium (Vectashield
with 4′,6-diamidino-2-phenylindole (DAPI); Vector Laboratories,
Burlingame, CA, USA). The tissues were observed under an LSM
510 Meta confocal fluorescence microscope (Carl Zeiss MicroImaging, Jena, Germany).
4.6. Immunofluorescent image analysis for NF-κB in cryostat
sections
To investigate the changes of expression of NF-κB after noise
exposure, we performed immunofluorescent image analysis of the
organs of Corti, lateral wall, and SGCs in all groups. We previously
reported that the peak expression of iNOS occurred at 1 h after
noise exposure for 5 h, and we also performed PBM within 1 h
after noise exposure (Tamura et al., 2015). Therefore, in the present
study, we evaluated NF-κB, which is an upstream protein of iNOS,
at 1 h after noise exposure to compare the effects of PBM between
the non-treatment group and the PBM group (165 mW/cm2,
30 min).
Animals were examined for the presence of NF-κB at 1 h after
noise exposure in the presence or absence of PBM. The PBM group
received a single PBM session (165 mW/cm2, 30 min) (Tamura
et al., 2015). We also set the spot size to 2 mm, the total power of
PBM to 2.9 mW, and the total energy to 5.2 J. The tissues were
removed and fixed as described for the iNOS and caspase-3 preparations, then decalcified in 0.1 M ethylenediaminetetraacetic
acid (pH 7.2) for 14 days at 4 °C. After the tissues were incubated in
15% sucrose solution for 36 h, 8-mm frozen sections were prepared.
Immunolabeling was performed the same as for iNOS and caspase3, but with a rabbit polyclonal NF-κB p65 antibody (1:50; #8242;
Cell Signaling Technology). The sections were similarly stained for
F-actin with 1% rhodamine–phalloidin, mounted on slides, and
observed with confocal fluorescence microscopy.
To quantify NF-κB-positive cells, we counted the total number
of NF-κB-positive OHCs and fibrocytes in each image (Kurioka
et al., 2014a). The number of NF-κB-positive SGCs was counted in a
100-mm square area at the center of the spiral ganglion in each
image (Mizutari et al., 2011). Counts were obtained from five cochleae. For each condition, the OHC, fibrocyte, and SGC counts
were obtained from three locations in the basal turn of each cochlea (Kurioka et al., 2014a).
474
A. Tamura et al. / Brain Research 1646 (2016) 467–474
4.7. Western blotting for NF-κB and p-Akt
Animals were examined for the presence of NF-κB and p-Akt at
1 h after noise exposure in the presence or absence of PBM. The PBM
group received a single PBM session (165 mW/cm2, 30 min) (Tamura
et al., 2015). We also set the spot size to 2 mm, the total power of
PBM to 2.9 mW, and the total energy to 5.2 J. The animals were
decapitated at 1 h after noise exposure. The cochlea tissues were
collected, and the tissues from the three cochleae were pooled to
generate one sample. The nuclear extracts were prepared following a
previous report (Nishi et al., 1997). The supernatants of the homogenates were subjected to sodium dodecyl sulfate polyacrylamide gel
electrophoresis, and the proteins in the gels were transferred onto
nitrocellulose membranes (Thermo Scientific, Waltham, MA, USA).
The blots were incubated with anti-NF-κB p65 (1:2000; #8242; Cell
Signaling Technology), anti-p-Akt (Ser472) (1:1000; #9271; Cell
Signaling Technology) and with a secondary antibody (1:5000; antirabbit IgG, horseradish peroxidase-linked antibody #7074; Cell Signaling Technology). A horseradish-peroxidase-conjugated anti-β-actin (1:5000; rabbit monoclonal #5125; Cell Signaling Technology)
antibody was used as the control for protein loading. The protein
bands were visualized using a chemiluminescence detection system
(ECL plus GE Healthcare, Little Chalfont, UK). The signals from the
immunoblots were analyzed with the LAS3000 digital imaging system (Fujifilm, Tokyo, Japan). The western blot assay was performed
four times with the same samples, and relative quantification analysis was performed using Image J (http://rsbweb.nih.gov/ij/down
load.html) (Mikuriya et al., 2008).
4.8. Statistical analysis
The ABR threshold shift, the number of NF-κB-positive OHCs,
fibrocytes, and SGCs, and the relative optical densities in the western blot analysis among the groups were compared using a oneway analysis of variance with Tukey–Kramer multiple comparison
tests. All data are presented as means with standard deviation (SD).
Differences with a p-valueo0.05 were considered significant.
Conflict of interest
All authors have no conflicts of interest to disclose.
Acknowledgment
This work was supported in part by the Japan Society for the
Promotion of Science KAKENHI Grant Number 23592504.
References
Assis, L., Moretti, A.I., Abrahão, T.B., Cury, V., Souza, H.P., Hamblin, M.R., Parizotto, N.
A., 2012. Low-level laser therapy (808 nm) reduces inflammatory response and
oxidative stress in rat tibialis anterior muscle after cryolesion. Lasers Surg. Med.
44, 726–735.
Chen, A.C., Arany, P.R., Huang, Y.Y., Tomkinson, E.M., Sharma, S.K., Kharkwal, G.B.,
Saleem, T., Mooney, D., Yull, F.E., Blackwell, T.S., Hamblin, M.R., 2011a. PLoS One
6, e22453.
Chen, A.C., Huang, Y.Y., Sharma, S.K., Hamblin, M.R., 2011b. Photomed. Laser Surg.
29, 383–389.
Chen, G.D., McWilliams, M.L., Fechter, L.D., 2000. Succinate dehydrogenase (SDH)
activity in hair cells: a correlate for permanent threshold elevations. Hear. Res.
145, 91–100.
Conlan, M.J., Rapley, J.W., Cobb, C.M., 1996. Biostimulation of wound healing by
low-energy laser irradiation: a review. J. Clin. Periodontol. 23, 492–496.
Hirose, K., Liberman, M.C., 2003. Lateral wall histopathology and endocochlear
potential in the noise-damaged mouse cochlea. J. Assoc. Res. Otolaryngol. 4,
339–352.
Hsu, W.C., Wang, J.D., Hsu, C.J., Lee, S.Y., Yeh, T.H., 2004. Expression of connexin 26
in the lateral wall of the rat cochlea after acoustic trauma. Acta Otolaryngol.
124, 459–463.
Huang, Y.Y., Chen, A.C., Carroll, J.D., Hamblin, M.R., 2009. Biphasic dose response in
low level light therapy. Dose Response 7, 358–383.
Jiang, H., Sha, S.H., Schacht, J., 2005. NF-κB pathway protects cochlear hair cells
from aminoglycoside-induced ototoxicity. J. Neurosci. Res. 79, 644–651.
Kurioka, T., Matsunobu, T., Niwa, K., Tamura, A., Satoh, Y., Shiotani, A., 2014a. Activated protein C rescues the cochlea from noise-induced hearing loss. Brain
Res. 1583, 201–210.
Kurioka, T., Matsunobu, T., Satoh, Y., Niwa, K., Shiotani, A., 2014b. Inhaled hydrogen
gas therapy for prevention of noise-induced hearing loss through reducing
reactive oxygen species. Neurosci. Res. 89, 69–74.
Le Prell, C.G., Yamashita, D., Minami, S.B., Yamasoba, T., Miller, J.M., 2007. Mechanisms of noise-induced hearing loss indicate multiple methods of prevention. Hear. Res. 226, 22–43.
Madrid, L.V., Wang, C.Y., Guttridge, D.C., Schottelius, A.J., Baldwin Jr., A.S., Mayo, M.
W., 2000. Akt suppresses apoptosis by stimulating the transactivation potential
of the RelA/p65 subunit of NF-kappa B. Mol. Cell Biol. 20, 1626–1638.
Masuda, M., Nagashima, R., Kanzaki, S., Fujioka, M., Ogita, K., Ogawa, K., 2006.
Nuclear factor-kappa B nuclear translocation in the cochlea of mice following
acoustic overstimulation. Brain Res. 1068, 237–247.
Mikuriya, T., Sugahara, K., Sugimoto, K., Fujimoto, M., Takemoto, T., Hashimoto, M.,
Hirose, Y., Shimogori, H., Hayashida, N., Inouye, S., Nakai, A., Yamashita, H., 2008.
Attenuation of progressive hearing loss in a model of age-related hearing loss by
a heat shock protein inducer, geranylgeranylacetone. Brain Res. 1212, 9–17.
Mizutari, K., Nakagawa, S., Mutai, H., Fujii, M., Ogawa, K., Matsunaga, T., 2011. Latephase recovery in the cochlear lateral wall following severe degeneration by
acute energy failure. Brain Res. 1419, 1–11.
Nagy, I., Monge, A., Albinger-Hegyi, A., Schmid, S., Bodmer, D., 2005. NF-κB is required for survival of immature auditory hair cell in vitro. J. Assoc. Res. Otolaryngol. 6, 260–268.
Nakamoto, T., Mikuriya, T., Sugahara, K., Hirose, Y., Hashimoto, T., Shimogori, H.,
Takii, R., Nakai, A., Yamashita, H., 2012. Geranylgeranylacetone suppresses
noise-induced expression of proinflammatory cytokines in the cochlea. Auris
Nasus Larynx 39, 270–274.
Nelson, D.I., Nelson, R.Y., Concha-Barrientos, M., Fingerhut, M., 2005. The global
burden of occupational noise-induced hearing loss. Am. J. Ind. Med. 48, 446–458.
Nicotera, T., Hu, B.H., Henderson, D., 2003. The caspase pathway in noise-induced
apoptosis of the chinchilla cochlea. J. Assoc. Res. Otolaryngol. 4, 466–477.
Nishi, A., Snyder, G.L., Greengard, P., 1997. Bidirectional regulation of DARPP-32
phosphorylation by dopamine. J. Neurosci. 7, 8147–8155.
Nuttall, A.L., 1999. Sound-induced cochlear ischemia/hypoxia as a mechanism of
hearing loss. Noise Health 2, 17–32.
Paez, J., Sellers, W.R., 2003. PI3K/PTEN/AKT pathway. A critical mediator of oncogenic signaling. Cancer Treat. Res. 115, 145–167.
Puel, J.L., Ruel, J., Gervais, d’Aldin, C., Pujol, R., 1998. Excitotoxicity and repair of cochlear
synapses after noise-trauma induced hearing loss. Neuroreport 9, 2109–2114.
Rhee, C.K., Bahk, C.W., Kim, S.H., Ahn, J.C., Jung, J.Y., Chung, P.S., Suh, M.W., 2012.
Effect of low-level laser treatment on cochlea hair-cell recovery after acute
acoustic trauma. J. Biomed. Opt. 17, 068002.
Rizzi, C.F., Mauriz, J.L., Freitas Correa, D.S., Moreira, A.J., Zettler, C.G., Filippin, L.I.,
Marroni, N.P., Gonzalez-Gallego, J., 2006. Effects of low-level laser therapy
(LLLT) on the nuclear factor (NF)-κB signaling pathway in traumatized muscle.
Lasers Surg. Med. 38, 704–713.
Romashkova, J.A., Makarov, S.S., 1999. NF-κB is a target of AKT in anti-apoptotic
PDGF signalling. Nature 401, 86–90.
Sha, S.H., Taylor, R., Forge, A., Schacht, J., 2001. Differential vulnerability of basal and
apical hair cells is based on intrinsic susceptibility to free radicals. Hear. Res.
155, 1–8.
Sha, S.H., Chen, F.Q., Schacht, J., 2010. PTEN attenuates PIP3/Akt signaling in the
cochlea of the aging CBA/J mouse. Hear. Res. 264, 86–92.
Streeter, J., Taboada, L.D., Oron, U., 2004. Mechanisms of action of light therapy for
stroke and acute myocardial infarction. Mitochondrion 4, 569–576.
Takumida, M., Anniko, M., 2002. Nitric oxide in the inner ear. Curr. Opin. Neurol. 15,
11–15.
Takumida, M., Anniko, M., Zhang, D.M., 2001. Pharmacological models for inner ear
therapy with emphasis on nitric oxide. Acta Otolaryngol. 121, 16–20.
Tamura, A., Matsunobu, T., Mizutari, K., Niwa, K., Kurioka, T., Kawauchi, S., Satoh, S.,
Hiroi, S., Satoh, Y., Nibuya, M., Tamura, R., Shiotani, A., 2015. Low-level laser
therapy for prevention of noise-induced hearing loss in rats. Neurosci. Lett. 595,
81–86.
Tata, D.B., Waynant, R.W., 2011. Laser therapy: a review of its mechanism of action
and potential medical application. Laser Photonics Rev. 5, 1–12.
Yamamoto, H., Omelchenko, I., Shi, X., Nuttall, A.L., 2009. The influence of NF-κB
signal-transduction pathways on the murine inner ear by acoustic overstimulation. J. Neurosci. Res. 87, 1832–1840.
Yamauchi, T., Sakurai, M., Abe, K., Takano, H., Sawa, Y., 2006. Neuroprotective effects
of activated protein C through induction of insulin-like growth factor-1 (IGF-1),
IGF-1 receptor, and its downstream signal phosphorylated serine-threonine
kinase after spinal cord ischemia in rabbits. Stroke 37, 1081–1086.