Round-window delivery of lithium chloride regenerates cochlear synapses damaged by noise-induced excitotoxic trauma via inhibition of the NMDA receptor in the rat

Ji Eun Choi; Nathaniel T Carpena; Jae-Hun Lee; So-Young Chang; Min Young Lee; Jae Yun Jung; Won-Ho Chung

doi:10.1371/journal.pone.0284626

. 2023 May 22;18(5):e0284626. doi: 10.1371/journal.pone.0284626

Round-window delivery of lithium chloride regenerates cochlear synapses damaged by noise-induced excitotoxic trauma via inhibition of the NMDA receptor in the rat

Ji Eun Choi ^1,^2,^#, Nathaniel T Carpena ^2,^#, Jae-Hun Lee ³, So-Young Chang ², Min Young Lee ^1,², Jae Yun Jung ^1,^2,^‡,^*, Won-Ho Chung ^4,^‡,^*

Editor: Alan G Cheng⁵

PMCID: PMC10202264 PMID: 37216352

Abstract

Noise exposure can destroy the synaptic connections between hair cells and auditory nerve fibers without damaging the hair cells, and this synaptic loss could contribute to difficult hearing in noisy environments. In this study, we investigated whether delivering lithium chloride to the round-window can regenerate synaptic loss of cochlea after acoustic overexposure. Our rat animal model of noise-induced cochlear synaptopathy caused about 50% loss of synapses in the cochlear basal region without damaging hair cells. We locally delivered a single treatment of poloxamer 407 (vehicle) containing lithium chloride (either 1 mM or 2 mM) to the round-window niche 24 hours after noise exposure. Controls included animals exposed to noise who received only the vehicle. Auditory brainstem responses were measured 3 days, 1 week, and 2 weeks post-exposure treatment, and cochleas were harvested 1 week and 2 weeks post-exposure treatment for histological analysis. As documented by confocal microscopy of immunostained ribbon synapses, local delivery of 2 mM lithium chloride produced synaptic regeneration coupled with corresponding functional recovery, as seen in the suprathreshold amplitude of auditory brainstem response wave 1. Western blot analyses revealed that 2 mM lithium chloride suppressed N-methyl-D-aspartate (NMDA) receptor expression 7 days after noise-exposure. Thus, round-window delivery of lithium chloride using poloxamer 407 reduces cochlear synaptic loss after acoustic overexposure by inhibiting NMDA receptor activity in rat model.

Introduction

Noise can cause temporary or permanent damage to the inner ear depending on exposure characteristics such as the sound pressure level (SPL), exposure time, and noise spectrum, as well as characteristics of the individual [1]. Exposure to loud sounds can destroy cochlear sensory hair cells, causing permanent elevation of the hearing threshold [2–4]. Even at an SPL too low to permanently damage cochlear hair cells, noise exposure can impair hearing by destroying the cochlear ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) [5, 6]. Although this cochlear synaptopathy does not elevate the threshold, it could contribute to difficulties in understanding speech in noisy environments, tinnitus, and hyperacusis [7, 8].

The accumulated evidence indicates that noise can cause excitotoxic trauma to cochlear synapses by triggering the excessive release of the neurotransmitter glutamate from auditory hair cells [9–12]. Direct application of glutamate agonist causes swelling of the SGN terminals in regions of synaptic contact with IHCs, similar to the morphological changes seen after acoustic overexposure [9, 13–15]. Conversely, a glutamate receptor blockade reduces swelling of the post-synaptic terminals during acoustic overexposure [10–12]. Although the downstream mechanisms of noise-induced damage are unknown, those observations suggest that the primary cause of noise-induced synaptic loss is the activation of glutamate receptors.

Recently, lithium has been reported to show neuroprotective effects against glutamate excitotoxicity, and that neuroprotective action has been associated with the inactivation of N-methyl-D-aspartate (NMDA) receptors [16–18]. Increasing evidence supports the notion that lithium-induced inhibition of phosphorylation of the NMDA receptor’s NR2B subunit is likely to result in its inactivation and contribute to neuroprotection against excitotoxicity [18, 19]. Prior studies have shown that lithium can protect against cisplatin-induced cytotoxicity in auditory cells [20] and attenuate aging-related auditory cortex apoptosis [21]. However, there is a lack of evidence about whether lithium can reverse noise-induced cochlear synaptopathy.

Our aim in the present study was to evaluate whether lithium has neuroprotective effects in noise-induced cochlear synaptopathy. We therefore used a thermoreversible hydrogel to deliver lithium directly to the round-window in a rat model of noise-induced cochlear synaptopathy and conducted functional and histological analyses to investigate whether lithium reduced synaptic loss after acoustic overexposure. In addition, using western blot analyses, we explored the role of changes in the NMDA receptor in mediating neuroprotection in noise-induced cochlear synaptopathy.

Methods

Animals

Male Sprague-Dawley rats (5 weeks old; 130–150 g body weight; Nara Biotech, South Korea) were used for this study. The animals were housed in the laboratory animal facility and given free access to food and water. Before starting the experimental procedures, all rats were acclimated to their housing conditions for 1 week and measured the baseline auditory brainstem response (ABR) threshold.

Based on the ABR measurement, forty rats with normal hearing threshold were arbitrarily assigned to one of five groups. The group 1 was not exposed to noise or surgery to assess normal cochlear synaptic counts (normal group, n = 8). The group 2 was exposed to a noise band designed to destroy cochlear synapses (noise-only group, n = 4). The three other groups underwent surgery 24 hours after noise exposure to place a solution containing only vehicle (Group 3: noise + vehicle, n = 14), 1 mM lithium chloride (LiCl; Group 4: noise + 1 mM LiCl, n = 10), or 2 mM LiCl (Group 5: noise + 2 mM LiCl, n = 14), onto the round-window membrane.

Fig 1 shows a schematic timeline of the experimental protocol. All experimentation was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Dankook (Protocol Number: DKU-17-041 and DKU-19-040). After the completion of all experiments, animals were euthanized by tiletamine–zolazepam/xylazine overdose followed by cervical dislocation.

Noise exposure

The awake and unrestrained animals were exposed to a narrow band of noise (16 kHz with 1 kHz of bandwidth) for 2 hours at an SPL of 105 dB. During that time, the animals were placed in individual cages to prevent defensive behaviors, and those cages were placed in custom-made soundproof acryl chambers equipped with a BEYMA CP800Ti speaker (Beyma, Valencia, Spain). The noise was generated with a type 1027 sine random generator (Bruel and Kjaer, Denmark) and amplified with an R300 plus amplifier (Inter-M, Seoul, Korea). The sound levels were verified in the floor of the chamber using a frequency-specific sound level meter (Sound Level Meter-Type 2250; Bruel and Kjaer, Copenhagen, Denmark) before and during each noise exposure.

Round-window delivery of LiCl

An 18% (w/v) stock solution was prepared by slowly dissolving poloxamer 407 (Sigma-Aldrich Cat# 16758) in sterile water. LiCl powder (42.39 g/mol) (Sigma-Aldrich Cat# L7026) was reconstituted in the 18% poloxamer 407 solution, yielding a final concentration of 1 mM as the low dose and 2 mM as the high dose, and stored overnight in a refrigerator at 4°C to ensure complete dissolution. The 18% poloxamer 407 solution is liquid in the refrigerator and room temperature but becomes highly viscous at body temperature.

The animals were anesthetized by an intraperitoneal injection of tiletamine–zolazepam (30 mg/kg) and xylazine (10 mg/kg) and positioned right ear-down. A postauricular skin incision was made, and the subcutaneous tissues and superficial fascia of the neck were bluntly dissected. After exposing the otic bulla, a tiny hole was made and enlarged until the round-window membrane was clearly visible (Fig 1). A 16G needle was positioned within the round-window niche, and 50 μl of the poloxamer solution was injected using a 1 mL syringe over the round-window membrane. The hole was sealed with muscle, and the wound was closed with 4–0 Vicryl sutures (Ethicon). Intramuscular injection of ketamine (10mg/kg) was applied after surgery (prior to anesthetic recovery) to minimize animal suffering.

Auditory brainstem response measurements

ABRs were measured using a Davis Technologies system (TDT system III, Alchua, Florida, USA) to identify the degrees of hearing loss and recovery. The animals were anesthetized by an intraperitoneal injection of tiletamine–zolazepam (30 mg/kg) and xylazine (10 mg/kg) and placed in a sound-proof chamber. Five millisecond tone-burst stimuli (0.5 ms cos2 rise-fall) at four frequencies (8, 12, 16, and 32 kHz) were delivered with alternating polarity. Electrical responses were collected via needle electrodes at the vertex (active) and ventral edge of each pinna (reference and ground), amplified 10,000X with a 0.3–3 kHz passband, and averaged using 1,024 responses at each SPL. The responses were collected for stimulus levels in 5 dB steps from 90 to 10 dB SPL. The ABR threshold was defined as the lowest sound level at which a reproducible waveform could be observed. The wave 1 amplitude was defined as the difference between the first positive peak and the following negative peak. Hearing thresholds and wave 1 amplitudes were obtained before noise exposure (baseline) and 3, 7, and 14 days after the treatment.

Cochlear processing and immunohistochemistry

Following the endpoint ABR measurements, thirty-eight animals were sacrificed (n = 2 for group 1 and 2, n = 5 for group 3–5 at 1 week and 2 weeks post-treatment). After anesthesia with intraperitoneal tiletamine/zolazepam (30 mg/kg)–xylazine (10 mg/kg), their cochleae were quickly harvested. The harvested cochleae were fixed in 4% paraformaldehyde overnight. After being washed with 0.1 M phosphate buffered saline (PBS), the cochleae were decalcified in 0.5 M EDTA (pH 8.0) and micro-dissected into 4–5 pieces for whole-mount cochlear processing. For immunostaining, the cochlear pieces were blocked with 5% normal goat serum (NGS) in PBS and 0.3% Triton X-100 (TX) at room temperature for 1 hour followed by overnight incubation at 4°C with the following primary antibodies diluted in 1% NGS with 0.3% TX: 1) mouse (IgG1) anti-CtBP2 (C-terminal binding protein) at 1:500 (BD Transduction Labs Cat# 612044, RRID: AB_399431) to quantify pre-synaptic ribbons and 2) rabbit anti-myosin 7a at 1:200 (Proteus Biosciences Cat# 25–6790, RRID: AB_10015251) to delineate hair cells. After being washed three times with PBS for 5 min, the cochlear pieces were incubated for 1 hour at room temperature in species-appropriate secondary antibodies: 1) Alexa Fluor 488-conjugated goat anti-mouse (IgG2a) at 1:1000 (Molecular Probes Cat# A-21131, RRID: AB_141618) or 2) Alexa Fluor 568-conjugated chicken anti-rabbit at 1:200 (Innovative Research Cat# A21443, RRID: AB_1500685). After being washed three times with PBS for 5 min, the stained cochlear pieces were slide-mounted using Vectashield (Vector Labs) and cover-slipped.

Synapse and hair cell counts

Images were obtained using a confocal microscope (Flow-View 3000, Olympus, Japan) with a glycerol-immersion 40X objective and 2X digital zoom. For each image stack, the z dimension was sampled at 25 μm with a resolution of 0.5 μm per slice, and the span was adjusted to include all synaptic elements in the xy field of view. One z-stack was obtained in each frequency location, and each x-stack included 8–12 adjacent IHCs. To identify and count hair cell synapses, image stacks were imported into image processing software (ImageJ ver. 1.43u, NIH, US). The outer hair cells (OHCs) and IHCs in each stack were counted using faint nuclear staining by anti-CtBP2 and hair-cell staining by anti-myosin 7a. To quantitatively assess pre-synaptic ribbons, pre-synaptic markers within 10–11 IHCs were counted and divided by the number of IHCs. The location of the basilar membrane at the relevant frequency was determined by computing a cochlear frequency map [22].

Western blot analysis

To perform the western blot, cochlear tissues were additionally dissected under the anesthesia with intraperitoneal tiletamine/zolazepam (30 mg/kg)–xylazine (10 mg/kg) from group 1 (normal), group 3 (noise + vehicle), and group 5 (noise + 2 mM LiCl). Four cochlear tissues per group were pooled with 3 replicates and then homogenized in radioimmunoprecipitation assay buffer (Biosesang, Seongnam, Gyeonggi, Korea) containing protease (1:100, Sigma-Aldrich Cat# P8340) and phosphatase inhibitor cocktails (1:100, Sigma-Aldrich Cat# P8340). The cochlear lysates were centrifuged at 12,000 rpm and 4°C for 15 min. The collected supernatants were collected and stored at -20°C until further use. Equal amounts of proteins were mixed with Laemmli loading buffer (Bio-Rad Cat# 161–0737) and β-mercaptoethanol (0.7 mM), boiled at 95°C for 5 min, and then separated using 6% SDS-polyacrylamide gel. After electrophoresis, the gel proteins were electro-transferred onto an immunoblot nitrocellulose membrane (Bio-Rad Laboratories, CA, USA). The membranes were blocked with 5% skimmed milk and incubated overnight at 4°C with primary antibodies to anti-NR2B (1:500; Abcam Cat# ab28373, RRID: AB_776810), anti-phospho-NR2B (Ser1303) (1:500; Abcam Cat# ab81271, RRID: AB_1640731), or anti-β-actin (1:2,000; Abcam Cat# ab6276, RRID: AB_2223210). After being washed with PBS and Tween-20 (PBST; 0.1M PBS, 0.1% Tween-20), the membranes were incubated with horseradish peroxidase–conjugated goat anti-rabbit (1:2000; Thermo Fisher Scientific Cat# A16104, RRID: AB_2534776) or goat anti-mouse secondary antibody (1:2000; Thermo Fisher Scientific Cat# A16072, RRID: AB_2534745) in PBS with 2.5% skimmed milk. After being thoroughly washed with PBST, the membranes were visualized by enhancing chemiluminescence substrate (Pierce, Rockford, IL) for 2 min, followed by chemiluminescence detection on ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA). The band intensities were quantified using image processing software (ImageJ ver. 1.43u, NIH, Bethesda, MD, US), and the relative expression of each protein was normalized to β-actin. Fold change was calculated by comparing to the normal group.

Statistical analysis

Statistical analyses were conducted using Prism software 8.4.3 (GraphPad Software, La Jolla, CA, USA, RRID:SCR_002798). All datasets were tested for normal distribution using the Shapiro-Wilk test and QQ-plots. Outlier were identified by ROUT test and dataset had no outliers. The ABR thresholds, ABR wave 1 amplitude, synapse counts, and western blot result were compared between experimental groups (Group 3–5). For multiple groups, nonparametric data were statistically analyzed using the Kruskal-Wallis H test and the two-way repeated measures analysis of variance (ANOVA). If there were significant differences among the experimental groups, post-hoc analysis with Dunn’s multiple comparisons test was performed to evaluate differences between two groups. Tests were considered statistically significant when p-value and adjusted p-value were less than 0.05.

Results

Early recovery of temporary noise-induced threshold shifts after round-window delivery of LiCl

ABRs were tested to examine the threshold of hearing before and after the noise exposure and round-window drug delivery. As observed by comparing the normal and noise-only groups, noise exposure caused a threshold shift of ~30 dB SPL at frequencies above the noise band, when measured 1 week after the treatment, (Fig 2). Two week later, ABR thresholds recovered up to 15 dB SPL. Finally, noise exposure caused a permanent ~15 dB SPL threshold shift at frequencies above the noise band. Compared to the noise-only group, the noise + vehicle group had worse ABR threshold at 8 kHz and 12 kHz at 1 week after the treatment, but ABR threshold in the noise + vehicle group had returned to that of the noise-only group.

The delivery of LiCl to the round-window produced early recovery of the temporary noise-induced threshold shift (Fig 2b). The Kruskal-Wallis H test showed significant threshold differences among the experimental groups (noise + vehicle, noise + 1 mM LiCl, and noise + 2 mM LiCl groups) one week after the treatment (8 kHz: χ²(2) = 11.859, p = 0.003; 12 kHz: χ²(2) = 12.071, p = 0.002; 16 kHz: χ²(2) = 14.256, p = 0.001; 32 kHz: χ²(2) = 19.090, p < 0.001). The post-hoc analysis with Dunn’s multiple comparisons test revealed that the hearing thresholds recovered earlier in the noise + 1 mM LiCl (8 kHz: p = 0.003, 12 kHz: p = 0.006, 16 kHz: p = 0.001, 32 kHz: p < 0.001) and noise + 2 mM LiCl (8 kHz: p = 0.031, 12 kHz: p = 0.010, 16 kHz: p = 0.009, 32 kHz; p = 0.002) groups than in the noise + vehicle group. However, there were no significant differences among the experimental groups two weeks after the treatment (8 kHz: χ²(2) = 1.056, p = 0.590; 12 kHz: χ²(2) = 2.491, p = 0.288; 16 kHz: χ²(2) = 3.583, p = 0.167; 32 kHz: χ²(2) = 4.154, p = 0.125).

Functional recovery of suprathreshold responses after round-window delivery of LiCl

Changes in the mean peak 1 amplitude after noise exposure are shown in Fig 3. The mean peak 1 amplitudes upon suprathreshold stimuli (all levels from 65–80 dB SPL) were reduced at frequencies above the noise band, where the maximal noise-induced threshold shift was seen. All experimental groups had statistically significant differences before and after noise exposure as determined by two-way ANOVA for all levels from 65–80 dB SPL (noise + vehicle group: F(3, 27) = 28.15, p < 0.001 for 16 kHz and F(3, 27) = 40.1, p < 0.001 for 32 kHz; noise + 1 mM LiCl: F(3, 27) = 41.4, p < 0.001 for 16 kHz and F(3, 27) = 24.3, p < 0.001 for 32 kHz; noise + 2 mM LiCl: F(3, 27) = 22.67, p < 0.001 for 16 kHz and F(1.674, 15.07) = 23.58, p < 0.001 for 32 kHz).

Two weeks after the treatment, the mean peak 1 amplitudes had not completely recovered in the noise + vehicle (all adjusted p < 0.001 by post-hoc analysis with Dunnett’s multiple comparisons test except for stimulus levels from 70- and 65-dB SPL at 16 Hz) and noise + 1 mM LiCl groups (all adjusted p < 0.001 by post-hoc analysis with Dunnett’s multiple comparisons test). However, the suprathreshold amplitudes of the ABRs had fully recovered in the noise + 2 mM LiCl group two weeks after the treatment. The suprathreshold peak 1 amplitude at 16 kHz and 32 kHz did not differ significantly between baseline and two weeks post-exposure treatment in the noise + 2 mM LiCl group (all adjusted p > 0.05 by post-hoc analysis with Dunnett’s multiple comparisons test).

Synaptic regeneration after round-window delivery of LiCl

Cochlear tissue immunostained with antibodies specific for hair cells (myosin 7a) and ribbon synapses (CtBP2) showed no loss of OHCs or IHCs; instead, there was a loss of IHC synapses after noise exposure (Fig 4). The loss of IHC synapses is clearly visible in the immunostained cochlear tissue from the noise + vehicle group compared with the normal group (Fig 4A and 4B). As quantitatively shown in Fig 4C and 4D, the mean synaptic count per IHC was reduced in the noise-exposed groups (noise-only and noise + vehicle groups), mainly in the frequencies above the noise band (16 kHz and 32 kHz).

Synaptic rescue can be seen in the noise + 2 mM LiCl group both one week (Fig 4A) and two weeks (Fig 4B) after the treatment. The Kruskal-Wallis H test showed a statistically significant difference among the experimental groups (noise + vehicle, noise + 1 mM LiCl, noise + 2 mM LiCl groups) in the mean pre-synaptic counts at 16 kHz and 32 kHz one week (16 kHz: χ²(3) = 10.50, p < 0.001; 32 kHz: χ²(3) = 12.50, p < 0.001) and two weeks (16 kHz: χ²(3) = 9.637, p = 0.002; 32 kHz: χ²(3) = 11.18, p < 0.001) after the treatment. The post-hoc analysis with Dunn’s multiple comparisons test revealed that the mean pre-synaptic counts in the noise + 2 mM LiCl group were significantly higher than those in the noise + vehicle group (all adjusted p < 0.5 for 16 kHz and 32 kHz when measured one week and two weeks after the treatment). However, the mean pre-synaptic counts did not differ significantly between the noise + vehicle and noise + 1 mM LiCl groups.

Neuroprotective effects of round-window delivery of LiCl against glutamate excitotoxicity

Fig 5 shows representative western blots and corresponding quantitative analyses of total NR2B and phospho-NR2B (pNR2B) expression. The Kruskal-Wallis test was conducted to compare the fold changes of total NR2B and pNR2B levels among the normal, noise + vehicle, and noise + 2 ml LiCl groups on days 1, 3, and 7 after the treatment. The levels of total NR2B and pNR2B were unchanged until 3 days after the noise exposure and/or the treatment [NR2B: χ²(3) = 0.368, p = 0.868 on day 1 and χ²(3) = 4.782, p = 0.104 on day 3, pNR2B: χ²(3) = 0.644, p = 0.818 on day 1 and χ²(3) = 0.644, p = 0.818 on day 3). However, the levels of total NR2B and pNR2B were significantly different among the normal, noise + vehicle, and noise + 2 ml LiCl groups on days 7 after the noise exposure and/or the treatment (NR2B 004 and pNR2B: χ²(3) = 7.448, p = 0.004). The levels of total NR2B and pNR2B were increased 7 days after the noise exposure, and that noise-induced increase in total NR2B and pNR2B expression were suppressed by round-window delivery of LiCl. The post-hoc analysis with Dunn’s multiple comparison test showed that the noise + 2 mM LiCl group had significantly lower NR2B and pNR2B expression than the noise + vehicle group 7 days after the treatment (NR2B and pNR2B: p = 0.0019).

Discussion

Our study has demonstrated that round-window delivery of high-dose lithium after a noise exposure rescued the noise-induced loss of presynaptic ribbons (Fig 4) and restored the suprathreshold amplitudes for ABR wave 1 at frequencies above the noise band (Fig 3). In addition, lithium delivery led to early recovery of the ABR threshold shifts after noise-induced damage (Fig 2). There is a robust literature on the delivery of potential therapeutic agents to prevent or regenerate noise-induced synaptic loss in the cochlea [7, 23–26]. However, few studies have successfully demonstrated regeneration of cochlear synapses when a therapeutic agent was administered after noise exposure, such as our study [27].

There is strong evidence that an excess release of the excitatory neurotransmitter glutamate from the IHC synapses in response to acoustic overstimulation may cause excitotoxicity with a loss of synaptic connections to the SGN [5, 6, 9–12]. Glutamate excitotoxicity can allow calcium influx which in turn can trigger cell death pathways eventually leading to auditory hair cell death [28]. Although the pharmacological mechanisms of lithium are not completely understood, lithium has been reported to have neuroprotective effects against the glutamate-induced excitotoxicity [16–18, 29]. The neuroprotective effect of lithium has been associated with the suppression of NR2B phosphorylation of the NMDA glutamate receptor, inhibition of NMDA receptor–mediated calcium influx, upregulation of anti-apoptotic Bcl-2, downregulation of pro-apoptotic p53 and Bax, and activation of the survival signaling pathway [18, 29, 30]. Our western blotting results indicate that noise exposure induced increases in the expression of the NR2B subunit and its phosphorylation at Ser1303 after 7 days, but round-window delivery of high-dose lithium suppressed these NR2B levels (Fig 4).

In the mammalian inner ear, SGNs express a variety of glutamate receptor types, including NMDA receptors and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors [31]. AMPA receptors mediate the large fast excitatory currents necessary for synaptic transmission and thereby maintain the temporal fidelity essential for hearing [32, 33], whereas the NMDA receptors are not involved in fast excitatory neurotransmission because they are blocked at resting potential by Mg²⁺. NMDA receptors are instead activated by excessive neurotransmitter released from IHCs that becomes excitotoxic to the primary auditory neuron [34–36]. NMDA receptors are not directly involved in glutamatergic transmission at IHC–SGN synapses [33], but they are thought to contribute to the regulation of AMPA receptor expression following acute insult [37, 38]. In mouse auditory neurons, the expression of surface AMPA receptors decreased in the presence of an NMDA receptor agonist, and that decrease was blocked by the application of an NMDA receptor antagonist [37]. Under physiological conditions, post-synaptic AMPA receptors are paired with pre-synaptic ribbons. However, studies of noise-induced hearing loss have shown that reorganization of synaptic ribbon locations causes orphan AMPA receptors that lack opposed pre-synaptic ribbons and the loss of synaptic ribbons [39, 40]. In normal mature mouse cochleae, most NMDA receptors are distributed on the modiolar side close to the nuclear region of the IHCs, whereas most synaptic ribbons and AMPA receptors are on neural terminals closer to the basal poles of the IHCs. After gentamicin exposure, AMPA receptors and NMDA receptors are relocated to nerve fiber terminals around the IHCs. The NMDA receptors move downward toward the basal poles of the IHCs, and the AMPA receptors move upward toward the bundle poles of the IHCs [41]. Those results suggest that the post-synaptic rearrangement of AMPA receptors, modulated by NMDA receptors, might affect the number and location of pre-synaptic ribbons. Therefore, inhibiting NMDA receptors might prevent noise-induced synaptic loss by preventing the relocation of AMPA and NMDA receptors on the dendrites of the SGNs and thus maintain the integrity of the ribbon synapses, preserving hearing function.

Although the function of NMDA receptors in noise-induced cochlear synaptopathy is unclear, several studies have suggested that NMDA receptors play a role in the excitotoxicity caused by noise trauma, and its antagonists have shown potential as treatments for noise-induced cochlear synaptopathy [34]. Calcium influx through NMDA receptor at excitatory synapse causes activation of post-synaptic calcium/calmodulin-dependent protein kinase type II (CaMKII) and CaMKII undergoes phosphorylation at Ser1301 by binding to the NR2B subunit of NMDA receptor [42]. Thus, lithium treatment likely has neuroprotective effects against noise-induced excitotoxicity by the suppression of inhibition of NMDA receptor–mediated calcium influx.

Our study has some limitations. First, round-window delivery of LiCl could yield inconsistent results. It was difficult to deliver a viscous gel into the small bulla opening required to minimize the surgically induced threshold shift. That constraint likely decreased the effective dose of lithium chloride delivered to the round-window. Nevertheless, lithium was administered locally in this study because it has a narrow therapeutic window and adverse effects on the kidneys, thyroid gland, and parathyroid glands, which necessitates close monitoring of its plasma concentration and the functioning of those organs. Second, we initially intended to make a rat model of noise-induced synaptic damage without causing permanent elevation of the hearing threshold. Prior work on this type noise-induced synaptic damage caused no significant permanent threshold elevation [43], but our model did cause permanent threshold elevations of ~15 dB SPL at 16 and 32 kHz without a significant loss of hair cells (Fig 2). Because noise-induced threshold shifts are sensitive to overall exposure energy, it seems necessary to adjust the exposure level or duration. Third, the pharmacological mechanisms by which lithium regenerates IHC synapses are still not fully understood. Lithium causes a wide range of intracellular responses that affect the neurotrophic response, autophagy, oxidative stress, inflammation, and mitochondrial function [44–49]. The blockade of NMDA receptor activity by lithium could occur through any of several mechanisms to prevent the loss of IHC synapses, so further study is needed. Fourthly, many key questions remain for clinical application. There is likely a significant clinical population with loss of cochlea synapse that could be addressed by a treatment analogous to that used here in the noise-exposed rat. Although the local delivery of lithium to the round window was effective in regenerating cochlear synapses 24 hours after the noise exposure, a key question would be how long this time window could be extended for effective synaptogenesis in a clinical setting.

Conclusion

The present experiments showed that round window delivery of lithium can be effective local access route to the inner ear, and regenerates cochlear synapses even when administered 24 hours post-exposure of noise.

Supporting information

S1 Fig. Western blot analysis of total NR2B, phospho-NR2B (pNR2B), β-actin protein expression levels in the cochlea.

A-C Original uncropped and unadjusted images representing for the β-actin (A), total NR2B (B), and phopho-NR2B (C). d = day, R = replicate.

(DOCX)

Click here for additional data file.^{(660.3KB, docx)}

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) to JEC [no. NRF-2020R1C1C1009849] and by the Basic Science Research Program through the NRF founded by the Ministry of Education in the form of funding to JEC [NRF-2020R1A6A1A03043283, RS-2023-00208177]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Ryan AF, Kujawa SG, Hammill T, Le Prell C, Kil J. Temporary and Permanent Noise-induced Threshold Shifts: A Review of Basic and Clinical Observations. Otol Neurotol. 2016;37(8):e271–5. Epub 2016/08/16. doi: 10.1097/MAO.0000000000001071 . [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Robertson D. Effects of acoustic trauma on stereocilia structure and spiral ganglion cell tuning properties in the guinea pig cochlea. Hear Res. 1982;7(1):55–74. Epub 1982/05/01. doi: 10.1016/0378-5955(82)90081-8 . [DOI] [PubMed] [Google Scholar]
3.Liberman MC, Dodds LW. Single-neuron labeling and chronic cochlear pathology. III. Stereocilia damage and alterations of threshold tuning curves. Hear Res. 1984;16(1):55–74. Epub 1984/10/01. doi: 10.1016/0378-5955(84)90025-x . [DOI] [PubMed] [Google Scholar]
4.Wang Y, Hirose K, Liberman MC. Dynamics of noise-induced cellular injury and repair in the mouse cochlea. J Assoc Res Otolaryngol. 2002;3(3):248–68. Epub 2002/10/17. doi: 10.1007/s101620020028 . [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Kujawa SG, Liberman MC. Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth. J Neurosci. 2006;26(7):2115–23. Epub 2006/02/17. doi: 10.1523/JNEUROSCI.4985-05.2006 . [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Kujawa SG, Liberman MC. Adding insult to injury: cochlear nerve degeneration after "temporary" noise-induced hearing loss. J Neurosci. 2009;29(45):14077–85. Epub 2009/11/13. doi: 10.1523/JNEUROSCI.2845-09.2009 . [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Kujawa SG, Liberman MC. Synaptopathy in the noise-exposed and aging cochlea: Primary neural degeneration in acquired sensorineural hearing loss. Hear Res. 2015;330(Pt B):191–9. Epub 2015/03/15. doi: 10.1016/j.heares.2015.02.009 . [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Plack CJ, Barker D, Prendergast G. Perceptual consequences of "hidden" hearing loss. Trends Hear. 2014;18. Epub 2014/09/11. doi: 10.1177/2331216514550621 . [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Pujol R, Lenoir M, Robertson D, Eybalin M, Johnstone BM. Kainic acid selectively alters auditory dendrites connected with cochlear inner hair cells. Hear Res. 1985;18(2):145–51. Epub 1985/05/01. doi: 10.1016/0378-5955(85)90006-1 . [DOI] [PubMed] [Google Scholar]
10.Pujol R, Puel JL, Gervais d’Aldin C, Eybalin M. Pathophysiology of the glutamatergic synapses in the cochlea. Acta Otolaryngol. 1993;113(3):330–4. Epub 1993/05/01. doi: 10.3109/00016489309135819 . [DOI] [PubMed] [Google Scholar]
11.Pujol R, Puel JL. Excitotoxicity, synaptic repair, and functional recovery in the mammalian cochlea: a review of recent findings. Ann N Y Acad Sci. 1999;884:249–54. Epub 2000/06/08. doi: 10.1111/j.1749-6632.1999.tb08646.x . [DOI] [PubMed] [Google Scholar]
12.Wang Q, Green SH. Functional role of neurotrophin-3 in synapse regeneration by spiral ganglion neurons on inner hair cells after excitotoxic trauma in vitro. J Neurosci. 2011;31(21):7938–49. Epub 2011/05/27. doi: 10.1523/JNEUROSCI.1434-10.2011 . [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Liberman MC. Single-neuron labeling in the cat auditory nerve. Science. 1982;216(4551):1239–41. Epub 1982/06/11. doi: 10.1126/science.7079757 . [DOI] [PubMed] [Google Scholar]
14.Robertson D. Functional significance of dendritic swelling after loud sounds in the guinea pig cochlea. Hear Res. 1983;9(3):263–78. Epub 1983/03/01. doi: 10.1016/0378-5955(83)90031-x . [DOI] [PubMed] [Google Scholar]
15.Puel JL, Ruel J, Gervais d’Aldin C, Pujol R. Excitotoxicity and repair of cochlear synapses after noise-trauma induced hearing loss. Neuroreport. 1998;9(9):2109–14. Epub 1998/07/23. doi: 10.1097/00001756-199806220-00037 . [DOI] [PubMed] [Google Scholar]
16.Friedlander RM. Apoptosis and caspases in neurodegenerative diseases. N Engl J Med. 2003;348(14):1365–75. Epub 2003/04/04. doi: 10.1056/NEJMra022366 . [DOI] [PubMed] [Google Scholar]
17.Mattson MP, Kroemer G. Mitochondria in cell death: novel targets for neuroprotection and cardioprotection. Trends Mol Med. 2003;9(5):196–205. Epub 2003/05/24. doi: 10.1016/s1471-4914(03)00046-7 . [DOI] [PubMed] [Google Scholar]
18.Hashimoto R, Hough C, Nakazawa T, Yamamoto T, Chuang DM. Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation. J Neurochem. 2002;80(4):589–97. Epub 2002/02/14. doi: 10.1046/j.0022-3042.2001.00728.x . [DOI] [PubMed] [Google Scholar]
19.Hashimoto R, Fujimaki K, Jeong MR, Christ L, Chuang DM. Lithium-induced inhibition of Src tyrosine kinase in rat cerebral cortical neurons: a role in neuroprotection against N-methyl-D-aspartate receptor-mediated excitotoxicity. FEBS Lett. 2003;538(1–3):145–8. Epub 2003/03/14. doi: 10.1016/s0014-5793(03)00167-4 . [DOI] [PubMed] [Google Scholar]
20.Park HJ, Kim HJ, Bae GS, Seo SW, Kim DY, Jung WS, et al. Selective GSK-3beta inhibitors attenuate the cisplatin-induced cytotoxicity of auditory cells. Hear Res. 2009;257(1–2):53–62. Epub 2009/08/12. doi: 10.1016/j.heares.2009.08.001 . [DOI] [PubMed] [Google Scholar]
21.Xia MY, Zhao XY, Huang QL, Sun HY, Sun C, Yuan J, et al. Activation of Wnt/beta-catenin signaling by lithium chloride attenuates d-galactose-induced neurodegeneration in the auditory cortex of a rat model of aging. FEBS Open Bio. 2017;7(6):759–76. Epub 2017/06/09. doi: 10.1002/2211-5463.12220 . [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Muller M. Developmental changes of frequency representation in the rat cochlea. Hear Res. 1991;56(1–2):1–7. Epub 1991/11/01. doi: 10.1016/0378-5955(91)90147-2 . [DOI] [PubMed] [Google Scholar]
23.Ohinata Y, Miller JM, Schacht J. Protection from noise-induced lipid peroxidation and hair cell loss in the cochlea. Brain Res. 2003;966(2):265–73. doi: 10.1016/s0006-8993(02)04205-1 . [DOI] [PubMed] [Google Scholar]
24.Chen H, Xing Y, Xia L, Chen Z, Yin S, Wang J. AAV-mediated NT-3 overexpression protects cochleae against noise-induced synaptopathy. Gene Ther. 2018;25(4):251–9. Epub 20180313. doi: 10.1038/s41434-018-0012-0 . [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Hu N, Rutherford MA, Green SH. Protection of cochlear synapses from noise-induced excitotoxic trauma by blockade of Ca(2+)-permeable AMPA receptors. Proc Natl Acad Sci U S A. 2020;117(7):3828–38. Epub 20200203. doi: 10.1073/pnas.1914247117 . [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Hashimoto K, Hickman TT, Suzuki J, Ji L, Kohrman DC, Corfas G, et al. Protection from noise-induced cochlear synaptopathy by virally mediated overexpression of NT3. Sci Rep. 2019;9(1):15362. Epub 20191025. doi: 10.1038/s41598-019-51724-6 . [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Suzuki J, Corfas G, Liberman MC. Round-window delivery of neurotrophin 3 regenerates cochlear synapses after acoustic overexposure. Sci Rep. 2016;6:24907. Epub 20160425. doi: 10.1038/srep24907 . [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Minami SB, Yamashita D, Schacht J, Miller JM. Calcineurin activation contributes to noise-induced hearing loss. J Neurosci Res. 2004;78(3):383–92. doi: 10.1002/jnr.20267 . [DOI] [PubMed] [Google Scholar]
29.Nonaka S, Hough CJ, Chuang DM. Chronic lithium treatment robustly protects neurons in the central nervous system against excitotoxicity by inhibiting N-methyl-D-aspartate receptor-mediated calcium influx. Proc Natl Acad Sci U S A. 1998;95(5):2642–7. Epub 1998/04/16. doi: 10.1073/pnas.95.5.2642 . [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Chuang DM, Wang Z, Chiu CT. GSK-3 as a Target for Lithium-Induced Neuroprotection Against Excitotoxicity in Neuronal Cultures and Animal Models of Ischemic Stroke. Front Mol Neurosci. 2011;4:15. Epub 2011/09/03. doi: 10.3389/fnmol.2011.00015 . [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Usami S, Matsubara A, Fujita S, Shinkawa H, Hayashi M. NMDA (NMDAR1) and AMPA-type (GluR2/3) receptor subunits are expressed in the inner ear. Neuroreport. 1995;6(8):1161–4. Epub 1995/05/30. doi: 10.1097/00001756-199505300-00022 . [DOI] [PubMed] [Google Scholar]
32.Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev. 2010;62(3):405–96. Epub 2010/08/19. doi: 10.1124/pr.109.002451 . [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Ruel J, Chen C, Pujol R, Bobbin RP, Puel JL. AMPA-preferring glutamate receptors in cochlear physiology of adult guinea-pig. J Physiol. 1999;518 (Pt 3):667–80. Epub 1999/07/27. doi: 10.1111/j.1469-7793.1999.0667p.x . [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Bing D, Lee SC, Campanelli D, Xiong H, Matsumoto M, Panford-Walsh R, et al. Cochlear NMDA receptors as a therapeutic target of noise-induced tinnitus. Cell Physiol Biochem. 2015;35(5):1905–23. Epub 2015/04/15. doi: 10.1159/000374000 . [DOI] [PubMed] [Google Scholar]
35.Ruel J, Chabbert C, Nouvian R, Bendris R, Eybalin M, Leger CL, et al. Salicylate enables cochlear arachidonic-acid-sensitive NMDA receptor responses. J Neurosci. 2008;28(29):7313–23. Epub 2008/07/18. doi: 10.1523/JNEUROSCI.5335-07.2008 . [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Chen GD, Kong J, Reinhard K, Fechter LD. NMDA receptor blockage protects against permanent noise-induced hearing loss but not its potentiation by carbon monoxide. Hear Res. 2001;154(1–2):108–15. Epub 2001/06/26. doi: 10.1016/s0378-5955(01)00228-3 . [DOI] [PubMed] [Google Scholar]
37.Chen Z, Kujawa SG, Sewell WF. Auditory sensitivity regulation via rapid changes in expression of surface AMPA receptors. Nat Neurosci. 2007;10(10):1238–40. Epub 2007/09/11. doi: 10.1038/nn1974 . [DOI] [PubMed] [Google Scholar]
38.Sanchez JT, Ghelani S, Otto-Meyer S. From development to disease: diverse functions of NMDA-type glutamate receptors in the lower auditory pathway. Neuroscience. 2015;285:248–59. Epub 2014/12/03. doi: 10.1016/j.neuroscience.2014.11.027 . [DOI] [PubMed] [Google Scholar]
39.Liberman LD, Suzuki J, Liberman MC. Dynamics of cochlear synaptopathy after acoustic overexposure. J Assoc Res Otolaryngol. 2015;16(2):205–19. Epub 2015/02/14. doi: 10.1007/s10162-015-0510-3 . [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Hickman TT, Hashimoto K, Liberman LD, Liberman MC. Synaptic migration and reorganization after noise exposure suggests regeneration in a mature mammalian cochlea. Sci Rep. 2020;10(1):19945. Epub 2020/11/19. doi: 10.1038/s41598-020-76553-w . [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Hong J, Chen Y, Zhang Y, Li J, Ren L, Yang L, et al. N-Methyl-D-Aspartate Receptors Involvement in the Gentamicin-Induced Hearing Loss and Pathological Changes of Ribbon Synapse in the Mouse Cochlear Inner Hair Cells. Neural Plast. 2018;2018:3989201. Epub 2018/08/21. doi: 10.1155/2018/3989201 . [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Raveendran R, Devi Suma Priya S, Mayadevi M, Steephan M, Santhoshkumar TR, Cheriyan J, et al. Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin-dependent protein kinase II. J Neurochem. 2009;110(1):92–105. Epub doi: 10.1111/j.1471-4159.2009.06108.x . [DOI] [PubMed] [Google Scholar]
43.Lee JH, Lee MY, Chung PS, Jung JY. Photobiomodulation using low-level 808 nm diode laser rescues cochlear synaptopathy after acoustic overexposure in rat. J Biophotonics. 2019:e201900145. Epub 2019/06/27. doi: 10.1002/jbio.201900145 . [DOI] [PubMed] [Google Scholar]
44.Rowe MK, Chuang DM. Lithium neuroprotection: molecular mechanisms and clinical implications. Expert Rev Mol Med. 2004;6(21):1–18. Epub 2004/10/19. doi: 10.1017/S1462399404008385 . [DOI] [PubMed] [Google Scholar]
45.Camins A, Verdaguer E, Junyent F, Yeste-Velasco M, Pelegri C, Vilaplana J, et al. Potential mechanisms involved in the prevention of neurodegenerative diseases by lithium. CNS Neurosci Ther. 2009;15(4):333–44. Epub 2009/11/06. doi: 10.1111/j.1755-5949.2009.00086.x . [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Chiu CT, Chuang DM. Neuroprotective action of lithium in disorders of the central nervous system. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2011;36(6):461–76. Epub 2011/07/12. doi: 10.3969/j.issn.1672-7347.2011.06.001 . [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Forlenza OV, De-Paula VJ, Diniz BS. Neuroprotective effects of lithium: implications for the treatment of Alzheimer’s disease and related neurodegenerative disorders. ACS Chem Neurosci. 2014;5(6):443–50. Epub 2014/04/29. doi: 10.1021/cn5000309 . [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Leeds PR, Yu F, Wang Z, Chiu CT, Zhang Y, Leng Y, et al. A new avenue for lithium: intervention in traumatic brain injury. ACS Chem Neurosci. 2014;5(6):422–33. Epub 2014/04/05. doi: 10.1021/cn500040g . [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Dell’Osso L, Del Grande C, Gesi C, Carmassi C, Musetti L. A new look at an old drug: neuroprotective effects and therapeutic potentials of lithium salts. Neuropsychiatr Dis Treat. 2016;12:1687–703. Epub 2016/07/29. doi: 10.2147/NDT.S106479 . [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0284626.r001

Decision Letter 0

Peter Rowland Thorne

31 Oct 2022

PONE-D-22-25555Round-window delivery of lithium chloride protects cochlear synapses from noise-induced excitotoxic trauma by inhibiting NMDA receptorPLOS ONE

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Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: This is a well written paper on the application of a LiCl treatment for the protection of the hair cell-auditory neuron synapse following a noise exposure in rats. The results would be of interest to researchers and clinicians in the field of hearing research.

I have a few specific comments that should be addressed:

The abstract mentions synaptic regeneration, but later talks about reducing synaptic loss. In this paper, it is more likely to be the latter that is occurring, since the treatment is given 24 hours after noise exposure. Please check the manuscript to make sure there are no other inconsistencies with respect to protection/regeneration.

The introduction was appropriately reference and introduced all relevant topics.

In this sentence in the methods: “The control group was not exposed to noise or surgery to access normal cochlear synaptic counts.” Do you mean assess here?

It’s not clear from the methods whether the lithium is injected through the RWM or into the niche. Please amend the text to clarify.

Figure 1 is a nice summary of the experimental protocol. Thanks for including this.

In this sentence in the results: “As quantitatively shown in Fig. 3C and D, the mean synaptic count per IHC was reduced”, do you mean Fig 4C and D?

And here as well: “Synaptic rescue can be seen in the noise + 2 mM LiCl group both one week (Fig. 3A) and two weeks (Fig. 3B) after the treatment.” I think you mean Fig 4A and B

In figure 4, there are some differences in the brightness of the green stain between images, particularly in Fig 4B noise + vehicle which seems dimmer than the ones on either side of it. It’s hard to see the synaptic puncta. Also in Fig 4A noise + vehicle and noise + 2 mM LiCl. Could these images be adjusted to be of similar intensity?

In figure 5 there are 2 bands in the NR2B image. Are they both relevant to the protein? Do you expect to see a reduction in NR2B compared to control? In the results section you only compare to noise-induced increase in total NR2Band pNR2B, but what would you find if you compared to control?

The first sentence in the discussion seems a little off topic, mentioning a therapeutic window in which to regenerate “peripheral axons”, whereas this study is looking at synapses.

In the discussion – Could you please include discussion on the timing of the experimental protocol, and the clinical applicability of delivering a therapeutic within 1 day of a noise exposure, particularly a noise exposure that only causes synaptopathy, as opposed to one that causes a significant threshold shift, and particularly where the treatment requires deliver of the agent in a clinical setting (as opposed to taking an oral medication for example). If synaptopathy in the human population occurs because of moderate noise exposure over a long period of time, how applicable is the animal model used in this study? How would the patient identify that a damaging noise exposure has occurred?

Depending on the requirements of the journal, a conclusion would be nice to have.

Reviewer #2: This manuscript investigated the effects of round-window delivery of lithium on noise-induced cochlear synaptopathy and hearing loss in a rat model. They also investigated the changes in NMDA receptor expression as potential underlying mechanisms. The experimental design is straightforward, although including a control + vehicle group would add more confidence for some of the conclusions (see specific comments below). The methods were appropriate and well described except the statistical analysis (see specific comments below). The results section could be improved with a more appropriate statistical analysis.

Since there is no page numbers and line numbers for the downloaded manuscript, the page numbers mentioned in the comments below is referred to the page number of the entire PDF file , which does not corresponding to the actual page numbers of the manuscript.

1. Title: The animal species needs to be included in the title

2. Page 9, Abstract:

a. Please provide the dose (or dose range) of lithium chloride and clarify whether it was a single treatment or multiple treatment

b. Throughout the whole abstract, there is no mentioning of what animals was used.

3. Page 10: “it could contribute to difficulties understanding …” should be “…difficulties in understanding…”.

4. Page 14, first line: Will the ABR testing at 3 days after treatment not only assess the “effects of the surgical manipulations”, but also the treatment effects?

5. Page 14, line 6: n = 2 for groups 1 and 2?

6. Page 15, line 1: What does “imaged at frequencies of 8, 12, 16 and 32 kHz” mean? Please clarify how the frequency location was determined.

7. Page 15, Western blot analysis: The statement “To assess NR2B activity” is not appropriate, as Western blot can only look at the protein expression of the receptor, but not the activity of the receptor.

8. Page 16, line 15: “followed by autoradiography”. “Autoradiography” is referred to the photochemical technique used to record the spatial distribution of radiolabeled compounds within a specimen or an object, which shouldn’t be used to describe the imaging analysis procedure for Western blot using ChemiDoc.

9. Pages 16 – 17: “To compare the ABR thresholds, synapse counts, and western blot result between experimental groups.” This is not a complete sentence.

10. Page 17: The sentence “parametric data were analyzed using the repeated measures one-way analysis of variance (ANOVA) was used with the Greenhouse-Geisser correction” is not grammatically correct.

11. Statistical analysis:

a. For the ABR data, you have 5 groups (control, noise-only, noise + vehicle, noise + 1mM and noise + 2mM), 4 time pints (baseline, 3 days, 1 week and 2 weeks) and 4 frequencies (8, 12, 16 and 32). A 3-way ANOVA (group x time x frequency) with repeated measures or a linear mixed model analysis should be used. If you want to analyse each frequency separately, you should use a two-way ANOVA (group x time) with repeated measures or a linear mixed model analysis. An one-way ANOVA is not appropriate for this data. In addition, why the baseline data was not included in the analysis?

b. For the wave I amplitude data (i.e., figure 3), no statistical method was mentioned. Assume that a one-way ANOVA was used, but looking at the data, at least a two-way ANOVA (time x stimulus level) with repeated measures should be used for each group instead, but a 3- way analysis would be even better.

12. Page 17, Results: The statement of “a temporary ~15 dB SPL threshold shift and a permanent ~15 dB SPL threshold shift” is not appropriate, as “temporary” means lasting for only a limited period of time; not permanent, but the threshold shift was “permanent” in your case. So, please rewrite this sentence.

13. Figure 2: The baseline ABR for all the groups should be included in order to see changes before and after noise exposure/drug treatment.

14. Page 17, Results: “The surgery for the round-window delivery of vehicle caused temporary increases in the 8 kHz and 12 kHz thresholds for only one week after the surgery, as observed by comparing the noise-only and noise + vehicle groups”. Without a control + vehicle group, the conclusion that “the surgery for the round-window delivery of vehicle caused temporary increases in the 8 kHz and 12 kHz thresholds” cannot be made. As shown in Fig 2, there seems no difference in the threshold between 3 days and 1 week for the noise + vehicle group (there seems slight improvement).

15. Page 18, Results: “All experimental groups had statistically significant differences before and after noise exposure as determined by one-way ANOVA with the Greenhouse-Geisser correction for all levels from 65 -80 dB SPL”. This statement is not backed up by the one-way ANOVA analysis, as you were talking about two factors here, i.e., time and levels. It is not clear how the one-way ANOVA could be run to tell you the differences for the two factors. As commented on the statistical analysis, a two-way ANOVA or 3-way ANOVA/linear mixed model should be used.

16. Figure 3 legend: what does the grey area mean? The exposed intensity? Please clearify.

17. Page 19, Results: For the synapses data, it is also better to do a 2-way ANOVA or linear mixed model analysis for similar reasons mentioned above. In addition, n = 2 seems a very small sample size for valid statistical analysis.

18. Figure 5: please add sample size for each group. From the western blot image, there seems only one sample in the control group and 2 samples in the other two groups. Please clarify.

19. The Supplementary figure seems not necessary.

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Reviewer #1: Yes: Rachael T Richardson

Reviewer #2: Yes: Yiwen Zheng

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PLoS One. 2023 May 22;18(5):e0284626. doi: 10.1371/journal.pone.0284626.r002

Author response to Decision Letter 0

30 Nov 2022

Review Comments to the Author:

I have a few specific comments that should be addressed:

#1. The abstract mentions synaptic regeneration, but later talks about reducing synaptic loss. In this paper, it is more likely to be the latter that is occurring, since the treatment is given 24 hours after noise exposure. Please check the manuscript to make sure there are no other inconsistencies with respect to protection/regeneration.

Author’s Response: Thank you for your comment. We made sure to use the term “regeneration” throughout the entire manuscript.

#2. The introduction was appropriately reference and introduced all relevant topics.

Author’s Response: Thank you for positive response.

#3. In this sentence in the methods: “The control group was not exposed to noise or surgery to access normal cochlear synaptic counts.” Do you mean assess here?

Author’s Response: It means negative control. Some modifications have been made to avoid confusion to readers as follows: The control group was not exposed to noise or surgery (Group 1: control group, n=8) (Line 83-84).

#4. It’s not clear from the methods whether the lithium is injected through the RWM or into the niche. Please amend the text to clarify.

Author’s Response: Thank you for your valid point. The sentence has been changes as follows: A 16G needle was positioned within the round-window niche, and 50 µl of the poloxamer solution was injected using a 1 mL syringe over the round-window membrane (Line 120-121).

#5. Figure 1 is a nice summary of the experimental protocol. Thanks for including this.

Author’s Response: Thank you for positive response.

#6. In this sentence in the results: “As quantitatively shown in Fig. 3C and D, the mean synaptic count per IHC was reduced”, do you mean Fig 4C and D?

And here as well: “Synaptic rescue can be seen in the noise + 2 mM LiCl group both one week (Fig. 3A) and two weeks (Fig. 3B) after the treatment.” I think you mean Fig 4A and B

Author’s Response: Thank you for your careful observation. We changed the number.

#7. In figure 4, there are some differences in the brightness of the green stain between images, particularly in Fig 4B noise + vehicle which seems dimmer than the ones on either side of it. It’s hard to see the synaptic puncta. Also in Fig 4A noise + vehicle and noise + 2 mM LiCl. Could these images be adjusted to be of similar intensity?

Author’s Response: Thank you for your comment. We increased the brightness of Noise + vehicle and noise + 2 mM LiCl in Fig. 4A and Noise + vehicle in Fig. 4B.

#8. In figure 5 there are 2 bands in the NR2B image. Are they both relevant to the protein? Do you expect to see a reduction in NR2B compared to control? In the results section you only compare to noise-induced increase in total NR2Band pNR2B, but what would you find if you compared to control?

Author’s Response: Thank you for your comment. The other papers also showed 2 bands in the NR2B image (see reference Neurosignals . 2013;21(1-2):42-54, PMID: 22377595). We expected that the levels of total NR2B and pNR2B would be increased after the noise exposure, and that noise-induced increase in total NR2B and pNR2B expression, and that noise-induced increase in total NR2B and pNR2B expression would be suppressed by lithium treatment. The post-hoc analysis with Dunn’s multiple comparison test showed that the noise + 2 mM LiCl group had significantly lower NR2B and pNR2B expression than the noise + vehicle group 7 days after the treatment (NR2B and pNR2B: p = 0.0019) (Line 292-295).

#9. The first sentence in the discussion seems a little off topic, mentioning a therapeutic window in which to regenerate “peripheral axons”, whereas this study is looking at synapses.

Author’s Response: Thank you for your valid point. We deleted the first sentence.

#10. In the discussion – Could you please include discussion on the timing of the experimental protocol, and the clinical applicability of delivering a therapeutic within 1 day of a noise exposure, particularly a noise exposure that only causes synaptopathy, as opposed to one that causes a significant threshold shift, and particularly where the treatment requires deliver of the agent in a clinical setting (as opposed to taking an oral medication for example). If synaptopathy in the human population occurs because of moderate noise exposure over a long period of time, how applicable is the animal model used in this study? How would the patient identify that a damaging noise exposure has occurred?

Author’s Response: Thank you for your comment. We included discussion on the timing of the treatment as follows: There is a robust literature on potential therapeutic agent to prevent or regenerate noise-induced synaptic loss in the cochlea [7, 23-26]. However, few studies have successfully demonstrated regeneration of cochlear synapses when a therapeutic agent was administered after noise exposure, such as our study [27] (Line 303-306). Also, we included discussion on the application to human hearing impairment at the end of the discussion as follows: Fourthly, many key questions remain for clinical application. There is likely a significant clinical population with loss of cochlea synapse that could be addressed by a treatment analogous that used here in noise-exposed rat. Although the success of local lithium delivery in regenerating cochlear synapses, most important is how long the therapeutic window can be elicited synaptogenesis after the noise exposure. (Line 371-375).

#11. Depending on the requirements of the journal, a conclusion would be nice to have.

Author’s Response: Thank you for your comment. We added the conclusion as follows: The present experiments showed that round window delivery of lithium can be effective local access route to the inner ear, and regenerates cochlear synapses even when administered 24 hours post-exposure of noise.

#1. Title: The animal species needs to be included in the title

Author’s Response: Thank you for your comment. The title has been changed as follows: Round-window delivery of lithium chloride protects rat cochlear synapses from noise-induced excitotoxic trauma by inhibiting NMDA receptor

#2. Page 9, Abstract:

a. Please provide the dose (or dose range) of lithium chloride and clarify whether it was a single treatment or multiple treatment

b. Throughout the whole abstract, there is no mentioning of what animals was used.

Author’s Response: Thank you for your valuable comment. We provided the dose and number of treatments of lithium chloride in the abstract as follows: We locally delivered a single treatment of poloxamer 407 (vehicle) containing lithium chloride (either 1 mM or 2 mM) to the round-window niche 24 hours after noise exposure (Line 28-29). Also, we mentioned what animal was used as follows: Our rat animal model of noise-induced cochlear synaptopathy caused about 50% loss of synapses in the cochlear basal region without damaging hair cells (Line 26-27).

#3. Page 10: “it could contribute to difficulties understanding …” should be “…difficulties in understanding…”.

Author’s Response: Thank you for your careful observation. We added the “in” in the line 49.

#4. Page 14, first line: Will the ABR testing at 3 days after treatment not only assess the “effects of the surgical manipulations”, but also the treatment effects?

Author’s Response: Thank you for this valid point. That sentence has been deleted because it can confuse readers. The ABR test after 3 days of treatment assessed the “effects of the surgical manipulations” as well as the “treatment effects”. (Line 126-138)

#5. Page 14, line 6: n = 2 for groups 1 and 2?

Author’s Response: Group 1 and 2 were assigned two rats each. We revised the wording as follows: n=2 for group 1 and 2, n=5 for group 3-5 at 1 week and 2 weeks post-treatment in the Line 141-142.

#6. Page 15, line 1: What does “imaged at frequencies of 8, 12, 16 and 32 kHz” mean? Please clarify how the frequency location was determined.

Author’s Response: Thank you for your comment. The location of the basilar membrane at the relevant frequency was determined by computing a cochlear frequency map (Muller, 1991 reference #22). Since the end of the sentence describes how the frequency location was determined, the first sentence has been modified as follows. Images were obtained using a confocal microscope (Flow-View 3000, Olympus, Japan) with a glycerol-immersion 40X objective and 2X digital zoom.

#7. Page 15, Western blot analysis: The statement “To assess NR2B activity” is not appropriate, as Western blot can only look at the protein expression of the receptor, but not the activity of the receptor.

Author’s Response: Thank you for your comment. The wording has been changed as follows: To perform the western blot (Line 175).

#8. Page 16, line 15: “followed by autoradiography”. “Autoradiography” is referred to the photochemical technique used to record the spatial distribution of radiolabeled compounds within a specimen or an object, which shouldn’t be used to describe the imaging analysis procedure for Western blot using ChemiDoc.

Author’s Response: Thank you for your valid point. The sentence has been changed as follows: After being thoroughly washed with PBST, the membranes were visualized by enhancing chemiluminescence substrate (Pierce, Rockford, IL) for 2 min, followed by chemiluminescence detection on ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA) (Line 195-197).

#9. Pages 16 – 17: “To compare the ABR thresholds, synapse counts, and western blot result between experimental groups.” This is not a complete sentence.

Author’s Response: The reviewer’s comments are appropriate. The sentence has been changed as follows: The ABR thresholds, ABR wave 1 amplitude, synapse counts, and western blot result were compared between experimental groups (Group 3-5) (Line 206-207).

#10. Page 17: The sentence “parametric data were analyzed using the repeated measures one-way analysis of variance (ANOVA) was used with the Greenhouse-Geisser correction” is not grammatically correct.

Author’s Response: The reviewer’s comments are appropriate. The sentence has been changed as follows: For multiple groups, nonparametric data were statistically analyzed using the Kruskal-Wallis H test and the repeated measures one-way analysis of variance (ANOVA) with the Greenhouse-Geisser correction.

#11. Statistical analysis:

Author’s Response: We fully understand the reviewer’s concerns. As mentioned in the statistical analysis, the ABR thresholds was compared between experimental groups (Group 3-5). Thus, we have 3 groups (noise + vehicle, noise + 1mM and noise + 2mM), 4 time points (baseline, 3 days, 1 week and 2 weeks) and 4 frequencies (8, 12, 16 and 32). For showing the ABR threshold result, usually, the Y-axis represents ABR thresholds, and the X-axis represents frequency of stimuli presented. To show the ABR thresholds between the experimental groups, audiograms for ABR threshold were separated by time point. All rats have normal hearing before surgery (baseline), so that the ABR threshold at baseline was omitted. Based on the figures, we analyzed the ABR threshold each frequency and each time point separately using Kruskal-Wallis H test.

Author’s Response: We fully understand the reviewer’s concerns. We mentioned the statistical method in line 243-244 as follows: All experimental groups had statistically significant differences before and after noise exposure as determined by one-way ANOVA with the Greenhouse-Geisser correction for all levels from 65–80 dB SPL. We want to see the changes of ABR wave 1 amplitude from 65-80dB SPL in each group, which were measured multiple times. Here, the dependent variable is one (time). If we used two-way ANOVA (4 times x 4 stimulus level), we could get the results of multiple comparison test depending on each stimulus level. As reviewer’s comment, we re-analyzed using two-way ANOVA.

#12. Page 17, Results: The statement of “a temporary ~15 dB SPL threshold shift and a permanent ~15 dB SPL threshold shift” is not appropriate, as “temporary” means lasting for only a limited period of time; not permanent, but the threshold shift was “permanent” in your case. So, please rewrite this sentence.

Author’s Response: We fully understand the reviewer’s concerns. As shown in figure 2, noise exposure caused 30 dB SPL threshold shift until one week after the treatment. Two weeks after the treatment, there was improvement of 15 dB SPL threshold. Thus, finally, noise exposure caused a permanent ~15 dB SPL threshold shift. The sentence has been changed as follows: As observed by comparing the control and noise-only groups, noise exposure caused a threshold shift of ~30 dB SPL at frequencies above the noise band, when measured 1 week after the treatment, (Fig. 2). Two week later, ABR thresholds recovered up to 15 dB SPL. Finally, noise exposure caused a permanent ~15 dB SPL threshold shift at frequencies above the noise band (Lone 216-220)

#13. Figure 2: The baseline ABR for all the groups should be included in order to see changes before and after noise exposure/drug treatment.

Author’s Response: The reviewer’s comments are appropriate. We added the baseline ABR threshold in the figure 2-A.

#14. Page 17, Results: “The surgery for the round-window delivery of vehicle caused temporary increases in the 8 kHz and 12 kHz thresholds for only one week after the surgery, as observed by comparing the noise-only and noise + vehicle groups”. Without a control + vehicle group, the conclusion that “the surgery for the round-window delivery of vehicle caused temporary increases in the 8 kHz and 12 kHz thresholds” cannot be made. As shown in Fig 2, there seems no difference in the threshold between 3 days and 1 week for the noise + vehicle group (there seems slight improvement).

Author’s Response: Thank you for your comment. The difference between the noise-only and noise + vehicle groups is whether the vehicle, a thermos-reversible gel, was surgically placed into the middle ear cavity. Therefore, it can be assumed that the difference in ABR threshold between the two groups is due to the presence of the thermos-reversible gel in the middle ear cavity. Conductive hearing loss could be caused by thermos-reversible gel by 1 week after the treatment. The sentence has been changed as follows: Compared to the noise-only group, the noise + vehicle group had worse ABR threshold at 8 kHz and 12 kHz at 1 week after the treatment, but ABR threshold in the noise + vehicle group had returned to that in the noise-only group (Line 223-224).

#15. Page 18, Results: “All experimental groups had statistically significant differences before and after noise exposure as determined by one-way ANOVA with the Greenhouse-Geisser correction for all levels from 65 -80 dB SPL”. This statement is not backed up by the one-way ANOVA analysis, as you were talking about two factors here, i.e., time and levels. It is not clear how the one-way ANOVA could be run to tell you the differences for the two factors. As commented on the statistical analysis, a two-way ANOVA or 3-way ANOVA/linear mixed model should be used.

Author’s Response: Thank you for your comment. As response to comment #11-b, we used a two-way ANOVA (Line 244-257).

#16. Figure 3 legend: what does the grey area mean? The exposed intensity? Please clearify.

Author’s Response: Thank you for your comment. The gray shaded area indicates suprathreshold stimuli (all levels from 65–80 dB SPL). We added the sentence in the figure 3 legend.

#17. Page 19, Results: For the synapses data, it is also better to do a 2-way ANOVA or linear mixed model analysis for similar reasons mentioned above. In addition, n = 2 seems a very small sample size for valid statistical analysis.

Author’s Response: We fully understand the reviewer’s concerns. As mentioned in the statistical analysis, the synaptic count was compared between experimental groups (Group 3-5). Also, synaptic count is not repeated measured data. We used a Kruskal-Wallis H test.

#18. Figure 5: please add sample size for each group. From the western blot image, there seems only one sample in the control group and 2 samples in the other two groups. Please clarify.

Author’s Response: Thank you for your comment. As mentioned in the method, four cochlear tissues per group were pooled with 3 replicates. We changed the figure 5.

#19. The Supplementary figure seems not necessary.

Author’s Response: Thank you for your comment. The supplementary figure was deleted.

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PLoS One. doi: 10.1371/journal.pone.0284626.r003

Decision Letter 1

Alan G Cheng

24 Feb 2023

PONE-D-22-25555R1Round-window delivery of lithium chloride regenerates rat cochlear synapses from noise-induced excitotoxic trauma by inhibiting NMDA receptorPLOS ONE

Dear Dr. Jung,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: Please address the minor changes recommended by both reviewers, including the comparison between control and experimental groups.

==============================

Please submit your revised manuscript by Apr 10 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Alan G. Cheng, M.D.

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Thank you for the changes you made to the manuscript.

I have a couple of minor revisions that I think need to be addressed prior to publication.

Suggested change to the title from "Round-window delivery of lithium chloride regenerates rat cochlear synapses from noise-induced excitotoxic trauma by inhibiting NMDA receptor" to one of the following:

"Round-window delivery of lithium chloride regenerates cochlear synapses damaged by noise-induced excitotoxic trauma via inhibition of the NMDA receptor in the rat"

"Regeneration of cochlear synapses in the rat following noise-induced excitotoxic trauma by round-window delivery of lithium chloride to inhibit the NMDA receptor" or something similar, because I don't think "regenerates rat cochlear synapses from noise-induced excitotoxic trauma" makes sense.

Line 208: "the two-way repeated measures one-way analysis of variance" Was it one way or two way?

Line 224 "in the noise + vehicle group had returned to that in the noise-only group." change to ...of the noise-only group.

Line 302 "There is a robust literature on potential therapeutic agent to prevent or regenerate noise-induced synaptic loss in the cochlea [7, 23-26]. " Change to "There is a robust literature on the delivery of potential therapeutic agents to prevent or regenerate noise-induced synaptic loss in the cochlea [7, 23-26].

Suggested change to Line 371 ""Fourthly, many key questions remain for clinical application. There is likely a significant clinical population with loss of cochlea synapse that could be addressed by a treatment analogous to that used here in the noise-exposed rat. Although the local delivery of lithium to the round window was effective in regenerating cochlear synapses 24 hours after the noise exposure, a key question would be how long this time window could be extended for effective synaptogenesis in a clinical setting."

Reviewer #2: The authors have addressed most of my comments to my satisfaction. However, for the ABR results, although the authors added the control group to Figure 2, the control group was still excluded from the statistical analysis. Without statistically analyse the difference between control and other experimental groups, especially, between control and noise + drug groups, a piece of very important information is missing. That is whether or not the drug treatment would have any therapeutic value/potential, i.e., to make the hearing threshold return to the control level. Therefore, control group should be included in the analysis and the results need to be discussed accordingly. In addition, in lines 208 – 208, please clarify what does “the two-way repeated measures one-way analysis of variance (ANOVA)” mean?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: Yes: Rachael Richardson

Reviewer #2: Yes: Yiwen Zheng

**********

PLoS One. 2023 May 22;18(5):e0284626. doi: 10.1371/journal.pone.0284626.r004

Author response to Decision Letter 1

19 Mar 2023

Rebuttal Letter

Manuscript ID: PONE-D-22-25555R1

Title of Manuscript: Round-window delivery of lithium chloride protects cochlear synapses from noise-induced excitotoxic trauma by inhibiting NMDA receptor

Authors’ Comment

We would like to thank the associate editor and the two reviewers for their valuable time and constructive comments. Below please find our response to the reviewers’ comments. Their comments are listed below followed by our responses summarizing how we addressed their concerns. We also included page numbers to indicate where changes were made in the manuscript. We hope that we have satisfactorily addressed the reviewers’ concerns. Due to insightful comments provided by the reviewers, the quality of our manuscript has been substantially improved. We want to thank the two reviewers and the editors for their valuable time and effort.

Reviewer #1: Thank you for the changes you made to the manuscript.

I have a couple of minor revisions that I think need to be addressed prior to publication.

"Round-window delivery of lithium chloride regenerates cochlear synapses damaged by noise-induced excitotoxic trauma via inhibition of the NMDA receptor in the rat"

Author’s Response: Thank you for your comment. We changed the title to "Round-window delivery of lithium chloride regenerates cochlear synapses damaged by noise-induced excitotoxic trauma via inhibition of the NMDA receptor in the rat".

Line 208: "the two-way repeated measures one-way analysis of variance" Was it one way or two way?

Author’s Response: Thank you for your careful observation. There was typo. We used a two-way repeated measures analysis of variance (ANOVA).

Line 224 "in the noise + vehicle group had returned to that in the noise-only group." change to ...of the noise-only group.

Author’s Response: Thank you for your careful observation. We changed to “of”.

Author’s Response: Thank you for your comment. We changed the sentence.

Author’s Response: Thank you for your comment. We changed the last sentences as reviewer’s comment.

Author’s Response: We fully understand the reviewer’s concerns. The purpose of this study was to compare the protective effects on cochlea synapses after noise exposure. To achieve this, the control group should have similar variables to the experimental group except for not receiving lithium administration. Therefore, controls included animals exposed to noise who received only the vehicle (see abstract line 29-30). To avoid confusion, we have changed the term ‘control group’ to ‘normal group’ in the manuscript and figures. Additionally, we have corrected a typographical error in the statistical description in line 208 as follows: the two-way repeated measures analysis of variance (ANOVA).

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PLoS One. doi: 10.1371/journal.pone.0284626.r005

Decision Letter 2

Alan G Cheng

5 Apr 2023

Round-window delivery of lithium chloride regenerates cochlear synapses damaged by noise-induced excitotoxic trauma via inhibition of the NMDA receptor in the rat

PONE-D-22-25555R2

Dear Dr. Jung,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Alan G. Cheng, M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Rachael Richardson

Reviewer #2: Yes: Yiwen Zheng

**********

PLoS One. doi: 10.1371/journal.pone.0284626.r006

Acceptance letter

Alan G Cheng

11 May 2023

PONE-D-22-25555R2

Round-window delivery of lithium chloride regenerates cochlear synapses damaged by noise-induced excitotoxic trauma via inhibition of the NMDA receptor in the rat

Dear Dr. Jung:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Alan G. Cheng

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Western blot analysis of total NR2B, phospho-NR2B (pNR2B), β-actin protein expression levels in the cochlea.

A-C Original uncropped and unadjusted images representing for the β-actin (A), total NR2B (B), and phopho-NR2B (C). d = day, R = replicate.

(DOCX)

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Data Availability Statement

All relevant data are within the paper and its Supporting information files.

[pone.0284626.ref001] 1.Ryan AF, Kujawa SG, Hammill T, Le Prell C, Kil J. Temporary and Permanent Noise-induced Threshold Shifts: A Review of Basic and Clinical Observations. Otol Neurotol. 2016;37(8):e271–5. Epub 2016/08/16. doi: 10.1097/MAO.0000000000001071 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref002] 2.Robertson D. Effects of acoustic trauma on stereocilia structure and spiral ganglion cell tuning properties in the guinea pig cochlea. Hear Res. 1982;7(1):55–74. Epub 1982/05/01. doi: 10.1016/0378-5955(82)90081-8 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref003] 3.Liberman MC, Dodds LW. Single-neuron labeling and chronic cochlear pathology. III. Stereocilia damage and alterations of threshold tuning curves. Hear Res. 1984;16(1):55–74. Epub 1984/10/01. doi: 10.1016/0378-5955(84)90025-x . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref004] 4.Wang Y, Hirose K, Liberman MC. Dynamics of noise-induced cellular injury and repair in the mouse cochlea. J Assoc Res Otolaryngol. 2002;3(3):248–68. Epub 2002/10/17. doi: 10.1007/s101620020028 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref005] 5.Kujawa SG, Liberman MC. Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth. J Neurosci. 2006;26(7):2115–23. Epub 2006/02/17. doi: 10.1523/JNEUROSCI.4985-05.2006 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref006] 6.Kujawa SG, Liberman MC. Adding insult to injury: cochlear nerve degeneration after "temporary" noise-induced hearing loss. J Neurosci. 2009;29(45):14077–85. Epub 2009/11/13. doi: 10.1523/JNEUROSCI.2845-09.2009 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref007] 7.Kujawa SG, Liberman MC. Synaptopathy in the noise-exposed and aging cochlea: Primary neural degeneration in acquired sensorineural hearing loss. Hear Res. 2015;330(Pt B):191–9. Epub 2015/03/15. doi: 10.1016/j.heares.2015.02.009 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref008] 8.Plack CJ, Barker D, Prendergast G. Perceptual consequences of "hidden" hearing loss. Trends Hear. 2014;18. Epub 2014/09/11. doi: 10.1177/2331216514550621 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref009] 9.Pujol R, Lenoir M, Robertson D, Eybalin M, Johnstone BM. Kainic acid selectively alters auditory dendrites connected with cochlear inner hair cells. Hear Res. 1985;18(2):145–51. Epub 1985/05/01. doi: 10.1016/0378-5955(85)90006-1 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref010] 10.Pujol R, Puel JL, Gervais d’Aldin C, Eybalin M. Pathophysiology of the glutamatergic synapses in the cochlea. Acta Otolaryngol. 1993;113(3):330–4. Epub 1993/05/01. doi: 10.3109/00016489309135819 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref011] 11.Pujol R, Puel JL. Excitotoxicity, synaptic repair, and functional recovery in the mammalian cochlea: a review of recent findings. Ann N Y Acad Sci. 1999;884:249–54. Epub 2000/06/08. doi: 10.1111/j.1749-6632.1999.tb08646.x . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref012] 12.Wang Q, Green SH. Functional role of neurotrophin-3 in synapse regeneration by spiral ganglion neurons on inner hair cells after excitotoxic trauma in vitro. J Neurosci. 2011;31(21):7938–49. Epub 2011/05/27. doi: 10.1523/JNEUROSCI.1434-10.2011 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref013] 13.Liberman MC. Single-neuron labeling in the cat auditory nerve. Science. 1982;216(4551):1239–41. Epub 1982/06/11. doi: 10.1126/science.7079757 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref014] 14.Robertson D. Functional significance of dendritic swelling after loud sounds in the guinea pig cochlea. Hear Res. 1983;9(3):263–78. Epub 1983/03/01. doi: 10.1016/0378-5955(83)90031-x . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref015] 15.Puel JL, Ruel J, Gervais d’Aldin C, Pujol R. Excitotoxicity and repair of cochlear synapses after noise-trauma induced hearing loss. Neuroreport. 1998;9(9):2109–14. Epub 1998/07/23. doi: 10.1097/00001756-199806220-00037 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref016] 16.Friedlander RM. Apoptosis and caspases in neurodegenerative diseases. N Engl J Med. 2003;348(14):1365–75. Epub 2003/04/04. doi: 10.1056/NEJMra022366 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref017] 17.Mattson MP, Kroemer G. Mitochondria in cell death: novel targets for neuroprotection and cardioprotection. Trends Mol Med. 2003;9(5):196–205. Epub 2003/05/24. doi: 10.1016/s1471-4914(03)00046-7 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref018] 18.Hashimoto R, Hough C, Nakazawa T, Yamamoto T, Chuang DM. Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation. J Neurochem. 2002;80(4):589–97. Epub 2002/02/14. doi: 10.1046/j.0022-3042.2001.00728.x . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref019] 19.Hashimoto R, Fujimaki K, Jeong MR, Christ L, Chuang DM. Lithium-induced inhibition of Src tyrosine kinase in rat cerebral cortical neurons: a role in neuroprotection against N-methyl-D-aspartate receptor-mediated excitotoxicity. FEBS Lett. 2003;538(1–3):145–8. Epub 2003/03/14. doi: 10.1016/s0014-5793(03)00167-4 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref020] 20.Park HJ, Kim HJ, Bae GS, Seo SW, Kim DY, Jung WS, et al. Selective GSK-3beta inhibitors attenuate the cisplatin-induced cytotoxicity of auditory cells. Hear Res. 2009;257(1–2):53–62. Epub 2009/08/12. doi: 10.1016/j.heares.2009.08.001 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref021] 21.Xia MY, Zhao XY, Huang QL, Sun HY, Sun C, Yuan J, et al. Activation of Wnt/beta-catenin signaling by lithium chloride attenuates d-galactose-induced neurodegeneration in the auditory cortex of a rat model of aging. FEBS Open Bio. 2017;7(6):759–76. Epub 2017/06/09. doi: 10.1002/2211-5463.12220 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref022] 22.Muller M. Developmental changes of frequency representation in the rat cochlea. Hear Res. 1991;56(1–2):1–7. Epub 1991/11/01. doi: 10.1016/0378-5955(91)90147-2 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref023] 23.Ohinata Y, Miller JM, Schacht J. Protection from noise-induced lipid peroxidation and hair cell loss in the cochlea. Brain Res. 2003;966(2):265–73. doi: 10.1016/s0006-8993(02)04205-1 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref024] 24.Chen H, Xing Y, Xia L, Chen Z, Yin S, Wang J. AAV-mediated NT-3 overexpression protects cochleae against noise-induced synaptopathy. Gene Ther. 2018;25(4):251–9. Epub 20180313. doi: 10.1038/s41434-018-0012-0 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref025] 25.Hu N, Rutherford MA, Green SH. Protection of cochlear synapses from noise-induced excitotoxic trauma by blockade of Ca(2+)-permeable AMPA receptors. Proc Natl Acad Sci U S A. 2020;117(7):3828–38. Epub 20200203. doi: 10.1073/pnas.1914247117 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref026] 26.Hashimoto K, Hickman TT, Suzuki J, Ji L, Kohrman DC, Corfas G, et al. Protection from noise-induced cochlear synaptopathy by virally mediated overexpression of NT3. Sci Rep. 2019;9(1):15362. Epub 20191025. doi: 10.1038/s41598-019-51724-6 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref027] 27.Suzuki J, Corfas G, Liberman MC. Round-window delivery of neurotrophin 3 regenerates cochlear synapses after acoustic overexposure. Sci Rep. 2016;6:24907. Epub 20160425. doi: 10.1038/srep24907 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref028] 28.Minami SB, Yamashita D, Schacht J, Miller JM. Calcineurin activation contributes to noise-induced hearing loss. J Neurosci Res. 2004;78(3):383–92. doi: 10.1002/jnr.20267 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref029] 29.Nonaka S, Hough CJ, Chuang DM. Chronic lithium treatment robustly protects neurons in the central nervous system against excitotoxicity by inhibiting N-methyl-D-aspartate receptor-mediated calcium influx. Proc Natl Acad Sci U S A. 1998;95(5):2642–7. Epub 1998/04/16. doi: 10.1073/pnas.95.5.2642 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref030] 30.Chuang DM, Wang Z, Chiu CT. GSK-3 as a Target for Lithium-Induced Neuroprotection Against Excitotoxicity in Neuronal Cultures and Animal Models of Ischemic Stroke. Front Mol Neurosci. 2011;4:15. Epub 2011/09/03. doi: 10.3389/fnmol.2011.00015 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref031] 31.Usami S, Matsubara A, Fujita S, Shinkawa H, Hayashi M. NMDA (NMDAR1) and AMPA-type (GluR2/3) receptor subunits are expressed in the inner ear. Neuroreport. 1995;6(8):1161–4. Epub 1995/05/30. doi: 10.1097/00001756-199505300-00022 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref032] 32.Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev. 2010;62(3):405–96. Epub 2010/08/19. doi: 10.1124/pr.109.002451 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref033] 33.Ruel J, Chen C, Pujol R, Bobbin RP, Puel JL. AMPA-preferring glutamate receptors in cochlear physiology of adult guinea-pig. J Physiol. 1999;518 (Pt 3):667–80. Epub 1999/07/27. doi: 10.1111/j.1469-7793.1999.0667p.x . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref034] 34.Bing D, Lee SC, Campanelli D, Xiong H, Matsumoto M, Panford-Walsh R, et al. Cochlear NMDA receptors as a therapeutic target of noise-induced tinnitus. Cell Physiol Biochem. 2015;35(5):1905–23. Epub 2015/04/15. doi: 10.1159/000374000 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref035] 35.Ruel J, Chabbert C, Nouvian R, Bendris R, Eybalin M, Leger CL, et al. Salicylate enables cochlear arachidonic-acid-sensitive NMDA receptor responses. J Neurosci. 2008;28(29):7313–23. Epub 2008/07/18. doi: 10.1523/JNEUROSCI.5335-07.2008 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref036] 36.Chen GD, Kong J, Reinhard K, Fechter LD. NMDA receptor blockage protects against permanent noise-induced hearing loss but not its potentiation by carbon monoxide. Hear Res. 2001;154(1–2):108–15. Epub 2001/06/26. doi: 10.1016/s0378-5955(01)00228-3 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref037] 37.Chen Z, Kujawa SG, Sewell WF. Auditory sensitivity regulation via rapid changes in expression of surface AMPA receptors. Nat Neurosci. 2007;10(10):1238–40. Epub 2007/09/11. doi: 10.1038/nn1974 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref038] 38.Sanchez JT, Ghelani S, Otto-Meyer S. From development to disease: diverse functions of NMDA-type glutamate receptors in the lower auditory pathway. Neuroscience. 2015;285:248–59. Epub 2014/12/03. doi: 10.1016/j.neuroscience.2014.11.027 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref039] 39.Liberman LD, Suzuki J, Liberman MC. Dynamics of cochlear synaptopathy after acoustic overexposure. J Assoc Res Otolaryngol. 2015;16(2):205–19. Epub 2015/02/14. doi: 10.1007/s10162-015-0510-3 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref040] 40.Hickman TT, Hashimoto K, Liberman LD, Liberman MC. Synaptic migration and reorganization after noise exposure suggests regeneration in a mature mammalian cochlea. Sci Rep. 2020;10(1):19945. Epub 2020/11/19. doi: 10.1038/s41598-020-76553-w . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref041] 41.Hong J, Chen Y, Zhang Y, Li J, Ren L, Yang L, et al. N-Methyl-D-Aspartate Receptors Involvement in the Gentamicin-Induced Hearing Loss and Pathological Changes of Ribbon Synapse in the Mouse Cochlear Inner Hair Cells. Neural Plast. 2018;2018:3989201. Epub 2018/08/21. doi: 10.1155/2018/3989201 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref042] 42.Raveendran R, Devi Suma Priya S, Mayadevi M, Steephan M, Santhoshkumar TR, Cheriyan J, et al. Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin-dependent protein kinase II. J Neurochem. 2009;110(1):92–105. Epub doi: 10.1111/j.1471-4159.2009.06108.x . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref043] 43.Lee JH, Lee MY, Chung PS, Jung JY. Photobiomodulation using low-level 808 nm diode laser rescues cochlear synaptopathy after acoustic overexposure in rat. J Biophotonics. 2019:e201900145. Epub 2019/06/27. doi: 10.1002/jbio.201900145 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref044] 44.Rowe MK, Chuang DM. Lithium neuroprotection: molecular mechanisms and clinical implications. Expert Rev Mol Med. 2004;6(21):1–18. Epub 2004/10/19. doi: 10.1017/S1462399404008385 . [DOI] [PubMed] [Google Scholar]

[pone.0284626.ref045] 45.Camins A, Verdaguer E, Junyent F, Yeste-Velasco M, Pelegri C, Vilaplana J, et al. Potential mechanisms involved in the prevention of neurodegenerative diseases by lithium. CNS Neurosci Ther. 2009;15(4):333–44. Epub 2009/11/06. doi: 10.1111/j.1755-5949.2009.00086.x . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref046] 46.Chiu CT, Chuang DM. Neuroprotective action of lithium in disorders of the central nervous system. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2011;36(6):461–76. Epub 2011/07/12. doi: 10.3969/j.issn.1672-7347.2011.06.001 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref047] 47.Forlenza OV, De-Paula VJ, Diniz BS. Neuroprotective effects of lithium: implications for the treatment of Alzheimer’s disease and related neurodegenerative disorders. ACS Chem Neurosci. 2014;5(6):443–50. Epub 2014/04/29. doi: 10.1021/cn5000309 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref048] 48.Leeds PR, Yu F, Wang Z, Chiu CT, Zhang Y, Leng Y, et al. A new avenue for lithium: intervention in traumatic brain injury. ACS Chem Neurosci. 2014;5(6):422–33. Epub 2014/04/05. doi: 10.1021/cn500040g . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0284626.ref049] 49.Dell’Osso L, Del Grande C, Gesi C, Carmassi C, Musetti L. A new look at an old drug: neuroprotective effects and therapeutic potentials of lithium salts. Neuropsychiatr Dis Treat. 2016;12:1687–703. Epub 2016/07/29. doi: 10.2147/NDT.S106479 . [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Round-window delivery of lithium chloride regenerates cochlear synapses damaged by noise-induced excitotoxic trauma via inhibition of the NMDA receptor in the rat

Ji Eun Choi

Nathaniel T Carpena

Jae-Hun Lee

So-Young Chang

Min Young Lee

Jae Yun Jung

Won-Ho Chung

Roles

Abstract

Introduction

Methods

Animals

Fig 1. The otic bulla was exposed via postauricular incision, and a tiny hole was made to visualize the round-window membrane (arrow) and stapedial artery (asterisk).

Noise exposure

Round-window delivery of LiCl

Auditory brainstem response measurements

Cochlear processing and immunohistochemistry

Synapse and hair cell counts

Western blot analysis

Statistical analysis

Results

Early recovery of temporary noise-induced threshold shifts after round-window delivery of LiCl

Fig 2. Mean plots of ABR thresholds measured at baseline (A), 3 days (B), one week (C), and two weeks (D) after the treatment.

Functional recovery of suprathreshold responses after round-window delivery of LiCl

Fig 3. Mean amplitude vs. level functions (± SEM) of ABR wave 1 at frequencies above the noise band measured in the noise + vehicle (A), noise + 1 mM LiCl (B), and noise + 2 mM LiCl (C) groups.

Synaptic regeneration after round-window delivery of LiCl

Fig 4. High-dose LiCl delivery can rescue the noise-induced loss of inner hair synapses.

Neuroprotective effects of round-window delivery of LiCl against glutamate excitotoxicity

Fig 5.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Peter Rowland Thorne

Roles

Author response to Decision Letter 0

Decision Letter 1

Alan G Cheng

Roles

Author response to Decision Letter 1

Decision Letter 2

Alan G Cheng

Roles

Acceptance letter

Alan G Cheng

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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