Use of caMKKbeta and its inhibitors for the preparation of products for the diagnosis, prevention, treatment of multiple types of hearing loss

Abstract

The application discloses a kind of CaMKK beta and its inhibitor in the purposes of preparation diagnosis, prevent, treat multiple types hearing loss product.CaMKK beta can be used as a kind of marker, to diagnose hair cell loss and hearing loss;By inhibiting CaMKK beta, can significantly protect outer hair cell, inner hair cell from the loss caused by noise exposure, prevent the effect on noise-induced hearing loss;In addition to noise-induced hearing loss, by inhibiting, silencing CAMKK beta, also can play the prevention, weakening effect to aminoglycoside ototoxicity side effect, can be applied to various types hearing loss to realize diagnosis, prevention and treatment effect.Thereby for prior art provides effective hearing loss prevention, treatment drug, more can cooperate aminoglycoside, overcome its ototoxicity side effect, promote aminoglycoside in various types of disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of CaMKKβ and its inhibitors in the preparation of products for the diagnosis, prevention, and treatment of various types of hearing loss. Background Technology

[0002] According to a World Health Organization report, 5% of the world's population currently suffers from hearing loss, and it is projected that by 2050, one in ten people will experience some degree of hearing loss. Acquired hearing loss accumulates over a lifetime and is typically caused by damage such as noise exposure or treatment with ototoxic drugs such as aminoglycoside antibiotics. Corresponding to the functional impairment, the loss of sensory hair cells is the primary pathology of acquired hearing loss. Because hair cells in mammals do not regenerate, this type of hearing loss is permanent. Hearing loss causes inconvenience and suffering for patients and requires significant financial investment in treatment; therefore, prevention and treatment of hearing loss is a pressing issue in personal health. However, there are currently no established drug treatments for preventing or mitigating acquired hearing loss. This is due, on the one hand, to the complexity of the sensory hair cell death pathways responding to harmful challenges and the uncertainty of potential molecular targets; on the other hand, the optimal delivery route for drugs to prevent hearing loss is limited by the blood-labyrinthine barrier (BLB) in the inner ear, which restricts the entry of compounds into the inner ear tissue (Nyberg et al., 2019). Therefore, there is an urgent need to develop a drug or treatment strategy to effectively prevent ototoxicity caused by noise exposure or aminoglycoside drugs. Summary of the Invention

[0003] This invention aims to overcome at least one deficiency of the prior art and provide the use of CaMKKβ and its inhibitors in the preparation of products for the diagnosis, prevention, and treatment of various types of hearing loss. CaMKKβ can serve as a biomarker for diagnosing hair cell loss and hearing loss. Furthermore, by inhibiting CaMKKβ, it can significantly protect outer and inner hair cells from noise-induced hearing loss, thus preventing noise-induced hearing loss. In addition to noise-induced hearing loss, inhibiting or silencing CaMKKβ can also prevent and reduce the ototoxic side effects of aminoglycoside drugs. Therefore, it can be applied to the diagnosis, prevention, and treatment of various types of hearing loss.

[0004] One object of the present invention is to provide a biomarker for the loss of outer hair cells and / or inner hair cells, namely, relatively upregulated CaMKKβ. In one or more embodiments of the present invention, it was found that traumatic noise exposure activates CaMKKβ in outer hair cells; traumatic noise exposure increases the mRNA and protein levels of CaMKKβ in outer hair cells. In fact, based on this upregulated mRNA and protein, it can be used as a biomarker for traumatic noise exposure and hair cell loss in the diagnostic process.

[0005] Another object of the present invention is to provide the use of CaMKKβ inhibitors in the preparation of products for the prevention and treatment of ototoxicity.

[0006] Furthermore, CaMKKβ inhibitors include siRNA, shRNA, and / or KN93.

[0007] Furthermore, the shRNA sequence includes 5'-CCGGGTATCCACTTGGGCATGGAATCTCGAGATTCCATGCCCAAGTGGATACTTTTTG-3'.

[0008] Another object of the present invention is to provide a drug for the prevention and treatment of ototoxicity, containing a CaMKKβ inhibitor.

[0009] Another object of the present invention is to provide the use of the above-mentioned medicament in the preparation of products for the prevention and treatment of noise-induced hair cell loss, noise-induced hearing loss and / or ototoxicity caused by aminoglycoside drugs.

[0010] Furthermore, noise-induced hair cell loss includes inner hair cell synapse loss and outer hair cell loss.

[0011] Furthermore, aminoglycoside drugs include gentamicin, natriuretic peptide, and / or furosemide.

[0012] Furthermore, it also includes a carrier for delivering CaMMKβ.

[0013] Furthermore, the CaMKKβ inhibitor comprises shRNA, and the vector comprises an adeno-associated virus vector. Even further, the adeno-associated virus vector comprises AAV2.7m8. In one embodiment of the invention, the AAV vector can infect sensory hair cells and safely transduce the corresponding CaMKKβ shRNA to protect the hair cells.

[0014] Furthermore, CaMKKβ inhibitors include siRNA, shRNA, and / or KN93. In one or more embodiments of the present invention, inhibition of CaMKKβ via siRNA or shRNA can be effectively delivered to the target site and exert its effect, with high safety. Local drug delivery offers even greater safety than conventional systemic compound therapy.

[0015] Another object of the present invention is to provide a pharmaceutical composition characterized by comprising a CaMKKβ inhibitor and an aminoglycoside drug.

[0016] Furthermore, aminoglycoside drugs include gentamicin, natriuretic peptide, and / or furosemide.

[0017] Another object of the present invention is to provide the use of CaMKKβ inhibitors in the preparation of products that inhibit noise-induced activation of AMPKα in outer hair cells.

[0018] Compared with existing technologies, the beneficial effects of this invention are as follows: CaMKKβ can serve as a biomarker for diagnosing hair cell loss and hearing loss; by inhibiting CaMKKβ, outer and inner hair cells can be significantly protected from noise-induced hearing loss, thus preventing noise-induced hearing loss; in addition to noise-induced hearing loss, inhibiting and silencing CaMKKβ can also prevent and reduce the ototoxic side effects of aminoglycoside drugs, and can be applied to various types of hearing loss for diagnosis, prevention, and treatment. This provides an effective drug for the prevention and treatment of hearing loss, and can be used in conjunction with aminoglycoside drugs to overcome their ototoxic side effects, promoting the use of aminoglycoside drugs in various diseases. Attached Figure Description

[0019] Figure 1The image shows the activation of CaMKI in basal transgenic OHCs by traumatic noise exposure. (A) Compared with unexposed control mice, in basal OHCs treated 1–3 hours after traumatic noise exposure, the epidermal plates of structurally damaged OHCs showed p-CaMKI (T177, red) immunomarking. Representative images show the sensitivity of basal transgenics to the 56, 48, and 40 kHz regions, with each image representing 6 mice; PTSN: Permanent Threshold Shift Noise Exposure; Scale bar = 10 μm. (A') Enlarged images of four OHCs show p-CaMKI (Th177) immunomarking in structurally damaged OHCs (2, 3), but not in intact OHCs (1) or scars with missing OHCs (4). The count of OHCs at the lower basal turn of the cochlea confirms a significant increase in the number of p-CaMKI (Thr177) immunomarked OHCs within 1–3 hours after PTSN. Data are presented as mean + SD and analyzed using unpaired t-tests. The number of animals in each group is shown in the bar chart. p<0.001.

[0020] Figure 2The images show that traumatic noise exposure increases CaMKKβ mRNA and protein levels in OHCs. (A) Representative images show the apex, middle, and basal turn between PTSN-exposed mice and unexposed controls: RNAscope FISH detected with CaMKKβ (red dots); immunolabeling with myosin-VIIa (Myo7a, green); PTSN-exposed mice stained with DAPI (blue) at 24 hours post-exposure to the PTSN-exposed group and the unexposed control group (ctrl). In the figures, for better visualization, the bottom panel of each row represents the OHCs in the white rectangular area of ​​the top panel. Images are compressed from 30 Z-stack projections. OHCs: outer hair cells, IHCs: inner hair cells. Scale bar = 10 μm. (B) Red dot counts show that CaMKKβ mRNA doubles at 24 hours post-PTSN exposure; the number of animals in each group is indicated in the labels. (C) Compared to the unexposed control group, the immunomarking of CaMKKβ (red) appeared stronger in OHCs treated 24 hours after PTSN exposure (24 hours post-PTSN exposure) corresponding to 30–32 kHz sensitivity; magnified OHCs allowed for better observation of the dotted markings of CaMKKβ; green: OHCs stained with phalloidin. Scale bar = 10 μm. (D) Semi-quantitative analysis of relative CaMKKβ marking (grayscale) in OHCs confirmed a significant increase at 24-hour examination but not at 1–3 hours post-PTSN examination. Bar charts show the number of animals per group. Data (B and D) are expressed as mean ± SD and analyzed by one-sample t-tests. p<0.05, p<0.01.

[0021] Figure 3The application of shCaMKKβ via adeno-associated virus (AAV) transfection significantly reduced CaMKKβ expression in the inner ear. (A) Representative images show the expression of eGFP (green) at the apical, middle, and basal turning points of AAV-infected sensory hair cells; colocalization by immunolabeling of myosin VIIa (Myo7a, red); cochlea harvested at p21 after microinjection of 2 μl AAV-shControl (shCtrl) or AAV-shCaMKKβ stock solution (2.5 × 10¹³ GCs / mL) into the left ear of FVB / NJ mice at p1–2. eGFP: enhanced green fluorescent protein; Myo7a: myosin VIIa antibody, used as a specific marker for sensory hair cells; shCtrl: out-of-order shRNA; AAV-shCtrl: AAV2.7m8-U6-shControl-CMV-eGFP; and AAV-shCaMKKβ: AAV2.7m8-U6-shCaMKKβ-CMV-eGFP. Scale bar = 10 μm. (B) Counts of eGFP-positive sensory hair cells showed no difference in infection rates of shCtrl and shCaMKKβ in OHCs and inner hair cells (IHCs), with an infection rate close to 100% in IHCs, 90% in intermediate and basal OHCs, and 40% in apical turns of OHCs. Data are presented as mean + SD and analyzed by unpaired t-tests. ns: no statistical significance. (C) Western blot results of inner ear homogenates showed that CaMKKβ expression was significantly reduced by 70% in the group transfected with the virus compared to shCtrl mice. GAPDH was used as a protein loading control, and GFP was used as a transfection marker. Data are presented as mean + SD and analyzed by one-way ANOVA and multiple comparisons. The number of animals in each group is shown in the label (B) or bar chart (C). (D) illustrates a schematic diagram of the AAV vector plasmid and the experimental timeline of FVB / NJ mice.

[0022] Figure 4The application of shCaMKKβ via adeno-associated virus transfection significantly protects FVB / NJ mice from noise-induced OHC and NIHL loss. (A) Application of shCaMKKβ via AAV2.7m8 significantly reduces noise-induced OHC loss; OHC loss is calculated along the entire length of the cochlear spiral. (B) Surface treatment shows AAV-shCtrl or aav-shCaMKKβ mice immunolabeled with myosin via (red) 14 days post-exposure. Images are taken from the cochlear epithelium 5.25 mm from the apex of the cochlea; scale bar = 20 μm. (C) 14 days post-exposure, AAV1153 shCaMKKβ mice showed a significantly reduced noise-induced auditory threshold shift compared to AAV-shCtrl mice, as measured by ABR. (D) 14 days post-exposure, the noise-induced reduction in DPOAE amplitude was significantly reversed by shCaMKKβ. Data (A, C, and D) are expressed as mean ± SD and analyzed by one-way ANOVA with multiple comparisons (C) and repeated measures ANOVA with post-hoc tests (A and D). In the figures, A and D... p-values ​​represent the comparison between shCtrl+ noise exposure and shCaMKKβ+ noise exposure. Detailed statistics are listed in Table 1 (for Figure 4A) and Table 2 (for Figure 4D). The number of animals in each group is indicated in the labels (A and D) or shown as a single point (C). p<0.05, p<0.01, p<0.001.

[0023] Figure 5Pretreatment with CaMKKβ siRNA via intratympanic delivery reduces noise-induced hearing loss (OHC), IHC synapse loss, and NIHL. (A) Representative image showing myosin-VIIa labeled, DAB-stained (brown) OHC 14 days after PTSN exposure in groups pretreated with siControl or siCaMKKβ; image taken from the basal bend 4 mm from the apex; scale bar = 10 μm. (B) OHC loss counts along the entire length of the cochlear duct indicate that pretreatment with siCaMKKβ significantly reduces noise-induced OHC loss. (C) Pretreatment with siCaMKKβ significantly reduces hearing threshold shifts induced by noise at 16 and 32 kHz. (D) Pretreatment with siCaMKKβ significantly attenuates the DPOAE amplitude reduced by PTS noise from 8–36 kHz. (E) Representative images show the immunomarking of CtBP2 (red) and GluA2 (green) in the 22 kHz region of cochlear epithelial cells 14 days after PTSN exposure. Images are compressed from 30 Z-stack projections. Scale bar = 10 μm. (F) Quantification of CtBP2 / GluA2 immunomarked synaptic granules in IHC corresponding to 6, 8, 16, 22, and 32 kHz shows a significant decrease in PTSN at 22 and 32 kHz, while pretreatment with siCaMKKβ prevents noise-induced IHC synaptic loss. Frequency correlations along the cochlear duct are shown in the lower part of F; data are presented as single pints (C) and mean ± SD (B) or ± SD (D and F), and compared by multiple analysis using repeated measures ANOVA with post-hoc tests (B and D) and one-way ANOVA (C). Detailed statistics are listed in Figure 5 Table S3 of B and Figure 5 In Table S4 of D, the labels (B, D, and F) indicate the number of animals in each group. In the diagram, B, D, and F... This represents the p-value of the comparison between siCtrl+PTSN and siCaMKKβ+PTSN. p<0.05, p<0.01, p<0.001, p<0.0001.

[0024] Figure 6The results show that, assessed 14 days after KM-FU treatment, pretreatment with siCaMKKβ delivered via PSC almost completely prevented KM-FU-induced OHC loss and hearing loss. (A) Representative images of the apical, intermediate, and basal turns in both the siCtrl and siCaMKKβ groups at 14 days after KM+FU treatment, showing myosin VIIa-labeled, DAB-stained sensory hair cells; images taken at 10x magnification; magnified images are shown at 63x for better visualization; scale bar = 10 μm. (B) OHC counts along the entire length of the cochlear spiral show that, in mice treated with siCaMKKβ, KM-FU-induced OHC loss was almost completely prevented; distance along the cochlear duct corresponds to sensitivity at 8, 16, and 32 kHz. (C) Assessing 14 days after KM-FU treatment, siCaMKKβ treatment (0.3 μg or 0.6 μg) almost completely prevented KM-FU-induced changes in auditory threshold. (D) 14-day evaluation after KM-FU treatment showed that siCaMKKβ pretreatment completely prevented the reduction in DPOAE magnitude caused by KM-FU. Data are presented as mean ± SD (B, C, and D) and analyzed by repeated measures ANOVA with post-hoc tests (B and D) and one-way ANOVA with multiple comparisons (C). Detailed statistics are listed in […]. Figure 6 Table S5 of B and Figure 6 In Table S6 of Figure D, the number of animals in each group is indicated by labels (B and D) or presented as a separate point (C). In Figure D, The p-value represents the comparison between siCtrl+KM+FU and shCaMKKβ+KM+FU. p<0.001, p<0.0001, ns: no statistical significance.

[0025] Figure 7The combination therapy with the CaMKKβ inhibitor KN93 completely prevented gentamicin-induced hair cell loss and blocked gentamicin-induced AIF translocation to the cochlear explant OHC nucleus. (A) Representative images show sensory hair cells in the apical, intermediate, and basal transitions 24 hours after administration of control, GM, KN93, and GM+KN93 in four groups. Red: Alexa-594 phalloidin staining for sensory hair cells. (B) OHC loss counts in the apical, intermediate, and basal transitions indicate that co-treatment with KN93 completely prevented GM-induced complete loss of intermediate and basal OHCs and 50% loss in the apical transition. Data are presented as mean ± SD, n = 5. (C) Dose-response curves for OHC loss after 24 hours of treatment with KN93. Data are presented as mean ± SD, n = 5. (D) Representative images obtained from the basal transfer of cochlear explants 18 hours after application of GM, KN93, and GM+KN93, compared to a control culture medium with nuclei stained with AIF (red) and DAPI (blue) immunolabeling. Examples of AIF labeling in dead OHC nuclei are indicated by arrows. These images represent 5 replicates. Scale bar = 10 μm. (E) Western blots of cochlear explant homogenates showing bands of AIF and GAPDH 18 hours after application of GM, KN93, and GM+KN93, compared to the control. There were no differences in band density among the four groups. Data are presented as mean ± SD, n = 4.

[0026] Figure 8 The image shows that CaMKKβ is an upstream kinase of AMPKα. (A) Representative images show an increase in p-AMPKα (T172) in OHCs 1–3 h post-exposure. (B) Semi-quantitative analysis of p-AMPKα (T172) in basal torus OHCs indicates a significant increase in OHCs after PTSN exposure. (C) Representative images show that siCaMKKβ reduces the PTSN-induced increase in the immunomarker (red) of p-AMPKα (T172) compared to siControl mice examined 1–3 h post-exposure. (D) Semi-quantitative analysis of p-AMPKα (T172) in basal torus confirms a significant reduction in cochlea treated with siCaMKKβ. All representative images are from basal torus, corresponding to a sensitivity of 30–32 kHz. Green: OHCs stained with phalloidin. Scale bar = 10 μm. All bar data are expressed as mean ± SD and analyzed by one-sample t-tests (B, D, and F). The number of animals in each group is represented in the bar chart. p<0.01.

[0027] Figure 9p-CaMKI (T177) is localized in the cilia and epidermal plates of the OHC, but no change in p-CaMKI (T177) or CaMKKβ was observed in whole cochlear tissue homogenates examined 1–3 hours after traumatic noise exposure; (A) confocal images (20 Z-stacks) were taken from the inferior basal rotation and reconstructed to localize p-CaMKI (T177) in OHCs. Side views were reconstructed using the Zeiss Aim Image Browser projection function. A' and A'' are side views of the horizontal portion of image A. White boxes in the three images (A, A', and A") mark the same OHCs shown in different views. White arrows highlight that p-CaMKI (T177) expression is located only in the cilia and epidermal plates of the OHC. Scale bar = 10 µm.

[0028] (B) Western blots of whole cochlear tissue homogenates showed specificity for CaMKKβ and p-CaMKI (T177), but the band density of CaMKKβ or p-CaMKI (T177) examined 1–3 hours after noise exposure did not change compared to unexposed controls. GAPDH was used as a loading control. Molecular weights are shown to the right of the bands. Dara is expressed as mean + SD and analyzed by unpaired t-tests (A: t4 = 0.959, p = 0.392; B: t4 = 0.063, p = 0.953). The number of animals in each group is shown in the bar chart.

[0029] Figure 10Injection of AAV-shCtrl and AAV-shCaMKKβ into p1-2 FVB / NJ mice did not affect auditory thresholds or DPOAE amplitude. (A) At weaning on day 21 postnatal day, no significant differences were found in auditory baseline thresholds among the three groups (uninjected control mice, mice injected with AAV shCtrl-, and mice injected with AAV shCaMKKβ-) at all test frequencies (8, 16, and 32 kHz). Data are presented as single points with mean ± SD and analyzed by one-way ANOVA and post-hoc tests (8 kHz: F2, 21 = 0.687, p = 0.514; 16 kHz: F2, 21 = 1.108, p = 0.349; 32 kHz: F2, 21 = 0.061, p = 0.941). ns: not significant. (B) In the three groups above, the DPOAE amplitudes showed similar waveforms between 4 and 36 kHz. Data are expressed as mean ± SD and analyzed by repeated measures ANOVA (F2,17 = 0.353, p = 0.707). The number of animals in each group is indicated in the labels.

[0030] Figure 11 PTSN-induced loss of OHCs was observed in the apical, basal, and hook regions of FVB / NJ mice 14 days post-exposure. Surface treatment with Myosin7a immunolabeling followed by DAB staining was used to visualize sensory hair cells. Representative images were taken from the apical, middle, basal, and hook regions, with each group representing six mice. Scale bar = 10 µm.

[0031] Figure 12 The silencing effect of siCaMKKβ delivered via PSC is higher than that delivered via WRM. (A) Representative images show that, compared with siControl mice, 72 hours after intratympanic delivery of siCaMKKβ to the left ear of mice, CaMKKβ-related immunoreactivity in the OHC was reduced (red). Scale bar = 10 µm.

[0032] (BC) Semi-quantitative analysis of CaMKKβ immunomarkers in basal transgenic OHCs corresponding to RWM (B) and PSC (C) administration, compared to the control group siControls, confirmed a significant reduction in CaMKKβ immunoreactivity after siCaMKKβ treatment (40% and 74% via RWM and PSC, respectively). Data are presented as mean + SD, n=5. p<0.01, p<0.0001 Figure 13 Immunomarking of CaMKKβ and p-AMPKα in cochlear OHCs increased 24 hours after kanamycin plus furosemide (KM-FU) treatment. (A) Immunomarking of CaMKKβ (red) increased in basal transgenic OHCs from KM-FU mice compared to untreated mice. Green: OHCs stained with phalloidin. Scale bar = 10 µm. (B) Semi-quantitative analysis of the gray density of CaMKKβ markers in OHCs, corresponding to a sensitivity of 30–32 kHz, confirmed a significant increase.

[0033] Figure 14 The study showed no difference in FM1-43 uptake in the cochlear OHC between siCtrl and siCaMKKβ treated mice. (A) Green fluorescence images show FM1-43 uptake in OHC; phalloidin-594 staining (red) reveals HC structures. MTC function in OHC was measured by delivery of siRNA via PSC 48 hours prior to FM1-43 uptake. Scale bar = 10 µm. (B) Semi-quantitative analysis of green fluorescence grayscale showed no significant difference between the siCtrl and siCaMKKβ groups. Data are presented as mean + SD and analyzed by a one-sample t-test, n = 5 per group, ns: not significant. Detailed Implementation

[0034] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0035] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0036] The present invention will now be further illustrated with specific examples. The following embodiments are only for explaining the present invention and do not constitute a limitation thereof. The test samples and test procedures used in the following embodiments include the following (if the specific experimental conditions are not specified in the embodiments, they are usually performed according to conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments can be obtained commercially unless otherwise specified).

[0037] Example 1 I. Materials and Methods (1) Animals Six-week-old male CBA / J mice (stock #00656) and four-week-old FVB / NJ mice (stock #001800) were purchased from Jackson Laboratory. All mice had free access to water and a standard mouse diet (Irradiated Lab Diet #5V75) and were housed in a specific pathogen-free animal facility at MUSC under standard 12:12 hour light-dark cycles, with the room maintained at 22 ± 1°C and the background ambient sound level maintained at approximately 50 dB SPL. CBA / J mice had a one-week acclimatization period prior to baseline auditory brainstem response (ABR) measurements. FVB / NJ mice were housed in the MUSC animal facility, and baseline ABR was measured 3–4 days prior to noise exposure. All study protocols were approved by the MUSC Institutional Animal Care and Use Committee. Animal care was conducted under the supervision of the MUSC Laboratory Animal Resources Department.

[0038] (2) Noise exposure In this study, 8-week-old CBA / J mice and 4-week-old FVB / NJ mice were exposed to broadband noise (BBN) at a sound pressure level (SPL) of 100 dB in the 2–20 kHz frequency range for 2 hours, inducing IHC synaptic band loss, OHC loss, and permanent threshold shift (PTS) 14 days post-exposure. These were referred to as our PTS noise condition (PTSN). Generally, four unrestrained mice were exposed to the sound chamber in the morning for 2 hours, one mouse per stainless steel wire cage (approximately 9 cm³). The sound level of the noise exposure was measured at multiple locations within the sound chamber using a sound level meter to ensure the homogeneity of the sound field, and measurements were taken before and after exposure to ensure stability. The sound exposure chamber was equipped with a loudspeaker (model 2450H; JBL) driven by a power amplifier (model XLS 202D; Crown Audio), which was powered by a CD player (model CD-200; Tascam TEAC American). The audio CD sound files were created and equalized using audio editing software (Audition 3; Adobe Systems, Inc.). The background sound intensity of the environment around the cages was 65 dB, measured using a sound level meter (Model 1200; Quest Technologies). Control group mice remained silent (without speakers) in cages within the same room for 2 hours.

[0039] (3) Kanamycin-Lasix (KM-FU) administration Kanamycin sulfate (KM) was purchased from Thermo Scientific (#25389-94-0), and pharmaceutical grade furosemide (FU) for injection (Baxter, #AIN00218 at 10 mg / mL) was purchased from the MUSC Hospital Pharmacy. At 6 weeks of age, CBA / J mice were immediately subcutaneously injected with KM dissolved in 0.9% saline at a dose (SQ) of 900 mg base per kilogram of body weight, immediately following an intraperitoneal injection of 200 mg / kg fluorouracil (IP). For example, a 20-gram mouse would receive 200 μL of KM and 400 μL of FU.

[0040] (4) The AAV vector was inserted into the inner ear of the FVB / NJ larva on the first or second day after birth. The AAV2.7m8 capsid plasmid, backbone vector plasmid, and helper plasmids were purchased from Addgene (#64839, #85451, and #112867). The sequences of the control and CaMKKβ shRNAs were obtained from Sigma Aldrich (control: 5'-CCGGGCGCGATAGCGCTAATAATTTCTCGAGAAATTATTAGCGCTATCGCGCTTTTTG-3', CaMKKβ: 5'-CCGGGTATCCACTTGGGCATGGAATCTCGAGATTCCATGCCCAAGTGGATACTTTTTG-3') and reconstructed into vector plasmids by Synbio Technologies. Final AAV production was performed using the vector core of the University of Pennsylvania at a titer of 2.5 × 10⁸. 13 GC / mL, and stored at -80°C before use. A schematic diagram of the AAV vector plasmid and experimental protocol is shown in Figure 3D. In short, FVB / NJ pups (p1–2) were injected with RWM using a slanted glass micropipette. The pipette was removed from the capillary glass (WPI, Sarasota, FL) using a P-2000 pipette (Sutter Instrument, Novato, CA). The pipette tip was cut at approximately a 30° angle. The puppies were anesthetized until unconscious by rapid hypothermia induction in ice / water for 2 minutes, and then placed on a cooling platform for approximately 10 minutes during the procedure. The surgical site (left ear region) was disinfected by wiping with 70% ethanol. A 0.6 cm postauricular incision was made behind the left ear to expose the transparent ear vesicle, and the tip of the glass micropipette was manually inserted through the ear vesicle, the covering fascia, and the RWM. Approximately 2 μL of the above-mentioned viral concentration (nanolithic syringe controlled by a MICRO4 controller, World Precision Instruments) was injected over 5 minutes. The skin incision is sutured with 5-0 nylon sutures. Allow the pups to recover from anesthesia for about 5 minutes on a warm mat (about 37°C), then return them to their mother for care until weaning.

[0041] (5) Delivery of siRNA into the tympanic cavity 72 hours before noise exposure As previously described, siCaMKKβ (Invitrogen, #n437808) or the disordered RNA control (siControl, Invitrogen, #4390843) is delivered topically to the RWN via intratympanic administration. (Oishi et al., 2013) In short, after anesthesia, the area behind the left ear is shaved and disinfected with 10% povidone-iodine. Under a surgical microscope, a postauricular incision (approximately 1.0 cm) is made close to the temporal bone. The auricular bulla is located ventral to the facial nerve, and a shallow hole is made in the thin portion of the bulla using a 30-G needle, enlarging it to a diameter of 2 mm to expose the RWN. A custom-made sterile micromedical tube is inserted into the hole directly above the RWN to slowly deliver 10 μL (0.6 μg or 0.3 μg per ear) of the pre-designed siCaMKKβ or siControl. Following siRNA delivery, the opening in the auricular bulla was covered by surrounding muscle and sealed with tissue adhesive (3M Vetbond Tissue Adhesive). Finally, the skin incision was closed with tissue adhesive, and the mouse was held in the surgical position during anesthesia recovery (approximately 1 hour). The animal was exposed to PTSN 72 hours after siRNA delivery.

[0042] (6) siRNA was delivered to the posterior semicircular canals 48 hours before KM-FU injection. siCaMKKβ (Invitrogen, #n437808), siAMPKα1 (Invitrogen, #s98535), and siControl (Invitrogen, #4390843) were delivered locally via the PSC. In short, after anesthesia, the area behind the left ear was shaved and disinfected with 10% povidone-iodine. Under a surgical microscope, a postauricular incision (approximately 1.0 cm) was made close to the temporal bone. The sternocleidomastoid muscle was dissected to expose the PSC. A small hole was made in the PSC with the tip of a 30-G needle and left in place for approximately 1–2 minutes until no significant endolymphatic leakage was observed. Then, a custom-made polyimide tube (HPC01862, ID: 0.0025 inch, OD: 0.0031 inch, HPC Medical) was inserted into the PSC with the tip pointing towards the ampulla, and 1 μL of siRNA (using a nanoliter syringe controlled by a MICRO4 controller, World Precision Instruments) was slowly microinjected over 2 minutes. After siRNA delivery, the tube was cut into lengths of approximately 1.5 mm. The remaining tube was sealed with heated forceps, covered with surrounding muscle, and secured with tissue adhesive (3M Vetbond Tissue Adhesive). Finally, the skin incision was sutured with 5-0 nylon sutures. The total procedure time was approximately 30 minutes. Forty-eight hours after siRNA delivery, the animal was injected with KM-FU.

[0043] (7) Measurement of auditory brainstem response and distortion product otoacoustic emissions Auditory brainstem response (ABR) measurements were performed one week before noise exposure or KM-FU treatment and 14 days after noise injury. Briefly, mice were anesthetized by intraperitoneal (IP) injection of a mixture of toluidine (10 mg / kg) and ketamine (100 mg / kg) and then placed in a soundproof and electrically shielded chamber (Acoustic Systems, Austin, Texas). The mice's body temperature was maintained at approximately 37°C using heating pads. Acoustic stimuli were delivered to a Bayer headset via a single ear, connected to a custom-made plastic endoscope inserted into the ear canal. Electrodes were inserted into the subcutaneous tissue at the apex of the skull and into the mastoid region below the left and right ears (ground level). ABR was measured at 8, 16, and 32 kHz. Tucker Davis Technology (TDT) System III hardware and SigGen / Biosig software were used to present stimuli (15 ms duration, pitch bursts with a rise-fall time of 1 ms) and record responses. An average of up to 1024 responses were recorded per stimulus level. Thresholds for each frequency were determined by decreasing the intensity increment by 10 dB until a response without tissue was detected. Thresholds were estimated between the lowest stimulus level at which a response was observed and the highest stimulus level at which no response was observed. ABR wave II was used to determine the thresholds for each frequency, which were specified by an expert unaware of the treatment conditions. The DPOAE test was performed after the ABR measurement. Measurements were taken using a TDT RZ6 system and SigGen software. An acoustic assembly containing an ER-10B+ microphone connected to two sensors was placed tightly in the ear canal. The tones were presented at fixed intensity levels of L1 = 65 dB SPL and L2 = 55 dB SPL, with an f2 / f1 ratio of 1.2. Physiological responses at individual frequencies were analyzed for each mouse, and averaging was performed on each of these frequencies from 4 kHz to 36 kHz.

[0044] (8) Cochlear surface treatment for hair cell counting: myosin VIIa labeling and DAB staining The surface treatment and diaminobenzidine (DAB) staining procedures for cochlear epithelial cells were as described previously (Chen et al., 2012; Fang et al., 2019).

[0045] (9) Images were captured from the top to the bottom of the DAB-stained surface treatment using a 20x magnifying glass on a Zeiss microscope. The length of the cochlear epithelium was measured and recorded in millimeters. Since there was no or only very slight inner hair cell (IHC) loss in our PTS noise conditions or KM-FU treatment, OHCs were counted only along the entire length of the mouse cochlear spiral from top to bottom. The percentage of OHC loss in each 0.25 or 0.5 mm length of epithelial cells was plotted as a function of cochlear length in a cellular cochlear map (Chen et al., 2012). The formula [d (%) = 156.5] from the Müller paper was used. [82.5 × log (f)] Calculate the frequency versus distance along the entire length of the cochlear spiral. (Muller et al., 2005) (10) Immunocytochemistry for cochlear surface treatment The surface treatment and sample mounting techniques are the same as those described in the section on "Myosin VIIa Labeling and DAB Staining of Cochlear Epithelial Cells" above. After blocking with 10% goat serum, the specimens were incubated with primary antibodies: 1:200 polyclonal rabbit anti-CaMKKβ (Invitrogen, #PA5-30399), 1:50 polyclonal rabbit anti-p-CaMKI (Thr 177) (Invitrogen, #PA5-38434), and monoclonal rabbit anti-p-AMPKα (Thr 172) at 1:200 (Cell Signaling Technology, # 2535) in the dark at 4 °C for 24 hours, then incubated with Alexa-Fluor-594-conjugated secondary antibody (Thermofisher Scientific, #A11012) overnight at 4 °C at a concentration of 1:200, and then incubated with Alexa-Flour-488-phalloidin (Invitrogen, #A12379) in the dark at room temperature for 1 hour. Secondary antibody controls were routinely prepared and not incubated with primary antibodies. For immunolabeling of IHC synapses, samples were incubated overnight at 37 °C in the dark with 1:200 primary monoclonal mouse anti-CtBP2 IgG1 (BD Biosciences, #612044) and 1:2,000 mouse anti-GluA2 IgG2a (Millipore, #MAB397), followed by Alexa-Fluor-594 goat anti-mouse IgG1 and Alexa-Fluor-488 goat anti-mouse IgG2a (1:1,000) at 37 °C in the dark for 1 hour. Immunolabeling images were captured using a Zeiss LSM 880 under the same Z-stack conditions and with a 63x magnifying glass.

[0046] (11) Semi-quantitative analysis of immunofluorescence signals from surface treatment Immunohistochemistry has been widely accepted as a semi-quantitative method when the practicality and semi-quantitative nature of these assays are carefully considered (Taylor & Levenson, 2006; Walker, 2006). We first assessed antibody specificity using Western blot analysis. Antibodies displaying only single bands with the correct molecular weight were used for quantification of surface-treated immunolabeling and OHC immunolabeling. Immunofluorescence of CaMKKβ, p-CaMKI (T177), and p-AMPKa (T172) on surface treatments was quantified from the original confocal images, each image taken under identical conditions using a 63x magnifying glass with identical laser gain and PMT gain settings, using ImageJ software (National Institutes of Health, Bethesda, MD). Cochleas from different groups were simultaneously fixed and stained with the same solution and processed in parallel. All surface treatments were counterstained with Alexa Fluor 488 phalloidin (green) to label hair cell structures for identification of comparable hair cell portions in the confocal images. The target region of a single OHC was delineated using a circular tool based on phalloidin staining. Gray values ​​were determined within the OHC to quantify changes in fluorescence intensity. Immunofluorescence of the target protein was measured in the apical (equivalent to sensitivity to 8–10 kHz), intermediate (equivalent to sensitivity to 16–18 kHz), and upper basal (equivalent to sensitivity to 30–32 kHz) regions of the cochlear epithelium, each region containing approximately 60 ohms. The background intensity was subtracted, and the mean gray intensity per cell was then calculated. For each repetition, the relative gray value was determined by normalizing the ratio to a control. Since there was no significant difference in the immunomarking of CaMKKβ and p-AMPKα in the cochlear OHCs of control mice 3 hours or 24 hours after PTS noise exposure or 24 hours after KM-FU treatment, and no p-CaMKI immunomarking was found in the apical and mid-transition OHCs, we only performed semi-quantitative analysis of the CaMKKβ and p-AMPKα immunomarking in OHCs and counted the OHCs with positive p-CaMKI markers in the basal transition.

[0047] (12) Use RNAscope for whole-body fluorescence in situ hybridization RNAscope Multiplex Fluorescence Kit v2 (cat #323100) and CaMKKβ probe (cat #529851) were purchased from ACD. The FISH procedure using RNAscope was performed under RNase-free conditions, following the manufacturer's instructions and as previously described (Kersigo et al., 2018; Salehi et al., 2018).

[0048] (13) Quantify the immunomarker bands or RNAscope puncta from the Z-projection on the surface treatment. We followed the procedure described above (Hill et al., 2016).

[0049] (14) Extract total protein from mouse cochlea or explants The cochlea containing intact Mini EDTA-free protease inhibitor cocktail tablets (Sigma Aldrich, #11836170001) was rapidly removed and dissected in ice-cold PBS (pH 7.4). To extract total protein from mouse cochleas or explants, cochlear tissue from a single mouse or four explants was homogenized for 5 minutes in ice-cold RIPA lysis buffer (Sigma Aldrich, #R0278) with cocktail protease inhibitors, phosphatase inhibitors cocktail II and III (Sigma Aldrich, #P5726 and #P0044). Tissue debris was removed by centrifugation at 10,000 × g for 10 minutes at 4 °C, and the supernatant was retained as the total protein fraction. Protein concentration was determined using the Bio-Rad Protein Assay dye reagent (Bio-Rad, #500-0114) with bovine serum albumin as the protein standard. Two ears or four explants were taken from each mouse and stored at -80°C.

[0050] (15) Western blot analysis Protein samples (30 μg) from mouse cochlear homogenate were separated by SDS-PAGE. After electrophoresis, the proteins were transferred to a PVDF membrane (Bio-rad, #1620177) and blocked with 5% bovine serum albumin (Sigma Aldrich, #A7906) in PBS-0.1% Tween-20 (PBS-T) (Sigma Aldrich, #P1379). The membrane was incubated overnight at 4 °C with anti-CaMKKβ (1:1,000, Invitrogen, #PA5-30399), anti-p-CaMKI (T177) (1:1,000, Invitrogen, #PA5-38434), or anti-GAPDH (1:5,000, Millipore, #ABS16), followed by washing three times with PBS-T buffer (10 min each time). The membrane was incubated with secondary antibody (Cell signaling technology, #7074 or #7076) at a concentration of 1:3,000 at room temperature for 1 hour at RT. After thorough washing of the membrane, the immunoreaction bands were observed using SuperSignal West Dura Extended Duration (Thermo Scientific, #34075). Protein blot bands were scanned using a LI-COR Odyssey FC imaging system and analyzed using ImageJ software. First, the background staining density of each band was subtracted from the band density. Next, the probe protein / GAPDH ratio was calculated based on the band density run on the same gel to normalize the difference in protein loading. Finally, the difference between the control and experimental band ratios was tested for statistical significance.

[0051] (16) FM1-43 intake experiment Mouse ear sacs were obtained via PSC delivery 48 hours after treatment with CaMKKβ or AMPKa1 siRNA in the control group and placed in Hanks balanced salt solution (HBSS). FM1-43 FX (Thermo Fisher, #F35355), a fixable analog of N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridine dibromide, was diluted to 5 μM in HBSS and slowly perfused for 1 minute per window at RT through oval and round windows. After three immediate washes with HBSS (10 seconds each), the cochlea was fixed overnight in 4% paraformaldehyde at 4°C, followed by decalcification with 4% EDTA at 4°C for 48 hours. Surface preparation and mounting were as described above. The samples were counterstained with Alexa Fluor 594phalloidin (Invitrogen, #A12381, 1:200) for 1 hour and observed using an LMS 510 confocal microscope equipped with ZEN software.

[0052] (17) Cochlear explant FVB / NJ mice on day 3 of life were used to prepare cochlear explants (Chen et al., 2009). Dissected cochlear epithelial cells were placed in pre-prepared rat tail collagen culture dishes containing 1 mL of serum-free basal medium (Eagle + serum-free supplement (Invitrogen), 1% BSA, 2 mM glutamine, and 5 mg / mL glucose). The explants were incubated for approximately 2 hours (37°C, 5% CO2), and then 1 mL of medium was added to completely submerge the explants. After overnight incubation, the medium was replaced with fresh medium containing the drugs, and the explants were incubated for 18 or 24 hours depending on the experiment. The cultures were fixed overnight at 4°C in 4% (vol / vol) paraformaldehyde, then infiltrated with 3% (vol / vol) Triton X-100 in PBS for 30 minutes, and washed three times with PBS. After incubation overnight at 4°C with Alexa-594 phalloidin or Alexa-488 phalloidin, explants were washed again with PBS at least three times for 10 minutes each time before mounting. The presence or loss of hair cells was determined by evaluating phalloidin-stained ciliary bundles and circumferential F-actin rings on the epidermal plate using a Zeiss fluorescence microscope. For AIF immunolabeling or Western blotting, explants were incubated with the drug for 18 hours and then fixed. Primary antibody for AIF (D39D2) was used (1:200 for immunolabeling, 1:1000 for Western blotting, Cell signaling technology #5318).

[0053] (18) Drug treatment for cochlear implants Gentamicin (GM) 10 mM (Sigma-Aldrich #G3632) or KN93 phosphate 3.3 mM (Selleckchem #S7423) stock solution was dissolved in double-distilled water (ddH2O), aliquoted, and stored at -80°C. Before administration to culture dishes, the stock solution was diluted with culture medium to a final concentration of 0.2 mM GM and 10 μM KN93. Treatment for 24 hours was used to assess the protective effect against GM-induced hair cell loss, or for 18 hours to assess AIF OHC nuclear translocation and AIF expression by Western blot analysis. KN93 dose profiles of 5–50 µM were evaluated within 24 hours of treatment.

[0054] (19) Statistical analysis Data were analyzed using SYSTAT 8.0 and GraphPad 5.0 software on Windows. Data on OHC loss and synaptic loss along the cochlear spiral length were analyzed using repeated measures ANOVA, with post-hoc comparisons performed using SYSTAT 8.0. The remaining analyses were performed using GraphPad 5.0. Differences in multiple comparisons were assessed using one-way ANOVA with multiple comparisons. Differences in one-to-one comparisons were analyzed using a two-tailed unpaired Student's t-test. Relative ratio data for one-to-one comparisons were analyzed using a one-sample t-test. A p-value <0.05 was considered statistically significant. Data are expressed as mean ± SD, depending on sample size and within-group variability. Sample size is indicated for each figure.

[0055] (20) Partial abbreviations AAV: Adeno-associated virus; ABR: Auditory brainstem response; AMPKα: AMP-dependent protein kinase α subunit; CaMKKβ: Ca2+ / calmodulin-dependent protein kinase kinase-β; DMSO: Dimethyl sulfoxide; DPOAE: Aberration-related otoacoustic emission; eGFP: Enhanced green fluorescent protein; FISH: Fluorescence in situ hybridization; FU: Furosemide; HC: Hair cells; IHC: Inner hair cells; IP: Intraperitoneal injection; KM: Kanamycin; KO: Knockout mouse; LKB1: Hepatokinase B1; MTC: Mechanosensor channel; NIHL: Noise-induced hearing loss; OHC: Outer hair cells; PBS: Phosphate-buffered saline; PBS-T: PBS containing 0.1% Tween 20; PSC: Posterior semicircular canal; PTS: Permanent threshold variation; PTSN: PTS-noise; ROS: Reactive oxygen species; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPL: Sound pressure level; siRNA: Small interfering RNA Silencing; shRNA: short hairpin RNA silencing; siControl: disordered siRNA; TDT: Tucker-Davis technology; RWM: round window membrane.

[0056] II. Traumatic noise exposure activates CaMKKβ in outer hair cells. Previous studies have shown that noise activates p-AMPKα T172, and silencing AMPKα1 can prevent NIHL. Since CaMKKβ is one of the upstream kinases of AMPKα, the inventors first evaluated the activation of CaMKKβ. Phosphorylated CaMKI (p-CaMKI T177) is one of the markers of CaMKKβ activation; therefore, p-CaMKI (T177) in OHCs at several time points after traumatic noise exposure was evaluated by immunohistochemistry. The results showed that the immunomarker representing p-CaMKI appeared in the styloid cilia and epidermal plates of structurally damaged OHCs, rather than in intact OHCs or in scars resulting from lost OHCs; detection at 1, 3, 5, and 8 hours after traumatic noise exposure revealed that p-CaMKI decreased over time after exposure, with no p-CaMKI-positive OHCs observed until 24 hours post-exposure. At each examination time point, p-CaMKI-positive OHCs were found only in the basal region of the cochlear epithelium between 40–56 kHz. Confocal images with 20 Z-stacks showed that p-CaMKI was localized to the OHC stenocilia and epidermal plate (Fig. 9A). Counting of p-CaMKI146-positive OHCs 1–3 hours after noise exposure showed that an average of 18% of OHCs were positively labeled in the region between 40–56 kHz at the lower basal turn (Fig. 1B, t10 = 5.791, p = 0.0002, analyzed by unpaired t-test). Similarly, after traumatic noise exposure, OHCs in both CBA / J and FVB / NJ mice exhibited a similar p-CaMKI immunomarking status to the aforementioned condition.

[0057] Furthermore, to determine the specificity of the antibodies in immunohistochemistry, Western blotting was performed using mouse whole cochlear tissue homogenates. Individual protein bands representing CaMKKβ and p-CaMKI had the correct molecular weights of 55 kDa and 45 kDa, respectively, corresponding to CaMKKβ and p-CaMKI, confirming the antibody specificity. Semi-quantitative results of band density showed no difference between control and traumatic noise-exposed mice examined 1–3 hours after exposure or sham exposure (Fig. 9B-C, p-CaMKI: t4 = 0.063, p = 0.953; CaMKKβ: t4 = 0.959, p = 0.392). These results indicate that traumatic noise exposure activates CaMKKβ in OHCs.

[0058] III. Exposure to traumatic noise increases the mRNA and protein levels of CaMKKβ in outer hair cells. To determine whether traumatic noise exposure alters CaMKKβ mRNA levels, FISH with RNAscope was used on cochlear surface formulations, as previously reported (Kersigo et al., 2018; Salehi et al., 2018). Typically, CaMKKβ mRNA expression levels are low in sensory hair cells in unexposed control mice. Since the loss of OHCs occurs at the inferior basal turn and immediately following the basotopic gradient at 1 hour and 24 hours post-exposure, the CaMKKβ mRNA level in the inferior basal turn of the OHCs could not be quantified. Therefore, the inventors analyzed CaMKKβ mRNA in the superior basal region (corresponding to a sensitivity of 32 kHz), middle region (corresponding to a sensitivity of 16 kHz), and apical region (corresponding to a sensitivity of 8 kHz) of the sOHCs 1–3 hours or 24 hours post-noise exposure. Figure 2 (As shown in A). We found that in the preliminary experiments, the spots representing CaMKKβ mRNA markers were more prominent at 24 hours than at 1–3 hours. Then, the inventors focused on comparing mice 24 hours after exposure with control mice not exposed to traumatic noise; they found that compared to control mice without noise exposure, mice 24 hours after exposure showed a significant approximately 2-fold increase in CaMKKβ mRNA expression levels at the apical (t6=3.528, p=0.0124), middle (t5=3.4612, p=0.018), and basal turn (t6=5.247, p=0.0019) OHCs. Figure 2 B, analyzed by a one-sample t-test). Then, CaMKKβ immunolabeling was performed on OHCs at checkpoints 1–3 hours and 24 hours after exposure. Strong CaMKKβ immunolabeling was found in the basal corner region (corresponding to sensitivity to 32 kHz) of OHCs at 24 hours post-exposure. Figure 2 C), while at checkpoints 1-3 hours post-exposure, CaMKKβ immunomarking showed only a weak increase. Semi-quantitative analysis of CaMKKβ grayscale intensity in basal OHCs confirmed that CaMKKβ levels significantly increased to twice that of the control group 24 hours post-exposure (t5 = 3.098, p = 0.0269), but there was no significant difference between the two groups at 1-3 hours post-exposure. Figure 2 (D, analyzed by one-sample t-test). These results indicate that the increase in CaMKKβ mRNA and CaMKKβ protein levels induced by traumatic noise is not related to CaMKKβ activation. Traumatic noise activation of CaMKKβ may be due to calcium overload entering OHCs.

[0059] IV. Transfection of shRNA with AAV2.7m8 to mediate CaMKKβ silencing significantly protects against hair cell loss and hearing loss caused by traumatic noise. To assess the role of increased CaMKKβ mRNA and protein levels in oculomotor hearing loss (OHCs) following traumatic noise exposure, CaMKKβ silencing was mediated by viral transduction of short hairpin RNA (shCaMKKβ) via AAV2.7m8. Since FVB / NJ mice exhibited normal baseline hearing at least 10 weeks of age and showed similar susceptibility to noise-induced basal turn OHC loss as CBA / J mice, FVB / NJ mice were used for shCaMKKβ manipulation in this experiment, with FVB / NJ mothers providing normal care for AAV-injected pups during the experiment. Based on reports of AAV2.7m8's efficient infection of cochlear IHCs and OHCs (Isgrig et al., 2019), this experiment used AAV2.7m8 transfection of shScrambled-RNA (shControl) or shCaMKKβ. Specifically, viral vectors carrying the corresponding hairpin RNA were injected into the left ear RWM of pups aged 1–2 days (p1–2) for transduction. Three weeks after viral injection, eGFP-positive sensory hair cell counts showed: 100% infection rate of IHCs along the entire cochlear spiral; 90% infection rate of OHCs in the basal and intermediate turning regions; and 40% infection rate of OHCs in the apical region. There was no difference in infection rates between shControl and shCaMKKβ in sensory hair cells (Fig. 3A-B, analyzed by unpaired t-test). Three weeks after viral injection, Western blot analysis of whole cochlear homogenates showed that shCaMKKβ exhibited approximately 70% CaMKKβ silencing efficiency compared to shControl mice (Fig. 3C, F2, 15 = 67.28, p < 0.001, analyzed by one-way ANOVA and multiple comparisons). There was no difference in CaMKKβ expression between shControl mice and uninjected control mice (p > 0.05). AAV transfection of mice with shControl and shCaMKKβ produced similar GFP band densities, confirming the lack of difference in infection rates. Furthermore, ABR and DPOAE measurements were performed within 3 weeks post-injection, and it was found that injecting the viral vector into p1-2 pups did not cause hearing loss. Among the control group (initial control group), shControl, and shCaMKKβ groups, the auditory thresholds at three test frequencies (8, 16, and 32 kHz) and the DPOAE amplitude at 4–36 kHz were similar. Figure 10(A: ABR data were analyzed using one-way ANOVA for multiple comparisons; B: DPOAE data were analyzed using repeated measures ANOVA). Noise conditions were then characterized by exposing 4-week-old FVB / NJ mice to noise intensities ranging from 96 to 100 dB SPL for 2 hours. At 14 days post-exposure assessment, mice exposed to 100 dB SPL exhibited permanent threshold shift (PTS) accompanied by loss of OHCs at the apex and base. Based on this PTS condition, in vivo experiments were conducted to assess whether shCaMKKβ attenuated noise-induced sensory hair cell loss and NIHL. FVB / NJ control mice without noise exposure showed no OHC loss at the mid-region or base, except for a 5% loss of OHCs at the apical turn (0–0.25 mm from the apex). In mice exposed to 100 dB noise for 14 days, OHC loss occurred at the apical and basal bends, with significantly greater OHC loss in the hook region; no OHC loss was observed at the middle bend. Figure 11OHC loss counting along the entire length of the cochlear spiral showed that noise-induced OHC loss in the basal turn began at 3.75 mm from the apex and gradually increased in the direction toward the hook region, reaching 65% OHC loss at 5.5 mm. Furthermore, post-exposure, approximately 20% OHC loss was also observed in the apical region at 0.25 mm from the apex (Fig. 4A). In shControl mice, the pattern of noise-induced OHC loss was similar to that in non-viral transduced mice (from 0.25–1.25 mm: F1,11 = 0.878, p = 0.369; from 3.75–5.5 mm: F1,11 = 0.232, p = 0.639), indicating that AAV2.7m8 infection alone had no effect on the susceptibility of FVB / NJ mice to noise-induced OHC loss. In contrast, in shCaMKKβ-silenced mice, noise-induced OHC loss was significantly reduced at both the apex (0.25–1.25 mm: F1,13 = 14.586, p = 0.002) and the basal bend (3.75–5.5 mm: F1,13 = 148.532, p < 0.001, Fig. 4A, analyzed by repeated measures ANOVA and post-hoc tests; see Table 1 for detailed statistics). In contrast, in shCaMKKβ-silenced mice, noise-induced OHC loss was significantly reduced at both the apex (0.25–1.25 mm: F1,13 = 14.586, p = 0.002) and basal turn (3.75–5.5 mm: F1,13 = 148.532), p < 0.001 (Figure 4A, analyzed by repeated measures ANOVA and post-hoc tests; see Table 1 for detailed statistics). Even in the lower basal hook region (5.25–5.5 mm from the apex), the percentage of noise-induced OHC loss decreased from a mean of 50% to less than 10%, as shown in the representative images. Figure 4 B).

[0060] Table 1. Post-hoc analysis of OHC loss (Fig. 4A)

[0061] Furthermore, shCaMKKβ mice showed significant protection against PTS noise-induced auditory threshold changes at all three tested frequencies (Fig. 4C, by one-way ANOVA and Bonferroni's multiple comparisons, AAV-shCtrl+PTS vs AAV-shCaMKKβ+PTS: 8 kHz: F2,21 = 8.895, p < 0.01; 16 kHz: F2,21 = 25.26, p < 0.0001; 32 kHz: F2,21 = 24.64, p < 0.0001). Additionally, at 14 days post-exposure, auditory threshold shifts induced by PTS noise exposure were similar between AAV-shControl mice and control mice without AAV transduction at all three measured frequencies (8, 16, and 32 kHz).

[0062] OHC functional testing using DPOAE measurements showed that in unexposed control mice, the amplitude peaked at 16 kHz, while at 14 days post-exposure, the peak disappeared and the wave flattened at an amplitude below -10 dBSPL across all test frequencies from 4 to 36 kHz. Figure 4 D, Ctrl vs Ctrl+PTSN, F1,13=211.077, p<0.001).

[0063] shControl mice showed similar DPOAE amplitudes to untransfected mice after PTS noise exposure (Fig. 4D, F1,12 = 0.099, p = 0.759); shCaMKKβ-silenced mice showed a significant reversal of the PTS noise-induced reduced DPOAE amplitude in the 8 kHz–36 kHz range, with a peak amplitude at 16 kHz, but lower than the unexposed control mice (Fig. 4D, AAV-shCtrl + PTSN vs. AAV-shCaMKKβ + PTSN, F1,13 = 56.405, p < 0.001, analyzed by repeated measures ANOVA; see Table 2 for detailed statistics).

[0064] All these results indicate that CaMKKβ silencing via the AAV vector has a significant preventive effect against NIHL in FVB / NJ mice.

[0065] Table 2 Post-hoc analysis of DPOAE amplitudes (Fig. 4D)

[0066] 5. Pretreatment with CaMKKβ small interfering RNA can reduce noise-induced synaptic loss in inner hair cells, loss in outer hair cells, and NIHL.

[0067] To test whether silencing CaMKKβ also attenuates NIHL in CBA / J mice, we applied out-of-order siRNA (siControl) or siCaMKKβ to the mice's RWM 72 hours before noise exposure. The siCaMKKβ treatment group, delivering 0.6 μg siCAMKKβ, showed approximately 40% silencing efficiency of OHC in mice compared to the siControl group (Fig. 12A, B, t5 = 5.1711, p = 0.0036, analyzed by one-sample t-test). The choice of 0.6 μg siCAMKKβ in this experiment was based on our preliminary results. Furthermore, we characterized the noise conditions in 12-week-old CBA / J mice in detail; to induce PTS in IHC synaptic loss and OHC loss, the mice were exposed to 101 dB for 2 hours. Myosin VIIa labeling and DAB staining were used to surface-treat the cochlear spiral. The OHC counts along the entire length of the cochlear spiral showed that pretreatment with siCaMKKβ significantly reduced noise-induced OHC loss (Fig. 5A, B, F1, I1 = 9.213, p = 0.011, analyzed by repeated measures ANOVA and post-hoc tests; see Table 3 for detailed statistics).

[0068] Table 3 Post-hoc analysis of OHC loss (Fig. 5B)

[0069] In siControl-treated mice, noise-induced OHC loss began at 3.0 mm from the vertex and gradually increased towards the base until complete loss of OHC at 5.5 mm from the vertex. In siCaMKKβ-treated mice, noise-induced OHC loss began at 4 mm from the vertex and decreased to an average of 40% at 5.5 mm from the vertex. OHC loss was significantly reduced at 3.5 mm (p = 0.044), 4 mm (p < 0.001), and 4.5 mm (p = 0.001) from the vertex. Similarly, compared to mice treated with siControl 14 days post-exposure, pretreatment with siCaMKKβ significantly reduced noise-induced auditory threshold shifts at 8 kHz (F2,37=4.106, p=0.0245), 16 kHz (F2,37=69.55, p<0.0001), and 32 kHz (F2,37=28.11, p<0.0001) (Fig. 5C, by one-way ANOVA and Bonferroni's multiple comparison test). Furthermore, noise-induced auditory threshold changes were similar between the siCtrl group and the untreated group (Fig. 5C). Furthermore, noise exposure significantly reduced the DPOAE amplitude in the 8–36 kHz range (F1, 9 = 111.894, p < 0.001), and this reduction was significantly mitigated by siCaMKKβ treatment (Fig. 5D, F1, 9 = 58.829, p < 0.001, measured by repeated analysis of variance; see Table 4 for detailed statistics).

[0070] Table 4. Post-hoc analysis of DPOAE amplitudes (Fig. 5D)

[0071] Noise-induced IHC synaptic loss has been well-documented through co-immunomarking of CtBP2 in the presynaptic band and Glu2A in the postsynaptic terminal (Wan et al., 2014; Hill et al., 2016; Fang et al., 2019). We evaluated whether pretreatment with siCaMKKβ 14 days after noise exposure could also prevent noise-induced IHC synaptic loss. Previously, we found that siControl treatment alone without noise exposure did not alter the number of IHC synapses compared to the initial group (control group) (Hill et al., 2016). In siControl-treated mice, noise-induced IHC synaptic loss occurred at 22 kHz and 32 kHz 14 days after exposure, without the presence of large orphan bands (marked by the separation of CtBP2 and GluA2 markers) (Figure 5E). Following noise exposure, IHC synapse counts at 0.4, 1.0, 2.4, 3.3, and 3.9 mm from the apex (corresponding to 6, 8, 16, 22, and 32 kHz) showed significant IHC synapse loss at 22 kHz (F2, 17 = 29.04, p < 0.0001) and 32 kHz (F2, 15 = 22.83, p < 0.0001) compared to unexposed siControl mice, but no significant loss at 6, 8, and 16 kHz (Fig. 5F, by one-way ANOVA analysis with multiple comparisons). Pretreatment with siCaMKKβ significantly reduced noise-induced synaptic loss in the 22-kHz and 32-kHz regions (p < 0.0001), and the number of IHC synapses in siCaMKKβ-treated mice was similar to that in unexposed control mice. All these results are consistent with shCaMKKβ treatment of FVB / NJ mice to prevent NIHL.

[0072] VI. siCaMKKβ pretreatment completely prevents ototoxicity induced by kanamycin and furosemide treatments. Since the loss of sensory hair cells is a common pathological feature of noise-induced and aminoglycoside-induced hearing loss, the inventors tested whether silencing CaMKKβ could prevent aminoglycoside-induced hearing loss. Based on literature reports (Taylor et al., 2008; Ruhl et al., 2019), we first tested KM-FU-induced hearing loss in 6-week-old CBA / J mice. Specifically, in the preliminary experiment, we used 600-900 mg / kg of KM base, followed by 100-400 mg / kg of FU. We found that the application of 900 mg / kg of KM base alone (SQ injection), followed by 200 mg / kg of FU (IP injection), resulted in significant hearing loss; the average hearing threshold at 8 and 16 kHz was 50 dB, and after 14 days of drug treatment, at 32 kHz, the intermediate and basal curves of the OHC were completely lost, and the apical turn of the cochlea was partially lost (death without drug treatment). Figure 6 A). We chose this dose to test whether siCaMKKβ pretreatment could prevent KM-FU-induced hair cell loss and hearing loss.

[0073] Before we assessed the protective effect, we found that KM-FU treatment also significantly increased the immunomarker of CaMKKβ in OHCs at 24 hours post-treatment compared with untreated control mice (t4=2.896, p=0.0443, analyzed by one-sample t-test). Figure 13 KM or FU alone did not alter the immunomarkers of CaMKKβ, nor did they result in OHC loss. We also found that the silencing effect of CaMKKβ (0.6 μg / ear) via the posterior semicircular canal (PSC) was 74%, while the silencing effect via RWM was 40%. Figure 12 We then opted to administer siCaMKKβ via PSC to assess whether siCaMKKβ pretreatment could prevent KM-FU-induced hearing loss. Pretreatment with siCaMKKβ at a dose of 0.3 μg or 0.6 μg / ear via PSC 48 hours prior to KM-FU administration almost completely prevented substantial KM-FU-induced hearing loss (OHC). Figure 6 AB, siCaMKKβ and siCtrl:F1,15=1593.468, p<0.001).

[0074] In the hook region of some mice, only a small amount of OHC loss was observed. KM-FU-induced hearing threshold shift was also completely blocked at all three measured frequencies (8, 16, and 32 kHz). Figure 6C, Data obtained through one-way ANOVA and multiple comparison analysis; detailed statistics are shown in Table 5). Furthermore, OHC function was measured by DPOAE, and the results showed that pretreatment with siCaMKKβ at doses of 0.3 μg or 0.6 μg completely blocked the DPOAE amplitude reduction of KM-FU at all test frequencies from 8 to 36 kHz, while pretreatment with siControl did not affect the DPOAE amplitude reduction of KM-FU (Figure 6D, analyzed by repeated measures ANOVA and post-hoc testing; detailed statistics are shown in Table 6). Since aminoglycoside antibiotics primarily enter OHCs via mechanotransduction channels (MTCs), to determine whether this protective effect of siCaMKKβ pretreatment is due to blocking KM-FU entry into OHCs, we evaluated OHC MTCs using FM1-43FX according to the literature (Meyers et al., 2003; Taylor et al., 2008; Nakanishi et al., 2018). Our results indicate that, compared with the control group, PSC-administered siCaMKKβ pretreatment did not alter the uptake of OHCs by FM1-43FX ( Figure 14 ).

[0075] These results indicate that siCaMKKβ pretreatment can completely prevent KM-FU-induced ototoxicity.

[0076] Table 5 Statistical analysis of ABR (Fig. 6C)

[0077] Table 6. Post-hoc analysis of DPOAE amplitudes (Fig. 6D)

[0078] VII. Combined treatment with CaMKKβ inhibitors can block the translocation of gentamicin-induced apoptosis-inducing factors to the outer hair cell nucleus and completely prevent gentamicin-induced loss of cochlear explant hair cells. Since the prevention of hair cell loss by silencing CaMKKβ in vivo is consistent between lesions exposed to noise and those treated with KM-FU, we evaluated whether combination therapy with the CaMKKβ inhibitor KN93 could block gentamicin (GM)-induced cochlear explant hair cell death and loss. Following our previous publication (Chen et al., 2009), we treated FVB / NJ mouse explants at 8, 16, 18, 20, and 24 hours postnatally (p3) with 0.2 mM GM. Hair cell loss occurred 18–24 hours after GM application, but not at 8 and 16 hours. Hair cell loss began at 18 hours of basal turning and progressed in a gradient from base to apex, reaching 85% loss at 24 hours (Fig. 7A–B). We then determined the appropriate dose of KN93 for combination therapy with GM. No hair cell loss was observed after 24 hours of treatment with 5-10 μM KN93 on p3 explants, but hair cell loss was observed with 20 μM KN93, and the loss reached 40% with 50 μM treatment. Figure 7 C).

[0079] Therefore, we selected 10 μM KN93 and 0.2 mM GM for co-treatment for 24 hours to evaluate its protective effect. We found that the combination treatment with KN93 almost completely prevented GM-induced hair cell loss. Figure 7 AB). Next, we assessed AIF expression by immunohistochemistry and Western blotting, as it is associated with noise- and aminoglycoside-induced hair cell death. Eighteen hours after GM treatment, AIF was strongly immunomarked in the nuclei of dead OHC cells (AB). Figure 7 D). Other groups (cell culture medium control group, KN93-only group, and GM+KN93 combined treatment group) did not have AIF nuclear markers ( Figure 7 D). Furthermore, immunoblotting using explant homogenates showed that, 18 hours after administration, a single band of AIF appeared at 67 kDa in each group, with no difference in band density. Figure 7 E). These results further support the view that inhibition of CaMKKβ can prevent hair cell death induced by acute inner ear trauma and are consistent with the literature on the involvement of AIF in aminoglycoside-induced hearing loss.

[0080] 8. Inhibition of CaMKKβ can reduce the activation of AMPKα in outer hair cells by noise. To assess whether inhibition of CaMKKβ would reduce inner ear AMPKα activation, we pretreated mice with siCaMKKβ via RWM delivery, following a previous report (Oishi et al., 2013). Although delivery of siCaMKKβ via PSC showed a higher silencing efficiency than delivery via RWM (…),… Figure 12 However, after PSC delivery, mice exhibited some vestibular dysfunction (recovering within 5–7 days). Therefore, to avoid this potential confounding factor of noise exposure, siRNA was delivered to RWM, and it was assessed whether noise-induced AMPKα (T172) activation in OHCs was attenuated by siCAMMKβ treatment within 1–3 hours post-exposure. Consistent with previous publications (Hill et al., 2016; Wu et al., 2020), traumatic noise exposure resulted in a significant increase in p-ampkα in OHCs (…). Figure 8 C, D, t7 = 5.095, p = 0.0014 (one-sample t-test analysis). Furthermore, the increase in p-AMPKα (T172) immunomarker in OHCS was inhibited by siCAMMKβ pretreatment. Figure 8 E). Semi-quantitative analysis using the immunomarker p-ampkα (T172) grayscale confirmed a 25% reduction in OHCs. Figure 8 F, t4 = 5.024, p = 0.0074 (analyzed by one-sample t-test).

[0081] These results support the view that CAMMKβ is an upstream kinase of AMPKα and that CAMMKβ is involved to some extent in noise-induced AMPKα activation in OHC.

[0082] Overall, this study found that acute inner ear injury, including exposure to traumatic noise or KM-FU treatment, activates CaMKKβ, leading to pathological signal changes associated with CaMKKβ cascade activation, such as the CaMKKβ-AMPKα pathway. Silencing CaMKKβ via AAV transduction or pretreatment via intratympanic delivery of siCaMKKβ significantly attenuated NIHL; pretreatment via PSC delivery of siCaMKKβ almost completely prevented significant KM-FU-induced OHC loss and hearing loss. Furthermore, co-treatment with the CaMKKβ inhibitor KN93 completely blocked GM-induced cochlear explant hair cell loss. All these results indicate that acute inner ear trauma activates CaMKKβ, leading to hair cell loss and hearing loss. In addition, this study used whole-body FISH in adult mice and in vivo AAV2.7m8 transduced shRNA to investigate noise and ototoxic drug-induced hearing loss, demonstrating the feasibility of applying these cutting-edge technologies to studies of acquired hearing loss in adult mice.

[0083] (1) Exposure to traumatic noise or kanamycin-furosemide treatment can activate CaMKKβ, while inhibition of CaMKKβ can protect mice from noise-induced and aminoglycoside-induced hearing loss. Our results indicate that CaMKKβ is activated in a time-dependent manner after exposure to traumatic noise and, as shown by the presence of p-CaMKI (T177) immunolabeled OHCs in the basal translocation 1–3 hours post-exposure, is associated with initial OHC death, and the number of labels decreases over time until no labeled OHCs are observed 24 days post-exposure.

[0084] We used functional and morphological evidence from various inner ear traumas, including exposure to noise, AAV transduction of shCaMKKβ during KM-FU treatment, in vivo silencing with siCaMKKβ, and explant co-treatment with GM and the specific CaMKKβ inhibitor KN93, to further support the view that CaMKKβ is a key target for preventing OHC death and hearing loss caused by acute inner ear injury. Furthermore, CaMKKβ activation is involved in noise-induced IHC synaptic and OHC pathological losses; however, the degree of protection differs between OHC and IHC synapses. For example, inhibiting CaMKKβ in OHC by 40% via RWM delivery reduces noise-induced OHC loss but completely prevents noise-induced synaptic loss. This difference in protection between OHC and IHC synapses suggests that completely preventing OHC loss is more difficult than protecting IHC synapses. Interestingly, OHC loss is not entirely correlated with the hearing threshold measured by ABR. For example, no OHC loss was observed at a distance of 2.4 mm from the peak after exposure (corresponding to a sensitivity of 16 kHz), while a significant hearing threshold shift was observed at 16 kHz. This difference may be due to morphologically intact but physiologically impaired OHC, as demonstrated by OHC functional measurements of DPOAE. Traumatic noise exposure caused the DPOAE amplitude to drop from around +20 dB SPL to below 0 in the 8–36 kHz range, while AAV transduction with shCaMKKβ treatment or sicamkβ pretreatment significantly increased the DPOAE amplitude, indicating that OHC function was preserved. Surprisingly, pretreatment with CaMKKβ via PSC delivery almost completely halted the substantial OHC and hearing loss induced by KM-FU, suggesting that the mechanism of OHC loss in acute KM-FU lesions primarily occurs through CaMKKβ activation. Furthermore, silencing CaMKKβ did not inhibit aminoglycoside antibiotic uptake of OHC because it had no effect on MTC with each assessment using FM1-43 (Meyers et al., 2003; Taylor et al., 2008; Nakanishi et al., 2018). Co-treatment with KN93 completely blocked GM-induced AIF translocation to the OHC nucleus in explants and completely prevented GM-induced hair cell loss, consistent with in vivo protective results and supporting the accepted view that caspase-independent cell death pathways are involved in noise- and aminoglycoside-induced hearing loss (Jiang et al., 2006; Zheng et al., 2014).

[0085] (2) RNAi-based therapy has the potential to prevent hearing loss caused by inner ear trauma. Our results support this view, demonstrating that AAV vector-mediated RNA silencing transduction of CaMKKβ can prevent NIHL. Since shRNAs are typically around 80 base pairs in size, the inherent limitation of AAV for small package size (approximately 4.5 kb) is not a problem. Our results indicate that the AAV2.7m8 vector showed very high infection rates in sensory hair cells of FVB / NJ mice, with 100% infection in IHCs, 90% in OHC basal and intermediate transductions, and a lower rate of 40% in OHC apical transduction; consistent with previous publications, injection of AAV2.7m8 into FVB / NJ p1-2 pups via the cochlear vestibule does not affect auditory thresholds (Wang et al., 2013; Yu et al., 2014; Landeg et al., 2017; Isgrig et al., 2019). Furthermore, we found that AAV2.7m8 infection alone or shControl transduction did not alter the sensitivity of OHCs to traumatic noise exposure. In this study, we selected FVB / NJ mice for shCaMKKβ AAV transduction because FVB / NJ mice exhibited normal baseline hearing at least 10 weeks of age and showed similar susceptibility to noise-induced basal-to-OHC loss as CBA / J mice, consistent with previous reports (Ho et al., 2014). Typically, pups have higher AAV infection rates than adults, and FVB / NJ mouse breeders are prolific, with maternal lactation continuing after AAV injection at p1-2. Furthermore, we did not observe eGFP-induced ototoxicity within 8 weeks of viral injection into FVB / NJ p1-2 pups, whereas a recent report showed that Anc80-driven GFP overexpression for 5 weeks induced ototoxicity in adult CBA / CaJ mice (Hashimoto et al., 2019). While traumatic noise exposure does induce OHC and hearing loss in FVB / NJ mice, it is worth noting that noise-induced apical turn OHC loss differs between FVB / NJ and CBA / J mice. Although we observed loss of apical turn OHCs in FVB / NJ mice, we did not observe it in CBA / J mice. This sensitivity of FVB / NJ mouse apical OHC to noise damage may be an inherent, broader vulnerability, as control FVB / NJ mice without noise exposure show some OHC loss (approximately 5%) at the apical turn initiation (0–0.25 mm), even at 4 weeks of age.

[0086] Consistent with the results showing that shCaMKKβ attenuates NIHL via AAV transduction, pretreatment with siCaMKKβ via intratympanic delivery of siRNA to the middle ear via RWM also significantly attenuated noise-induced IHC synaptic and OHC and NIHL losses. Furthermore, pretreatment with siCaMKKβ via PSC delivery almost completely prevented the significant OHC losses induced by KM-FU treatment, demonstrating the therapeutic potential of siRNA in the treatment of acute inner ear injury.

[0087] In summary, our study highlights the pathological consequences of CaMKKβ activation. The responses of different inner ear injury models (OHCs) were examined. Transduction or delivery of siCaMKKβ via shCaMKKβ AAV significantly attenuated the functional deficits and morphological losses of IHC synapses and OHCs induced by traumatic noise, indicating that CaMKKβ is a key factor contributing to NIHL. Furthermore, pretreatment with siCaMKKβ via PSC delivery completely prevented significant KM-FU-induced OHC loss and hearing loss, demonstrating the importance of targeting CaMKKβ for treating inner ear injury. Co-treatment with the CaMKKβ inhibitor KN93 to counteract GM-induced cochlear explant hair cell loss further supports this view. Based on the results of these embodiments, future development of drugs and better treatment strategies for preventing inner ear injury is beneficial.

[0088] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. Use of KN93 in the manufacture of a product for preventing ototoxicity caused by gentamicin.

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