ABR/DPOAE publications

Large-scale epidemiological surveys suggest that hearing loss (HL) is a significant risk factor for dementia. We previously showed that noise-induced HL (NIHL) impairs hippocampal cognitive function and decreases hippocampal neurogenesis and neuronal complexity, suggesting a causal role of HL in dementia. To further investigate the influence of acquired peripheral HL on hippocampal neurogenesis with the aging process as well as the underlying mechanism, we produced NIHL in male CBA/J mice and assessed hippocampal neurogenesis and microglial morphology in the auditory brain and hippocampus at 4 days post-noise exposure (DPN) or 1, 3, 6, or 12 months post-noise exposure (MPN) by immunofluorescence labeling. We found that the age-related decline in hippocampal neurogenesis was accelerated in mice with NIHL. Furthermore, in mice with NIHL, prolonged microglial activation occurred from 1 MPN to 12 MPN across multiple auditory nuclei, while aggravated microglial deterioration occurred in the hippocampus and correlated with the age-related decline in hippocampal neurogenesis. These results suggest that acquired peripheral HL accelerates the age-related decline in hippocampal neurogenesis and that hippocampal microglial degeneration may contribute to the development of neurodegeneration following acquired peripheral HL.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.

The effect of dextromethorphan (DXM) use in sensorineural hearing loss (SNHL) has not been fully examined. We conducted an animal model and nationwide retrospective matched-cohort study to explore the association between DXM use and SNHL. Eight-week-old CBA/CaJ hearing loss was induced by a white noise 118 dB sound pressure level for 3 h. DXM (30 mg/kg) was administered intraperitoneally for 5 days and boost once round window DXM socking. In population-based study, we examined the medical records over 40 years old in Taiwan’s National Health Insurance Research Database between 2000 and 2015 to establish retrospective matched-cohort to explore the correlation between DXM use and SNHL. Using click auditory brainstem response (ABR), hearing threshold was measured as 48.6 ± 2.9 dB in control mice compared with 42.6 ± 7.0 dB in DXM mice, which differed significantly (p = 0.002) on day 60 after noise exposure with a larger ABR wave I amplitude in DXM mice. In human study, we used a Cox regression hazard model to indicate that a significantly lower percentage individuals developed SNHL compared with and without DXM use (0.44%, 175/39,895 vs. 1.05%, 1675/159,580, p < 0.001). After adjustment for age and other variables [adjusted hazard ratio: 0.725 (95% confidence interval: 0.624–0.803, p < 0.001)], this study also demonstrated that DXM use appeared to reduce the risk of developing SNHL. This animal study demonstrated that DXM significantly attenuated noise-induced hearing loss. In human study, DXM use may have a protective effect against SNHL.

Tinnitus is a common auditory disease worldwide; it is estimated that more than 10% of all individuals experience this hearing disorder during their lifetime. Tinnitus is sometimes accompanied by hearing loss. However, hearing loss is not acquired in some other tinnitus generations. In this study, we injected adult rats with salicylate sodium (SS) (200 mg/kg/day for 10 days) and found no significant hearing threshold changes at 2, 4, 8, 12, 14, 16, 20, or 24 kHz (all p > 0.05 ). Tinnitus was confirmed in the treated rats via Behaviour Testing of Acoustic Startle Response (ASR) and Gap Prepulse Inhibition Test of Acoustic Startle Reflex (GPIAS). A immunostaining study showed that there is significant loss of anti-CtBP2 puncta (a marker of cochlear inner hair cell (HC) ribbon synapses) in treated animals in apical, middle, and basal turns (all p < 0.05 ). The ABR wave I amplitudes were significantly reduced at 4, 8, 12, 14, 16, and 20 kHz (all p < 0.05 ). No significant losses of outer HCs, inner HCs, or HC cilia were observed (all p > 0.05 ). Thus, our study suggests that loss of cochlear inner HC ribbon synapse after SS exposure is a contributor to the development of tinnitus without changing hearing threshold.

The objective of this study was to explore the molecular mechanisms of acute noise-induced hearing loss and recovery of steady-state noise-induced hearing loss using miniature pigs. We used miniature pigs exposed to white noise at 120 dB (A) as a model. Auditory brainstem response (ABR) measurements were made before noise exposure, 1 day and 7 days after noise exposure. Proteomic Isobaric Tags for Relative and Absolute Quantification (iTRAQ) was used to observe changes in proteins of the miniature pig inner ear following noise exposure. Western blot and immunofluorescence were performed for further quantitative and qualitative analysis of proteomic changes. The average ABR-click threshold of miniature pigs before noise exposure, 1 day and 7 days after noise exposure, were 39.4 dB SPL, 67.1 dB SPL, and 50.8 dB SPL, respectively. In total, 2,158 proteins were identified using iTRAQ. Both gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses showed that immune and metabolic pathways were prominently involved during the impairment stage of acute hearing loss. During the recovery stage of acute hearing loss, most differentially expressed proteins were related to cholesterol metabolism. Western blot and immunofluorescence showed accumulation of reactive oxygen species and nuclear translocation of NF- κ B (p65) in the hair cells of miniature pig inner ears during the acute hearing loss stage after noise exposure. Nuclear translocation of NF- κ B (p65) may be associated with overexpression of downstream inflammatory factors. Apolipoprotein (Apo) A1 and Apo E were significantly upregulated during the recovery stage of hearing loss and may be related to activation of cholesterol metabolic pathways. This is the first study to use proteomics analysis to analyze the molecular mechanisms of acute noise-induced hearing loss and its recovery in a large animal model (miniature pigs). Our results showed that activation of metabolic, inflammatory, and innate immunity pathways may be involved in acute noise-induced hearing loss, while cholesterol metabolic pathways may play an important role in recovery of hearing ability following noise-induced hearing loss.

The round window membrane (RWM) is the most common entryway for local drug and gene delivery into the inner ear, but its permeability can change the treatment outcome. We previously demonstrated a feasible and highly efficient approach using ultrasound-aided microbubble (USMB) cavitation to enhance the permeability of the RWM. Here, we investigated the safety of USMB exposure and the association between temporal changes in RWM permeability and ultrastructure. Experimental guinea pigs were divided into two treatment groups: a control group receiving round window soaking (RWS) with MBs and treatment (USM) groups undergoing 3 (USM-3) or 5 (USM-5) consecutive USMB exposures (1 min/exposure) at an acoustic intensity of 3 W/cm2 and 1 MHz frequency. The trans-RWM delivery efficiency of biotin-fluorescein isothiocyanate conjugates, used as permeability tracers, revealed a greater than 7-fold higher delivery efficiency for the USM groups immediately after 3 or 5 exposures than for the RWS group. After 24 h, the delivery efficiency was 2.4-fold higher for the USM-3 group but was 6.6-fold higher for the USM-5 group (and 3.7-fold higher after 48 h), when compared to the RWS group. Scanning electron microscopy images of the RWM ultrastructure revealed USMB-induced sonoporation effects that could include the formation of heterogeneous pore-like openings with perforation diameters from 100 nm to several micrometers, disruption of the continuity of the outer epithelial surface layer, and loss of microvilli. These ultrastructural features were associated with differential permeability changes that depended on the USMB exposure course. Fourteen days after treatment, the pore-like openings had significantly decreased in number and the epithelial defects were healed either by cell expansion or by repair by newly migrated epithelial cells. The auditory brainstem response recordings of the animals following the 5-exposure USMB treatment indicated no deterioration in the hearing thresholds at a 2-month follow-up and no significant hair cell damage or apoptosis, based on scanning electron microscopy, surface preparations, and TUNEL assays. USMBs therefore appear to be safe and effective for inner ear drug delivery. The mechanism of enhanced permeability may involve a disruption of the continuity of the outer RWM epithelial layer, which controls transmembrane transport of various substances.

The formation of reactive oxygen species (ROS) is a well-documented process in noise-induced hearing loss (NIHL). We have also previously shown that activation of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPKα) at its catalytic residue T172 is one of the key reactions triggering noise-induced outer hair cell (OHC) death. In this study, we are addressing the link between ROS formation and activation of AMPKα in OHCs after noise exposure. In-vivo treatment of CBA/J mice with the antioxidant N-acetyl cysteine (NAC) reduced noise-induced ROS formation (as assessed by the relative levels of 4-hydroxynonenal and 3-nitrotyrosine) and activation of AMPKα in OHCs. Forskolin, an activator of adenylyl cyclase (AC) and an antioxidant, significantly increased cyclic adenosine monophosphate (cAMP) and decreased ROS formation and noise-induced activation of AMPKα. Consequently, treatment with forskolin attenuated noise-induced losses of OHCs and NIHL. In HEI-OC1 cells, H2O2-induced activation of AMPKα and cell death were inhibited by the application of forskolin. The sum of our data indicates that noise activates AMPKα in OHCs through formation of ROS and that noise-exposure-induced OHC death is mediated by a ROS/AMPKα-dependent pathway. Forskolin may serve as a potential compound for prevention of NIHL.

In this study, we investigated whether the curcuminoids, CLEFMA and EF24, improved cisplatin efficacy and reduced cisplatin ototoxicity. We used the lung cancer cell line, A549, to determine the effects of the curcuminoids and cisplatin on cell viability and several apoptotic signaling mechanisms. Cellular viability was measured using the MTT assay. A scratch assay was used to measure cell migration and fluorescent spectrophotometry to measure reactive oxygen species (ROS) production. Western blots and luminescence assays were used to measure the expression and activity of apoptosis-inducing factor (AIF), caspases-3/7, -8, -9, and -12, c-Jun N-terminal kinases (JNK), mitogen-activated protein kinase (MAPK), and proto-oncogene tyrosine-protein kinase (Src). A zebrafish model was used to evaluate auditory effects. Cisplatin, the curcuminoids, and their combinations had similar effects on cell viability (IC50 values: 2–16 μM) and AIF, caspase-12, JNK, MAPK, and Src expression, while caspase-3/7, -8, and -9 activity was unchanged or decreased. Cisplatin increased ROS yield (1.2-fold), and curcuminoid and combination treatments reduced ROS (0.75–0.85-fold). Combination treatments reduced A549 migration (0.51–0.53-fold). Both curcuminoids reduced auditory threshold shifts induced by cisplatin. In summary, cisplatin and the curcuminoids might cause cell death through AIF and caspase-12. The curcuminoids may potentiate cisplatin’s effect against A549 migration, but may counteract cisplatin’s effect to increase ROS production. The curcuminoids might also prevent cisplatin ototoxicity.

Hearing sensitivity is of general interest from the perspective of understanding the functionality and evolution of vertebrate auditory systems. Sexual dimorphism of auditory systems has been reported in several species of vertebrates, but little is known about this phenomenon in turtles. Some morphological characteristics, such as middle ear and tympanic membrane that influence the hearing sensitivity of animals can result in hearing sexual dimorphism. To examine whether sexual dimorphism in hearing sensitivity occurs in turtles and to compare hearing characteristics with respect to the shape of the tympanic membrane, we measured the hearing sensitivity and tympanum diameter in both sexes of Trachemys scripta elegans . The results showed that, with the exception of 0.9 kHz, auditory brainstem response thresholds were significantly lower in females than in males for frequencies in the 0.2–1.1 kHz range, indicating that the hearing of females shows greater sensitivity. No significant differences were detected in the tympanum diameter of both sexes. These results showed that sexually dimorphic hearing sensitivity has evolved in turtles; however, this difference does not appear to be related to differences in the size of the tympanic membrane. The possible origin and function of the sexual differences in auditory characteristic are discussed.

Foxg1 is one of the forkhead box genes that are involved in morphogenesis, cell fate determination, and proliferation, and Foxg1 was previously reported to be required for morphogenesis of the mammalian inner ear. However, Foxg1 knock-out mice die at birth, and thus the role of Foxg1 in regulating hair cell (HC) regeneration after birth remains unclear. Here we used Sox2 CreER/+ Foxg1 loxp/loxp mice and Lgr5-EGFP CreER/+ Foxg1 loxp/loxp mice to conditionally knock down Foxg1 specifically in Sox2+ SCs and Lgr5+ progenitors, respectively, in neonatal mice. We found that Foxg1 conditional knockdown (cKD) in Sox2+ SCs and Lgr5+ progenitors at postnatal day (P)1 both led to large numbers of extra HCs, especially extra inner HCs (IHCs) at P7, and these extra IHCs with normal hair bundles and synapses could survive at least to P30. The EdU assay failed to detect any EdU+ SCs, while the SC number was significantly decreased in Foxg1 cKD mice, and lineage tracing data showed that much more tdTomato+ HCs originated from Sox2+ SCs in Foxg1 cKD mice compared to the control mice. Moreover, the sphere-forming assay showed that Foxg1 cKD in Lgr5+ progenitors did not significantly change their sphere-forming ability. All these results suggest that Foxg1 cKD promotes HC regeneration and leads to large numbers of extra HCs probably by inducing direct trans-differentiation of SCs and progenitors to HCs. Real-time qPCR showed that cell cycle and Notch signaling pathways were significantly down-regulated in Foxg1 cKD mice cochlear SCs. Together, this study provides new evidence for the role of Foxg1 in regulating HC regeneration from SCs and progenitors in the neonatal mouse cochlea.

Aminoglycoside ototoxicity is common in clinical practice but reliable protective agents currently do not exist. Aminoglycoside regimens causing ototoxicity in different laboratory animals are under investigation. The assessment method used most commonly to determine auditory effects is the auditory brainstem response (ABR). Distortion product otoacoustic emissions (DPOAE) have been used less frequently. A precise recommendation on the specific method to assess peripheral auditory function before and after aminoglycoside toxicity in mice does not exist. In order to evaluate various mouse models for ototoxic injury caused by various aminoglycoside regimens, there is a need for performing preliminary tests in small cohorts before large experiments. The aim of our study was to investigate different aminoglycoside regimens that cause substantial ototoxic damage in vivo. Aminoglycosides are safe and produce a detectable hearing threshold shift in a small cohort of mice that can be used as a model for preliminary tests. Different ototoxic regimens were assessed by ABR and DPOAE measurements pre- and post-treatment. Further, the sensory cell loss was quantified by counting hair cells in the cochlea. It was revealed that an ototoxic regimen with kanamycin twice daily for 15 consecutive days is safe, well tolerated and produces an early significant hearing threshold shift detected by DPOAE in a small cohort of mice. The study compared ABR and DPOAE in mentioned regimens for the first time and illustrated that DPOAE is well suited for detecting hearing threshold shifts in high frequencies before ABR threshold shifts occur in accordance with predominating outer hair cell damage mainly in the basal turn of the cochlea.

Infection and inflammation of the middle ears that characterizes acute and chronic otitis media (OM), is a major reason for doctor visits and antibiotic prescription, particularly among children. Nasopharyngeal pathogens that are commonly associated with OM in humans do not naturally colonize the middle ears of rodents, and experimental models in most cases involve directly injecting large numbers of human pathogens into the middle ear bullae of rodents, where they induce a short-lived acute inflammation but fail to persist. Here we report that Bordetella pseudohinzii, a respiratory pathogen of mice, naturally, efficiently and rapidly ascends the eustachian tubes to colonize the middle ears, causing acute and chronic histopathological changes with progressive decrease in hearing acuity that closely mimics otitis media in humans. Laboratory mice experimentally inoculated intranasally with very low numbers of bacteria consistently have their middle ears colonized and subsequently transmit the bacterium to cage mates. Taking advantage of the specifically engineered and well characterized immune deficiencies available in mice we conducted experiments to uncover different roles of T and B cells in controlling bacterial numbers in the middle ear during chronic OM. The iconic mouse model provides significant advantages for elucidating aspects of host-pathogen interactions in otitis media that are currently not possible using other animal models. This natural model of otitis media permits the study of transmission between hosts, efficient early colonization of the respiratory tract, ascension of the eustachian tube, as well as colonization, pathogenesis and persistence in the middle ear. It also allows the combination of the powerful tools of mouse molecular immunology and bacterial genetics to determine the mechanistic basis for these important processes.

Background and objectives: The antioxidant ebselen will be able to limit or prevent the ototoxicity arising from 2-hydroxypropyl-β-cyclodextrin (HPβCD). Niemann-Pick Type C (NPC) disease is a disorder of lysosomal storage manifested in sphingolipidosis. Recently, it was noted that experimental use of HPβCD could partially resolve the symptoms in both animals and human patients. Despite its desirable effect, HPβCD can induce hearing loss, which is the only major side effect noted to date. Understanding of the pathophysiology of hearing impairment after administration of HPβCD and further development of preventive methods are essential to reduce the ototoxic side effect. The mechanisms of HPβCD-induced ototoxicity remain unknown, but the resulting pathology bears some resemblance to other ototoxic agents, which involves oxidative stress pathways. To indirectly determine the involvement of oxidative stress in HPβCD-induced ototoxicity, we tested the efficacy of an antioxidant reagent, ebselen, on the extent of inner ear side effects caused by HPβCD. Materials and methods: Ebselen was applied prior to administration of HPβCD in mice. Auditory brainstem response thresholds and otopathology were assessed one week later. Bilateral effects of the drug treatments also were examined. Results: HPβCD-alone resulted in bilateral, severe, and selective loss of outer hair cells from base to apex with an abrupt transition between lesions and intact areas. Ebselen co-treatment did not ameliorate HPβCD-induced hearing loss or alter the resulting histopathology. Conclusions: The results indirectly suggest that cochlear damage by HPβCD is unrelated to reactive oxygen species formation. However, further research into the mechanism(s) of HPβCD otopathology is necessary.

Inherited hearing loss is associated with gene mutations that result in sensory hair cell (HC) malfunction. HC structure is defined by the cytoskeleton, which is mainly composed of actin filaments and actin-binding partners. LIM motif-containing protein kinases (LIMKs) are the primary regulators of actin dynamics and consist of two members: LIMK1 and LIMK2. Actin arrangement is directly involved in the regulation of cytoskeletal structure and the maturation of synapses in the central nervous system, and LIMKs are involved in structural plasticity by controlling the activation of the actin depolymerization protein cofilin in the olfactory system and in the hippocampus. However, the expression pattern and the role of LIMKs in mouse cochlear development and synapse function also need to be further studied. We show here that the Limk genes are expressed in the mouse cochlea. We examined the morphology and the afferent synapse densities of HCs and measured the auditory function in Limk1 and Limk2 double knockout (DKO) mice. We found that the loss of Limk1 and Limk2 did not appear to affect the overall development of the cochlea, including the number of HCs and the structure of hair bundles. There were no significant differences in auditory thresholds between DKO mice and wild-type littermates. However, the expression of p-cofilin in the DKO mice was significantly decreased. Additionally, no significant differences were found in the number or distribution of ribbon synapses between the DKO and wild-type mice. In summary, our data suggest that the Limk genes play a different role in the development of the cochlea compared to their role in the central nervous system.

Aminoglycoside antibiotics-induced hearing loss is a common sensorineural impairment. Spiral ganglion neurons (SGNs) are first-order neurons of the auditory pathway and are critical for the maintenance of normal hearing. In the present study, we investigated the time-course of morphological changes and the degeneration process of spiral ganglion cells (SGCs) following chronic kanamycin-induced deafness and determined whether the endoplasmic reticulum (ER) stress was involved in the degeneration of SGNs. We detected density changes in SGCs and the expressions of Bip, inositol requirement 1 (IRE1)α, activating transcription factor-6α, p-PERK, p-eIF2α, CHOP, and caspase-12 at each time point after kanamycin treatment. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was also performed. The number of SGC deletions reached ∼50% at the 70th day after kanamycin administration and the ER of most SGCs were dilated. The expression of p-PERK, p-eIF2α, p-IRE1α, Bip, caspase-12, and Chop was significantly unregulated after kanamycin treatment. The number of SGCs that were positive for both TUNEL and caspase-12 increased from day 7 to 28. Taken together, these data demonstrate that ER stress was involved in kanamycin-induced apoptosis of SGNs. Kanamycin-induced SGN apoptosis is mediated, at least in part, by ER stress-induced upregulation of CHOP and caspase-12.

Mitochondria modulate cellular calcium homeostasis by the combined action of the mitochondrial calcium uniporter (MCU), a selective calcium entry channel, and the sodium calcium exchanger (NCLX), which extrudes calcium from mitochondria. In this study, we investigated MCU and NCLX in noise-induced hearing loss (NIHL) using adult CBA/J mice and noise-induced alterations of inner hair cell (IHC) synapses in MCU knockout mice. Following noise exposure, immunoreactivity of MCU increased in cochlear sensory hair cells of the basal turn, while immunoreactivity of NCLX decreased in a time- and exposure-dependent manner. Inhibition of MCU activity via MCU siRNA pretreatment or the specific pharmacological inhibitor Ru360 attenuated noise-induced loss of sensory hair cells and synaptic ribbons, wave I amplitudes, and NIHL in CBA/J mice. This protection was afforded, at least in part, through reduced cleavage of caspase 9 (CC9). Furthermore, MCU knockout mice on a hybrid genetic CD1 and C57/B6 background showed resistance to noise-induced seizures compared to wild-type littermates. Owing to the CD1 background, MCU knockouts and littermates suffer genetic high frequency hearing loss, but their IHCs remain intact. Noise-induced loss of IHC synaptic connections and reduction of auditory brainstem response (ABR) wave I amplitude were recovered in MCU knockout mice. These results suggest that cellular calcium influx during noise exposure leads to mitochondrial calcium overload via MCU and NCLX. Mitochondrial calcium overload, in turn, initiates cell death pathways and subsequent loss of hair cells and synaptic connections, resulting in NIHL.

Introduction: There are several key differences between impulse and continuous noise: the nature of the noise itself, the cochlear and neuronal structures affected, the severity to which they damage the auditory system, and the period of time in which damage occurs. Notably, no work on hidden hearing loss after impulse noise exposure has been done to this point, though it has been extensively studied after continuous noise. Hidden hearing loss manifests physiologically with reductions in suprathreshold amplitudes of the first wave of the auditory brainstem response, while auditory thresholds can remain relatively normal. Objective: This study aimed to assess the extent to which, if at all, hidden hearing loss is present after exposure to impulse noise in C57BL6/J mice. Methods: Thirty-one C57BL6/J mice were used in the experiment, in accordance with IACUC protocols. Auditory brainstem responses were recorded before and after noise exposures. The noise exposures consisted of 500 impulses at 137 dB peSPL. Results: Suprathreshold amplitude reductions in the P1 wave of the mouse auditory brainstem response were seen, but only at frequencies with significant threshold shift. Conclusion: These amplitude changes were consistent with hidden hearing loss, and we conclude that impulse noise can cause hidden hearing loss, but future studies are required to determine the specific mechanisms involved and if they parallel those of hidden hearing loss after continuous noise.

Introduction: To detect congenital hearing loss in mice, an objective measure is needed other than mouse behavioral observation. This study aimed to refine the methodology of auditory brainstem response measurement and identify potential congenital hearing loss models in laboratory mouse strains. Methods: Mice were anesthetized and fitted with head electrodes. Each mouse underwent four ABR measurements according to four testing conditions: A - no chamber; conventional tone; B - chamber; conventional tone; C - no chamber; short tone; D - chamber; short tone. Potential congenital hearing loss models were identified using 10 mice from each strain (C57BL, BALB/c, CH3, ICR, and ddY) through sound-attenuated ABR measurements with short-tone bursts. Potential congenital hearing loss models exhibited hearing thresholds ≥30 dB in both ears. Data were analyzed for normal distribution and variance homogeneity using the D'Agostino-Pearson/Kolmogorov-Smirnov and F value tests, respectively. One-way analysis of variance (ANOVA), with the Tukey-Kramer test, was used to conduct parametric analysis, and the Kruskal-Wallis/Friedman test, with a Dunn's test for post hoc analysis, was used to perform non-parametric analysis. Results: The simultaneous use of a sound-attenuating chamber and short-tone bursts provided clearly defined wave patterns, even at lower sound intensities. Inbred strains, especially C57BL/6 sub-strains, constitute suitable congenital hearing loss models. Conclusions: Our study shows that environmental factors should be addressed in animal studies of hearing function. Potential congenital hearing loss models may be found amongst commercially available inbred strains.

ARHGEF6 belongs to the family of guanine nucleotide exchange factors (GEFs) for Rho GTPases, and it specifically activates Rho GTPases CDC42 and RAC1. Arhgef6 is the X-linked intellectual disability gene also known as XLID46, and clinical features of patients carrying Arhgef6 mutations include intellectual disability and, in some cases, sensorineural hearing loss. Rho GTPases act as molecular switches in many cellular processes. Their activities are regulated by binding or hydrolysis of GTP, which is facilitated by GEFs and GTPase-activating proteins, respectively. RAC1 and CDC42 have been shown to play important roles in hair cell (HC) stereocilia development. However, the role of ARHGEF6 in inner ear development and hearing function has not yet been investigated. Here, we found that ARHGEF6 is expressed in mouse cochlear HCs, including the HC stereocilia. We established Arhgef6 knockdown mice using the clustered regularly interspaced short palindromic repeat-associated Cas9 nuclease (CRISPR-Cas9) genome editing technique. We showed that ARHGEF6 was indispensable for the maintenance of outer hair cell (OHC) stereocilia, and loss of ARHGEF6 in mice caused HC stereocilia deficits that eventually led to progressive HC loss and hearing loss. However, the loss of ARHGEF6 did not affect the synapse density and did not affect the mechanoelectrical transduction currents in OHCs at postnatal day 3. At the molecular level, the levels of active CDC42 and RAC1 were dramatically decreased in the Arhgef6 knockdown mice, suggesting that ARHGEF6 regulates stereocilia maintenance through RAC1/CDC42.

The use of cisplatin (CDDP), the most common chemotherapy drug for head and neck cancer, is limited by its undesirable side effects, especially nephrotoxicity. We investigated ultrasound microbubbles (USMB) as a tool to increase the local intra-tumoral CDDP level while decreasing systemic CDDP cytotoxicity. We allowed CDDP to interact with human serum albumin and then sonicated the resulting CDDP‒albumin complex to generate CDDP-loaded MBs (CDDP-MBs). We then established a head-and-neck tumor-bearing mouse model by implanting FaDu-fLuc/GFP cells into severe combined immunodeficiency mice and used IVIS® bioluminescence imaging to determine the tumor xenograft formation and size. Twice weekly (until Day 33), we administered CDDP only, CDDP + MBs + US, CDDP-MBs, or CDDP-MBs + US intravenously by tail-vein injection. The US treatment was administered at the tumor site immediately after injection. The in vivo systemic distribution of CDDP indicated that the kidney was the most vulnerable organ, followed by the liver, and then the inner ear. However, CDDP uptake into the kidney and liver was significantly decreased in both the CDDP-MBs and CDDP-MBs + US groups, suggesting that MB binding significantly reduced the systemic toxicity of CDDP. The CDDP-MBs + US treatment reduced the tumor size as effectively as conventional CDDP-only chemotherapy. Therefore, the combination of CDDP-MBs with ultrasound is effective and significantly attenuates CDDP-associated nephrotoxicity, indicating a promising clinical potential for this approach.