ABR/DPOAE publications

Here, we present a protocol for evaluating auditory function and synaptic molecular-structural integrity in mice, specifically female GluA3-knockout (GluA3KO) mice raised in quiet or ambient sound levels. We describe steps for combining auditory brainstem response recordings, immunofluorescence, and electron microscopy. We detail procedures for assessing hearing function, the expression and localization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits, and the ultrastructure of cochlear-ribbon synapses. This protocol provides a reproducible framework for studying molecular-structural synaptopathy in mouse models of cochlear dysfunction. For complete details on the use and execution of this protocol, please refer to Pal et al.1.

Age-related hearing loss (ARHL) is a major public health concern, driven by the interplay of multiple factors. Here, we reveal spatiotemporal metabolic shifts in murine inner ears mirroring erythrocytes and plasma. Isotope-labeled glucose tracing demonstrates a metabolic rerouting favoring glycolysis over the pentose phosphate pathway, alongside downregulation of the tricarboxylic acid cycle, indicating impaired energy production and redox homeostasis. Accumulation of medium- and long-chain acylcarnitines further exacerbates lipotoxicity. Notably, age-dependent depletion of arginine, lysine, proline, and glycine disrupts the arginine-polyamine-urea cycle. Translationally, UK Biobank plasma metabolomics links omega-6 fatty acids, linoleic acid, glycine, and albumin to ARHL resilience, while branched-chain amino acids, tyrosine, creatinine, glycoprotein acetyls and urea confer risk. Sex differences in ARHL were linked to fatty acid metabolism divergence. These bioenergetic disruptions in the inner ear are mirrored in erythrocytes and plasma, highlighting potential biomarkers for early ARHL diagnosis and treatment.

Adeno-associated virus-based gene therapy offers a promising treatment paradigm for inner ear diseases; however, the genetic heterogeneity of hereditary deafness requires gene-specific strategies and optimization of current approaches to identify the range of treatable conditions and improve therapeutic outcomes. To consider the therapeutic potential for a hearing loss gene not previously explored, we investigated the gene encoding the chloride intracellular channel protein CLIC5, mutations in which lead to DFNB103 in humans and deafness and circling behavior in a Clic5 -deficient mouse model. In this study, we utilized two constructs to deliver the wild-type Clic5 coding sequence into Clic5 -deficient mice: single-stranded and self-complementary adeno-associated virus, the latter known for rapid onset of transgene expression. We report a robust restoration of CLIC5 expression using either construct, including prevention of morphological degeneration and preserving auditory and vestibular function. Interestingly, the self-complementary construct achieved comparable functional recovery to single-stranded construct but at a lower titer. These findings highlight the potential of self-complementary adeno-associated virus to reduce dose requirements, minimize toxicity and broaden clinical utility for inner ear therapies.

Niemann-Pick disease type C (NPC) is a life-threatening neurodegenerative disease caused by impaired intracellular cholesterol trafficking. A cyclic heptasaccharide, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), is currently under clinical investigation for treating NPC, but its ototoxicity remains problematic. We previously reported that β- and γ-forms of cyclodextrin (CD) derivatives with various substituents capable of accommodating/solubilizing unesterified cholesterol (UC) exerted more pronounced therapeutic and ototoxic effects when administered intracerebroventricularly than subcutaneously for NPC treatment. However, the impact of substituent variations on these effects is unconfirmed for cyclic hexasaccharides, α-CD derivatives. Here, we investigated these effects for α-CD derivatives with various replaced substituents from the perspective of their interaction with UC. Even when administered intracerebroventricularly, a representative α-CD derivative had no impact on survival in NPC model mice. The normalization of intracellular cholesterol trafficking in NPC model cells and the auditory dysfunction in wild-type mice, both caused by HP-β-CD, were not seen for the α-CD derivatives, regardless of their substituent variations. Solubility and molecular simulation analyses showed negligible UC-solubilizing ability of α-CD derivatives because of their insufficient capacity to accommodate the UC molecule, rather than their substituent variations. These results underscore the importance of UC accommodation by CD for both efficacy and ototoxicity in NPC treatment.

Background: Age related hearing loss (ARHL) is a sensorineural hearing disease caused by multiple factors and its pathogenesis is still unclear. This work aims to investigate the precise role of AIMP1 in ARHL. Methods: HEI-OC1 cells were treated with 1 mM H2O2 for 2 h to induce cell damage. CCK-8, flow cytometry and TUNEL staining examined cell viability and apoptosis. DCFH-DA fluorescence probe assessed ROS. The levels of MDA, SOD and copper were detected by kits. The expression of proteins related to mitophagy, cuproptosis were examined by western blotting and immunofluorescence. Finally, D-galactose was administered to mice to establish an ARHL model for verifying the functional role of AIMP1 in ARHL. Results: AIMP1 and SIRT1 were down-regulated in H2O2-treated HEI-OC1 cells. AIMP1 overexpression promoted cell viability, reduced ROS and MDA levels, and increased SOD levels in H2O2-treated HEI-OC1 cells. Moreover, the levels of copper and apoptosis were decreased in H2O2-treated HEI-OC1 cells in the presence of AIMP1 overexpression. AIMP1 overexpression caused a down-regulation of cuproptosis-related proteins FDX1, DLAT, DLST, and an up-regulation of mitophagy-related proteins PINK1, Parkin, Mfn2 and Drp1 in H2O2-treated HEI-OC1 cells. Knockdown PINK1 or SIRT1 reversed the influence of AIMP1 overexpression on H2O2 induced HEI-OC1 cell damage. In vivo, AIMP1 overexpression reduced damage of cochlear tissues and partially restored hearing in ARHL mice. Conclusion: AIMP1 up-regulated SIRT1 to promote PINK1/Parkin-mediated mitophagy and inhibit cuproptosis of cochlear hair cells in ARHL mice. Thus, AIMP1 may be a potential target for ARHL treatment.

Background: Hearing loss is known to cause structural and functional abnormalities in the central auditory pathways. Interventions with hearing aids that amplify acoustic signals have been developed to combat hearing loss. However, little is known about how such devices may affect the brain and mitigate the progression of hearing loss. We hypothesized that timely intervention that amplifies acoustic signals would delay further progression of hearing loss by maintaining central auditory activity and neural structure. Method: To that end, we provided eight weeks of acoustic stimulation tailored to compensate for subject-specific patterns of frequency loss in two mouse models of progressive hearing loss. We evaluated the effects of sound amplification on endbulb of Held anatomy at different ages of intervention in mice with early-onset (DBA/2) and late-onset (C57Bl/6) hearing loss. Results: We observed in both strains that endbulbs undergo rapid and progressive atrophy in untreated control subjects exposed to a baseline, unamplified, sound environment. In contrast, endbulb atrophy was significantly slowed in treated mice (p < 0.05). Conclusions: These data provide a possible explanation for how the brain benefits from sound amplification via hearing aid devices.

Sensorineural hearing loss (SNHL) is characterized by cochlear inflammation, macrophage activation, and degeneration of hair cells, synapses, and neurons. Macrophage-mediated inflammation in the damaged cochlea is regulated via CX3CR1-CX3CL1 signaling, where the fractalkine ligand CX3CL1 serves as a chemotactic and calming signal for macrophage activation. Furthermore, disrupted CX3CR1-CX3CL1 signaling in CX3CR1-KO and CX3CL1-KO mice leads to reduced macrophage numbers, exacerbated inflammation, and loss of hair cells, ribbon synapses, and neurons in the damaged cochlea. Notably, ~ 25% of the human population carries single-nucleotide polymorphisms (SNPs) in the CX3CR1 gene, CX3CR1I249/M280, which results in a receptor with lower binding affinity for CX3CL1, while most individuals carry the common wild-type CX3CR1V249/T280 allele. Although these polymorphisms are associated with various CNS neurodegenerative disorders, their impact on SNHL, cochlear degeneration and the macrophage response remains largely unknown. Here, we used a humanized mouse model expressing human CX3CR1 SNPs in lieu of its murine counterpart to investigate the effects of I249/M280 polymorphisms on cochlear function and structure following noise trauma. Young CX3CR1 WT, CX3CR1 KO, and human CX3CR1I249/M280 mice of both sexes were exposed to a noise level of 93 decibel sound pressure for 2 h at an octave band (8-16 kHz). Cochlear function was assessed prior to exposure and at 1 day and 2 weeks postexposure. Also, the densities of inner and outer hair cells, ribbon synapses, and macrophages in Rosenthal's canal were examined after two weeks of exposure and compared among the three genotypes. We found that at 2 weeks postexposure, hearing thresholds were elevated and input‒output function was impaired in hCX3CR1I249/M280 and CX3CR1 KO, whereas mice carrying WT alleles showed functional recovery. A significant synaptic loss (~ 30%) in hCX3CR1I249/M280 and CX3CR1 KO mice was observed relative to that in WT, which exhibited synaptic repair. hCX3CR1I249/M280 resulted in a ~ 17% loss of outer hair cells, which correlated with reduced otoacoustic emissions in the basal cochlear region. Noise led to increased macrophage numbers in the spiral ganglion and lateral wall of the WT; however, this response was attenuated in the CX3CR1 KO and hCX3CR1I249/M280 strains. Additionally, macrophages from CX3CR1 KO and hCX3CR1I249/M280 mice presented altered morphology, increased CD68 expression, and inflammation. Compared with those of mice carrying the CX3CR1 WT or KO allele, young hCX3CR1I249/M280 mice fostered under ambient noise presented early elevations in hearing thresholds at basal frequencies. Together, these findings reveal that human CX3CR1 variant-mediated macrophage dysregulation strongly correlates with worsening of hearing loss and cochlear degeneration after noise trauma. Our work proposes a novel immune-related genetic polymorphism that may aid in the identification of individuals with increased vulnerability to SNHL.

Purpose Present-day cochlear implants (CIs) can deliver usable speech reception in quiet surroundings. Most CI users, however, show impaired sensitivity to temporal fine structure, which hampers their use of pitch contours and spatial cues to segregate competing talkers. In previous short-term animal studies, we used intraneural (IN) electrodes to stimulate pathways originating from various cochlear turns. Neurons in the inferior colliculus synchronized to apical stimulation at higher rates than to stimulation of the middle-to-basal pathways that are stimulated primarily by today’s CIs. Here, we use non-invasive recordings to test the safety and efficacy of up to 6 months of IN implantation and stimulation in cats. Methods Deafened cats (ten female, two male) were implanted with IN and/or conventional CI electrodes. The IN electrodes were single activated-iridium shanks that targeted apical-turn fibers. Scalp recordings were made from sedated animals at 2–3-week intervals. Auditory brainstem responses to single electrical pulses (eABR) tracked sensitivity and growth of responses. Frequency following responses to electrical pulse trains (eFFR) assessed brainstem temporal transmission at varying pulse rates. Results Thresholds for eABR were lower for IN than for CI stimulation, dynamic ranges were wider, and (by inference) spread of activation was more restricted. The eFFR evaluated at latencies comparable to those of inferior-colliculus spikes synchronized at maximum pulse rates averaging > 360 pulses/s for IN compared to ~ 240 pulses/s for CI stimulation. The eABR thresholds and eFFR cutoff rates were stable out to 6 months after implantation. Conclusions The results demonstrate the safety and efficacy of chronic IN stimulation in an animal model. In a future clinical device, an IN electrode could augment cochlear-implant performance by enhancing temporal acuity, thereby improving speech reception amid competing sounds.

Objective Cisplatin is a chemotherapeutic agent with the undesirable side effect of ototoxicity. Transtympanic injections of antioxidant formulations may provide local otoprotection. We tested a novel antioxidant‐eluting microparticle for its otoprotective capability from systemic cisplatin as measured by cochlear electrophysiology. Study Design Basic science. Setting Translational research laboratory. Methods Eighteen mice were assigned to three groups. All mice underwent baseline click‐evoked auditory brainstem response (ABR) audiometry and right ear microparticle injections before beginning 42‐day intraperitoneal administration regimens of either saline (healthy control empty microparticle [HCEMP] group) or cisplatin (cisplatin empty microparticle [CEMP] group and cisplatin N ‐acetylcysteine microparticle [CNAC] group). These regimens consisted of three 4‐day cycles of intraperitoneal saline or cisplatin administration followed by 10 rest days. HCEMP and CEMP received right‐sided transtympanic empty microparticles, and CNAC received transtympanic N ‐acetylcysteine eluting microparticles. On day 43, all mice underwent posttreatment ABR. ABR thresholds and threshold shifts were analyzed with mixed‐effects models and Tukey's post hoc tests and were compared across pretreatment/posttreatment ears, treatment groups, and injected and non‐injected ears. Results We found that threshold shifts in the ears that received a transtympanic injection of N ‐acetylcysteine and three cycles of intraperitoneal cisplatin were similar to the paired ears of mice that received no cisplatin. Mice that received a transtympanic injection without N ‐acetylcysteine and intraperitoneal cisplatin had increased thresholds compared to mice that received a transtympanic injection of N ‐acetylcysteine and cisplatin. Conclusion Transtympanic N ‐acetylcysteine microparticle injections provided functional otoprotection in cisplatin‐exposed mice.

Hearing loss (HL) in patients with systemic lupus erythematosus (SLE) has been widely reported, though the underlying mechanisms remain unclear. In this study, we demonstrate significant HL in an imiquimod-induced mouse model of SLE, accompanied by blood–labyrinth barrier (BLB) damage in the stria vascularis (SV). We found that cytotoxic CD8 + T lymphocytes and natural killer cells mediate BLB disruption, with granzyme b (Gzmb) acting as the primary factor inducing cell death of the cochlear capillary endothelial cells. Remarkably, inhibiting Gzmb by Serpinb9 significantly alleviates the elevations of hearing thresholds in imiquimod-induced SLE model mice, suggesting a therapeutic target for autoimmune-related HL. Our findings provide insights into the pathogenesis of SLE-related HL and propose Gzmb inhibition as a potential therapeutic strategy.

Hearing loss is one of the most prevalent sensory disorders, but no commercial biological treatments are currently available. Here, we identify an East Asia-specific founder mutation, the homozygous c.220 C > T mutation in MPZL2, that contributes to a significant proportion of hereditary deafness cases in our cohort study. We find that the disease-causing mutation can be targetable by adenine base editors (ABEs) that enable A·T-to-G·C base corrections without DNA double-strand breaks. To demonstrate this, we develop a humanized mouse model (hMPZL2Q74X/Q74X) that recapitulates human MPZL2 deafness and leads to progressive hearing loss. A PAM-flexible ABE variant with reduced bystander and off-target effects (ABE8eWQ-SpRY:sgRNA3) is packaged in dual adeno-associated viruses (AAVs) and injected into the inner ear of hMPZL2Q74X/Q74X mice and effectively corrects the mutation. This treatment significantly restores hearing function, improves inner ear structural integrity, and reverses altered gene expression. Base editing may hold therapeutic potential for hereditary deafness, including most cases of MPZL2 deafness.

Diverse biological factors, such as sex and age, confer heterogeneity on sensory processing challenges in autism. These factors result in major difficulties in the processing of contextual information in social and non-social situations. To assess divergence in autistic traits, it is critical to consider sex- and age-related variability. Nevertheless, these differences remain largely elusive. Animal models of autism offer the possibility to examine contextual processing at the single-neuron level. Here, we investigated predictive processing of contextual auditory cues in the auditory midbrain of control and prenatally valproic acid-induced rats, a well-established animal model of autism. The rats were prepubertal and adult female and male animals. We performed single-unit recordings in the inferior colliculus of control and prenatally, or in utero , exposed rats under the classical oddball paradigm and non-repetitive cascade control sequences to study neuronal mismatch. This is the neuronal correlate of mismatch negativity, the brain’s automatic response to interruptions in environmental regularity. When comparing control and exposed rats, our results demonstrated a reduction in neuronal mismatch in rats exposed to valproic acid. However, exposed adult females exhibited an increased neuronal mismatch compared to their control counterparts. With respect to sex distinctions, valproic acid induced sex differences in neuronal mismatch of prepubertal and adult rats that are not observable in control animals. Moreover, we detected an age-dependent refinement in prediction error that is not affected by the drug. But valproic acid altered typical developmental trajectory of neuronal mismatch in both sexes. Such observations support sex- and age-related effects of in utero valproic acid exposure in contextual auditory processing at the neural level of the inferior colliculus. In autism, atypical predictive processing of environmental regularities underlies unusual responses to novel experiences. The present study highlights the importance of sex and age, that confer heterogeneity to these challenges.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by complex sensory processing deficits, which continue to elude comprehensive mechanistic understanding. A key unresolved question is how alterations in neural connectivity and communication translate into the behavioral manifestations seen in ASD. Here, we investigate how oligodendrocyte dysfunction alters myelin plasticity and neuronal activity, leading to auditory processing disorder associated with ASD. We focus on the SCN2A gene, an ASD-risk factor, to understand its role in myelination and neural processing within the auditory nervous system. Transcriptional profiling suggests alterations in the expression of myelin-associated genes in Scn2a conditional knockout mice, highlighting the cellular consequences engendered by Scn2a deletion in oligodendrocytes. The results reveal a nuanced interplay between oligodendrocytes and axons, where Scn2a deletion causes alterations in the intricate process of myelination. This disruption instigates changes in axonal properties, presynaptic excitability, and synaptic plasticity at the single cell level. Furthermore, oligodendrocyte-specific Scn2a deletion compromises the integrity of neural circuitry within auditory pathways, leading to auditory hypersensitivity. Our findings reveal a pathway linking myelin deficits to synaptic activity and sensory abnormalities in ASD.

Noise can induce hearing loss. In particular, noise can induce cochlear synapse degeneration leading to hidden hearing loss, which is the most common type of hearing disorders in the clinic. Currently, there is no pharmacological treatment, especially no post-exposure (i.e., therapeutic) treatment available in the clinic. Here, we report that systemic administration of K+ channel blockers by intraperitoneal injection before or after noise exposure significantly ameliorated noise-induced hearing loss (NIHL) and cochlear synapse degeneration. After systemic administration of a general K+-channel blocker tetraethylammonium (TEA) before or after noise exposure, the noise-induced threshold elevation of auditory brainstem responses (ABRs) and reduction of suprathreshold responses were significantly attenuated. The impairment on the active cochlear mechanics measured by distortion product otoacoustic emission (DPOAE) was also reduced. The therapeutic effect was further improved as the post-exposure administration time extended. BK channel is a predominant K+ channel in the inner hair cell. We further found that post-exposure administration of a BK channel blocker GAL-021 could ameliorate NIHL and improved the hearing behavioral responses examined by acoustic startle response (ASR). Finally, both TEA and GAL-021 significantly reduced noise-induced ribbon synapse degeneration. These findings demonstrate a promise of systemic administration of K+ channel blockers to treat synapse degeneration and NIHL after noise exposure, which currently is urgently required in the clinic.

Machine learning (ML) techniques are increasingly being used to improve disease diagnosis and treatment. However, the application of these computational approaches to the early diagnosis of age-related hearing loss (ARHL), the most common sensory deficit in adults, remains underexplored. Here, we demonstrate the potential of ML for identifying early signs of ARHL in adult mice. We used auditory brainstem responses (ABRs), which are non-invasive electrophysiological recordings that can be performed in both mice and humans, as a readout of hearing function. We recorded ABRs from C57BL/6N mice (6N), which develop early-onset ARHL due to a hypomorphic allele of Cadherin23 (Cdh23ahl), and from co-isogenic C57BL/6NTacCdh23+ mice (6N-Repaired), which do not harbour the Cdh23ahl allele and maintain good hearing until later in life. We evaluated several ML classifiers across different metrics for their ability to distinguish between the two mouse strains based on ABRs. Remarkably, the models accurately identified mice carrying the Cdh23ahl allele even in the absence of obvious signs of hearing loss at 1 month of age, surpassing the classification accuracy of human experts. Feature importance analysis using Shapley values indicated that subtle differences in ABR wave 1 were critical for distinguishing between the two genotypes. This superior performance underscores the potential of ML approaches in detecting subtle phenotypic differences that may elude manual classification. Additionally, we successfully trained regression models capable of predicting ARHL progression rate at older ages from ABRs recorded in younger mice. We propose that ML approaches are suitable for the early diagnosis of ARHL and could potentially improve the success of future treatments in humans by predicting the progression of hearing dysfunction.

Background: Although several evidence demonstrates a "gut-microbiota-brain axis", suggesting a bidirectional communication between gut microbiota and the central nervous system, less is known about a possible link between the gut and the peripheral nervous system, including the inner ear. Methods: Here, we investigated the impact of intestinal inflammation and the modulation of gut microbiota through fecal microbiota transplantation on hearing sensitivity. Female C57BL/6 mice were assigned to four groups: control (Ctrl), DSS-induced colitis (DSS), FMT from patients with active ulcerative colitis (FMT aUC), and FMT from patients with ulcerative colitis in remission (FMT rUC). Auditory function was evaluated by auditory brainstem responses (ABR). Morphological and molecular analyses on cochlear tissues were performed using immunofluorescence, histological staining, and Western blot to assess inflammation, oxidative stress, and blood-labyrinth barrier integrity. Donor microbiota composition was characterized by 16S rRNA sequencing, and systemic inflammation was evaluated by measuring serum lipopolysaccharide (LPS) levels. Results: We found that intestinal dysbiosis is associated with functional, morphological, and molecular alterations in the cochlea, such as increased oxidative stress, inflammation, and altered blood-labyrinth barrier permeability. This leads to macrophage infiltration and immune response activation through the MyD88/NF-κB pathway. Notably, these effects were exacerbated by FMT from subjects with aUC, while FMT from patients with rUC provided a protective effect on cochlear functions. Conclusions: Overall, our findings suggest that gut inflammation, microbiota alteration, or its therapeutic modulation can impact inner ear pathology: worsening gut inflammatory status negatively affects hearing sensitivity, while the restoration of gut microbiota positively impacts auditory function.

Age-related hearing loss is common in the human population but it is highly heterogeneous in aetiology which has hampered efforts to develop ways of stopping its progression. Three major sites of the initial dysfunction are the sensory hair cells, their innervation, and the stria vascularis which generates the high potassium endolymph maintained at a high endocochlear potential bathing the apical surface of hair cells. Treatments aimed at the initial site-of-lesion may be useful, and diagnostic tools to distinguish the primary site would help stratify clinical trials and facilitate selection of the most suitable treatment for each person. Here we report a new non-invasive test that distinguishes mutant mice with known stria vascularis dysfunction and reduced endocochlear potential from mice with sensory hair cell or neural defects but with normal endocochlear potential. It is based on measuring inter-trial coherence in auditory brainstem responses to individual stimuli. Mice with reduced strial function show good inter-trial coherence compared with the amplitude of the averaged response, while mice with sensory or neural deficits show poor coherence. This method might be useful in humans to predict whether they have reduced strial function, which cannot be measured directly, and identify who would benefit from treatments aimed at boosting strial function.

Middle age represents a critical period of accelerated brain changes and provides a window for early detection and intervention in age-related neurological decline. Hearing loss is a key early marker of such decline and is linked to numerous comorbidities in older adults. Yet, ~10% of middle-aged individuals who report hearing difficulties show normal audiograms. Cochlear neural degeneration (CND) could contribute to these hidden hearing deficits, though its role remains unclear due to a lack of objective diagnostics and uncertainty regarding its perceptual outcomes. Here, we employed a cross-species design to examine neural and behavioral signatures of CND. We measured envelope following responses (EFRs) – neural ensemble responses to sound originating from the peripheral auditory pathway – in young and middle-aged adults with normal audiograms and compared these responses to young and middle-aged Mongolian gerbils, where CND was histologically confirmed. We observed near-identical changes in EFRs across species that were associated with CND. Behavioral assessments revealed age-related speech-in-noise deficits under challenging conditions, while pupil-indexed listening effort increased with age even when behavioral performance was matched. Together, these results demonstrate that CND contributes to speech perception difficulties and elevated listening effort in midlife, which may ultimately lead to listening fatigue and social withdrawal.

Circadian sensitivity significantly influences the severity of noise-induced hearing loss (NIHL), but the underlying mechanisms remain unclear. Here, we applied single-cell RNA sequencing to 97,043 cochlear cells, identifying macrophages as the primary immune responders to acoustic trauma, with a notable increase in their proportion in the cochlea. Immunofluorescence confirmed significant recruitment and activation of cochlear macrophages following noise exposure, while in vivo macrophage depletion resulted in the recovery of hearing. Furthermore, analyses of differentially-expressed genes and pathways revealed pronounced activation of NLRP3 inflammasome signaling in macrophages during night-time noise exposure. Measurements of elevated IL-1β and IL-18 expression in cochlear macrophages by multiplex immunohistochemistry correlated with heightened inflammation in the night-time exposure group. These findings were further confirmed by the administration of the selective NLRP3 inhibitor CY-09, which mitigated inflammasome activation, preserved synaptic integrity, and protect against hearing loss. In conclusion, our findings underscore the role of macrophage-driven NLRP3 inflammasome activation in mediating circadian variations in cochlear damage, offering a potential therapeutic target for mitigating NIHL.

Hearing hinges upon the ear’s ability to enhance its responsiveness by means of an energy-expending active process that amplifies the very mechanical inputs that it detects. This process is defined by four properties that, although seemingly unrelated, consistently occur together: amplification, sharp frequency tuning, compressive nonlinearity, and spontaneous otoacoustic emission. In nonmammal tetrapods, the active process is evident in individual hair cells. The hair bundles of the bullfrog, for example, exhibit all four attributes by operating near a Hopf bifurcation—a critical regime in which these properties naturally coalesce. In mammals, however, the delicate nature of the cochlea has restricted the evidence for an active process to studies in vivo, where it is generally attributed to the collective effort of the outer hair cells that energize the traveling wave along the cochlear spiral. As a result, the cellular mechanisms that underlie the properties of mammalian hearing remain contested, with uncertainty about whether criticality plays a role in the cochlea’s active process. Here we show that, when placed in a recording chamber that closely mimics the in vivo physiological environment, a segment of the mammalian cochlea ex vivo displays the features of the active process—amplification, frequency tuning, compressive nonlinearity, and the generation of distortion products. We show that this process operates locally, independently of traveling waves, and that the sensory epithelium achieves active amplification by operating near criticality at a Hopf bifurcation. The results reveal the existence of a unified biophysical principle that underlies auditory processing across species and even phyla.