Posterior Semicircular Canal Ouabain Injection Induces Early Synaptic Loss and Delayed Cochlear Nerve Degeneration

Posterior Semicircular Canal Ouabain Injection Induces Early Synaptic Loss and Delayed Cochlear Nerve Degeneration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Posterior Semicircular Canal Ouabain Injection Induces Early Synaptic Loss and Delayed Cochlear Nerve Degeneration Yoshihiro Nitta, Takaomi Kurioka, Sachiyo Mogi, Lingshuai Kong, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8897404/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Auditory neuropathy (AN) and cochlear synaptopathy (CS) are characterized by impaired neural transmission despite preserved hair cell (HC) function. Ouabain (OB), a Na⁺/K⁺-ATPase inhibitor, has been used as a selective auditory nerve injury model via round window membrane delivery. However, this approach is technically variable and invasive. Objective To establish a minimally invasive and stable cochlear nerve injury model by delivering OB through the posterior semicircular canal (PSC) and characterizing short- and long-term pathological consequences. Methods Seven-week-old CBA/J mice received PSC injections of saline, 1 mM OB, or 5 mM OB. Auditory brainstem responses (ABRs) were evaluated before treatment and at 7 and 56 days post-treatment. Cochlear pathology was assessed through immunohistochemical and histological analyses of HCs, ribbon synapses, spiral ganglion neurons (SGNs), cochlear nerve fiber density, and stria vascularis morphology. Results PSC-delivered OB elevated ABR thresholds and reduced wave I amplitudes without HC loss. At 7 days, significant SGN loss occurred only in the 5 mM OB group. Both OB doses induced ribbon synapse loss and increased orphan ribbons. Cochlear nerve fiber density was significantly reduced as early as 7 days after 1 mM OB administration, despite preserved SGN cell bodies. Stria vascularis morphology remained unchanged. Conclusion PSC-delivered OB induces synaptic uncoupling and peripheral cochlear nerve fiber loss, preceding SGN degeneration. This minimally invasive model recapitulates key pathological features of CS progressing toward AN and provides a platform for studying cochlear neuropathy and therapeutic interventions. Ouabain Posterior semicircular canal Cochlear neuropathy Spiral ganglion neuron loss Ribbon synapse Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction In recent years, increasing attention has been directed toward patients who experience difficulty in speech perception and hearing in noise, even when conventional audiometric measurements do not fully account for their listening difficulties. The underlying pathologies of such cases have been conceptualized as auditory neuropathy (AN) and cochlear synaptopathy (CS). AN is defined as a disorder in which outer hair cell (OHC) function is preserved, whereas synchronous signal transmission between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) or along the auditory nerve fibers is impaired [ 1 ] [ 2 ]. Clinically, patients typically show preserved otoacoustic emissions (OAEs) and cochlear microphonics, whereas auditory brainstem responses (ABRs) are absent or markedly abnormal, often accompanied by elevated ABR thresholds and disproportionately poor speech discrimination relative to the behavioral hearing thresholds. In contrast, CS is characterized by the selective degeneration of ribbon synapses connecting IHCs and SGNs. Animal studies have shown that this pathology can occur early after noise exposure or with aging [ 3 ]. In this condition, despite preserved hair cells (HCs) and relatively maintained auditory sensitivity at low stimulus levels, the amplitude of ABR wave I is reduced, reflecting a decrease in the number of synchronously firing auditory nerve fibers (ANFs). This dissociation between auditory sensitivity and neural output has been proposed as the basis of so-called “hidden hearing loss.” Low-spontaneous-rate auditory nerve fibers are particularly vulnerable, potentially contributing to impaired speech perception and reduced hearing ability in noisy environments [ 4 ]. Previous basic studies have implicated several mechanisms underlying cochlear synaptic damage, including glutamate excitotoxicity driven by excessive glutamate release and overactivation of AMPA receptors [ 5 ], as well as oxidative stress associated with mitochondrial dysfunction [ 6 ]. Moreover, irreversible loss of IHC–SGN synapses is thought to precede the degeneration of auditory nerve fibers and SGN cell bodies, which progresses in a distal-to-proximal “dying-back” pattern [ 3 ]. As synaptic injury becomes more extensive, functional deficits may extend beyond reduced neural output to include elevated ABR thresholds, reflecting progression toward a more overt AN–like phenotype. Nevertheless, several important questions remain unresolved, including the extent to which synaptic injury leads to irreversible SGN loss and the potential for synaptic regeneration or plastic recovery following such damage. Thus, establishing a reproducible and stable animal model is critical for elucidating the pathophysiology of the disease and promoting the development of novel therapeutic approaches. Traditionally, the local application of ouabain (OB), a Na⁺/K⁺-ATPase inhibitor, onto the round window membrane (RWM) has been widely used as a selective auditory nerve injury model [ 7 ]. OB preferentially affects type I SGNs, which express the α3 subunit of Na⁺/K⁺-ATPase, inducing a reduction in ABR wave I amplitude and synaptic loss while preserving the OHC function. However, RWM application requires opening the middle ear bulla, which carries risks, including conductive hearing loss secondary to otitis media, interindividual variability in drug absorption, and fatality due to drug leakage into the middle or inner ear. In contrast, posterior semicircular canal (PSC) administration has been reported to facilitate a more efficient distribution of agents throughout the cochlea via hydrodynamic flow compared with RWM application [ 8 ]. Indeed, viral vector studies have demonstrated that PSC injection enables widespread and efficient transduction across the cochlea [ 9 ]. In addition, PSC administration does not require opening the middle ear cavity, thereby minimizing the risk of conductive hearing loss or inflammation associated with middle-ear manipulation. Moreover, this approach allows precise control of the volume and concentration of agents delivered to the inner ear, ensuring more consistent and reproducible inner ear drug exposure across animals. Based on these findings, we investigated whether local administration of OB via the PSC could serve as a less invasive and more stable approach to establish a selective cochlear nerve injury model in mice. Methods Animals and study approval Seven-week-old male CBA/J mice (Oriental Yeast Co., Ltd., Tokyo, Japan) were used in this study. CBA/J mice have been validated as a model for OB-induced hearing loss in previous studies [ 7 ] [ 10 ]. All animal procedures were performed in accordance with the institutional guidelines of the Animal Experimentation and Ethics Committee of Kitasato University School of Medicine and were approved by the committee (Approval No. M2025-121). Ototoxic drug administration and surgical procedure Mice were anesthetized via intraperitoneal injection of medetomidine (0.75 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg). Under a stereomicroscope (Leica S9E; Leica Microsystems, Tokyo, Japan), a postauricular skin incision was made on the left side, and the overlying muscles were separated to expose the PSC. A small fenestration was created in the canal wall using a 32-gauge needle. After confirming perilymph leakage, fluid efflux was allowed to subside for approximately 5 min. A fine glass micropipette connected to a microsyringe pump was gently inserted into the fenestration opening. The insertion site was sealed with a small amount of tissue adhesive to prevent fluid leakage. Sterile saline (control), 1 mM ouabain (RSD, 1076/100), or 5 mM ouabain (dissolved in saline) was injected into the PSC at a rate of 1 µL/min for a total volume of 1 µL (Fig. 1 ). Following the injection, the micropipette was left in place for 5 min to facilitate diffusion. Thereafter, the micropipette was removed, and the fenestration site was sealed with small muscle fragments and a tissue adhesive. Finally, the skin incision was closed using nylon sutures. Auditory brainstem response (ABR) ABR thresholds were evaluated prior to OB administration and 7 and 56 days post-treatment. The mice were anesthetized as described above and placed on a warming pad in a sound-attenuating chamber. Subcutaneous needle electrodes were positioned at the nose (reference), mastoid region (recording), and tail (ground) [ 11 ] [ 12 ]. Tone burst stimuli at 4, 8, 16, and 32 kHz were delivered in 5-dB steps. Responses were averaged over 512 sweeps using a Neuropack Sigma system (Nihon Kohden, Tokyo, Japan). Thresholds were defined as the lowest stimulus levels that produced a reproducible waveform above the noise floor. Cochlear immunohistochemistry To examine cochlear expression of Na⁺/K⁺-ATPase, immunofluorescence staining was performed on paraffin-embedded cochlear sections. The mice were transcardially perfused with 4% paraformaldehyde (PFA) in phosphate buffer (PB), and the cochleae were removed and post-fixed overnight at 4°C. The specimens were decalcified in 5% EDTA for 1 week, embedded in paraffin, and sectioned to a thickness of 4 µm. After deparaffinization, rehydration, and antigen retrieval, the sections were incubated with a primary antibody against the α3 subunit of sodium–potassium ATPase (alpha 3 Na + /K + -ATPase/ATP1A3, clone H-4; sc-365744, Santa Cruz Biotechnology). Phalloidin-iFluor 488 Reagent (Abcam) was used to label the filamentous actin. Appropriate fluorescent secondary antibodies were applied, and the sections were mounted with an antifade mounting medium for imaging. For synaptic evaluation, separate cochleae were processed for whole-mount immunostaining of the organ of Corti. The mice were transcardially perfused with 4% PFA in PB, and the cochleae were harvested and post-fixed in 4% PFA in PB for 2 h. After decalcification in 5% EDTA overnight, the cochleae were dissected by removing the lateral wall and tectorial membrane. Whole-mount preparations were incubated overnight at 4°C with the following primary antibodies: anti-Myosin7A (rabbit IgG; Invitrogen, Grand Island, NY), anti-GluR2 (mouse IgG2a; Millipore, Billerica, MA), and anti-CtBP2 (mouse IgG1; BD Transduction Laboratories, San Jose, CA). Secondary antibodies (Alexa Fluor, 1:200 in blocking buffer; Molecular Probes, Eugene, OR, USA) were applied for 2 h at room temperature. Assessment of hair cells and synapses Seven days after OB administration, HC and synapse survival were assessed in the cochleae of the experimental mice (saline group, n = 4; 1 mM OB group, n = 4; 5 mM OB group, n = 3). The specimens were mounted on glass slides and imaged using a confocal laser-scanning microscope (LSM710; Zeiss, Jena, Germany). The total cochlear length was measured, and a cochlear frequency map was generated to localize the 4-, 8-, 16-, and 32-kHz regions. IHC and OHC survival was quantified and expressed as percentages. For synaptic evaluation, confocal z-stack images were acquired from the IHC region at the same frequency locations using a 63× oil-immersion objective (3× digital zoom; 0.25-µm z-steps). CtBP2/GluR2 puncta were counted to determine the number of paired synaptic ribbons per IHC, and unpaired CtBP2-positive puncta without corresponding GluR2 immunoreactivity were quantified as orphan ribbons. Assessment of SGN survival At 7 and 56 days post-administration, SGN survival was evaluated (normal group, n = 4; saline group, n = 4; 1 mM OB group, n = 4; and 5 mM OB group, n = 3). Following transcardial perfusion with 4% PFA, the cochleae were removed and post-fixed overnight. The specimens were decalcified in 5% EDTA for 1 week, embedded in paraffin, and sectioned at 4 µm. The sections were stained with hematoxylin and eosin (HE). The regions containing the largest SGN areas were analyzed. The spiral ganglion was divided into apical and basal turns, and the SGN density was calculated as the number of neurons per unit area. Assessment of cochlear nerve fibers in the osseous spiral lamina To quantify peripheral cochlear nerve integrity, the density of auditory nerve fibers within the osseous spiral lamina (OSL) was assessed as previously described, with minor modifications [ 13 ] [ 14 ]. Cochlear nerve fiber density was assessed using the same paraffin-embedded cochlear sections prepared for the SGN analysis. HE-stained mid-modiolar sections were used for the quantitative analysis of cochlear nerve fibers within the OSL. Analyses were restricted to the middle turn of the cochlea to ensure consistency among the specimens. Sections containing well-defined openings of the habenula perforata were selected, and all clearly identifiable cochlear nerve fibers within the OSL were counted manually under high magnification. The cochlear nerve fiber density was calculated as the number of nerve fibers per unit area (fibers per 100 µm²). Assessment of stria vascularis area To determine whether OB administration induces structural alterations in the stria vascularis (SV), which could potentially contribute to auditory dysfunction independently of neural injury, the SV morphology was quantitatively evaluated. This analysis was performed using the same paraffin-embedded mid-modiolar cochlear sections prepared for SGN analysis, as vascular atrophy and metabolic dysfunction of the SV have been implicated in certain forms of sensorineural hearing loss [ 15 ] [ 16 ]. A quantitative assessment of the SV area was conducted in the apical and basal turns of the cochlea. In selected mid-modiolar sections, the SV was identified along the lateral wall based on its characteristic trilaminar epithelial structure. Statistical analysis Data are presented as mean ± standard error. Normality was assessed using the Shapiro–Wilk test. Normally distributed data were evaluated using two-way ANOVA, followed by Tukey’s post hoc test; non-normally distributed data were analyzed using the Kruskal–Wallis test, followed by Dunn’s post hoc test. Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software Inc., La Jolla, CA, USA). Statistical significance was set at p < 0.05. Results Expression of Na⁺/K⁺-ATPase in the cochlea Immunofluorescence staining revealed prominent expression of the Na⁺/K⁺-ATPase α3 subunit within the cochlea. At low magnification (Fig. 2 A), strong Na⁺/K⁺-ATPase immunoreactivity was observed in SGN somata within the modiolus and along afferent nerve fiber bundles extending toward the organ of Corti. Higher-magnification imaging of the organ of Corti region (Fig. 2 B) demonstrated Na⁺/K⁺-ATPase α3 expression in afferent nerve fibers traversing the osseous spiral lamina and afferent terminals located beneath the IHCs. The immunoreactive signal was distributed in a punctate and linear pattern in the vicinity of the IHC basal pole, consistent with localization near the IHC–SGN synaptic region. In contrast, no appreciable Na⁺/K⁺-ATPase immunoreactivity was detected in the IHCs, OHCs, or surrounding supporting cells. Assessment of ABR thresholds Baseline ABR measurements obtained at 8 weeks of age, immediately prior to OB administration, revealed no significant differences in hearing thresholds among the groups (Fig. 3 A, n = 17). At 7 days after administration, the 1mM OB (n = 4 ears) and 5 mM OB groups (n = 3 ears) demonstrated significantly elevated ABR thresholds compared with the saline group (n = 4 ears) (two-way ANOVA, p < 0.0001; Fig. 3 B). To determine whether the PSC injection procedure alone affected auditory function, we compared the ABR thresholds between saline-injected mice and untreated controls (normal group). No significant differences were found (two-way ANOVA, p > 0.05; Fig. 3 B), indicating that PSC injection did not adversely affect hearing. At 56 days after 1 mM OB administration (n = 4 ears), ABR thresholds were significantly elevated compared to those in the saline group (n = 4 ears) (two-way ANOVA, p 0.05). Peak wave-I amplitudes at 80 dB SPL were also evaluated at 7 and 56 days after OB treatment. At 7 days, both the 1 mM and 5 mM OB groups showed reduced wave I amplitudes compared to the normal group (Fig. 3 E). At 56 days, wave-I amplitudes remained significantly reduced in the 1 mM OB group compared to the control group (two-way ANOVA, p < 0.0001; Fig. 3 F). Because the ABR wave-I amplitude reflects cochlear nerve activity, these reductions suggest OB-induced cochlear nerve degeneration. Assessment of HC survival Previous reports have shown that OB administration via the RWM induces AN without HC loss [ 7 ]. Consistent with these findings, no loss of IHCs or OHCs was observed 7 days after PSC administration of 1 mM or 5 mM OB (Fig. 4 B). Assessment of SGN survival Prior studies reported that SGN degeneration began on day 7 and continued through day 30 following OB application to the RWM in adult mice [ 10 ]. Based on these insights, we examined SGN survival at both early (7 days) and late (56 days) time points after PSC OB administration. At 7 days after treatment, HE staining revealed a significant loss of SGNs in the apical and basal turns of the cochlea in the 5 mM OB group compared to the saline group (two-way ANOVA, p < 0.0001;Fig. 5 B), whereas SGN loss in the 1 mM OB group was minimal and did not reach statistical significance at this time point. In contrast, at 56 days after 1 mM OB administration, a marked and statistically significant reduction in SGN density was observed in both the apical and basal turns compared to the saline group (two-way ANOVA, p < 0.0001;Fig. 5 B). No significant SGN loss was detected in the saline group at any time point. Assessment of synapses Seven days after OB administration, the ABR wave-I amplitudes were significantly reduced, whereas the SGN loss was minimal. Therefore, we evaluated cochlear synapses by quantifying the number of CtBP2/GluR2 puncta per IHC [ 17 ]. Previous studies on CS have demonstrated that synaptic injury is frequently accompanied not only by a reduction in paired pre- and postsynaptic elements but also by an increase in the so-called orphan ribbons, defined as presynaptic ribbons lacking apposed postsynaptic glutamate receptor labeling, reflecting synaptic uncoupling rather than complete synapse elimination [ 3 ] [ 18 ]. Compared with the saline group, the 1 mM OB and 5 mM OB groups showed a significant reduction in synapses across all frequency regions examined (two-way ANOVA, p < 0.001; Fig. 6 B). In contrast, the number of orphan synapses was significantly increased in the 1 mM OB and 5 mM OB groups (two-way ANOVA, Fig. 6 C). Assessment of cochlear nerve fibers in the osseous spiral lamina In the 1 mM OB group, no significant loss of SGN somata was observed in either the apical or basal turns compared with saline-treated controls 7 days after administration, indicating preserved SGN cell bodies at this early time point. In contrast, analysis of peripheral auditory nerve integrity revealed a significant reduction in cochlear nerve fiber density within the OSL of the middle turn in the 1 mM OB group 7 days after administration (two-way ANOVA, p < 0.001; Fig. 7 B). Together, these findings indicate that peripheral cochlear nerve fibers are selectively affected at an early stage following low-dose OB administration, preceding the detectable degeneration of SGN cell bodies. Assessment of stria vascularis area To determine whether ouabain administration affected SV morphology, the cross-sectional area of the SV was quantified in the apical and basal turns using HE-stained sections. Quantitative analysis revealed no significant differences in the stria vascularis area between the control and ouabain-treated groups in either the apical or basal turn (two-way ANOVA, p > 0.05; Fig. 8 ). These findings indicate that PSC-delivered ouabain did not induce detectable structural changes in the stria vascularis under the examined conditions. Discussion AN remains an incompletely characterized disorder in which synchronous neural signaling is disrupted despite the preservation of OHC function[ 1 , 2 ]. The intracochlear delivery of OB, a potent Na⁺/K⁺-ATPase inhibitor, has been widely used over the past decade to model AN in rodents. In particular, the RWM application of OB selectively injures type I SGNs while sparing HCs, thereby reproducing a hallmark pathological feature of AN [ 7 ] [ 19 ]. However, OB exposure via the RWM requires opening the middle ear bulla and is technically challenging. This approach is associated with variability in drug delivery and the risk of postoperative complications, including conductive hearing loss. To overcome these limitations, the present study evaluated whether OB administration through PSC could reproducibly induce AN-like pathology. We demonstrated that PSC delivery of OB produced selective SGN injury while preserving HC morphology, thereby establishing a reliable SGN-specific lesion model. Functionally, this model was characterized not only by a reduction in ABR wave I amplitude but also by a significant elevation in ABR thresholds, indicating impaired neural synchrony and reduced effective neural recruitment. Short-term evaluation (7 days) following 1 mM OB administration revealed significant loss of ribbon synapses despite minimal loss of SGN soma, consistent with CS, in which IHC–SGN synapses degenerate prior to neuronal loss [ 3 ]. Importantly, this early synaptic pathology was accompanied by a significant increase in orphan ribbons, defined as presynaptic ribbons lacking apposed postsynaptic glutamate receptor puncta, indicating synaptic uncoupling rather than ribbon elimination. Thus, this PSC-based OB model not only recapitulates the SGN pathology characteristic of AN but also captures early synaptic dysfunction associated with progression from CS toward an AN-like phenotype rather than pure “hidden hearing loss.” OB-induced neuropathy is thought to arise from the selective inhibition of the Na⁺/K⁺-ATPase α3 subunit, which is highly expressed in type I SGNs but sparse in IHCs and OHCs [ 7 ]. Immunohistochemical analyses have demonstrated that α3 protein is distributed not only in SGN somata but also along afferent fibers adjacent to the IHC–SGN synapse [ 20 ], suggesting that OB may directly perturb neuronal excitability and axonal homeostasis in the cochlea. In the present study, a significant loss of ribbon synapses was observed 7 days after exposure to 1 mM OB, whereas degeneration of SGN cell bodies was minimal at this time point. In contrast, a marked loss of SGN cell bodies became evident after 56 days. This temporal pattern supports a stepwise degenerative process in which synaptic terminals constitute the most vulnerable compartments. The presence of orphan ribbons suggests the destabilization and functional disconnection of IHC–SGN synapses, which likely contributes to desynchronized auditory nerve firing and elevation of ABR thresholds despite preserved HC morphology. Comparable findings have been reported in noise-induced CS, in which synaptic injury is the earliest event, followed by delayed axonal retraction and SGN cell body loss in a distal-to-proximal “dying-back” pattern [ 3 ] [ 4 ]. Several mechanisms may contribute to early synaptic vulnerability. Inhibition of Na⁺/K⁺-ATPase disrupts ionic homeostasis and reduces the driving force for glutamate transport. High-affinity glutamate transporters, including GLAST and GLT-1, depend on Na + gradients to clear glutamate from the synaptic cleft [ 21 ]. Within the cochlea, GLAST is localized to the supporting cells adjacent to the IHC–SGN synapse and plays a major role in glutamate clearance [ 22 ]. Pharmacological inhibition of GLAST markedly delays the recovery of cochlear afferent responses after noise exposure, demonstrating that impaired glutamate uptake contributes to synaptic excitotoxicity [ 23 ]. Although these downstream pathways have not been fully characterized in OB-treated cochleae, similar mechanisms are likely to be involved. Synaptic uncoupling and orphan ribbon formation may represent an intermediate pathological state preceding the complete elimination of synapses. Following synaptic elimination, SGNs may lose trophic support from IHCs, promoting axonal retraction and ultimately, neuronal death. This sequence aligns with the delayed SGN loss observed at 56 days in our study and with prior reports demonstrating progressive degeneration following synaptic disconnection. PSC delivery offers several advantages. First, PSC injection yielded a more consistent and widespread intracochlear distribution than RWM application. Dye-tracking studies have shown that PSC delivery produces stronger and more uniform labeling across both apical and basal turns, likely due to direct access to the perilymphatic space and a reduced dependence on RWM permeability [ 8 ]. Additionally, PSC injection of AAV vectors enables efficient and near pan-cochlear transduction in adult mice [ 24 ]. Importantly, unlike RWM-based approaches, PSC delivery does not require opening the middle ear bulla, thereby avoiding surgical manipulation of the middle ear space and reducing the risk of conductive hearing loss associated with otitis media or bulla inflammation. Second, RWM-based drug delivery is influenced by variability in RWM thickness, mucosal coverage, and middle ear conditions, which can lead to inconsistent intracochlear drug concentrations [ 25 ]. By directly accessing the perilymphatic lumen, PSC administration minimizes this variability and improves reproducibility. Third, recent refinements in PSC injection techniques have demonstrated that inner ear delivery can be achieved with minimal postoperative auditory dysfunction when the injection volume and perilymph leakage are controlled carefully [ 26 ]. Collectively, these findings highlight PSC delivery as a practical and reproducible alternative to RWM for selective cochlear manipulation. Although this study demonstrated that PSC-delivered OB can reproduce SGN degeneration while preserving HCs, several limitations should be recognized. The intracochlear pharmacokinetics of OB remain unclear; therefore, the relative contributions of drug concentration gradients versus intrinsic cellular susceptibility to the observed patterns of synaptic and neuronal loss remain uncertain. Furthermore, although the sequence of early ribbon synapse loss with orphan ribbon formation, followed by delayed SGN degeneration, is consistent with the progression from CS toward AN, this study cannot determine whether synaptic disconnection alone is sufficient to initiate subsequent neuronal death or whether the OB exerts direct cytotoxic effects on SGN soma via Na⁺/K⁺-ATPase α3 inhibition [ 16 ]. In addition, the downstream molecular pathways leading to SGN loss, including oxidative stress, mitochondrial dysfunction, and inflammatory responses, remain poorly defined and were not examined in this study. Further studies are required to determine how early synaptic injury progresses to irreversible neuronal degeneration. Conclusion In this study, we demonstrated that local administration of OB via PSC effectively creates a selective cochlear neuropathy model in mice. Early synaptic pathology is characterized by ribbon synapse loss and a significant increase in orphan ribbons, accompanied by reduced ABR wave I amplitude and elevated ABR thresholds. Substantial SGN degeneration appeared only at later time points, suggesting a stepwise degenerative process consistent with CS progression toward AN. Importantly, this approach preserved HC morphology and did not induce hearing changes when saline was administered, indicating that PSC injection is selective and minimally invasive. Compared with traditional RWM delivery, PSC administration offers an improved intralabyrinthine distribution and reduced technical variability. This model will be valuable for elucidating the mechanisms underlying SGN degeneration and evaluating therapeutic strategies aimed at synaptic protection and neuronal preservation. Declarations Clinical Trial Number not applicable Consent to Publish declaration : not applicable Ethics and Consent to Participate declarations not applicable Funding This research was supported by the following grants: JSPS KAKENHI grant (Grant Number 25K20171). Author Contribution Y.N. and T.K. designed the experiments. Y.N., S.M. and L.K.performed the experiments. Y.N. analyzed the data. Y.N. and T.K. co-wrote the manuscript. T.Y. reviewed the manuscript. Acknowledgement The authors would like to thank Dr. Teppei Noda, Dr. Takahiro Wakizono, and Dr. Takahiro Manabe from the Department of Otorhinolaryngology–Head and Neck Surgery, Kyushu University, and Dr. Koji Nishimura from the Department of Otorhinolaryngology–Head and Neck Surgery, Kyoto University, for their valuable advice and support in establishing the mouse model of hearing loss used in this study. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Starr A et al. Auditory neuropathy. Brain, 1996. 119 (Pt 3): pp. 741 – 53. Rance G, Starr A. Pathophysiological mechanisms and functional hearing consequences of auditory neuropathy. Brain. 2015;138(Pt 11):3141–58. Kujawa SG, Liberman MC. Adding insult to injury: cochlear nerve degeneration after temporary noise-induced hearing loss. J Neurosci. 2009;29(45):14077–85. Liberman MC, Kujawa SG. Cochlear synaptopathy in acquired sensorineural hearing loss: Manifestations and mechanisms. Hear Res. 2017;349:138–47. Puel JL, et al. Excitotoxicity and repair of cochlear synapses after noise-trauma induced hearing loss. NeuroReport. 1998;9(9):2109–14. Henderson D, et al. The role of oxidative stress in noise-induced hearing loss. Ear Hear. 2006;27(1):1–19. Yuan Y, et al. Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy. J Assoc Res Otolaryngol. 2014;15(1):31–43. Talaei S, et al. Dye Tracking Following Posterior Semicircular Canal or Round Window Membrane Injections Suggests a Role for the Cochlea Aqueduct in Modulating Distribution. Front Cell Neurosci. 2019;13:471. Suzuki J, et al. Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction. Sci Rep. 2017;7:45524. Zhang ZJ, et al. Dose-dependent effects of ouabain on spiral ganglion neurons and Schwann cells in mouse cochlea. Acta Otolaryngol. 2017;137(10):1017–23. Naganuma H, et al. Effects of arginine vasopressin on auditory brainstem response and cochlear morphology in rats. Auris Nasus Larynx. 2014;41(3):249–54. Nitta Y, et al. Suppression of the TGF-beta signaling exacerbates degeneration of auditory neurons in kanamycin-induced ototoxicity in mice. Sci Rep. 2024;14(1):10910. Kurioka T, et al. Selective hair cell ablation and noise exposure lead to different patterns of changes in the cochlea and the cochlear nucleus. Neuroscience. 2016;332:242–57. Eggink MC, et al. Human vestibular schwannoma reduces density of auditory nerve fibers in the osseous spiral lamina. Hear Res. 2022;418:108458. Gratton MA, Schulte BA, Smythe NM. Quantification of the stria vascularis and strial capillary areas in quiet-reared young and aged gerbils. Hear Res. 1997;114(1–2):1–9. Kaur C, et al. Predicting Atrophy of the Cochlear Stria Vascularis from the Shape of the Threshold Audiogram. J Neurosci. 2023;43(50):8801–11. Zhang Y, et al. Engineering olivocochlear inhibition to reduce acoustic trauma. Mol Ther Methods Clin Dev. 2023;29:17–31. Liberman MC, et al. Toward a Differential Diagnosis of Hidden Hearing Loss in Humans. PLoS ONE. 2016;11(9):e0162726. Lang H, Schulte BA, Schmiedt RA. Ouabain induces apoptotic cell death in type I spiral ganglion neurons, but not type II neurons. J Assoc Res Otolaryngol. 2005;6(1):63–74. McLean WJ, et al. Distribution of the Na,K-ATPase alpha subunit in the rat spiral ganglion and organ of corti. J Assoc Res Otolaryngol. 2009;10(1):37–49. Rose EM, et al. Glutamate transporter coupling to Na,K-ATPase. J Neurosci. 2009;29(25):8143–55. Glowatzki E, et al. The glutamate-aspartate transporter GLAST mediates glutamate uptake at inner hair cell afferent synapses in the mammalian cochlea. J Neurosci. 2006;26(29):7659–64. Chen Z, Kujawa SG, Sewell WF. Functional roles of high-affinity glutamate transporters in cochlear afferent synaptic transmission in the mouse. J Neurophysiol. 2010;103(5):2581–6. Suzuki J, et al. Corrigendum: Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction. Sci Rep. 2017;7:46827. Borkholder DA, Zhu X, Frisina RD. Round window membrane intracochlear drug delivery enhanced by induced advection. J Control Release. 2014;174:171–6. Zhu J, et al. Refining surgical techniques for efficient posterior semicircular canal gene delivery in the adult mammalian inner ear with minimal hearing loss. Sci Rep. 2021;11(1):18856. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8897404","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":600310790,"identity":"ac7d3fc0-a982-46aa-bc68-a60a250021c1","order_by":0,"name":"Yoshihiro Nitta","email":"data:image/png;base64,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","orcid":"","institution":"Kitasato University school of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yoshihiro","middleName":"","lastName":"Nitta","suffix":""},{"id":600310791,"identity":"21411d8a-0f62-47d4-84f5-b6cdeb9dfef1","order_by":1,"name":"Takaomi Kurioka","email":"","orcid":"","institution":"Kitasato University school of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takaomi","middleName":"","lastName":"Kurioka","suffix":""},{"id":600310792,"identity":"1b9ddb94-3da5-4fad-83af-0fa7ffcc3405","order_by":2,"name":"Sachiyo Mogi","email":"","orcid":"","institution":"Kitasato University school of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sachiyo","middleName":"","lastName":"Mogi","suffix":""},{"id":600310794,"identity":"0371c2ed-fca6-4b8f-8cd9-749567bac0da","order_by":3,"name":"Lingshuai Kong","email":"","orcid":"","institution":"Kitasato University school of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lingshuai","middleName":"","lastName":"Kong","suffix":""},{"id":600310795,"identity":"1564a085-bf5f-43b3-bd12-a53ff738ea11","order_by":4,"name":"Taku Yamashita","email":"","orcid":"","institution":"Kitasato University school of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Taku","middleName":"","lastName":"Yamashita","suffix":""}],"badges":[],"createdAt":"2026-02-17 04:24:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8897404/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8897404/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104094742,"identity":"0612706e-1883-42df-8109-41902211bed7","added_by":"auto","created_at":"2026-03-06 17:19:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30458,"visible":true,"origin":"","legend":"\u003cp\u003eSchedule of the experimental procedures\u003c/p\u003e\n\u003cp\u003eSchematic timeline illustrating the experimental schedule of the study. PSC administration of saline, 1 mM OB, or 5 mM OB was performed at 7 weeks of age. ABRs were recorded before treatment and 7 and 56 days after administration. Cochlear tissues were harvested at the indicated time points for histological and immunohistochemical analyses, including the assessment of hair cells, synapses, spiral ganglion neurons, cochlear nerve fibers, and stria vascularis morphology.\u003c/p\u003e\n\u003cp\u003ePSC: Posterior semicircular canal, OB: ouabain, ABRs: Auditory brainstem responses\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8897404/v1/1fb11c61a6ab5a5e2187f506.png"},{"id":104403933,"identity":"dd5bac2a-87cd-4c08-9bc1-6360631f3bae","added_by":"auto","created_at":"2026-03-11 12:19:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1120157,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of Na⁺/K⁺-ATPase in the cochlea\u003c/p\u003e\n\u003cp\u003e(A) Low-magnification immunofluorescence image showing strong expression of the Na⁺/K⁺-ATPase α3 subunit in spiral ganglion neuron somata within the modiolus and along the afferent nerve fiber bundles.\u003c/p\u003e\n\u003cp\u003e(B) High-magnification view of the organ of Corti region demonstrating Na⁺/K⁺-ATPase α3 immunoreactivity in afferent nerve fibers traversing the osseous spiral lamina and in afferent terminals beneath the inner hair cells. No appreciable labeling was observed in the hair or supporting cells. Red, Na⁺/K⁺-ATPase α3; green, phalloidin. Scale bars are indicated.\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8897404/v1/43c6c3529e75861c40b45cc7.png"},{"id":105033196,"identity":"7be2eaea-ee37-4710-ae43-ae288933b786","added_by":"auto","created_at":"2026-03-20 07:14:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":551180,"visible":true,"origin":"","legend":"\u003cp\u003eAuditory brainstem response thresholds and wave I amplitudes\u003c/p\u003e\n\u003cp\u003e(A) Baseline ABR thresholds measured prior to ouabain administration, showing no significant differences among groups.\u003c/p\u003e\n\u003cp\u003e(B) ABR thresholds 7 days after PSC administration, demonstrating significant threshold elevations in both the 1 mM OB and 5 mM OB groups compared with the saline group.\u003c/p\u003e\n\u003cp\u003e(C) ABR thresholds at 56 days after administration showing sustained threshold elevation in the 1 mM OB group.\u003c/p\u003e\n\u003cp\u003e(D–F) Peak ABR wave I amplitudes measured at 80 dB SPL. Wave I amplitudes were significantly reduced in the OB-treated groups at 7 days and remained reduced in the 1 mM OB group at 56 days. Data are presented as mean ± SEM.\u003c/p\u003e\n\u003cp\u003eABR: Auditory brainstem response, OB: ouabain, PSC: Posterior semicircular canal\u003c/p\u003e","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8897404/v1/d7f896f6d6cba7528a95b758.png"},{"id":104094750,"identity":"cc099a3a-ddc1-41f3-8b9b-4d6ba4b8ed2c","added_by":"auto","created_at":"2026-03-06 17:19:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1491876,"visible":true,"origin":"","legend":"\u003cp\u003eHair cell survival\u003c/p\u003e\n\u003cp\u003e(A) Representative confocal images of IHCs and OHCs 7 days after PSC administration of saline, 1 mM OB, or 5 mM OB. Scale bars are indicated.\u003c/p\u003e\n\u003cp\u003e(B) Quantitative analysis of IHC and OHC survival 7 days after PSC administration. No loss of IHCs or OHCs was observed in any of the OB-treated groups, consistent with the preserved hair cell morphology.\u003c/p\u003e\n\u003cp\u003eIHCs: inner hair cells, OHCs: outer hair cells, PSC: posterior semicircular canal, OB: ouabain,\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8897404/v1/9fb0d9a684484f41b8807c7f.png"},{"id":104094747,"identity":"40337b6f-b07a-43c2-8203-c9cd7d2c55ff","added_by":"auto","created_at":"2026-03-06 17:19:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2061776,"visible":true,"origin":"","legend":"\u003cp\u003eSpiral ganglion neuron survival\u003c/p\u003e\n\u003cp\u003e(A) Representative hematoxylin and eosin–stained mid-modiolar sections illustrating the SGNs in the apical and basal turns.\u003c/p\u003e\n\u003cp\u003e(B) Quantification of SGN density showing significant SGN loss in the 5 mM OB group at 7 days, whereas the SGN density in the 1 mM OB group was preserved at this time point. At 56 days after 1 mM OB administration, a significant reduction in SGN density was observed in both the apical and basal turns. Data are presented as mean ± SEM.\u003c/p\u003e\n\u003cp\u003eSGN: Spiral ganglion neuron, PSC: posterior semicircular canal, OB: ouabain\u003c/p\u003e","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8897404/v1/35c006fc83e8a4c91f084b3d.png"},{"id":104403849,"identity":"e35418f7-078f-4624-8269-5c9a8f1d2de9","added_by":"auto","created_at":"2026-03-11 12:19:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1315329,"visible":true,"origin":"","legend":"\u003cp\u003eCochlear synapse loss and orphan ribbon formation\u003c/p\u003e\n\u003cp\u003e(A) Representative confocal images of IHC synapses labeled with CtBP2 (presynaptic ribbons) and GluR2 (postsynaptic receptors).\u003c/p\u003e\n\u003cp\u003e(B) Quantification of paired synaptic ribbons per IHC demonstrating significant synapse loss in both the 1 mM and 5 mM OB groups across all frequency regions examined.\u003c/p\u003e\n\u003cp\u003e(C) Quantification of orphan ribbons showing a significant increase in CtBP2-positive puncta lacking apposed GluR2 labeling in the OB-treated groups. Data are presented as mean ± SEM.\u003c/p\u003e\n\u003cp\u003eIHCs: inner hair cells, OB: ouabain,\u003c/p\u003e","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8897404/v1/4825f6659f183451b577371a.png"},{"id":104094745,"identity":"7cccbe1c-f7d0-459c-8d18-f344c3c76775","added_by":"auto","created_at":"2026-03-06 17:19:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":621616,"visible":true,"origin":"","legend":"\u003cp\u003eEarly reduction of cochlear nerve fiber density in the osseous spiral lamina\u003c/p\u003e\n\u003cp\u003e(A) Representative hematoxylin and eosin–stained sections of the OSL in the middle turn, illustrating cochlear nerve fibers traversing the habenula perforata.\u003c/p\u003e\n\u003cp\u003e(B) Quantitative analysis showing a significant reduction in cochlear nerve fiber density at 7 days after 1 mM OB administration, despite the preservation of SGN cell bodies at the same time point. Data are presented as mean ± SEM.\u003c/p\u003e\n\u003cp\u003eOSL: osseous spiral lamina; OB: ouabain; SGN: Spiral ganglion neuron.\u003c/p\u003e","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-8897404/v1/45dc0a244366a33cb91238db.png"},{"id":104094749,"identity":"cc4470b4-fc3d-4921-a5e4-02461c76bb9c","added_by":"auto","created_at":"2026-03-06 17:19:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1820154,"visible":true,"origin":"","legend":"\u003cp\u003eStria vascularis morphology\u003c/p\u003e\n\u003cp\u003e(A) Representative hematoxylin and eosin–stained sections of the SV in the apical and basal turns.\u003c/p\u003e\n\u003cp\u003e(B) Quantification of the SV cross-sectional area, demonstrating no significant differences between the control and OB-treated groups. These findings indicate that PSC-delivered OB does not induce detectable structural changes in SV. Data are presented as mean ± SEM.\u003c/p\u003e\n\u003cp\u003eSV: stria vascularis, OB: ouabain\u003c/p\u003e","description":"","filename":"OnlineFig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-8897404/v1/93ab83989859cf6042069419.png"},{"id":105896163,"identity":"3d524c1c-1f34-4a10-a9a6-3aa0bbfaf942","added_by":"auto","created_at":"2026-04-01 08:43:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1896523,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8897404/v1/85012900-a632-4edf-bc9f-441f7cbb094b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Posterior Semicircular Canal Ouabain Injection Induces Early Synaptic Loss and Delayed Cochlear Nerve Degeneration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, increasing attention has been directed toward patients who experience difficulty in speech perception and hearing in noise, even when conventional audiometric measurements do not fully account for their listening difficulties. The underlying pathologies of such cases have been conceptualized as auditory neuropathy (AN) and cochlear synaptopathy (CS). AN is defined as a disorder in which outer hair cell (OHC) function is preserved, whereas synchronous signal transmission between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) or along the auditory nerve fibers is impaired [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Clinically, patients typically show preserved otoacoustic emissions (OAEs) and cochlear microphonics, whereas auditory brainstem responses (ABRs) are absent or markedly abnormal, often accompanied by elevated ABR thresholds and disproportionately poor speech discrimination relative to the behavioral hearing thresholds.\u003c/p\u003e \u003cp\u003eIn contrast, CS is characterized by the selective degeneration of ribbon synapses connecting IHCs and SGNs. Animal studies have shown that this pathology can occur early after noise exposure or with aging [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In this condition, despite preserved hair cells (HCs) and relatively maintained auditory sensitivity at low stimulus levels, the amplitude of ABR wave I is reduced, reflecting a decrease in the number of synchronously firing auditory nerve fibers (ANFs). This dissociation between auditory sensitivity and neural output has been proposed as the basis of so-called \u0026ldquo;hidden hearing loss.\u0026rdquo; Low-spontaneous-rate auditory nerve fibers are particularly vulnerable, potentially contributing to impaired speech perception and reduced hearing ability in noisy environments [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Previous basic studies have implicated several mechanisms underlying cochlear synaptic damage, including glutamate excitotoxicity driven by excessive glutamate release and overactivation of AMPA receptors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], as well as oxidative stress associated with mitochondrial dysfunction [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, irreversible loss of IHC\u0026ndash;SGN synapses is thought to precede the degeneration of auditory nerve fibers and SGN cell bodies, which progresses in a distal-to-proximal \u0026ldquo;dying-back\u0026rdquo; pattern [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As synaptic injury becomes more extensive, functional deficits may extend beyond reduced neural output to include elevated ABR thresholds, reflecting progression toward a more overt AN\u0026ndash;like phenotype. Nevertheless, several important questions remain unresolved, including the extent to which synaptic injury leads to irreversible SGN loss and the potential for synaptic regeneration or plastic recovery following such damage.\u003c/p\u003e \u003cp\u003eThus, establishing a reproducible and stable animal model is critical for elucidating the pathophysiology of the disease and promoting the development of novel therapeutic approaches. Traditionally, the local application of ouabain (OB), a Na⁺/K⁺-ATPase inhibitor, onto the round window membrane (RWM) has been widely used as a selective auditory nerve injury model [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. OB preferentially affects type I SGNs, which express the α3 subunit of Na⁺/K⁺-ATPase, inducing a reduction in ABR wave I amplitude and synaptic loss while preserving the OHC function.\u003c/p\u003e \u003cp\u003eHowever, RWM application requires opening the middle ear bulla, which carries risks, including conductive hearing loss secondary to otitis media, interindividual variability in drug absorption, and fatality due to drug leakage into the middle or inner ear. In contrast, posterior semicircular canal (PSC) administration has been reported to facilitate a more efficient distribution of agents throughout the cochlea via hydrodynamic flow compared with RWM application [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Indeed, viral vector studies have demonstrated that PSC injection enables widespread and efficient transduction across the cochlea [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, PSC administration does not require opening the middle ear cavity, thereby minimizing the risk of conductive hearing loss or inflammation associated with middle-ear manipulation. Moreover, this approach allows precise control of the volume and concentration of agents delivered to the inner ear, ensuring more consistent and reproducible inner ear drug exposure across animals.\u003c/p\u003e \u003cp\u003eBased on these findings, we investigated whether local administration of OB via the PSC could serve as a less invasive and more stable approach to establish a selective cochlear nerve injury model in mice.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and study approval\u003c/h2\u003e \u003cp\u003eSeven-week-old male CBA/J mice (Oriental Yeast Co., Ltd., Tokyo, Japan) were used in this study. CBA/J mice have been validated as a model for OB-induced hearing loss in previous studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. All animal procedures were performed in accordance with the institutional guidelines of the Animal Experimentation and Ethics Committee of Kitasato University School of Medicine and were approved by the committee (Approval No. M2025-121).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOtotoxic drug administration and surgical procedure\u003c/h3\u003e\n\u003cp\u003eMice were anesthetized via intraperitoneal injection of medetomidine (0.75 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg). Under a stereomicroscope (Leica S9E; Leica Microsystems, Tokyo, Japan), a postauricular skin incision was made on the left side, and the overlying muscles were separated to expose the PSC. A small fenestration was created in the canal wall using a 32-gauge needle. After confirming perilymph leakage, fluid efflux was allowed to subside for approximately 5 min. A fine glass micropipette connected to a microsyringe pump was gently inserted into the fenestration opening. The insertion site was sealed with a small amount of tissue adhesive to prevent fluid leakage. Sterile saline (control), 1 mM ouabain (RSD, 1076/100), or 5 mM ouabain (dissolved in saline) was injected into the PSC at a rate of 1 \u0026micro;L/min for a total volume of 1 \u0026micro;L (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Following the injection, the micropipette was left in place for 5 min to facilitate diffusion. Thereafter, the micropipette was removed, and the fenestration site was sealed with small muscle fragments and a tissue adhesive. Finally, the skin incision was closed using nylon sutures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAuditory brainstem response (ABR)\u003c/h3\u003e\n\u003cp\u003eABR thresholds were evaluated prior to OB administration and 7 and 56 days post-treatment. The mice were anesthetized as described above and placed on a warming pad in a sound-attenuating chamber. Subcutaneous needle electrodes were positioned at the nose (reference), mastoid region (recording), and tail (ground) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Tone burst stimuli at 4, 8, 16, and 32 kHz were delivered in 5-dB steps. Responses were averaged over 512 sweeps using a Neuropack Sigma system (Nihon Kohden, Tokyo, Japan). Thresholds were defined as the lowest stimulus levels that produced a reproducible waveform above the noise floor.\u003c/p\u003e\n\u003ch3\u003eCochlear immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eTo examine cochlear expression of Na⁺/K⁺-ATPase, immunofluorescence staining was performed on paraffin-embedded cochlear sections. The mice were transcardially perfused with 4% paraformaldehyde (PFA) in phosphate buffer (PB), and the cochleae were removed and post-fixed overnight at 4\u0026deg;C. The specimens were decalcified in 5% EDTA for 1 week, embedded in paraffin, and sectioned to a thickness of 4 \u0026micro;m. After deparaffinization, rehydration, and antigen retrieval, the sections were incubated with a primary antibody against the α3 subunit of sodium\u0026ndash;potassium ATPase (alpha 3 Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e-ATPase/ATP1A3, clone H-4; sc-365744, Santa Cruz Biotechnology). Phalloidin-iFluor 488 Reagent (Abcam) was used to label the filamentous actin. Appropriate fluorescent secondary antibodies were applied, and the sections were mounted with an antifade mounting medium for imaging.\u003c/p\u003e \u003cp\u003eFor synaptic evaluation, separate cochleae were processed for whole-mount immunostaining of the organ of Corti. The mice were transcardially perfused with 4% PFA in PB, and the cochleae were harvested and post-fixed in 4% PFA in PB for 2 h. After decalcification in 5% EDTA overnight, the cochleae were dissected by removing the lateral wall and tectorial membrane. Whole-mount preparations were incubated overnight at 4\u0026deg;C with the following primary antibodies: anti-Myosin7A (rabbit IgG; Invitrogen, Grand Island, NY), anti-GluR2 (mouse IgG2a; Millipore, Billerica, MA), and anti-CtBP2 (mouse IgG1; BD Transduction Laboratories, San Jose, CA). Secondary antibodies (Alexa Fluor, 1:200 in blocking buffer; Molecular Probes, Eugene, OR, USA) were applied for 2 h at room temperature.\u003c/p\u003e\n\u003ch3\u003eAssessment of hair cells and synapses\u003c/h3\u003e\n\u003cp\u003eSeven days after OB administration, HC and synapse survival were assessed in the cochleae of the experimental mice (saline group, n\u0026thinsp;=\u0026thinsp;4; 1 mM OB group, n\u0026thinsp;=\u0026thinsp;4; 5 mM OB group, n\u0026thinsp;=\u0026thinsp;3). The specimens were mounted on glass slides and imaged using a confocal laser-scanning microscope (LSM710; Zeiss, Jena, Germany). The total cochlear length was measured, and a cochlear frequency map was generated to localize the 4-, 8-, 16-, and 32-kHz regions. IHC and OHC survival was quantified and expressed as percentages. For synaptic evaluation, confocal z-stack images were acquired from the IHC region at the same frequency locations using a 63\u0026times; oil-immersion objective (3\u0026times; digital zoom; 0.25-\u0026micro;m z-steps). CtBP2/GluR2 puncta were counted to determine the number of paired synaptic ribbons per IHC, and unpaired CtBP2-positive puncta without corresponding GluR2 immunoreactivity were quantified as orphan ribbons.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of SGN survival\u003c/h2\u003e \u003cp\u003eAt 7 and 56 days post-administration, SGN survival was evaluated (normal group, n\u0026thinsp;=\u0026thinsp;4; saline group, n\u0026thinsp;=\u0026thinsp;4; 1 mM OB group, n\u0026thinsp;=\u0026thinsp;4; and 5 mM OB group, n\u0026thinsp;=\u0026thinsp;3). Following transcardial perfusion with 4% PFA, the cochleae were removed and post-fixed overnight. The specimens were decalcified in 5% EDTA for 1 week, embedded in paraffin, and sectioned at 4 \u0026micro;m. The sections were stained with hematoxylin and eosin (HE). The regions containing the largest SGN areas were analyzed. The spiral ganglion was divided into apical and basal turns, and the SGN density was calculated as the number of neurons per unit area.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAssessment of cochlear nerve fibers in the osseous spiral lamina\u003c/h3\u003e\n\u003cp\u003eTo quantify peripheral cochlear nerve integrity, the density of auditory nerve fibers within the osseous spiral lamina (OSL) was assessed as previously described, with minor modifications [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Cochlear nerve fiber density was assessed using the same paraffin-embedded cochlear sections prepared for the SGN analysis. HE-stained mid-modiolar sections were used for the quantitative analysis of cochlear nerve fibers within the OSL. Analyses were restricted to the middle turn of the cochlea to ensure consistency among the specimens. Sections containing well-defined openings of the habenula perforata were selected, and all clearly identifiable cochlear nerve fibers within the OSL were counted manually under high magnification. The cochlear nerve fiber density was calculated as the number of nerve fibers per unit area (fibers per 100 \u0026micro;m\u0026sup2;).\u003c/p\u003e\n\u003ch3\u003eAssessment of stria vascularis area\u003c/h3\u003e\n\u003cp\u003eTo determine whether OB administration induces structural alterations in the stria vascularis (SV), which could potentially contribute to auditory dysfunction independently of neural injury, the SV morphology was quantitatively evaluated. This analysis was performed using the same paraffin-embedded mid-modiolar cochlear sections prepared for SGN analysis, as vascular atrophy and metabolic dysfunction of the SV have been implicated in certain forms of sensorineural hearing loss [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A quantitative assessment of the SV area was conducted in the apical and basal turns of the cochlea. In selected mid-modiolar sections, the SV was identified along the lateral wall based on its characteristic trilaminar epithelial structure.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Normality was assessed using the Shapiro\u0026ndash;Wilk test. Normally distributed data were evaluated using two-way ANOVA, followed by Tukey\u0026rsquo;s post hoc test; non-normally distributed data were analyzed using the Kruskal\u0026ndash;Wallis test, followed by Dunn\u0026rsquo;s post hoc test. Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software Inc., La Jolla, CA, USA). Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExpression of Na⁺/K⁺-ATPase in the cochlea\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining revealed prominent expression of the Na⁺/K⁺-ATPase α3 subunit within the cochlea. At low magnification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), strong Na⁺/K⁺-ATPase immunoreactivity was observed in SGN somata within the modiolus and along afferent nerve fiber bundles extending toward the organ of Corti. Higher-magnification imaging of the organ of Corti region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) demonstrated Na⁺/K⁺-ATPase α3 expression in afferent nerve fibers traversing the osseous spiral lamina and afferent terminals located beneath the IHCs. The immunoreactive signal was distributed in a punctate and linear pattern in the vicinity of the IHC basal pole, consistent with localization near the IHC\u0026ndash;SGN synaptic region. In contrast, no appreciable Na⁺/K⁺-ATPase immunoreactivity was detected in the IHCs, OHCs, or surrounding supporting cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of ABR thresholds\u003c/h2\u003e \u003cp\u003eBaseline ABR measurements obtained at 8 weeks of age, immediately prior to OB administration, revealed no significant differences in hearing thresholds among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, n\u0026thinsp;=\u0026thinsp;17). At 7 days after administration, the 1mM OB (n\u0026thinsp;=\u0026thinsp;4 ears) and 5 mM OB groups (n\u0026thinsp;=\u0026thinsp;3 ears) demonstrated significantly elevated ABR thresholds compared with the saline group (n\u0026thinsp;=\u0026thinsp;4 ears) (two-way ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To determine whether the PSC injection procedure alone affected auditory function, we compared the ABR thresholds between saline-injected mice and untreated controls (normal group). No significant differences were found (two-way ANOVA, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating that PSC injection did not adversely affect hearing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt 56 days after 1 mM OB administration (n\u0026thinsp;=\u0026thinsp;4 ears), ABR thresholds were significantly elevated compared to those in the saline group (n\u0026thinsp;=\u0026thinsp;4 ears) (two-way ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In contrast, no significant differences were detected between the normal (n\u0026thinsp;=\u0026thinsp;4 ears) and saline groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Peak wave-I amplitudes at 80 dB SPL were also evaluated at 7 and 56 days after OB treatment. At 7 days, both the 1 mM and 5 mM OB groups showed reduced wave I amplitudes compared to the normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). At 56 days, wave-I amplitudes remained significantly reduced in the 1 mM OB group compared to the control group (two-way ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Because the ABR wave-I amplitude reflects cochlear nerve activity, these reductions suggest OB-induced cochlear nerve degeneration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of HC survival\u003c/h2\u003e \u003cp\u003ePrevious reports have shown that OB administration via the RWM induces AN without HC loss [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consistent with these findings, no loss of IHCs or OHCs was observed 7 days after PSC administration of 1 mM or 5 mM OB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of SGN survival\u003c/h2\u003e \u003cp\u003ePrior studies reported that SGN degeneration began on day 7 and continued through day 30 following OB application to the RWM in adult mice [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Based on these insights, we examined SGN survival at both early (7 days) and late (56 days) time points after PSC OB administration. At 7 days after treatment, HE staining revealed a significant loss of SGNs in the apical and basal turns of the cochlea in the 5 mM OB group compared to the saline group (two-way ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001;Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), whereas SGN loss in the 1 mM OB group was minimal and did not reach statistical significance at this time point. In contrast, at 56 days after 1 mM OB administration, a marked and statistically significant reduction in SGN density was observed in both the apical and basal turns compared to the saline group (two-way ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001;Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). No significant SGN loss was detected in the saline group at any time point.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of synapses\u003c/h2\u003e \u003cp\u003eSeven days after OB administration, the ABR wave-I amplitudes were significantly reduced, whereas the SGN loss was minimal. Therefore, we evaluated cochlear synapses by quantifying the number of CtBP2/GluR2 puncta per IHC [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Previous studies on CS have demonstrated that synaptic injury is frequently accompanied not only by a reduction in paired pre- and postsynaptic elements but also by an increase in the so-called orphan ribbons, defined as presynaptic ribbons lacking apposed postsynaptic glutamate receptor labeling, reflecting synaptic uncoupling rather than complete synapse elimination [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Compared with the saline group, the 1 mM OB and 5 mM OB groups showed a significant reduction in synapses across all frequency regions examined (two-way ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In contrast, the number of orphan synapses was significantly increased in the 1 mM OB and 5 mM OB groups (two-way ANOVA, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of cochlear nerve fibers in the osseous spiral lamina\u003c/h2\u003e \u003cp\u003eIn the 1 mM OB group, no significant loss of SGN somata was observed in either the apical or basal turns compared with saline-treated controls 7 days after administration, indicating preserved SGN cell bodies at this early time point. In contrast, analysis of peripheral auditory nerve integrity revealed a significant reduction in cochlear nerve fiber density within the OSL of the middle turn in the 1 mM OB group 7 days after administration (two-way ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Together, these findings indicate that peripheral cochlear nerve fibers are selectively affected at an early stage following low-dose OB administration, preceding the detectable degeneration of SGN cell bodies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of stria vascularis area\u003c/h2\u003e \u003cp\u003eTo determine whether ouabain administration affected SV morphology, the cross-sectional area of the SV was quantified in the apical and basal turns using HE-stained sections. Quantitative analysis revealed no significant differences in the stria vascularis area between the control and ouabain-treated groups in either the apical or basal turn (two-way ANOVA, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings indicate that PSC-delivered ouabain did not induce detectable structural changes in the stria vascularis under the examined conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAN remains an incompletely characterized disorder in which synchronous neural signaling is disrupted despite the preservation of OHC function[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The intracochlear delivery of OB, a potent Na⁺/K⁺-ATPase inhibitor, has been widely used over the past decade to model AN in rodents. In particular, the RWM application of OB selectively injures type I SGNs while sparing HCs, thereby reproducing a hallmark pathological feature of AN [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, OB exposure via the RWM requires opening the middle ear bulla and is technically challenging. This approach is associated with variability in drug delivery and the risk of postoperative complications, including conductive hearing loss. To overcome these limitations, the present study evaluated whether OB administration through PSC could reproducibly induce AN-like pathology. We demonstrated that PSC delivery of OB produced selective SGN injury while preserving HC morphology, thereby establishing a reliable SGN-specific lesion model. Functionally, this model was characterized not only by a reduction in ABR wave I amplitude but also by a significant elevation in ABR thresholds, indicating impaired neural synchrony and reduced effective neural recruitment. Short-term evaluation (7 days) following 1 mM OB administration revealed significant loss of ribbon synapses despite minimal loss of SGN soma, consistent with CS, in which IHC\u0026ndash;SGN synapses degenerate prior to neuronal loss [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Importantly, this early synaptic pathology was accompanied by a significant increase in orphan ribbons, defined as presynaptic ribbons lacking apposed postsynaptic glutamate receptor puncta, indicating synaptic uncoupling rather than ribbon elimination. Thus, this PSC-based OB model not only recapitulates the SGN pathology characteristic of AN but also captures early synaptic dysfunction associated with progression from CS toward an AN-like phenotype rather than pure \u0026ldquo;hidden hearing loss.\u0026rdquo;\u003c/p\u003e \u003cp\u003eOB-induced neuropathy is thought to arise from the selective inhibition of the Na⁺/K⁺-ATPase α3 subunit, which is highly expressed in type I SGNs but sparse in IHCs and OHCs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Immunohistochemical analyses have demonstrated that α3 protein is distributed not only in SGN somata but also along afferent fibers adjacent to the IHC\u0026ndash;SGN synapse [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], suggesting that OB may directly perturb neuronal excitability and axonal homeostasis in the cochlea. In the present study, a significant loss of ribbon synapses was observed 7 days after exposure to 1 mM OB, whereas degeneration of SGN cell bodies was minimal at this time point. In contrast, a marked loss of SGN cell bodies became evident after 56 days.\u003c/p\u003e \u003cp\u003eThis temporal pattern supports a stepwise degenerative process in which synaptic terminals constitute the most vulnerable compartments. The presence of orphan ribbons suggests the destabilization and functional disconnection of IHC\u0026ndash;SGN synapses, which likely contributes to desynchronized auditory nerve firing and elevation of ABR thresholds despite preserved HC morphology. Comparable findings have been reported in noise-induced CS, in which synaptic injury is the earliest event, followed by delayed axonal retraction and SGN cell body loss in a distal-to-proximal \u0026ldquo;dying-back\u0026rdquo; pattern [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral mechanisms may contribute to early synaptic vulnerability. Inhibition of Na⁺/K⁺-ATPase disrupts ionic homeostasis and reduces the driving force for glutamate transport. High-affinity glutamate transporters, including GLAST and GLT-1, depend on Na\u0026thinsp;+\u0026thinsp;gradients to clear glutamate from the synaptic cleft [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Within the cochlea, GLAST is localized to the supporting cells adjacent to the IHC\u0026ndash;SGN synapse and plays a major role in glutamate clearance [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Pharmacological inhibition of GLAST markedly delays the recovery of cochlear afferent responses after noise exposure, demonstrating that impaired glutamate uptake contributes to synaptic excitotoxicity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Although these downstream pathways have not been fully characterized in OB-treated cochleae, similar mechanisms are likely to be involved. Synaptic uncoupling and orphan ribbon formation may represent an intermediate pathological state preceding the complete elimination of synapses. Following synaptic elimination, SGNs may lose trophic support from IHCs, promoting axonal retraction and ultimately, neuronal death. This sequence aligns with the delayed SGN loss observed at 56 days in our study and with prior reports demonstrating progressive degeneration following synaptic disconnection.\u003c/p\u003e \u003cp\u003ePSC delivery offers several advantages. First, PSC injection yielded a more consistent and widespread intracochlear distribution than RWM application. Dye-tracking studies have shown that PSC delivery produces stronger and more uniform labeling across both apical and basal turns, likely due to direct access to the perilymphatic space and a reduced dependence on RWM permeability [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, PSC injection of AAV vectors enables efficient and near pan-cochlear transduction in adult mice [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Importantly, unlike RWM-based approaches, PSC delivery does not require opening the middle ear bulla, thereby avoiding surgical manipulation of the middle ear space and reducing the risk of conductive hearing loss associated with otitis media or bulla inflammation. Second, RWM-based drug delivery is influenced by variability in RWM thickness, mucosal coverage, and middle ear conditions, which can lead to inconsistent intracochlear drug concentrations [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. By directly accessing the perilymphatic lumen, PSC administration minimizes this variability and improves reproducibility. Third, recent refinements in PSC injection techniques have demonstrated that inner ear delivery can be achieved with minimal postoperative auditory dysfunction when the injection volume and perilymph leakage are controlled carefully [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Collectively, these findings highlight PSC delivery as a practical and reproducible alternative to RWM for selective cochlear manipulation.\u003c/p\u003e \u003cp\u003eAlthough this study demonstrated that PSC-delivered OB can reproduce SGN degeneration while preserving HCs, several limitations should be recognized. The intracochlear pharmacokinetics of OB remain unclear; therefore, the relative contributions of drug concentration gradients versus intrinsic cellular susceptibility to the observed patterns of synaptic and neuronal loss remain uncertain. Furthermore, although the sequence of early ribbon synapse loss with orphan ribbon formation, followed by delayed SGN degeneration, is consistent with the progression from CS toward AN, this study cannot determine whether synaptic disconnection alone is sufficient to initiate subsequent neuronal death or whether the OB exerts direct cytotoxic effects on SGN soma via Na⁺/K⁺-ATPase α3 inhibition [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, the downstream molecular pathways leading to SGN loss, including oxidative stress, mitochondrial dysfunction, and inflammatory responses, remain poorly defined and were not examined in this study. Further studies are required to determine how early synaptic injury progresses to irreversible neuronal degeneration.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we demonstrated that local administration of OB via PSC effectively creates a selective cochlear neuropathy model in mice. Early synaptic pathology is characterized by ribbon synapse loss and a significant increase in orphan ribbons, accompanied by reduced ABR wave I amplitude and elevated ABR thresholds. Substantial SGN degeneration appeared only at later time points, suggesting a stepwise degenerative process consistent with CS progression toward AN. Importantly, this approach preserved HC morphology and did not induce hearing changes when saline was administered, indicating that PSC injection is selective and minimally invasive. Compared with traditional RWM delivery, PSC administration offers an improved intralabyrinthine distribution and reduced technical variability. This model will be valuable for elucidating the mechanisms underlying SGN degeneration and evaluating therapeutic strategies aimed at synaptic protection and neuronal preservation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eClinical Trial Number\u003c/h3\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edeclaration\u003c/strong\u003e: not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was supported by the following grants: JSPS KAKENHI grant (Grant Number 25K20171).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eY.N. and T.K. designed the experiments. Y.N., S.M. and L.K.performed the experiments. Y.N. analyzed the data. Y.N. and T.K. co-wrote the manuscript. T.Y. reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank Dr. Teppei Noda, Dr. Takahiro Wakizono, and Dr. Takahiro Manabe from the Department of Otorhinolaryngology\u0026ndash;Head and Neck Surgery, Kyushu University, and Dr. Koji Nishimura from the Department of Otorhinolaryngology\u0026ndash;Head and Neck Surgery, Kyoto University, for their valuable advice and support in establishing the mouse model of hearing loss used in this study.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStarr A et al. \u003cem\u003eAuditory neuropathy.\u003c/em\u003e Brain, 1996. 119 (Pt 3): pp. 741\u0026thinsp;\u0026ndash;\u0026thinsp;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRance G, Starr A. Pathophysiological mechanisms and functional hearing consequences of auditory neuropathy. Brain. 2015;138(Pt 11):3141\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKujawa SG, Liberman MC. Adding insult to injury: cochlear nerve degeneration after temporary noise-induced hearing loss. J Neurosci. 2009;29(45):14077\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiberman MC, Kujawa SG. Cochlear synaptopathy in acquired sensorineural hearing loss: Manifestations and mechanisms. Hear Res. 2017;349:138\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuel JL, et al. Excitotoxicity and repair of cochlear synapses after noise-trauma induced hearing loss. NeuroReport. 1998;9(9):2109\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHenderson D, et al. The role of oxidative stress in noise-induced hearing loss. Ear Hear. 2006;27(1):1\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan Y, et al. Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy. J Assoc Res Otolaryngol. 2014;15(1):31\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalaei S, et al. Dye Tracking Following Posterior Semicircular Canal or Round Window Membrane Injections Suggests a Role for the Cochlea Aqueduct in Modulating Distribution. Front Cell Neurosci. 2019;13:471.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki J, et al. Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction. Sci Rep. 2017;7:45524.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang ZJ, et al. Dose-dependent effects of ouabain on spiral ganglion neurons and Schwann cells in mouse cochlea. Acta Otolaryngol. 2017;137(10):1017\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaganuma H, et al. Effects of arginine vasopressin on auditory brainstem response and cochlear morphology in rats. Auris Nasus Larynx. 2014;41(3):249\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNitta Y, et al. Suppression of the TGF-beta signaling exacerbates degeneration of auditory neurons in kanamycin-induced ototoxicity in mice. Sci Rep. 2024;14(1):10910.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurioka T, et al. Selective hair cell ablation and noise exposure lead to different patterns of changes in the cochlea and the cochlear nucleus. Neuroscience. 2016;332:242\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEggink MC, et al. Human vestibular schwannoma reduces density of auditory nerve fibers in the osseous spiral lamina. Hear Res. 2022;418:108458.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGratton MA, Schulte BA, Smythe NM. Quantification of the stria vascularis and strial capillary areas in quiet-reared young and aged gerbils. Hear Res. 1997;114(1\u0026ndash;2):1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaur C, et al. Predicting Atrophy of the Cochlear Stria Vascularis from the Shape of the Threshold Audiogram. J Neurosci. 2023;43(50):8801\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, et al. Engineering olivocochlear inhibition to reduce acoustic trauma. Mol Ther Methods Clin Dev. 2023;29:17\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiberman MC, et al. Toward a Differential Diagnosis of Hidden Hearing Loss in Humans. PLoS ONE. 2016;11(9):e0162726.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLang H, Schulte BA, Schmiedt RA. Ouabain induces apoptotic cell death in type I spiral ganglion neurons, but not type II neurons. J Assoc Res Otolaryngol. 2005;6(1):63\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcLean WJ, et al. Distribution of the Na,K-ATPase alpha subunit in the rat spiral ganglion and organ of corti. J Assoc Res Otolaryngol. 2009;10(1):37\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRose EM, et al. Glutamate transporter coupling to Na,K-ATPase. J Neurosci. 2009;29(25):8143\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlowatzki E, et al. The glutamate-aspartate transporter GLAST mediates glutamate uptake at inner hair cell afferent synapses in the mammalian cochlea. J Neurosci. 2006;26(29):7659\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Z, Kujawa SG, Sewell WF. Functional roles of high-affinity glutamate transporters in cochlear afferent synaptic transmission in the mouse. J Neurophysiol. 2010;103(5):2581\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki J, et al. Corrigendum: Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction. Sci Rep. 2017;7:46827.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorkholder DA, Zhu X, Frisina RD. Round window membrane intracochlear drug delivery enhanced by induced advection. J Control Release. 2014;174:171\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu J, et al. Refining surgical techniques for efficient posterior semicircular canal gene delivery in the adult mammalian inner ear with minimal hearing loss. Sci Rep. 2021;11(1):18856.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ouabain, Posterior semicircular canal, Cochlear neuropathy, Spiral ganglion neuron loss, Ribbon synapse","lastPublishedDoi":"10.21203/rs.3.rs-8897404/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8897404/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAuditory neuropathy (AN) and cochlear synaptopathy (CS) are characterized by impaired neural transmission despite preserved hair cell (HC) function. Ouabain (OB), a Na⁺/K⁺-ATPase inhibitor, has been used as a selective auditory nerve injury model via round window membrane delivery. However, this approach is technically variable and invasive.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo establish a minimally invasive and stable cochlear nerve injury model by delivering OB through the posterior semicircular canal (PSC) and characterizing short- and long-term pathological consequences.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSeven-week-old CBA/J mice received PSC injections of saline, 1 mM OB, or 5 mM OB. Auditory brainstem responses (ABRs) were evaluated before treatment and at 7 and 56 days post-treatment. Cochlear pathology was assessed through immunohistochemical and histological analyses of HCs, ribbon synapses, spiral ganglion neurons (SGNs), cochlear nerve fiber density, and stria vascularis morphology.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePSC-delivered OB elevated ABR thresholds and reduced wave I amplitudes without HC loss. At 7 days, significant SGN loss occurred only in the 5 mM OB group. Both OB doses induced ribbon synapse loss and increased orphan ribbons. Cochlear nerve fiber density was significantly reduced as early as 7 days after 1 mM OB administration, despite preserved SGN cell bodies. Stria vascularis morphology remained unchanged.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003ePSC-delivered OB induces synaptic uncoupling and peripheral cochlear nerve fiber loss, preceding SGN degeneration. This minimally invasive model recapitulates key pathological features of CS progressing toward AN and provides a platform for studying cochlear neuropathy and therapeutic interventions.\u003c/p\u003e","manuscriptTitle":"Posterior Semicircular Canal Ouabain Injection Induces Early Synaptic Loss and Delayed Cochlear Nerve Degeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 17:19:46","doi":"10.21203/rs.3.rs-8897404/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"611e9da6-cff0-4350-837c-c6962490ec43","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T08:42:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 17:19:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8897404","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8897404","identity":"rs-8897404","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}