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

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.