Abstract <p>Aminoglycoside antibiotics remain crucial for the treatment of severe infections caused by multidrug-resistant bacteria; however, their clinical use is limited by the risk of irreversible hearing loss. Previous studies have primarily attributed aminoglycoside-induced hearing loss to cochlear hair cell loss and subsequent spiral ganglion degeneration, while the contribution of the stria vascularis remains unclear. In the present study, we demonstrate that gentamicin administration induces apoptosis of stria vascularis pericytes, leading to stria vascularis dysfunction and consequent hearing loss in mice. Mechanistically, gentamicin exposure activated macrophages within the stria vascularis and significantly increased local expression of tumor necrosis factor-α (TNF-α). In vitro, we found that TNF-α triggered stria vascularis pericyte apoptosis via activation of the NF-κB signaling pathway. Importantly, in vivo pharmacological blockade of TNF-α with infliximab (IFX) or inhibition of NF-κB signaling with JSH-23 effectively preserved pericyte survival, attenuated stria vascularis damage, and mitigated gentamicin-induced hearing loss in mice.</p> <p>Collectively, these findings uncover TNF-α/NF-κB-dependent vascular-inflammatory mechanism underlying gentamicin ototoxicity and identify stria vascularis pericytes as a novel and druggable therapeutic target for the prevention of gentamicin-induced hearing loss.</p> Graphical abstract <p></p>