FIGURE 3.

Cellular mechanisms and proposed timeline of aminoglycoside ototoxicity in cochlear outer hair cells. The main schematic depicts a stylised outer hair cell with five temporally ordered mechanistic stages, each color-coded and labeled on the left margin with approximate time windows derived from in vitro cochlear explant and in vivo rodent models. (1) Entry (∼0–6 h): aminoglycosides (AGs) enter outer hair cells primarily through mechanoelectrical transduction (MET) channels at stereocilia tips and via a secondary endocytotic route; AG interaction with TRP channels produces altered ion conductance,* disrupting K⁺ and Ca²⁺ flux and impairing mitochondrial ribosomal protein synthesis. (2) Mitochondrial Targeting (∼2–12 h): AGs accumulate in mitochondria and bind the 12S rRNA of the mitochondrial ribosome, causing translational errors and impaired oxidative phosphorylation, with consequent early ROS generation. (3) Oxidative Stress (∼6–24 h): iron–AG complexes catalyze Fenton-type free radical production; NADPH oxidase activation amplifies the oxidative burden; lipid peroxidation produces membrane damage and nuclear DNA fragmentation. (4) Calcium Dysregulation (∼12–36 h): oxidative injury to the endoplasmic reticulum (ER) impairs Ca²⁺ sequestration, producing pathological cytosolic Ca²⁺ elevation; sustained Ca²⁺ overload triggers cytochrome c release,‡ committing the cell to intrinsic apoptosis. (5) Cell Death (∼24–72 h): hair cell demise proceeds via three converging pathways -- intrinsic apoptosis (casp9/casp3 activation), RIPK1/RIPK3/MLKL-dependent necroptosis, and iron-dependent ferroptosis driven by unrestricted lipid peroxidation. The inset (upper right) illustrates the MET channel entry mechanism at the stereocilium tip. Panel 1 (left) shows the resting/closed state: the tip link is intact, the MET channel is closed, and no cation flux or AG entry occurs. Panel 3 (right) shows the open/depolarized state: stereociliary bundle deflection tensions the tip link, mechanically gating the MET channel open; K⁺, Ca²⁺, and AGs enter through the open pore driven by the endocochlear potential, constituting the principal Stage 1 entry mechanism; the resulting Ca²⁺ influx also seeds the dysregulation cascade of Stage 4. Panel 2 of the original MET transduction diagram (mechanical displacement) was omitted as it describes the normal auditory stimulus rather than the drug entry mechanism. The dashed connector links the inset to the MET channel zone of the main schematic. The horizontal timeline bar (base of figure) depicts the five colour-matched stage segments across the ∼72-h progression. The solid green bar (∼0–24 h) demarcates the proposed intervention window within which preclinical otoprotective strategies demonstrate maximal efficacy; the pale green zone (∼24–72 h) reflects progressively declining otoprotective efficacy as cell death commitment becomes irreversible. All timeframes are approximate and should not be extrapolated to clinical contexts, in which pharmacokinetic variability, patient age, renal function, aminoglycoside compound, and concurrent ototoxic exposures substantially modify the ototoxicity timeline. All depicted interventions are preclinical and have not been validated in randomised clinical trials.* “Altered Ion Conductance” replaces the previously used term “K⁺ Channel Dysfunction” to reflect mechanistic evidence implicating TRP channels and non-selective cation conductances in addition to K⁺-selective pathways in early AG-mediated electrophysiological disruption. ‡ Cytochrome c release is positioned at the Stage 4→5 boundary to reflect the temporal sequence observed in vitro models, wherein mitochondrial outer membrane permeabilization follows sustained Ca²⁺ dysregulation. Abbreviations: AG, aminoglycoside; Ca²⁺, calcium ion; casp3, caspase-3; casp9, caspase-9; ER, endoplasmic reticulum; Fe–AG, iron–aminoglycoside complex; MET, mechanoelectrical transduction; MLKL, mixed lineage kinase domain-like protein; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); OHC, outer hair cell; RIPK1, receptor-interacting protein kinase 1; RIPK3, receptor-interacting protein kinase 3; ROS, reactive oxygen species; TRP, transient receptor potential. Created in BioRender. Sherwin C. (2026) https://BioRender.com/265z8w8.