Aminoglycoside antibiotics are critical in clinical use for treating severe infections, but they can occasionally cause irreversible sensorineural hearing loss. To establish a rational pathway for otoprotectant discovery, we provide an integrated, three-tier methodology—comprising cell-model selection, transcriptomic analysis, and a gentamicin–Texas Red (GTTR) uptake assay—to guide the development of otoprotective strategies. We first utilized two murine auditory cell lines—UB/OC-2 and HEI-OC1. We focused on TMC1 and OCT2 and further explored the underlying mechanisms of ototoxicity. UB/OC-2 exhibited a higher sensitivity to gentamicin, which correlated with elevated OCT2 expression confirmed via RT-PCR and Western blot. Transcriptomic analysis revealed upregulation of PI3K-Akt, calcium, and GPCR-related stress pathways in gentamicin-treated HEI-OC1 cells. Protein-level analysis further confirmed that gentamicin suppressed phosphorylated Akt while upregulating ER stress markers (GRP78, CHOP) and apoptotic proteins (cleaved caspase 3, PARP). Co-treatment with PI3K inhibitors (LY294002, wortmannin) further suppressed Akt phosphorylation, supporting the role of PI3K-Akt signaling in auditory cells. To visualize drug entry, we used GTTR to evaluate its applicability as a fluorescence-based uptake assay in these cell lines, which were previously employed mainly in cochlear explants. Sodium thiosulfate (STS) and N-acetylcysteine (NAC) significantly decreased GTTR uptake, suggesting a protective effect against gentamicin-induced hair cell damage. In conclusion, our findings showed a complex ototoxic cascade involving OCT2- and TMC1-mediated drug uptake, calcium imbalance, ER stress, and disruption of PI3K-Akt survival signaling. We believe that UB/OC-2 cells serve as a practical in vitro model for mechanistic investigations and screening of otoprotective compounds. Additionally, GTTR may be a simple, effective method for evaluating protective interventions in auditory cell lines. Overall, this study provides molecular-level insights into aminoglycoside-induced ototoxicity and introduces a platform for protective strategies.

Background/Objectives: Temporary artery ligation or compression is commonly used to reduce intraoperative blood loss in various surgeries, including uterine procedures. In head and neck surgery, the external carotid artery (ECA) typically branches into eight vessels, supplying most of the head and neck except for the brain. Severe and uncontrolled bleeding can occur if these branches are inadvertently damaged during surgery. However, limited research exists on temporary arterial ligation during head and neck surgeries. This study aimed to evaluate the effects of temporary ECA restriction and internal maxillary artery (IMA) ligation on minimizing intraoperative blood loss during head and neck surgery. Methods: This study involved 25 patients with terminal-stage maxillary tumors who underwent total maxillectomy. The effectiveness of IMA ligation and ECA restriction using a Rummel tourniquet in controlling intraoperative bleeding was compared. Results: The average blood loss was significantly lower in the ECA restriction (467 mL) and IMA ligation (461 mL) groups than in the control group (794 mL). However, no significant difference was observed between the IMA ligation and ECA restriction methods. Conclusions: Overall, our results suggest that either method is effective; however, ECA restriction is preferred for tumors involving the infratemporal fossa.