Simple SummaryVestibular schwannomas are benign tumors of the eighth cranial nerve, caused by mutations in theNF2gene encoding the tumor suppressor, merlin. A standard treatment for vestibular schwannoma is radiation therapy; however, radiation can cause sudden or progressive hearing loss in patients over time. Most commonly, vestibular schwannomas are treated with stereotactic radiation in one or multiple fractions. In this study, we investigated the effect of different radiation fractionation regimens on hearing in rats, loss of auditory hair cells, and control of tumor growth. Our work suggests that hypofractionation may improve the therapeutic ratio by increasing the likelihood of hearing preservation whilst equally reducing proliferation of tumor cells in comparison with single fraction. Further investigations are needed to understand radiation mechanisms and identify new therapeutic options to reduce hearing loss and enhance tumor control in vestibular schwannoma.AbstractBackground: Vestibular schwannomas (VS) are benign intracranial tumors caused by loss of function of the merlin tumor suppressor. We tested three hypotheses related to radiation, hearing loss (HL), and VS cell survival: (1) radiation causes HL by injuring auditory hair cells (AHC), (2) fractionation reduces radiation-induced HL, and (3) single fraction and equivalent appropriately dosed multi-fractions are equally effective at controlling VS growth. We investigated the effects of single fraction and hypofractionated radiation on hearing thresholds in rats, cell death pathways in rat cochleae, and viability of human merlin-deficient Schwann cells (MD-SC). Methods: Adult rats received cochlear irradiation with single fraction (0 to 18 Gray [Gy]) or hypofractionated radiation. Auditory brainstem response (ABR) testing was performed for 24 weeks. AHC viabilities were determined using immunohistochemistry. Neonatal rat cochleae were harvested after irradiation, and gene- and cell-based assays were conducted. MD-SCs were irradiated, and viability assays and immunofluorescence for DNA damage and cell cycle markers were performed. Results: Radiation caused dose-dependent and progressive HL in rats and AHC losses by promoting expression of apoptosis-associated genes and proteins. When compared to 12 Gy single fraction, hypofractionation caused smaller ABR threshold and pure tone average shifts and was more effective at reducing MD-SC viability. Conclusions: Investigations into the mechanisms of radiation ototoxicity and VS radiobiology will help determine optimal radiation regimens and identify potential therapies to mitigate radiation-induced HL and improve VS tumor control.