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Background: The vestibular schwannoma (VS) secretome can initiate monocyte recruitment and macrophage polarization to M1 (proinflammatory) and/or M2 (protumorigenic) phenotypes, which in turn secrete additional cytokines that contribute to the tumor microenvironment. Profiling cyst fluid and cerebrospinal fluid (CSF) in cystic VS provides a unique opportunity to understand mechanisms that may contribute to tumor progression and cyst formation., Hypothesis: Cystic VSs secrete high levels of cytokines into cyst fluid and express abundant M1 and M2 macrophages., Methods: Tumor, CSF, and cyst fluid were prospectively collected from 10 cystic VS patients. Eighty cytokines were measured in fluid samples using cytokine arrays and compared with normal CSF from normal donors. Immunofluorescence was performed for CD80 + M1 and CD163 + M2 macrophage markers. Demographic, audiometric, and radiographic information was obtained through retrospective chart review., Results: Cyst fluid expressed more osteopontin and monocyte chemotactic protein-1 (MCP-1; p < 0.0001), when compared with normal CSF. Cyst fluid also expressed more protein ( p = 0.0020), particularly MCP-1 ( p < 0.0001), than paired CSF from the same subjects. MCP-1 expression in cyst fluid correlated with CD80 + staining in VS tissue ( r = 0.8852; p = 0.0015) but not CD163 + staining., Conclusion: Cyst fluid from cystic VS harbored high levels of osteopontin and MCP-1, which are cytokines important in monocyte recruitment and macrophage polarization. MCP-1 may have a significant role in molding the tumor microenvironment, by polarizing monocytes to CD80 + M1 macrophages in cystic VS. Further investigations into the role of cytokines and macrophages in VS may lead to new avenues for therapeutic intervention., Competing Interests: The authors disclose no conflicts of interest., (Copyright © 2023, Otology & Neurotology, Inc.)

Hypothesis: AR42, a histone deacetylase (HDAC) inhibitor, reduces viability of primary vestibular schwannoma (VS) cells and delays tumor progression and hearing loss (HL) in a xenograft model of VS., Background: The impact of HDAC expression on AR42 response in primary VS cells is unknown, as well as the effects of AR42 on VS-associated HL and imbalance., Methods: Primary human VS cells (n = 7) were treated with AR42 (0-3.0 μM), and viability assays were conducted. Immunohistochemistry and western blotting for phosphorylated-HDAC2 (pHDAC2) were performed on tumor chunks. Pharmacokinetic studies were conducted in Fischer rats using mass spectrometry. Merlin-deficient Schwann cells were grafted onto cochleovestibular nerves of immunodeficient rats and treated with vehicle (n=7) or AR42 (25 mg/kg/day for 4weeks; n=12). Tumor bioluminescence imaging, auditory brainstem response (ABR), and rotarod tests were conducted to 6weeks. Final tumor weight and toxicities were measured., Results: AR42 caused dose-dependent reductions in viability of VS cells. Tumors with higher pHDAC2:HDAC2 ratios had greater reductions in viability with AR42. On pharmacokinetic studies, AR42 reached peak levels in nerve ~24 hours after oral administration. Although AR42-treated rats demonstrated mean ABR threshold shifts ~10 to 20 dB lower than controls, this did not persist nor reach significance. When compared to controls, AR42 did not affect tumor bioluminescence, tumor weight, and rotarod measurements., Conclusions: Response of primary VS cells to AR42 may be influenced by pHDAC2 expression in tumor. Although AR42 may delay HL in our xenograft model, it did not halt tumor growth or vestibular dysfunction. Further investigations are warranted to evaluate the AR42 effectiveness in NF2-associated VS., Competing Interests: The authors disclose no conflicts of interest., (Copyright © 2022, Otology & Neurotology, Inc.)

Objectives: Determine whether asymmetric hearing loss (AHL) affects postoperative speech outcomes in cochlear implant (CI) patients., Study Design: Retrospective cohort study., Setting: Tertiary care hospital., Patients: Adult English-speaking patients with unilateral CIs implanted between 2014 and 2018 were stratified into NonAHL and AHL groups based on preoperative AzBio scores in quiet from the nonimplanted ear (0-50% vs. 51-100%, respectively)., Interventions: CI surgery in the poorer performing ear., Main Outcome Measures: Postoperative consonant-nucleusconsonant (CNC) word and AzBio sentence test scores in quiet and/or noise at +5 dB signal-to-noise ratio (SNR)., Results: Of 512 patients, 33 non-AHL and 27 AHL patients were included. Average ages were 65.6 and 63.6 years, respectively. As expected, preoperative AzBio scores in quiet from the nonimplanted ear were higher in the AHL group (95% confidence interval [95%CI]: 66.4-76.4%) than the non-AHL group at baseline (95%CI: 12.3-23.6%). In both cohorts, AzBio scores in quiet from the implanted ear improved from baseline, with 24-month scores (95%CI: 73.8 - 84.9%) being higher than preoperative scores (95%CI: 13.2-23.1%). There were also significant differences in AzBio scores in quiet between cohorts overall (p  = 0.0120) on mixed model analysis, with the AHL group performing ∼6.4% better than the non-AHL group; however, differences were not significant when scores were stratified by time. In addition, there were no significant differences in CNC in quiet and AzBio scores in noise at +5 dB SNR between cohorts (p  = 0.1786 and p  = 0.6215, respectively)., Conclusions: After CI, patients with AHL can achieve scores on word and sentence tests at least comparable to traditional CI candidates, supporting the expansion of CI candidacy to include patients with AHL., Competing Interests: Carly Misztal, Stefanie Peña, Diane Martinez, Stefania Goncalves, Ruixuan Ma, and Christine Dinh have no conflicts of interest. Sandra Velandia: Consultant for Cochlear Corporation. Meredith Holcomb: Consultant for Advanced Bionics, Cochlear Corporation, MED-EL, and Institute for Cochlear Implant Training. Simon Angeli: Consultant for Advanced Bionics. Fred Telischi: Consultant for Advanced Bionics, Cochlear Corporation, and MED-EL. The authors disclose no conflicts of interest., (Copyright © 2022, Otology & Neurotology, Inc.)

Hypothesis: Vestibular Schwannoma (VS) can avoid cell death following radiation injury by entering cell cycle arrest and activating RAD51-related DNA repair., Background: Although the radiobiology of various cancers is well-studied, the radiobiological effects in VS are poorly understood. In this study, we describe how VS cells enter cell cycle arrest (through p21 expression), activate DNA repair (through RAD51 upregulation), and avoid cell death after radiation-induced double-stranded breaks (DSB) in DNA (as measured by γ-H2AX)., Methods: Primary human VS cells were cultured on 96-well plates and 16-well culture slides at 10,000 cells/well and exposed to either 0 or 18 Gray of radiation. Viability assays were performed at 96 h in vitro. Immunofluorescence for γ-H2AX, RAD51, and p21 was performed at 6 h., Results: Radiation (18 Gy) induced the expression of γ-H2AX, p21, and RAD51 in six cultured VS, suggesting that irradiated VS acquire DSBs, enter cell cycle arrest, and initiate RAD51 DNA repair to evade cell death. However, viability studies demonstrate variable responses in individual VS cells with 3 of 6 VS showing radiation resistance to 18 Gy. On further analyses, radiation-resistant VS cells expressed significantly more p21 than radiation-responsive tumors., Conclusions: In response to radiation-induced DNA damage, primary VS cells can enter cell cycle arrest and express RAD51 DNA repair mechanisms to avoid cell death. Radioresistant VS cells may mount a more robust p21 response to ensure sufficient time for DNA repair. Further investigation into DNA repair proteins and cell cycle checkpoints may provide important insight on the radiobiology of VS and mechanisms for resistance., Competing Interests: No relevant conflicts of interest., (Copyright © 2021, Otology & Neurotology, Inc.)

Objectives: To review the current state of knowledge about the influence of specific genetic mutations that cause sensorineural hearing loss (SNHL) on cochlear implant (CI) functional outcomes, and how this knowledge may be integrated into clinical practice. A multistep and sequential population-based genetic algorithm suitable for the identification of congenital SNHL mutations before CI placement is also examined., Data Sources, Study Selection: A review was performed of the English literature from 2000 to 2019 using PubMed regarding the influence of specific mutations on CI outcomes and the use of next-generation sequencing for genetic screening of CI patients., Conclusion: CI is an effective habilitation option for patients with severe-profound congenital SNHL. However, it is well known that CI outcomes show substantial inter-patient variation. Recent advances in genetic studies have improved our understanding of genotype-phenotype relationships for many of the mutations underlying congenital SNHL, and have explored how these relationships may account for some of the variance seen in CI performance outcomes. A sequential genetic screening strategy utilizing next-generation sequencing-based population-specific gene panels may allow for more efficient mutation identification before CI placement. Understanding the relationships between specific mutations and CI outcomes along with integrating routine comprehensive genetic testing into pre-CI evaluations will allow for more effective patient counseling and open the door for the development of mutation-specific treatment strategies.

Objectives: In this review, we discuss current knowledge about the genetics and epigenetics of vestibular schwannoma (VS) in relation to hearing loss. A multistep and sequential genetic algorithm suitable for the identification of Neurofibromatosis Type 2 (NF2) constitutional and somatic mutations is discussed., Data Sources, Study Selection: A review was performed of the English literature from 1990 to 2019 using PubMed regarding genetics and epigenetics of vestibular schwannoma and NF2., Conclusion: NF2 is a genetic disorder characterized by NF2 mutations that affect the function of a tumor suppressor called merlin. In particular, individuals with NF2 develop bilateral VS that can lead to hearing loss and even deafness. Recent advances in genetic and epigenetic studies have improved our understanding of the genotype-phenotype relationships that affect hearing in NF2 patients. Specific constitutional NF2 mutations including particular truncating, deletion, and missense mutations have been associated with poorer hearing outcomes and more severe clinical manifestations. Epigenetic events, such as DNA methylation and histone modifications, also contribute to the development and progression of hearing loss in NF2 patients. Furthermore, the accumulation of multiple NF2 and non-NF2 genetic and epigenetic abnormalities at the level of the tumor may contribute to worse hearing outcomes. Understanding genetic and epigenetic signatures in individual NF2 patients and particularly in each VS will allow us to develop novel gene therapies and precision medicine algorithms to preserve hearing in NF2 individuals.

Hypothesis: Merlin-deficient Schwann cells (MD-SC) and primary human vestibular schwannoma (VS) cells exhibit selective uptake of sodium-fluorescein (SF), allowing for fluorescent detection and improved visualization of tumor cells, when compared with Schwann cells (SC)., Background: SF is a fluorescent compound used for fluorescence-guided resection of gliomas. The utility of SF for VS surgery has not been assessed., Methods: Mouse MD-SCs and rat SCs were cultured on 96-well plates at different cell densities and treated with SF at several drug concentrations and durations. Relative fluorescence units (RFU) were measured using a fluorometer to determine optimal treatment parameters in vitro. Subsequently, a four-point Likert scale for fluorescence visualization of pelleted cells was created and validated. Blinded observers rated SF-treated primary human VS and SC cultures, which were developed from deidentified specimens obtained from live and cadaveric donors, respectively., Results: In contrast to SCs that showed low levels of fluorescence, MD-SCs demonstrated dose-dependent increases in RFUs when treated with incremental dosages of SF as well as longer treatment and fluorescent excitation times. In addition, RFUs were higher at greater MD-SC densities. The Likert scale for fluorescence visualization was validated using nine blinded observers and there were excellent inter- and intrarater reliabilities (intraclass coefficients of 0.989 and >0.858, respectively). Using the Likert scale, human VS treated with SF received higher scores than human SCs (p < 0.001)., Conclusion: Mouse MD-SC and human VS cells demonstrate preferential uptake of SF when compared with normal primary SCs. Observers detected differences in fluorescence using the validated Likert scale. Further investigations into the utility of SF-guidance in VS surgery are warranted.

Hypothesis: Microsurgical implantation of mouse merlin-deficient Schwann cells (MD-SC) into the cerebellopontine angle of immunodeficient rats will initiate tumor formation, hearing loss, and vestibular dysfunction., Background: The progress in identifying effective drug therapies for treatment of Neurofibromatosis type II (NF2) is limited by the availability of animal models of VS that develop hearing loss and imbalance., Methods: A microsurgical technique for implanting MD-SCs onto the cochleovestibular nerve of rats was developed. Ten Rowett Nude rats were implanted with either ∼10 MD-SCs expressing luciferase (N = 5) or vehicle (N = 5). Rats received bioluminescence imaging, auditory brainstem response testing, and were observed for head tilt every 2 weeks after surgery, for a total of 6 weeks. Tumors were harvested and processed with hematoxylin & eosin staining and immunohistochemistry was performed for S100., Results: Rats implanted with MD-SCs developed significantly higher tumor bioluminescence measurements and hearing threshold shifts at multiple frequencies by the 4th and 6th weeks post-implantation, compared with control rats. Rats implanted with MD-SCs also developed gross tumor. The tumor volume was significantly greater than nerve volumes obtained from rats in the control group. All rats with tumors developed a head tilt, while control rats had no signs of vestibular dysfunction. Tumors demonstrated histological features of schwannoma and express S100., Conclusion: Using this microsurgical technique, this xenograft rat model of VS develops tumors involving the cochleovestibular nerve, shifts in hearing thresholds, and vestibular dysfunction. This animal model can be used to investigate tumor-mediated hearing loss and perform preclinical drug studies for NF2.

Background: Stereotactic radiosurgery for lateral skull base tumors can cause hearing loss when the cochleae are exposed to high doses of single-fraction radiation. Currently, there are no known nondosimetric preventative treatments for radiation-induced ototoxicity., Hypothesis: Intratympanic (IT) dexamethasone (DXM), a synthetic steroid, protects against radiation-induced auditory hair cell (HC) and hearing losses in rats in vivo., Methods: Seven rats received radiation (12 Gy) to both cochleae. In irradiated rats and six nonirradiated rats, IT DXM was randomized to one ear, while tympanic puncture without DXM was performed on the contralateral ear. Baseline and 4-week postradiation auditory-evoked potential tests were performed. The cochleae were processed for HC viability., Results: Cochleae exposed to radiation demonstrated more outer HC (OHC) loss in all turns than nonirradiated ears (p <0.05). OHCs were more susceptible to radiation injury than inner HCs in the middle and basal turns (p <0.05). In irradiated cochleae, there was a nonsignificant trend for less OHC loss with IT DXM in the basal turn when compared with placebo. IT DXM did not improve radiation-induced hearing threshold shifts; however, a high rate of tympanic membrane perforations occurred with irradiated ears which may contribute to this finding., Conclusion: Radiation induced loss of OHCs in all turns of the cochlea. IT DXM reduced OHC loss in the basal turn of irradiated ears; however, this finding did not achieve statistical significance. Although IT DXM did not affect radiation-induced hearing threshold shifts in adult rats in vivo, this may be due to a high rate of tympanic membrane perforations.

Hypothesis: Dexamethasone (DXM) protects against radiation-induced loss of auditory hair cells (HCs) in rat organ of Corti (OC) explants by reducing levels of oxidative stress and apoptosis., Background: Radiation-induced sensorineural hearing loss (HL) is progressive, dose-dependent, and irreversible. Currently, there are no preventative therapeutic modalities for radiation-induced HL. DXM is a synthetic steroid that can potentially target many of the pathways involved in radiation-induced ototoxicity., Methods: Whole OC explants were dissected from 3-day-old rat cochleae exposed to specific dosages of single-fraction radiation (0, 2, 5, 10, or 20 Gy), were either untreated or treated with DXM (75, 150, 300 μg/mL), and then cultured for 48 or 96 hours. Confocal microscopy for oxidative stress (CellRox, 48 h) and apoptosis (TUNEL assay, 96 h) and fluorescent microscopy for viable HC counts (fluorescein isothiocyanate-phalloidin, 96 h) were performed. Analysis of variance and Tukey post hoc testing were used for statistical analysis., Results: Radiation exposure initiated dose-dependent losses of inner and outer HCs, predominantly in the basal turns of the OC explants. DXM protected against radiation-induced HC losses in a dose-dependent manner. DXM significantly reduced levels of oxidative stress and apoptosis in radiation-injured OC explants (p < 0.001)., Conclusions: Radiation-initiated HC losses were dose-dependent in OC explants. DXM treatment protected explant HCs against radiation-initiated losses by decreasing the levels of oxidative stress and apoptosis. DXM may potentially be a therapeutic modality for preventing radiation-induced HL; further in vivo studies are necessary.

Hypothesis: Dexamethasone (DXM) protects against cisplatin-induced auditory hair cell (HC) loss in rat organ of Corti (OC) explants in vitro by reducing levels of oxidative stress and NADPH-Oxidase-3 (NOX-3)., Background: Intratympanic DXM has demonstrated protective effects against cisplatin-induced hearing loss in a few animal studies and one clinical trial. However, levels of protection with intratympanic DXM vary significantly between studies, which may not be a result of the intrinsic properties of DXM but rather reflect the diffusion of DXM into the cochlea. The molecular mechanisms and degree of DXM protection against cisplatin ototoxicity are currently unknown., Methods: OC explants from 3-day-old rats were cultured with no treatment or various concentrations of cisplatin (2, 5, or 10 μM) and DXM (75, 150, or 300 μg/mL) in vitro. HC viability and TUNEL assay were performed after 72 hours in vitro and levels of oxidative stress and NOX-3 were evaluated with confocal microscopy after 48 hours in vitro. Analysis of variance with Tukey's post hoc testing was performed., Results: Cisplatin initiated dose-dependent losses of outer HCs (OHCs) in the basal turns of exposed explants (p < 0.001). DXM protected against cisplatin (2 μM)-induced OHC loss in a dose-dependent manner with complete protection at 300 μg/mL of DXM (p < 0.001). DXM (150 μg/mL) significantly reduced levels of oxidative stress, NOX-3, and apoptosis in the basal turn of explants exposed to cisplatin (2 μM)., Conclusions: DXM protects against cisplatin-induced loss of OHCs in the basal turn of rat OC explants as demonstrated by reductions in oxidative stress and NOX-3 production and decreased levels of apoptotic cell death.